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Journal of Bacteriology, August 1998, p. 3793-3798, Vol. 180, No. 15
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Species-Dependent Phenotypes of Replication-Temperature-Sensitive
trfA Mutants of Plasmid RK2: a Codon-Neutral Base
Substitution Stimulates Temperature Sensitivity by Leading to
Reduced Levels of trfA Expression
Ponniah
Karunakaran,
Janet
Martha
Blatny,
Helga
Ertesvåg, and
Svein
Valla*
UNIGEN Center for Molecular Biology and
Department for Biotechnology, Norwegian University of Science and
Technology, 7005 Trondheim, Norway
Received 3 September 1997/Accepted 15 May 1998
 |
ABSTRACT |
TrfA is the only plasmid-encoded protein required for initiation of
replication of the broad-host-range plasmid RK2. Here we describe the
isolation of four trfA mutants temperature sensitive for
replication in Pseudomonas aeruginosa. One of the mutations led to substitution of arginine 247 with cysteine. This mutant has been
previously described to be temperature sensitive for replication, but
poorly functional, in Escherichia coli. The remaining three
mutants were identical, and each of them carried two mutations, one
leading to substitution of arginine 163 with cysteine (mutation 163C)
and the other a codon-neutral mutation changing the codon for glycine
235 from GGC to GGU (mutation 235). Neither of the two mutations caused
a temperature-sensitive phenotype alone in P. aeruginosa,
and the effect of the neutral mutation was caused by its ability to
strongly reduce the trfA expression level. The double
mutant and mutant 163C could not be stably maintained in E. coli, but mutant 235 could be established and, surprisingly, displayed a temperature-sensitive phenotype in this host. Mutation 235 strongly reduced the trfA expression level also in E. coli. The glycine 85 codon in trfA mRNA is GGU, and a
change of this to GGC did not significantly affect expression. In
addition, we found that wild-type trfA was expressed at
much lower levels in E. coli than in P. aeruginosa, indicating that this level is a key parameter in the
determination of the temperature-sensitive phenotypes in different
species. The E. coli lacZ gene was translationally fused at
the 3' end and internally in trfA, in both cases leading to
elimination of the effect of mutation 235 on expression. We therefore
propose that this mutation acts through an effect on mRNA structure or
stability.
| |
INTRODUCTION |
RK2 is a 60-kb self-transmissible
plasmid capable of replicating in a large number of bacterial species
(25). The plasmid is present in the cell at a copy number of
four to seven per chromosome in Escherichia coli
(9). Replication is known to be initiated at a single
origin, oriV (26), and the only other plasmid
locus essential for replication is the trfA gene
(8). Minireplicons of RK2 containing only
oriV and trfA were found to replicate in at least
12 gram-negative bacterial species (1, 12, 20).
The trfA gene of RK2 specifies two replication initiation
proteins, a 44-kDa protein (TrfA-44) and a 33-kDa protein (TrfA-33), resulting from alternative translational starts within the same open
reading frame (21, 24). In E. coli, either
TrfA-44 or TrfA-33 alone is sufficient for replication, whereas in
Pseudomonas aeruginosa, there is a specific requirement for
TrfA-44 in the stable maintenance of the RK2 replicon (6).
However, TrfA-33 can drive replication in P. aeruginosa if
this protein is expressed at a sufficiently high level (22).
The TrfA protein can be supplied in cis or in
trans (with respect to oriV) to an RK2 origin
plasmid, and it initiates replication by binding to the iterons at the origin (16-18).
A large number of mutations in the trfA gene have been shown
to lead to enhanced copy numbers (4, 7, 11, 12), but the
highest-copy-number mutants isolated in E. coli could not be
established in other tested bacterial species. We later found that the
reason for this is that each species has a specific upper maximum
tolerable copy number tolerance, and none of the tested species
tolerated as high copy numbers as E. coli (12).
Similarly, we have found that trfA mutants temperature
sensitive for replication do not display this phenotype in other
species (11, 28). These observations have consequences for
the construction of broad-host-range mini-RK2 cloning and expression
vectors (1, 2) and are also important for the understanding
of the nature of the broad-host-range properties of this replicon. In
this report, we show that a codon-neutral trfA mutation can
act in combination with a substitution mutation to generate a
replication-temperature-sensitive phenotype in P. aeruginosa. The codon-neutral mutation acts by reducing the
trfA expression level, and it therefore seems clear that
this parameter is important for the determination of the
species-specific phenotypes of replication-temperature-sensitive
trfA mutants.
| |
MATERIALS AND METHODS |
Bacterial strains and plasmids.
The bacterial strains and
plasmids used in this study are listed in Table
1.
Growth of bacteria, conjugative matings, electrotransformations,
and standard molecular biology techniques.
E. coli and
P. aeruginosa strains were grown in L broth or on L agar at
37°C unless otherwise stated (19). Antibiotics were used
at the following concentrations: ampicillin, 200 µg/ml;
carbenicillin, 400 µg/ml; and tetracycline, 15 µg/ml. Plasmids were
transferred to P. aeruginosa either by electrotransformation
(in all cases except for the construction of the pFF1 bank containing
the mutagenized trfA genes and for the establishment of the
trfA single mutants) as described for E. coli
(10) or by conjugative matings from E. coli as
described by Haugan et al. (12). Plasmids were transferred to E. coli by the method of Chung et al. (5).
Preparation of plasmid DNA, restriction endonuclease digestions, and
agarose gel electrophoresis were performed according
to standard
protocols (
19).
DNA sequencing was performed with the primers described by Haugan et
al. (
11) on an Applied Biosystems model 373A DNA sequencing
system, using a TaqPRISM Ready Reaction Dye Deoxy Terminator Cycle
Sequencing kit (Applied Biosystems). The conditions for the 30
thermal
cycles were 30 s at 94°C, 15 s at 50°C, and 4 min at
60°C.
Sequence assembly was performed with the Auto assembler
software
(Applied Biosystems).
Isolation of temperature-sensitive trfA mutants in
P. aeruginosa.
The pFF1 bank containing the mutagenized
trfA genes in E. coli S17.1 (12) was
transferred into P. aeruginosa, and the resulting pool of
about 120,000 transconjugants was used as a source for the isolation of
temperature-sensitive mutants. The transconjugants were incubated on
selective (carbenicillin) agar medium at 23°C until small colonies
appeared, and the plates were then shifted to 42°C. Colonies that
failed to increase in size at 42°C were characterized further. A
total of about 100,000 colonies were inspected, leading to the
identification of four plasmid mutants temperature sensitive for
replication. The molecular analysis of the mutants was performed by
localizing the mutations to a smaller fragment of the trfA
gene as described by Haugan et al. (11), followed by DNA
sequencing.
Site-directed mutagenesis and translational fusions.
A
codon-neutral mutation at glycine 85 of the trfA gene (from
GGT to GGC) (mutation 85) was made by modified restriction site PCR
(13). The unique restriction sites EcoRI and
SfiI at the 5' end of the trfA gene were used for
this purpose. The four primers used in the experiment were
5'-GCAGGGGATCAAGATCGACG-3', 5'-ATCCGGGTAATTCCGGGGCA-3', 5'-TGATCTGCTGCTTCGTGTGT-3', and
5'-CTTCGCCAAGCCTGCCGCCT-3'. The trfA-lacZ
translational fusions were made by first PCR amplifying lacZ
from plasmid pMC1871. Two different fragments were made, one containing
a BsmI and another containing a SexAI restriction endonuclease site at the 5' end of lacZ. The primers used to
generate these 5' ends were 5'-GCCGATGAATGCCCCGGGGATCCCGTC-3'
(BsmI site) and
5'-GCCGATACCTGGTTCCCGGGGATCCCGTC-3' (SexAI site).
For both fragments, the same 3' primer
(5'-GAATGAAGCCATACCAAACG-3') was used. This primer
corresponds to sequences downstream of the PstI site 3' of
lacZ. The BsmI and SexAI sites are
naturally present in trfA, allowing in-frame translational
fusions at positions corresponding to codons 245 and 379, respectively.
There is also a PstI site downstream of trfA in
pRD110-34 and pPK34, simplifying insertions of the PCR fragments in the
appropriate positions.
Western blotting and quantification of 
-galactosidase activity
and total protein.
Samples for Western analysis were prepared by
boiling the cells for 2 to 5 min in the loading buffer
(18a). The samples were subjected to electrophoresis in 10%
sodium dodecyl sulfate-polyacrylamide gels according to the procedure
of Laemmli (14). The proteins were transferred from the gel
to Immobilon-P transfer membranes (Millipore) as described by Towbin et
al. (27). The Immobilon sheets were incubated with a
polyclonal anti-TrfA antibody diluted 1:1,000 in Tris-buffered saline
(20 mM Tris chloride [pH 7.5], 150 mM NaCl, 3% bovine serum albumin,
0.2% Triton X-100) for 2 h at 37°C, followed by incubation with
peroxidase-conjugated goat anti-rabbit immunoglobulin diluted 1:5,000
in Tris-buffered saline for 1 h at room temperature (25°C). The
blots were then processed for detection of the TrfA antibody complexes
by using the Pierce SuperSignal Western blotting kit, which uses a
chemiluminescent reaction for visualization.
-Galactosidase activities were measured as described by Sambrook et
al. (
19).
For quantification of protein, the soluble fractions obtained by
centrifugation of sonicated cells were analyzed by the Bio-Rad
Coomassie brilliant blue-based protein assay (as described in
the
Bio-Rad manual).
| |
RESULTS |
Isolation and characterization of mutants temperature sensitive for
replication in P. aeruginosa.
E. coli S17.1 cells
containing plasmid pFF1 with mutagenized trfA genes were
conjugated into P. aeruginosa, and the transconjugants were
pooled. The use of strain S17.1 prevents loss of plasmids nonfunctional
in E. coli because S17.1 expresses wild-type TrfA from the
chromosome. Screening of the transconjugants indicated that the
frequency of temperature-sensitive mutants was about 4 × 10
5, and four such mutants were identified and
characterized further (Table 2). The
mutants fell into two phenotype classes. One mutant (mutant 1) allowed
cell growth in the presence of carbenicillin up to 37°C, while the
remaining three strains grew at 23°C but not at any of the higher
tested temperatures.
All four mutations were found to map between the
SfiI and
NdeI restriction endonuclease sites in
trfA, and
the corresponding
DNA fragments were therefore sequenced in all four
mutants (Fig.
1). The results showed that
the mutation in mutant 1 leads to
replacement of arginine 247 with
cysteine. This mutation has previously
been described for
E. coli, and its phenotype was characterized
in this host and to some
extent in
P. aeruginosa (
11,
28).
In
E. coli the mutant was found to display a temperature-sensitive
phenotype, and functional replication at the permissive temperature
was
found to require high TrfA expression levels. The phenotype
of the
mutant was also tested in
P. aeruginosa, and it was found
to
be able to replicate at 42°C at low but not at high concentrations
of
carbenicillin (100 and 800 µg/ml, respectively). In the present
work
400 µg/ml was used, and based on these data this mutant was
not
studied further.
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FIG. 1.
Map of trfA mutations leading to a
replication-temperature-sensitive phenotype. Mutant number designations
correspond to amino acid residues affected by the mutations (the first
methionine from the amino-terminal end in the 44-kDa protein represents
residue 1). The three-letter designations following the numbers
indicate the new DNA sequence at the corresponding codon. Arrowheads
indicates the locations of the mutations previously reported to lead to
a replication-temperature-sensitive phenotype in E. coli
(11).
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The sequence data of the remaining three mutants (mutants 2, 3, and 4)
showed that they were identical, consistent with their
indistinguishable phenotypes (Fig.
1). These mutants contained
two
mutations, one of which involved substitution of arginine
163 with
cysteine (mutation 163C). This mutation has to our knowledge
not been
previously reported. The second mutation involves a base
substitution
(C to T) at the position corresponding to glycine
235 (mutation 235).
Surprisingly, this mutation does not lead
to an amino acid
substitution.
The phenotype of double mutant 163C,235 in
P. aeruginosa is
consistent with the assumption that replication is temperature
sensitive; to confirm that this is the case, we carried out a
stability
test in which plasmid loss at a nonpermissive temperature
was monitored
(Fig.
2). The data show that a switch
from growth
at 23°C to growth at 37°C leads to a rapid loss of the
plasmids,
consistent with a replication deficiency.
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FIG. 2.
Plasmid loss kinetics of P. aeruginosa cells
containing the mutant plasmid pFF1 163C,235. Cells were grown
exponentially at 23°C in the presence of carbenicillin, diluted
104-fold (0 generations), and then incubated further at
37°C in the absence of carbenicillin. At the times indicated, the
cells were diluted and plated on agar medium without antibiotics. After
incubation at 23°C, individual colonies were picked and tested for
carbenicillin resistance at 23°C. Wild-type pFF1 is not lost at
significant frequencies over such short incubation periods.
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Separation of the mutations in the double mutant and
characterization of phenotypes in P. aeruginosa and
E. coli.
The two mutations in the double mutant could easily
be separated by the unique PflMI site localized between the
mutations. The single mutations were later moved to pFF1 as
EcoRI-PstI fragments and were established in
E. coli S17.1. The phenotypes of the corresponding mutants
(163C and 235, respectively) were then analyzed in P. aeruginosa (Table 3). Surprisingly,
none of the two mutants displayed a temperature-sensitive phenotype,
and it could therefore be concluded that the codon-neutral mutation
235, together with mutation 163C, is somehow involved in determining
the temperature-sensitive phenotype.
To study this further, we also attempted to transfer the mutants to
E. coli DH5
, which does not express wild-type TrfA. The
double mutant could not be established at any temperature, and
the same
was true for mutant 163C (Table
4).
However, mutant
235 could be established at 23°C but not at higher
temperatures.
Thus, both single mutations display a strong negative
effect on
replication in
E. coli. We have previously
observed that overexpression
of the
trfA gene may lead to a
reduction of temperature sensitivity
for replication, and we therefore
also analyzed the phenotypes
of the mutants in a two-plasmid system
which is known to lead
to an approximately 10-times-higher
trfA expression level (
7,
28). In this system,
trfA is expressed from the ColE1 replicon
pPK34 (a
derivative of plasmid pBR322), while
oriV is on a replicon
(pSV16) lacking the
trfA gene. The results showed that the
double
mutant could still not be established in
E. coli,
while cells
containing mutant 235 could now grow also at 30°C in the
presence
of pSV16 selection (Table
4). We were also able to get some
transformants
at 23°C with mutant 163C, but the plasmids in these
cells could
not be stably maintained. Thus, overexpression of
trfA reduces
but does not eliminate the temperature
sensitivity.
Mutation 235 acts by reducing the trfA expression
level.
One possible way of explaining the puzzling effect of
mutation 235 is to assume that it somehow acts by reducing the
trfA expression level. To study this, we used Western
blotting and a TrfA polyclonal antibody (Fig.
3). The data showed that trfA in mutant 163C is expressed at levels similar to wild-type levels in
P. aeruginosa, while the expression levels in the double
mutant and in mutant 235 were severely reduced (Fig. 3A). This finding strongly supports the above hypothesis concerning the effect of mutation 235, and it therefore also follows that single mutant 163C
displays a temperature-sensitive phenotype at low but not high
trfA expression levels in P. aeruginosa.
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FIG. 3.
Western blot analysis of TrfA protein produced by
different plasmid mutants. (A) Derivatives of pFF1 in P. aeruginosa. Lanes: 1, 235; 2, 163C; 3, 163C,235; 4, wild type. (B)
Derivatives of pPK34 in E. coli DH5. Lanes: 1, 85; 2, 235; 3, 163C; 4, 163C,235; 5, wild type. (C) Comparison of
trfA expression in E. coli and P. aeruginosa. Lanes: 1, pFF1 in P. aeruginosa; 2, pFF1 in
E. coli DH5; 3, pPK34 in E. coli DH5. The
amounts of lysed cells loaded on the gel correspond to the following
quantities of soluble protein: 6 µg (A), 20 µg (B), and 7.5 µg
(C). Note that signal intensities cannot be compared between panels, as
film exposure times varied.
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|
We were interested in studying the effect of mutation 235 also in
E. coli; by expressing
trfA from plasmid pPK34,
the expression
is decoupled from replication. The results indicate that
the relative
expression levels are similar to those in
P. aeruginosa. Mutation
235 leads to a severe reduction of expression
relative to wild-type
trfA, while this is not the case for
mutation 163C (Fig.
3B).
Another very interesting observation is that
wild-type
trfA is
expressed from pFF1 at much lower levels
in
E. coli than in
P. aeruginosa (Fig.
3C).
Furthermore, relative to total soluble protein,
the expression level
from pPK34 in
E. coli was also lower than
that from pFF1 in
P. aeruginosa.
The mutant 235-mediated effect on the trfA expression
level is site specific and is probably the result of an effect on mRNA
structure.
The glycine codon GGU in mutant 235 has been reported
to be used about three times less frequently in P. aeruginosa than the original GGC codon (29). In
E. coli, the GGU codon is used at a frequency similar to
that of GGC (29). We also recently showed that the
celB gene (encoding phosphoglucomutase) from the bacterium Acetobacter xylinum could be expressed at very high levels
in E. coli (1, 2), and inspection of the
celB sequence showed that it contains six GGU codons
(3). This made us suspect that the effect of the GGU codon
in 235 is due to a more specific effect than, for instance,
insufficient supplies of the relevant tRNA. To test this further, we
inspected the trfA gene sequence and found that glycine 85 is encoded by GGU, and the possible effect of changing this codon to
GGC could thus be tested. The mutant was made by PCR and the mutation
did not significantly affect the trfA expression level (Fig.
3B). We also constructed a double mutant of 85 and 235, and
trfA expression was then similar to that of mutant 235 (results not shown). Thus, mutation 235 appeared to act by some
site-specific mechanism.
To further analyze the mechanism by which mutation 235 reduces
trfA expression we constructed two pairs of in-frame
translational
lacZ fusions with
trfA. In the
first of these pairs,
lacZ was
fused at the position
corresponding to codon 245 in the wild type
and mutant 235, generating
plasmids pJB1002 and pJB1004, respectively.
In the second pair, the
fusions were near the 3' terminus of
trfA,
such that only
four of the carboxy-terminal amino acids in the
TrfA protein are
missing (constructs pJB1006 and pJB1008). The
constructs were used for
Western blotting with the TrfA antibody,
and the results confirmed that
the fusion proteins were being
produced, as the molecular masses had
increased as expected (Fig.
4A).
Surprisingly, there was no significant difference between
the
expression levels of any of the constructs. Thus, both fusions
eliminate the effect of mutation 235. To confirm these data by
an
independent method, we also analyzed the
-galactosidase activities
in extracts prepared from the same strains. All activities were
similar
(980, 1,060, 1,020, and 1,010 U for pJB1002, pJB1004,
pJB1006, and
pJB1008, respectively), confirming the Western blot
data.
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FIG. 4.
Western blot analysis of expression of TrfA-LacZ fusion
proteins in E. coli DH5 (A) and of TrfA in an E. coli rho-deficient host mutant (B). (A) Lanes: 1, pJB1002; 2, pJB1004; 3, pJB1006; 4, pJB1008. (B) Lanes: 1, K37(pRD110-34); 2, K7487(pRD110-34); 3, K37(pPK34 235); 4, K7487(pPK34 235). In each lane, lysed cells corresponding to 25 µg of
soluble protein were loaded. The numbers represent deduced molecular
masses in kilodaltons. The intensities of the bands from the pPK34
extracts were found to be similar to those from the
trfA-lacZ fusions (not shown).
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One possible way by which mutation 235 might act is by creating a site
for
rho-dependent transcriptional termination. This
could be
tested by comparing expression in the
rho-deficient
E. coli mutant K7487 and its parent strain K37 (Fig.
4B). The results
showed that significantly more TrfA was produced in K7487 than
in K37,
but this difference was not affected by mutation 235.
We noted that
strain K37 grew much faster than strain K7487, which
is probably the
reason for the mutation 235-independent differences
in the expression
levels. Thus, mutation 235 probably does not
act by generating a
rho-dependent transcription termination site.
| |
DISCUSSION |
The trfA mutant bank used to isolate the
temperature-sensitive mutants from P. aeruginosa originated
from a previously constructed E. coli mutant bank estimated
to contain about 60,000 original clones (12). The frequency
of temperature-sensitive mutants in P. aeruginosa was found
to be as low as about 1 in 25,000, which probably explains the repeated
(three times) isolation of the double mutant. By using a more complex
mutant bank, it might therefore have been possible to identify other
temperature-sensitive mutants in P. aeruginosa.
Based on results previously reported, it appears that
temperature-sensitive trfA mutants isolated from E. coli in most cases display a wild-type phenotype in P. aeruginosa. Furthermore, mutants that show some degree of reduced
functionality or are temperature sensitive tend to be those that are
particularly poorly functional in E. coli (11,
28). The temperature-sensitive mutants isolated in P. aeruginosa are poorly (247C) or not (163C,235) functional in
E. coli. Another way of stating this is to propose that
poorly functional TrfA proteins generally works better in supporting the replication of RK2 minireplicons in P. aeruginosa than
in E. coli. The observation that trfA is
expressed (from Pneo) at much higher levels in
P. aeruginosa than in E. coli may partly explain
why such a pattern appears to exist. The reason is simply that
functionality can be improved by expressing more protein. This is also
consistent with our previous observation that increased expression
levels lead to a less temperature-sensitive phenotype in E. coli (11). Even more striking is the observation that mutation 235 can make the substitution mutant 163C temperature sensitive by leading to reduced levels of trfA expression.
Despite all this evidence, we believe that there most probably also
exist other factors that influence the phenotypes. The results
presented in Fig. 3 indicate that the expression level of mutant
163C,235 in P. aeruginosa is much lower than that of 163C in
E. coli. The same protein is presumably produced in both
cases, but functionality is still observed only in P. aeruginosa.
Another interesting problem raised by the results reported here is how
mutation 235 acts to reduce the trfA expression level. It
seems clear that it is not the codon as such that leads to reduced
expression but rather the location of it. Early transcriptional rho-dependent termination also appears unlikely since the
relative difference between the trfA expression levels in a
rho-deficient strain relative to its parent wild-type strain
was not selectively affected by mutation 235. In contrast, the effect
of codon 235 was eliminated by two different trfA-lacZ
fusions. The most likely explanation for this sensitivity to
lacZ fusions appears to be that the mutation 235 effect
depends on the generation of some particular mRNA structure. Such a new
structure could in principle lead to reduced mRNA stability or
translatability (15). We have tried to model the folding
patterns by a computer program (the Zuker-Turner RNA folding package;
Washington University Medical School, St. Louis, Mo.). The program
predicted a significant difference in the folding of wild-type and
mutant 235 mRNAs. However, such effects were also observed by some
randomly tested single base changes in the sequence, and we therefore
feel that the biological significance of such analyses is doubtful. To
fully understand the effect of mutation 235 it will therefore be
necessary to study this system in much more detail.
We also found it surprising that the codon-neutral mutation alone
displayed a temperature-sensitive phenotype in E. coli. We
do not know the reason for this, but it could be that the expression level becomes reduced beyond a subcritical level at elevated
temperatures, possibly due to an effect of temperature on the mechanism
by which mutation 235 acts. It could also be that TrfA is intrinsically slightly temperature sensitive, such that more protein is needed at
higher temperatures or that expression is somewhat reduced at elevated
temperatures for reasons unrelated to mutation 235.
| |
ACKNOWLEDGMENTS |
This work was supported by grants from The Norwegian Research
Council and by the Norwegian University of Science and Technology.
We thank D. R. Helinski for his generous gift of the TrfA
antibody.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: UNIGEN Center
for Molecular Biology and Department for Biotechnology, Norwegian
University of Science and Technology, 7005 Trondheim, Norway. Phone: 47 735998690. Fax: 47 73598705. E-mail:
svein.valla{at}unigen.ntnu.no.
Present address: Department of Microbiology and Immunology,
University of British Columbia, Vancouver, B.C. V6T 1Z3, Canada.
| |
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Journal of Bacteriology, August 1998, p. 3793-3798, Vol. 180, No. 15
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
