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Journal of Bacteriology, August 2001, p. 4771-4778, Vol. 183, No. 16
Division of Biological Sciences, The
University of Montana, Missoula, Montana 59812,1
and Laboratory of Human Bacterial Pathogenesis, Rocky
Mountain Laboratories, National Institute of Allergy and Infectious
Diseases, National Institutes of Health, Hamilton, Montana
598402
Received 16 February 2001/Accepted 16 May 2001
We previously described a bacteriophage of the Lyme disease agent
Borrelia burgdorferi designated Borrelia
burgdorferi, the causative agent of Lyme disease
(5, 32), has a complex genome that contains both
linear and circular DNA (7, 13). A growing number of tools
have been developed for studying the molecular biology of this
organism, although elaborating a convenient manipulatable genetic
system will require further effort. Despite indirect evidence for
lateral gene transfer in these bacteria (19, 33, 38),
natural mechanisms for the transfer of genetic material (including
conjugative plasmids and transducing bacteriophages) remain elusive for
B. burgdorferi and related spirochetes.
In a number of other bacteria, bacteriophages are well characterized,
naturally occurring mechanisms for lateral gene transfer (22). Bacteriophages have been observed in association
with many spirochetes (reviewed in references 10 and
11), including Borrelia spp. (3, 12, 14,
23), Leptospira biflexa (29), and
Brachyspira hyodysenteriae (15, 27). VSH-1, a
generalized transducing phage of B. hyodysenteriae, has been
used to demonstrate the transfer of antibiotic resistance markers
between two different strains of this spirochete (16).
Recently, a prophage from L. biflexa was developed into a
shuttle vector (28). To our knowledge, no transduction or
any other naturally occurring mechanism for the introduction of
exogenous DNA has been demonstrated in B. burgdorferi or
related species.
We previously reported the preliminary characterization of a temperate
bacteriophage (Fig. 1)
(10-12), designated
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183.16.4771-4778.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Transduction by
BB-1, a Bacteriophage of
Borrelia burgdorferi
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
BB-1. This phage
packages the host complement of the 32-kb circular plasmids (cp32s), a group of homologous molecules found throughout the genus
Borrelia. To demonstrate the ability of BB-1 to
package and transduce DNA, a kanamycin resistance cassette was
inserted into a cloned fragment of phage DNA, and the resulting
construct was transformed into B. burgdorferi CA-11.2A
cells. The kan cassette recombined into a resident cp32 and
was stably maintained. The cp32 containing the kan cassette
was packaged by BB-1 released from this B.
burgdorferi strain. BB-1 has been used to transduce this
antibiotic resistance marker into naive CA-11.2A cells, as well as two
other strains of B. burgdorferi. This is the first
direct evidence of a mechanism for lateral gene transfer in B.
burgdorferi.
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
BB-1, which packages the
32-kb circular plasmids (cp32s) of the B. burgdorferi genome. The cp32s are a family of extrachromosomal
elements, several of which can be maintained in a single cell (7,
8, 35). There are three regions of variability on these
homologous plasmids: two that encode various lipoproteins, and one that
may encompass a possible partitioning region (1, 6-8, 17, 24,
25, 33-36, 40, 42). Sequence analysis of these molecules
suggests that they have evolutionarily undergone recombination,
possibly from both endogenous and exogenous sources (6-8, 20,
33, 35, 38). Many features of the cp32 family are consistent
with the hypothesis that these plasmids are temperate prophage genomes (7-11).
View larger version (148K):
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FIG. 1.
B. burgdorferi phage
particles. Samples were collected from polyethylene
glycol-precipitated cell supernatants of an MNNG-treated culture of
B. burgdorferi CA-11.2A and viewed by transmission
electron microscopy. Although previously reported as having a simple
noncontractile tail (12), subsequent modifications to the
purification and preparation protocol (10, 11) reveal that
all phage particles observed have intact contractile sheaths, seen here
either extended (left) or contracted (right). Phosphotungstic acid
stain. Bar, 45 nm.
To elucidate a possible biological role for BB-1 as well as evaluate
its use as a potential molecular tool, we have extended our
characterization of this phage. Using a DNA fragment from a partial
library of BB-1 DNA, we have constructed a recombinant cp32 (BB-1
prophage) carrying a kanamycin resistance cassette. Subsequently,
BB-1 was used to transduce this recombinant cp32 to other strains of
B. burgdorferi. The introduction of an antibiotic resistance
marker into naive cells by BB-1 is the first direct demonstration of
lateral gene transfer in B. burgdorferi.
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MATERIALS AND METHODS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Bacterial strains and bacteriophage recovery.
B. burgdorferi sensu stricto strains CA-11.2A (21) and
high-passage B31-UM were part of our collection. High-passage B. burgdorferi sensu stricto strain 1A7, a clone of Sh2-82, was
generously provided by Tom Schwan (Rocky Mountain Laboratories).
Bacterial isolates were routinely cultivated in modified
Barbour-Stoenner-Kelly (BSK-H) complete medium (Sigma, St. Louis, Mo.)
at 34°C with a 5% CO2 atmosphere. Culture
densities were determined, and BB-1 particles were recovered from
cultures induced with 1-methyl-3-nitro-1-nitrosoguanidine(MNNG) as
described previously (12). Phage particles were
concentrated, stained, and visualized by transmission electron
microscopy using a protocol described elsewhere (10).
Agarose gel electrophoresis.
DNA for restriction digests was
extracted as described previously (12) and digested with
different restriction enzymes as instructed by the manufacturer (New
England Biolabs, Beverly, Mass.). DNA samples were resolved in 0.8%
agarose gels (SeaKem GTG; Bio*Whittaker Molecular Applications;
Walkersville, Md.) in TBE (45 mM Tris-borate, 2 mM EDTA) at 80 V (4.3 V
cm
1). Field-inversion gel electrophoresis
(FIGE) was performed at room temperature using a PPI-200 programmable
power inverter (MJ Research, Waltham, Mass.) with optimal programs and
times for separating the desired range of fragments determined using
the GelTimes software supplied by the manufacturer. The running buffer was supplemented with 100 mM glycine (as suggested by MJ Research), and
the buffer was recirculated during electrophoresis. After electrophoresis, gels were stained with 0.5 µg of ethidium bromide (EtBr) ml1 for 0.5 to 1 h and destained in
water for 1 to 2 h. The DNA was visualized on a UV
transilluminator, and images were captured on a Gel Doc 1000 system
(Bio-Rad, Hercules, Calif.). Two-dimensional gel electrophoresis and
genomic DNA extraction were performed essentially as described
previously (12, 31).
Southern hybridization. Agarose gels to be blotted were prepared and run as described above. After visualization, the gels were destained in water and then vacuum blotted to Immobilon-Ny+ (Millipore, Bedford, Mass.) as described elsewhere (18). Southern hybridization was performed as previously described (12). Approximate sizes of the major fragments were determined by comparison to markers of known sizes. Blots to be reprobed were stripped in increasingly stringent solutions as described elsewhere (2).
The conserved cp32-specific probes used for Southern hybridizations were probe 4 (8) and a probe that flanks an NdeI site (cp32SK12NdeI) in the paralogous family (PF) gene 50. Probes were generated from B. burgdorferi genomic DNA by PCR with Taq polymerase as described by the manufacturer (Sigma) using an annealing temperature of either 50°C (probe 4) or 44°C (probe cp32SK12NdeI). Additionally, the pOK12 plasmid was used as a probe for the kan cassette. All probes were labeled using the Prime-it II kit as instructed by the manufacturer (Stratagene, La Jolla, Calif.).Variable region PCR.
A ClustalW alignment (MacVector 6.5.1;
Oxford Molecular, Madison, Wis.) of the available B. burgdorferi B31 cp32 sequences from GenBank (7) was
performed to identify possible diagnostic variable regions.
Oligonucleotides to highly conserved sequences flanking one variable
region, designated VR1, were designed using MacVector (Table
1). The VR1s were amplified from either
cellular cp32s or phage DNA using Taq polymerase and the
cp32-VR1 primers with 25 cycles of 92°C for 1 min, 50°C for 1 min,
and 72°C for 3 min. The PCR products were resolved on 0.8% agarose
gels subjected to FIGE, as described above. The PPI-200 power inverter
was programmed with a maximum resolution in the range of 2 to 6 kb. The
sizes of the amplified VR1s were determined using Multi-Analyst
Software 1.0 (Bio-Rad).
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Construction of pCE210.
HindIII-digested
BB-1 DNA was ligated into the HindIII site of
pBluescript II SK+ (Stratagene, La Jolla,
Calif.), generating a library that contained random phage DNA fragments
of less than or equal to 4 kb. A plasmid with a 4-kb insert was
selected and designated pCE100. The insert was found to contain one
NdeI site approximately 1.1 kb from one end (Fig.
2A).
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) control.
Transformation of B. burgdorferi CA-11.2A
with the kan cassette.
pCE210 was digested with
BssHII, and the insert containing the recombinant BB-1
DNA-kan cassette was gel purified with a Qiaex gel
extraction kit (Qiagen, Valencia, Calif.). Purified insert DNA (10 µl, ~2 µg) was electroporated into competent B. burgdorferi CA-11.2A cells and plated in solid medium as described previously (30). Transformants were selected in solid BSK
at 34°C in the presence of 500 µg of kanamycin per ml. Colonies
were screened by PCR using the cp32SK12NdeI primers (Table 1 and Fig. 2) as described above. PCR products were resolved on 1% agarose gels
and stained with GelStar nucleic acid stain (Bio*Whittaker Molecular Applications).
Transduction assays. Phage particles were purified from the cell supernatants of the CA-11.2A/kanR transformant (12) and treated with an additional 10% chloroform (CHCl3) to eliminate possible cellular contamination, incubated at room temperature for 15 min, and then centrifuged at 14,000 × g, and the aqueous phase was recovered. Sterile 1 M MgCl2 was added to the sample to bring the final Mg2+ concentration to 16 mM, and 1 µl of RQ1 DNase (Promega, Madison, Wis.) was added per 250 µl of volume. After 0.5 h at 37°C, 100 µl of the prepared phage sample (~35 ng of phage DNA; the equivalent of ~109 phage) was mixed with 107 cells (100:1), and BSK-complete was added for a final volume of 1 ml.
After 16 h of incubation at 34°C, the phage-cell mixtures were plated, and potential transductants were selected on solid medium with 500 µg of kanamycin per ml. Colonies were picked and screened by PCR with the cp32SK12NdeIF and KanR1207F primers (Table 1 and Fig. 2A) using the same parameters as for the cp32SK12NdeI primer pair. Positive clones were transferred into 10 ml of BSK-complete with 500 µg of kanamycin per ml. The frequency of transduction was determined as the number of positive transductants divided by the total number of CFU. When the putative transductants reached approximately 5 × 107 cells ml1, DNA was
extracted from the culture and the clones were screened again by
PCR for the kan cassette using the above primers.
Additionally, using the KanR1207F primer and
the cp32-VR1R primer, a 7.5-kb region of DNA was amplified (25 cycles
of 92°C for 30 s, 50°C for 30 s, and 72°C for 6 min)
with TaqPlus long (Stratagene) from each transductant.
Analysis of recombinants. Plasmid DNA was extracted from log-phase cultures of B. burgdorferi using Wizard Plus Midi preps (Promega). The concentration of DNA was determined by measuring the absorbance at 260 nm. A total of 500 ng of DNA was digested with either EcoRV or XbaI, and the DNA was resolved by FIGE. Following electrophoresis and staining, the gels were vacuum blotted, probed, stripped, and reprobed as described above.
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RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Determining the minimum number of cp32s present in a population of
cells or phage.
Specific probes to individual B. burgdorferi B31 cp32s (8) did not hybridize to
B. burgdorferi CA-11.2A cp32s (data not shown). To
determine an approximate number of cp32s in both B. burgdorferi cells and BB-1 capsids, oligonucleotides that flank a diagnostic variable region (designated VR1) were designed (Table 1).
VR1 encompasses a portion of each cp32 where the
ospE/ospF/elp genes (erp loci) are located
(1, 6, 7, 20, 25, 35). The cp32-VR1R primer (27 nucleotides) is conserved on all the B. burgdorferi B31
cp32s and the cp32-like molecule integrated into lp56 (7).
The cp32-VR1F primer (26 nucleotides) has one mismatch on cp32-9 and
two mismatches on the cp32-like molecule integrated into lp56, but is
conserved on all the other B31 cp32s. PCR amplification of VR1 with
these oligonucleotides generates products of different sizes for
several of the B. burgdorferi B31 cp32s whose sequences
are available (Table 2).
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BB-1 phages released from CA-11.2A (Fig. 3, lane 2) and B31 cells (Fig. 3, lane 4) package cp32s that are
represented by only three of the VR1s amplified from the respective
hosts. The absence of the ~2.8-kb band (Table 3) from both BB-1
samples suggests that the phage does not package the cp32 that is
integrated into lp56 (or any DNA that is packaged from the lp56 does
not include the targeted region).
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Inserting the kanamycin resistance cassette into a cp32.
A
construct containing a 1.3-kb kan gene
integrated into a fragment of phage DNA (see Fig. 2A) was transformed
into B. burgdorferi CA-11.2A cells. Colonies selected
in 500 µg of kanamycin per ml were screened by PCR using the
cp32SK12NdeI primers (Table 1) that flank the site into which the
kan cassette had been inserted (Fig. 2A and B). Partial
sequence obtained from the fragment of phage DNA (data not shown)
indicates that the kan gene was inserted into the PF50 paralog of cp32. The cp32SK12NdeI primers amplify a
product from both the recombined cp32 (~1.4 kb) and the other homologous cp32 loci in the cells that do not contain the insert (~100 bp) (Fig. 2B). Both products are expected in a population of
B. burgdorferi cells containing more than one cp32 if,
as anticipated, the fragment bearing the kan cassette does
not recombine with all of the cp32s. The BB-1 capsids collected from
the supernatants of these CA-11.2A transformants include genomes
containing the kan gene as well as
uninserted parental phage genomes (Fig. 2C). These data are consistent
with the results from the variable-region analysis, indicating that
more than one cp32 is packaged in a population of BB-1 phage heads
(Fig. 3, lanes 2 and 4).
BB-1 DNA packaged and released by
CA-11.2A/kanR (Fig. 5C).
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Transduction.
To evaluate the ability of BB-1 to transduce
the kan gene into kanamycin-susceptible
cells, bacteriophage from the CA-11.2A/kanR
transformant were incubated with CA-11.2A cells at an approximately 100:1 multiplicity of infection. After an overnight incubation, the
cells were plated in 500 µg of kanamycin per ml. We screened 10 of
~100 colonies by PCR. All the colonies screened contained the
kan gene integrated into a cp32 (Fig.
6A and data not shown).
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BB-1 between CA-11.2A cells is
~105 to 106 per cell.
Alternatively, the frequency of transduction by BB-1 is
~107 per phage particle (~100 transductants
from ~109 phage particles). No colonies grew
when either CA-11.2A cells without
BB-1/kanR or
BB-1/kanR phage without cells were
plated in kanamycin. The addition of proteinase K to
BB-1/kanR prior to incubation with
cells abrogated the transduction of the antibiotic resistance marker.
Incubating washed, CHCl3-killed CA-11.2A/kanR cells with live CA-11.2A
cells resulted in 0 to 3 colonies. However, no colonies grew when
proteinase K was added to the dead transformant cells prior to mixing
them with susceptible cells, implying that even the small amount of
transfer between dead CA-11.2A/kanR cells
and live CA-11.2A cells requires protein. These data indicate that
phage BB-1 mediated the lateral gene transfer.
BB-1/kanR isolated from the CA-11.2A
transductant (CA-11.2A/TR3) was incubated with several other strains of
B. burgdorferi. The kan cassette was
transduced by BB-1/kanR (CA-11.2A) into
B. burgdorferi strains B31 and 1A7 (a high-passage clone of B. burgdorferi Sh2-82) (Fig. 6A). No
demonstrable DNA transfer occurred when dead
CA-11.2A/kanR cells were incubated with
1A7 and B31 cells. Long-range PCR using an internal kan
primer (KanR1207F) and the
cp32-VR1R primer, which lies outside the cloned fragment approximately
7.5 kb from the insertion site, was done to verify the integration of
the kan cassette into the cp32 molecule (Fig. 6B). A partial
sequence of the ospC gene from the transductant B31/TR1
confirmed that the strain (or at least the cp26) was derived from B31
(data not shown).
The frequency of transduction by
BB-1/kanR (CA-11.2A) into B31 is
~106 to 107 and into
1A7 is ~105 to 106
for individual clones of these strains. Neither the 1A7 transductant (1A7/TR5) nor the B31 transductant (B31/TR1) assumes the prolific CA-11.2A phage-producing phenotype, although both B31 and B31/TR1 spontaneously produce low levels of BB-1. BB-1 (B31) is also capable of packaging the kan cassette from B31/TR1 (data not
shown). The kan marker can be efficiently
transduced back into naive B31 using
BB-1/kanR (B31), but we have not yet
been able to transduce the gene back into susceptible CA-11.2A cells
using BB-1/kanR (B31).
Variable-region analysis of the transduced cp32s.
We have
demonstrated a mechanism for lateral gene transfer in B. burgdorferi via transduction by BB-1. To analyze possible changes in the cp32 population of the transductants, the VR1s of the
uncloned parent strains CA-11.2A, B31, and 1A7, as well as the
transduced clones CA-11.2A/TR3, B31/TR1, and 1A7/TR5, were amplified
and resolved by FIGE (Fig. 7A). The
region of the cp32 that we have designated VR1 is located ~5 kb from
the site of the kan insertion
(7).
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BB-1/kanR (CA-11.2A) prophage is not
known. We have confirmed that the 2,537-bp VR1 is linked to the
kan cassette on the transduced
BB-1/kanR (CA-11.2A) genome by
amplifying the VR1 region from the
kanR/cp32-VR1R long-range PCR product
(Fig. 6B and data not shown).
Restriction mapping of transductants.
Plasmid DNA from
CA-11.2A, CA-11.2A/kanR (transformant),
and CA-11.2A/TR3 (transductant) was extracted and digested with
EcoRV to analyze changes in the restriction pattern of the
cp32 molecules. The location of the kan cassette on cp32 was
mapped using Southern hybridization (Fig.
8A).
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BB-1 DNA probed
with the cp32SK12NdeI probe (data not shown).
To compare the kan insertion site in the
other transductants, plasmid DNA was extracted from parental CA-11.2A,
B31, and 1A7 and the transductant CA-11.2A/TR3, B31/TR1, and 1A7/TR5
cells. The plasmids were digested with XbaI, and the DNA was
resolved by FIGE. A blot of the gel was probed with both the
cp32SK12NdeI PCR product and the kan cassette (Fig. 8B).
When probed with the cp32SK12NdeI product (Fig. 8B, left panel), there
is hybridization to a ~6.6-kb XbaI fragment that is found
exclusively in the parental CA-11.2A (Fig. 8B, lane 1; hatched arrow)
and not in the parental B31 or 1A7. However, a ~8-kb fragment is
present in all the transductant lanes (Fig. 8B, lanes 2, 4, and 6, black arrow). Hybridization with kan (Fig. 8B, right panel)
verified that the ~8-kb fragment contained the kan
cassette in all three transductants. This suggests that the
kan cassette is on a cp32 in the transductants that is not a
member of either of the parental B31 or 1A7 populations. The B31 parent
has three bands that do not appear in B31/TR1 (Fig. 8B, left panel,
lanes 3 and 4, asterisk), possibly due either to displacement of a cp32
or loss of plasmids (cp32 or lp56) during cloning. There are also three
bands that are present in the B31 parent but absent from the
transductant when plasmid DNA from these isolates is digested with
EcoRV and probed with the cp32SK12NdeI probe (data not shown).
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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We recently identified BB-1, a temperate bacteriophage of
B. burgdorferi (Fig. 1), whose prophage is in the cp32
family of circular plasmids (10-12). In this study we
demonstrated that BB-1 can package all of the free cp32s within a
population of cells (Fig. 3). To date, no known strain of B. burgdorferi carries fewer than three cp32s (7, 8,
26), and any or all of these cp32s may be prophages (associated
with all of the necessary functions for lysogeny, progeny formation,
and cellular lysis), or perhaps cryptic prophages, requiring other
factors in trans for the successful propagation of BB-1.
The effect that the protein products of the various cp32s (such as
repressors) may have on each other in trans has not been studied.
Prior to this work, no mechanism for lateral gene transfer had been demonstrated in Borrelia. Previous evidence of recombination in at least one locus (erp, containing the ospE, ospF, and elp homologs) on the cp32s (33, 38) suggested that lateral gene transfer has occurred among these plasmids. Gene transfer could be between different cp32s within the same cell, by phage transduction of alternate cp32s within a population of cells, or by some other, as yet undefined, mechanism. In this study, the kan cassette was inserted into a fragment of phage DNA and electroporated into CA-11.2A cells, generating a recombinant cp32. To our knowledge, this is the first such integration into a member of the cp32 plasmid family.
Mapping and sequence data (not shown) indicate that the site of the kan cassette insertion is within the cp32-3 PF50 paralog (BBS34) of a CA-11.2A cp32, located near the variable region that includes the putative paralogous partitioning genes of the cp32s (7, 8, 36, 41). The cp32 containing the kan gene is stable over 20 passages (~150 generations) in the absence of selection, suggesting that the disrupted open reading frame (ORF) (BBS34 homolog) either has no critical role in the molecular metabolism of this plasmid or has a function that can be supplied in trans by another cellular cp32. The PF50 paralog (BBC02) of the 9-kb circular plasmid (cp9), a derivative of cp32, is critical for the replication of a cp9-based vector (37), suggesting that the replication requirements for this smaller plasmid are different than those of the cp32s.
We have demonstrated transduction of the kan cassette by
BB-1 into three different strains of B. burgdorferi
(Fig. 6-8). This represents the first direct evidence of lateral gene
transfer in B. burgdorferi. The ability of BB-1 to
transduce the antibiotic resistance marker into a strain is apparently
unrelated to the ability of that strain to produce phage, since we have
found that both low-passage B31 and high-passage 1A7 are transducible
but not inducible (12; data not shown). Additionally,
introducing the BB-1/kanR (CA-11.2A)
into these transducible strains does not confer the phage-producing
phenotype of CA-11.2A on these strains.
The evidence suggests that the
BB-1/kanR genome is being introduced as
a discrete plasmid into the cell during transduction. The host cell
loses a VR1 marker concomitantly with gaining the BB-1 (CA-11.2A)
VR1, suggesting the displacement of a host plasmid (Fig. 7). However,
there are no obvious candidates discernible from the restriction maps
(Fig. 8B). The PF50 paralog into which the kan cassette has
been inserted is most similar to the PF50 paralog on cp32-3 of B31,
the plasmid that is apparently absent in the transductant as determined
by VR1 analysis. We hypothesize that the transduced
cp32/kanR from CA-11.2A phage displaced the
resident cp32 homologs (carrying the smallest VR1s) in strains B31 and
1A7 (no cp32 data are currently available for the 1A7 parental strain
Sh2-82). An alternate hypothesis is that prior loss of these cp32
homologs created populations of cells that were susceptible to
transduction. B31 cp32-3 can be lost during cloning, and the loss of
this plasmid has little or no effect on the infectivity of this isolate
(26). Analysis of the plasmid profiles of several recent
clones of CA-11.2A, B31, and 1A7 by PCR amplification of the VR1s
suggests that the prior absence of the smallest VR1 is not required for
transduction to occur, particularly in strain 1A7 (C. H. Eggers,
B. J. Kimmel, and D. S. Samuels, unpublished data).
In this study, we have demonstrated a mechanism for lateral gene
transfer that should be explored to establish its role within the
infectious cycle or during the course of disease. Further analysis of
BB-1, the first bacteriophage of B. burgdorferi
described at a molecular level, could play a critical role in the
investigation of plasmid genetics, the development of a genetic system,
and the analysis of metabolic processes in these bacteria as a whole.
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ACKNOWLEDGMENTS |
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We thank Mike Minnick, Thad Stanton, Sherwood Casjens, and Sharyl Fyffe for thoughtful and critical reading of the manuscript; S. Casjens, Brian Stevenson, Kit Tilly, Fred Hayes, Claude Garon, Lori Lubke, Melissa Caimano, and Justin Radolf for useful discussions; B. Stevenson, Chris Damman, and Don Oliver for cp32-specific probes; Tom Schwan for bacterial strains; and Gary Hettrick for assistance with figure preparation.
This work was supported by grants from the National Institutes of Health (AI41559), Arthritis Foundation, National Science Foundation (MCB-9722408), and the University of Montana University Grant Program to D.S.S. C.H.E. is a recipient of a Predoctoral Honors Fellowship from the University of Montana.
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FOOTNOTES |
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* Corresponding author. Mailing address: Division of Biological Sciences, The University of Montana, 32 Campus Dr #4824, Missoula, MT 59812-4824. Phone: (406) 243-6145. Fax: (406) 243-4304. E-mail: samuels{at}selway.umt.edu.
Present address: Center for Microbial Pathogenesis, University of
Connecticut Health Center, Farmington, CT 06030.
Present address: Center for Vascular Biology, Department of
Physiology, University of Connecticut Health Center, Farmington, CT 06030.
§ Present address: US Meat Animal Research Center, USDA, ARS, Clay Center, NE 68933.
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REFERENCES |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Journal of Bacteriology, August 2001, p. 4771-4778, Vol. 183, No. 16
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183.16.4771-4778.2001
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