U.S. patent application number 09/805681 was filed with the patent office on 2002-10-03 for gene conferring resistance to the antibacterial 4,5-dihydroxy-2-cyclopente- n-1-one (dhcp), the protein encoded by same, and applications thereof.
This patent application is currently assigned to The University of Medicine and Dentistry of New Jersey. Invention is credited to Inouye, Masayori, Kato, Ikunoshin, Phadtare, Sangita, Yamanaka, Kunitoshi.
Application Number | 20020143163 09/805681 |
Document ID | / |
Family ID | 22858353 |
Filed Date | 2002-10-03 |
United States Patent
Application |
20020143163 |
Kind Code |
A1 |
Phadtare, Sangita ; et
al. |
October 3, 2002 |
Gene conferring resistance to the antibacterial
4,5-dihydroxy-2-cyclopente- n-1-one (DHCP), the protein encoded by
same, and applications thereof
Abstract
The invention relates to a gene, dep, which confers resistance
to the antibacterial activity of 4,5-dihydroxy-2-cyclopenten-1-one
(DHCP). The invention further relates to the putative protein
encoded by dep, which is a hydrophobic, transmembrane efflux
protein specific for DHCP. The invention further relates to
plasmids containing the dep gene, and to bacterial cells expressing
dep. Furthermore, the invention provides applications for use in
conferring resistance to antibacterial activity in organisms. The
dep gene can be used to identify compounds which inhibit the efflux
activity responsible for the resistance to DHCP or to compounds
which are functionally equivalent to DHCP.
Inventors: |
Phadtare, Sangita; (Highland
Park, NJ) ; Yamanaka, Kunitoshi; (Highland Park,
NJ) ; Kato, Ikunoshin; (Kyoto, JP) ; Inouye,
Masayori; (Piscataway, NJ) |
Correspondence
Address: |
SCHNADER HARRISON SEGAL & LEWIS, LLP
1600 MARKET STREET
SUITE 3600
PHILADELPHIA
PA
19103
|
Assignee: |
The University of Medicine and
Dentistry of New Jersey
|
Family ID: |
22858353 |
Appl. No.: |
09/805681 |
Filed: |
March 14, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60228727 |
Aug 29, 2000 |
|
|
|
Current U.S.
Class: |
536/23.1 ;
530/350 |
Current CPC
Class: |
C07K 14/245 20130101;
Y02A 50/30 20180101; A61P 31/00 20180101 |
Class at
Publication: |
536/23.1 ;
530/350 |
International
Class: |
C07H 021/02; C07H
021/04; C07K 001/00; C07K 014/00; C07K 017/00 |
Claims
We claim:
1. A DHCP efflux protein, which is specific for
4,5-dihydroxy-2-cyclopente- n-1-one (DHCP).
2. The protein of claim 1, which is a transmembrane protein that
forms a cytoplasmic channel specific for efflux transport of
DHCP.
3. The protein of claim 1, which confers resistance to DHCP.
4. The protein of claim 1, which protein is from E. coli.
5. The protein of claim 1, which protein does not confer
cross-resistance to the any of the following antibiotics:
chloramphenicol, spectinomycin and tetracycline.
6. The protein of claim 1, which protein possesses 13 predicted
transmembrane-spanning .alpha.-helices.
7. A gene encoding dep, the DHCP efflux protein.
8. The dep gene of claim 7, wherein the dep gene is from E.
coli.
9. The gene of claim 7, wherein said gene confers resistance to
DHCP or a functionally equivalent compound when present in multiple
copies in a bacterial cell.
10. A plasmid comprising the dep gene, which plasmid confers
expression of multiple copies of the dep gene in bacteria cells
that have been transformed with said plasmid.
11. The plasmid of claim 10, which plasmid confers resistance to
DHCP and does not confer cross-resistance to any of the following
antibiotics: chloramphenicol, spectinomycin and tetracycline.
12. Bacteria cells containing multiple copies of the plasmid of
claim 10.
13. The bacteria cells of claim 12, which bacteria cells are
resistant to DHCP.
14. A method which uses the gene of claim 7 to identify a compound
which inhibits efflux activity responsible for resistance to DHCP
or a functionally equivalent compound.
Description
[0001] This patent application claims the benefit of U.S.
Provisional Application No. 60/228,727, filed Aug. 29, 2000. This
earlier provisional application is hereby incorporated by
reference.
[0002] In spite of a number of antibiotics available against a
variety of bacteria, due to emergence of multiple drug resistant
strains, the search for newer and more effective antibacterial
compounds has continued. 4,5-dihydroxy-2-cyclopenten-1-one (DHCP)
(see FIG. 1) is a compound having antibacterial activity against a
variety of gram-positive and -negative bacteria, such as
Escherichia coli, Bacillus, Salmonella, Staphylococcus etc. The
process for manufacture and the properties of DHCP have been
patented (Koyama et al., 1999). It is prepared by the
heat-treatment of uronic acid or its derivatives, wherein uronic
acid is galacturonic acid, glucuronic acid, mannuronic acid or
iduronic acid. It is also produced from roasted or parched
vegetables, fruits, cereals, mushrooms, sea algae, cortex or
cartilage. It has been demonstrated that this compound induces
cancer cell differentiation and apoptosis. It has potential
application as therapeutic or preventive agent against cancer and
also as an antibacterial agent in antiseptics, dentrifices,
cosmetics and bathing agents (Koyama et al., 1999).
[0003] We have isolated a multicopy suppressor from an E. coli
genomic library for the DHCP toxicity. The putative protein encoded
by this gene showed high homology to known efflux proteins
conferring resistance to a number of antibiotics including
chloramphenicol, bicyclomycin and tetracycline. The gene was mapped
at 37.5 min on the E. coli chromosome. It is designated as dep for
DHCP efflux protein. However, the Dep protein does not confer
cross-resistance to any of the antibiotics tested.
PRIOR ART REFERENCES
[0004] U.S. Pat. No. 6,087,401 to Koyama, et al. Cyclopentones,
process for preparing same, and the use thereof.
[0005] This patent discloses a method of manufacturing
4,5-dihydroxy-2-cyclopenten-1-one (DHCP). It also describes the
antibacterial activity of DHCP.
[0006] In contrast, the invention disclosed in the present
provisional application relates to a gene, dep, that, when present
in multiple copies in bacterial cells, confers resistance to the
antibacterial activity of DHCP, thus rendering the bacteria
resistant to killing by DHCP. The present application also
describes the protein encoded by the dep gene.
[0007] European Patent EP 0 941 981 A1 to Koyama, et al.
Cyclopentones, process for preparing same, and the use thereof.
[0008] This patent application relates to essentially the same
subject matter as that described in U.S. Pat. No. 6,087,401 to
Koyama, et al.
[0009] U.S. Pat. No. 6,111,145 to Kobayashi et al. Cyclopentenone
derivative.
[0010] This patent relates to functionally equivalent ether
derivatives of DHCP and discloses the biological activity of these
derivatives.
[0011] European Patent Publication EP 1 000 923 A1 to Kobayashi et
al. Cyclopentenone derivatives.
[0012] This patent application relates to essentially the same
subject matter as that described in U.S. Pat. No. 6,111,145 to
Kobayashi et al.
[0013] U.S. Pat. No. 6,136,854 to Koyama et al. Cyclopentenone
derivative.
[0014] This patent relates to functionally equivalent ester
derivatives of DHCP and discloses the biological activity of these
derivatives.
[0015] European Patent Publication EP 0 976 717 A1 to Koyama et al.
Cyclopentenone derivatives.
[0016] This patent application relates to essentially the same
subject matter as that described in U.S. Pat. No. 6,136,854 to
Koyama et al.
[0017] Clinical significance of P-glycoprotein expression and
function for response to induction chemotherapy, relapse rate and
overall survival in acute leukemia. C. Wuchter, et al.
Haematologica 85(7):711-21 (2000).
[0018] In acute leukemia, a multidrug-resistance (MDR) phenotype
mediated by P-glycoprotein (P-gp) contributes to chemotherapy
failure. This study investigated whether P-gp expression levels or
functional P-gp activity was a better predictor of response to
induction chemotherapy, relapse rate and overall survival in acute
leukemia. The data demonstrated that the functional rhodamine-123-
( rh123)-efflux assay was preferred over P-gp expression analysis
by monoclonal antibodies in acute leukemia.
[0019] Increased drug delivery to the brain by P-glycoprotein
inhibition. A. J. Sadeque, et al. Clinical Pharmacology &
Therapeutics 68(3):231-7 (2000).
[0020] In vitro studies had demonstrated that the antidiarrheal
drug loperamide is a substrate for the efflux membrane transporter
P-glycoprotein. Although loperamide is a potent opiate drug, it
does not opioid central nervous system effects, such as respiratory
depression, when given to patients at usual doses. This study
tested the hypothesis that inhibition of P-glycoprotein with
quinidine would increase the entry of loperamide into the central
nervous system, thus causing respiratory depression. The results
demonstrated that although loperamide produced no respiratory
depression when used alone, respiratory depression was seen when
loperamide was administered with quinidine.
[0021] Expression of the multidrug-resistance-associated protein in
myelodysplastic syndromes. S. Poulain, et al. British Journal of
Haematology 110(3):591-8 (2000).
[0022] In myelodysplastic syndromes (MDS), P-glycoprotein (P-gp)
expression is associated with drug resistance, while the clinical
significance of the multidrug resistance-associated protein (MRP1)
is unclear. In this study of bone marrow from patients with MDS,
expression of MRP1 was correlated with disease stage in MDS. With
respect to P-gp, discordant expression/function of MRP1 was found
in some cases, suggesting the existence of nonfunctional transport
proteins in MDS. MRP1 expression did not appear to be a prognostic
factor in MDS.
[0023] Soft tissue leiomyosarcomas and malignant gastrointestinal
stromal tumors: differences in clinical outcome and expression of
multidrug resistance proteins. B. E. Plaat, et al. Journal of
Clinical Oncology 18(18):3211-20 (2000).
[0024] In this study, parameters associated with multidrug
resistance (MDR) were compared between soft tissue leiomyosarcomas
(LMS) and malignant gastrointestinal stromal tumors (GIST).
Immunohistochemistry was used to detect P-glycoprotein (P-gp),
multidrug resistance protein (MRP(1)), lung resistance protein
(LRP), and c-kit. The results demonstrate that LMS patients have
better survival rates compared to GIST patients, and the pattern of
metastasis differs between the two patient groups. The expression
of the MDR proteins tested is less pronounced in LMS than in
GIST.
[0025] Quorum sensing in Escherichia coli, Salmonella typhimurium,
and Vibrio harveyi: A new family of genes responsible for
autoinducer production. M. G. Surette, et al. Proc. Natl. Acad.
Sci. 96:1639-44 (1999).
[0026] In bacteria, the regulation of gene expression in response
to changes in cell density, called quorum sensing, is dependent on
hormone-like molecules known as autoinducers that are produced by
the bacteria and accumulate in the external environment as the
bacterial cell population increases. The marine bacterium Vibrio
harveyi has been shown to have two parallel quorum sensing systems,
each composed of a sensor-autoinducer pair. The two different
autoinducers belonging to each system have been termed autoinducer
1 (AI-1) and autoinducer 2 (AI-2). The identification and analysis
of the genes responsible for AI-2 production in E. coli, S.
typhimurium, and V. harveyi is reported.
[0027] Quorum sensing in Vibrio fischeri: Probing autoinducer-LuxR
interactions with autoinducer analogs. A. L. Schaefer, et al.
Journal of Bacteriology 178:2897-2901 (1996).
[0028] In Vibrio fischeri, luminescence genes are activated by the
transcription factor LuxR in combination with a diffusible signal
compound known as the autoinducer. This study analyzed the ability
of a number of autoinducer analogs to interact with LuxR.
[0029] Regulation of quorum sensing in Vibrio harveyi by LuxO and
Sigma-54. B. N. Lilley and B. L. Bassler. Molecular Microbiology
36(4):940-954 (2000).
[0030] The bioluminescent marine bacterium Vibrio harveyi controls
light production (lux) by a quorum-sensing circuit. This study
demonstrates that the response regulator protein LuxO functions as
an activator protein via interaction with the alternative sigma
factor, .sigma..sup.54. Since LuxO is responsible for repression of
the luciferase structural operon (luxCDABEGH), these results
suggest that LuxO, together with .sigma..sup.54, functions to
activate a negative regulator of luminescence.
[0031] Bentley, J., Hyatt, L. S., Ainley, K., Parish, J. H.,
Herbert, R. B., and White, G. R. 1993. Cloning and sequence
analysis of an Escherichia coli gene conferring bicyclomycin
resistance. Gene 127: 117-120.
[0032] Berlyn, M. K. B., Low, K. B., and Rudd. K. E. 1996. Linkage
map of Escherichia coli K-12, Ed. 9. Pages 1715-1902. In Neidhardt,
F. C., Curtiss III, R., Ingraham, J. L., Lin, E.C.C., et al. (ed)
Escherichia coli and Salmonella. Cellular and Molecular Biology,
Vol. 2, 2nd Ed., ASM Press, Washington DC.
[0033] Desomer, J., Vereecke, D., Crespi, M., and Van Montagu, M.
1992. The plasmid-encoded chloramphenicol-resistance protein of
Rhodococcus fascians is homologous to the transmembrane
tetracycline efflux proteins. Mol. Microbiol. 6: 2377-2385.
[0034] Dittrich, W., Betzler, M., and Schrempf, H. 1991. An
amplifiable and deletable chloramphenicol-resistance determinant of
Streptomyces lividans 1326 encodes a putative transmembrane
protein. Mol. Microbiol. 5:2789-2797.
[0035] Hiraga S., Niki, H., Ogura, T., Ichinose, C., Mori, H.,
Ezaki, B., and Jaffe, A. 1989. Chromosome partitioning in
Escherichia coli: novel mutants producing anucleate cells. J.
Bacteriol. 171:1496-505.
[0036] Koyama, N., Sagawa, H., Kobayashi, E., Enoki, T., Wu, H-K.,
Nishiyama, E., Ikai, K., and Kato, I. 1999. Cyclopentanones,
process for preparing the same, and the use thereof. European
Patent (EP 0941 981 A1, date of publication: Sep. 15, 1999).
[0037] Lu Q., and Inouye, M. 1998. The gene for 16S rRNA
methyltransferase (ksgA) functions as a multicopy suppressor for a
cold-sensitive mutant of Era, an essential RAS-like GTP-binding
protein in Escherichia coli. J. Bacteriol. 180:5243-5246.
[0038] Mosher, R. H., Camp, D. J., Yang, K., Brown, M. P., Shawl,
W. V., and Vining, L. C. 1995. Inactivation of chloramphenicol by
O-phosphorylation. J. Biol. Chem. 270:27000-27006.
[0039] Nagy, I., Schoofs, G., Vanderleyden, J., and De Mot, R.
1997. Transposition of the IS21-related element IS1415 in
Rhodococcus erythropolis. J. Bacteriol. 179:4635-4638.
[0040] Ohki, R., and Murata, M. 1997. bmr3, a third multidrug
transporter gene of Bacillus subtilis. J. Bacteriol.
179:1423-1427.
[0041] Rouch, D. A., Cram, D. S., DiBerardino, D., Littlejohn, T.
G., and Skurray, R. A. 1990. Efflux-mediated antiseptic resistance
gene qacA from Staphylococcus aureus: common ancestry with
tetracycline- and sugar-transport proteins. Mol. Microbiol.
4:2051-2062.
[0042] Schwarz, S., Cardoso, M., and Wegener, H. C. 1992.
Nucleotide sequence and phylogeny of the tet(L) tetracycline
resistance determinant encoded by plasmid pSTE1 from Staphylococcus
hyicus. Antimicrob. Agents Chemother. 36:580-588.
[0043] Yanisch-Perron, C., Vieira, J., and Messing, J. 1985.
Improved M13 phage cloning vectors and host strains: Nucleotide
sequences of the M13mp18 and pUC19 vectors. Gene 33: 103-119.
[0044] All references cited herein are incorporated by
reference.
BRIEF DESCRIPTION OF THE DRAWINGS
[0045] FIG. 1 is the chemical structure of
4,5-dihydroxy-2-cyclopenten- 1 -one (DHCP).
[0046] FIG. 2A is a graphical representation of the effect of DHCP
concentration on the growth of E. coli.
[0047] FIG. 2B is a graphical representation of the effect of DHCP
concentration on the survival of E. coli.
[0048] FIG. 3 is a restriction mapping of the plasmid pSP001
showing the DNA fragments conferring resistance to DHCP.
[0049] FIG. 4 is a comparison of the amino acid sequence of the
polypeptide encoded by dep with the proteins encoded by cmr, cmrA,
cmx, cmlv, bcr, bmr3, yjcC, and tet.
[0050] FIG. 5 is a comparison of the hydropathic profiles of the
putative proteins encoded by dep, cmr, and cml.
[0051] FIG. 6 is a nucleotide sequence showing the DNA sequence of
a region of the E. coli genome containing the sequence of the dep
gene.
[0052] FIG. 7 is a nucleotide sequence showing the isolated DNA
sequence of the dep gene.
BRIEF DESCRIPTION OF THE INVENTION
[0053] The provisional application describes the cloning of a gene
encoding a transmembrane protein from E. coli. This protein, when
expressed from a multi-copy plasmid, functions to transport
4,5-dihydroxy-2-cyclopenten-1-one (DHCP) out of the cell.
[0054] DHCP and functionally equivalent compounds are represented
by the formulas [I] and [II] and include optically active compounds
thereof. In Formula I, R.sup.1 and R.sup.2 are the same or
different and each of them is hydrogen, a straight or branched
alkyl group, a straight or branched alkenyl group, an aromatic
group, an aromatic-aliphatic group, with the proviso that R.sup.1=a
benzyl group and R.sup.2=H is excluded.
[0055] See References 3 and 4. R.sup.3-R.sup.6 are independently
hydrogen or an alkyl group, preferably a lower alkyl group such as
a C.sub.1-C.sub.6 alkyl. 1
[0056] In Formula II, R.sup.1 and R.sup.2 are the same or different
and each of them is hydrogen, a straight or branched alkyl group, a
straight or branched alkenyl group, an aromatic group, an
aromatic-aliphatic group, with the proviso that the case where
R.sup.1=R.sup.2=CH.sub.3 is excluded. See References 5 and 6.
R.sup.3-R.sup.6 are independently hydrogen or an alkyl group,
preferably a lower alkyl group such as a C.sub.1-C.sub.6 alkyl.
[0057] DHCP is shown to possess anti-bacterial activity; it
inhibits cell growth at a concentration of 350 .mu.M or higher. At
lower concentrations, it causes cells to elongate and grow poorly.
To determine if E. coli is naturally resistant to DHCP, a library
of E. coli genomic DNA fragments was transformed into strain JM83
and grown on agar plates containing 400 .mu.M DHCP. Colonies that
were capable of growing on this medium were isolated. DNA was
isolated from these colonies to identify and sequence the cloned
genomic fragment that specified resistance. Four genes were found
in the fragment that conferred resistance. Inactivation of various
combinations of these four genes led to the conclusion that ORF389
was responsible for conferring resistance. This was confirmed by
cloning ORF389 by itself into pUC19 (a multi-copy plasmid) and
transforming strain JM83. The resultant cells were resistant to
DHCP.
[0058] Comparison of the nucleotide sequence of ORF389 with the E.
coli gene database showed that it was similar to known efflux
proteins involved in conferring resistance to chloramphenicol and
other antibiotics. Further analysis of the predicted structure of
the protein encoded by ORF389 suggested that it was a membrane
protein; it possesses multiple transmembrane domains and shares
structural similarity with the aforementioned chloramphenicol
efflux polypeptides.
[0059] To determine if ORF389 was capable of conferring resistance
to other antibiotics such as chloramphenicol, spectinomycin, and
tetracycline, the transformed JM83 cells containing the
pUC19/ORF389 plasmid were plated on media containing these
antibiotics. The presence of ORF389 failed to confer resistance to
any antibiotic other than DHCP, suggesting that the efflux activity
of the Dep protein is specific for DHCP.
[0060] It is important to note that ORF389 confers resistance to
DHCP only when it is present in multiple copies in the cell. The
gene is naturally found in the genome of E. coli cells, but it is
present in single copy. Such cells are susceptible to the
antimicrobial activity of DHCP. When ORF389 is cloned into pUC19
and introduced into JM83 cells, it is present in multiple copies
(up to several hundred copies of the gene per cell), since pUC19 is
maintained in up to several hundred copies per cell. Only when the
gene dosage is increased, is resistance to DHCP found. The
mechanism of resistance is simply increased efflux activity arising
from the increased expression of the efflux protein in the
transformed cells.
[0061] It should be noted that due to the degeneneracy of the
genetic code, the nucleotide sequence encoding an efflux protein
that is responsible for conferring resistance to DHCP or a compound
functionally equivalent to DHCP may vary from the nucleic acid
sequence disclosed herein.
Additional Description OF THE Invention
Mode of Action
[0062] DHCP is a compound that exhibits antimicrobial and
anti-tumor activity. It is made by heating various uronic acids
(e.g., glucuronic acid, galacturonic acid, mannuronic acid). The
inventors have cloned a gene from E. coli that encodes a protein
which is capable of transporting DHCP out of the cell. This
transport protein shows sequence similarity with known efflux
proteins that function to transport antibiotics such as
chloramphenicol out of the cell. It has been shown that organisms
which overexpress the transport protein become resistant to DHCP,
probably because they are able to efficiently transport DHCP.
Overexpression of the transport protein arises from the presence of
multiple copies of the gene, rather than increased expression from
the endogenous gene in E. coli. In other words, all E. coli possess
a single copy of the transport gene. However, the level of
transport protein expression from a single copy of the gene is
insufficient to confer resistance to DHCP. The inventors have
cloned the gene into a high copy number plasmid, pUC19, which is
maintained in E. coli cells at 200-500 copies per cell. Thus,
transformed E. coli containing this plasmid construct will possess
200-500 copies of the transport gene, and protein expression from
multiple copies is greater than from a single copy. These
transformed cells are resistant to DHCP.
[0063] The general mode of action of DHCP requires that it enter
the target cell. Resistance to DHCP can occur if DHCP is
transported out of the cell as fast as or faster than it enters the
cell. Given that, the concentration of DHCP within the cell can
never accumulate to a toxic dose and the cell is resistant to the
antimicrobial effects of the compound. Apparently, the transport
protein encoded by gene disclosed does not transport DHCP very
efficiently, or the amount of transport protein expressed from the
endogenous gene is very low. In either case, the presence of more
transport protein (arising from many copies of the gene) will
result in more efficient transfer of DHCP out of the cell.
Applications
[0064] An important application of the gene of the invention will
be its use in studies to identify inhibitors of efflux activity.
Such inhibitory compounds will function to block the transport
activity. Thus a microbe or a tumor cell that is resistant to DHCP
can be made to be more sensitive to the compound by preventing the
resistant cell from transporting the compound back out. It is also
conceivable that inhibitors of the transport gene of the invention
may also be active in blocking transport of other efflux proteins
such as the efflux proteins that transport chloramphenicol, or the
P glycoprotein family of multiple drug resistant proteins. The P
glycoproteins are expressed in many tumor cells, making these
tumors resistant to chemotherapy agents. Abstracts regarding
studies of P glycoproteins are referenced above.
DETAILED DESCRIPTION OF THE DRAWINGS
[0065] FIG. 1 is the chemical structure of
4,5-dihydroxy-2-cyclopenten-1-o- ne (DHCP).
[0066] FIG. 2 Effect of DHCP concentrations on the growth of E.
coli.
[0067] A. The JM83 cells were grown in LB medium up to Klett unit
of 50 and DHCP was added at various concentrations (0-400 .mu.M).
After the growth reached to Klett unit of 90-100, cells were
diluted with medium containing respective concentrations of DHCP
and growth was further monitored. DHCP concentration: 0 .mu.M, open
squares; 50 .mu.M, closed diamonds; 100 .mu.M, open circles; 250
.mu.M, open diamonds; 400 .mu.M closed squares.
[0068] B. Overnight grown cells of E. Coli JM83 were diluted
appropriately and plated on LB plates containing different
concentrations of DHCP (0-350 .mu.M). The number of colonies on the
plate without DHCP was taken as 100% and the other numbers were
expressed as relative percentages.
[0069] FIG. 3 Restriction mapping of the plasmid pSP001 conferring
resistance to DHCP. Four ORFs comprising the DNA fragment (5.2 kb)
conferring resistance to DHCP and the flanking ORFs are shown. The
orientation of each ORF is marked with an arrow. The restriction
enzyme sites are also shown. The ORFs are not drawn to scale. The
plasmid pSP001 containing the DNA fragment conferring resistance to
DHCP was digested with restriction enzymes to disrupt each of four
ORFs, religated and transformed into JM83 cells. The transformants
were then examined for their sensitivity to DHCP (400 .mu.M). The
enzymes used for digestion were: forpurR: MluI for ydhB;
NruI-Eco47III, for ORF389, purR , and ydhB: NruI and SmaI, for
ORF389: Aval and forpurR and ydhB: MluI and NruI. For construction
of plasmid with ORF389 (dep), the plasmid pSP001 was digested with
SmaI and MscI, the fragment was purified and cloned into pUC19 to
yield plasmid pSP007.
[0070] FIG. 4 The sequence homology between Dep, Cmr, CmrA, Cmx,
CmlV, BcR, Bmr3, YjcC and Tet. Identical and similar sequences are
marked with black and gray boxes, respectively. The consensus
sequences for transmembrane proteins are marked with dotted lines
and are represented as I, II, and III stretches.
[0071] FIG. 5 Hydropathic profiles of Dep (A), Cmr from
Rhodococcusfaciens (B) (6) and Cml from Streptomyces lividans (C)
(8). Horizontal bars indicate predicted transmembrane regions.
[0072] FIG. 6 is a nucleotide sequence showing the DNA sequence of
a region of the E. coli genome containing the sequence of the dep
gene. This region of the E. coli genome is available at Accession
No. AE000261 U00096. The sequence shown is that of nucleotides
4381-8280. The dep gene is encoded by nucleotides 4627-5838. The
dep sequence is shown in brackets.
[0073] FIG. 7 is a nucleotide sequence showing the isolated DNA
sequence of the dep gene. The plasmid pSP007 was confirmed to
contain the dep gene by obtaining DNA sequence data from one end of
the 1.7 kb insert. Sequence data obtained in this manner matched
the first.
Detailed Description of Experimental Work
Effect of DHCP on the Growth of E. coli
[0074] The E. coli wild-type strain JM83 [F.sup.-ara.DELTA.
(lac-proAB) rpsL(str')](Yanisch-Perron et al., 1985) was grown in
Luria broth (LB). Media were supplemented with ampicillin (final
concentration of 50 .mu.g/ml) whenever required. To check the
effect of DHCP on the growth of E. coli, cells grown overnight in
LB medium were diluted into fresh LB medium. After the growth
reached to the Klett unit of 50, DHCP was added at various
concentrations (0-400 .mu.M) and growth was further monitored.
After it reached to the Klett unit of 90-100, it was diluted
10-fold into media containing respective concentrations of DHCP.
FIG. 2A shows the effect of different concentrations of DHCP on E.
coli. The growth was slowed after 3 h of incubation in the presence
of 50 .mu.M DHCP, but it reached the maximum density after 8 h,
similar to that without DHCP. The cells grew more slowly after 3 h
incubation with 100 .mu.M of DHCP and the maximum cell density was
lower than that without DHCP. In the presence of 250 .mu.M DHCP,
growth was severely impaired after 3 h of incubation and cells
stopped growing after 5 h. In the presence of 400 .mu.M DHCP, cell
growth stopped after 4 h of incubation. Microscopic examination of
the cells grown with 250 .mu.M DHCP for 8 h showed that the cells
were elongated forming filaments, which are approximately 15-fold
longer than the control cells. DAPI (diamidino phenylindole)(Hiraga
et al., 1989) staining of these cells showed that the chromosomal
condensation of the cells might be impaired by DHCP (data not
shown).
[0075] To check the colony formation ability of E. coli at various
concentrations of DHCP, cells grown overnight in LB medium were
diluted appropriately and plated on LB plates containing DHCP
(0-350 .mu.M). After incubation at 37.degree. C, the number of
colonies on each plate were counted. The number of colonies on the
control plate without DHCP was taken as 100% and the other numbers
were expressed as relative percentages (FIG. 2B). In the presence
of 300 .mu.M DHCP, 100-fold decrease in the colony numbers was
observed. When 1.times.10.sup.4 cells were plated on LB medium
containing 350 .mu.M DHCP, no colonies were obtained.
Screening of an E. coli Genomic Library for Genes Conferring
Resistance to DHCP
[0076] In order to examine if E. coli contains a gene(s) that
confers resistance to DHCP, the E. coli genomic library was
screened. The construction of E. coli genomic library was described
previously (Lu and Inouye, 1998). The partially digested Sau3AI
chromosomal DNA fragments from E. coli JM83 were cloned into the
BamHI site of pUC19. The JM83 cells were transformed with the
genomic library. Transformants were isolated for their ability to
grow on DHCP (400 .mu.M) containing LB plates at 37.degree. C.
Plasmid DNA was isolated from the resistant colonies, purified and
retransformed into JM83 cells to confirm its ability to confer
resistance to DHCP. The plasmid was designated as pSP001 and was
found to contain a 5.2-kb DNA fragment. This fragment was sequenced
from both ends using Sequenase and BLAST search was carried out for
the analysis of homology of this fragment with the entire E. coli
genome. It was found that this DNA fragment is located at 37.5 min
on the E. coli chromosome and contains four ORFs (FIG. 3): ORF389,
purR encoding purine synthesis repressor, ydhB encoding a homologue
of the cyn operon transcriptional activator and ydhC encoding a
homologue of bicyclomycin resistance protein (Berlyn et al.,
1996).
[0077] To determine which gene is responsible for conferring
resistance to DHCP, several deletion constructs were prepared as
shown in FIG. 3. Disruption of purR, ydhB and both purR and ydhB
had no effect on the resistance to DHCP (constructs pSP002, pSP003
and pSP006, respectively). However, disruption of ORF389 with purR
and ydhB (pSP004) as well as disruption of ORF389 alone (pSP005)
resulted in loss of DHCP resistance. We thus cloned ORF389
separately in pUC19 (pSP007), transformed the resultant plasmid in
JM83 and checked sensitivity to DHCP. This plasmid conferred
resistance to DHCP. These results clearly demonstrate that ORF389
is responsible for resistance to DHCP when cloned in a multicopy
plasmid and further work was carried out using the plasmid pSP007.
The ORF389 was named as dep-DHCP efflux protein (see below).
Homology Analysis of ORF389 with Other Genes Conferring Drug
Resistance
[0078] Using BLAST-homology search computer program, we carried out
a homology search for the putative protein encoded by dep. FIG. 4
shows nine proteins showing significantly high homology with Dep.
Half of these proteins confer resistance to chloramphenicol. The
proteins showing the highest degree of homology include: Cmr from
Rhodococcus fasciens (Desomer et al., 1992), CmrA from R.
erythropolis (Nagy et al., 1997), Cml from Streptomyces lividans
1326 (Dittrich et al., 1991), Cmx from Corynebacterium striatum
(Accession no. U72639), and CmlV from S. venezuelae ISP5230 (Mosher
et al., 1995). As seen from FIG. 4, Dep has the highest degree of
homology with Cmr, product of chloramphenicol resistant gene (cmr)
as compared to other proteins. Cmr protein was shown to contain
three consensus sequences defined by Rouch et al. (1990) for
transmembrane proteins. These sequences are at similar positions
with respect to the predicted transmembrane domains. These are
marked in FIG. 6 with dotted lines and are designated as I, II,
III. In case of Dep, the first stretch (I) comprising of LP is
completely homologous with the stretch defined by these authors.
The second stretch (II) shows 50% similarity with that of Cmr
protein and the third stretch (III) is homologous between these two
proteins except for one residue. According to the model proposed by
Rouch et al. (1990), the stretches I and III are located on the
outside of the cytoplasmic membrane and the stretch II is located
on the inside of the membrane. The positions of the membrane loops
for the putative protein encoded by qacA were ascertained by
inspecting the antigenic index profile and turn prediction. Such
regions have a high antigenic index and turn probability (Rouch et
al., 1990).
[0079] In addition to homology in the primary sequences, the
hydropathic profile of Dep (FIG. 5A) is significantly similar to
those of Cmr of R. faciens (Desomer et al., 1992) (FIG. 5B) and Cml
of S. lividans (Dittrich et al., 1991) (FIG. 5C). Dep is
predominantly hydrophobic and probably contains 12 predicted
transmembrane .alpha.-helices (FIG. 5A).
[0080] The other proteins homologous to Dep include BcR
(bicyclomycin- resistance protein) from E. coli (Bentley et al.,
1993), Bmr3 from B. subtilis involved in the multiple drug efflux
pump conferring resistance to puromycin, tosufloxacin, norfloxacin
(Ohki and Murata, 1997), Tet from Staphylococcus hyicus conferring
tetracycline resistance (Schwarz et al., 1992) and YjcC conferring
tetracenomycin-resistance (Accession no. D90826) (FIG. 4). All of
these are efflux proteins, which is one of the most common
mechanisms for drug resistance. We speculate that dep encodes a
putative efflux protein that forms a cytoplasmic channel specific
for DHCP. The homologies are more prominent towards the N-terminal
end of the proteins, which also is a common feature for efflux
proteins (Desomer et al., 1992).
Measurement of Minimum Inhibitory Concentrations for Cells
Harboring pUC19 and pSP007
[0081] Since Dep shows homology to efflux proteins for multiple
drug resistance, we checked if it confers resistance to other
antibiotics as well. The E. coli wild-type cells harboring pUC 19
or pSP007 plasmid were grown overnight in LB medium containing
ampicillin. The cells were diluted 10- and 1000- times, and 5 .mu.l
of each dilution (corresponding to 3.5.times.10.sup.5 cells and
3.5.times.10.sup.3 cells, respectively) was spotted on LB plates
containing serial dilutions of kanamycin, chloramphenicol,
spectinomycin, tetracycline and DHCP. Plates were incubated at
37.degree. C. for 20 h. As seen from Table 1, pSP007 did not confer
significant cross-resistance to any of the antibiotics tested. The
MIC values for cells harboring pUC19 and pSP007 were same for
spectinomycin, chloramphenicol and tetracycline. The MIC value was
two times higher for kanamycin for the cells harboring pSP007 than
the cells with pUC19. The MIC value for DHCP on the other hand was
8 times higher for the cells harboring pSP007 than that for the
cells with pUC 19. It is interesting that Dep did not confer
resistance to chloramphenicol, in spite of the high homology to
cmr.
1TABLE 1 Minimum inhibitory concentrations (MICs) of various
antibiotics for E. coli JM83 cells harboring pUC19 and pSP007. MICs
(.mu.g/ml) chloram- tetra- kanamycin spectinomycin phenicol cycline
DHCP cells with 25 12.5 6.25 3.125 25 pUC19 cells with 50 12.5 6.25
3.125 200 pSP007 MICs for both dilutions of the cells (3.5 .times.
10.sup.5 and 3.5 .times. 10.sup.3 cells) were the same.
[0082]
Sequence CWU 1
1
2 1 3900 DNA Escherichia coli 1 gccagccact cttccagctg acgcacggta
tagctgaccg cagaaggaac gcgatgcagc 60 tcctgtgccg cagcgctaaa
actaccatta cgcgctaccg catcaacaac ttcgagtgaa 120 tattctgacc
acatagtctg cctgcaaaat ttttgaaacc agtcatcaaa tattaccgtt 180
tcacaacact aatttcactc cctacacttt gcggcggtgt ttaattgaga gatttagaga
240 atatacatgc aacctgggaa aagattttta gtctggctgg cgggtttgag
cgtactcggt 300 tttctggcaa ccgatatgta tctgcctgct ttcgccgcca
tacaggccga cctgcaaacg 360 cctgcgtctg ctgtcagtgc cagccttagt
ctgttccttg ccggttttgc cgcagcccag 420 cttctgtggg ggccgctctc
cgaccgttat ggtcgtaaac cggtattatt aatcggcctg 480 acaatttttg
cgttaggtag tctggggatg ctgtgggtag aaaacgccgc tacgctgctg 540
gtattgcgtt ttgtacaggc tgtgggtgtc tgcgccgcgg cggttatctg gcaagcatta
600 gtgacagatt attatccttc acagaaagtt aaccgtattt ttgcggccat
catgccgctg 660 gtgggtctat ctccggcact ggctcctctg ttaggaagct
ggctgctggt ccatttttcc 720 tggcaggcga ttttcgccac cctgtttgcc
attaccgtgg tgctgattct gcctattttc 780 tggctcaaac ccacgacgaa
ggcccgtaac aatagtcagg atggtctgac ctttaccgac 840 ctgctacgtt
ctaaaaccta tcgcggcaac gtgctgatat acgcagcctg ttcagccagt 900
ttttttgcat ggctgaccgg ttcaccgttc atccttagtg aaatgggcta cagcccggca
960 gttattggtt taagttatgt cccgcaaact atcgcgtttc tgattggtgg
ttatggctgt 1020 cgcgccgcgc tgcagaaatg gcaaggcaag cagttattac
cgtggttgct ggtgctgttt 1080 gctgtcagcg tcattgcgac ctgggctgcg
ggcttcatta gccatgtgtc gctggtcgaa 1140 atcctgatcc cattctgtgt
gatggcgatt gccaatggcg cgatctaccc tattgttgtc 1200 gcccaggcgc
tgcgtccctt cccacacgca actggtcgcg ccgcagcgtt gcagaacact 1260
cttcaactgg gtctgtgctt cctcgcaagt ctggtagttt cctggctgat cagtatcagc
1320 acgccattgc tcaccaccac cagcgtgatg ttatcaacag taatgctggt
cgcgctgggt 1380 tacatgatgc aacgttgtga agaagttggc tgccagaatc
atggcaatgc cgaagtcgct 1440 catagcgaat cacactgacc tatatcgata
tacttatact taggctgcta acaaaatttt 1500 gttgtatgat tgaaattagc
ggcctatact aatttcgagt tgttaaagct acgataaata 1560 ttatgttttt
acggggacag gatcgttccc gactcactat ggatagtcat ttcggcaagg 1620
gttcctcctt tccctctgtt ctacgtcgga ttatagactc gcggtttttt ctgcgagatt
1680 tctcacaaag cccaaaaagc gtctacgctg ttttaaggtt ctgatcaccg
accagtgatg 1740 gagaaactat gagttcatcg tgtatagaag aagtcagtgt
accggatgac aactggtacc 1800 gtatcgccaa cgaattactt agccgtgccg
gtatagccat taacggttct gccccggcgg 1860 atattcgtgt gaaaaacccc
gattttttta aacgcgttct gcaagaaggc tctttggggt 1920 taggcgaaag
ttatatggat ggctggtggg aatgtgaccg actggatatg ttttttagca 1980
aagtcttacg cgcaggtctc gagaaccaac tcccccatca tttcaaagac acgctgcgta
2040 ttgccggcgc tcgtctcttc aatctgcaga gtaaaaaacg tgcctggata
gtcggcaaag 2100 agcattacga tttgggtaat gacttgttca gccgcatgct
tgatcccttc atgcaatatt 2160 cctgcgctta ctggaaagat gccgataatc
tggaatctgc ccagcaggcg aagctcaaaa 2220 tgatttgtga aaaattgcag
ttaaaaccag ggatgcgcgt actggatatt ggctgcggct 2280 ggggcggact
ggcacactac atggcatcta attatgacgt aagcgtggtg ggcgtcacca 2340
tttctgccga acagcaaaaa atggctcagg aacgctgtga aggcctggat gtcaccattt
2400 tgctgcaaga ttatcgtgac ctgaacgacc agtttgatcg tattgtttct
gtggggatgt 2460 tcgagcacgt cggaccgaaa aattacgata cctattttgc
ggtggtggat cgtaatttga 2520 aaccggaagg catattcctg ctccatacta
tcggttcgaa aaaaaccgat ctgaatgttg 2580 atccctggat taataaatat
atttttccga acggttgcct gccctctgta cgccagattg 2640 ctcagtccag
cgaaccccac tttgtgatgg aagactggca taacttcggt gctgattacg 2700
atactacgtt gatggcgtgg tatgaacgat tcctcgccgc atggccagaa attgcggata
2760 actatagtga acgctttaaa cgaatgttta cctattatct gaatgcctgt
gcaggtgctt 2820 tccgcgcccg tgatattcag ctctggcagg tcgtgttctc
acgcggtgtt gaaaacggcc 2880 ttcgagtggc tcgctaaagg ctattctatc
gccccctctc cgggggcgat ttcagatcag 2940 gcttctgtgc ctggttgatt
catggcattt tctcgtgccg ccagcacacg ttctaccgta 3000 tctaccactg
cctgagtttg tggatcgatt tcaatgttga cgcgtgcgcc aagttttttc 3060
ttcccaagag tcgtgcgttc cagtgtttcc ggaattaaat ggacgcaaaa acgcgttggc
3120 gtgacttcgc cgacggtcag gctaataccg tcgatgccaa taaatccttt
gtacagaata 3180 tatttcatca actgactatc ctggacttta aaccagatct
ggcgattatt ttctgaggtt 3240 aatattttcg ccacttcagc agtggtcata
atatgacctg acattaagtg tccgccaatt 3300 tcatcactga atttcgccgc
acgctcaacg tttacccaat cccccacttt taaatcgcca 3360 agattggtaa
tgcgtaacgt ttctttcatc aggtcaaaac tgacatggtt gccgttaatt 3420
tccgtcacgg tcaggcagca accgttatgc gccacggaag caccggtttc caggccgtcc
3480 agcatgtggt cgggtaactc caccacatgc gtacgaaaat ttggtttctc
gtcaatcgac 3540 accagttttg cggtgccctg tacaatcccc gtaaacatac
ttacaactcc tgaaatcagt 3600 taagacattc tgttcagcac aatagcaggt
ggaaaacgcc cttaccagtg aaggggtaag 3660 aatggctatt ttttcactgg
agaattaata aatcctcgct acaatagact gaatttcccc 3720 tgcttcttct
ttttgctgcc cattcaggcg gctttttagt ctctcatata actacaaata 3780
aaaggtgttc acgtgcagaa gtatatcagt gaagcgcgtc tgttattagc attagcaatc
3840 ccggtgattc tcgcgcaaat cgcccaaact gcgatgggtt ttgtcagtac
cgtgatggcg 3900 2 1212 DNA Escherichia coli 2 atgcaacctg ggaaaagatt
tttagtctgg ctggcgggtt tgagcgtact cggttttctg 60 gcaaccgata
tgtatctgcc tgctttcgcc gccatacagg ccgacctgca aacgcctgcg 120
tctgctgtca gtgccagcct tagtctgttc cttgccggtt ttgccgcagc ccagcttctg
180 tgggggccgc tctccgaccg ttatggtcgt aaaccggtat tattaatcgg
cctgacaatt 240 tttgcgttag gtagtctggg gatgctgtgg gtagaaaacg
ccgctacgct gctggtattg 300 cgttttgtac aggctgtggg tgtctgcgcc
gcggcggtta tctggcaagc attagtgaca 360 gattattatc cttcacagaa
agttaaccgt atttttgcgg ccatcatgcc gctggtgggt 420 ctatctccgg
cactggctcc tctgttagga agctggctgc tggtccattt ttcctggcag 480
gcgattttcg ccaccctgtt tgccattacc gtggtgctga ttctgcctat tttctggctc
540 aaacccacga cgaaggcccg taacaatagt caggatggtc tgacctttac
cgacctgcta 600 cgttctaaaa cctatcgcgg caacgtgctg atatacgcag
cctgttcagc cagttttttt 660 gcatggctga ccggttcacc gttcatcctt
agtgaaatgg gctacagccc ggcagttatt 720 ggtttaagtt atgtcccgca
aactatcgcg tttctgattg gtggttatgg ctgtcgcgcc 780 gcgctgcaga
aatggcaagg caagcagtta ttaccgtggt tgctggtgct gtttgctgtc 840
agcgtcattg cgacctgggc tgcgggcttc attagccatg tgtcgctggt cgaaatcctg
900 atcccattct gtgtgatggc gattgccaat ggcgcgatct accctattgt
tgtcgcccag 960 gcgctgcgtc ccttcccaca cgcaactggt cgcgccgcag
cgttgcagaa cactcttcaa 1020 ctgggtctgt gcttcctcgc aagtctggta
gtttcctggc tgatcagtat cagcacgcca 1080 ttgctcacca ccaccagcgt
gatgttatca acagtaatgc tggtcgcgct gggttacatg 1140 atgcaacgtt
gtgaagaagt tggctgccag aatcatggca atgccgaagt cgctcatagc 1200
gaatcacact ga 1212
* * * * *