U.S. patent number RE45,170 [Application Number 14/015,881] was granted by the patent office on 2014-09-30 for streptococcus suis vaccines and diagnostic tests.
This patent grant is currently assigned to Stichting Dienst Landbouwkundig Onderzoek. The grantee listed for this patent is Stichting Dienst Landbouwkundig Onderzoek. Invention is credited to Hilda Elizabeth Smith.
United States Patent |
RE45,170 |
Smith |
September 30, 2014 |
Streptococcus suis vaccines and diagnostic tests
Abstract
The invention relates to Streptococcus suis infection in pigs,
vaccines directed against those infections and tests for diagnosing
Streptococcus suis infections. The invention provides an isolated
or recombinant nucleic acid encoding a capsular gene cluster of
Streptococcus suis or a gene or gene fragment derivated thereof.
The invention further provides a nucleic acid probe or primer
allowing species or serotype-specific detection of Streptococcus
suis. The invention also provides a Streptococcus suis antigen and
vaccine derived thereof.
Inventors: |
Smith; Hilda Elizabeth
(Lelystad, NL) |
Applicant: |
Name |
City |
State |
Country |
Type |
Stichting Dienst Landbouwkundig Onderzoek |
Wageningen |
N/A |
NL |
|
|
Assignee: |
Stichting Dienst Landbouwkundig
Onderzoek (Wageningen, NL)
|
Family
ID: |
26150559 |
Appl.
No.: |
14/015,881 |
Filed: |
August 30, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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PCT/NL99/00460 |
Jul 19, 1999 |
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Reissue of: |
09767041 |
Jan 22, 2001 |
7125548 |
Oct 24, 2006 |
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Foreign Application Priority Data
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Jul 22, 1998 [EP] |
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98202465 |
Jul 22, 1998 [EP] |
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98202467 |
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Current U.S.
Class: |
424/93.2;
424/244.1; 424/200.1; 424/93.44; 435/252.3; 435/253.4 |
Current CPC
Class: |
A61P
37/04 (20180101); A61P 31/04 (20180101); A61K
39/092 (20130101); C07K 14/315 (20130101); C12N
9/1048 (20130101); C12N 1/205 (20210501); A61K
2039/522 (20130101); C12R 2001/46 (20210501); A61K
39/00 (20130101) |
Current International
Class: |
A01N
63/00 (20060101); A61K 48/00 (20060101) |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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0 750 043 |
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Dec 1996 |
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EP |
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WO 92/16630 |
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Oct 1992 |
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WO |
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WO 92/21465 |
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Dec 1992 |
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WO |
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WO 93/18164 |
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Sep 1993 |
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WO |
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WO 95/06732 |
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Mar 1995 |
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WO |
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WO 95/31548 |
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Nov 1995 |
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WO |
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WO 96/21465 |
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Jul 1996 |
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WO |
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WO 96/23073 |
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Aug 1996 |
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WO |
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WO 98/19689 |
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May 1998 |
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WO |
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WO 00/05378 |
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Feb 2000 |
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WO |
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WO 00/06738 |
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Feb 2000 |
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WO |
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WO 00/37105 |
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Jun 2000 |
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WO |
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WO 02/38597 |
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May 2002 |
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WO |
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WO 02/061070 |
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Aug 2002 |
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WO |
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WO 02/061070 |
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Aug 2002 |
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WO |
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WO 2009/020391 |
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Aug 2008 |
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WO |
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|
Primary Examiner: Duffy; Patricia A
Attorney, Agent or Firm: TraskBritt, P.C.
Claims
What is claimed is:
1. A composition, comprising: a Streptococcus suis serotype 2
knockout mutant wherein the knockout mutation is in the capsular
polysaccharide (cps) gene cluster as set forth in SEQ ID NO: 9,
wherein the knockout mutation is in the cpsB gene encoding the cpsB
protein as set forth in SEQ ID NO: 13, the cpsE gene encoding the
cpsE protein as set for in SEQ ID NO:16, or the cpsF gene encoding
the cpsF protein as set forth in SEQ ID NO:17 or a combination
thereof, the knockout mutation causing a deficiency in cellular
capsular expression, and a pharmaceutically acceptable carrier or
adjuvant.
2. The composition of claim 1, wherein said Streptococcus suis
serotype 2 knockout mutant is capable of surviving in an
immune-competent host.
3. The composition of claim 2, wherein said Streptococcus suis
serotype 2 knockout mutant is capable of surviving at least 4-5
days in said immune-competent host.
4. The composition of claim 1, wherein said Streptococcus suis
serotype 2 knockout mutant expresses a Streptococcus virulence
factor or antigenic determinant.
5. The composition of claim 1, wherein said Streptococcus suis
serotype 2 knockout mutant expresses a non-Streptococcus
protein.
6. The composition of claim 5, wherein said non-Streptococcus
protein has been derived from a pathogen.
7. The composition of claim 2, wherein said Streptococcus suis has
been produced by homologous recombination.
8. The composition of claim 2, wherein said Streptococcus suis is
capable of surviving at least 8-10 days in said host.
9. The composition of claim 1, wherein the knockout mutation is in
the cpsB gene encoding the cpsB protein as set forth in SEQ ID NO:
13.
10. The composition of claim 1, wherein the knockout mutation is in
the cpsE gene encoding the cpsE protein as set forth in SEQ ID
NO:16.
11. The composition of claim 1, wherein the knockout mutation is in
the cpsF gene encoding the cpsF protein as set forth in SEQ ID
NO:17.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims priority to, and is a continuation of,
International Application No. PCT/NL99/00460, filed on Jul. 19,
1999, designating the United States of America, the contents of
which are incorporated herein by this reference, the PCT
International Patent Application itself claiming priority from
European Patent Office Application Ser. No. 98202465.5 filed Jul.
22, 1998 and European Patent Office Application Ser. No. 98202467.1
filed Jul. 22, 1998.
TECHNICAL FIELD
The invention relates to Streptococcus infections in pigs, vaccines
directed against those infections, tests for diagnosing
Streptococcus infections and bacterial vaccines. More particularly,
the invention relates to vaccines directed against Streptococcus
infections.
BACKGROUND OF THE INVENTION
Streptococcus species, of which a large variety cause infections in
domestic animals and man, are often grouped according to
Lancefield's groups. Typing according to Lancefield occurs on the
basis of serological determinants or antigens that are, among
others, present in the capsule of the bacterium, and allows for
only an approximate determination. Often, bacteria from different
groups show cross-reactivity with each other, while other
Streptococci cannot be assigned a group-determinant at all. Within
groups, further differentiation is often possible on the basis of
serotyping. These serotypes further contribute to the large
antigenic variability of Streptococci, a fact that creates an array
of difficulties within diagnosis of and vaccination against
Streptococcal infections.
Lancefield group A Streptococcus species (Group A streptococci
"GAS", Streptococcus pyogenes) are common in children, causing
nasopharyngeal infections and complications thereof. Among animals,
cattle are especially susceptible to GAS, and the resulting
mastitis.
Group A streptococci are the etiologic agents of streptococcal
pharyngitis and impetigo, two of the most common bacterial
infections in children, as well as a variety of less common, but
potentially life-threatening, infections including soft tissue
infections, bacteremia, and pneumonia. In addition, GAS are
uniquely associated with the post-infectious autoimmune syndromes
of acute rheumatic fever and post streptococcal
glomerulonephritis.
Several recent reports suggest that the incidence of both serious
infections due to GAS and acute rheumatic fever has increased
during the past decade, focusing renewed interest on defining the
attributes or virulence factors of the organism that may play a
role in the pathogenesis of these diseases.
GAS produce several surface components and extracellular products
that may be important in virulence. The major surface protein, M
protein, has been studied in the most detail and has been
convincingly shown to play a role in both virulence and immunity.
Isolates rich in M protein are able to grow in human blood, a
property thought to reflect the capacity of M protein to interfere
with phagocytosis, and these isolates tend to be virulent in
experimental animals.
Lancefield group B Streptococcus ("GBS") are most often seen in
cattle, causing mastitis; however, human infants are susceptible as
well, often with fatal consequences. Group B streptococci (GBS)
constitute a major cause of bacterial sepsis and meningitis among
human neonates born in the United States and Western Europe and are
emerging as significant neonatal pathogens in developing countries
as well.
It is estimated that GBS strains are responsible for 10,000 to
15,000 cases of invasive infection in neonates in the United States
alone. Despite advances in early diagnosis and treatment, neonatal
sepsis due to GBS continues to carry a mortality rate of 15 to 20%.
In addition, survivors of GBS meningitis have 30 to 50% incidence
of long-term neurologic sequelae. Over the past two decades,
increasing recognition of GBS as an important pathogen for human
infants has generated renewed interest in defining the bacterial
and host factors important in virulence of GBS and in the immune
response to GBS infection.
Particular attention has focused on the capsular polysaccharide as
the predominant surface antigen of the organisms. In a modification
of the system originally developed by Rebecca Lancefield, GBS
strains are serotyped on the basis of antigenic differences in
their capsular polysaccharides and the presence or absence of
serologically defined C proteins. While GBS isolated from nonhuman
sources often lack a serologically detectable capsule, a large
majority of strains associated with neonatal infection belong to
one of four major capsular serotypes, 1a, 1b, II or III. The
capsular polysaccharide forms the outermost layer around the
exterior of the bacterial cell, superficial to the cell wall. The
capsule is distinct from the cell wall-associated group B
carbohydrate. It has been suggested that the presence of sialic
acid, in the capsule of bacteria that causes meningitis, is
important for allowing these bacteria to breach the blood-brain
barrier. Indeed, in S. agalactiae, sialic acid has been shown to be
critical for the virulence function of the type III capsule. The
capsule of S. suis serotype is composed of glucose, galactose,
N-acetylglucosamine, rhamnose and sialic acid.
The group B polysaccharide, in contrast to the type-specific
capsule, is present on all GBS strains and is the basis for
serogrouping the organisms into Lancefield's group B. Early studies
by Lancefield and co-workers showed that antibodies raised in
rabbits against whole GBS organisms protected mice against
challenge with strains of homologous capsular type, demonstrating
the central role of the capsular polysaccharide as a protective
antigen. Studies in the 1970s by Baker and Kasper demonstrated that
cord blood of human infants with type III GBS sepsis uniformly had
low or undetectable levels of antibodies directed against the type
III capsule, suggesting that a deficiency of anticapsular antibody
was a key factor in susceptibility of human neonates to GBS
disease.
Lancefield group C infections, such as those with S. equi, S.
zooepidemicus, S. dysgalactiae, and others, are mainly seen in
horses, cattle and pigs, but can also cross the species barrier to
humans. Lancefield group D (S. bovis) infections are found in all
mammals and some birds, sometimes resulting in endocarditis or
septicemia.
Lancefield groups E, G, L, P, U and V (S. porcinus, S. canis, S.
dysgalactiae) are found in various hosts, causing neonatal
infections, nasopharyngeal infections or mastitis.
Within Lancefield groups R, S, and T (and with ungrouped types),
Streptococcus suis is an important cause of meningitis, septicemia,
arthritis and sudden death in young pigs (4, 46). Incidentally, it
can also cause meningitis in man (1). S. suis strains are usually
identified and classified by their morphological, biochemical and
serological characteristics (58, 59, 46). Serological
classification is based on the presence of specific antigenic
polysaccharides. So far, 35 different serotypes have been described
(9, 56, 14). In several European countries. S. suis serotype 2 is
the most prevalent type isolated from diseased pigs, followed by
serotypes 9 and 1. Serological typing of S. suis is performed using
different types of agglutination tests. In these tests, isolated
and biochemically characterized S. suis cells are agglutinated with
a panel of 35 specific sera. These methods are very laborious and
time-consuming.
Little is known about the pathogenesis of the disease caused by S.
suis, let alone about its various serotypes such as type 2. Various
bacterial components, such as extracellular and cell-membrane
associated proteins, fimbriae, hemagglutinins, and hemolysin have
been suggested as virulence factors (9, 10, 11, 15, 16, 47, 49).
However, the precise role of these protein components in the
pathogenesis of the disease remains unclear (37). It is well known
that the polysaccharide capsule of various Streptococci and other
Gram-positive bacteria plays an important role in pathogenesis (3,
6, 35, 51, 52). The capsule enables these microorganisms to resist
phagocytosis and is therefore regarded as an important virulence
factor. Recently, a role of the capsule of S. suis in the
pathogenesis was suggested as well (5). However, the structure,
organization and function of the genes responsible for capsule
polysaccharide synthesis ("cps") in S. suis is unknown. Within S.
suis, serotype 1 and 2, strains can differ in virulence for pigs
(41, 45, 49). Some type 1 and 2 strains are virulent, other strains
are not. Because both virulent and nonvirulent strains of serotype
1 and 2 strains are fully encapsulated, it may even be that the
capsule is not a relevant factor required for virulence.
Attempts to control S. suis infections or disease are still
hampered by the lack of knowledge about the epidemiology of the
disease and the lack of effective vaccines and sensitive
diagnostics. It is well known and generally accepted that the
polysaccharide capsule of various Streptococci and other
gram-positive bacteria plays an important role in pathogenesis. The
capsule enables these microorganisms to resist phagocytosis and is
therefore regarded as an important virulence factor.
Compared to encapsulated S. suis strains, non-encapsulated S. suis
strains are phagocytosed by murine polymorphonuclear leucocytes to
a greater degree. Moreover, an increase in thickness of capsule was
noted for in vivo grown virulent strains while no increase was
observed for avirulent strains. Therefore, these data again
demonstrate the role of the capsule in the pathogenesis for S. suis
as well.
Ungrouped Streptoccus species, such as S. mutans, causing caries in
humans, S. uberis.causing mastitis in cattle, and S. pneumonia,
causing major infections in humans, and Enterococcus faecilalis and
E. faecium, further contribute to the large group of
Streptococci.
Streptococcus pneumoniae (the pneumococcus) is a human pathogen
causing invasive diseases, such as pneumonia, bacteremia, and
meningitis. Despite the availability of antibiotics, pneumococcal
infections remain common and can still be fatal, especially in
high-risk groups, such as young children and elderly people.
Particularly in developing countries, many children under the age
of five years die each year from pneumococcal pneumonia. S.
pneumoniae is also the leading cause of otitis media and sinusitis.
These infections are less serious, but nevertheless incur
substantial medical costs, especially when leading to
complications, such as permanent deafness. The normal ecological
niche of the pneumococcus is the nasopharynx of man. The entire
human population is colonized by the pneumococcus at one time or
another, and at a given time, up to 60% of individuals may be
carriers. Nasopharyngeal carriage of pneumococci by man is often
accompanied by the development of protection against infection by
the same serotype. Most infections do not occur after prolonged
carriage but follow exposure to recently acquired strains. Many
bacteria contain surface polysaccharides that act as a protective
layer against the environment. Surface polysaccharides of
pathogenic bacteria usually make the bacteria resistant to the
defense mechanisms of the host, for example, the lytic action of
serum or phagocytosis. In this respect, the serotype-specific
capsular polysaccharide ("CP") of Streptococcus pneumoniae, is an
important virulence factor. Unencapsulated strains are avirulent,
and antibodies directed against the CP are protective. Protection
is serotype specific; each serotype has its own, specific CP
structure. Ninety different capsular serotypes have been
identified. Currently, CPs of 23 sero-types are included in a
vaccine.
Vaccines directed against Streptococcus infections typically aim to
utilize an immune response directed against the polysaccharide
capsule of the various Streptococcus species. especially since the
capsule is considered a primary virulence factor for these
bacteria. During infection, the capsule provides resistance against
phagocytosis and thus protects the bacteria from the immune system
of the host, and from elimination by macrophages and
neutrophils.
The capsule particularly confers the bacterium resistance to
complement-mediated opsonophagocytosis. In addition, some bacteria
express capsular polysaccharides (CPs) that mimic host molecules,
thereby avoiding the immune system of the host. Also, even when the
bacteria have been phagocytosed, intracellular killing is hampered
by the presence of a capsule.
It is generally thought that the bacterium will be recognized by
the immune system through the anticapsular-antibodies or
serum-factors bound to its capsule, and will, through opsonization,
be phagocytosed and killed only when the host has antibodies or
other serum factors directed against capsule antigens.
However, these antibodies are serotype-specific, and will often
only confer protection against only one of the many serotypes known
within a group of Streptococci.
For example, current commercially available S. suis vaccines, which
are generally based on whole-cell-bacterial preparations, or on
capsule-enriched fractions of S. suis, confer only limited
protection against heterologous strains. Also, the current
pneumococcal vaccine, which was licensed in the United states in
1983, consists of purified CPs of 23 pneumococcal serotypes whereas
at least 90 CP types exist.
The composition of this pneumococcal vaccine was based on the
frequency of the occurrence of disease isolates in the US and
cross-reactivity between various serotypes. Although this vaccine
protects healthy adults against infections caused by serotypes
included in the vaccine, it fails to raise a protective immune
response in infants younger than 18 months and it is less effective
in elderly people. In addition, the vaccine confers only limited
protection in patients with immunodeficiencies and hematology
malignancies.
Thus, improved vaccines are needed against Streptococcus
infections. Much attention is directed toward producing CP vaccines
by producing the relevant polysaccharides via chemical or
recombinant means. However, chemical synthesis of polysaccharides
is costly, and capsular polysaccharide synthesis by recombinant
means necessitates knowledge about the relevant genes, which is not
always available, and needs to be determined for every relevant
serotype.
DISCLOSURE OF THE INVENTION
The invention provides an isolated or recombinant nucleic acid
encoding a capsular (cps) gene cluster of Streptococcus suis.
Biosynthesis of capsule polysaccharides has generally been studied
in a number of Gram-positive and Gram-negative bacteria (32). In
Gram-negative bacteria, but also in a number of Gram-positive
bacteria, genes which are involved in the biosynthesis of
polysaccharides are clustered at a single locus.
Streptococcus suis capsular genes, as provided by the invention,
show a common genetic organization involving three distinct
regions. The central region is serotype specific and encodes
enzymes responsible for the synthesis and polymerization of the
polysaccharides. The central region is flanked by two regions
conserved in Streptococcus suis which encode proteins for common
functions, such as transport of the polysaccharide across the
cellular membrane. However, between species, only low homologies
exist, hampering easy comparison and detection of seemingly similar
genes. Knowing the nucleic acid encoding the flanking regions
allows type-specific determination of nucleic acid of the central
region of Streptococcus suis serotypes, as, for example, described
herein.
The invention provides an isolated or recombinant nucleic acid
encoding a capsular gene cluster of Streptococcus suis or a gene or
gene fragment derived thereof. Such a nucleic acid is, for example,
provided by hybridizing chromosomal DNA derived from any one of the
Streptococcus suis serotypes to a nucleic acid encoding a gene
derived from a Streptococcus suis serotype 1, 2 or 9 capsular gene
cluster, as provided by the invention (see for example, Tables 4
and 5) and cloning of (type-specific) genes as, for example,
described herein. At least 14 open reading frames are identified.
Most of the genes belong to a single transcriptional unit,
identifying a coordinate control of these genes. The genes and the
enzymes and proteins they encode, act in concert to provide the
capsule with the relevant polysaccharides.
The invention provides cps genes and proteins encoded thereof
involved in regulation (CpsA), chain length determination (CpsB,
C), export (CpsC) and biosynthesis (CpsE, F, G, H, J, K). Although,
at first glance, the overall organization seemed to be similar to
that of the cps and eps gene clusters of a number of Gram-positive
bacteria (19, 32, 42), overall homologies are low (see, table 3).
The region involved in biosynthesis is located at the center of the
gene cluster and is flanked by two regions containing genes with
more common functions.
The invention provides an isolated or recombinant nucleic acid
encoding a capsular gene cluster of Streptococcus suis serotype 2,
or a gene or gene fragment derived thereof, preferably as
identified in FIG. 3. Genes in this gene cluster are involved in
polysaccharide biosynthesis of capsular components and antigens.
For a further description of such genes see, for example, Table 2.
For example, a cpsA gene is provided functionally encoding
regulation of capsular polysaccharide synthesis, whereas cpsB and
cpsC are functionally involved in chain-in-chain length
determination. Other genes, such as cpsD, E, F, G, H, I, J, K and
related genes, are involved in polysaccharide synthesis,
functioning, for example, as glucosyl or glycosyltransferase. The
cpsF, G, H, I, J genes encode more type-specific proteins than the
flanking genes which are found more-or-less conserved throughout
the species and can serve as a base for selection of primers or
probes in PCR-amplification or cross-hybridization experiments for
subsequent cloning.
The invention further provides an isolated or recombinant nucleic
acid encoding a capsular gene cluster of Streptococcus suis
serotype 1 or a gene or gene fragment derived thereof, preferably
as identified in FIG. 4.
In addition, the invention provides an isolated or recombinant
nucleic acid encoding a capsular gene cluster of Streptococcus suis
serotype 9 or a gene or gene fragment derived thereof, preferably
as identified in FIG. 5.
Furthermore, the invention provides, for example, a fragment of the
cps locus or parts thereof, involved in the capsular polysaccharide
biosynthesis, of S. suis, exemplified herein for serotypes 1, 2 or
9, and allows easy identification or detection of related fragments
derived of other serotypes of S. suis.
The invention provides a nucleic acid probe or primer derived from
a nucleic acid according to the invention allowing species or
.[.serotype specific.]. .Iadd.serotype-specific .Iaddend.detection
of Streptococcus suis. Such a probe or primer (used interchangeably
herein) is, for example, a DNA, RNA or PNA (peptide nucleic acid)
probe hybridizing with capsular nucleic acid as provided by the
invention. Species-specific detection is provided preferably by
selecting a probe or primer sequence from a species-specific region
(e.g. flanking region) whereas serotype-specific detection is
provided preferably by selecting a probe or primer sequence from a
type-specific region (e.g. central region) of a capsular gene
cluster as provided by the invention. Such a probe or primer can be
used in a further unmodified form, for example, in
cross-hybridization or polymerase-chain reaction (PCR) experiments
as, for example, described in the experimental part herein. The
invention provides the isolation and molecular characterization of
additional type-specific cps genes of S. suis types 1 and 9. In
addition, we describe the genetic diversity of the cps loci of
serotypes 1, 2 and 9 among the 35 S. suis serotypes known.
Type-specific probes are identified. Also, a type-specific PCR, for
example, for serotype 9, is provided, being a rapid, reliable and
sensitive assay used directly on nasal or tonsillar swabs or other
samples of infected or carrier animals.
The invention also provides a probe or primer according to the
invention with at least one reporter molecule. Examples of reporter
molecules are manifold and known in the art; for example, a
reporter molecule can include additional nucleic acid provided with
a specific sequence (e.g. oligo-dT) hybridizing to a corresponding
sequence in which hybridization can easily be detected, for
example, because it has been immobilized to a solid support.
Yet other reporter molecules include chromophores, e.g.
fluorochromes for visual detection, for example, by light
microscopy or fluorescent in situ hybridization ("FISH")
techniques, or include an enzyme such as horseradish peroxidase for
enzymatic detection, for example in enzyme-linked assays ("EIA").
Yet other reporter molecules include radioactive compounds for
detection in radiation-based assays.
In a preferred embodiment of the invention, at least one probe or
primer according to the invention is provided (labeled) with a
reporter molecule and a quencher molecule, together with an
unlabeled probe or primer in a PCR-based test allowing rapid
detection of specific hybridization.
The invention further provides a diagnostic test or test kit
including a probe or primer as provided by the invention. Such a
test or test kit is, for example, a cross-hybridization test or
PCR-based test advantageously used in rapid detection and/or
serotyping of Streptococcus suis.
The invention further provides a protein or fragment thereof
encoded by a nucleic acid according to the invention. Examples of
such a protein or fragment are proteins described in Table 2. For
example, a cpsA protein is provided that functionally encodes
regulation of capsular polysaccharide synthesis, whereas cpsB and
cpsC are functionally involved in chain-in-chain length
determination. Other proteins or functional fragments thereof, as
provided by the invention, such as cpsD, E, F, G, H, I, J, K and
related proteins, are involved in polysaccharide biosynthesis,
functioning, for example, as glucosyl or glycosyltransferase in
polysaccharide biosynthesis of Streptococcus suis capsular
antigen.
The invention also provides a method of producing a Streptococcus
suis capsular antigen including using a protein or functional
fragment thereof as provided by the invention, and provides
therewith a Streptococcus suis capsular antigen obtainable by such
a method.
A comparison of the predicted amino acid sequences of the cps2
genes with sequences found in the databases allowed the assignment
of functions to the open reading frames. The central region
contains the type-specific glycosyltransferases and the putative
polysaccharide polymerase. This region is flanked by two regions
encoding for proteins with common functions, such as regulation and
transport of polysaccharide across the membrane. Biosynthesis of
Streptococcus capsular polysaccharide antigen using a protein or
functional fragment thereof is advantageously used in
chemo-enzymatic synthesis and the development of vaccines which
offer protection against serotype-specific Streptococcal disease,
and is also advantageously used in the synthesis and development of
multivalent vaccines against Streptococcal infections. Such
vaccines elicit ariticapsular antibodies which confer
protection.
Furthermore, the invention provides an acapsular Streptococcus
mutant for use in a vaccine, a vaccine strain derived thereof and a
vaccine derived thereof. Surprisingly, and against the grain of
common doctrine, the invention provides use of a Streptococcus
mutant deficient in capsular expression in a vaccine.
Acapsular Streptococcus mutants have long been known in the art and
can be found in nature. Griffith (J. Hyg. 27:113-159, 1928)
demonstrated that pneumococci could be transformed from one type to
another. If he injected live rough (acapsular or unencapsulated)
type 2 pneumococci into mice, the mice would survive. If, however,
he injected the same dose of live rough type 2 mixed with
heat-killed smooth (encapsulated) type 1 into a mouse, the mouse
would die, and, from the blood, he could isolate live smooth type 1
pneumococci. At that time, the significance of this transforming
principle was not understood. However, understanding came when it
was shown that DNA constituted the genetic material responsible for
phenotypic changes during transformation.
Streptococcus mutants deficient in capsular expression are found in
several forms. Some are fully deficient and have no capsule at all,
others form a deficient capsule, characterized by a mutation in a
capsular gene cluster. Deficiency can, for instance, include
capsular formation wherein the organization of the capsular
material has been rearranged, as, for example, demonstrable by
electron microscopy. Yet others have a nearly fully developed
capsule which is only deficient in a particular sugar
component.
Now, after much advance of biotechnology and despite the fact that
little is still known about the exact localization and sequence of
genes involved in capsular synthesis in Streptococci, it is
possible to create mutants of Streptococci, for example, by
homologous recombination or transposon mutagenesis, which has, for
example, been done for GAS (Wessels et al., PNAS 88:8317-8321,
1991), for GBS (Wessels et al., PNAS 86: 8983-8987, 1989), for S.
suis (Smith, ID-DLO Annual report 1996, page 18-19; Charland et
al., Microbiol. 144:325-332, 1998) and S. pneumoniae (Kolkman et
al., J. Bact. 178:3736-3741, 1996). Such recombinant derived
mutants, or isogenic mutants, can easily be compared with the
wild-type strains from which they have been derived.
In a preferred embodiment, the invention provides use of a
recombinant-derived Streptococcus mutant deficient in capsular
expression in a vaccine. Recombinant techniques useful in producing
such mutants are, for example, homologous recombination, transposon
mutagenesis, and others, wherein deletions, insertions or (point)
mutations are introduced in the genome. Advantages of using
recombinant techniques include the stability of the obtained
mutants (especially with homologous recombination and double
crossover techniques), and the knowledge about the exact site of
the deletion, mutation or insertion.
In another embodiment, the invention provides a stable mutant
deficient in capsular expression obtained, for example, through
homologous recombination or crossover integration events. Examples
of such a mutant can be found herein, for example, mutants 1OcpsB
or 10cpsEF are stable mutants as provided by the invention.
The invention also provides a Streptococcus vaccine strain and
vaccine that has been derived from a Streptococcus mutant deficient
in capsular expression. In general, the strain or vaccine is
applicable within the whole range of Streptococcal infections,
including animals or man or with zoonotic infections. It is, of
course, now possible to first select a common vaccine strain and
derive a Streptococcus mutant deficient in capsular expression
thereof for the selection of a vaccine strain and use in a vaccine
according to the invention.
In a preferred embodiment, the invention provides use of a
Streptococcus mutant deficient in capsular expression in a vaccine
wherein the Streptococcus mutant is selected from the group
composed of Streptococcus group A, Streptococcus group B,
Streptococcus suis and Streptococcus pneumoniae. Herewith the
invention provides vaccine strains and vaccines for use with these
notoriously heterologous Streptococci, of which a multitude of
serotypes exist. With a vaccine, as provided by the invention, that
is derived from a specific Streptococcus mutant that is deficient
in capsular expression, the difficulties relating to lack of
heterologous protection can be circumvented since these mutants do
not rely on capsular antigens, per se, to induce protection.
In a preferred embodiment, the vaccine strain is selected for its
ability to survive, or even replicate, in an immune-competent host
or host cells and thus can persist for a certain period, varying
from 1-2 days to more than one or two weeks, in a host, despite its
deficient character.
Although an immunodeficient host will support replication of a wide
range of bacteria that are deficient in one or more virulence
factors, in general, it is considered a characteristic of
pathogenicity of Streptococci that they can survive for certain
periods or replicate in a normal host or host cells such as
macrophages. For example, Wiliams and Blakemore (Neuropath. Appl.
Neurobiol.: 16, 345-356, 1990; Neuropath. Appl. Neurobiol.: 16,
377-392, 1990; J. Infect. Dis.: 162, 474-481, 1990) show that both
polymorphonuclear cells and macrophage cells are capable of
phagocytosing pathogenic S. suis in pigs lacking anti-S. suis
antibodies; only pathogenic bacteria could survive and multiply
inside macrophages and the pig.
In a preferred embodiment, the invention, however, provides a
deficient or avirulent mutant or vaccine strain which is capable of
surviving at least 4-5 days, preferably at least 8-10 days in the
host, thereby allowing the development of a solid immune response
to subsequent Streptococcus infection.
Due to its persistent but avirulent character, a Streptococcus
mutant or vaccine strain, as provided by the invention, is well
suited to generate specific and/or long-lasting immune responses
against Streptococcal antigens. Moreover, possible specific immune
responses of the host directed against a capsule are relatively
irrelevant because a vaccine strain, as provided by the invention,
is typically not recognized by such antibodies.
In addition, the invention provides a Streptococcus vaccine strain
according to the invention, which strain includes a mutant capable
of expressing a Streptococcus virulence factor or antigenic
determinant.
In a preferred embodiment, the invention provides a Streptococcus
vaccine strain, according to the invention, which includes a mutant
capable of expressing a Streptococcus virulence factor wherein the
virulence factor or antigenic determinant is selected from a group
of cellular components, such as muramidase-released protein
("MRP"), extracellular factor ("EF") and cell-membrane associated
proteins 60kDA heat shock protein, pneumococcal surface protein A
(Psp A), pneumolysin, C protein, protein M, fimbriae,
hemagglutinins and hemolysin or components functionally related
thereto.
In a preferred embodiment, the invention provides a Streptococcus
vaccine strain including a mutant capable of over-expressing the
virulence factor. In this way, the invention provides a vaccine
strain for incorporation in a vaccine which specifically causes a
host immune response directed against antigenically important
determinants of virulence (listed above), thereby providing
specific protection against the determinants. Over-expression can,
for example, be achieved by cloning the gene involved behind a
strong promoter, which is, for example, constitutionally expressed
in a multicopy system, either in a plasmid or via intergration in a
genome.
In yet another embodiment, the invention provides a Streptococcus
vaccine strain, according to the invention, including a mutant
capable of expressing a non-Streptococcus protein. Such a
vector-Streptococcus vaccine strain allows, when used in a vaccine,
protection against pathogens other than Streptococcus.
Due to its persistent but avirulent character, a Streptococcus
vaccine strain or mutant as provided by the invention is well
suited to generate specific and long-lasting immune responses, not
only against Streptococcal antigens, but also against other
antigens expressed by the strain. Specifically, antigens derived
from another pathogen are now expressed without the detrimental
effects of the antigen or pathogen which would otherwise have
harmed the host.
An example of such a vector is a Streptococcus vaccine strain or
mutant wherein the antigen is derived from a pathogen, such as
Actinobacillus pleuropneumonia, Mycoplasmatae, Bordetella,
Pasteurella, E. coli, Salmonella, Campylobacter, Serpulina and
others.
The invention also provides a vaccine including a Streptococcus
vaccine strain or mutant according to the invention and a
pharmaceutically acceptable carrier or adjuvant. Carriers or
adjuvants are well known in the art; examples are phosphate
buffered saline, physiological salt solutions, (double-)
oil-in-water emulsions, aluminumhydroxide, Specol, block- or
co-polymers, and others.
A vaccine according to the invention can include a vaccine strain
either in a killed or live form. For example, a killed vaccine
including a strain having (over) expressed a Streptococcal or
heterologous antigen or virulence factor is very well suited for
eliciting an immune response. In a preferred embodiment, the
invention provides a vaccine wherein the strain is live, due to its
persistent but avirulent character; a Streptococcus vaccine strain,
as provided by the invention, is well suited to generate specific
and long-lasting immune responses.
The invention also provides a method for controlling or eradicating
a Streptococcal disease in a population comprising vaccinating
subjects in the population with a vaccine according to the
invention.
In a preferred embodiment, a method for controlling or eradicating
a Streptococcal disease is provided including testing a sample,
such as a blood sample, or nasal or throat swab, feces, urine, or
other samples such as can be sampled at or after slaughter,
collected from at least one subject, such as an infant or a pig, in
a population partly or wholly vaccinated with a vaccine according
to the invention for the presence of encapsulated Streptococcal
strains or mutants. Since a vaccine strain or mutant according to
the invention is not pathogenic, and can be distinguished from
wild-type strains by capsular expression, the detection of (fully)
encapsulated Streptococcal strains indicates that wild-type
infections are still present. Such wild-type infected subjects can
then be isolated from the remainder of the population until the
infection has passed. With domestic animals, such as pigs, it is
even possible to remove the infected subject from the population as
a whole by culling. Detection of wild-type strains can be achieved
via traditional culturing techniques, or by rapid detection
techniques such as PCR detection.
In yet another embodiment, the invention provides a method for
controlling or eradicating a Streptococcal disease including
testing a sample collected from at least one subject in a
population partly or wholly vaccinated with a vaccine according to
the invention for the presence of capsule-specific antibodies
directed against Streptococcal strains. Capsule specific antibodies
can be detected with classical techniques known in the art, such as
used for Lancefield's group typing or serotyping.
A preferred embodiment for controlling or eradicating a
Streptococcal disease in a population includes vaccinating subjects
in the population with a vaccine according to the invention and
testing a sample collected from at least one subject in the
population for the presence of encapsulated Streptococcal strains
and/or for the presence of capsule-specific antibodies directed
against Streptococcal strains.
For example, a method is provided wherein the Streptococcal disease
is caused by Streptococcus suis.
The invention also provides a diagnostic assay for testing a sample
for use in a method according to the invention including at least
one means for the detection of encapsulated Streptococcal strains
and/or for the detection of capsule-specific antibodies directed
against Streptococcal strains.
The invention further provides a vaccine including an antigen
according to the invention and a suitable carrier or adjuvant. The
immunogenicity of a capsular antigen provided by the invention is,
for example, increased by linking to a carrier (such as a carrier
protein), allowing the recruitment of T-cell help in developing an
immune response.
The invention further provides a recombinant microorganism provided
with at least a part of a capsular gene cluster derived from
Streptococcus suis. The invention provides, for example, a lactic
acid bacterium provided with at least a part of a capsular gene
cluster derived from Streptococcus suis. Various food-grade lactic
acid bacteria (Lactococcus lactis, Lactobacillus casei,
Lactobacillus plantarium and Streptococcus gordonii) have been used
as delivery systems for mucosal immunization. It has now been shown
that oral (or mucosal) administration of recombinant L. lactis,
Lactobacillus, and Streptococcus gordonii can elicit local IgA
and/or IgG antibody responses to an expressed antigen. The use of
oral routes for immunization against infective diseases is
desirable because oral vaccines are easier to administer and have
higher compliance rates, and because mucosal surfaces are the
portals of entry for many pathogenic microbial agents. It is within
the skill of the artisan to provide such micro-organisms with
(additional) genes.
The invention further provides a recombinant Streptococcus suis
mutant provided with a modified capsular gene cluster. It is within
the skill of the artisan to swap genes within a Species. In a
preferred embodiment, an avirulent Streptococcus suis mutant is
selected to be provided with at least a part of a modified capsular
gene cluster according to the invention.
The invention further provides a vaccine including a microorganism
or a mutant provided by the invention. An advantage of such a
vaccine over currently used vaccines is that they include
accurately defined microorganisms and well-characterized antigens,
allowing accurate determination of immune responses against various
antigens of choice.
The invention is further explained in the experimental part of this
description without limiting the invention thereto.
DESCRIPTION OF THE FIGURES
FIG. 1 illustrates the organization of the cps2 gene cluster of S.
suis type 2. (A) Genetic map of the cps2 gene cluster. The shadowed
arrows represent potential ORFs. Interrupted ORFs indicate the
presence of stop codons or frame-shift mutations. Gene designations
are indicated below the ORFs. The closed arrows indicate the
position of the potential promoter sequences. I indicates the
position of the potential transcription regulator sequence. III
indicates the position of the 100-bp repeated sequence. (B)
Physical map of the cps2 locus. Restriction sites are as follows:
A: Alul; C: ClaI, E: EcoRI; H: HindIII; K: KpnI; M: MluI; N: NsiI;
P: PstI; S: SnaBI; Sa: SacI; X: XbaI. (C) The DNA fragments cloned
in the various plasmids.
FIG. 2 illustrates ethidium bromide stained agarose gel showing PCR
products obtained with chromosomal DNA of S. suis strains belonging
to the serotypes 1,2, 1/2, 2, 9 and 14 and cps2J, cpsII, and cps9H
primer sets as described herein. (A) cpsII primers; (B) cps2J
primers and (C) cps9H primers. Lanes 1-3: serotype 1 strains; lanes
4-6: serotype 2 strains; lanes 7-9: serotype 1/2 strains; lanes
10-12: serotype 9 strains and lanes 13-15: serotype 14 strains. (B)
Ethidium bromide stained agarose gel showing PCR products obtained
with tonsillar swabs collected from pigs carrying S. suis type 2,
type 1 or type 9 strains and cps2J, cpsII and cpsH primer sets as
described in Materials and Methods. Bacterial DNA suitable for PCR
was prepared by using the multiscreen methods as described
previously (20). (C) cpsII primers. (B) cps2J primers and (C) cps9H
primers. Lanes 1-3: PCR products obtained with tonsillar swabs
collected from pigs carrying S. suis type 1 strains; lanes 4-6: PCR
products obtained with tonsillar swabs collected from pigs carrying
S. suis type 2 strains; lanes 7-9: PCR products obtained with
tonsillar swabs collected from pigs carrying S. suis type 9
strains; lanes 10-12: PCR products obtained with chromosomal DNA
from serotype 9, 2 and 1 strains respectively; lane 13: negative
control, no DNA present.
FIG. 3 illustrates the CPS2 nucleotide sequences and corresponding
amino acid sequences from the open reading frames.
FIG. 4 illustrates the CPS 1 nucleotide sequences and
corresponding, amino acid sequences from the open reading
frames.
FIG. 5 illustrates the CPS9 nucleotide sequences and corresponding
amino acid sequences from the open reading frames.
FIG. 6 illustrates the CPS7 nucleotide sequences and corresponding
amino acid sequences from the open reading frames.
FIG. 7 illustrates alignment of the N-terminal parts of Cps2J and
Cps2K. Identical amino acids are marked by bars. The amino acids
shown in bold are also conserved in CPS14I Cps14J of S. pneumoniae
and several other glycosyltransferases (19). The aspartate residues
marked by asterisks are strongly conserved.
FIG. 8 illustrates transmission electron micrographs of thin
sections of various S. suis strains.
(A) wild type strain 10;
(B) mutant strain 10cpsB;
(C) mutant strain 10cpsEF.
Bar=100 nm
FIG. 9 illustrates the kinetics of phagocytosis of wild type and
mutant S. suis strains. (A) Kinetics of phagocytosis of wild type
and mutant S. suis strains by porcine alveolar macrophages.
Phagocytosis was determined as described herein. The Y-axis
represents the number of CFU per milliliter in the supernatant
fluids as determined by plate counting, the X-axis represents time
in minutes.
wild type strain 10;
o mutant strain 10cpsB;
.DELTA. mutant strain 10cpsEF. (B) Kinetics of intracellular
killing of wild type and mutant S. suis strains by porcine AM. The
intracellular killing was determined as described herein. The
Y-axis represents the number of CFU per ml in the supernatant
fluids after lysis of the macrophages as determined by plate
counting, the X-axis represents time in minutes.
wild type strain 10;
o mutant strain 10cpsB;
.DELTA. mutant strain 10cpsEF.
FIG. 10 illustrates the nucleotide sequence alignment of the highly
conserved 100-bp repeated element.
(1) 100-bp repeat between cps2G and cps2H
(2) 100-bp repeat within "cps2M"
(3) 100-bp repeat between cps2O and cps2P
FIG. 11 illustrates the cps2, cps9 and cps7 gene clusters of S.
suis serotypes 2, 9 and 7. (A) Genetic organization of the cps2
gene cluster [84]. The large arrows represent potential ORFs. Gene
designations are indicated below the ORFs. Identically filled
arrows represent ORFs which showed homology. The small closed
arrows indicate the position of the potential promoter sequences. |
indicates the position of the potential transcription regulator
sequence. (B) Physical map and genetic organization of the cps9
gene cluster [15]. Restriction sites are as follows: B: BamHI; P:
PstI; H: HindIII; X: XbaI. The DNA fragments cloned in the various
plasmids are indicated. The open arrows represent potential ORFs.
(C) Physical map and genetic organization of the cps7 gene cluster.
Restriction sites are as follows: C: Clal; P: PstI; Sc: ScaI. The
DNA fragments cloned in the various plasmids are indicated. The
open arrows represent potential ORFs.
FIG. 12 illustrates ethidium bromide stained agarose gel showing
PCR products. (A) Ethidium bromide stained agarose gel showing PCR
products obtained with chromosomal DNA of S. suis strains belonging
to the serotypes 1, 2, 9 and 7 and the cps7H primer set. Strain
designations are indicated above the lanes. C: negative control, no
DNA present. M: molecular size marker (lambda digested with EcoRI
and HindIII). (B) Ethidium bromide stained agarose gel showing PCR
products obtained with serotype 7 strains collected in different
countries and from different organs. Bacterial DNA suitable for PCR
was prepared by using the multiscreen method as described herein
[89]. Strain designations are indicated above the lanes. M:
molecular size marker (lambda digested with EcoRI and HindIII).
DETAILED DESCRIPTION OF THE INVENTION
Experimental part
Material and Methods
Bacterial strains and growth conditions.
The bacterial strains and plasmids used in this study are listed in
Table 1. S. suis strains were grown in Todd-Hewitt broth (code
CM189, Oxoid), and plated on Columbia agar blood base (code CM331,
Oxoid) containing 6% (v/v) horse blood. E. coli strains were grown
in Luria broth (28) and plated on Luria broth containing 1.5% (w/v)
agar. If required, antibiotics were added to the plates at the
following concentrations: spectinomycin: 100 .mu.g/ml for S. suis
and 50 .mu.g/ml for E. coli and ampicillin, 50 .mu.g/ml.
Serotyping. The S. suis Strains were serotyped by the slide
agglutination test with serotype-specific antibodies (44).
DNA techniques. Routine DNA manipulations were performed as
described by Sambrook et al. (36).
Alkaline phosphatase activity. To screen for PhoA fusions in E.
coli, plasmid libraries were constructed. Therefore, chromosomal
DNA of S. suis type 2 was digested with AluI. The 300-500-bp
fragments were ligated to Smal-digested pPHOS2. Ligation mixtures
were transformed to the PhoA E. coli strain CC118. Transformants
were plated on LB media supplemented with
5-Bromo-4-chloro-3-indolylfosfaat (BCIP, 50 .mu.g/ml, Boehringer,
Mannheim, Germany). Blue colonies were purified on fresh LB/BCIP
plates to verify the blue phenotype.
DNA sequence analysis. DNA sequences were determined on a 373A DNA
Sequencing System (Applied Biosystems, Warrington, GB). Samples
were prepared by using an ABI/PRISM dye terminator cycle sequencing
ready reaction kit (Applied Biosystems). Sequencing data were
assembled and analyzed using the MacMollyTetra program. Custom-made
sequencing primers were purchased from Life Technologies.
Hydrophobic stretches within proteins were predicted by the method
of Klein et al. (17). The BLAST program available on Netscape
Navigator.TM. was used to search for protein sequences related to
the deduced amino acid sequences.
Construction of gene-specific knock-out mutants of S. suis. To
construct the mutant strains 10cpsB and 10cpsEF, we
electrotransformed the pathogenic serotype 2 strain 10 (45, 49) of
S. suis with pCPS 11 and pCPS28 respectively. In these plasmids,
the cpsB and cpsEF genes were disturbed by the insertion of a
spectinomycin-resistance gene. To create pCPS11, the internal 400
by PstIBamHI fragment of the cpsB gene in pCPS7 was replaced by the
Spc.sup.R gene. For this purpose, pCPS7 was digested with PstI and
BamHI and ligated to the 1,200-bp PstI-BamHi fragment, containing
the Spc.sup.R gene; from pIC-spc. To construct pCPS28, we have used
pIC20R. In this plasmid we inserted the KpnI-SalI fragment from
pCPS17 (resulting in pCPS25) and the XbaI-ClaI fragment from pCPS20
(resulting in pCPS27). pCPS27 was digested with PstI and XhoI and
ligated to the 1,200-bp Pstl-Xhol fragment, containing the
Spc.sup.R gene of pIC-spc. The electrotransformation to S. suis was
carried out as described before (38).
Southern blotting and hybridization. Chromosomal DNA was isolated
as described by Sambrook et al. (36). DNA fragments were separated
on 0.8% agarose gels and transferred to Zeta-Probe GT membranes
(Bio-Rad) as described by Sambrook et al. (36). DNA probes were
labeled with [(.sup.-32p] dCTP (3000 Ci mmol.sup.-1; Amersham) by
use of a random primed labeling kit (Boehringer). The DNA on the
blots was hybridized at 65.degree. C. with appropriate DNA probes
as recommended by the supplier of the Zeta-Probe membranes. After
hybridization, the membranes were washed twice with a solution of
40 mM sodium phosphate, pH 7.2, 1 mM EDTA, 5% SDS for 30 min at
65.degree. C. and twice with a solution of 40 mM sodium phosphate,
pH 7.2, 1 mM EDTA, 1% SDS for 30 min at 65.degree. C.
PCR. The primers used in the cps2J PCR correspond to the positions
13791-13813 and 14465-14443 in the S. suis cps2 locus. The
sequences were: 5'-CAAACGCAAGGAATTACGGTATC-3' (SEQ. ID. No. 1) and
5'-GAGTATCTAAAGAATGCCTATTG-3' (SEQ. ID. No. 2). The primers used
for the cpslI PCR correspond to the positions 4398-4417 and
4839-4821 in the S. suis cps1 sequence. The sequences were:
5'-GGCGGTCTAGCAGATGCTCG-3' (SEQ. ID. No. 3) and
5'-GCGAACTGTTAGCAATGAC-3' (SEQ. ID. No. 4). The primers used in the
cps9H PCR correspond to the positions 4406-4126 and 4494-4475 in
the S. suis cps9 sequence. The sequences were:
5'-GGCTACATATAATGGAAGCCC3' (SEQ. ID No. 5) and
5'-CGGAAGTATCTGGGCTACTG-3' (SEQ. ID. No. 6).
Construction of gene-specific knock-out mutants of S. suis. To
construct the mutant strains 10cpsB and 10cpsEF, we
electrotransformed the pathogenic serotype 2 strain 10 of S. suis
with pCPS11 and pCPS28 respectively. In these plasmids, the cpsB
and cpsEF genes were disturbed by the insertion of a
spectinomycin-resistance gene. To create pCPS11, the internal 400
bp PstI-BamHI fragment of the cpsB gene in pCPS7 was replaced by
the Spc.sup.R gene. For this purpose, pCPS7 was digested with PstI
and BamHI and ligated to the 1,200-bp PstI-BamHI fragment,
containing the Spc.sup.R gene, from pIC-spc. To construct pCPS28,
we have used pIC20R. In this plasmid, we inserted the KpnI-SalI
fragment from pCPS17 (resulting in pCPS25) and the XbaI-ClaI
fragment from pCPS20 (resulting in pCPS27). pCPS27 was digested
with PSI and XhoI and ligated to the 1,200-bp PstI-XhoI fragment,
containing the Spc.sup.R gene of pIC-spc. The electrotransformation
to S. suis was carried out as described before (38).
Phagocytosis assay. Phagocytosis assays were performed as described
by Leij et al. (23). Briefly, to opsonize the cells, 10.sup.7 S.
suis cells were incubated with 6% SPF-pig serum for 30 min at
37.degree. C. in a head-over-head rotor at 6 rpm. 10.sup.7 AM and
10.sup.7 opsonized S. suis cells were combined and incubated at
37.degree. C. under continuous rotation at 6 rpm. At 0, 30, 60 and
90 min, 1- ml samples were collected and mixed with 4 ml of
ice-cold EMEM to stop phagocytosis. Phagocytes were removed by
centrifugation for 4 min at 110.times.g and 4.degree. C. The number
of colony-forming units, ("CFU") in the supernatants was
determined. Control experiments were carried out simultaneously by
combining 10.sup.7 opsonized S. suis cells with EMEM (without
AM).
Killing assays. AM (10.sup.7/ml) and opsonized S. suis cells
(10.sup.7/ml) were mixed 1:1 and incubated for 10 min at 37.degree.
C. under continuous rotation at 6 rpm. Ice-cold EMEM was added to
stop further phagocytosis and killing. To remove extracellular S.
suis cells, phagocytes were washed twice (4 min, 110.times.g,
4.degree. C.) and resuspended in 5 ml EMEM containing 6% SPF serum.
The tubes were incubated at 37.degree. C. under rotation at 6 rpm.
After 0, 15, 30, 60 and 90 min, samples were collected and mixed
with ice-cold EMEM to stop further killing. The samples were
centrifuged for 4 min at 110.times.g at 4.degree. C. and the
phagocytic cells were lysed in EMEM containing 1% saponine for 20
min at room temperature. The number of CFU in the suspensions was
determined.
Pigs. Germfree pigs, crossbreeds of Great Yorkshire and Dutch
Landrace, were obtained from sows by caesarian sections. The
surgery was performed in sterile flexible film isolators. Pigs were
allotted to groups, each consisting of 4 pigs, and were housed in
sterile stainless steel incubators.
Experimental infections. Pigs were inoculated intranasally with S.
suis type 2 as described before. To predispose the pigs for
infection with S. suis, five-day old pigs were inoculated
intranasally with about 10.sup.7 CFU of Bordetella bronchiseptica
strain 92932. Two days later, the pigs were inoculated intranasally
with S. suis type 2 (10.sup.6 CFU). Pigs were monitored twice daily
for clinical signs of disease, such as fever, nervous signs and
lameness. Blood samples were collected three times a week from each
pig. White blood cells were counted with a cell counter. To monitor
infection with S. suis and B. bronchiseptica and to check for
absence of contaminants, we collected swabs of nasopharynx and
feces daily. The swabs were plated directly onto Columbia agar
containing 6% horse blood. After three weeks, the pigs were killed
and examined for pathological changes. Tissue specimens from the
central nervous system, serosae, and joints were examined
bacteriologically and histologically as described herein (45, 49).
Colonization of the serosae was scored positively when S. suis was
isolated from the pericardium, thoracal pleura or the peritoneum.
Colonization of the joints was scored positively when S. suis was
isolated from one or more joints (12 joints per animal were
scored).
Vaccination and challenge. One week old pigs were vaccinated
intravenously with a dosage of 106 cfu of the S. suis strains
10cpsEF or 10cpsB. Three weeks later, the pigs were challenged
intravenously with the pathogenic Serotype 2 strain 10 (107 cfu).
Disease monitoring, hematological, serological and bacteriological
examinations as well as post-mortum examinations were as described
before under experimental infections.
Electron Microscopy. Bacteria were prepared for electron microscopy
as described by Wagenaar et al. (50). Shortly, bacteria were mixed
with agarose ND (Boehringer) of 37.degree. C. to a concentration of
0.7%. The mixture was immediately cooled on ice. Upon gelifying,
samples were cut into 1 to 1.5 mm slices and incubated in a
fixative containing 0.8% glutaraldehyde and 0.8% osmiumtetraoxide.
Subsequently, the samples were fixed and stained with uranyl
acetate by microwave stimulation, dehydrated and imbedded in
eponaraldite resin. Ultra-thin sections were counterstained with
lead citrate and examined with a Philips CM 10 electron microscope
at 80 kV (FIG. 8).
Isolation of porcine alveolar macrophages (AM). Porcine AM were
obtained from the lungs of specific pathogen free ("SPF") pigs.
Lung lavage samples were collected as described by van Leengoed et
al. (43). Cells were suspended in EMEM containing 6% (v/v). SPF-pig
serum and adjusted to 10.sup.7 cells per ml.
RESULTS
Identification of the cps locus.
The cps locus of S. suis type 2 was identified through a strategy
developed for the genetic identification of exported proteins (13,
31). In this system, we used a plasmid (pPHOS2) containing a
truncated alkaline phosphatase gene (13). The gene lacked the
promoter sequence, the translational start site and the signal
sequence. The truncated gene is preceded by a unique Smal
restriction site. Chromosomal DNA of S suis type 2, digested with
AluI, was randomly cloned in this restriction site. Because
translocation of PhoA across the cytoplasmic membrane of E. coli is
required for enzymatic activity, the system can be used to select
for S. suis fragments containing a promoter sequence, a
translational start site and a functional signal sequence. Among
560 individual E. coli clones tested, 16 displayed a dark blue
phenotype when plated on media containing BCIP. DNA sequence
analysis of the inserts from several of these plasmids was
performed (results not shown) and the deduced amino acid sequences
were analyzed. The hydrophobicity profile of one of the clones
(pPHOS7, results not shown) showed that the N-terminal part of the
sequence resembled the characteristics of a typical signal peptide:
a short hydrophilic N-terminal region is followed by a hydrophobic
region of 38 amino acids. These data indicate that the phoA system
was successfully used for the selection of S. suis genes encoding
exported proteins. Moreover, the sequences were analyzed for
similarities present in the databases. The sequence of pPHOS7
showed a high similarity (37% identity) with the protein encoded by
the cps14C gene of Streptococcus pneumoniae (19). This strongly
suggests that pPHOS7 contains a part of the cps operon of S. suis
type 2.
Cloning of the flanking cps genes. In order to clone the flanking
cps genes of S. suis type 2, the insert of pPHOS7 was used as a
probe to identify chromosomal DNA fragments which contain flanking
cps genes. A 6-kb HindIII fragment was identified and cloned in
pKUN19. This yielded clone pCPS6 (FIG. 1, part C). Sequence
analysis of the insert of pCPS6 revealed that pCPS6 most probably
contained the 5'-end of the cps locus, but still lacked the 3'-end.
Therefore, sequences of the 3'-end of pCPS6 were in turn used as a
probe to identify chromosomal fragments containing cps sequences
located further downstream. These fragments were also cloned in
pKUN19, resulting in pCPS17. Using the same system of chromosomal
walking, we subsequently generated the plasmids pCPS18, pCPS20,
pCPS23 and pCPS26, containing downstream cps sequences.
Analysis of the cps operon. The complete nucleotide sequence of the
cloned fragments was determined (FIG. 4). Examination of the
compiled sequence revealed the presence of at least 13 potential
open reading frames (Orfs), which were designated as Orf 2Y, Orf2X
and Cps2A-Cps2K (FIG. 1, part A; .Iadd.FIG. 11, part A.Iaddend.).
Moreover, a 14th, incomplete Orf (Orf 2Z) was located at the 5'-end
of the sequence. Two potential promoter sequences were identified.
One was located 313 bp (locations 1885-1865 and 1884-1889) upstream
of Orf2X. The other potential promoter sequence was located 68 bp
upstream of Orf2Y (locations 2241-2236 and 2216-2211). Orf2Y is
expressed in opposite orientation. Between Orfs 2Y and 2Z, the
sequence contained a potential stem-loop structure, which could act
as a transcription terminator. Each Orf is preceded by a
ribosome-binding site and the majority of the Orfs are very closely
linked. The only significant intergenic gap was found between Cps2G
and Cps2H (389 nucleotides). However, no obvious promoter sequences
or potential stem-loop structures were found in this region. These
data suggest that Orf2X and Cps2A-Cps2K are arranged as an
operon.
An overview of all Orfs with their properties is shown in Table 2.
The majority of the predicted gene products is related to proteins
involved in polysaccharide biosynthesis. Orf2Z showed some
similarity with the YitS protein of Bacillus subtilis. YitS was
identified during the sequence analysis of the complete genome of
B. subtilis. The function of the protein is unknown.
Orf2Y showed similarity with the YcxD protein of B. subtilis (53).
Based on the similarity between YcxD and MocR of Rhizohium meliloti
(33), YcxD was suggested to be a regulatory protein.
Orf2X showed similarity with the hypothetical YAAA proteins of
Haemophilus influenzae and E. coli. The function of these proteins
is unknown.
The gene products encoded by the cps2A, cps2B, cps2C and cps2D
genes showed approximate similarity with the CpsA, CpsC, CpsD and
CpsB proteins of several serotypes of Streptococcus pneumoniae
(19), respectively. This suggests similar functions for these
proteins. Hence, Cps2A may have a role in the regulation of the
capsular polysaccharide synthesis. Cps2B and Cps2C could be
involved in the chain length determination of the type 2 capsule
and Cps2C can play an additional role in the export of the
polysaccharide. The Cps2D protein of S. suis is related to the CpsB
protein of S. pneumoniae and to proteins encoded by genes of
several other Gram-positive bacteria involved in polysaccharide or
exopolysaccharide synthesis, but their function is unknown
(19).
The protein encoded by the cps2E gene showed similarity to several
bacterial proteins with glycosyltransferase activities Cps14E and
Cps19fE of S. pneumoniae serotypes 14 and 19F (18, 19, 29), CpsE of
Streptococcus salvarius (X94980) and CpsD of Streptococcus
agalactiae (34). Recently. Kolkman et al. (18) showed that Cps14E
is a glucosyl-1-phosphate transferase that links glucose to a lipid
carrier, the first step in the biosynthesis of the S. pneumoniae
type 14 repeating unit. Based on these data, a similar function may
be fulfilled by Cps2E of S. suis.
The protein encoded by the cps2F gene showed similarity to the
protein encoded by the rfbU gene of Salmonella enteritica.(25).
This similarity is most pronounced in the C-terminal regions of
these proteins. The rfbU gene was shown to encode
mannosyltransferase activity (25).
The cps2G gene encoded a protein that showed moderate similarity
with the rfbF gene product of Campylohacter hyoilei (22), the epsF
gene product of S. thermophilus (40) and the capM gene product of
S. aureus (24). On the basis of similarity, the rfbF, epsF and capM
genes are suggested to encode galactosyltransferase activities.
Hence, a similar glycosyltransferase activity could be fulfilled by
the cps2G gene product.
The cps2H gene encodes a protein that is similar to the N-terminal
region of the lgtD gene product of Haemophilus influenzae (U32768).
Moreover, the hydrophobicity plots of Cps2H and LgtD looked very
similar in these regions (data not shown). Based on sequence
similarity, the .[.IgtD.]. .Iadd.lgtD .Iaddend.gene product was
suggested to have glycosyltransferase activity (U32768).
The gene product encoded by the cps2I gene showed some similarity
with a protein of Actinobacillus actinomycetemcomitans (AB002668).
This protein is part of the gene cluster responsible for the
serotype-b-specific antigen of A. actinomycetemcomitans. The
function of the protein is unknown.
The gene products encoded by the cps2J and cps2K genes showed
significant similarities to the Cps14J protein of S. pneumoniae.
The cps14J gene of S. pneumoniae was shown to encode a
.beta.-1,4-galactosyltransferase activity. In S. pneumoniae, CpsJ
is responsible for the addition of the fourth (i.e. last) sugar in
the synthesis of the S. pneumoniae serotype 14 polysaccharide (20).
Even some similarity was found between Cps2J and Cps2K (FIG. 2,
25.5% similarity). This similarity was most pronounced in the
N-terminal regions of the proteins (FIG. 7). Recently, two small
conserved regions were identified in the N-terminus of Cps14J and
Cps14I and their homologues (20). These regions were predicted to
be important for catalytic activity. Both regions, DXS and DXDD
(FIG. 2), were also found in Cps2J and Cps2K.
Distribution of the cps2 genes in other S. suis serotypes. To
examine the relationship between the cps2 genes and cps genes in
the other S. suis serotypes, we performed crosshybridization
experiments. DNA fragments of the individual cps2 genes were
amplified by PCR, labeled with .sup.32P, and used to probe Southern
blots of chromosomal DNA of the reference strains of the 35
different S. suis serotypes. Large variations in the hybridization
patterns were observed (Table 4). As a positive control, we used a
probe specific for 16S rRNA. The 16S rRNA probe hybridized with all
serotypes tested. However, none of the other genes tested were
common in all serotypes. Based on the genetic organization of the
genes, we previously suggested that orfX and cpsA-cpsK genes are
part of one operon and that the proteins encoded by these genes are
all involved in polysaccharide biosynthesis. OrfY and OrfZ are not
a part of this operon, and their role in the polysaccharide
biosynthesis is unclear. Based on sequence similarity data, OrfY
may be involved in regulation of the cps2 genes. OrfZ is proposed
to be unrelated to polysaccharide biosynthesis. Probes specific for
the orfZ, orfY, orfX, cpsA, cpsB, cpsC and cpsD genes hybridized
with most other serotypes. This suggests that the proteins encoded
by these genes are not type-specific, but may perform more common
functions in biosynthesis of the capsular polysaccharide. This
confirms previous data which showed that the cps2A-cps2D genes
showed strong similarity to cps genes of several serotypes of
Streptococcus pneumoniae. Based on this similarity, Cps2A is
possibly a regulatory protein, whereas Cps2B and Cps2C may play a
role in length determination and export of polysaccharide. The
cps2E gene hybridized with DNA of Serotypes 1, 2, 14 and 1/2. The
cps2E gene showed a strong similarity to the cps14E gene of S.
pneumoniae (18). T enzyme was shown to have a glucosyl-1-phosphate
activity and catalyzed the transfer of glucose to a lipid carrier
(18). These data indicate that a glycosyltransferase closely
related to Cps14E may be responsible for the first step in the
biosynthesis of polysaccharide in the S. suis serotypes 1, 2, 14
and 1/2. The cps2F, cps2G, cps2H, cps2I and cps2J genes hybridized
with chromosomal DNA of serotypes 2 and 1/2 only. The cps2G gene
showed an additional weak hybridization signal with DNA of serotype
34. In agglutination tests, serotype 1/2 showed agglutination with
sera specific for serotype 2 as well as with sera specific for
serotype 1. This suggests that serotype 1/2 shares antigenic
determinants with both types 1 and 2. The hybridization data
confirmed these data. All putative glycosyltransferases present in
serotype 2 are also present in serotype 1/2. The cps2K gene showed
a hybridization pattern similar to the cps2E gene. Hybridization
was observed with DNA of serotypes 1, 2, 14 and 1/2. Taken
together, these hybridization data show that the cps2 gene cluster
can be divided into three regions: a central region containing the
type-specific genes is flanked by two regions containing common
genes for various serotypes.
Cloning of the type-specific cps genes of serotypes 1 and 9. To
clone the type-specific cps genes of S. suis serotype 1, we used
the cps2E gene as a probe to identify chromosomal DNA fragments of
type 1 which contain flanking cps genes. A 5 kb EcoRV fragment was
identified and cloned in pKUN19. This yielded pCPS1-1 (FIG. 1, part
B). This fragment was in turn used as a probe to identify an
overlapping 2.2 kb HindIII fragment. pKUN19 containing this HindIII
fragment was designated pCPS1-2. The same strategy was followed to
identify and clone the type-specific cps genes of serotype 9. In
this case, we used the cps2D gene as a probe. A 0.8 kb HindIII-XbaI
fragment was identified and cloned, yielding pCPS9-1 (FIG. 1, part
C). This fragment was in turn used as a probe to identify a 4 kb
XbaI fragment. pKUN19 containing this 4 kb XbaI fragment was
designated pCPS9-2.
Analysis of the cloned cpsl genes. The complete nucleotide sequence
of the inserts of pCPS1-1 and pCPS1-2 was determined (FIG. 5).
Examination of the sequence revealed the presence of five complete
and two incomplete Orfs (FIG. 1, part B). Each Orf is preceded by a
ribosome-binding site. In accord with data obtained for the cps2
genes of serotype 2, the majority of the Orfs is very closely
linked. The only significant gap (718 bp) was found between Cps1G
and Cps1H. No obvious promoter sequences or potential stem-loop
structures could be found in this region. This suggests that, as in
serotype 2, the cps genes in serotype 1 are arranged in an
operon.
An overview of the Orfs and their properties is shown in Table 2.
As expected on the basis of the hybridization data (Table 4), the
protein encoded by the cps1E gene was related to Cps2E of S. suis
type 2 (identity of 86%). The fragment cloned in pCPS1-1 lacked the
coding region for the first 7 amino acids of the cps1E gene.
The protein encoded by the cps1F and cps1G genes showed strong
similarity to the Cps14F and Cps14G proteins of Streptococcus
pneumoniae serotype 14, respectively (20). The function of the
Cps14F is not completely clear, but it has been suggested that
Cps14F has a role in glycosyltransferase activity. The cps14G gene
of S. pneumoniae was shown to encode .beta.-1,
4-galactosyltransferase activity. In S. pneumoniae type 14, this
activity is required for the second step in the biosynthesis of the
oligosaccharide subunit (20). Based on the similarity of the data,
similar glyco syltransferase and enhancing activities are suggested
for the cps1G and cps1F genes of S. suis type 1.
The protein encoded by the cps1H gene showed similarity to the
Cps14M protein of S. pneumoniae (20). Based on sequence similarity,
Cps14H was proposed to be the polysaccharide polymerase (20).
The protein encoded by the cps1I gene showed some similarity with
the Cps14J protein of S. pneumoniae (19). The cps14J gene was shown
to encode a .beta.-1, 4-galactosyltransferase activity, responsible
for the addition of the fourth (i.e. last) sugar in the synthesis
of the S. pneumoniae serotype 14 polysaccharide.
Between Cps1G and Cps1H, a gap of 718 bp was found. This region
revealed three small Orfs. The three Orfs were expressed in three
different reading frames and were not preceded by potential
ribosome binding sites, nor contained potential start sites.
However, the three potential gene products encoded by this region
showed some similarity with three successive regions of the
C-terminal part of the EpsK protein of Streptococcus thermophilus
(27% identity, 40). The region related to the first 82 amino acids
is lacking.
Analysis of the cloned cps9 genes. We also determined the complete
nucleotide sequence of the inserts of pCPS9-1 and pCPS9-2 (FIG. 6).
Examination of the sequence revealed the presence of three complete
and two incomplete Orfs (FIG. 1, part C). As in serotypes 1 and 2,
all Orfs are preceded by a ribosome-binding site and are very
closely coupled. As suggested by the hybridization data (Table 4),
the Cps2D and Cps9D proteins were highly related (Table 2). Based
on sequence comparisons, pCPS9-1 lacked the first 27 amino acids of
the Cps9D protein.
The protein encoded by the cps9E gene showed some similarity with
the CapD protein of Staphylococcus aureus serotype 1 (24). Based on
sequence similarity data, the Cap1D protein was suggested to be an
epimerase or a dehydratase involved in the synthesis of
N-acetylfiuctosamine or N-acetylgalactosamine (63).
Cps9F showed some similarity to the CapM proteins of S. aureus
serotypes 5 and 8 (61, 64, 65). Based on sequence similarity data,
Cap5M and Cap8M are proposed to be glycosyltransferases (63).
The protein encoded by the cps9G gene showed some similarity to a
protein of Actinobacillus actinomycetemcomitans (AB002668.sub.--4).
This protein is part of a gene cluster responsible for the
.[.serotype-b specific.]. .Iadd.serotype b-specific
.Iaddend.antigens of Actinobacillus actinomycetemcomitans. The
function of the protein is unknown.
The protein encoded by the cps9H gene showed some similarity to the
rfbB gene of Yersinia enterolitica (68). The RfbB protein was shown
to be essential for O-antigen synthesis, but the function of the
protein in the synthesis of the 0:3 lipopolysaccharide is
unknown.
Serotype 1 and serotype .[.9 specific.]. .Iadd.9-specific
.Iaddend.cps genes. To determine whether the cloned fragments in
pCPS1-1, pCPS1-2, pCPS9-1 and pCPS9-2 contained the type-specific
genes for serotype 1 and 9, respectively, cross-hybridization
experiments were performed. DNA fragments of the individual cps1
and cps9 genes were amplified by PCR, labeled with .sup.32P, and
used to probe Southern blots of chromosomal DNA of the reference
strains of the 35 different S. suis serotypes. The results are
shown in Table 5. Based on the data obtained with the cps2E probe
(Table 4), the cps1E probe was expected to hybridize with
chromosomal DNA of S. suis serotypes 1, 2, 14, 27 and 1/2. The
cps1H, cps9E and cps9F probes hybridized with most other serotypes
However, the cps1F and cps1G and cps1I probes hybridized with
chromosomal DNA of serotypes 1 and 14 only. The cps9G and cps9H
probes hybridized with serotype 9 only. These data suggest that the
cps9G and cps9H probes are specific for serotype 9 and, therefore,
could be useful tools for the development of rapid and sensitive
diagnostic tests for S. suis type 9 infections.
.[.Type specific.]. .Iadd.Type-specific .Iaddend.PCR. So far, the
probes were tested on the 35 different reference strains only. To
test the diagnostic value of the type-specific cps probes further,
several other S. suis serotype 1, 2, 1/2, 9 and 14 strains were
used. Moreover, since a PCR-based method would be even more rapid
and sensitive than a hybridization test, we tested, whether we
could use a PCR for the serotyping of the S. suis strains. The
oligonucleotide primer sets were chosen within the cps2J, cps1I and
cps9H genes. Amplified fragments of 675 bp, 380 bp and 390 bp were
expected, respectively. The results show that 675 bp fragments were
amplified on type 2 and 1/2 strains using cps2J printers; 380 bp
fragments were amplified on type 1 and 14 strains using cps1I
primers and 390 bp fragments were amplified on type 9 strains using
cps9H primers.
Construction of mutants impaired in capsule production. To evaluate
the role of the capsule of S. suis type 2 in pathogenesis, we
constructed two isogenic mutants in which capsule production was
disturbed. To construct mutant 10cpsB, pCPS11 was used. In this
plasmid, a part of the cps2B gene was replaced by the
spectinomycin-resistance gene. To construct mutant strain 10cpsEF,
the plasmid pCPS28 was used. In pCPS28, the 3'-end of cps2E gene,
as well as the 5'-end, of cps2F gene, were replaced by the
spectinomycin-resistance gene. pCPS 11 and pCPS28 were used to
electrotransform strain 10 of S suis type 2 and
spectinomycin-resistant colonies were selected. Southern blotting
and hybridization experiments were used to select double crossover
integration events (results not shown). To test whether the
capsular structure of the strains 10cpsB and 10cpsEF was disturbed,
we used a slide agglutination test using a suspension of the mutant
strains in hyperimmune anti-S. suis type 2 serum (44). The results
showed that even in the absence of .[.serotype specific.].
.Iadd.serotype-specific .Iaddend.antisera, the bacteria
agglutinated. This indicates that, in the mutant strains, the
capsular structure was disturbed. To confirm this, thin sections of
wild type and mutant strains were compared by electron microscopy.
The results showed that, compared to the wild type (FIG. 3, part
A), the amount of capsule produced by the mutant strains was
greatly reduced (FIG. 3, parts B and C). Almost no capsular
material could be detected on the surface of the mutant
strains.
Capsular mutants are sensitive to phagocytosis and killing by
porcine alveolar macrophages ("PAM"). The capsular mutants were
tested for their ability to resist phagocytosis by PAM in the
presence of porcine SPF serum. The wild type strain 10 seemed to be
resistant to phagocytosis under these conditions (FIGS. 9A and 9B).
In contrast, the mutant strains were efficiently ingested by
macrophages (FIGS. 9A and 9B). After 90 min., more than 99.7%
(strain 10cpsB) and 99.8% (strain 10cpsEF) of the mutant cells were
ingested by the macrophages. Moreover, as shown in FIGS. 9A and 9B
the ingested strains were efficiently killed by the macrophages.
90-98% of all ingested cells were killed within 90 min. No
differences could be observed between wild type and mutant strains.
These data indicate that the capsule of S. suis type 2 efficiently
protects the organism from uptake by macrophages in vitro.
Capsular mutants are less virulent for germfree piglets. The
virulence properties of the wild-type and mutant strains were
tested after experimental infection of newborn germ-free pigs (45,
49). Table 1 shows that specific and nonspecific signs of disease
could be observed in all pigs inoculated with the wild type strain.
Moreover, all pigs inoculated with the wild type strain died during
the course of the experiment or were killed because of serious
illness or nervous disorders (Table 3). In contrast, the pigs
inoculated with strains 10cpsB and 10cpsEF showed no specific signs
of disease and all pigs survived until the end of the experiment
(Table 6). The temperature of the pigs inoculated with the wild
type strain increased 2 days after inoculation and remained high
until day 5 (Table 3). The temperature of the pigs inoculated with
the mutant strains sometimes exceeded 40.degree. C., however, we
could observe significant differences in the fever index (i.e.
percent of observations in an experimental group during which pigs
showed fever (>40.degree. C.)) between pigs inoculated with wild
type and mutant strains. All pigs showed increased numbers of
polymorphonuclear leucocytes (PMLs) (>10.times.10.sup.9 PMLs per
litre) (Table 3). However, in pigs inoculated with the mutant
strains, the percentage of samples with increased numbers of PMLs
was considerably lower. S. suis strains and B. bronchiseptica could
be isolated from the nasopharynx and feces swab samples of all pigs
from 1 day post-infection until the end of the experiment (Table
3). Postmortem, the wild type strain could frequently be isolated
from the central nervous system ("CNS"), kidney, heart, liver,
spleen, serosae, joints and tonsils. Mutant strains could easily be
recovered from the tonsils, but were never recovered from the
kidney, liver or spleen. Interestingly, low numbers of the mutant
strains were isolated from the CNS, the serosae, the joints, the
lungs and the heart. Taken together, these data strongly indicated
that mutant S. suis strains, impaired in capsule production, are
not virulent for young germfree pigs.
We describe the identification and the molecular characterization
of the cps locus, involved in the capsular polysaccharide
biosynthesis, of S. suis. Most of the genes seemed to belong to a
single transcriptional unit, suggesting a coordinate control of
these genes. We assigned functions to most of the gene products. We
thereby identified regions involved in regulation (Cps2A), chain
length determination (Cps2B, C), export (Cps2C) and biosynthesis
(Cps2E, F, G, H, J, K). The region involved in biosynthesis is
located at the center of the gene cluster and is flanked by two
regions containing genes with more common functions. The incomplete
orf2Z gene was located at the 5'-end of the cloned fragment. Orf2Z
showed some similarity with the YitS protein of B. subtilis.
However, because the function of the YitS protein is unknown, this
did not give us any information about the possible function of
Orf2Z. Because the orf2Z gene is not a part of the cps operon, a
role of this gene in polysaccharide biosynthesis is not expected.
The Orf2Y protein showed some similarity with the YcxD protein of
B. subtilis (53). The YcxD protein was suggested to be a regulatory
protein. Similarly, Orf2Y may be involved in the regulation of
polysaccharide biosynthesis. The Orf2X protein showed similarity
with the YAAA proteins of H. influenzae and E. coli. The function
of these proteins is unknown. In S. suis type 2, the orf2X gene
seemed to be the first gene in the cps2 operon. This suggests a
role of Orf2X in the polysaccharide biosynthesis. In H. influenzae
and E. coli, however, these proteins are not associated with
capsular gene clusters. The analysis of isogenic mutants impaired
in the expression of Orf2X should give more insight in the presumed
role of Orf2X in the polysaccharide biosynthesis of S. suis type
2.
The gene products encoded by the cps2E, cps2F, cps2G, cps2H, cps2J
and cps2K genes showed little similarity with glycosyltransferases
of several Gram-positive or Gram-negative bacteria (18, 19, 20, 22,
25). The cps2E gene product shows some similarity with the Cps14E
protein of S. pneumoniae (18, 19). Cps14E is a glucosyl-1-phosphate
transferase that links glucose to a lipid carrier (18). In S.
pneumoniae, this is the first step in the biosynthesis of the
oligosaccharide repeating unit. The structure of the S. suis
serotype 2 capsule contains glucose, galactose, rhamnose, N-acetyl
glucosamine and sialic acid in a ratio of 3:1:1:1:1 (7). Based on
these data, we conclude that Cps2E of S. suis has
glucosyltransferase activity and is involved in the linkage of the
first sugar to the lipid carrier.
The C-terminal region of the cps2F gene product showed some
similarity with the RfbU of Salmonella enteritica. RfbU was shown
to have mannosyltransferase activity (24). Because mannosyl is not
a component of the S. suis type 2 polysaccharide, a
mannosyltransferase activity is not expected in this organism.
Nevertheless, cps2F encodes a glycosyltransferase with another
sugar specificity.
Cps2G showed moderate similarity to a family of gene products
suggested to encode galactosyltransferase activities (22, 24, 40).
Hence, a similar activity is shown for Cps2G.
Cps2H showed some similarity with LgtD of H. influenzae (U32768).
Because LgtD was proposed to have glycosyltransferase activity, a
similar activity is fulfilled by Cps2H.
Cps2J and Cps2K showed similarity to Cps14J of S. pneumoniae (20).
Cps2J showed similarity with Cps14I of S. pneumoniae as well.
Cps14I was shown to have N-acetyl glucosaminyltransferase activity,
whereas Cps14J has a .beta.-1,4-galactosyltransferase activity
(20). In S. pneumoniae, Cps14I is responsible for the addition of
the third sugar and Cps14J for the addition of the last sugar in
the synthesis of the type 14 repeating unit (20). Because the
capsule of S. suis type 2 contains galactose as well as N-acetyl
glucosamine components, galactosyltransferase as well as N-acetyl
glucoaminyltransferase activities could be envisaged for the cps2J
and cps2K gene products, respectively. As was observed for Cps14I
and Cps14J, the N-termini of Cps2J and Cps2K showed a significant
degree of sequence similarity. Within the N-terminal domains of
Cps14I and Cps14J, two small regions were identified, which were
also conserved in several other glycosyltransferases (22). Within
these two regions, two Asp residues were proposed to be important
for catalytic activity. The two conserved regions, DXS and DXDD,
were also found in Cps2J and Cps2K.
The function of Cps2I remains unclear. Cps2I showed some similarity
with a protein of A. actinomycetemcomitans. Although this protein
part is of the gene cluster responsible for the serotype-B-specific
antigens, the function of the protein is unknown.
We further describe the identification and characterization of the
cps genes specific for S. suis serotypes 1, 2 and 9. After the
entire cps2 locus of S. suis serotype 2 was cloned and
characterized, functions for most of the cps2 gene products could
be assigned by sequence homologies. Based on these data, the
glycosyltransferase activities, required for type specificity,
could be located in the center of the operon. Cross-hybridization
experiments, using the individual cps2 genes as probes on
chromosomal DNAs of the 35 different serotypes, confirmed this
idea. The regions containing the type-specific genes of serotypes 1
and 9 could be cloned and characterized, showing that an identical
genetic organization of the CpS operons of other S. suis serotypes
exists. The cps1E, cps1F, cps1G, cps1H, and cps1I genes revealed a
striking similarity with cps14E, cps14F, cps14G, cps14H and cps14J
genes of S. pneumoniae. Interestingly, S. pneumoniae serotype 14 is
the serotype most commonly associated with pneumococcal infections
in young children (54), whereas S. suis serotype 1 strains are most
commonly isolated from piglets younger than 8 weeks (46). In S.
pneumoniae, the cps14E, cps14G, cps14I and cps14J encode the
glycosyltransferases required for the synthesis of the type 14
tetrameric repeating unit, showing that the cps1E, cps1G and cps1I
genes encoded glycosyltransferases. The precise functions of these
genes as well as the substrate specificities of the enzymes can be
established. In S. pneumoniae, the cps14E gene was shown to encode
a glucosyl-1-phosphate transferase catalyzing the transfer of
glucose to a lipid carrier. Moreover, cpsE-like genes were found in
S. pneumoniae serotypes 9N, 13, 14, 15B, 15C, 18F, 18A and 19F
(60). CpsE mutants were constructed in the serotypes 9N, 13, 14 and
15B. All mutant strains lacked glucosyltransferase activity (60).
Moreover, in all these S. pneumoniae serotypes, the cpsE gene
seemed to be responsible for the addition of glucose to the lipid
carrier. Based on these data, we suggest that in S. suis type 1,
the cps1E gene may fulfil a similar function. The structure of the
S. suis type 1 capsule is unknown, but it is composed of glucose,
galactose, N-acetyl glucosamine, N-acetyl galactosamine and sialic
acid in a ratio of 1:2.4:1:1:1.4 (5). Therefore, a role of a
cpsE-like glucosyltransferase activity can easily be envisaged.
CpsE-like sequences were also found in serotypes 2, 1/2 and 14.
For polysaccharide biosynthesis in S. pneumoniae type 14, transfer
of the second sugar of the repeating unit to the first lipid-linked
sugar is performed by the gene products of cps14F and cps14G (20).
Similar to Cps14F and Cps14G, the S. suis type 1 prot Cps1G may act
as one glycosyltransferase performing the same reaction. Cps14F and
Cps14G of S. pneumoniae showed similarity to the N-terminal half
and C-terminal half of the SpsK protein of Sphingomonas (20, 67),
respectively. This suggests a combined function for both proteins.
Moreover, cps14F- and cps14G-like sequences were found in several
serotypes of S. pneumoniae and these genes always seemed to exist
together (60). The same was observed for S. suis type 1. The cps1F
and cps1G probes hybridized with type 1 and type 14 strains.
According to the similarity found between the cps1H gene and the
cps14H gene of S. pneumoniae (20), cps1H is expected to encode a
polysaccharide polymerase.
The protein encoded by the cps1I gene showed some similarity with
the Cps14J protein of S. pneumoniae (19). The cps14J gene was shown
to encode a .beta.-1, 4-galactosyltransferase activity, responsible
for the addition of the fourth (i.e. last) sugar in the synthesis
of the S. pneumoniae serotype 14 polysaccharide. In S. suis type 2,
the proteins encoded by the cps2J and cps2K genes showed similarity
to the Cps14J protein. However, no significant homologies were
found between Cps2J, Cps2K and Cps1I. In the N-terminal regions of
Cps14J and Cps14I, two small conserved regions, DXS and DXDD, were
identified (19). These regions seemed to be important for catalytic
activity (13). At the same positions in the sequence, Cps2I
contained the regions DXS and DXED.
In the region between Cps1G and Cps1H, three small Orfs were
identified. Since the Orfs were expressed in three different
reading frames, and did not contain potential start sites,
expression is not expected. However, the three potential gene
products encoded by this region showed some similarity with three
successive regions of the C-terminal part of the EpsK protein of
Streptococcus thermophilus (27% identity, 40). The region related
to the first 82 amino acids is lacking. The EpsK protein was
suggested to play a role in the export of the exopolysaccharide by
rendering the polymerized exopolysaccharide more hydrophobic
through a lipid modification. These data could suggest that the
sequences in the region between Cps1G and Cps1H originated from
epsK-like sequence. Hybridization experiments showed that this
epsK-like region is also present in other serotype 1 strains as
well as in serotype 14 strains (results not shown).
The function of most of the cloned serotype 9 genes can be
established. Based on sequence similarity data, the cps9E and cps9F
genes could be glycosyltransferases (61, 24, 63, 64, 65). Moreover,
the cps9G and cps9H genes showed similarity to genes located in
regions involved in polysaccharide biosynthesis, but the function
of these genes is unknown (68).
Cross-hybridization experiments using the individual cps2, cps1 and
cps9 genes as probes showed that the cps9G and cps9H probes
specifically hybridized with serotype 9 strains.
Therefore, these are useful as tools for the identification of S.
suis type 9 strains both for diagnostic purposes as well as in
epidemiological and transmission studies. We previously developed a
PCR method which can be used to detect S. suis strains in nasal and
tonsil swabs of pigs (62). The method was used to identify
pathogenic (EF-positive) strains of S. suis serotype 2. Besides S.
suis type 2 strains, serotype 9 strains are frequently isolated
from organs of diseased pigs. However, until now, a rapid and
sensitive diagnostic test was not available for type 9 strains.
Therefore, the type .[.9 specific probes.]. .Iadd.9-specific probes
.Iaddend.or the type .[.9 specific.]. .Iadd.9-specific .Iaddend.PCR
is of great diagnostic value. The cps1F, cps1G and cps1I probes
hybridized with serotype 1 as well as with serotype 14 strains. In
coagglutination tests, type 1 strains react with the anti-type 1 as
well as with the anti-type 14 antisera (56). This suggests the
presence of common epitopes between these serotypes. On the other
hand, type 1 strains agglutinated only with anti-type 1 serum (56,
57), indicating that it is possible to detect differences between
those serotypes.
The cps2F, cps2G, cps2H, cps2.[.J.]..Iadd.I .Iaddend.and cps2J
probes hybridized with serotypes 2 and 1/2 only. Serotype 34 showed
a weak hybridizing signal with the cps2G probe. As shown in
agglutination tests, type 1/2 strains react with sera directed
against type 1 as well as with sera directed against type 2 strains
(46). Therefore, type 1/2 shared antigens with both types 1 and 2.
Based on the hybridization patterns of serotype 1/2 strains with
the .[.cps1 and cps2 specific.]. .Iadd.cps1- and cps2-specific
.Iaddend.genes, serotype 1/2 seemed to be more closely related to
type 2 strains than to type 1 strains. In our current studies, we
identify type-specific genes, primers or probes which are used for
the discrimination of serotypes 1, 14 and 2 and 1/2 and others of
the 35 serotypes yet known. Furthermore, type-specific genes,
primers or probes can now easily be developed for yet unknown
serotypes, once they become isolated.
Cloning and characterization of a further part of the cps2
locus.
Based on the established sequence, 11 genes, designated cps2L to
cps2T, orf2U and orf2V, were identified. A gene homologous to genes
involved in the polymerization of the repeating oligosaccharide
unit (cps2O) as well as genes involved in the synthesis of sialic
acid (cps2P to cps2T) were identified. Moreover, hybridization
experiments showed that the genes involved in the sialic acid
synthesis are present in S. suis serotypes 1, 2, 14, 27 and 1/2.
The "cps2M" and "cps2N" regions showed similarity to proteins
involved in the polysaccharide biosynthesis of other Gram-positive
bacteria. However, these regions seemed to be truncated or were
nonfunctional as the result of frame-shift or point mutations. At
its 3'-end, the cps2 locus contained two insertional elements
("orf2U" and "orf2V"), both of which seemed to be
non-functional.
To clone the remaining part of the cps2 locus, sequences of the
3'-end of pCPS26 (FIG. 1, part C) were used to identify a
chromosomal fragment containing cps2 sequences located further
downstream. This fragment was cloned in pKUN19, resulting in
pCPS29. Using a similar approach, we subsequently isolated the
plasmids pCPS30 and pCPS34 containing downstream cps2 sequences
(FIG. 1, part C).
Analysis of the cps2 operon.
The complete nucleotide sequence of the cloned fragments was
determined. Examination of the compiled sequence revealed the
presence of: a sequence encoding the C-terminal part of Cps2K, six
apparently functional genes (designated cps2O-cps2T) and the
remnants of 5 different ancestral genes (designated "cps2L"
"cps2M", "cps2N", "orf2U" and "orf2V"). The latter genes seemed to
be truncated or incomplete as the result of the presence of stop
codons or frame-shift mutations (FIG. 1, part A). Neither potential
promoter sequences nor potential stem-loop structures could be
identified within the sequenced region. A ribosome-binding site
precedes each ORF and the majority of the ORFs are very closely
linked. Three intergenic gaps were found: one between "cps2M" and
"cps2N" (176 nucleotides), one between cps2O and cps2P (525
nucleotides), and one between cps2T and "orf2U" (200 nucleotides).
These and our above data show that Orf2X and Cps2A-Orf2T are part
of a single operon.
A list of all loci and their properties is shown in Table 4. The
"cps2L" region contained three potential ORFs of 103, 79 and 152
amino acids, respectively, which were only separated from each
other by stop codons. Only the first ORF is preceded by a potential
ribosomal binding site and contained a methionine start codon. This
suggests that "cps2L" originates from an ancestral cps2L gene,
which coded for a protein of 339 amino acids. The function of this
hypothetical Cps2L protein remains unclear so far: no significant
homologies were found between Cps2L and proteins present in the
data libraries. It is not clear whether the first ORF of the
"cps2L" region is expressed into a protein of 103 amino acids. The
"cps2M" region showed homology to the N-terminal 134 amino acids of
the NeuA proteins of Streptococcus agalactiae and Escherichia coli
(AB017355, 32). However, although the "cps2 M" region contained a
potential ribosome binding site, a methionine start codon was
absent. Compared with the S. agalactiae sequence, the ATG start
codon was replaced by a lysin encoding AAG codon. Moreover, the
region homologous to the first 58 amino acids of the S. agalactiae
NeuA (identity 77%) was separated from the region homologous to
amino acids 59-134 of NeuA by a repeated DNA sequence of 100-bp
(see, herein). In addition, the region homologous to amino acids 59
to 95 of NeuA (identity 32%) and the region homologous to the amino
acids 96 to 134 of NeuA (identity 50%) were present in different
reading frames. Therefore, the partial and truncated NeuA homologue
is probably nonfunctional in S. suis. The "cps2N" region showed
homology to CpsJ of S. agalactiae (accession no. AB017355).
However, sequences homologous to the first 88 amino acids of CpsJ
were lacking in S. suis. Moreover, the homologous region was
present in two different reading frames. The protein encoded by the
cps2O gene showed homology to proteins of several streptococci
involved in the transport of the oligosaccharide repeating unit
(accession no. AB017355), suggesting a similar function for Cps2O.
The proteins encoded by the cps2P, cps2S and cps2T genes showed
homology to the NeuB, NeuD and NeuA proteins of S. agalactiae and
E. coli (accession no. AB017355). Because the "cps2M" region also
showed homology to NeuA of E. coli, the S. suis cps2 locus contains
a functional neuA gene (cps2T) as well as a nonfunctional ("cps2M")
gene. The mutual homology between these two regions showed an
identity of 77% at the amino acid level over amino acids 1-58 and
49% over the amino acids 59-134. Cps2Q and Cps2R showed homology to
the N-terminal and C-terminal parts of the NeuC protein of S.
agalactiae and E. coli, respectively. This suggests that the
function of the S. agalactiae NeuC protein in S. suis is likely
fulfilled by two different proteins. In E. coli, the neu genes are
known to be involved in the synthesis of sialic acid. NeuNAc is
synthesized from N-acetylmannosamine and phosphoenolpyruvate by
NeuNAc synthetase. Subsequently, NeuNAc is converted to CMP-NeuNAc
by the enzyme CMP-NeuNAc synthetase. CMP-NeuNAc is the substrate
for the synthesis of polysaccharide. In E. coli, K1 NeuB is the
NeuNAc synthetase, and NeuA is the CMP-NeuNAc synthetase. NeuC has
been implicated in the NeuNAc synthesis, but its precise role is
not known. The precise role of NeuD is not known. A role of the
Cps2P-Cps2T proteins in the synthesis of sialic acid can easily be
envisaged, since the capsule of S. suis serotype 2 is rich in
sialic acid. In S. agalactiae, sialic acid has been shown to be
critical to the virulence function of the type III capsule.
Moreover, it has been suggested that the presence of sialic acid in
the capsule of bacteria which can cause meningitis may be important
for these bacteria to breach the blood-brain barrier. So far,
however, the requirement of the sialic acid for virulence of S.
suis remains unclear.
"Orf2U" and "Orf2V" showed homology to proteins located on two
different insertional elements. "Orf2U" is homologous to IS1194 of
Streptococcus thermophilus, whereas "Orf2V" showed homology to a
putative transposase of Streptococcus pneumoniae. This putative
transposase was recently found to be associated with the type 2
capsular locus of S. pneunioniae. Compared with the original
insertional elements in S. thermophilus and S. pneumoniae, both
"Orf2U" and "Orf2V" are likely to be nonfunctional due to frame
shift mutations within their coding regions.
A striking observation was the presence of a sequence of 100 bp
(FIG. 10) which was repeated three times within the cps2 operon.
The sequence is highly conserved (between 94% and 98%) and was
found in the intergenic regions between cps2G and cps2H, within
"cps2M" and between cps2O and cps2P. No significant homologies were
found between this 100-bp direct repeat sequence and sequences
present in the data libraries, suggesting that the sequence is
unique for S. suis.
Distribution of the cps2 sequences among the 35 S. suis
serotypes.
To examine the presence of sialic acid encoding genes in other S.
suis serotypes, we performed cross-hybridization experiments. DNA
fragments of the individual cps2 genes were amplified by PCR,
radiolabeled with 32P and hybridized to chromosomal DNA of the
reference strains of the 35 different S. suis serotypes. As a
positive control, we used a probe specific for S. suis 16S rRNA.
The 16S rRNA probe hybridized with almost equal intensities to all
serotypes tested (Table 4). The "cps2L" sequence hybridized with
DNA of serotypes 1, 2, 14 and 1/2. The "cps2M", cps2O, cps2P,
cps2Q, cps2R, cps2S and cps2T genes hybridized with DNA of
serotypes 1, 2, 14, 27 and 1/2. Because the cps2P-cps2T genes are
most likely involved in the synthesis of sialic acid, these results
suggest that sialic acid is also a part of the capsule in the S.
suis serotypes 1, 2, 14, 27 and 1/2. This is in agreement with the
finding that the serotypes 1, 2 and 1/2 possess a capsule that is
rich in sialic acid. Although the chemical compositions of the
capsules of serotypes 14 and 27 are unknown, recent agglutination
studies using sialic acid-binding lectins suggested the presence of
sialic acid in S. suis serotype 14, but not in serotype 27. In
these studies, sialic acid was also detected in serotypes 15 and
16. Since the latter observation is not in agreement with our
hybridization studies, it might be that other genes, not homologous
to the cps2P-cps2T genes, are responsible for the sialic acid
synthesis in serotypes 15 and 16.
A probe based on "cps2N" sequences hybridized with DNA from
serotypes 1, 2, 14 and 1/2. A probe specific for "orf2U" hybridized
with serotypes 1, 2, 7, 14, 24, 27, 32, 34, and 1/2, whereas a
probe specific for "orf2V" hybridized with many different
serotypes. In addition, we prepared a probe specific for the 100-bp
direct repeat sequence. This probe hybridized with the serotypes 1,
2, 13, 14, 22, 24, 27, 29, 32, 34 and 1/2 (Table 4). To analyze the
number of copies of the direct repeat sequence within the S. suis
serotype 2 chromosome, a Southern blot hybridization and analysis
was performed. Therefore, chromosomal DNA of S. suis serotype 2 was
digested with NcoI and hybridized with a 32P-labeled direct repeat
sequence. Only one hybridizing fragment, containing the three
direct repeats present on the cps2 locus, was found (results not
shown). This indicates that the 100-bp direct repeat sequence is
only associated with the cps2 locus. In S. pneumoniae, a 115-bp
long repeated sequence was found to be associated with the capsular
genes of serotypes 1, 3, 14 and 19F. In S. pneumoniae, this 115-bp
sequence was also found in the vicinity of other genes involved in
pneumococcal virulence (hyaluronidase and neuraminidase genes). A
regulatory role of the 115-bp sequence in coordinate control of
these virulence-related genes was suggested.
To study the role of the capsule in resistance to phagocytosis and
in virulence, we constructed two isogenic mutants in which capsule
synthesis was disturbed. In 10cpsB, the cps2B gene was disturbed by
the insertion of an antibiotic-resistance gene, whereas in 10cpsEF,
parts of the cps2E and cps2F genes were replaced. Both mutant
strains seemed to be completely unencapsulated. Because the cps2
genes seemed to be part of an operon, polar effects cannot be
excluded. Therefore, these data did not give any information about
the role of Cps2B, Cps2E or Cps2F in the polysaccharide
biosynthesis. However, the results clearly show that the capsular
polysaccharide of S. suis type 2 is a surface component with
antiphagocytic activity. In vitro wild type encapsulated bacteria
are ingested by phagocytes at a very low frequency, whereas the
mutant unencapsulated bacteria are efficiently ingested by porcine
macrophages. Within 2 hours, over 99.6% of mutant bacteria were
ingested and over 92% of the ingested bacteria were killed.
Intracellularly, wild type as well as mutant strains seemed to be
killed with the same efficiency. This suggests that the loss of
capsular material is associated with loss of capacity to resist
uptake by macrophages. This loss of resistance to in vitro
phagocytosis was associated with a substantial attenuation of the
virulence in germfree pigs. All pigs inoculated with the mutant
strains survived the experiment and did not show any specific
clinical signs of disease. Only some aspecific clinical signs of
disease could be observed. Moreover, mutant bacteria could be
reisolated from the pigs. This supports the idea that, as in other
pathogenic Streptococci, the capsule of S. suis acts as an
important virulence factor. Transposon mutants prepared by Charland
impaired in the capsule production showed a reduced virulence in
pigs and mice. To construct these mutants, the type 2 reference
strain S735 was used. We previously showed that this strain is only
weakly virulent for young pigs. Moreover, the insertion site of the
transposon is unsolved so far.
As a further example herein, a rapid PCT test for Streptococcus
suis type 7 is described.
Recent epidemiological studies on Streptococcus suis infections in
pigs indicated that, besides serotypes 1, 2 and 9, serotype 7 is
also frequently associated with diseased animals. For the latter
serotype, however, no rapid and sensitive diagnostic methods are
available. This hampers prevention and control programs. Here we
describe the development of a type-specific PCR test for the rapid
and sensitive detection of S. suis serotype 7. The test is based on
DNA sequences of capsular (cps) genes specific for serotype 7.
These sequences could be identified by cross-hybridization of
several individual cps genes with the chromosomal DNAs of 35
different S. suis serotypes.
Streptococcus suis is an important cause of meningitis, septicemia,
arthritis and sudden death in young pigs (69, 70). It can however,
also cause meningitis in man (71). Attempts to control the disease
are still hampered by the lack of sufficient knowledge about the
epidemiology of the disease and the lack of effective vaccines and
sensitive diagnostics.
S. suis strains can be identified and classified by their
morphological, biochemical and serological characteristics (70, 73,
74). Serological classification is based on the presence of
specific antigenic determinants. Isolated and biochemically
characterized S. suis cells are agglutinated with a panel of
specific sera. These typing methods are very laborious and
time-consuming and can only be performed on isolated colonies.
Moreover, it has been reported that non-specific cross-reactions
may occur among different types of S. suis (75, 76).
So far, 35 different serotypes have been described (7, 78, 79). S.
suis serotype 2 is the most prevalent type isolated from diseased
pigs, followed by serotypes 9 and 1. However, recently, serotype 7
strains were also frequently isolated from diseased pigs (80, 81,
82). This suggests that infections with S. suis serotype 7 strains
seem to be an increasing problem. Moreover, the virulence of S.
suis serotype 7 strains was confirmed by experimental infection of
young pigs (83).
Recently, rapid and sensitive PCR assays specific for serotypes 2
(and 1/2), 1 (and 14) and 9 were developed (84). These assays were
based on the cps loci of S. suis serotypes 2, 1 and 9 (84, 85).
However, until now, no rapid and sensitive diagnostic test was
available for S. suis serotype 7. Herein we describe the
development of a PCR test for the rapid and sensitive detection of
S. suis serotype 7 strains. The test is based on DNA sequences
which form a part of the cps locus of S. suis serotype 7. Compared
with the serological serotyping methods, the PCR assay was a rapid,
reliable and sensitive assay. Therefore, this test, in combination
with the PCR tests which we previously developed for serotypes 1, 2
and 9, will undoubtedly contribute to a more rapid and reliable
diagnosis of S. suis and may facilitate control and eradication
programs.
Materials and Methods
Bacterial strains, growth conditions and serotyping.
The bacterial strains and plasmids used in this study are listed in
Table 7. The S. suis reference strains were obtained from M.
Gottschalk, Canada. S. suis strains were grown in Todd-Hewitt broth
(code CM189, Oxoid), and plated on Columbia agar blood base (code
CM331, Oxoid) containing 6% (v/v) horse blood. E. Coli strains were
grown in Luria broth (86) and plated on Luria broth containing 1.5%
(w/v) agar. If required, ampicillin was added to the plates. The S.
suis strains were serotyped by the slide agglutination test with
serotype-specific antibodies (70).
DNA techniques.
Routine DNA manipulations and PCR reactions were performed as
described by Sambrook et al. (88). Blotting and hybridization were
performed as described previously (84, 86).
DNA sequence analysis.
DNA sequences were determined on a 373A DNA Sequencing System
(Applied Biosystems, Warrington, GB). Samples were prepared by use
of an ABI/PRISM dye terminator hcycle sequencing ready reaction kit
(Applied Biosystems). Custom-made sequencing primers were purchased
from Life Teclmologies. Sequencing data were assembled and analyzed
using the McMollyTetra program. The BLAST program was used to
search for protein sequences homologous to the deduced amino acid
sequences.
PCR.
The primers used for the cps7H PCR correspond to the positions
3334-3354 and 3585-3565 in the S. suis cps7 locus.
The sequences were:
TABLE-US-00001 5'-AGCTCTAACACGAAATAAGGC-3' (SEQ. ID. No. 7) and
5'-GTCAAACACCCTGGATAGCCG3' (SEQ. ID. No. 8).
The reaction mixtures contained 10 mM Tris-HCl, pH 8.3; 1.5 mnM
MgCl2; 50 mM KCl; 0.2 mM of each of the four deoxynucleotide
triphosphates; 1 microM of each of the primers and 1U of AmpliTaq
Gold DNA polymerase (Perkin Elmer Applied Biosystems, N.J.). DNA
amplification was carried out in a Perkin Elmer 9600 thermal cycler
and the program consisted of an incubation for 10 min at 95.degree.
C. and 30 cycles of 1 min at 95.degree. C., 2 min at 56.degree. C.
and 2 min at 72.degree. C.
Results and discussion
Cloning of the seroytpe 7-specific cps genes.
To isolate the type-specific cps genes of S. suis serotype 7, we
used the cps9E gene of serotype 9 as a probe to identify
chromosomal DNA fragments of type 7 containing homologous DNA
sequences (84). A 1.6-kb PstI fragment was identified and cloned in
pKUN19. This yielded pCPS7-1 (FIG. 11, part C). In turn, this
fragment was used as a probe to identify an overlapping 2.7 kb
ScaI-ClaI fragment. pGEM7 containing the latter fragment was
designated pCPS7-2 (FIG. 11, part C).
Analysis of the cloned cps7 genes.
The complete nucleotide sequences of the inserts of pCPS7-1,
pCPS7-2 were determined. Examination of the cps7 sequence revealed
the presence of two complete and two incomplete open reading frames
(ORFs) (FIG. 11, part C). All ORFs are preceded by a
ribosome-binding Site. In accord with the data obtained for the
cps1, cps2 and cps9 genes of serotypes 1, 2 and 9, respectively,
the type 7 ORFs are very closely linked to each other. The only
significant intergenic gap was that found between cps7E and cps7F
(443 nucleotides). No obvious promoter sequences or potential
stem-loop structures were found in this region. This suggests that,
as in serotypes 1, 2 and 9, the cps genes in serotype 7 form part
of an operon.
An overview of the ORFs and their properties is shown in Table 8.
As expected on the basis of the hybridization data (84), the Cps9E
and Cps7E proteins showed a high similarity (identity 99%. Table
8). Based on sequence comparisons between Cps9E and Cps7E, the PstI
fragment of pCPS7-1 lacks the region encoding the first 371 codons
of Cps7E. The C-terminal part of the protein encoded by the cps7F
gene showed some similarity with the Bp1G protein of Bordetella
pertussis (88), as well as with the C-terminal part of S. suis
Cps2E (85). Both Bp1G and Cps2E were suggested to have
glycosyltransferase activity and are probably involved in the
linkage of the first sugar to the lipid carrier (85, 88). The
protein encoded by the cps7G gene showed similarity with the Bp1F
protein of Bordetella pertussis (88). B1pF is likely to be involved
in the biosynthesis of an amino sugar, suggesting a similar
function for Cps7G. The protein encoded by the cps7H gene showed
similarity with the WbdN protein of E. coli (89) as well as with
the N-terminal part of the Cps2K protein of S. suis (81). Both WbdN
and Cps2K were suggested to have glycosyltransferase activity (85,
89).
Serotype .[.7 specific.]. .Iadd.7-specific .Iaddend.cps genes.
To determine whether the cloned fragments in pCPS7-1 and pCPS7-2
contained serotype 7-specific DNA sequences, cross-hybridization
experiments were performed. DNA fragments of the individual cps7
genes were amplified by PCR, labeled with 32P, and used to probe
spot blots of chromosomal DNA of the reference strains of 35
different S. suis serotypes. The results are summarized in Table 9.
As expected, based on the data obtained with the cps9E probe (84),
the cps7E probe hybridized with chromosomal DNA of many different
S. suis serotypes. The cps7F and cps7G probes showed hybridization
with chromosomal DNA of S. suis serotypes 4, 5, 7, 17, and 23.
However, the cps7H probe hybridized with chromosomal DNA of
serotype 7 only, indicating that this gene is specific for serotype
7.
.[.Type specific.]. .Iadd.Type-specific .Iaddend.PCR.
We tested whether we could use PCR instead of hybridization for the
typing of the S. suis serotype 7 strains. For that purpose, we
selected an oligonucleotide primer set within the cps7H gene with
which an amplified fragment of 251-bp was expected. In addition, we
included in our analysis several S. suis serotype 7 strains, other
than the reference strain. These strains were obtained from
different countries and were isolated from different organs (Table
7). The results show that indeed a fragment of about 250-bp was
amplified with all type 7 strains used (FIG. 12, part B), whereas
no PCR products were obtained with serotype 1, 2 and 9 strains
(FIG. 12, part A). This suggests that the PCR test, as described
here, is a rapid diagnostic tool for the identification of S. suis
serotype 7 strains. Until now, such a diagnostic test was not
available for serotype 7 .[.Strains.]. .Iadd.strains.Iaddend..
Together with the recently developed PCR assays for serotypes 1, 2,
1/2, 14 and 9, this assay may be an important diagnostic tool to
detect pigs carrying serotype 2, 1/2, 1, 14, 9 and 7 strains and
may facilitate control and eradication programs.
TABLE-US-00002 TABLE 1 Bacterial strains and plasmids relevant
strain/plasmid characteristics source/reference Strain E coli CC118
PhoA (28) XL2 blue Stratagene E. coli XL2 blue Stratagene S. suis
10 virulent serotype 2 strain (49) 3 serotype 2 (63) 17 serotype 2
(63) 735 reference strain serotype 2 (63) T15 serotype 2 (63) 6555
reference strain serotype 1 (63) 6388 serotype 1 (63) 6290 serotype
1 (63) 5637 serotype 1 (63) 5673 serotype 1/2 (63) 5679 serotype
1/2 (63) 5928 serotype 1/2 (63) 5934 serotype 1/2 (63) 5209
reference strains serotype 1/2 (63) 5218 reference strain, serotype
9 (63) 5973 serotype 9 (63) 6437 serotype 9 (63) 6207 serotype 9
(63) reference strains serotypes 1-34 (9, 56, 14) S. suis 10
virulent serotype 2 strain (51) 10cpsB isogenic cpsB mutant of
strain 10 this work 10cpsEF isogenic cpsEF mutant of strain 10 this
work Plasmid pKUN19 replication functions pUC, Amp.sup.R (23)
pGEM7Zf(+) replication functions pUC, Amp.sup.R Promega Corp.
pIC19R replication functions pUC, Amp.sup.R (29) pIC20R replication
functions pUC, Amp.sup.R (29) pIC-spc pIC19R containing spc.sup.R
gene labcollection of pDL282 pDL282 replication functions of pBR322
(43) and pVT736-1, Amp.sup.R, Spc.sup.R pPHOS2 pIC-spc containing
the truncated this work phoA gene of pPHO7 as a PstI-BamHI fragment
pPHO7 contains truncated phoA gene (15) pPHOS7 pPHOS2 containing
chromosomal this work S. suis DNA pCPS6 pKUN19 containing 6 kb
HindIII this work (FIG. 1) fragment of cps operon pCPS7 pKUN19
containing 3,5 kb EcoRI- this work (FIG. 1) HindIII fragment of cps
operon pcPS11 pCPS7 in which 0.4 kb PstI- this work (FIG. 1) BamHI
fragment of cpsB gene is replaced by Spc.sup.R gene of pIC-spc
pCPS17 pKUN19 containing 3.1 kb KpnI this work (FIG. 1) fragment of
cps operon pCPS18 pKUN19 containing 1.8 kb SnaBI this work. (FIG.
1) fragment of cps operon pCPS20 pKUN19 containing 3.3 kb XbaI-
this work (FIG. 1) HindIII fragment of cps operon pCPS23 pGEM7Zf(+)
containing 1.5 kb this work (FIG. 1) Mini fragment of cps operon
pCPS25 pIC20R containing 2.5 kb this work (FIG. 1) KpnI-SalI
fragment of pCPS17 pCPS26 pKUN19 containing 3.0 kb HindIII this
work (FIG. 1) fragment of cps operon pCPS27 pCPS25 containing 2.3
kb XbaI this work (FIG. 1) (blunt)-ClaI fragment of pCPS20 pCPS28
pCPS27 containing the 1.2 kb this work (FIG. 1) PstI-XhoI Spc.sup.R
gene of pIC-spc pCPS29 pKUN19 containing 2.2 kb SacI- this work
(FIG. 1) PstI fragment of cps operon pCPS1-1 pKUN19 containing 5 kb
EcoRV this work (FIG. 1) fragment of cps operon of type I pCPS1-2
pKUN19 containing 2.2 kb HindIII this work (FIG. 1) fragment of cps
operon of type I pCPS9-1 pKUN19 containing 1 kb HindIII- this work
(FIG. 1) XbaI fragment of cps operon of serotype 9 pCPS9-2 pKUN19
containing 4.0 kb this work (FIG. 1) XbaI-XbaI fragment of cps
operon of serotype 9 Amp.sup.R: ampicillin resistant Spc.sup.R:
spectinomycin resistant cps: capsular polysaccharide
TABLE-US-00003 TABLE 2 Properties of Orfs in the cps locus of S.
suis serotype 2 and similarities to gene product other bacteria
nucleotide number position in of amino GC proposed function similar
gene product ORF sequence acids % of gene product.sup.1 (%
identity) Orf2Z 1-719 240 44 Unknown B. subtilis YitS (26%) Orf2Y
2079-822 419 38 Transcription B. subtilis YcxD (39%) regulation
Orf2X 2202-2934 244 39 Unknown H. influenzae YAAA (24%) Cps2A
3041-4484 481 39 Regulation S. pneumoniae Cps19fA (58%) Cps2B
4504-5191 229 40 Chain length S. pneumoniae type 3 Orfl (58%)
determination Cps2C 5203-5878 225 40 Chain length S. pneumoniae
Cps23fD (63%) determination/Export Cps2D 5919-6648 243 38 Unknown
S. pneumoniae CpsB (62%) Cps2E 6675-8052 459 33 Glycosyltransferase
S. pneumoniae Cps14E (56%) Cps2F 8089-9256 389 32
Glycosyltransferase S. pneumoniae Cps23fT Cps2G 9262-10417 385 36
Glycosyltransferase S. thermophilus EpsF (25%) Cps2H 10808-12176
457 31 Glycosyltransferase S. mutans RGPEC,.sup.N (29%) Cps2I
12213-13443 410 29 CP polymerase S. pneumoniae Cps23f1 (48%) Cps2J
13583-14579 332 29 Glycosyltransferase S. pneumoniae Cps14J (31%)
Cps2K 14574-15576 334 37 Glycosyltransferase S. pneumoniae Cps14J
(40%) "Cps2L" 15618-16635 103 37 Unknown -- "Cps2M" 16811-17322 --
38 -- S. agalactiae CpsF.sup.N (77%) E. coli NeuA,.sup.N (47%)
"Cps2N" 17559-18342 -- 39 -- S. agalactiae CpsJ (43%) Cps2O
18401-19802 476 40 Repeat unit S. agalactiae CpsK (41%) transporter
Cps2P 20327-21341 338 39 Sialic acid synthesis S. agalactiae NeuB
(80%) E. coli NeuB (59%) Cps2Q 21355-21865 170 42 Sialic acid
synthesis S. agalactiae NeuC.sup.N (61%) E. coli NeuC.sup.N (54%)
Cps2R 21933-22483 184 40 Sialic acid synthesis S. agalactiae
NeuC.sup.c (55%) E. coli NeuC.sup.c (40%) Cps2S 22501-23125 208 42
Sialic acid synthesis E. coli NeuD (32%) Cps2T 23136-24366 395 40
CMP-NeuNAc S. agalactiae CpsF (49%) synthetase E. coli NeuA (34%)
"Orf2U" 24566-25488 168 42 Transposase S. thermophilus IS1194 (51%)
"Orf2V" 25691-26281 116 37 Transposase S. pneumoniae orfl (85%)
.sup.1Predicted by sequence similarity .sup.NSimilarity refers to
the amino-terminal part of the gene product .sup.cSimilanty refers
to the carboxy-terminal part of the gene product ORFs between " "
are truncated or non-functional as the result of frame-shift or
point mutations
TABLE-US-00004 TABLE 3 Properties of Orfs in the cps genes of S.
suis serotypes 1 and 9 and similarities to gene products of other
bacteria nucleotide number (kDa) position in of amino predicted
predicted proposed function similar gene product reference/ ORF
sequence G + C% acids mol. mass pI of gene product.sup.1 (%
identity) accession nr. Cps1E.sup.2 1-1363 34% 454 52.2 8.0
Glucosyltransferase Streptococcus suis Cps2E (26) (86%)
Streptococcus pneumoniae Cps14E (12) (48%) Cps1F 1374-1821 33% 149
17.3 8.2 Unknown Streptococcus pneumoniae Cps14F (14) (83%) Cps1G
1823-2315 25% 164 19.5 7.5 Glycosyltransferase Streptococcus
pneumoniae Cps14G (14) (50%) Cps1H 3035-4202 24% 389 45.5 8.4 CP
polymerase Streptococcus pneumoniae Cps14H (14) (30%) Cps1I 4197-
Glycosyltransferase Streptococcus pneumoniae Cps14J (13) (38%)
Lactococus lactis EpsG (29) (31%) Streptococcus thermophilus EpsI
(28) (33%) Cps1J Glycosyltransferase Streptococcus pneumoinae
Cps14J (13) ( ) Cps1K.sup.3 37% 278 32.5 7.8 Glycosyltransferase
Streptococcus pneumoniae Cps14J (13) (44%) Cps9D.sup.2 1-646 37%
215 24.9 8.1 Unknown Streptococcus suis Cps2D (26) (89%) Cps9E 680-
Glycosyltransferase Staphylococcus aureus Cap1D (18) (27%) Cps9F
36% 200 22.3 8.2 Glycosyltransferase Staphylococcus aureus Cap5M
(17) (52%) Cps9G 35% 269 31.5 8.0 Unknown Actinobacillus
acunomycetemcomitans (A8002668_4) (43%) Haemophilus influenzae Lsg
(005081) (43%) Cps9H.sup.3 30% 143 16.5 7.2 Unknown Yersinia
enterolitica RfbB (33) (28%) .sup.1Predicted by sequence similarity
.sup.2N-terminal part of protein is lacking .sup.3C-terminal part
of protein is lacking
TABLE-US-00005 TABLE 4 Hybridization of serotype 2 cps genes and
neighboring sequences with chromosomal DNA of other serotypes DNA
serotypes probes 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 orf2Z
+ + + + + + + + + + + + .+-. + + + + + orf2Y + + + + + + + + + + +
+ .+-. + + + + + orf2X + + + + + + + + + + + + .+-. + + + + + cps2A
+ + + + + + + + + + + + + + + + + + cps2B + + + + + + + + + + - -
.+-. + - - .+-. .+-. cps2C + + + + + + + + + + + - .+-. + - .+-. -
- cps2D + + + + + + + + + + + .+-. .+-. + - .+-. + + cps2E + + - -
- - - - - - - - - - - - - - cps2F - + - - - - - - - - - - - - - - -
- cps2G - + - - - - - - - - - - - - - - - - cps2H - + - - - - - - -
- - - - - - - - - cps2I - + - - - - - - - - - - - - - - - - cps2J -
+ - - - - - - - - - - - - - - - - cps2K + + - - - - - - - - - - - +
- - - - "cps2L" + + - - - - - - - - - - - + - - - - "cps2M" + + - -
- - - - - - - - - + - - - - "cps2N" + + - - - - - - - - - - - + - -
- - cps2O + + - - - - - - - - - - - + - - - - cps2P + + - - - - - -
- - - - - + - - - - cps2Q + + - - - - - - - - - - - + - - - - cps2R
+ + - - - - - - - - - - - + - - - - cps2S + + - - - - - - - - - - -
+ - - - - cps2T + + - - - - - - - - - - - + - - - - "orf2U" + + - -
- - + - - - - - - + - - - - "orf2V" + + .+-. .+-. .+-. - .+-. - - -
- - - + + - + + 100-bp repeat + + - - - - - - - - - - + + - - - -
16SrRNA + + + + + + + + + + + + + + + + + + serotypes DNA probes 19
20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 1/2 orf2Z + - + - + +
+ - + + + + + - - - + orf2Y + .+-. + .+-. + + + + + + + + + - - - +
orf2X + - + - + + + - + + + + + - - - + cps2A + - + - + + + - + + +
+ + - - - + cps2B .+-. - .+-. - + + + - - - + .+-. + - .+-. - +
cps2C - - - - + + + - + .+-. - - + - .+-. - + cps2D + - .+-. - + +
+ - + + + .+-. + - - - + cps2E - - - - - - - - + - - - - - - - +
cps2F - - - - - - - - - - - - - - - - + cps2G - - - - - - - - - - -
- - - - .+-. + cps2H - - - - - - - - - - - - - - - - + cps2I - - -
- - - - - - - - - - - - - + cps2J - - - - - - - - - - - - - - - - +
cps2K - - - - - - - - - - - - - - - - + "cps2L" - - - - - - - - - -
- - - - - - + "cps2M" - - - - - - - - + - - - - - - - + "cps2N" - -
- - - - - - - - - - - - - - + cps2O - - - - - - - - + - - - - - - -
+ cps2P - - - - - - - - + - - - - - - - + cps2Q - - - - - - - - + -
- - - - - - + cps2R - - - - - - - - + - - - - - - - + cps2S - - - -
- - - - + - - - - - - - + cps2T - - - - - - - - + - - - - - - - +
"orf2U" - - - - - + - - + - - - - + - + + "orf2V" .+-. - - .+-. + -
- + - - - - + + - .+-. + 100-bp repeat - - - + - + - - + - - - - +
- + + 16SrRNA + + + + + + + + + + + + + + + + +
TABLE-US-00006 TABLE 5 Hybridization of serotypes 1 and 9 cps genes
with chromosomal DNA of other S. suis serotypes DNA probes Serotype
cps1E cps1F cps1G cps1H cps1I cps9E cps9F cps9G cps9H 16rRNA 1 + +
+ + + - - - - + 2 + - - - - - - - - + 3 - - - + - + - - - + 4 - - -
+ - + - - - + 5 - - - + - + - - - + 6 - - - - - - - - - + 7 - - - +
- + - - - + 8 - - - - - - - - - + 9 - - - + - + + + + + 10 - - - +
- + + - - + 11 - - - + - + .+-. - - + 12 - - - .+-. - + .+-. - - +
13 - - - + - + - - - + 14 - - - + + - - - - + 15 - - - - - - - - -
+ 16 - - - - - - - - - + 17 - - - + - + - - - + 18 - - - + - + - -
- + 19 - - - + - + - - - + 20 - - - - - - - - - + 21 - - - + - +
.+-. - - + 22 - - - - - - - - - + 23 - - - + - + - - - + 24 - - - +
- + + - - + 25 - - - - - - - - - + 26 - - - - - - .+-. - - + 27 + -
- - - - - - - + 28 - - - + - + .+-. - - + 29 - - - + - + - - - + 30
- - - + - + .+-. - - + 31 - - - + - + - - - + 32 - - - - - - - - -
+ 33 - - - - - - .+-. - - + 34 - - - - - - - - - + 1/2 + - - - - -
- - - +
TABLE-US-00007 TABLE 6 Virulence of wild type and capsular mutant
S. suis strains, in germfree pigs clinical index of isolation of
pigs/ the group leuco- S suis in pigs S. suis group mortality.sup.2
morbidity.sup.3 spec non-spec. fever cyte [n- ] per group in
strains.sup.1 [n] [%] [%] symptoms.sup.5 symptoms.sup.6 index.sup.7
index.- sup.8 CNS serosae joints 10 4 100 100 11 88 43 44 2 3 4
10cpsB 4 0 0 0 10 1 3 1 3 2 10cpsEF 4 0 0 0 0 1 0 1 3 2
.sup.1strain10 in the wild type strain, strains 10cpsB and 10cpsEF
are isogenic capsular mutant strains .sup.2piglets which died
spontaneously or had to be killed for animal welfare reasons
.sup.3only considering pigs with specific symptoms .sup.4clinical
index: % of observations which matched the described criteria
.sup.5specific symptoms: ataxia, lameness on at least one joint,
stiffness .sup.6non-specific symptoms: inappetance, depression
.sup.7% of observations in the experimental group with a body
temperature > 40.degree. C. .sup.8% of blood samples in the
group in which nunber of granulocytes > 10.sup.10/1
TABLE-US-00008 TABLE 7 Bacterial strains and plasmids
strain/plasmid relevant characteristics Strain E. coli XL2 blue S.
suis reference strains serotypes 1-34 5667 serotype 7, tonsil
(1993) 7037 serotype 7, organs (1994) 7044 serotype 7, brains
(1994) 7068 serotype -7 (1994) 7646 serotype 7 (1994) 7744 serotype
7, lungs (1996) 7759 serotype 7, joints (1996) 8169 serotype 7
(1997) 15913 serotype 7, meninges (1998) Plasmid pKUN19replication
functions pUC, Amp.sup.R pGEM7Zf(+) replication functions MIC,
Amp.sup.R pCPS9-1 pKUN19 containing 1 kb HindIII-XbaI fragment of
cps operon of serotype 9 pCPS9-2 pKUN19 containing 4.0 kb XbaI-XbaI
fragment of cps operon of serotype 9 pCPS7-1 pKUN19 containing
1.6-kb PstI fragment of cps operon of type 7 pCPS7-2 pGEM7
containing 2.7-kh ScaI-C1aI fragment of cps operon of type 7
Amp.sup.R: ampicillin resistant cps: capsular polysaccharide
TABLE-US-00009 TABLE 8 Properties of Orfs in the cps genes of S.
suis serotype 7 and similarities to gene products of other bacteria
nucleotide position in proposed function similar gene product Orf
sequence of gene product (% identity) Cps7E 1-719
Glycosyltransferase Streptococcus suis Cps9E (99%) Cps7F 1164-1863
Glycosyltransferase Bordetella pertussis Bp1G.sup.1 (43%)
Streptococcus suis Cps2E.sup.1- (33%) Cps7G 1872-3086 Biosynthesis
amino sugar Bordetella pertussis Bp1F (48%) Cps7H 3104-3737
Glycosyltransferase Escherichia coli WbdN (35%) Streptococcus suis
Cps2K.sup.2 (31%) .sup.1similarity refers to the Cdenninal part of
the gene product .sup.2similarity refers to the N-terminal part of
the gene product
TABLE-US-00010 TABLE 9 Hybridization of serotype 7 cps probes with
chromosomal DNA of S. suis serotypes serotypes DNA probes 1 2 3 4 5
6 7 8 9 10 11 12 13 14 15 16 17 18 cps7E - - + + + - + - + + + + +
- - - + + cps7F - - - + + - + - - - - - - - - - + - cps7G - - - + +
- + - - - - - - - - - + - cps7H - - - - - - + - - - - - - - - - - -
16SrRNA + + + + + + + + + + + + + + + + + + serotypes DNA probes 19
20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 1/2 cps7E + - + - + +
- - - - + + + - - - - cps7F - - - - + - - - - - - - - - - - - cps7G
- - - - + - - - - - - - - - - - - cps7H - - - - - - - - - - - - - -
- - - 16SrRNA + + + + + + + + + + + + + + + + +
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SEQUENCE LISTINGS
1
53123DNAArtificialPrimer 1caaacgcaag gaattacggt atc
23223DNAArtificialprimer 2gagtatctaa agaatgccta ttg
23320DNAArtificialprimer 3ggcggtctag cagatgctcg
20419DNAArtificialprimer 4gcgaactgtt agcaatgac
19521DNAArtificialprimer 5ggctacatat aatggaagcc c
21620DNAArtificialprimer 6cggaagtatc tgggctactg
20721DNAArtificialprimer 7agctctaaca cgaaataagg c
21821DNAArtificialprimer 8gtcaaacacc ctggatagcc g
21926281DNAStreptococcus suis 9aagcttggat attgatcaca tgatggaggt
gatggaagca tctaagtctg cagcggggtc 60ggcgtgccca agtccgcagg cttatcaggc
agcttttgag ggagctgaga acattatcgt 120tgtgacgatt acaggtgggc
tatcgggtag ttttaatgcg gcacgtgtag ctagggatat 180gtatatcgaa
gagcatccga atgtcaatat ccatttgata gatagtttgt cagccagtgg
240ggaaatggat ttacttgtac accaaatcaa tcgcttaatt agtgcaggat
tagattttcc 300acaagtagta gaagcgataa ctcactatcg ggaacacagt
aagctcctct ttgttttagc 360gaaagttgat aatcttgtta agaatggaag
actgagcaaa ttggtaggca ctgtcgttgg 420tcttctcaat atccgtatgg
ttggtgaggc aagtgctgaa ggaaaattag agttgcttca 480aaaggcgcgt
ggtcataaga aatctgtgac agcagccttt gaagaaatga aaaaagcagg
540ctatgatggt ggtcgaattg ttatggccca ccgcaacaat gctaagttct
tccaacaatt 600ctcagagttg gtaaaagcaa gttttccaac ggctgttatt
gacgaagttg caacatcagg 660tctatgcagt ttttatgctg aagaaggtgg
acttttgatg ggctacgaag tgaaagcgtg 720attcacagag taataatttt
gggctgtaat ttccgctata gaataatccc cctcttcttc 780taagttcgag
ggggattgtt tgtatgagac tattggattt cattcattca aatatcttac
840gaattgctcc agtttatctg caaaatcttg ttcaaagaag atctgtaaga
aatcagcttt 900ctgtccgctg aaataataac attttccaaa catgtgttgg
atgctaggag aaagaatccc 960cttgcttagc tgaaaggtca cgctcccctt
tggaattcga tacgggatgt ttaaagcgta 1020tttctctaga cagtctttta
ttttattcca ttgagcgtga taaatgtgat gaagatgctg 1080tgtgttccgc
gcaaacatac cgttatcaat gtagagcgag agagcttttt gcatgataag
1140attggtatcg tagtcgatta gactcttatg tttgatgaag atatcacgta
gctgattagg 1200aaggctgatt gcaccgattc ggagggcagg aaagagtgtc
ggtgtaaaag attttatata 1260gatgacgcga ttatctgtat caagatagtg
taaaggtagg ctatgactag agtcgaaatc 1320tgctaaatag tcatcctcaa
tgatgtagac atcgtattgc tttgctaatt ttacgatggc 1380tgtttttgtt
gctatatcat aggttgaacc gagagggttg tgcaagcgag gaattgtgta
1440gaaaaactta atttttccag tttggaagat actttccaat tcttctaggt
caattccatc 1500taaattccgt tcaattgttt gataggggat tccttgatgt
cgaatgagct ctatcattcg 1560tgaataggta gggttctcta tcaagatttc
cgtttttcca gccaaggttt ccatttgtgt 1620gagaatatat agagcttgtt
gactaccagc tgtgataacc agctggtctt tttttgtata 1680gacatgatag
tccattaaca gactttgaac ggaggaaatc aattctgcca atccctcttg
1740ctggtgatag tagttgaata ggtaattttc ccgcccaata agactttctt
ttagacaaat 1800ccgaaaatct tcataggtaa ttcttgaaag tctgtaggat
tgagctctac aggtatggtc 1860ttggaaatct ctatcctcta agatataata
accgcttttt tcgacagcgt agatcttatt 1920ttggtatttt aattccaaca
tagccttttg gacagtgtct ttgctacaat gatattgctc 1980gcggagttga
cggatagaag gtaatttctc tccacgtttg aatcgatgtt cctctattcc
2040agtcaaaata tcttggatga taacttgata ttttttcatc taggtcccct
tttttataga 2100ctatgttact agctagtata tagaaaaaat tgaagaaaga
caatatatga ataatggggt 2160tgaggttcag gaattaagct actctatggt
ataattaagt gatgaaaata attataccta 2220atgcaaaaga agtaaataca
aatctagaga atgcctcgtt ttatctcctg tctgatcgaa 2280gcaagccggt
gctggatgcc ataagtcaat ttgatgtaaa aaagatggct gccttttata
2340aattgaatga agcaaaggct gagttagaag ctgaccgttg gtatcgaatc
aggacaggtc 2400aagcaaaaac ctatccagcc tggcagttat atgatggtct
catgtatcgt tatatggata 2460ggcgaggtat agattcgaaa gaagaaaatt
atttacgtga ccacgttcgt gtagcgacag 2520ccttatacgg attgattcat
ccttttgaat tcatttcacc tcaccgctta gattttcaag 2580ggagcttaaa
gataggcaat cagtctttga aacagtactg gcgaccgtat tatgaccaag
2640aagttggtga tgatgaactg attctctcac tggcttcgtc agaatttgag
caggtgtttt 2700ctccccagat tcagaaaaga ttagttaaaa ttcttttcat
ggaagaaaaa gcaggtcagc 2760taaaagttca ctcgactata tcaaaaaaag
gcagaggaag attgctgtcc tggttggcta 2820agaacaatat tcaggaatta
tcggacattc aagattttaa ggtggatggc tttgaatatt 2880gtacttccga
atcaacggca aaccaactta ccttcatacg atcaataaaa atgtgaaatt
2940atgaaaaaga taacgttttc cagcgctaaa aagggtagaa aaatattaat
ttctatgata 3000taatggatgc gttataggta aaagtctagg aaggttgttt
atgaaaaaga gaagcggacg 3060aagtaagtcg tccaagttca aattggtaaa
ttttgcgctt ttgggacttt attccattac 3120tctatgtttg ttcttagtga
ccatgtatcg ctataacatc ctagatttcc ggtatttaaa 3180ctatattgtg
acgcttttgc tagtaggagt ggcagtattg gctggattat tgatgtggcg
3240taagaaagcg cgcatattta cagcgctctt acttgttttt tcactggtca
tcacgtctgt 3300tgggatctat ggaatgcaag aagttgtaaa attttcaaca
cgactaaatt caaattcgac 3360attttcagaa tatgaaatga gtatccttgt
cccagcaaat agtgatatta cggacgttcg 3420tcagcttact agtatccttg
ctccagccga atacgaccaa gataacatca ccgctttatt 3480ggatgacata
tccaaaatgg aatctactca actagcaact agccccggga cttcttacct
3540gacagcatat caatctatgt tgaatggcga gagtcaagcg atggtgttca
acggagtttt 3600taccaatatt ttagaaaatg aagatccagg cttttcttca
aaagtgaaaa aaatatatag 3660tttcaaagtg actcagactg ttgaaacagc
tactaagcag gtgagtggag atagctttaa 3720tatctatatt agtggtattg
atgcttatgg accgatttct acggtctctc gttcagatgt 3780caatatcatt
atgactgtca atcgtgcgac acataagatt ttattgacaa ctactccacg
3840agattcatac gttgctttcg cagatggcgg gcaaaatcaa tacgataaac
taacacatgc 3900tggtatttac ggtgtcaatg cttctgtgca caccttagaa
aatttttatg ggattgacat 3960tagcaattat gtgcggttga acttcatttc
cttccttcaa ttaatcgact tggtgggtgg 4020aattgatgta tataacgatc
aagaatttac aagtttacat gggaattatc atttccctgt 4080tggacaagtt
catttaaact cagaccaagc attaggcttc gttcgagagc gctactcttt
4140aacagggggt gacaatgacc gtggtaaaaa ccaggaaaaa gtgattgctg
ccttgattaa 4200aaagatgagt acgccagaga atctaaaaaa ttaccaggca
atcctatctg gattggaagg 4260ctcaattcaa acggatttga gcttagaaac
gattatgagt ttagtgaata cccaactaga 4320atcaggaaca caatttacag
tagagtcaca agcattgaca ggaacaggac gctcagactt 4380atcttcttat
gcgatgcctg gatcacaact ttatatgatg gaaattaacc aagatagtct
4440ggagcaatca aaggcagcga ttcagtccgt acttgttgaa aaataaagat
tttaggagaa 4500aatatgaaca atcaagaagt aaatgcaatc gaaatcgatg
ttttattctt actaaaaaca 4560atttggagaa agaaattttt aattctctta
actgcagtgt tgactgcggg gttggcattt 4620gtctacagta gttttttagt
gacacctcaa tatgactcca ctacccgtat ctatgtagtg 4680agtcaaaatg
ttgaagccgg tgcgggcttg actaaccaag agttacaagc gggtacctat
4740ttggcaaaag actatcggga aattatccta tcacaagatg tattgacaca
agtagcaacg 4800gaattgaatc tgaaagagag tttgaaagaa aaaatatcag
tttctattcc tgttgatact 4860cgtatcgttt ctatttctgt gcgtgatgcg
gatccaaatg aagcggcacg tattgcaaat 4920agccttcgca cctttgcagt
gcaaaaggtt gttgaggtca ccaaggtaag cgatgtgacg 4980acacttgaag
aagcagtccc agcggaagaa ccaaccactc caaatacaaa acgaaatatc
5040ttgcttggtt tattagctgg aggtatcttg gcaacaggtc ttgtactggt
tatggaggtt 5100ttggatgacc gtgtaaaacg tcctcaggac atcgaagagg
taatgggatt gacattgcta 5160ggtatagtac cagattcgaa gaaattaaaa
taggagaaca atatggcgat gttagaaatt 5220gcacgtacaa aaagagaggg
agtaaataaa accgaggagt atttcaatgc tatccgtacc 5280aatattcagc
ttagcggagc agatattaag gttgttggta ttacctctgt taaatcgaat
5340gaaggtaaga gtacaactgc ggctagtctc gctattgcct atgctcgttc
aggttataag 5400accgtcttgg tggatgcaga tatccgaaat tcagtcatgc
ctggtttctt caagccaatt 5460acaaagatta caggtttgac ggattaccta
gcagggacaa cagacttgtc tcaaggatta 5520tgcgatacag atattccaaa
cttgaccgta attgagtcag gaaaggtttc tcccaaccct 5580actgcccttt
tacaaagtaa gaattttgaa aatctacttg cgactcttcg tcgctattat
5640gattatgtta tcgttgactg tccaccatta ggactggtaa ttgatgcagc
tatcattgca 5700caaaaatgtg atgcgatggt tgcagtagta gaagcaggca
atgttaagtg ctcatctttg 5760aaaaaagtaa aagagcagtt ggaacaaaca
ggcacaccgt tcttaggcgt tatcttgaac 5820aaatatgata ttgccactga
gaagtatagt gaatacggaa attacggcaa aaaagcctaa 5880tttctcagat
aacataagtt tgataagtag gtattaatat gattgatatc cattcgcata
5940tcatatttgg tgtggatgac ggtcccaaaa ctattgaaga gagcctgagt
ttgataagcg 6000aagcttatcg tcaaggtgtt cgctatatcg tagcgacatc
tcatagacga aaagggatgt 6060ttgaaacacc agaaaaaatc atcatgatta
actttcttca acttaaagag gcagtagcag 6120aagtttatcc tgaaatacga
ttgtgctatg gtgctgaatt gtattatagt aaagatatct 6180taagcaaact
tgaaaaaaag aaagtaccaa cacttaatgg ctcgtgctat attctcttgg
6240agttcagtac ggatactcct tggaaagaga ttcaagaagc agtgaacgaa
atgacgctac 6300ttgggctaac tcccgtactt gcccatatag agcgttatga
tgctctggca tttcagtcag 6360agagagtaga aaagctaatt gacaagggat
gctacactca ggtaaatagt aaccatgtgt 6420tgaagcctgc tttaattggc
gaacgagcaa aagaatttaa aaaacgtact cgatattttt 6480tagagcagga
tttagtacat tgtgttgcta gcgatatgca taatttatat agtagacctc
6540cgtttatgag ggaggcgtat cagcttgtaa aaaaagagta tggtgaggat
agagcgaagg 6600ctttgttcaa gaaaaatcct ttgttgatat tgaaaaatca
agtacagtaa cctcatagaa 6660atagtggagg agctatgaat attgaaatag
gatatcgcca aacgaaattg gcattgtttg 6720atatgatagc agttacgatt
tctgcaatct taacaagtca tataccaaat gctgatttaa 6780atcgttctgg
aatttttatc ataatgatgg ttcattattt tgcatttttt atatctcgta
6840tgccggttga atttgagtat agaggtaatc tgatagagtt tgaaaaaaca
tttaactata 6900gtataatatt tgtaattttt cttatggcag tttcatttat
gttagagaat aatttcgcac 6960tttcaagacg tggtgccgtg tatttcacat
taataaactt cgttttggta tacctattta 7020acgtaattat taagcagttt
aaggatagct ttctattttc gacaacctat caaaaaaaga 7080cgattctaat
tacaacggct gaactatggg aaaatatgca agttttattt gaatcagata
7140tactatttca aaaaaatctt gttgcattgg taattttagg tacagaaata
gataaaatta 7200atttaccatt accgctctat tattctgttg aagaagctat
agggttttca acaagggaag 7260tggtcgacta cgtctttata aatttaccaa
gtgaatattt tgacttaaag caattagttt 7320cagactttga gttgttaggt
attgatgtag gcgttgatat taattcattc ggttttactg 7380tgttgaagaa
taaaaaaatc caaatgctag gtgaccatag catcgtcact ttttccacaa
7440atttttataa gcctagtcac atctggatga aacgactttt agatatactt
ggagcagtag 7500tcgggttaat tattagtggt atagtttcta ttttgttaat
tccaattatt cgtagagatg 7560gtgggccagc catttttgct cagaaacgag
ttggacagaa tggacgcata tttacattct 7620acaagtttcg ttcgatgttt
gttgatgccg aggtacgtaa gaaagaatta atggctcaaa 7680accagatgca
aggtgggatg ttcaaaatgg acaacgatcc tagaattact ccaattggac
7740acttcatacg aaaaacaagt ttagatgagt taccacaatt ttataatgtt
ctaattggag 7800atatgagtct agtcggtacc cgtccgccta cagttgatga
atttgaaaaa tatactccta 7860gtcaaaagag aagattgagt tttaaaccag
ggattacagg tctttggcaa gtgagcggaa 7920gaagtgatat cacagatttt
aatgaagtcg ttaggctgga cctaacatac attgataatt 7980ggaccatctg
gtcagacatt aagattttat tgaagacagt gaaagttgta ttgttgagag
8040agggaggtca gtaagactcc tttaaaacaa agaatagtag taggggatat
gagaacagtt 8100tatattattg gttcaaaagg aataccagca aagtatggtg
gtttcgagac tttcgtagaa 8160aaattaactg agtatcagaa agataaatca
attaattatt ttgttgcatg tacaagagaa 8220aattcagcaa aatcagatat
tacaggagaa gtttttgaac ataatggagc aacatgtttt 8280aatattgatg
tgccaaatat tggttcagca aaagccattc tttatgatat tatggctctc
8340aagaaatcta ttgaaattgc caaagataga aatgatacct ctccaatttt
ctacattctt 8400gcttgtcgga ttggtccttt catttatctt tttaagaagc
agattgaatc aattggaggt 8460caacttttcg taaacccaga cggtcatgaa
tggctacgtg aaaagtggag ttatcccgtc 8520cgacagtatt ggaaattttc
tgagagtttg atgttaaaat acgctgattt actaatttgt 8580gatagcaaaa
atattgaaaa atatattcat gaagattatc gaaaatatgc tcctgaaaca
8640tcttatattg cttatggaac agacttagat aaatcacgcc tttctccgac
agatagtgta 8700gtacgtgagt ggtataagga gaaggaaatt tcagaaaatg
attactattt ggttgttgga 8760cgatttgtgc ctgaaaataa ctatgaagta
atgattcgag agtttatgaa atcatattca 8820agaaaagatt ttgttttgat
aacgaatgta gagcataatt ccttttatga gaaattgaaa 8880aaagaaacag
ggttcgataa agataagcgt ataaagtttg ttggaacagt ctataatcag
8940gagctgttaa aatatattcg tgaaaatgca tttgcttatt ttcatggtca
cgaggttgga 9000ggaacgaacc catctttact tgaagcactt tcttctacta
aactaaatct tcttctagat 9060gtgggcttta atagagaagt aggggaagaa
ggagcgaaat actggaataa agataatctt 9120cacagagtta ttgacagttg
tgagcaatta tcacaagaac aaattaatga tatggatagt 9180ttatcaacaa
aacaagtcaa agaaagattt tcttgggatt ttattgttga tgagtatgag
9240aagttgttta aaggataagt tatgaaaaag attctatatc tccatgctgg
agcagaatta 9300tatggggcag ataaggttct cttggaactt ataaaaggct
tagataagaa tgaatttgaa 9360gcgcatgtta tcctacctaa tgatggagtc
ctagtgccag cattaagaga agttggtgcg 9420caagttgaag ttattaacta
tccaattcta cgtaggaaat attttaatcc aaaagggatt 9480tttgactact
tcatatcata tcatcactat tctaaacaga ttgctcaata tgccatagaa
9540aataaggttg acataattca caataatact accgctgtct tagaaggcat
ttatctgaag 9600cgaaaactca aattaccttt gttgtggcat gttcatgaga
ttattgtcaa acctaaattc 9660atctctgatt cgatcaattt tttaatgggg
cgttttgctg ataagattgt gacagtttca 9720caggctgtgg caaaccatat
aaaacaatca cctcatatca aagatgacca aatcagtgta 9780atctacaatg
gggtagataa taaagtgttt tatcagtccg atgctcggtc tgttcgagaa
9840agatttgaca ttgacgaaga ggctcttgtc attggtatgg tcggtcgagt
caatgcgtgg 9900aaaggacaag gagatttttt agaagcagtt gctcctatac
tcgaacagaa tccaaaagct 9960atcgccttta tagcaggaag tgcttttgaa
ggagaagagt ggcgagtagt agaattagaa 10020aagaagattt ctcaattaaa
ggtctcttct caagtcagac gaatggatta ttatgcaaat 10080accactgaat
tatataatat gtttgatatt tttgtacttc caagtactaa tccagaccct
10140ctaccaacgg ttgtactaaa agcaatggca tgcggtaaac ctgttgtcgg
ttaccgacat 10200ggtggtgttt gtgagatggt gaaagaaggt gttaacggtt
tcttagtcac tccgaactca 10260ccgttaaatt tatcaaaagt aattcttcag
ttatcggaaa atataaatct cagaaaaaaa 10320attggtaata attctataga
acgtcaaaaa gaacattttt cgttaaaaag ctatgtaaaa 10380aatttttcga
aagtctacac ctccctcaaa gtatactgat tggctgaagt gaatgcttta
10440gtatagcgat ttatcgtatt ctcattcgat aaaacaaatg ttcagaaaca
gttataagtt 10500atttctaaag ggcacctcta taaactccca aaattgcgaa
tttggagtta cgaaagcctt 10560gttaaatcaa cattttaaat tttagaaaat
tagtttttag agctccccta aaatagaaga 10620taacagaagg gagccttcaa
aaacttcatt tttaattgga ttgtagaaaa actgttaaat 10680caatatttag
atttttagga gttcagtttt tggggggaga gcttaataat ctatgcacta
10740tatttcgaaa aatatatggt gtaaaatcag aactgatggt cgtggcaaaa
aagagaatga 10800ggaatttatg aaaattattt cttttacaat ggttaataac
gaaagtgaga taatagagtc 10860atttatacgg tataattata actttattga
cgagatggtc attattgata atggttgtac 10920agataacacg atgcaaatta
tttttaattt gattaaagag ggatataaaa tatccgtata 10980tgatgagtct
ttagaggcat ataatcagta tcgacttgat aataaatatc taacgaaaat
11040aattgctgaa aaaaatccag atttgataat acctttggat gcggatgaat
ttttaacagc 11100cgattcaaat ccacggaaac ttttggaaca actggactta
gaaaagatac attatgtgaa 11160ttggcaatgg tttgttatga ctaaaaaaga
tgatattaat gattcgttta taccacgtag 11220aatgcaatat tgttttgaaa
aacctgtttg gcatcattct gatggtaaac cagttactaa 11280atgtataatt
tccgctaagt attacaaaaa aatgaattta aagctatcga tgggacatca
11340cactgttttt ggtaacccaa atgtaaggat agaacatcat aatgatttga
aatttgcaca 11400ttatcgagct attagccaag agcaattaat ttataaaaca
atttgttaca ctattcgcga 11460tattgctact atggagaaca atatcgaaac
agctcaaaga acaaatcaga tggcgctcat 11520tgaatctggc gtggatatgt
gggaaacggc gagagaagcc tcttattcag gttatgattg 11580taatgttata
catgcaccaa ttgatttaag tttttgtaaa gaaaatattg taataaaata
11640taacgaacta tccagagaaa cagtagcaga acgcgtgatg aaaacgggaa
gagaaatggc 11700tgttcgtgca tataatgtgg agcgaaaaca aaaagaaaag
aaatttctaa aacctattat 11760atttgtatta gatgggttaa aaggagatga
gtatattcat cccaatccat caaatcattt 11820gacgatctta actgaaatgt
ataacgtcag aggcttactt accgataatc accaaattaa 11880atttctcaaa
gttaattata gattaattat aactccagat tttgctaagt ttttaccgca
11940tgaatttatt gttgtaccag ataccttgga tatagagcaa gttaaaagcc
agtatgttgg 12000tacaggtgta gacttgtcaa agattatttc tttaaaagag
tatcgaaaag agataggctt 12060tattggtaat ttgtatgcgc ttttaggatt
tgttccgaat atgctcaata gaatttatct 12120atatattcag agaaacggta
ttgcaaacac tattataaaa atcaagtcga gattgtgaga 12180gttgtttact
tttatttgta attttaaaag taatgcaggc agataggaga aaaacgtttg
12240gaaaaatgag aataagaatt aataatttgt tttttgttgc catagcgttt
atgggcataa 12300ttattagtaa ttcgcaagtt gttctagcga taggcaaagc
ttctgtgatt cagtatctat 12360cttatttagt tttgatttta tgtatagtta
atgatttatt aaaaaataac aaacatattg 12420tagtttataa attagggtat
ttgtttctta ttatattttt atttactatc ggaatatgtc 12480agcaaattct
tcctataaca actaaaatat atttatcaat ttcaatgatg attatttcag
12540ttttagcaac gttgccaata agtttgataa aagatattga tgattttaga
cggatttcaa 12600atcatttgtt attcgctctt tttataactt cgatattagg
aataaagatg ggggcaacga 12660tgttcacggg ggcagtagaa ggtatcggtt
ttagtcaggg ttttaatgga ggattgacgc 12720ataagaactt ttttggaata
actattttaa tggggttcgt attaacttac ttggcgtata 12780agtatggttc
ctataaaaga acggatcgtt ttattttagg attagaattg tttttgattc
12840ttatttcaaa cacacgctca gtttatttaa tactattgct ttttctattt
cttgttaatc 12900ttgacaaaat caaaatagaa caaagacaat ggagtacgct
taaatatatt tccatgctat 12960tttgtgctat ttttttatac tatttctttg
gttttttaat aacacatagt gattcttacg 13020ctcatcgcgt taatggtctt
attaattttt ttgagtatta tagaaatgat tggttccatc 13080taatgtttgg
tgcagcggat ttggcatatg gggatttaac tttagactat gctataaggg
13140ttagacgcgt tttaggttgg aatggaacgc ttgaaatgcc cttactgagt
attatgttaa 13200aaaatggttt tatcggtctg gtagggtatg ggattgtttt
atataaactt tatcgtaatg 13260taagaatatt aaaaacagat aatataaaaa
caataggaaa gtctgtattt atcattgtag 13320tcctatctgc aacagtagaa
aattatattg taaatttaag ttttgtattt atgccaatat 13380gtttttgttt
attaaattct atatctacta tggaatcaac tattaacaaa caactgcaaa
13440cataaattgg caggaataga gttttgagtt gctattaatt tggtagagca
tatgttctat 13500aggtggcaag ataaagatag tattttttac atgatgattt
ttatgatagc aaagcaagtt 13560acggcataaa aggaattaga ggatggaaaa
agtcagcatt attgtaccta tttttaatac 13620ggaaaagtac ttaagagagt
gtttagatag cattatttcc caatcgtata ctaatctaga 13680gattcttttg
atagatgacg gttcttcaga ttcatcaacg gatatatgtt tggaatacgc
13740agagcaagat ggtagaataa aacttttccg gttaccaaat ggtggtgttt
caaacgcaag 13800gaattacggt atcaaaaata gcacagcaaa ttatattatg
tttgtagatt ctgatgatat 13860tgttgacggc aacattgttg agtccttata
cacctgttta aaagagaatg atagtgattt 13920gtcgggaggg ttacttgcta
cttttgatgg aaattatcaa gaatctgagc tgcaaaagtg 13980tcaaattgat
ttggaagaga taaaagaggt gcgagactta ggaaatgaaa attttcccaa
14040tcattatatg agcggtatct ttaatagccc ttgttgcaaa ctttataaga
atatatatat 14100aaaccaaggt tttgacactg aacagtggtt aggagaggac
ttattattta atctaaatta 14160tttaaagaat ataaaaaaag tccgctatgt
taacagaaat ctttattttg ccagaagaag 14220tttacaaagt actacaaata
cgtttaaata tgatgttttt attcaattag aaaatttaga 14280agaaaaaact
tttgatttgt ttgttaaaat atttggtgga caatatgaat tttctgtttt
14340taaagagacg ctacagtggc atattattta ttatagctta ttaatgttca
aaaatggaga
14400tgaatcgctt ccaaagaaat tgcatatatt taagtattta tacaataggc
attctttaga 14460tactctaagt attaaacgaa cgtcctctgt ttttaaaaga
atatgtaaat taattgttgc 14520taataatttg tttaaaattt ttttaaatac
tttaattagg gaagaaaaaa ataatgatta 14580acatttctat catcgtccca
atttacaatg ttgaacaata tctatccaag tgtataaata 14640gcattgtaaa
tcagacctac aaacatatag agattcttct ggtgaatgac ggtagtacgg
14700ataattcgga agaaatttgt ttagcatatg cgaagaaaga tagtcgcatt
cgttatttta 14760aaaaagagaa cggcgggcta tcagatgccc gtaattatgg
cataagtcgc gccaagggtg 14820actacttagc ttttatagac tcagatgatt
ttattcattc ggagttcatc caacgtttac 14880acgaagcaat tgagagagag
aatgcccttg tggcagttgc tggttatgat agggtagatg 14940cttcggggca
tttcttaaca gcagagccgc ttcctacaaa tcaggctgtt ctgagcggca
15000ggaatgtttg taaaaagctg ctagaggcgg atggtcatcg ctttgtggtg
gcctggaata 15060aactctataa aaaagaacta tttgaagatt ttcgatttga
aaagggtaag attcatgaag 15120atgaatactt cacttatcgc ttgctctatg
agttagaaaa agttgcaata gttaaggagt 15180gcttgtacta ttatgttgac
cgagaaaata gtatcataac ttctagtatg actgaccatc 15240gcttccattg
cctactggaa tttcaaaatg aacgaatgga cttctatgaa agtagaggag
15300ataaagagct cttactagag tgttatcgtt catttttagc ctttgctgtt
ttgtttttag 15360gcaaatataa tcattggttg agcaaacagc aaaagaagct
tctccaaacg ctatttagaa 15420ttgtatataa acaattgaag caaaataagc
gacttgcttt actaatgaat gcttattatt 15480tggtagggtg tcttcatctt
aattttagtg tctttctgaa aacggggaaa gataaaattc 15540aagaaagatt
gagaagaagt gaaagtagta ctcggtaaga atgttgtaat aaatggttga
15600aagaaaaggg gattaaaatg aatccaacaa atagtagaat agcactcttt
gatacgatta 15660aatgtatcat ggtactttgt gttattttta cacatctgga
ttggtctgtt gagcagcgtc 15720aatggtttat ctttccgtat ttcgttgaca
tggctgttcc aatttttctg ttgctttctg 15780cctattttcg aacgaataag
tggaatacaa aacaagagac gctaaagctc aagttcagca 15840gtggtataaa
agaaagtata aacatgcttt gtctctatgc tatcgtgatg gctgttaatg
15900ttttattgag ctattcgaga accatctgat aggagtaaag cctttttcag
gttcttcatc 15960gctccgttca tttgtcctgt ggctactttc tggagaatcg
ggtccaggga gttgggagtt 16020actatgttcc gttgttgatt caggtagttt
ttttattacc aattttgtat gttcttttcg 16080agaaaaataa atggttgggc
ttgcttactt gttttttagt aaacttttca gtggatgcca 16140tatttgctaa
catggctgaa cacggcatat atatatagac taatatcact tcgttatctt
16200tttgttctag ggcttggttt tttctttcaa agcaggatgt gcgttccaag
gtagatactt 16260tcattgcgac cctatttggg attattggag caattctgat
ttttgtgaat cattctatag 16320agcccttctc ctggttttat ggttggaagt
ctacttcctt tctatgcgtc ccatttgcgt 16380atgctatgct attttttatg
ataaagtatg gacagaagat tccagcaata ctgttgtcaa 16440aattgggagt
tgcttcttat catatctact tgacccagat gctgtatttt tcagtagtcg
16500caccattttt agcagtgcaa tttaaggtat cttcgttgaa tttgtggaac
ggcttgttta 16560cctttctaat ttgcctgttt ggtggctata ttttctacaa
agtggatctg tttatgagag 16620tacgtggaaa acgataatga ctcatttcag
attagcagat gccatttcgt ttattagcag 16680attcgcatgt taatattccg
acaaagaaat tcaaataggt tgacgagaga ggagtggtat 16740ctgtttctaa
accccagtat ccccctttat tttcaaagct atatttatta actgaacaag
16800gagaattttt aagagaactg tttgtttaat cccagcacga tctggttcga
aaggcttacc 16860gaataaaaac atgctatttt tggacgggaa acccatgatt
tttcacacga ttgatgtggc 16920aattgaatca ggttgttttg agaaagaaga
catctatgtc agtacggatt cagaaatgta 16980taaggggggc acctctataa
attcccaaaa ttgcgaattt ggagttacga aagccttgtt 17040aaatcaacat
cttaaatttt agaaaattag tttttagagg tccccaaggg gatttgcgag
17100acaagaggca tcaatgtatt gttaagaccc aaagaactat ctacttatca
tactccatcg 17160aatgaagtca gtacgcactt ttttacgaat ctggatttta
tgaagattgt atatttgttc 17220ttctgcaagt cacctcaccg ttacggactg
gcgaacagat aaaagaagcc atgaatatgt 17280acttacaggg ggactcagaa
aatgttttgc atttcaatga tgaagggcaa gaaagagtga 17340atcagtacat
tatcgaagct gtacaggggt tataaaaagg ggttacttat ccttaaagtc
17400tgtatgtaga aggagaaaaa ttgagacgaa tttatatttg ccatacgatg
tatcagatcc 17460tgatttcctt gttaaagatg gacgttgaga gagatagttt
gatgtccgtt gatatcatcg 17520ggcattttcc agatgtcagg gagcaactgc
agcagcatgt tcatctaatc gagggagacg 17580gagcgttcat ttgatctata
ttctttgata gctagatcaa aaacaaaaga acgcctttcc 17640ttgttacaga
gctatgacga ggtgatcatt tttcaagatc accgtcaagt cggtcatttt
17700ttaaataaac atcggattcc ctattctctt ttggaggatg gttataattt
tttcaaggat 17760aaaagagtgt gcgatttgga gtcaattcaa tcatctgtct
ggaaaagact cttttatcaa 17820tggtatttta aaccaacata tttgattggt
tcaagtctct attgtcaatc cattgaggtc 17880aatgatctgt cgctcgtaca
atttgactag gcttataaac cctttgtaga agttccgaga 17940aagcaattat
ttgatcaagc atcgccagag aaggtgcaag cgctgctgca gatatttgga
18000gcaagggcga tagtagcgga tgaagagtct tctcaaaaac gattgctatt
attgacccag 18060cccttgtctt gggattatca tgtgaccgaa gagagttgtt
ggagatttat gtagcaggtc 18120ttgcccctta tcgggaagac tatacaatct
acataaaacc gcacccacga gatggggttg 18180attattcatt tctgggtaag
gctgtggtgc ttctgcctca aggtattccg tttgagttgt 18240tcgaaatggc
aggtaatatc cgttttgata tcggtatgac ctatagttcg tctgctttag
18300attttttaaa ttgttttgaa gagaaagtgt atttaaagga cacttttcct
cttctttcaa 18360aaaatgatat tttgcgtgag gggatagaat aggaggattc
atgtctaaaa aatcaatagt 18420tgtctcaggt ctcgtctata cgattggaac
catcctcgtt cagggattag ccttcattac 18480cctccccatc tatactcgtg
tcatttctca ggaagtatat gggcagttta gcttgtataa 18540ttcgtgggtg
gggctagttg gtctctttat cggtctacag ttaggtgggg cttttggccc
18600gggatgggta cacttccgcg agaaatttga tgatttcgta tccaccttga
tggtctcttc 18660tatcgctttc tttttaccaa tttttgggct atcttttctc
ctcagtcagc ccctatcgct 18720cctatttggt ttgcctgatt gggtcgttcc
gctttacttt ttgcaaagtt ttatgagtgt 18780tgtgcaagga ttttttacga
cctatttagt gcagcggcag cagtccatgt ggactttact 18840cctatcggta
ctgagcgctg ttatcaacac tgctttatct ttatttctca tcttttcgat
18900ggagaatgat ttcatcgctc gtgtaatggc aaactcggca acgactggtg
tttttgcttg 18960tgtgtccttg ttgtttttct ataagaagat tgggcttcat
tttcgaaagg actatcttcg 19020gtatggttta agtatatcga ttcctcttat
ttttcatgga ttaggtcata atgtactcaa 19080tcaatttgac agaatcatgc
tcggcaagat gctaacactg tcagatgtag ccctatacag 19140tttcggctac
acacttgcgt ctatcttaca aattgtgttt tcgagcttga atacggtatg
19200gtgtccgtgg tattttgaga aaaagagagg tgcagataaa gatttgctca
gttatgtccg 19260ttactatctg gcgattggcc tgtttgtgac ttttggattt
ctaacaattt accctgaatt 19320agcgatgttg ttaggtggat ctgagtatcg
tttcagtatg ggatttattc ccatgattat 19380tgtcggggtg ttctttgtat
ttctttatag ttttccagcc aatatccagt tttatagtgg 19440aaatacaaag
tttttgccaa ttggtacttt tatagcaggt gtactaaata tttccgtcca
19500ctttgttttg ataccgacaa agaatttatg gtgctgcttt gcaacgactg
cttcctatct 19560gttgttgcta gtcttgcatt attttgttgc taagaaaaag
tatgcttacg atgaagttgc 19620gatttcaaca tttgttaagg taattgctct
tgttgtcgtc tatacaggct tgatgacagt 19680atttgtcggt tcaatctgga
ttcgttggtc actaggaata gcggttctag tcgtttatgc 19740ctacattttt
agaaaggaat taacagttgc cctcaataca ttcagggaaa aacggtctaa
19800ataagggcac ctctataaac tcccaaaatt gcgaatttgg agttacgaaa
gccttgttaa 19860atcaaacatt ttaaatttta gaaaattagt ttttagaggt
ccccatataa aaacgtccca 19920aatgagaggt gctcataaga attgaccatc
actgccatct acccaaagtt caagtattct 19980ctaccatgaa aattgtgcta
taatcaagta taaagaaggg aatgtttctt aaaggacgta 20040tgcgcctctg
cttatgccag aagtcatgag gtaaatctcc ctaaaaattg ggtagaaaag
20100cagattaaac ttccaccaat ctattgaaga tcgtgttgaa gagcaggctt
tagaagcaac 20160aagccctgag actattcgaa agaaatctag ggctattttt
tctaatcggc tatcagaagt 20220gaagtagcga tctttattag tgttctttta
ctacttaagg aaaaccaagc tgctccctca 20280agactttatg ggagcgattt
acagtcattt ttagaaagga aataaaatgg tttatattat 20340tgcagaaatt
ggttgtaatc acaacggtga tgttcatcta gcacggaaaa tggtagaagt
20400tgccgttgat tgtggtgtgg atgccgttaa atttcagaca tttaaggcag
atttgttgat 20460ttcaaaatac gcaccaaagg ccgaatacca aaaaattaca
acaggagagt cagattctca 20520gctcgaaatg actcgtcgtt tggaattgag
ctttgaagag tatcttgatt tgcgtgatta 20580ctgtcttgaa aagggagttg
atgtgttttc gacacctttt gatgaggaat cattggactt 20640cttgattagc
acagatatgc ccgtttataa gattccatct ggtgagatta ccaatcttcc
20700ctatttggaa aaaattggtc gtcaagctaa gaaagttatt ctttcaactg
gtatggctgt 20760tatggatgaa attcatcaag cggtgaagat tttgcaggaa
aatggaacga ccgatatttc 20820gattttgcat tgtacaaccg agtatccaac
cccttaccct gctttgaatt tgaatgtctt 20880gcataccttg aaaaaagaat
ttccaaactt aacaattggc tattcagacc atagtgttgg 20940ttcagaagta
cccatcgctg ctgcagcaat gggagctgaa ttgattgaaa agcactttac
21000tctggacaat gaaatggaag gaccagatca taaagcgagt gctactcctg
atatcttagc 21060agccttggta aaaggagtga ggatagtgga acaatctctt
ggtaaatttg aaaaagagcc 21120agaagaagtt gaagtacgaa ataaaattgt
agctagaaaa tctattgttg ccaaaaaagc 21180aattgctaaa ggcgaagtct
ttacagaaga aaacatcact gtcaaaagac caggaaatgg 21240aatttcgcca
atggaatggt acaaagtctt ggggcaggtg agtgagcagg attttgagga
21300agaccaaaat atttgccata gtgcttttga aaatcaaatg taagcggagt
aaggatgaaa 21360aaaatttgtt ttgtgacagg ctctcgtgcc gaatatggga
ttatgcgtcg cttattgagc 21420tatctacagg atgatccaga aatggagctg
gatcttgtag tgacagccat gcatctagaa 21480gaaaaatatg ggatgacggt
caaagacatc gaagcggaca agcgtaggat tgtcaagcgg 21540attccattgc
atttgacgga tacgtctaag cagacaatcg tcaaatcttt agcgaccttg
21600acagagcaac tcacggttct ttttgaagaa gtccagtatg acttggtgtt
gattctgggg 21660gatcgctatg agatgctacc agttgccaat gctgcgttgc
tttataatat tcctatttgc 21720catattcatg gtggtgaaaa aaccatggga
aattttgatg agtcgattcg ccatgccatt 21780accaagatga gtcaccttca
tctgacatca acggatgaat ttagaaatcg tgtcattcaa 21840ctaggagaaa
atccaaccat gtactgaaca tcggagctat gggtgttgaa aatgttttaa
21900aacaagactt tttgacaaga gaagagttgg cgatggaact tggaattgat
tttgccgagg 21960attactatgt tgtactcttt caccctgtta ccttggagga
taacacagcc gaagaacaaa 22020cgcaggcctt attagatgct ctaaaagaag
atggtagcca gtgtttgata attggatcca 22080attcggatac acatgccgat
aagataatgg aattgatgca tgaatttgta aaacaagact 22140ctgattctta
catctttact tcgcttccaa ctcgttatta ccattccttg gtcaagcatt
22200cacaaggttt aatagggaat tcttcgtcag gtttgattga agtgccctca
ttacaggttc 22260cgaccttaaa tattggaaat cgccaatttg gacgtttgtc
aggaccgagt gtggtacatg 22320ttggaacttc taaggaagcg attgttggtg
gtttggggca attacgtgat gtgatagatt 22380ttaccaatcc atttgaacaa
cctgattctg ctttacaagg ttatcgagct atcaaggaat 22440ttttatctgt
acaggcctca accatgaaag agttttatga tagatagggg agaaagtttg
22500atgaaaaaag tagcctttct aggagcgggt accttttcag atggtgtcct
tccttggttg 22560gatagaactc gatatgaact cattggatat tttgaagata
aaccgatcag tgactatcgt 22620ggctatcctg tatttggtcc cttgcaagat
gtcctaacct atttggatga tggaaaagta 22680gatgctgtct tcgtcactat
aggtgacaat gtcaagcgca aggaaatctt tgacttgctt 22740gccaaagatc
attatgatgc tttgttcaac atcattagcg agcaagccaa tattttttcc
22800ccagatagta tcaagggacg aggggttttc ataggttttt caagttttgt
aggagccgat 22860tcctatgtct atgacaattg tatcatcaat acgggtgcca
ttgtggaaca tcataccacg 22920gtggaggccc attgtaacat tactccagga
gtgaccataa atggcttgtg ccgtatcgga 22980gaaagcactt atattggaag
tggttcaaca gtgattcaat gtatcgagat tgcaccttat 23040acaacattgg
gggcagggac agttgttttg aaatcgttga cggagtcagg gacctatgtt
23100ggtgtacctg ctagaaagat taaataggtg aattgatgga accaatttgt
ctgattcctg 23160ctcggtcagg atcaaaaggt ttaccaaata aaaacatgtt
atttttagat ggtgtaccga 23220tgattttcca taccattcga gctgcgattg
agtctggatg ttttaagaaa gaaaatatat 23280atgtcagtac tgattcagag
gtttacaagg aaatttgtga aacaactggg gttcaagtcc 23340tcatgcgtcc
agctgacttg gcgacagatt ttacaacctc ttttcaactg aacgaacatt
23400ttttacaaga tttttctgat gaccaagtat ttgttctcct gcaagttacg
tccccattaa 23460gatcgggaaa acatgtcaag gaggcgatgg agttatatgg
gaaaggtcaa gctgaccacg 23520ttgttagctt taccaaagtc gataagtctc
caacattgtt ttcaacttta gacgaaaacg 23580gattcgctaa ggatattgca
ggattaggtg gcagttatcg tcgtcaagat gagaaaacac 23640tctactatcc
taatggagcg atttatattt cttctaagca ggcttattta gcggataaaa
23700cttatttttc tgaaaaaaca gcggcctatg tgatgacgaa ggaagattcg
attgatgtag 23760atgatcactt tgattttact ggtgttattg gtcgaattta
ctttgattac cagcgtcgtg 23820agcaacaaaa caaaccattt tataaaagag
agttaaagcg tttatgtgag caacgagtcc 23880atgatagtct tgtgattggc
gatagtcgtc tgttagcctt gttactggat ggtttcgata 23940atatcagcat
cggtgggatg acagcttcga cagcacttga aaaccaaggt ctctttttgg
24000ctactccgat aaagaaagtt ttgctttctc ttggtgtgaa tgatttgatt
actgactatc 24060ccttgcatat gattgaggat actattcgcc agctgatgga
aagtcttgtt tccaaagcag 24120agcaggtttt tgtgacgacg attgcctaca
cgctgtttcg tgatagcgtt tccaatgaag 24180aaattgtgca gctgaatgac
gttattgttc agtcagcaag tgaactgggt atttcagtga 24240ttgatctaaa
tgaagttgtt gaaaaagagg cgatgcttga ctatcagtat accaatgatg
24300gattgcattt caatcagatt ggacaagagc gtgtgaatca gctgattttg
acaagtttga 24360caagataatt tggtgataga agctatttca gtggctagac
tatgttggta tgtgttttag 24420agcccaggaa taacatctgt agaggatgct
agccttgaga attgacaacc atttagttgt 24480tttaattata taaggggacc
tctaaaaact ccctaaattt cccaaaaatg agataataga 24540ataaaaagta
atgaggagag ctgtcatgca tttattcaca gacgatgaaa aaatcttgtc
24600aaaactatca gagaaaggca atcccttaga acgtttggat gccgttatgg
attggaatat 24660ctttcttcca ttgttgtcag agttattcag tcgtaaagat
aaagtcatca gtcgtggcgg 24720tcgtcctcac ctagactatc tcatgatgtt
caaagcgctc ttgcttcaac gtcttcataa 24780cctatctgac gatgccatgg
aatatcaact gctggatcgt atatcttttc gtcgttttgt 24840tggttgtcat
gaagacactg ttcccgatgc gaaaactatc tggctctatc gtgagaaatt
24900aaccaagtca ggtcgtgaaa aggagttgtt cgatttgttc tatgcccatc
tcacagatga 24960aggggtgatt gcccattcag gtcagattgt ggatgctacc
tttgtcgaat gccctaaaca 25020acgcaattca cgtgaggaca atcagaaaat
caaaacttat cgaaaattat gaggtcacaa 25080cagctagtgt acacgactcc
aatgtcctag ctcctctttg tgatgccaat gaagcggttt 25140ttgatgacag
tgcttatgtt ggaaaatcag taccagaagg ttgtcgccac cacacgattc
25200gtcgtgcttt tagaaataaa ccgttgactg agactgataa ggtcattaat
cgacatatta 25260ccaaagtccg ttgtcgcgtt gagcatggtt ttggcttcat
tgaaactaac atgaaaggta 25320acatctgtcg agcaattggg aaggcacgag
ctgaaaccaa tgtgacctta accaacctgc 25380tctacaatat ctgtcgtttt
gagcaaatca aacgactggg attaccatcc gtgggcttag 25440tgcgcccaaa
aaataggaaa ataagcaaaa agaggctggg caaaaactag tttctcacaa
25500taaaaaaacg gctctttgtc aactgtagtg ggtagacgaa aagctaacac
ctagagagga 25560cgaaattcgt tctctcattt ttgatgttta aagcgtaacc
gcctaataac aaggtatcta 25620tccaatcaca cattcctcca ttatatagtt
aaatgaaaca aaaacagtac atctatgata 25680taatgtattt atggcatatt
cattagattt tcgtaaaaaa gttctcgcat actgtgagaa 25740aaccggcagt
attactgaag catcagctat tttccaagtt tcacgtaaca ctatctatca
25800atggctaaaa ttaaaagaga aaaccggcga gcttcatcac caagttaaag
gaaccaagcc 25860aagaaaagtg gatagagata aattaaagaa ttatcttgaa
actcatccag atgcttattt 25920gactgaaata gcttctgaat ttgactgtca
tccaacagct attcattacc ccctcaaagc 25980tatgggatat actcgaaaaa
aaagagctgt acctactatg aacaagaccc tgaaaaagta 26040gaactgttcc
ttaaagaatt gaataactta agccacttga ctcctgttta tattgacgag
26100acagggtttg agacatattt tcatcgaaaa tatggtcgct ctttgaaagg
tcagttgata 26160aaaggtaagg tctctggaag aagataccag cggatatctt
tagtagcagg tctcataaat 26220ggtgcgctta tagccccgat gacatacaaa
gatactatga cgagtggctt tttcgaagct 26280t 2628110239PRTStreptococcus
suismisc_featureORF2Z 10Ser Leu Asp Ile Asp His Met Met Glu Val Met
Glu Ala Ser Lys Ser 1 5 10 15 Ala Ala Gly Ser Ala Cys Pro Ser Pro
Gln Ala Tyr Gln Ala Ala Phe 20 25 30 Glu Gly Ala Glu Asn Ile Ile
Val Val Thr Ile Thr Gly Gly Leu Ser 35 40 45 Gly Ser Phe Asn Ala
Ala Arg Val Ala Arg Asp Met Tyr Ile Glu Glu 50 55 60 His Pro Asn
Val Asn Ile His Leu Ile Asp Ser Leu Ser Ala Ser Gly 65 70 75 80 Glu
Met Asp Leu Leu Val His Gln Ile Asn Arg Leu Ile Ser Ala Gly 85 90
95 Leu Asp Phe Pro Gln Val Val Glu Ala Ile Thr His Tyr Arg Glu His
100 105 110 Ser Lys Leu Leu Phe Val Leu Ala Lys Val Asp Asn Leu Val
Lys Asn 115 120 125 Gly Arg Leu Ser Lys Leu Val Gly Thr Val Val Gly
Leu Leu Asn Ile 130 135 140 Arg Met Val Gly Glu Ala Ser Ala Glu Gly
Lys Leu Glu Leu Leu Gln 145 150 155 160 Lys Ala Arg Gly His Lys Lys
Ser Val Thr Ala Ala Phe Glu Glu Met 165 170 175 Lys Lys Ala Gly Tyr
Asp Gly Gly Arg Ile Val Met Ala His Arg Asn 180 185 190 Asn Ala Lys
Phe Phe Gln Gln Phe Ser Glu Leu Val Lys Ala Ser Phe 195 200 205 Pro
Thr Ala Val Ile Asp Glu Val Ala Thr Ser Gly Leu Cys Ser Phe 210 215
220 Tyr Ala Glu Glu Gly Gly Leu Leu Met Gly Tyr Glu Val Lys Ala 225
230 235 11244PRTStreptococcus suismisc_featureORF2X 11Met Lys Ile
Ile Ile Pro Asn Ala Lys Glu Val Asn Thr Asn Leu Glu 1 5 10 15 Asn
Ala Ser Phe Tyr Leu Leu Ser Asp Arg Ser Lys Pro Val Leu Asp 20 25
30 Ala Ile Ser Gln Phe Asp Val Lys Lys Met Ala Ala Phe Tyr Lys Leu
35 40 45 Asn Glu Ala Lys Ala Glu Leu Glu Ala Asp Arg Trp Tyr Arg
Ile Arg 50 55 60 Thr Gly Gln Ala Lys Thr Tyr Pro Ala Trp Gln Leu
Tyr Asp Gly Leu 65 70 75 80 Met Tyr Arg Tyr Met Asp Arg Arg Gly Ile
Asp Ser Lys Glu Glu Asn 85 90 95 Tyr Leu Arg Asp His Val Arg Val
Ala Thr Ala Leu Tyr Gly Leu Ile 100 105 110 His Pro Phe Glu Phe Ile
Ser Pro His Arg Leu Asp Phe Gln Gly Ser 115 120 125 Leu Lys Ile Gly
Asn Gln Ser Leu Lys Gln Tyr Trp Arg Pro Tyr Tyr 130 135 140 Asp Gln
Glu Val Gly Asp Asp Glu Leu Ile Leu Ser Leu Ala Ser Ser 145 150 155
160 Glu Phe Glu Gln Val Phe Ser Pro Gln Ile Gln Lys Arg Leu Val Lys
165 170 175 Ile Leu Phe Met Glu Glu Lys Ala Gly Gln Leu Lys Val His
Ser Thr 180 185 190 Ile Ser Lys Lys Gly Arg Gly Arg Leu Leu Ser Trp
Leu Ala Lys Asn 195 200 205 Asn Ile Gln Glu Leu Ser Asp Ile Gln Asp
Phe Lys Val Asp Gly Phe 210 215 220 Glu Tyr Cys Thr Ser Glu
Ser Thr Ala Asn Gln Leu Thr Phe Ile Arg 225 230 235 240 Ser Ile Lys
Met 12481PRTStreptococcus suismisc_featureCPS2A 12Met Lys Lys Arg
Ser Gly Arg Ser Lys Ser Ser Lys Phe Lys Leu Val 1 5 10 15 Asn Phe
Ala Leu Leu Gly Leu Tyr Ser Ile Thr Leu Cys Leu Phe Leu 20 25 30
Val Thr Met Tyr Arg Tyr Asn Ile Leu Asp Phe Arg Tyr Leu Asn Tyr 35
40 45 Ile Val Thr Leu Leu Leu Val Gly Val Ala Val Leu Ala Gly Leu
Leu 50 55 60 Met Trp Arg Lys Lys Ala Arg Ile Phe Thr Ala Leu Leu
Leu Val Phe 65 70 75 80 Ser Leu Val Ile Thr Ser Val Gly Ile Tyr Gly
Met Gln Glu Val Val 85 90 95 Lys Phe Ser Thr Arg Leu Asn Ser Asn
Ser Thr Phe Ser Glu Tyr Glu 100 105 110 Met Ser Ile Leu Val Pro Ala
Asn Ser Asp Ile Thr Asp Val Arg Gln 115 120 125 Leu Thr Ser Ile Leu
Ala Pro Ala Glu Tyr Asp Gln Asp Asn Ile Thr 130 135 140 Ala Leu Leu
Asp Asp Ile Ser Lys Met Glu Ser Thr Gln Leu Ala Thr 145 150 155 160
Ser Pro Gly Thr Ser Tyr Leu Thr Ala Tyr Gln Ser Met Leu Asn Gly 165
170 175 Glu Ser Gln Ala Met Val Phe Asn Gly Val Phe Thr Asn Ile Leu
Glu 180 185 190 Asn Glu Asp Pro Gly Phe Ser Ser Lys Val Lys Lys Ile
Tyr Ser Phe 195 200 205 Lys Val Thr Gln Thr Val Glu Thr Ala Thr Lys
Gln Val Ser Gly Asp 210 215 220 Ser Phe Asn Ile Tyr Ile Ser Gly Ile
Asp Ala Tyr Gly Pro Ile Ser 225 230 235 240 Thr Val Ser Arg Ser Asp
Val Asn Ile Ile Met Thr Val Asn Arg Ala 245 250 255 Thr His Lys Ile
Leu Leu Thr Thr Thr Pro Arg Asp Ser Tyr Val Ala 260 265 270 Phe Ala
Asp Gly Gly Gln Asn Gln Tyr Asp Lys Leu Thr His Ala Gly 275 280 285
Ile Tyr Gly Val Asn Ala Ser Val His Thr Leu Glu Asn Phe Tyr Gly 290
295 300 Ile Asp Ile Ser Asn Tyr Val Arg Leu Asn Phe Ile Ser Phe Leu
Gln 305 310 315 320 Leu Ile Asp Leu Val Gly Gly Ile Asp Val Tyr Asn
Asp Gln Glu Phe 325 330 335 Thr Ser Leu His Gly Asn Tyr His Phe Pro
Val Gly Gln Val His Leu 340 345 350 Asn Ser Asp Gln Ala Leu Gly Phe
Val Arg Glu Arg Tyr Ser Leu Thr 355 360 365 Gly Gly Asp Asn Asp Arg
Gly Lys Asn Gln Glu Lys Val Ile Ala Ala 370 375 380 Leu Ile Lys Lys
Met Ser Thr Pro Glu Asn Leu Lys Asn Tyr Gln Ala 385 390 395 400 Ile
Leu Ser Gly Leu Glu Gly Ser Ile Gln Thr Asp Leu Ser Leu Glu 405 410
415 Thr Ile Met Ser Leu Val Asn Thr Gln Leu Glu Ser Gly Thr Gln Phe
420 425 430 Thr Val Glu Ser Gln Ala Leu Thr Gly Thr Gly Arg Ser Asp
Leu Ser 435 440 445 Ser Tyr Ala Met Pro Gly Ser Gln Leu Tyr Met Met
Glu Ile Asn Gln 450 455 460 Asp Ser Leu Glu Gln Ser Lys Ala Ala Ile
Gln Ser Val Leu Val Glu 465 470 475 480 Lys 13229PRTStreptococcus
suismisc_featureCPS2B 13Met Asn Asn Gln Glu Val Asn Ala Ile Glu Ile
Asp Val Leu Phe Leu 1 5 10 15 Leu Lys Thr Ile Trp Arg Lys Lys Phe
Leu Ile Leu Leu Thr Ala Val 20 25 30 Leu Thr Ala Gly Leu Ala Phe
Val Tyr Ser Ser Phe Leu Val Thr Pro 35 40 45 Gln Tyr Asp Ser Thr
Thr Arg Ile Tyr Val Val Ser Gln Asn Val Glu 50 55 60 Ala Gly Ala
Gly Leu Thr Asn Gln Glu Leu Gln Ala Gly Thr Tyr Leu 65 70 75 80 Ala
Lys Asp Tyr Arg Glu Ile Ile Leu Ser Gln Asp Val Leu Thr Gln 85 90
95 Val Ala Thr Glu Leu Asn Leu Lys Glu Ser Leu Lys Glu Lys Ile Ser
100 105 110 Val Ser Ile Pro Val Asp Thr Arg Ile Val Ser Ile Ser Val
Arg Asp 115 120 125 Ala Asp Pro Asn Glu Ala Ala Arg Ile Ala Asn Ser
Leu Arg Thr Phe 130 135 140 Ala Val Gln Lys Val Val Glu Val Thr Lys
Val Ser Asp Val Thr Thr 145 150 155 160 Leu Glu Glu Ala Val Pro Ala
Glu Glu Pro Thr Thr Pro Asn Thr Lys 165 170 175 Arg Asn Ile Leu Leu
Gly Leu Leu Ala Gly Gly Ile Leu Ala Thr Gly 180 185 190 Leu Val Leu
Val Met Glu Val Leu Asp Asp Arg Val Lys Arg Pro Gln 195 200 205 Asp
Ile Glu Glu Val Met Gly Leu Thr Leu Leu Gly Ile Val Pro Asp 210 215
220 Ser Lys Lys Leu Lys 225 14225PRTStreptococcus
suismisc_featureCPS2C 14Met Ala Met Leu Glu Ile Ala Arg Thr Lys Arg
Glu Gly Val Asn Lys 1 5 10 15 Thr Glu Glu Tyr Phe Asn Ala Ile Arg
Thr Asn Ile Gln Leu Ser Gly 20 25 30 Ala Asp Ile Lys Val Val Gly
Ile Thr Ser Val Lys Ser Asn Glu Gly 35 40 45 Lys Ser Thr Thr Ala
Ala Ser Leu Ala Ile Ala Tyr Ala Arg Ser Gly 50 55 60 Tyr Lys Thr
Val Leu Val Asp Ala Asp Ile Arg Asn Ser Val Met Pro 65 70 75 80 Gly
Phe Phe Lys Pro Ile Thr Lys Ile Thr Gly Leu Thr Asp Tyr Leu 85 90
95 Ala Gly Thr Thr Asp Leu Ser Gln Gly Leu Cys Asp Thr Asp Ile Pro
100 105 110 Asn Leu Thr Val Ile Glu Ser Gly Lys Val Ser Pro Asn Pro
Thr Ala 115 120 125 Leu Leu Gln Ser Lys Asn Phe Glu Asn Leu Leu Ala
Thr Leu Arg Arg 130 135 140 Tyr Tyr Asp Tyr Val Ile Val Asp Cys Pro
Pro Leu Gly Leu Val Ile 145 150 155 160 Asp Ala Ala Ile Ile Ala Gln
Lys Cys Asp Ala Met Val Ala Val Val 165 170 175 Glu Ala Gly Asn Val
Lys Cys Ser Ser Leu Lys Lys Val Lys Glu Gln 180 185 190 Leu Glu Gln
Thr Gly Thr Pro Phe Leu Gly Val Ile Leu Asn Lys Tyr 195 200 205 Asp
Ile Ala Thr Glu Lys Tyr Ser Glu Tyr Gly Asn Tyr Gly Lys Lys 210 215
220 Ala 225 15243PRTStreptococcus suismisc_featureCPS2D 15Met Ile
Asp Ile His Ser His Ile Ile Phe Gly Val Asp Asp Gly Pro 1 5 10 15
Lys Thr Ile Glu Glu Ser Leu Ser Leu Ile Ser Glu Ala Tyr Arg Gln 20
25 30 Gly Val Arg Tyr Ile Val Ala Thr Ser His Arg Arg Lys Gly Met
Phe 35 40 45 Glu Thr Pro Glu Lys Ile Ile Met Ile Asn Phe Leu Gln
Leu Lys Glu 50 55 60 Ala Val Ala Glu Val Tyr Pro Glu Ile Arg Leu
Cys Tyr Gly Ala Glu 65 70 75 80 Leu Tyr Tyr Ser Lys Asp Ile Leu Ser
Lys Leu Glu Lys Lys Lys Val 85 90 95 Pro Thr Leu Asn Gly Ser Cys
Tyr Ile Leu Leu Glu Phe Ser Thr Asp 100 105 110 Thr Pro Trp Lys Glu
Ile Gln Glu Ala Val Asn Glu Met Thr Leu Leu 115 120 125 Gly Leu Thr
Pro Val Leu Ala His Ile Glu Arg Tyr Asp Ala Leu Ala 130 135 140 Phe
Gln Ser Glu Arg Val Glu Lys Leu Ile Asp Lys Gly Cys Tyr Thr 145 150
155 160 Gln Val Asn Ser Asn His Val Leu Lys Pro Ala Leu Ile Gly Glu
Arg 165 170 175 Ala Lys Glu Phe Lys Lys Arg Thr Arg Tyr Phe Leu Glu
Gln Asp Leu 180 185 190 Val His Cys Val Ala Ser Asp Met His Asn Leu
Tyr Ser Arg Pro Pro 195 200 205 Phe Met Arg Glu Ala Tyr Gln Leu Val
Lys Lys Glu Tyr Gly Glu Asp 210 215 220 Arg Ala Lys Ala Leu Phe Lys
Lys Asn Pro Leu Leu Ile Leu Lys Asn 225 230 235 240 Gln Val Gln
16459PRTStreptococcus suismisc_featureCPS2E 16Met Asn Ile Glu Ile
Gly Tyr Arg Gln Thr Lys Leu Ala Leu Phe Asp 1 5 10 15 Met Ile Ala
Val Thr Ile Ser Ala Ile Leu Thr Ser His Ile Pro Asn 20 25 30 Ala
Asp Leu Asn Arg Ser Gly Ile Phe Ile Ile Met Met Val His Tyr 35 40
45 Phe Ala Phe Phe Ile Ser Arg Met Pro Val Glu Phe Glu Tyr Arg Gly
50 55 60 Asn Leu Ile Glu Phe Glu Lys Thr Phe Asn Tyr Ser Ile Ile
Phe Val 65 70 75 80 Ile Phe Leu Met Ala Val Ser Phe Met Leu Glu Asn
Asn Phe Ala Leu 85 90 95 Ser Arg Arg Gly Ala Val Tyr Phe Thr Leu
Ile Asn Phe Val Leu Val 100 105 110 Tyr Leu Phe Asn Val Ile Ile Lys
Gln Phe Lys Asp Ser Phe Leu Phe 115 120 125 Ser Thr Thr Tyr Gln Lys
Lys Thr Ile Leu Ile Thr Thr Ala Glu Leu 130 135 140 Trp Glu Asn Met
Gln Val Leu Phe Glu Ser Asp Ile Leu Phe Gln Lys 145 150 155 160 Asn
Leu Val Ala Leu Val Ile Leu Gly Thr Glu Ile Asp Lys Ile Asn 165 170
175 Leu Pro Leu Pro Leu Tyr Tyr Ser Val Glu Glu Ala Ile Gly Phe Ser
180 185 190 Thr Arg Glu Val Val Asp Tyr Val Phe Ile Asn Leu Pro Ser
Glu Tyr 195 200 205 Phe Asp Leu Lys Gln Leu Val Ser Asp Phe Glu Leu
Leu Gly Ile Asp 210 215 220 Val Gly Val Asp Ile Asn Ser Phe Gly Phe
Thr Val Leu Lys Asn Lys 225 230 235 240 Lys Ile Gln Met Leu Gly Asp
His Ser Ile Val Thr Phe Ser Thr Asn 245 250 255 Phe Tyr Lys Pro Ser
His Ile Trp Met Lys Arg Leu Leu Asp Ile Leu 260 265 270 Gly Ala Val
Val Gly Leu Ile Ile Ser Gly Ile Val Ser Ile Leu Leu 275 280 285 Ile
Pro Ile Ile Arg Arg Asp Gly Gly Pro Ala Ile Phe Ala Gln Lys 290 295
300 Arg Val Gly Gln Asn Gly Arg Ile Phe Thr Phe Tyr Lys Phe Arg Ser
305 310 315 320 Met Phe Val Asp Ala Glu Val Arg Lys Lys Glu Leu Met
Ala Gln Asn 325 330 335 Gln Met Gln Gly Gly Met Phe Lys Met Asp Asn
Asp Pro Arg Ile Thr 340 345 350 Pro Ile Gly His Phe Ile Arg Lys Thr
Ser Leu Asp Glu Leu Pro Gln 355 360 365 Phe Tyr Asn Val Leu Ile Gly
Asp Met Ser Leu Val Gly Thr Arg Pro 370 375 380 Pro Thr Val Asp Glu
Phe Glu Lys Tyr Thr Pro Ser Gln Lys Arg Arg 385 390 395 400 Leu Ser
Phe Lys Pro Gly Ile Thr Gly Leu Trp Gln Val Ser Gly Arg 405 410 415
Ser Asp Ile Thr Asp Phe Asn Glu Val Val Arg Leu Asp Leu Thr Tyr 420
425 430 Ile Asp Asn Trp Thr Ile Trp Ser Asp Ile Lys Ile Leu Leu Lys
Thr 435 440 445 Val Lys Val Val Leu Leu Arg Glu Gly Gly Gln 450 455
17389PRTStreptococcus suismisc_featureCPS2F 17Met Arg Thr Val Tyr
Ile Ile Gly Ser Lys Gly Ile Pro Ala Lys Tyr 1 5 10 15 Gly Gly Phe
Glu Thr Phe Val Glu Lys Leu Thr Glu Tyr Gln Lys Asp 20 25 30 Lys
Ser Ile Asn Tyr Phe Val Ala Cys Thr Arg Glu Asn Ser Ala Lys 35 40
45 Ser Asp Ile Thr Gly Glu Val Phe Glu His Asn Gly Ala Thr Cys Phe
50 55 60 Asn Ile Asp Val Pro Asn Ile Gly Ser Ala Lys Ala Ile Leu
Tyr Asp 65 70 75 80 Ile Met Ala Leu Lys Lys Ser Ile Glu Ile Ala Lys
Asp Arg Asn Asp 85 90 95 Thr Ser Pro Ile Phe Tyr Ile Leu Ala Cys
Arg Ile Gly Pro Phe Ile 100 105 110 Tyr Leu Phe Lys Lys Gln Ile Glu
Ser Ile Gly Gly Gln Leu Phe Val 115 120 125 Asn Pro Asp Gly His Glu
Trp Leu Arg Glu Lys Trp Ser Tyr Pro Val 130 135 140 Arg Gln Tyr Trp
Lys Phe Ser Glu Ser Leu Met Leu Lys Tyr Ala Asp 145 150 155 160 Leu
Leu Ile Cys Asp Ser Lys Asn Ile Glu Lys Tyr Ile His Glu Asp 165 170
175 Tyr Arg Lys Tyr Ala Pro Glu Thr Ser Tyr Ile Ala Tyr Gly Thr Asp
180 185 190 Leu Asp Lys Ser Arg Leu Ser Pro Thr Asp Ser Val Val Arg
Glu Trp 195 200 205 Tyr Lys Glu Lys Glu Ile Ser Glu Asn Asp Tyr Tyr
Leu Val Val Gly 210 215 220 Arg Phe Val Pro Glu Asn Asn Tyr Glu Val
Met Ile Arg Glu Phe Met 225 230 235 240 Lys Ser Tyr Ser Arg Lys Asp
Phe Val Leu Ile Thr Asn Val Glu His 245 250 255 Asn Ser Phe Tyr Glu
Lys Leu Lys Lys Glu Thr Gly Phe Asp Lys Asp 260 265 270 Lys Arg Ile
Lys Phe Val Gly Thr Val Tyr Asn Gln Glu Leu Leu Lys 275 280 285 Tyr
Ile Arg Glu Asn Ala Phe Ala Tyr Phe His Gly His Glu Val Gly 290 295
300 Gly Thr Asn Pro Ser Leu Leu Glu Ala Leu Ser Ser Thr Lys Leu Asn
305 310 315 320 Leu Leu Leu Asp Val Gly Phe Asn Arg Glu Val Gly Glu
Glu Gly Ala 325 330 335 Lys Tyr Trp Asn Lys Asp Asn Leu His Arg Val
Ile Asp Ser Cys Glu 340 345 350 Gln Leu Ser Gln Glu Gln Ile Asn Asp
Met Asp Ser Leu Ser Thr Lys 355 360 365 Gln Val Lys Glu Arg Phe Ser
Trp Asp Phe Ile Val Asp Glu Tyr Glu 370 375 380 Lys Leu Phe Lys Gly
385 18385PRTStreptococcus suismisc_featureCPS2G 18Met Lys Lys Ile
Leu Tyr Leu His Ala Gly Ala Glu Leu Tyr Gly Ala 1 5 10 15 Asp Lys
Val Leu Leu Glu Leu Ile Lys Gly Leu Asp Lys Asn Glu Phe 20 25 30
Glu Ala His Val Ile Leu Pro Asn Asp Gly Val Leu Val Pro Ala Leu 35
40 45 Arg Glu Val Gly Ala Gln Val Glu Val Ile Asn Tyr Pro Ile Leu
Arg 50 55 60 Arg Lys Tyr Phe Asn Pro Lys Gly Ile Phe Asp Tyr Phe
Ile Ser Tyr 65 70 75 80 His His Tyr Ser Lys Gln Ile Ala Gln Tyr Ala
Ile Glu Asn Lys Val 85 90 95 Asp Ile Ile His Asn Asn Thr Thr Ala
Val Leu Glu Gly Ile Tyr Leu 100 105 110 Lys Arg Lys Leu Lys Leu Pro
Leu Leu Trp His Val His Glu Ile Ile 115 120 125 Val Lys Pro Lys Phe
Ile Ser Asp Ser Ile Asn Phe Leu Met Gly Arg 130 135 140 Phe Ala Asp
Lys Ile Val Thr Val Ser Gln Ala Val Ala Asn His Ile 145 150 155 160
Lys Gln Ser Pro His Ile Lys Asp Asp Gln Ile Ser Val Ile Tyr Asn 165
170 175 Gly Val Asp Asn Lys Val Phe Tyr Gln Ser Asp Ala Arg Ser Val
Arg 180 185 190 Glu Arg Phe Asp Ile Asp Glu Glu Ala Leu Val Ile Gly
Met Val Gly 195 200 205 Arg Val Asn Ala Trp Lys Gly Gln Gly Asp Phe
Leu Glu Ala Val Ala 210 215 220
Pro Ile Leu Glu Gln Asn Pro Lys Ala Ile Ala Phe Ile Ala Gly Ser 225
230 235 240 Ala Phe Glu Gly Glu Glu Trp Arg Val Val Glu Leu Glu Lys
Lys Ile 245 250 255 Ser Gln Leu Lys Val Ser Ser Gln Val Arg Arg Met
Asp Tyr Tyr Ala 260 265 270 Asn Thr Thr Glu Leu Tyr Asn Met Phe Asp
Ile Phe Val Leu Pro Ser 275 280 285 Thr Asn Pro Asp Pro Leu Pro Thr
Val Val Leu Lys Ala Met Ala Cys 290 295 300 Gly Lys Pro Val Val Gly
Tyr Arg His Gly Gly Val Cys Glu Met Val 305 310 315 320 Lys Glu Gly
Val Asn Gly Phe Leu Val Thr Pro Asn Ser Pro Leu Asn 325 330 335 Leu
Ser Lys Val Ile Leu Gln Leu Ser Glu Asn Ile Asn Leu Arg Lys 340 345
350 Lys Ile Gly Asn Asn Ser Ile Glu Arg Gln Lys Glu His Phe Ser Leu
355 360 365 Lys Ser Tyr Val Lys Asn Phe Ser Lys Val Tyr Thr Ser Leu
Lys Val 370 375 380 Tyr 385 19456PRTStreptococcus
suismisc_featurecps2h 19Met Lys Ile Ile Ser Phe Thr Met Val Asn Asn
Glu Ser Glu Ile Ile 1 5 10 15 Glu Ser Phe Ile Arg Tyr Asn Tyr Asn
Phe Ile Asp Glu Met Val Ile 20 25 30 Ile Asp Asn Gly Cys Thr Asp
Asn Thr Met Gln Ile Ile Phe Asn Leu 35 40 45 Ile Lys Glu Gly Tyr
Lys Ile Ser Val Tyr Asp Glu Ser Leu Glu Ala 50 55 60 Tyr Asn Gln
Tyr Arg Leu Asp Asn Lys Tyr Leu Thr Lys Ile Ile Ala 65 70 75 80 Glu
Lys Asn Pro Asp Leu Ile Ile Pro Leu Asp Ala Asp Glu Phe Leu 85 90
95 Thr Ala Asp Ser Asn Pro Arg Lys Leu Leu Glu Gln Leu Asp Leu Glu
100 105 110 Lys Ile His Tyr Val Asn Trp Gln Trp Phe Val Met Thr Lys
Lys Asp 115 120 125 Asp Ile Asn Asp Ser Phe Ile Pro Arg Arg Met Gln
Tyr Cys Phe Glu 130 135 140 Lys Pro Val Trp His His Ser Asp Gly Lys
Pro Val Thr Lys Cys Ile 145 150 155 160 Ile Ser Ala Lys Tyr Tyr Lys
Lys Met Asn Leu Lys Leu Ser Met Gly 165 170 175 His His Thr Val Phe
Gly Asn Pro Asn Val Arg Ile Glu His His Asn 180 185 190 Asp Leu Lys
Phe Ala His Tyr Arg Ala Ile Ser Gln Glu Gln Leu Ile 195 200 205 Tyr
Lys Thr Ile Cys Tyr Thr Ile Arg Asp Ile Ala Thr Met Glu Asn 210 215
220 Asn Ile Glu Thr Ala Gln Arg Thr Asn Gln Met Ala Leu Ile Glu Ser
225 230 235 240 Gly Val Asp Met Trp Glu Thr Ala Arg Glu Ala Ser Tyr
Ser Gly Tyr 245 250 255 Asp Cys Asn Val Ile His Ala Pro Ile Asp Leu
Ser Phe Cys Lys Glu 260 265 270 Asn Ile Val Ile Lys Tyr Asn Glu Leu
Ser Arg Glu Thr Val Ala Glu 275 280 285 Arg Val Met Lys Thr Gly Arg
Glu Met Ala Val Arg Ala Tyr Asn Val 290 295 300 Glu Arg Lys Gln Lys
Glu Lys Lys Phe Leu Lys Pro Ile Ile Phe Val 305 310 315 320 Leu Asp
Gly Leu Lys Gly Asp Glu Tyr Ile His Pro Asn Pro Ser Asn 325 330 335
His Leu Thr Ile Leu Thr Glu Met Tyr Asn Val Arg Gly Leu Leu Thr 340
345 350 Asp Asn His Gln Ile Lys Phe Leu Lys Val Asn Tyr Arg Leu Ile
Ile 355 360 365 Thr Pro Asp Phe Ala Lys Phe Leu Pro His Glu Phe Ile
Val Val Pro 370 375 380 Asp Thr Leu Asp Ile Glu Gln Val Lys Ser Gln
Tyr Val Gly Thr Gly 385 390 395 400 Val Asp Leu Ser Lys Ile Ile Ser
Leu Lys Glu Tyr Arg Lys Glu Ile 405 410 415 Gly Phe Ile Gly Asn Leu
Tyr Ala Leu Leu Gly Phe Val Pro Asn Met 420 425 430 Leu Asn Arg Ile
Tyr Leu Tyr Ile Gln Arg Asn Gly Ile Ala Asn Thr 435 440 445 Ile Ile
Lys Ile Lys Ser Arg Leu 450 455 20410PRTStreptococcus
suismisc_featureCPS2I 20Met Gln Ala Asp Arg Arg Lys Thr Phe Gly Lys
Met Arg Ile Arg Ile 1 5 10 15 Asn Asn Leu Phe Phe Val Ala Ile Ala
Phe Met Gly Ile Ile Ile Ser 20 25 30 Asn Ser Gln Val Val Leu Ala
Ile Gly Lys Ala Ser Val Ile Gln Tyr 35 40 45 Leu Ser Tyr Leu Val
Leu Ile Leu Cys Ile Val Asn Asp Leu Leu Lys 50 55 60 Asn Asn Lys
His Ile Val Val Tyr Lys Leu Gly Tyr Leu Phe Leu Ile 65 70 75 80 Ile
Phe Leu Phe Thr Ile Gly Ile Cys Gln Gln Ile Leu Pro Ile Thr 85 90
95 Thr Lys Ile Tyr Leu Ser Ile Ser Met Met Ile Ile Ser Val Leu Ala
100 105 110 Thr Leu Pro Ile Ser Leu Ile Lys Asp Ile Asp Asp Phe Arg
Arg Ile 115 120 125 Ser Asn His Leu Leu Phe Ala Leu Phe Ile Thr Ser
Ile Leu Gly Ile 130 135 140 Lys Met Gly Ala Thr Met Phe Thr Gly Ala
Val Glu Gly Ile Gly Phe 145 150 155 160 Ser Gln Gly Phe Asn Gly Gly
Leu Thr His Lys Asn Phe Phe Gly Ile 165 170 175 Thr Ile Leu Met Gly
Phe Val Leu Thr Tyr Leu Ala Tyr Lys Tyr Gly 180 185 190 Ser Tyr Lys
Arg Thr Asp Arg Phe Ile Leu Gly Leu Glu Leu Phe Leu 195 200 205 Ile
Leu Ile Ser Asn Thr Arg Ser Val Tyr Leu Ile Leu Leu Leu Phe 210 215
220 Leu Phe Leu Val Asn Leu Asp Lys Ile Lys Ile Glu Gln Arg Gln Trp
225 230 235 240 Ser Thr Leu Lys Tyr Ile Ser Met Leu Phe Cys Ala Ile
Phe Leu Tyr 245 250 255 Tyr Phe Phe Gly Phe Leu Ile Thr His Ser Asp
Ser Tyr Ala His Arg 260 265 270 Val Asn Gly Leu Ile Asn Phe Phe Glu
Tyr Tyr Arg Asn Asp Trp Phe 275 280 285 His Leu Met Phe Gly Ala Ala
Asp Leu Ala Tyr Gly Asp Leu Thr Leu 290 295 300 Asp Tyr Ala Ile Arg
Val Arg Arg Val Leu Gly Trp Asn Gly Thr Leu 305 310 315 320 Glu Met
Pro Leu Leu Ser Ile Met Leu Lys Asn Gly Phe Ile Gly Leu 325 330 335
Val Gly Tyr Gly Ile Val Leu Tyr Lys Leu Tyr Arg Asn Val Arg Ile 340
345 350 Leu Lys Thr Asp Asn Ile Lys Thr Ile Gly Lys Ser Val Phe Ile
Ile 355 360 365 Val Val Leu Ser Ala Thr Val Glu Asn Tyr Ile Val Asn
Leu Ser Phe 370 375 380 Val Phe Met Pro Ile Cys Phe Cys Leu Leu Asn
Ser Ile Ser Thr Met 385 390 395 400 Glu Ser Thr Ile Asn Lys Gln Leu
Gln Thr 405 410 21332PRTStreptococcus suismisc_featureCPS2J 21Met
Glu Lys Val Ser Ile Ile Val Pro Ile Phe Asn Thr Glu Lys Tyr 1 5 10
15 Leu Arg Glu Cys Leu Asp Ser Ile Ile Ser Gln Ser Tyr Thr Asn Leu
20 25 30 Glu Ile Leu Leu Ile Asp Asp Gly Ser Ser Asp Ser Ser Thr
Asp Ile 35 40 45 Cys Leu Glu Tyr Ala Glu Gln Asp Gly Arg Ile Lys
Leu Phe Arg Leu 50 55 60 Pro Asn Gly Gly Val Ser Asn Ala Arg Asn
Tyr Gly Ile Lys Asn Ser 65 70 75 80 Thr Ala Asn Tyr Ile Met Phe Val
Asp Ser Asp Asp Ile Val Asp Gly 85 90 95 Asn Ile Val Glu Ser Leu
Tyr Thr Cys Leu Lys Glu Asn Asp Ser Asp 100 105 110 Leu Ser Gly Gly
Leu Leu Ala Thr Phe Asp Gly Asn Tyr Gln Glu Ser 115 120 125 Glu Leu
Gln Lys Cys Gln Ile Asp Leu Glu Glu Ile Lys Glu Val Arg 130 135 140
Asp Leu Gly Asn Glu Asn Phe Pro Asn His Tyr Met Ser Gly Ile Phe 145
150 155 160 Asn Ser Pro Cys Cys Lys Leu Tyr Lys Asn Ile Tyr Ile Asn
Gln Gly 165 170 175 Phe Asp Thr Glu Gln Trp Leu Gly Glu Asp Leu Leu
Phe Asn Leu Asn 180 185 190 Tyr Leu Lys Asn Ile Lys Lys Val Arg Tyr
Val Asn Arg Asn Leu Tyr 195 200 205 Phe Ala Arg Arg Ser Leu Gln Ser
Thr Thr Asn Thr Phe Lys Tyr Asp 210 215 220 Val Phe Ile Gln Leu Glu
Asn Leu Glu Glu Lys Thr Phe Asp Leu Phe 225 230 235 240 Val Lys Ile
Phe Gly Gly Gln Tyr Glu Phe Ser Val Phe Lys Glu Thr 245 250 255 Leu
Gln Trp His Ile Ile Tyr Tyr Ser Leu Leu Met Phe Lys Asn Gly 260 265
270 Asp Glu Ser Leu Pro Lys Lys Leu His Ile Phe Lys Tyr Leu Tyr Asn
275 280 285 Arg His Ser Leu Asp Thr Leu Ser Ile Lys Arg Thr Ser Ser
Val Phe 290 295 300 Lys Arg Ile Cys Lys Leu Ile Val Ala Asn Asn Leu
Phe Lys Ile Phe 305 310 315 320 Leu Asn Thr Leu Ile Arg Glu Glu Lys
Asn Asn Asp 325 330 22332PRTStreptococcus suismisc_featureCPS2K
22Met Ile Asn Ile Ser Ile Ile Val Pro Ile Tyr Asn Val Glu Gln Tyr 1
5 10 15 Leu Ser Lys Cys Ile Asn Ser Ile Val Asn Gln Thr Tyr Lys His
Ile 20 25 30 Glu Ile Leu Leu Val Asn Asp Gly Ser Thr Asp Asn Ser
Glu Glu Ile 35 40 45 Cys Leu Ala Tyr Ala Lys Lys Asp Ser Arg Ile
Arg Tyr Phe Lys Lys 50 55 60 Glu Asn Gly Gly Leu Ser Asp Ala Arg
Asn Tyr Gly Ile Ser Arg Ala 65 70 75 80 Lys Gly Asp Tyr Leu Ala Phe
Ile Asp Ser Asp Asp Phe Ile His Ser 85 90 95 Glu Phe Ile Gln Arg
Leu His Glu Ala Ile Glu Arg Glu Asn Ala Leu 100 105 110 Val Ala Val
Ala Gly Tyr Asp Arg Val Asp Ala Ser Gly His Phe Leu 115 120 125 Thr
Ala Glu Pro Leu Pro Thr Asn Gln Ala Val Leu Ser Gly Arg Asn 130 135
140 Val Cys Lys Lys Leu Leu Glu Ala Asp Gly His Arg Phe Val Val Ala
145 150 155 160 Trp Asn Lys Leu Tyr Lys Lys Glu Leu Phe Asp Phe Arg
Phe Glu Lys 165 170 175 Gly Lys Ile His Glu Asp Glu Tyr Phe Thr Tyr
Arg Leu Leu Tyr Glu 180 185 190 Leu Glu Lys Val Ala Ile Val Lys Glu
Cys Leu Tyr Tyr Tyr Val Asp 195 200 205 Arg Glu Asn Ser Ile Ile Thr
Ser Ser Met Thr Asp His Arg Phe His 210 215 220 Cys Leu Leu Glu Phe
Gln Asn Glu Arg Met Asp Phe Tyr Glu Ser Arg 225 230 235 240 Gly Asp
Lys Glu Leu Leu Leu Glu Cys Tyr Arg Ser Phe Leu Ala Phe 245 250 255
Ala Val Leu Phe Leu Gly Lys Tyr Asn His Trp Leu Ser Lys Gln Gln 260
265 270 Lys Lys Leu Gln Thr Leu Phe Arg Ile Val Tyr Lys Gln Leu Lys
Gln 275 280 285 Asn Lys Arg Leu Ala Leu Leu Met Asn Ala Tyr Tyr Leu
Val Gly Cys 290 295 300 Leu His Leu Asn Phe Ser Val Phe Leu Lys Thr
Gly Lys Asp Lys Ile 305 310 315 320 Gln Glu Arg Leu Arg Arg Ser Glu
Ser Ser Thr Arg 325 330 23467PRTStreptococcus suismisc_featureCPS2O
23Met Ser Lys Lys Ser Ile Val Val Ser Gly Leu Val Tyr Thr Ile Gly 1
5 10 15 Thr Ile Leu Val Gln Gly Leu Ala Phe Ile Thr Leu Pro Ile Tyr
Thr 20 25 30 Arg Val Ile Ser Gln Glu Val Tyr Gly Gln Phe Ser Leu
Tyr Asn Ser 35 40 45 Trp Val Gly Leu Val Gly Leu Phe Ile Gly Leu
Gln Leu Gly Gly Ala 50 55 60 Phe Gly Pro Gly Trp Val His Phe Arg
Glu Lys Phe Asp Asp Phe Val 65 70 75 80 Ser Thr Leu Met Val Ser Ser
Ile Ala Phe Phe Leu Pro Ile Phe Gly 85 90 95 Leu Ser Phe Leu Leu
Ser Gln Pro Leu Ser Leu Leu Phe Gly Leu Pro 100 105 110 Asp Trp Val
Val Pro Leu Ile Phe Leu Gln Ser Leu Met Ile Val Val 115 120 125 Gln
Gly Phe Phe Thr Thr Tyr Leu Val Gln Arg Gln Gln Ser Met Trp 130 135
140 Thr Leu Pro Leu Ser Val Leu Ser Ala Val Ile Asn Thr Ala Leu Ser
145 150 155 160 Leu Phe Leu Thr Phe Pro Met Glu Asn Asp Phe Ile Ala
Arg Val Met 165 170 175 Ala Asn Pro Ala Thr Thr Gly Val Leu Ala Cys
Val Ser Xaa Trp Phe 180 185 190 Ser Gln Lys Lys Asn Gly Leu His Phe
Arg Lys Asp Tyr Leu Arg Tyr 195 200 205 Gly Leu Ser Ile Ser Ile Pro
Leu Ile Phe His Gly Leu Gly His Asn 210 215 220 Val Leu Asn Gln Phe
Asp Arg Ile Met Leu Gly Lys Met Leu Thr Leu 225 230 235 240 Ser Asp
Val Ala Leu Tyr Ser Phe Gly Tyr Thr Leu Ala Ser Ile Leu 245 250 255
Gln Ile Val Phe Ser Ser Leu Asn Thr Val Trp Cys Pro Trp Tyr Phe 260
265 270 Glu Lys Lys Arg Gly Ala Asp Lys Asp Leu Leu Ser Tyr Val Arg
Tyr 275 280 285 Tyr Leu Ala Ile Gly Leu Phe Val Thr Phe Gly Phe Leu
Thr Ile Tyr 290 295 300 Pro Arg Leu Ala Met Leu Leu Gly Gly Ser Glu
Tyr Arg Phe Ser Met 305 310 315 320 Gly Phe Ile Pro Met Ile Ile Val
Gly Val Phe Phe Val Phe Leu Tyr 325 330 335 Ser Phe Pro Ala Asn Ile
Gln Phe Tyr Ser Gly Asn Thr Lys Phe Leu 340 345 350 Pro Ile Gly Thr
Phe Ile Ala Gly Val Leu Asn Ile Ser Val His Phe 355 360 365 Val Leu
Ile Pro Thr Lys Asn Leu Trp Cys Cys Phe Ala Thr Thr Ala 370 375 380
Ser Tyr Leu Leu Leu Leu Val Leu His Tyr Phe Val Ala Lys Lys Lys 385
390 395 400 Tyr Ala Tyr Asp Glu Val Ala Ile Ser Thr Phe Val Lys Val
Ile Ala 405 410 415 Leu Val Val Val Tyr Thr Gly Leu Met Thr Val Phe
Val Gly Ser Ile 420 425 430 Trp Ile Arg Trp Ser Leu Gly Ile Ala Val
Leu Val Val Tyr Ala Ile 435 440 445 Tyr Phe Arg Lys Glu Leu Thr Val
Ala Leu Asn Thr Phe Arg Glu Lys 450 455 460 Arg Ser Lys 465
24338PRTStreptococcus suismisc_featureCPS2P 24Met Val Tyr Ile Ile
Ala Glu Ile Gly Cys Asn His Asn Gly Asp Val 1 5 10 15 His Leu Ala
Arg Lys Met Val Glu Val Ala Val Asp Cys Gly Val Asp 20 25 30 Ala
Val Lys Phe Gln Thr Glu Lys Ala Asp Leu Leu Ile Ser Lys Tyr 35 40
45 Ala Pro Lys Ala Glu Tyr Gln Lys Ile Thr Thr Gly Glu Ser Asp Ser
50 55 60 Gln Leu Glu Met Thr Arg Arg Leu Glu Leu Ser Phe Glu Glu
Tyr Leu 65 70 75 80 Asp Leu Arg Asp Tyr Cys Leu Glu Lys Gly Val Asp
Val Phe Ser Thr 85 90 95 Pro Glu Asp Glu Glu Ser Leu Asp Phe Leu
Ile Ser Thr Asp Met Pro 100 105
110 Val Tyr Lys Ile Pro Ser Gly Glu Ile Thr Asn Leu Pro Tyr Leu Glu
115 120 125 Lys Ile Gly Arg Gln Ala Lys Lys Val Ile Leu Ser Thr Gly
Met Ala 130 135 140 Val Met Asp Glu Ile His Gln Ala Val Lys Ile Leu
Gln Glu Asn Gly 145 150 155 160 Thr Thr Asp Ile Ser Ile Leu His Cys
Thr Thr Glu Tyr Pro Thr Pro 165 170 175 Tyr Pro Ala Leu Asn Leu Asn
Val Leu His Thr Leu Lys Lys Glu Phe 180 185 190 Pro Asn Leu Thr Ile
Gly Tyr Ser Asp His Ser Val Gly Ser Glu Val 195 200 205 Pro Ile Ala
Ala Ala Ala Met Gly Ala Glu Leu Ile Glu Lys His Phe 210 215 220 Thr
Leu Asp Asn Glu Met Glu Gly Pro Asp His Lys Ala Ser Ala Thr 225 230
235 240 Pro Asp Ile Leu Ala Ala Leu Val Lys Gly Val Arg Ile Val Glu
Gln 245 250 255 Ser Leu Gly Lys Phe Glu Lys Glu Pro Glu Glu Val Glu
Val Arg Asn 260 265 270 Lys Ile Val Ala Glu Lys Ser Ile Val Ala Lys
Lys Ala Ile Ala Lys 275 280 285 Gly Glu Val Phe Thr Glu Glu Asn Ile
Thr Val Lys Arg Pro Gly Asn 290 295 300 Gly Ile Ser Pro Met Glu Trp
Tyr Lys Val Leu Gly Gln Val Ser Glu 305 310 315 320 Gln Asp Phe Glu
Glu Asp Gln Asn Ile Cys His Ser Ala Phe Glu Asn 325 330 335 Gln Met
25170PRTStreptococcus suismisc_featureCPS2Q 25Met Lys Lys Ile Cys
Phe Val Thr Gly Ser Arg Ala Glu Tyr Gly Ile 1 5 10 15 Met Arg Arg
Leu Leu Ser Tyr Leu Gln Asp Asp Pro Glu Met Glu Leu 20 25 30 Asp
Leu Val Val Ala Thr Met His Leu Glu Glu Lys Tyr Gly Met Thr 35 40
45 Val Lys Asp Ile Glu Ala Asp Lys Arg Arg Ile Val Lys Arg Ile Pro
50 55 60 Leu His Leu Thr Asp Thr Ser Lys Gln Thr Ile Val Lys Ser
Leu Ala 65 70 75 80 Thr Leu Thr Glu Gln Leu Thr Val Leu Phe Glu Glu
Val Gln Tyr Asp 85 90 95 Leu Val Leu Ile Leu Gly Asp Arg Tyr Glu
Met Leu Pro Val Ala Asn 100 105 110 Ala Ala Leu Leu Tyr Asn Ile Pro
Ile Cys His Ile His Gly Gly Glu 115 120 125 Lys Thr Met Gly Asn Phe
Asp Glu Ser Ile Arg His Ala Ile Thr Lys 130 135 140 Met Ser His Leu
His Leu Thr Ser Thr Asp Glu Phe Arg Asn Arg Val 145 150 155 160 Ile
Gln Leu Gly Glu Asn Pro Thr Met Tyr 165 170 26184PRTStreptococcus
suismisc_featureCPS2R 26Met Glu Leu Gly Ile Asp Phe Ala Glu Asp Tyr
Tyr Val Val Leu Phe 1 5 10 15 His Pro Val Thr Leu Glu Asp Asn Thr
Ala Glu Glu Gln Thr Gln Ala 20 25 30 Leu Leu Asp Ala Leu Lys Glu
Asp Gly Ser Gln Cys Leu Ile Ile Gly 35 40 45 Ser Asn Ser Asp Thr
His Ala Asp Lys Ile Met Glu Leu Met His Glu 50 55 60 Phe Val Lys
Gln Asp Ser Asp Ser Tyr Ile Phe Thr Ser Leu Pro Thr 65 70 75 80 Arg
Tyr Tyr His Ser Leu Val Lys His Ser Gln Gly Leu Ile Gly Asn 85 90
95 Ser Ser Ser Gly Leu Ile Glu Val Pro Ser Leu Gln Val Pro Thr Leu
100 105 110 Asn Ile Gly Asn Arg Gln Phe Gly Arg Leu Ser Gly Pro Ser
Val Val 115 120 125 His Val Gly Thr Ser Lys Glu Ala Ile Val Gly Gly
Leu Gly Gln Leu 130 135 140 Arg Asp Val Ile Asp Phe Thr Asn Pro Phe
Glu Gln Pro Asp Ser Ala 145 150 155 160 Leu Gln Gly Tyr Arg Ala Ile
Lys Glu Phe Leu Ser Val Gln Ala Ser 165 170 175 Thr Met Lys Glu Phe
Tyr Asp Arg 180 27208PRTStreptococcus suismisc_featureCPS2S 27Met
Lys Lys Val Ala Phe Leu Gly Ala Gly Thr Phe Ser Asp Gly Val 1 5 10
15 Leu Pro Trp Leu Asp Arg Thr Arg Tyr Glu Leu Ile Gly Tyr Phe Glu
20 25 30 Asp Lys Pro Ile Ser Asp Tyr Arg Gly Tyr Pro Val Phe Gly
Pro Leu 35 40 45 Gln Asp Val Leu Thr Tyr Leu Asp Asp Gly Lys Val
Asp Ala Val Phe 50 55 60 Val Thr Ile Gly Asp Asn Val Lys Arg Lys
Glu Ile Phe Asp Leu Leu 65 70 75 80 Ala Lys Asp His Tyr Asp Ala Leu
Phe Asn Ile Ile Ser Glu Gln Ala 85 90 95 Asn Ile Phe Ser Pro Asp
Ser Ile Lys Gly Arg Gly Val Phe Ile Gly 100 105 110 Phe Ser Ser Phe
Val Gly Ala Asp Ser Tyr Val Tyr Asp Asn Cys Ile 115 120 125 Ile Asn
Thr Gly Ala Ile Val Glu His His Thr Thr Val Glu Ala His 130 135 140
Cys Asn Ile Thr Pro Gly Val Thr Ile Asn Gly Leu Cys Arg Ile Gly 145
150 155 160 Glu Ser Thr Tyr Ile Gly Ser Gly Ser Thr Val Ile Gln Cys
Ile Glu 165 170 175 Ile Ala Pro Tyr Thr Thr Leu Gly Ala Gly Thr Val
Val Leu Lys Ser 180 185 190 Leu Thr Glu Ser Gly Thr Tyr Val Gly Val
Pro Ala Arg Lys Ile Lys 195 200 205 28410PRTStreptococcus
suismisc_featureCPS2T 28Met Glu Pro Ile Cys Leu Ile Pro Ala Arg Ser
Gly Ser Lys Gly Leu 1 5 10 15 Pro Asn Lys Asn Met Leu Phe Leu Asp
Gly Val Pro Met Ile Phe His 20 25 30 Thr Ile Arg Ala Ala Ile Glu
Ser Gly Cys Phe Lys Lys Glu Asn Ile 35 40 45 Tyr Val Ser Thr Asp
Ser Glu Val Tyr Lys Glu Ile Cys Glu Thr Thr 50 55 60 Gly Val Gln
Val Leu Met Arg Pro Ala Asp Leu Ala Thr Asp Phe Thr 65 70 75 80 Thr
Ser Phe Gln Leu Asn Glu His Phe Leu Gln Asp Phe Ser Asp Asp 85 90
95 Gln Val Phe Val Leu Leu Gln Val Thr Ser Pro Leu Arg Ser Gly Lys
100 105 110 His Val Lys Glu Ala Met Glu Leu Tyr Gly Lys Gly Gln Ala
Asp His 115 120 125 Val Val Ser Phe Thr Lys Val Asp Lys Ser Pro Thr
Leu Phe Ser Thr 130 135 140 Leu Asp Glu Asn Gly Phe Ala Lys Asp Ile
Ala Gly Leu Gly Gly Ser 145 150 155 160 Tyr Arg Arg Gln Asp Glu Lys
Thr Leu Tyr Tyr Pro Asn Gly Ala Ile 165 170 175 Tyr Ile Ser Ser Lys
Gln Ala Tyr Leu Ala Asp Lys Thr Tyr Phe Ser 180 185 190 Glu Lys Thr
Ala Ala Tyr Val Met Thr Lys Glu Asp Ser Ile Asp Val 195 200 205 Asp
Asp His Phe Asp Phe Thr Gly Val Ile Gly Arg Ile Tyr Phe Asp 210 215
220 Tyr Gln Arg Arg Glu Gln Gln Asn Lys Pro Phe Tyr Lys Arg Glu Leu
225 230 235 240 Lys Arg Leu Cys Glu Gln Arg Val His Asp Ser Leu Val
Ile Gly Asp 245 250 255 Ser Arg Leu Leu Ala Leu Leu Leu Asp Gly Phe
Asp Asn Ile Ser Ile 260 265 270 Gly Gly Met Thr Ala Ser Thr Ser Leu
Glu Asn Gln Gly Leu Phe Leu 275 280 285 Ala Thr Pro Ile Lys Lys Val
Leu Leu Ser Leu Gly Val Asn Asp Leu 290 295 300 Ile Thr Asp Tyr Pro
Leu His Met Ile Glu Asp Thr Ile Arg Gln Leu 305 310 315 320 Met Glu
Ser Leu Val Ser Lys Ala Glu Gln Val Glu Val Thr Thr Ile 325 330 335
Ala Tyr Thr Leu Phe Arg Asp Ser Val Ser Asn Glu Glu Thr Val Gln 340
345 350 Leu Asn Asp Val Ile Val Gln Ser Ala Ser Glu Leu Gly Ile Ser
Val 355 360 365 Ile Asp Leu Asn Glu Val Val Glu Lys Glu Ala Met Leu
Asp Tyr Gln 370 375 380 Tyr Thr Asn Asp Gly Leu His Phe Asn Gln Ile
Gly Gln Glu Arg Val 385 390 395 400 Asn Gln Leu Ile Leu Thr Ser Leu
Thr Arg 405 410 296992DNAStreptococcus suismisc_featureCPS1
29atcgccaaac gaaattggca ttatttgata tgatagcagt tgcaatttct gcaatcttaa
60caagtcatat accaaatgct gatttaaatc gttctggaat ttttatcata atgatggttc
120attattttgc attttttata tctcgtatgc cagttgaatt tgagtataga
ggtaatctga 180tagagtttga aaaaacattt aactatagta taatatttgc
aatttttctt acggcagtat 240catttttgtt ggagaataat ttcgcacttt
caagacgtgg tgccgtgtat ttcacattaa 300taaacttcgt tttggtatac
ctatttaacg taattattaa gcagtttaag gatagctttc 360tattttcgac
aatctatcaa aaaaagacga ttctaattac aacggctgaa cgatgggaaa
420atatgcaagt tttatttgaa tcacataaac aaattcaaaa aaatcttgtt
gcattggtag 480ttttaggtac agaaatagat aaaattaatt tatcattacc
gctctattat tctgtggaag 540aagctataga gttttcaaca agggaagtgg
tcgaccacgt ctttataaat ctaccaagtg 600agtttttaga cgtaaagcaa
ttcgtttcag attttgagtt gttaggtatt gatgtaagcg 660ttgatattaa
ttcattcggt tttactgcgt tgaaaaacaa aaaaatccaa ctgctaggtg
720accatagcat tgtaactttt tccacaaatt tttataagcc tagtcatatc
atgatgaaac 780gacttttgga tatactcgga gcggtagtcg ggttaattat
ttgtggtata gtttctattt 840tgttagttcc aattattcgt agagatggtg
gaccggctat ttttgctcag aaacgagttg 900gacagaatgg acgcatattt
acattctaca agtttcgatc gatgtatgtt gatgctgagg 960agcgcaaaaa
agacttgctc agccaaaacc agatgcaagg gtgggtatgt tttaaaatgg
1020gaaaaacgat cctagaatta ctccaattgg acatttcata cgcaaaaaca
agtttagacg 1080agttaccaca gttttataat gttttaattg gcgatatgag
tctagttggt acacgtccac 1140ctacagttga tgaatttgaa aaatatactc
ctggtcaaaa gagacgattg agttttaaac 1200cagggattac aggtctctgg
caggttagtg gtcgtagtaa tatcacagac ttcgacgacg 1260tagttcggtt
ggacttagca tacattgata attggactat ctggtcagat attaaaattt
1320tattaaagac agtgaaagtt gtattgttga gagagggaag taagtaaaag
tatatgaaag 1380tttgtttggt cggttcttca gggggacatt tgactcactt
gtatttgtta aaaccgtttt 1440ggaaggaaga agaacgtttt tgggtaacat
ttgataaaga ggatgcaaga agtcttttga 1500agaatgaaaa aatgtatcca
tgttactttc caacaaatcg caatctcatt aatttagtga 1560aaaatacttt
cttagctttc aaaattttac gtgatgagaa accagatgtt attatttcat
1620ctggtgcggc cgttgctgtc cccttctttt acatcggaaa actatttgga
gcaaagacga 1680tttatattga agtatttgat cgagttaata aatctacatt
aactggaaaa ctagtttatc 1740ccgtaacaga tatttttatt gttcagtggg
aagaaatgaa gaaggtatat cctaaatcta 1800ttaacttggg gagtattttt
taatgatttt tgtaacagta ggaactcatg aacaacagtt 1860taatcgattg
ataaaagaga ttgatttatt gaaaaaaaat ggaagtataa ccgacgaaat
1920atttattcaa acaggatatt ctgactatat tccagaatat tgcaagtata
aaaaatttct 1980cagttacaaa gaaatggaac aatatattaa caaatcagaa
gtagttattt gccacggagg 2040ccccgctact tttatgaatt cattatccaa
aggaaaaaaa caattattgt ttcctagaca 2100aaaaaagtat ggtgaacatg
taaatgatca tcaagtagag tttgtaagaa gaattttaca 2160agataataat
attttattta tagaaaatat agatgatttg tttgaaaaaa ttattgaagt
2220ttctaagcaa actaacttta catcaaataa taattttttt tgtgaaagat
taaaacaaat 2280agttgaaaaa tttaatgagg atcaagaaaa tgaataataa
aaaagatgca tatttgataa 2340tggcttatca taatttttct cagattttac
tggagaggga tacagatatt atcatcttct 2400ctcaggagaa tgcacaccat
tagttccttc agaatacctg tataattatt ttaaatattc 2460tcaggattta
tatgttgaat ttacaaaaga tgagcaaaaa tataaagaaa ataggatata
2520tgaacgagtt aaatgttaca gattatttcc taatatatca gaaaaaacta
ttgataatgt 2580actgtttaga attttattaa gaatgtatcg agcttttgaa
tactatttac aaagattgtt 2640gtttattgat agaataaaaa acatggtcta
agaataagat ttggttctaa ttgggtttcg 2700cttccacatg attttgtggc
aattctttta tcaaatgaaa acgaaacagc ttatttattt 2760aagtaatcta
aatgtccaga tgaactattt atacagacaa ttatagaaaa atatgaattt
2820tcaaatagat tatctaaata tggaaattta agatatataa agtggaaaaa
atcaacatct 2880tctcctattg tctttacaga tgattctatt gatgaattgc
taaatgcaag aaatttaggt 2940tttttatttg ctagaaagtt aaaaatagaa
aataaatcta aatttaaaga aattattact 3000aaaaaataaa atagttgatt
ttgtgagagt aatgtatgtt taaattattt aaatatgacc 3060cggaatattt
tatttttaag tacttctggt tgattatttt tattccagag caaaagtatg
3120tatttttatt aatttttatg aatttaattt tatttcatat aaaatttttg
aaaactaagc 3180taatattaaa aaatgaaatt ttattgtttt tattatggtc
tatattatgt tttgtttcag 3240tagtcacaag tatgtttgtt gaaataaatt
ttgaaagatt atttgcagat tttactgctc 3300ccataatttg gattattgca
ataatgtatt ataatttgta ttcatttata aatattgatt 3360ataaaaaatt
aaaaaatagt atctttttta gttttttagt tttattaggt atatctgcat
3420tgtatattat tcaaaatggg aaagatattg tatttttaga cagacacctt
ataggactag 3480actatcttat aacaggcgtc aaaacaaggt tggttggctt
tatgaactat cctacgttaa 3540ataccactac aattatagtt tcaattccgt
taatctttgc acttataaaa aataaaatgc 3600aacaattttt tttcttgtgt
cttgctttta taccgatcta tttaagtgga tcgagaattg 3660gtagtttatc
gctagcaata ttaattatat gcttgttatg gagatatata ggtggaaaat
3720ttgcttggat aaaaaagcta atagtaatat ttgtaatact acttattatt
ttaaatactg 3780aattgcttta ccatgaaatt ttggctgttt ataattctag
agaatcaagt aacgaagcta 3840gatttattat ttatcaagga agtattgata
aagtattaga aaacaatatt ttatttggat 3900atggaatatc cgaatattca
gttacgggaa cttggctcgg aagtcattca ggctatatat 3960cattttttta
taaatcagga atagttgggt tgattttact gatgttttct tttttttatg
4020ttataaaaaa aagttatgga gttaatgggg aaacagcact attttatttt
acatcattag 4080ccatattttt catatatgaa acaatagatc cgattattat
tatattagta ctattctttt 4140cttcaatagg tatttggaat aatataaatt
ttaaaaagga tatggagaca aaaaatgaat 4200gatttaattt cagttattgt
accaatttat aatgtccaag attatcttga taaatgtatt 4260aacagtatta
ttaaccaaac atatactaat ttagaggtta ttctcgtaaa tgatggaagt
4320actgatgatt ctgagaaaat ttgcttaaac tatatgaaga acgatggaag
aattaaatat 4380tacaagaaaa ttaatggcgg tctagcagat gctcgaaatt
tcggactaga acatgcaaca 4440ggtaaatata ttgcttttgt cgattctgat
gactatatag aagttgcaat gttcgagaga 4500atgcatgata atataactga
gtataatgcc gatatagcag agatagattt ttgtttagta 4560gacgaaaacg
ggtatacaaa gaaaaaaaga aatagtaatt ttcatgtctt aacgagagaa
4620gagactgtaa aagaattttt gtcaggatct aatatagaaa ataatgtttg
gtgcaagctt 4680tattcacgag atattataaa agatataaaa ttccaaatta
ataatagaag tattggtgag 4740gatttgcttt ttaatttgga ggtcttgaac
aatgtaacac gtgtagtagt tgatactaga 4800gaatattatt ataattatgt
cattcgtaac agttcgctta ttaatcagaa attctctata 4860aataatattg
atttagtcac aagattggag aattacccct ttaagttaaa aagagagttt
4920agtcattatt ttgatgcaaa agttattaaa gagaaggtta aatgtttaaa
caaaatgtat 4980tcaacagatt gtttggataa tgagttcttg ccaatattag
agtcttatcg aaaagaaata 5040cgtagatatc catttattaa agcgaaaaga
tatttatcaa gaaagcattt agttacgttg 5100tatttgatga aattttcgcc
taaactatat gtaatgttat ataagaaatt tcaaaagcag 5160tagaggtaaa
aatggataaa attagtgtta ttgttccagt ttataatgta gataaatatt
5220taagtagttg tatagaaagc attattaatc aaaattataa aaatatagaa
atattattga 5280tagatgatgg ctctgtagat gattctgcta aaatatgcaa
ggaatatgca gaaaaagata 5340aaagagtaaa aatttttttc actaatcata
gtggagtatc aaatgctaga aatcatggaa 5400taaagcggag tacagctgaa
tatattatgt ttgttgactc tgatgatgtt gttgatagta 5460gattagtaga
aaaattatat tttaatatta taaaaagtag aagtgattta tctggttgtt
5520tgtacgctac tttttcagaa aatataaata attttgaagt gaataatcca
aatattgatt 5580ttgaagcaat taataccgtg caggacatgg gagaaaaaaa
ttttatgaat ttgtatataa 5640ataatatttt ttctactcct gtttgtaaac
tatataagaa aagatacata acagatcttt 5700ttcaagagaa tcaatggtta
ggagaagatt tactttttaa tctgcattat ttaaagaata 5760tagatagagt
tagttatttg actgaacatc tttattttta taggagaggt atactaagta
5820cagtaaattc ttttaaagaa ggtgtgtttt tgcaattgga aaatttgcaa
aaacaagtga 5880tagtattgtt taagcaaata tatggtgagg attttgacgt
atcaattgtt aaagatacta 5940tacgttggca agtattttat tatagcttac
taatgtttaa atacggaaaa cagtctattt 6000ttgacaaatt tttaattttt
agaaatcttt ataaaaaata ttattttaac ttgttaaaag 6060tatctaacaa
aaattctttg tctaaaaatt tttgtataag aattgtttcg aacaaagttt
6120ttaaaaaaat attatggtta taataggaag atatcatgga tactattagt
aaaatttcta 6180taattgtacc tatatataat gtagaaaaat atttatctaa
atgtatagat agcattgtaa 6240atcagaccta caaacatata gagattcttc
tggtgaatga cggtagtacg gataattcgg 6300aagaaatttg tttagcatat
gcgaagaaag atagtcgcat tcgttatttt aaaaaagaga 6360acggcgggct
atcagatgcc cgtaattatg gcataagtcg cgccaagggt gactacttag
6420cttttataga ctcagatgat tttattcatt cggagttcat ccaacgttta
cacgaagcaa 6480ttgagagaga gaatgccctt gtggcagttg ctggttatga
tagggtagat gcttcggggc 6540atttcttaac agcagagccg cttcctacaa
atcaggctgt tctgagcggc aggaatgttt 6600gtaaaaagct gctagaggcg
gatggtcatc gctttgtggt ggcctgtaat aaactctata 6660aaaaagaact
atttgaagat tttcgatttg aaaagggtaa gattcatgaa gatgaatact
6720tcacttatcg cttgctctat gagttagaaa aagttgcaat agttaaggag
tgcttgtact 6780attatgttga ccgagaaaat agtatcacaa cttctagcat
gactgaccat cgcttccatt 6840gcctactgga atttcaaaat gaacgaatgg
acttctatga aagtagagga gataaagagc 6900tcttactaga gtgttatcgt
tcatttttag cctttgctgt tttgttttta ggcaaatata 6960atcattggtt
gagcaaacag caaaagaagc tt
699230454PRTStreptococcus suismisc_featureCPS1E 30Arg Gln Thr Lys
Leu Ala Leu Phe Asp Met Ile Ala Val Ala Ile Ser 1 5 10 15 Ala Ile
Leu Thr Ser His Ile Pro Asn Ala Asp Leu Asn Arg Ser Gly 20 25 30
Ile Phe Ile Ile Met Met Val His Tyr Phe Ala Phe Phe Ile Ser Arg 35
40 45 Met Pro Val Glu Phe Glu Tyr Arg Gly Asn Leu Ile Glu Phe Glu
Lys 50 55 60 Thr Phe Asn Tyr Ser Ile Ile Phe Ala Ile Phe Leu Thr
Ala Val Ser 65 70 75 80 Phe Leu Leu Glu Asn Asn Phe Ala Leu Ser Arg
Arg Gly Ala Val Tyr 85 90 95 Phe Thr Leu Ile Asn Phe Val Leu Val
Tyr Leu Phe Asn Val Ile Ile 100 105 110 Lys Gln Phe Lys Asp Ser Phe
Leu Phe Ser Thr Ile Tyr Gln Lys Lys 115 120 125 Thr Ile Leu Ile Thr
Thr Ala Glu Arg Trp Glu Asn Met Gln Val Leu 130 135 140 Phe Glu Ser
His Lys Gln Ile Gln Lys Asn Leu Val Ala Leu Val Val 145 150 155 160
Leu Gly Thr Glu Ile Asp Lys Ile Asn Leu Ser Leu Pro Leu Tyr Tyr 165
170 175 Ser Val Glu Glu Ala Ile Glu Phe Ser Thr Arg Glu Val Val Asp
His 180 185 190 Val Phe Ile Asn Leu Pro Ser Glu Phe Leu Asp Val Lys
Gln Phe Val 195 200 205 Ser Asp Phe Glu Leu Leu Gly Ile Asp Val Ser
Val Asp Ile Asn Ser 210 215 220 Phe Gly Phe Thr Ala Leu Lys Asn Lys
Lys Ile Gln Leu Leu Gly Asp 225 230 235 240 His Ser Ile Val Thr Phe
Ser Thr Asn Phe Tyr Lys Pro Ser His Ile 245 250 255 Met Met Lys Arg
Leu Leu Asp Ile Leu Gly Ala Val Val Gly Leu Ile 260 265 270 Ile Cys
Gly Ile Val Ser Ile Leu Leu Val Pro Ile Ile Arg Arg Asp 275 280 285
Gly Gly Pro Ala Ile Phe Ala Gln Lys Arg Val Gly Gln Asn Gly Arg 290
295 300 Ile Phe Thr Phe Tyr Lys Phe Arg Ser Met Tyr Val Asp Ala Glu
Glu 305 310 315 320 Arg Lys Lys Asp Leu Leu Ser Gln Asn Gln Met Gln
Gly Trp Val Cys 325 330 335 Phe Lys Met Gly Lys Thr Ile Leu Glu Leu
Leu Gln Leu Asp Ile Ser 340 345 350 Tyr Ala Lys Thr Ser Leu Asp Glu
Leu Pro Gln Phe Tyr Asn Val Leu 355 360 365 Ile Gly Asp Met Ser Leu
Val Gly Thr Arg Pro Pro Thr Val Asp Glu 370 375 380 Phe Glu Lys Tyr
Thr Pro Gly Gln Lys Arg Arg Leu Ser Phe Lys Pro 385 390 395 400 Gly
Ile Thr Gly Leu Trp Gln Val Ser Gly Arg Ser Asn Ile Thr Asp 405 410
415 Phe Asp Asp Val Val Arg Leu Asp Leu Ala Tyr Ile Asp Asn Trp Thr
420 425 430 Ile Trp Ser Asp Ile Lys Ile Leu Leu Lys Thr Val Lys Val
Val Leu 435 440 445 Leu Arg Glu Gly Ser Lys 450
31149PRTStreptococcus suismisc_featureCPS1F 31Met Lys Val Cys Leu
Val Gly Ser Ser Gly Gly His Leu Thr His Leu 1 5 10 15 Tyr Leu Leu
Lys Pro Phe Trp Lys Glu Glu Glu Arg Phe Trp Val Thr 20 25 30 Phe
Asp Lys Glu Asp Ala Arg Ser Leu Leu Lys Asn Glu Lys Met Tyr 35 40
45 Pro Cys Tyr Phe Pro Thr Asn Arg Asn Leu Ile Asn Leu Val Lys Asn
50 55 60 Thr Phe Leu Ala Phe Lys Ile Leu Arg Asp Glu Lys Pro Asp
Val Ile 65 70 75 80 Ile Ser Ser Gly Ala Ala Val Ala Val Pro Phe Phe
Tyr Ile Gly Lys 85 90 95 Leu Phe Gly Ala Lys Thr Ile Tyr Ile Glu
Val Phe Asp Arg Val Asn 100 105 110 Lys Ser Thr Leu Thr Gly Lys Leu
Val Tyr Pro Val Thr Asp Ile Phe 115 120 125 Ile Val Gln Trp Glu Glu
Met Lys Lys Val Tyr Pro Lys Ser Ile Asn 130 135 140 Leu Gly Ser Ile
Phe 145 32164PRTStreptococcus suismisc_featureCPS1G 32Met Ile Phe
Val Thr Val Gly Thr His Glu Gln Gln Phe Asn Arg Leu 1 5 10 15 Ile
Lys Glu Ile Asp Leu Leu Lys Lys Asn Gly Ser Ile Thr Asp Glu 20 25
30 Ile Phe Ile Gln Thr Gly Tyr Ser Asp Tyr Ile Pro Glu Tyr Cys Lys
35 40 45 Tyr Lys Lys Phe Leu Ser Tyr Lys Glu Met Glu Gln Tyr Ile
Asn Lys 50 55 60 Ser Glu Val Val Ile Cys His Gly Gly Pro Ala Thr
Phe Met Asn Ser 65 70 75 80 Leu Ser Lys Gly Lys Lys Gln Leu Leu Phe
Pro Arg Gln Lys Lys Tyr 85 90 95 Gly Glu His Val Asn Asp His Gln
Val Glu Phe Val Arg Arg Ile Leu 100 105 110 Gln Asp Asn Asn Ile Leu
Phe Ile Glu Asn Ile Asp Asp Leu Phe Glu 115 120 125 Lys Ile Ile Glu
Val Ser Lys Gln Thr Asn Phe Thr Ser Asn Asn Asn 130 135 140 Phe Phe
Cys Glu Arg Leu Lys Gln Ile Val Glu Lys Phe Asn Glu Asp 145 150 155
160 Gln Glu Asn Glu 33388PRTStreptococcus suismisc_featureCPS1H
33Met Phe Lys Leu Phe Lys Tyr Asp Pro Glu Tyr Phe Ile Phe Lys Tyr 1
5 10 15 Phe Trp Leu Ile Ile Phe Ile Pro Glu Gln Lys Tyr Val Phe Leu
Leu 20 25 30 Ile Phe Met Asn Leu Ile Leu Phe His Ile Lys Phe Leu
Lys Thr Lys 35 40 45 Leu Ile Leu Lys Asn Glu Ile Leu Leu Phe Leu
Leu Trp Ser Ile Leu 50 55 60 Cys Phe Val Ser Val Val Thr Ser Met
Phe Val Glu Ile Asn Phe Glu 65 70 75 80 Arg Leu Phe Ala Asp Phe Thr
Ala Pro Ile Ile Trp Ile Ile Ala Ile 85 90 95 Met Tyr Tyr Asn Leu
Tyr Ser Phe Ile Asn Ile Asp Tyr Lys Lys Leu 100 105 110 Lys Asn Ser
Ile Phe Phe Ser Phe Leu Val Leu Leu Gly Ile Ser Ala 115 120 125 Leu
Tyr Ile Ile Gln Asn Gly Lys Asp Ile Val Phe Leu Asp Arg His 130 135
140 Leu Ile Gly Leu Asp Tyr Leu Ile Thr Gly Val Lys Thr Arg Leu Val
145 150 155 160 Gly Phe Met Asn Tyr Pro Thr Leu Asn Thr Thr Thr Ile
Ile Val Ser 165 170 175 Ile Pro Leu Ile Phe Ala Leu Ile Lys Asn Lys
Met Gln Gln Phe Phe 180 185 190 Phe Leu Cys Leu Ala Phe Ile Pro Ile
Tyr Leu Ser Gly Ser Arg Ile 195 200 205 Gly Ser Leu Ser Leu Ala Ile
Leu Ile Ile Cys Leu Leu Trp Arg Tyr 210 215 220 Ile Gly Gly Lys Phe
Ala Trp Ile Lys Lys Leu Ile Val Ile Phe Val 225 230 235 240 Ile Leu
Leu Ile Ile Leu Asn Thr Glu Leu Leu Tyr His Glu Ile Leu 245 250 255
Ala Val Tyr Asn Ser Arg Glu Ser Ser Asn Glu Ala Arg Phe Ile Ile 260
265 270 Tyr Gln Gly Ser Ile Asp Lys Val Leu Glu Asn Asn Ile Leu Phe
Gly 275 280 285 Tyr Gly Ile Ser Glu Tyr Ser Val Thr Gly Thr Trp Leu
Gly Ser His 290 295 300 Ser Gly Tyr Ile Ser Phe Phe Tyr Lys Ser Gly
Ile Val Gly Leu Ile 305 310 315 320 Leu Leu Met Phe Ser Phe Phe Tyr
Val Ile Lys Lys Ser Tyr Gly Val 325 330 335 Asn Gly Glu Thr Ala Leu
Phe Tyr Phe Thr Ser Leu Ala Ile Phe Phe 340 345 350 Ile Tyr Glu Thr
Ile Asp Pro Ile Ile Ile Ile Leu Val Leu Phe Phe 355 360 365 Ser Ser
Ile Gly Ile Trp Asn Asn Ile Asn Phe Lys Lys Asp Met Glu 370 375 380
Thr Lys Asn Glu 385 34322PRTStreptococcus suismisc_featureCPS1I
34Met Asn Asp Leu Ile Ser Val Ile Val Pro Ile Tyr Asn Val Gln Asp 1
5 10 15 Tyr Leu Asp Lys Cys Ile Asn Ser Ile Ile Asn Gln Thr Tyr Thr
Asn 20 25 30 Leu Glu Val Ile Leu Val Asn Asp Gly Ser Thr Asp Asp
Ser Glu Lys 35 40 45 Ile Cys Leu Asn Tyr Met Lys Asn Asp Gly Arg
Ile Lys Tyr Tyr Lys 50 55 60 Lys Ile Asn Gly Gly Leu Ala Asp Ala
Arg Asn Phe Gly Leu Glu His 65 70 75 80 Ala Thr Gly Lys Tyr Ile Ala
Phe Val Asp Ser Asp Asp Tyr Ile Glu 85 90 95 Val Ala Met Phe Glu
Arg Met His Asp Asn Ile Thr Glu Tyr Asn Ala 100 105 110 Asp Ile Ala
Glu Ile Asp Phe Cys Leu Val Asp Glu Asn Gly Tyr Thr 115 120 125 Lys
Lys Lys Arg Asn Ser Asn Phe His Val Leu Thr Arg Glu Glu Thr 130 135
140 Val Lys Glu Phe Leu Ser Gly Ser Asn Ile Glu Asn Asn Val Trp Cys
145 150 155 160 Lys Leu Tyr Ser Arg Asp Ile Ile Lys Asp Ile Lys Phe
Gln Ile Asn 165 170 175 Asn Arg Ser Ile Gly Glu Asp Leu Leu Phe Asn
Leu Glu Val Leu Asn 180 185 190 Asn Val Thr Arg Val Val Val Asp Thr
Arg Glu Tyr Tyr Tyr Asn Tyr 195 200 205 Val Ile Arg Asn Ser Ser Leu
Ile Asn Gln Lys Phe Ser Ile Asn Asn 210 215 220 Ile Asp Leu Val Thr
Arg Leu Glu Asn Tyr Pro Phe Lys Leu Lys Arg 225 230 235 240 Glu Phe
Ser His Tyr Phe Asp Ala Lys Val Ile Lys Glu Lys Val Lys 245 250 255
Cys Leu Asn Lys Met Tyr Ser Thr Asp Cys Leu Asp Asn Glu Phe Leu 260
265 270 Pro Ile Leu Glu Ser Tyr Arg Lys Glu Ile Arg Arg Tyr Pro Phe
Ile 275 280 285 Lys Ala Lys Arg Tyr Leu Ser Arg Lys His Leu Val Thr
Leu Tyr Leu 290 295 300 Met Lys Phe Ser Pro Lys Leu Tyr Val Met Leu
Tyr Lys Lys Phe Gln 305 310 315 320 Lys Gln 35322PRTStreptococcus
suismisc_featureCPS1J 35Met Asp Lys Ile Ser Val Ile Val Pro Val Tyr
Asn Val Asp Lys Tyr 1 5 10 15 Leu Ser Ser Cys Ile Glu Ser Ile Ile
Asn Gln Asn Tyr Lys Asn Ile 20 25 30 Glu Ile Leu Leu Ile Asp Asp
Gly Ser Val Asp Asp Ser Ala Lys Ile 35 40 45 Cys Lys Glu Tyr Glu
Lys Asp Lys Arg Val Lys Ile Phe Phe Thr Asn 50 55 60 His Ser Gly
Val Ser Asn Ala Arg Asn His Gly Ile Lys Arg Ser Thr 65 70 75 80 Ala
Glu Tyr Ile Met Phe Val Asp Ser Asp Asp Val Val Asp Ser Arg 85 90
95 Leu Val Glu Lys Leu Tyr Phe Asn Ile Ile Lys Ser Arg Ser Asp Leu
100 105 110 Ser Gly Cys Leu Tyr Ala Thr Phe Ser Glu Asn Ile Asn Asn
Phe Glu 115 120 125 Val Asn Asn Pro Asn Ile Asp Phe Glu Ala Ile Asn
Thr Val Gln Asp 130 135 140 Met Gly Glu Lys Asn Phe Met Asn Leu Tyr
Ile Asn Asn Ile Phe Ser 145 150 155 160 Thr Pro Val Cys Lys Leu Tyr
Lys Lys Arg Tyr Ile Thr Asp Leu Phe 165 170 175 Gln Glu Asn Gln Trp
Leu Gly Glu Asp Leu Leu Phe Asn Leu His Tyr 180 185 190 Leu Lys Asn
Ile Asp Arg Val Ser Tyr Leu Thr Glu His Leu Tyr Phe 195 200 205 Tyr
Arg Arg Gly Ile Leu Ser Thr Val Asn Ser Phe Lys Glu Gly Val 210 215
220 Phe Leu Gln Leu Glu Asn Leu Gln Lys Gln Val Ile Val Leu Phe Lys
225 230 235 240 Gln Ile Tyr Gly Glu Asp Phe Asp Val Ser Ile Val Lys
Asp Thr Ile 245 250 255 Arg Trp Gln Val Phe Tyr Tyr Ser Leu Leu Met
Phe Lys Tyr Gly Lys 260 265 270 Gln Ser Ile Phe Asp Lys Phe Leu Ile
Phe Arg Asn Leu Tyr Lys Lys 275 280 285 Tyr Tyr Phe Asn Leu Leu Lys
Val Ser Asn Lys Asn Ser Leu Ser Lys 290 295 300 Asn Phe Cys Ile Arg
Ile Val Ser Asn Lys Val Phe Lys Lys Ile Leu 305 310 315 320 Trp Leu
36278PRTStreptococcus suismisc_featureCPS1K 36Met Asp Thr Ile Ser
Lys Ile Ser Ile Ile Val Pro Ile Tyr Asn Val 1 5 10 15 Glu Lys Tyr
Leu Ser Lys Cys Ile Asp Ser Ile Val Asn Gln Thr Tyr 20 25 30 Lys
His Ile Glu Ile Leu Leu Val Asn Asp Gly Ser Thr Asp Asn Ser 35 40
45 Glu Glu Ile Cys Leu Ala Tyr Ala Lys Lys Asp Ser Arg Ile Arg Tyr
50 55 60 Phe Lys Lys Glu Asn Gly Gly Leu Ser Asp Ala Arg Asn Tyr
Gly Ile 65 70 75 80 Ser Arg Ala Lys Gly Asp Tyr Leu Ala Phe Ile Asp
Ser Asp Asp Phe 85 90 95 Ile His Ser Glu Phe Ile Gln Arg Leu His
Glu Ala Ile Glu Arg Glu 100 105 110 Asn Ala Leu Val Ala Val Ala Gly
Tyr Asp Arg Val Asp Ala Ser Gly 115 120 125 His Phe Leu Thr Ala Glu
Pro Leu Pro Thr Asn Gln Ala Val Leu Ser 130 135 140 Gly Arg Asn Val
Cys Lys Lys Leu Leu Glu Ala Asp Gly His Arg Phe 145 150 155 160 Val
Val Ala Cys Asn Lys Leu Tyr Lys Lys Glu Leu Phe Glu Asp Phe 165 170
175 Arg Phe Glu Lys Gly Lys Ile His Glu Asp Glu Tyr Phe Thr Tyr Arg
180 185 190 Leu Leu Tyr Glu Leu Glu Lys Val Ala Ile Val Lys Glu Cys
Leu Tyr 195 200 205 Tyr Tyr Val Asp Arg Glu Asn Ser Ile Thr Thr Ser
Ser Met Thr Asp 210 215 220 His Arg Phe His Cys Leu Leu Glu Phe Gln
Asn Glu Arg Met Asp Phe 225 230 235 240 Tyr Glu Ser Arg Gly Asp Lys
Glu Leu Leu Leu Glu Cys Tyr Arg Ser 245 250 255 Phe Leu Ala Phe Ala
Val Leu Phe Leu Gly Lys Tyr Asn His Trp Leu 260 265 270 Ser Lys Gln
Gln Lys Lys 275 374519DNAStreptococcus suismisc_featureCPS9
37aagcttatcg tcaaggtgtt cgctatatcg tggcgacatc tcatagacga aaagggatgt
60ttgaaacacc agaaaaagtt atcatgacta actttcttca atttaaagac gcagtagcag
120aagtttatcc tgaaatacga ttgtgctatg gtgctgaatt gtattatagt
aaagatatat 180taagcaaact tgaaaaaaag aaagtaccca cacttaatgg
ctcgcgctat attcttttgg 240agttcagtag tgatactcct tggaaagaga
ttcaagaagc agtgaacgaa gtgacgctac 300ttgggctaac tcccgtactt
gcccatatag aacgatatga cgccctagcg tttcatgcag 360agagagtaga
agagttaatt gacaagggat gctatactca ggtaaatagt aatcatgtgc
420tgaagcccac tttaattggt gatcgagcaa aagaatttaa aaaacgtact
cggtattttt 480tagagcagga tttagtacat tgtgttgcta gcgatatgca
taatttatct agtagacctc 540cgtttatgag ggaggcttat aagttgctaa
cagaggaatt tggcaaagat aaagcgaaag 600cgttgctaaa aaagaatcct
cttatgctat taaaaaacca ggcgatttaa actggttact 660ctagattgtg
gagagaaaaa tggatttagg aactgttact gataaactgt tagaacgcaa
720cagtaaacga ttgatactcg tgtgcatgga tacgtgtctt cttatagttt
ccatgatttt 780gagcagactg tttttggatg ttattattga cataccagat
gaacgcttca ttcttgcagt 840tttattcgta tcaattttat atttgattct
atcgtttaga ttaaaagtct tttcattaat 900tacgcgttac acagggtatc
agagttatgt aaaaatagga cttagtttaa tatctgcgca 960ttcattgttt
ttaattatct caatggtgtt gtggcaggct tttagttatc gtttcatctt
1020agtatcctta tttttgtcgt atgtaatgct cattactccg aggattgttt
ggaaagtctt 1080acatgagacg agaaaaaatg ctatccgtaa gaaggatagc
ccactaagaa tcttagtagt 1140aggtgctgga gatggtggta atatttttat
caatactgtc aaagatcgaa aattgaattt 1200tgaaattgtc
ggtatcgttg atcgtgatcc aaataaactt ggaacattta tccgtacggc
1260taaagtttta ggaaaccgta atgatattcc acgactggta gaggaattag
ctgttgacca 1320agtgacgatt gccatccctt ctttaaatgg taaggagcga
gagaagattg ttgaaatctg 1380taacactaca ggagtgaccg tcaataatat
gccgagtatt gaagacatta tggcggggaa 1440catgtctgtc agtgcctttc
aggaaattga cgtagcagac cttcttggtc gaccagaggt 1500tgttttggat
caggatgaat tgaatcagtt tttccaaggg aaaacaatcc ttgtcacagg
1560agcaggtggc tctatcggtt cagagctatg tcgtcaaatt gctaagttta
cgcctaaacg 1620cttgttgttg cttggacatg gagaaaattc aatctatctc
attcatcgag agttactgga 1680aaagtaccaa ggtaagattg agttggtccc
tctcattgca gatattcaag atagagaatt 1740gatttttagc ataatggctg
aatatcaacc cgatgttgtt tatcatgctg cagcacataa 1800gcatgttcct
ttgatggaat ataatccaca tgaagcagtg aagaataata tttttggaac
1860gaagaatgtg gctgaggcgg ctaaaactgc aaaggttgcc aaatttgtta
tggtttcaac 1920agataaagct gttaatccac caaatgtcat gggagcgact
aaacgtgttg cagaaatgat 1980tgttacaggt ttaaacgagc caggtcagac
tcaatttgcg gcagtccggt ttgggaatgt 2040tctaggtagt cgtggaagtg
ttgttccgct attcaaagag caaattagaa aaggtggacc 2100tgttacggtt
accgacttta ggatgactcg ttatttcatg acgattcctg aggcaagtcg
2160tttggttatc caagctggac atttggcaaa aggtggagaa atatttgtct
tggatatggg 2220cgagccagta caaatcctgg aattggcaag aaaagttatc
ttgttaagtg gacacacaga 2280ggaagaaatc gggattgtag aatctggaat
cagaccaggc gagaaactct acgaggaatt 2340attatcaaca gaagaacgtg
tcagcgaaca gattcatgaa aaaatatttg tgggtcgcgt 2400tacaaataag
cagtcggaca ttgtcaattc atttatcaat ggattactcc aaaaagatag
2460aaatgaatta aaaaatatgt tgattgaatt tgcaaaacaa gaataagaaa
gtaaaaaata 2520tttttacttt cctagagttt aaacgatgtt taagttctag
gaaggttaga atacctaatt 2580aacaacaata ttactattta ttaagagtca
gataatagca actaagtgct acaaactatc 2640tttataataa gtatatttgg
tcaaaaggga gatgtgaaat gtatccaatt tgtaaacgta 2700ttttagcaat
tattatctca gggattgcta ttgttgttct gagtccaatt ttattattga
2760ttgcattggc aattaaatta gattctaaag gtccggtatt atttaaacaa
aagcgggttg 2820gtaaaaacaa gtcatacttt atgatttata aattccgttc
tatgtacgtt gacgcaccaa 2880gtgatatgcc gactcatcta ttaaaggatc
ctaaggcgat gattaccaag gtgggcgcgt 2940ttctcagaaa aacaagttta
gatgaactgc cacagctttt taatattttt aaaggtgaaa 3000tggcgattgt
tggtccacgc ccagccttat ggaatcaata tgacttaatt gaagagcgag
3060ataaatatgg tgcaaatgat attcgtcctg gactaaccgg ttgggctcaa
attaatggtc 3120gtgatgaatt ggaaattgat gaaaagtcaa aattagatgg
atattatgtt caaaatatga 3180gtctaggttt ggatattaaa tgtttcttag
gtacattcct cagtgtagcc agaagcgaag 3240gtgttgttga aggtggaaca
gggcagaaag gaaaaggatg aaattttcag tattaatgtc 3300ggtctatgag
aaagaaaaac cagagtttct tagggaatct ttggaaagca tccttgtcaa
3360tcaaacaatg attccaacgg aggttgtctt ggtagaggat gggccactca
atcagagctt 3420atatagtatt ttagaagaat ttaaaagtcg attttcattt
tttaaaacga tagccttgga 3480aaagaattcg ggtttaggaa ttgcactgaa
tgaaggtttg aaacattgta attatgagtg 3540ggtttgcacg aaatggattc
tgatgatgtt gcatatacat acacgttttg aaaagcaagt 3600taactttata
aaacaaaacc cgactataga tattgagata gatgagttct taaattctac
3660tagtgaaata gtttctcata aaaatgttcc aacccagcac gatgaaatat
taaagatggc 3720aaggcgggag aaatccatgt gccacatgac tgtaatgttt
aaaaagaaaa gtgtcgagag 3780agcagggggg tatcaaacac ttccgtacgt
agaagattat ttcctttggg tgcgcatgat 3840tgcttcagga tcgaaatttg
caaacattga tgaaacacta gttcttgcac gtgttggaaa 3900tgggatgttc
aataggaggg ggaacagaga acaaattaac agttggacat tactaattga
3960atttatgtta gctcaaggaa ttgttacacc actagatgta tttattaatc
aaatttacat 4020tagggtcttt gtttatatgc caacttggat aaagaaactc
atttatggaa aaatcttaag 4080gaaatagtat gattacagta ttgatggcta
catataatgg aagcccattt ataataaaac 4140agttagattc aattcgaaat
caaagtgtat cagcagacaa agttattatt tgggatgatt 4200gctcgacaga
tgatacaata aaaataataa aagattatat aaaaaaatat tctttggatt
4260catgggttgt ctctcaaaat aaatctaatc aggggcatta tcaaacattt
ataaatttga 4320caaagttagt tcaggaagga atagtctttt tttcagatca
agatgatatt tgggactgtc 4380ataaaattga gacaatgctt ccaatctttg
acagagaaaa tgtatcaatg gtgttttgca 4440aatccagatt gattgatgaa
aacggaaata ttatcagtag cccagatact tcggatagaa 4500tcaatacgta
ctctctaga 451938215PRTStreptococcus suismisc_featureCPS9D 38Ala Tyr
Arg Gln Gly Val Arg Tyr Ile Val Ala Thr Ser His Arg Arg 1 5 10 15
Lys Gly Met Phe Glu Thr Pro Glu Lys Val Ile Met Thr Asn Phe Leu 20
25 30 Gln Phe Lys Asp Ala Val Ala Glu Val Tyr Pro Glu Ile Arg Leu
Cys 35 40 45 Tyr Gly Ala Glu Leu Tyr Tyr Ser Lys Asp Ile Leu Ser
Lys Leu Glu 50 55 60 Lys Lys Lys Val Pro Thr Leu Asn Gly Ser Arg
Tyr Ile Leu Leu Glu 65 70 75 80 Phe Ser Ser Asp Thr Pro Trp Lys Glu
Ile Gln Glu Ala Val Asn Glu 85 90 95 Val Thr Leu Leu Gly Leu Thr
Pro Val Leu Ala His Ile Glu Arg Tyr 100 105 110 Asp Ala Leu Ala Phe
His Ala Glu Arg Val Glu Glu Leu Ile Asp Lys 115 120 125 Gly Cys Tyr
Thr Gln Val Asn Ser Asn His Val Leu Lys Pro Thr Leu 130 135 140 Ile
Gly Asp Arg Ala Lys Glu Phe Lys Lys Arg Thr Arg Tyr Phe Leu 145 150
155 160 Glu Gln Asp Leu Val His Cys Val Ala Ser Asp Met His Asn Leu
Ser 165 170 175 Ser Arg Pro Pro Phe Met Arg Glu Ala Tyr Lys Leu Leu
Thr Glu Glu 180 185 190 Phe Gly Lys Asp Lys Ala Lys Ala Leu Leu Lys
Lys Asn Pro Leu Met 195 200 205 Leu Leu Lys Asn Gln Ala Ile 210 215
39608PRTStreptococcus suismisc_featureCPS9E 39Met Asp Leu Gly Thr
Val Thr Asp Lys Leu Leu Glu Arg Asn Ser Lys 1 5 10 15 Arg Leu Ile
Leu Val Cys Met Asp Thr Cys Leu Leu Ile Val Ser Met 20 25 30 Ile
Leu Ser Arg Leu Phe Leu Asp Val Ile Ile Asp Ile Pro Asp Glu 35 40
45 Arg Phe Ile Leu Ala Val Leu Phe Val Ser Ile Leu Tyr Leu Ile Leu
50 55 60 Ser Phe Arg Leu Lys Val Phe Ser Leu Ile Thr Arg Tyr Thr
Gly Tyr 65 70 75 80 Gln Ser Tyr Val Lys Ile Gly Leu Ser Leu Ile Ser
Ala His Ser Leu 85 90 95 Phe Leu Ile Ile Ser Met Val Leu Trp Gln
Ala Phe Ser Tyr Arg Phe 100 105 110 Ile Leu Val Ser Leu Phe Leu Ser
Tyr Val Met Leu Ile Thr Pro Arg 115 120 125 Ile Val Trp Lys Val Leu
His Glu Thr Arg Lys Asn Ala Ile Arg Lys 130 135 140 Lys Asp Ser Pro
Leu Arg Ile Leu Val Val Gly Ala Gly Asp Gly Gly 145 150 155 160 Asn
Ile Phe Ile Asn Thr Val Lys Asp Arg Lys Leu Asn Phe Glu Ile 165 170
175 Val Gly Ile Val Asp Arg Asp Pro Asn Lys Leu Gly Thr Phe Ile Arg
180 185 190 Thr Ala Lys Val Leu Gly Asn Arg Asn Asp Ile Pro Arg Leu
Val Glu 195 200 205 Glu Leu Ala Val Asp Gln Val Thr Ile Ala Ile Pro
Ser Leu Asn Gly 210 215 220 Lys Glu Arg Glu Lys Ile Val Glu Ile Cys
Asn Thr Thr Gly Val Thr 225 230 235 240 Val Asn Asn Met Pro Ser Ile
Glu Asp Ile Met Ala Gly Asn Met Ser 245 250 255 Val Ser Ala Phe Gln
Glu Ile Asp Val Ala Asp Leu Leu Gly Arg Pro 260 265 270 Glu Val Val
Leu Asp Gln Asp Glu Leu Asn Gln Phe Phe Gln Gly Lys 275 280 285 Thr
Ile Leu Val Thr Gly Ala Gly Gly Ser Ile Gly Ser Glu Leu Cys 290 295
300 Arg Gln Ile Ala Lys Phe Thr Pro Lys Arg Leu Leu Leu Leu Gly His
305 310 315 320 Gly Glu Asn Ser Ile Tyr Leu Ile His Arg Glu Leu Leu
Glu Lys Tyr 325 330 335 Gln Gly Lys Ile Glu Leu Val Pro Leu Ile Ala
Asp Ile Gln Asp Arg 340 345 350 Glu Leu Ile Phe Ser Ile Met Ala Glu
Tyr Gln Pro Asp Val Val Tyr 355 360 365 His Ala Ala Ala His Lys His
Val Pro Leu Met Glu Tyr Asn Pro His 370 375 380 Glu Ala Val Lys Asn
Asn Ile Phe Gly Thr Lys Asn Val Ala Glu Ala 385 390 395 400 Ala Lys
Thr Ala Lys Val Ala Lys Phe Val Met Val Ser Thr Asp Lys 405 410 415
Ala Val Asn Pro Pro Asn Val Met Gly Ala Thr Lys Arg Val Ala Glu 420
425 430 Met Ile Val Thr Gly Leu Asn Glu Pro Gly Gln Thr Gln Phe Ala
Ala 435 440 445 Val Arg Phe Gly Asn Val Leu Gly Ser Arg Gly Ser Val
Val Pro Leu 450 455 460 Phe Lys Glu Gln Ile Arg Lys Gly Gly Pro Val
Thr Val Thr Asp Phe 465 470 475 480 Arg Met Thr Arg Tyr Phe Met Thr
Ile Pro Glu Ala Ser Arg Leu Val 485 490 495 Ile Gln Ala Gly His Leu
Ala Lys Gly Gly Glu Ile Phe Val Leu Asp 500 505 510 Met Gly Glu Pro
Val Gln Ile Leu Glu Leu Ala Arg Lys Val Ile Leu 515 520 525 Leu Ser
Gly His Thr Glu Glu Glu Ile Gly Ile Val Glu Ser Gly Ile 530 535 540
Arg Pro Gly Glu Lys Leu Tyr Glu Glu Leu Leu Ser Thr Glu Glu Arg 545
550 555 560 Val Ser Glu Gln Ile His Glu Lys Ile Phe Val Gly Arg Val
Thr Asn 565 570 575 Lys Gln Ser Asp Ile Val Asn Ser Phe Ile Asn Gly
Leu Leu Gln Lys 580 585 590 Asp Arg Asn Glu Leu Lys Asn Met Leu Ile
Glu Phe Ala Lys Gln Glu 595 600 605 40200PRTStreptococcus
suismisc_featureCPS9F 40Met Tyr Pro Ile Cys Lys Arg Ile Leu Ala Ile
Ile Ile Ser Gly Ile 1 5 10 15 Ala Ile Val Val Leu Ser Pro Ile Leu
Leu Leu Ile Ala Leu Ala Ile 20 25 30 Lys Leu Asp Ser Lys Gly Pro
Val Leu Phe Lys Gln Lys Arg Val Gly 35 40 45 Lys Asn Lys Ser Tyr
Phe Met Ile Tyr Lys Phe Arg Ser Met Tyr Val 50 55 60 Asp Ala Pro
Ser Asp Met Pro Thr His Leu Leu Lys Asp Pro Lys Ala 65 70 75 80 Met
Ile Thr Lys Val Gly Ala Phe Leu Arg Lys Thr Ser Leu Asp Glu 85 90
95 Leu Pro Gln Leu Phe Asn Ile Phe Lys Gly Glu Met Ala Ile Val Gly
100 105 110 Pro Arg Pro Ala Leu Trp Asn Gln Tyr Asp Leu Ile Glu Glu
Arg Asp 115 120 125 Lys Tyr Gly Ala Asn Asp Ile Arg Pro Gly Leu Thr
Gly Trp Ala Gln 130 135 140 Ile Asn Gly Arg Asp Glu Leu Glu Ile Asp
Glu Lys Ser Lys Leu Asp 145 150 155 160 Gly Tyr Tyr Val Gln Asn Met
Ser Leu Gly Leu Asp Ile Lys Cys Phe 165 170 175 Leu Gly Thr Phe Leu
Ser Val Ala Arg Ser Glu Gly Val Val Glu Gly 180 185 190 Gly Thr Gly
Gln Lys Gly Lys Gly 195 200 41269PRTStreptococcus
suismisc_featureCPS2G 41Met Lys Phe Ser Val Leu Met Ser Val Tyr Glu
Lys Glu Lys Pro Glu 1 5 10 15 Phe Leu Arg Glu Ser Leu Glu Ser Ile
Leu Val Asn Gln Thr Met Ile 20 25 30 Pro Thr Glu Val Val Leu Val
Glu Asp Gly Pro Leu Asn Gln Ser Leu 35 40 45 Tyr Ser Ile Leu Glu
Glu Phe Lys Ser Arg Phe Ser Phe Phe Lys Thr 50 55 60 Ile Ala Leu
Glu Lys Asn Ser Gly Leu Gly Ile Ala Leu Asn Glu Gly 65 70 75 80 Leu
Lys His Cys Asn Tyr Glu Trp Val Cys Thr Lys Trp Ile Leu Met 85 90
95 Met Leu His Ile His Thr Arg Phe Glu Lys Gln Val Asn Phe Ile Lys
100 105 110 Gln Asn Pro Thr Ile Asp Ile Glu Ile Asp Glu Phe Leu Asn
Ser Thr 115 120 125 Ser Glu Ile Val Ser His Lys Asn Val Pro Thr Gln
His Asp Glu Ile 130 135 140 Leu Lys Met Ala Arg Arg Glu Lys Ser Met
Cys His Met Thr Val Met 145 150 155 160 Phe Lys Lys Lys Ser Val Glu
Arg Ala Gly Gly Tyr Gln Thr Leu Pro 165 170 175 Tyr Val Glu Asp Tyr
Phe Leu Trp Val Arg Met Ile Ala Ser Gly Ser 180 185 190 Lys Phe Ala
Asn Ile Asp Glu Thr Leu Val Leu Ala Arg Val Gly Asn 195 200 205 Gly
Met Phe Asn Arg Arg Gly Asn Arg Glu Gln Ile Asn Ser Trp Thr 210 215
220 Leu Leu Ile Glu Phe Met Leu Ala Gln Gly Ile Val Thr Pro Leu Asp
225 230 235 240 Val Phe Ile Asn Gln Ile Tyr Ile Arg Val Phe Val Tyr
Met Pro Thr 245 250 255 Trp Ile Lys Lys Leu Ile Tyr Gly Lys Ile Leu
Arg Lys 260 265 42143PRTStreptococcus suismisc_featureCPS9H 42Met
Ile Thr Val Leu Met Ala Thr Tyr Asn Gly Ser Pro Phe Ile Ile 1 5 10
15 Lys Gln Leu Asp Ser Ile Arg Asn Gln Ser Val Ser Ala Asp Lys Val
20 25 30 Ile Ile Trp Asp Asp Cys Ser Thr Asp Asp Thr Ile Lys Ile
Ile Lys 35 40 45 Asp Tyr Ile Lys Lys Tyr Ser Leu Asp Ser Trp Val
Val Ser Gln Asn 50 55 60 Lys Ser Asn Gln Gly His Tyr Gln Thr Phe
Ile Asn Leu Thr Lys Leu 65 70 75 80 Val Gln Glu Gly Ile Val Phe Phe
Ser Asp Gln Asp Asp Ile Trp Asp 85 90 95 Cys His Lys Ile Glu Thr
Met Leu Pro Ile Phe Asp Arg Glu Asn Val 100 105 110 Ser Met Val Phe
Cys Lys Ser Arg Leu Ile Asp Glu Asn Gly Asn Ile 115 120 125 Ile Ser
Ser Pro Asp Thr Ser Asp Arg Ile Asn Thr Tyr Ser Leu 130 135 140
433738DNAStreptococcus suismisc_featureCPS7 43ctgcagcaca taagcatgtt
ccattgatgg aatataatcc acatgaagca gtgaagaata 60atatttttgg aacgaagaat
gtggctgagg cggctaaaac tgcaaaggtt gccaaatttg 120ttatggtttc
aacagataaa gctgttaatc cgccaaatgt catgggagcg actaaacgtg
180ttgcagaaat gattgtaaca ggtttaaacg agccaggtca gactcaattt
gcggcagtcc 240gttttgggaa tgttctaggt agtcgtggaa gtgttgttcc
gctattcaaa gagcaaatta 300gaaaaggtgg acctgttacg gttaccgact
ttaggatgac tcgttatttc atgacgattc 360ctgaggcaag tcgtttggtt
atccaagctg gacatttggc aaaaggtgga gaaatctttg 420tcttggatat
gggtgagcca gtacaaatcc tggaattggc aagaaaagtt atcttgttaa
480gcggacatac agaggaagaa atcgggattg tagaatctgg aatcagacca
ggcgagaaac 540tctacgagga attgttatca acagaagaac gtgtcagcga
acagattcat gaaaaaatat 600ttgtgggtcg cgttacaaat aagcagtcgg
acattgtcaa ttcatttatc aatggattac 660tccaaaaaga tagaaatgaa
ttaaaagata tgttgattga atttgcaaaa caagaataag 720aaagtaaaaa
atatttttac tttcctagag tttaaacgat gtttaagttc taggaaggtt
780ggaattgctt tcgtggaggt gatagataga aacctatata tttgtagaag
aaaggatatt 840aaactaaagg tgaatcggaa cataaagttt agatagagtt
ggtatttaat gccaaacagg 900tgaatgcaac ctctcgctcg ttactaagca
ggagatagta aagttgcttg aaagagagtt 960tgttaatcag tataagtagg
ctaaagtgag aatatatatc tattattatc ggtaatgata 1020ctattattga
gaattattgt agtggggata aaaataattt ttggtgattt tatcgtccga
1080cttaaaggtg ggttaaaaaa gtacttatat tcttttagaa ttgatgaaaa
atatggggga 1140atataatatt tataggagat acgatgacta gagtagagtt
gattactaga gaatttttta 1200agaagaatga agcaaccagt aaatattttc
agaagataga atcaagaaga ggtgaattat 1260ttattaaatt ctttatggat
aagttacttg cgcttatcct attattgcta ttatccccag 1320taatcattat
attagctatt tggataaaat tagatagtaa ggggccaatt ttttatcgcc
1380aagaacgtgt tacgagatat ggtcgaattt ttagaatatt taagtttaga
acaatgattt 1440ctgatgcgga taaagtcgga agtcttgtca cagtcggtca
agataatcgt attacgaaag 1500tcggtcacat tatcagaaaa tatcggctgg
acgaagtgcc ccaacttttt aatgttttaa 1560tgggggatat gagctttgta
ggtgtaagac cagaagtaca aaaatatgta aatcagtata 1620ctgatgaaat
gtttgcgacg ttacttttac ctgcaggaat tacttcacca gcgagtattg
1680catataagga tgaagatatt gttttagaag aatattgttc tcaaggctat
agtcctgatg 1740aagcatatgt tcaaaaagta ttaccagaaa aaatgaagta
caatttggaa tatatcagaa 1800actttggaat tatttctgat tttaaagtaa
tgattgatac agtaattaaa gtaataaaat 1860aggagattaa aatgacaaaa
agacaaaata ttccattttc accaccagat attacccaag 1920ctgaaattga
tgaagttatt gacacactaa aatctggttg gattacaaca ggaccaaaga
1980caaaagagct agaacgtcgg ctatcagtat ttacaggaac caataaaact
gtgtgtttaa 2040attctgctac tgcaggattg
gaactagtct tacgaattct tggtgttgga cccggagatg 2100aagttattgt
tcctgctatg acctatactg cctcatgtag tgtcattact catgtaggag
2160caactcctgt gatggttgat attcaaaaaa acagctttga gatggaatat
gatgctttgg 2220aaaaagcgat tactccgaaa acaaaagtta tcattcctgt
tgatctagct ggtattcctt 2280gtgattatga taagatttat accatcgtag
aaaacaaacg ctctttgtat gttgcttctg 2340ataataaatg gcagaaactt
tttgggcgag ttattatcct atctgatagt gcacactcac 2400taggtgctag
ttataaggga aaaccagcgg gttccctagc agattttacc tcattttctt
2460tccatgcagt taagaatttt acaactgctg aaggaggtag tgtgacatgg
agatcacatc 2520ctgatttgga tgacgaagag atgtataaag agtttcagat
ttactctctt catggtcaga 2580caaaggatgc attagctaag acacaattag
ggtcatggga atatgacatt gttattcctg 2640gttacaagtg taatatgaca
gatattatgg caggtatcgg tcttgtgcaa ttagaacgtt 2700acccatcttt
gttgaatcgt cgcagagaaa tcattgagaa atacaatgct ggctttgagg
2760ggacttcgat taagccgttg gtacacctga cggaagataa acaatcgtct
atgcacttgt 2820atatcacgca tctacaaggc tatactttag aacaacgaaa
tgaagtcatt caaaaaatgg 2880ctgaagcagg tattgcgtgc aatgttcact
acaaaccatt acctcttctc acagcctaca 2940agaatcttgg ttttgaaatg
aaagattttc cgaatgccta tcagtatttt gaaaatgaag 3000ttacactgcc
tcttcatacc aacttgagtg atgaagatgt ggagtatgtg atagaaatgt
3060ttttaaaaat tgttagtaga gattagttat tttggaagga gatatggtgg
aaagagatat 3120ggtggaaaga gacacgttgg tatctataat aatgccctcg
tggaatacag ctaagtatat 3180atctgaatca atccagtcag tgttggacca
aacacaccaa aattgggaac ttataatcgt 3240tgatgattgt tctaatgacg
aaactgaaaa agttgtttcg catttcaaag attcaagaat 3300aaagtttttt
aaaaattcga ataatttagg ggcagctcta acacgaaata aggcactaag
3360aaaagctaga ggtaggtgga ttgcgttctt ggattcagat gatttatggc
acccgagtaa 3420gctagaaaaa cagcttgaat ttatgaaaaa taatggatat
tcatttactt atcacaattt 3480tgaaaagatt gatgaatcta gtcagtcttt
acgtgtcctg gtgtcaggac cagcaattgt 3540gactagaaaa atgatgtaca
attacggcta tccagggtgt ttgactttca tgtatgatgc 3600agacaaaatg
ggtttaattc agataaaaga tataaagaaa aataacgatt atgcgatatt
3660acttcaattg tgtaagaagt atgactgtta tcttttaaat gaaagtttag
cttcgtatcg 3720aattagaaaa aaatcgat 373844238PRTStreptococcus
suismisc_featureCPS7E 44Ala Ala His Lys His Val Pro Leu Met Glu Tyr
Asn Pro His Glu Ala 1 5 10 15 Val Lys Asn Asn Ile Phe Gly Thr Lys
Asn Val Ala Glu Ala Ala Lys 20 25 30 Thr Ala Lys Val Ala Lys Phe
Val Met Val Ser Thr Asp Lys Ala Val 35 40 45 Asn Pro Pro Asn Val
Met Gly Ala Thr Lys Arg Val Ala Glu Met Ile 50 55 60 Val Thr Gly
Leu Asn Glu Pro Gly Gln Thr Gln Phe Ala Ala Val Arg 65 70 75 80 Phe
Gly Asn Val Leu Gly Ser Arg Gly Ser Val Val Pro Leu Phe Lys 85 90
95 Glu Gln Ile Arg Lys Gly Gly Pro Val Thr Val Thr Asp Phe Arg Met
100 105 110 Thr Arg Tyr Phe Met Thr Ile Pro Glu Ala Ser Arg Leu Val
Ile Gln 115 120 125 Ala Gly His Leu Ala Lys Gly Gly Glu Ile Phe Val
Leu Asp Met Gly 130 135 140 Glu Pro Val Gln Ile Leu Glu Leu Ala Arg
Lys Val Ile Leu Leu Ser 145 150 155 160 Gly His Thr Glu Glu Glu Ile
Gly Ile Val Glu Ser Gly Ile Arg Pro 165 170 175 Gly Glu Lys Leu Tyr
Glu Glu Leu Leu Ser Thr Glu Glu Arg Val Ser 180 185 190 Glu Gln Ile
His Glu Lys Ile Phe Val Gly Arg Val Thr Asn Lys Gln 195 200 205 Ser
Asp Ile Val Asn Ser Phe Ile Asn Gly Leu Leu Gln Lys Asp Arg 210 215
220 Asn Glu Leu Lys Asp Met Leu Ile Glu Phe Ala Lys Gln Glu 225 230
235 45232PRTStreptococcus suismisc_featureCPS7F 45Met Thr Arg Val
Glu Leu Ile Thr Arg Glu Phe Phe Lys Lys Asn Glu 1 5 10 15 Ala Thr
Ser Lys Tyr Phe Gln Lys Ile Glu Ser Arg Arg Gly Glu Leu 20 25 30
Phe Ile Lys Phe Phe Met Asp Lys Leu Leu Ala Leu Ile Leu Leu Leu 35
40 45 Leu Leu Ser Pro Val Ile Ile Ile Leu Ala Ile Trp Ile Lys Leu
Asp 50 55 60 Ser Lys Gly Pro Ile Phe Tyr Arg Gln Glu Arg Val Thr
Arg Tyr Gly 65 70 75 80 Arg Ile Phe Arg Ile Phe Lys Phe Arg Thr Met
Ile Ser Asp Ala Asp 85 90 95 Lys Val Gly Ser Leu Val Thr Val Gly
Gln Asp Asn Arg Ile Thr Lys 100 105 110 Val Gly His Ile Ile Arg Lys
Tyr Arg Leu Asp Glu Val Pro Gln Leu 115 120 125 Phe Asn Val Leu Met
Gly Asp Met Ser Phe Val Gly Val Arg Pro Glu 130 135 140 Val Gln Lys
Tyr Val Asn Gln Tyr Thr Asp Glu Met Phe Ala Thr Leu 145 150 155 160
Leu Leu Pro Ala Gly Ile Thr Ser Pro Ala Ser Ile Ala Tyr Lys Asp 165
170 175 Glu Asp Ile Val Leu Glu Glu Tyr Cys Ser Gln Gly Tyr Ser Pro
Asp 180 185 190 Glu Ala Tyr Val Gln Lys Val Leu Pro Glu Lys Met Lys
Tyr Asn Leu 195 200 205 Glu Tyr Ile Arg Asn Phe Gly Ile Ile Ser Asp
Phe Lys Val Met Ile 210 215 220 Asp Thr Val Ile Lys Val Ile Lys 225
230 46404PRTStreptococcus suismisc_featureCPS7G 46Met Thr Lys Arg
Gln Asn Ile Pro Phe Ser Pro Pro Asp Ile Thr Gln 1 5 10 15 Ala Glu
Ile Asp Glu Val Ile Asp Thr Leu Lys Ser Gly Trp Ile Thr 20 25 30
Thr Gly Pro Lys Thr Lys Glu Leu Glu Arg Arg Leu Ser Val Phe Thr 35
40 45 Gly Thr Asn Lys Thr Val Cys Leu Asn Ser Ala Thr Ala Gly Leu
Glu 50 55 60 Leu Val Leu Arg Ile Leu Gly Val Gly Pro Gly Asp Glu
Val Ile Val 65 70 75 80 Pro Ala Met Thr Tyr Thr Ala Ser Cys Ser Val
Ile Thr His Val Gly 85 90 95 Ala Thr Pro Val Met Val Asp Ile Gln
Lys Asn Ser Phe Glu Met Glu 100 105 110 Tyr Asp Ala Leu Glu Lys Ala
Ile Thr Pro Lys Thr Lys Val Ile Ile 115 120 125 Pro Val Asp Leu Ala
Gly Ile Pro Cys Asp Tyr Asp Lys Ile Tyr Thr 130 135 140 Ile Val Glu
Asn Lys Arg Ser Leu Tyr Val Ala Ser Asp Asn Lys Trp 145 150 155 160
Gln Lys Leu Phe Gly Arg Val Ile Ile Leu Ser Asp Ser Ala His Ser 165
170 175 Leu Gly Ala Ser Tyr Lys Gly Lys Pro Ala Gly Ser Leu Ala Asp
Phe 180 185 190 Thr Ser Phe Ser Phe His Ala Val Lys Asn Phe Thr Thr
Ala Glu Gly 195 200 205 Gly Ser Val Thr Trp Arg Ser His Pro Asp Leu
Asp Asp Glu Glu Met 210 215 220 Tyr Lys Glu Phe Gln Ile Tyr Ser Leu
His Gly Gln Thr Lys Asp Ala 225 230 235 240 Leu Ala Lys Thr Gln Leu
Gly Ser Trp Glu Tyr Asp Ile Val Ile Pro 245 250 255 Gly Tyr Lys Cys
Asn Met Thr Asp Ile Met Ala Gly Ile Gly Leu Val 260 265 270 Gln Leu
Glu Arg Tyr Pro Ser Leu Leu Asn Arg Arg Arg Glu Ile Ile 275 280 285
Glu Lys Tyr Asn Ala Gly Phe Glu Gly Thr Ser Ile Lys Pro Leu Val 290
295 300 His Leu Thr Glu Asp Lys Gln Ser Ser Met His Leu Tyr Ile Thr
His 305 310 315 320 Leu Gln Gly Tyr Thr Leu Glu Gln Arg Asn Glu Val
Ile Gln Lys Met 325 330 335 Ala Glu Ala Gly Ile Ala Cys Asn Val His
Tyr Lys Pro Leu Pro Leu 340 345 350 Leu Thr Ala Tyr Lys Asn Leu Gly
Phe Glu Met Lys Asp Phe Pro Asn 355 360 365 Ala Tyr Gln Tyr Phe Glu
Asn Glu Val Thr Leu Pro Leu His Thr Asn 370 375 380 Leu Ser Asp Glu
Asp Val Glu Tyr Val Ile Glu Met Phe Leu Lys Ile 385 390 395 400 Val
Ser Arg Asp 47210PRTStreptococcus suismisc_featureCPS7H 47Met Val
Glu Arg Asp Met Val Glu Arg Asp Thr Leu Val Ser Ile Ile 1 5 10 15
Met Pro Ser Trp Asn Thr Ala Lys Tyr Ile Ser Glu Ser Ile Gln Ser 20
25 30 Val Leu Asp Gln Thr His Gln Asn Trp Glu Leu Ile Ile Val Asp
Asp 35 40 45 Cys Ser Asn Asp Glu Thr Glu Lys Val Val Ser His Phe
Lys Asp Ser 50 55 60 Arg Ile Lys Phe Phe Lys Asn Ser Asn Asn Leu
Gly Ala Ala Leu Thr 65 70 75 80 Arg Asn Lys Ala Leu Arg Lys Ala Arg
Gly Arg Trp Ile Ala Phe Leu 85 90 95 Asp Ser Asp Asp Leu Trp His
Pro Ser Lys Leu Glu Lys Gln Leu Glu 100 105 110 Phe Met Lys Asn Asn
Gly Tyr Ser Phe Thr Tyr His Asn Phe Glu Lys 115 120 125 Ile Asp Glu
Ser Ser Gln Ser Leu Arg Val Leu Val Ser Gly Pro Ala 130 135 140 Ile
Val Thr Arg Lys Met Met Tyr Asn Tyr Gly Tyr Pro Gly Cys Leu 145 150
155 160 Thr Phe Met Tyr Asp Ala Asp Lys Met Gly Leu Ile Gln Ile Lys
Asp 165 170 175 Ile Lys Lys Asn Asn Asp Tyr Ala Ile Leu Leu Gln Leu
Cys Lys Lys 180 185 190 Tyr Asp Cys Tyr Leu Leu Asn Glu Ser Leu Ala
Ser Tyr Arg Ile Arg 195 200 205 Lys Lys 210 48101DNAStreptococcus
suismisc_feature(1)..(101)N may be any nucleotide 48aagggcacct
ctataaactc ccaaaattgc gaatttggag ttacgaaagc cttgttaaat 60caancatttt
aaattttaga aaattagttt ttagagctcc c 10149101DNAStreptococcus
suismisc_feature(1)..(101)N may be any nucleotide 49gggggcacct
ctataaattc ccaaaattgc gaatttggag ttacgaaagc cttgttaaat 60caancatctt
aaattttaga aaattagttt ttagaggtcc c 10150101DNAStreptococcus
suismisc_feature100 base pair repeat between CPS2O and CPS2P
50aagggcacct ctataaactc ccaaaattgc gaatttggag ttacgaaagc cttgttaaat
60caaacatttt aaattttaga aaattagttt ttagaggtcc c
10151120PRTStreptococcus suismisc_featureN terminal part of CPS2J
51Met Ala Lys Val Ser Ile Ile Val Pro Ile Phe Asn Thr Glu Lys Tyr 1
5 10 15 Leu Arg Glu Cys Leu Asp Ser Ile Ile Ser Gln Ser Tyr Thr Asn
Leu 20 25 30 Glu Ile Leu Leu Ile Asp Asp Gly Ser Ser Asp Ser Ser
Thr Asp Ile 35 40 45 Cys Leu Glu Tyr Ala Glu Gln Asp Gly Arg Ile
Lys Leu Phe Arg Leu 50 55 60 Pro Asn Gly Gly Val Ser Asn Ala Arg
Asn Tyr Gly Ile Lys Asn Ser 65 70 75 80 Thr Ala Asn Tyr Ile Met Phe
Val Asp Ser Asp Asp Ile Val Asp Gly 85 90 95 Asn Ile Val Glu Ser
Leu Tyr Thr Cys Leu Lys Glu Asn Asp Ser Asp 100 105 110 Leu Ser Gly
Gly Leu Leu Ala Thr 115 120 52120PRTStreptococcus suismisc_featureN
terminal part of CPS2K 52Met Ile Asn Ile Ser Ile Ile Val Pro Ile
Tyr Asn Val Glu Gln Tyr 1 5 10 15 Leu Ser Lys Cys Ile Asn Ser Ile
Val Asn Gln Thr Tyr Lys His Ile 20 25 30 Glu Leu Leu Val Asn Asp
Gly Ser Ser Thr Asp Asn Ser Glu Glu Ile 35 40 45 Cys Leu Ala Tyr
Ala Lys Lys Asp Ser Arg Ile Arg Tyr Phe Lys Lys 50 55 60 Glu Asn
Gly Gly Leu Ser Asp Ala Arg Asn Tyr Gly Ile Ser Arg Ala 65 70 75 80
Lys Gly Asp Tyr Leu Ala Phe Ile Asp Ser Asp Asp Phe Ile His Ser 85
90 95 Glu Phe Ile Gln Arg Leu Xaa His Glu Ala Ile Glu Arg Glu Asn
Ala 100 105 110 Leu Xaa Xaa Val Ala Val Ala Gly 115 120
53419PRTStreptococcus suismisc_featureORF2Y 53Met Lys Lys Tyr Gln
Val Ile Ile Gln Asp Ile Leu Thr Gly Ile Glu 1 5 10 15 Glu His Arg
Phe Lys Arg Gly Glu Lys Leu Pro Ser Ile Arg Gln Leu 20 25 30 Arg
Glu Gln Tyr His Cys Ser Lys Asp Thr Val Gln Lys Ala Met Leu 35 40
45 Glu Leu Lys Tyr Gln Asn Lys Ile Tyr Ala Val Glu Lys Ser Gly Tyr
50 55 60 Tyr Ile Leu Glu Asp Arg Asp Phe Gln Asp His Thr Cys Arg
Ala Gln 65 70 75 80 Ser Tyr Arg Leu Ser Arg Ile Thr Tyr Glu Asp Phe
Arg Ile Cys Leu 85 90 95 Lys Glu Ser Leu Ile Gly Arg Glu Asn Tyr
Leu Phe Asn Tyr Tyr His 100 105 110 Gln Gln Glu Gly Leu Ala Glu Leu
Ile Ser Ser Val Gln Ser Leu Leu 115 120 125 Met Asp Tyr His Val Tyr
Thr Lys Lys Asp Gln Leu Val Ile Thr Ala 130 135 140 Gly Ser Gln Gln
Ala Leu Tyr Ile Leu Thr Gln Met Glu Thr Leu Ala 145 150 155 160 Gly
Lys Thr Glu Ile Leu Ile Glu Asn Pro Thr Tyr Ser Arg Met Ile 165 170
175 Glu Leu Ile Arg His Gln Gly Ile Pro Tyr Gln Thr Ile Glu Arg Asn
180 185 190 Leu Asp Gly Ile Asp Leu Glu Glu Leu Glu Ser Ile Phe Gln
Thr Gly 195 200 205 Lys Ile Lys Phe Phe Tyr Thr Ile Pro Arg Leu His
Asn Pro Leu Gly 210 215 220 Ser Thr Tyr Asp Ile Ala Thr Lys Thr Ala
Ile Val Lys Leu Ala Lys 225 230 235 240 Gln Tyr Asp Val Tyr Ile Ile
Glu Asp Asp Tyr Leu Ala Asp Phe Asp 245 250 255 Ser Ser His Ser Leu
Pro Leu His Tyr Leu Asp Thr Asp Asn Arg Val 260 265 270 Ile Tyr Ile
Lys Ser Phe Thr Pro Thr Leu Phe Pro Ala Leu Arg Ile 275 280 285 Gly
Ala Ile Ser Leu Pro Asn Gln Leu Arg Asp Ile Phe Ile Lys His 290 295
300 Lys Ser Leu Ile Asp Tyr Asp Thr Asn Leu Ile Met Gln Lys Ala Leu
305 310 315 320 Ser Leu Tyr Ile Asp Asn Gly Met Phe Ala Arg Asn Thr
Gln His Leu 325 330 335 His His Ile Tyr His Ala Gln Trp Asn Lys Ile
Lys Asp Cys Leu Glu 340 345 350 Lys Tyr Ala Leu Asn Ile Pro Tyr Arg
Ile Pro Lys Gly Ser Val Thr 355 360 365 Phe Gln Leu Ser Lys Gly Ile
Leu Ser Pro Ser Ile Gln His Met Phe 370 375 380 Gly Lys Cys Tyr Tyr
Phe Ser Gly Gln Lys Ala Asp Phe Leu Gln Ile 385 390 395 400 Phe Phe
Glu Gln Asp Phe Ala Asp Lys Leu Glu Gln Phe Val Arg Tyr 405 410 415
Leu Asn Glu
* * * * *
References