Previous Article | Next Article 
Infect Immun, April 1998, p. 1521-1526, Vol. 66, No. 4
0019-9567/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
A New Member of the S-Layer Protein Family:
Characterization of the crs Gene from
Campylobacter rectus
Beinan
Wang,1
Ellen
Kraig,2 and
David
Kolodrubetz1,*
Departments of
Microbiology1 and
Cellular and
Structural Biology,2 University of Texas Health
Science Center, San Antonio, Texas 78284
Received 30 July 1997/Returned for modification 1 October
1997/Accepted 31 December 1997
 |
ABSTRACT |
Strains of the periodontal pathogen Campylobacter
rectus express a 150- to 166-kDa protein on their cell surface.
This protein forms a paracrystalline lattice, called the surface layer
(S-layer), on the outer membrane of this gram-negative bacterium. To
initiate a genetic analysis of the function of the S-layer in the
pathogenesis of C. rectus, we have cloned and characterized
its gene. The S-layer gene (crs) from C. rectus
314 encodes a cell surface protein which does not have a cleaved signal
peptide at its amino terminus. Although the amino acid sequence deduced
from the crs gene has 50% identity with the amino-terminal
30 amino acids of the four S-layer proteins from Campylobacter
fetus, the similarity decreases to less than 16% over the rest
of the protein. Thus, the crs gene from C. rectus encodes a novel S-layer protein whose precise role in
pathogenesis may differ from that of S-layer proteins from other
organisms. Southern and Northern blot analyses with probes from
different segments of the crs gene indicate that the
S-layer gene is a single-copy, monocistronic gene in C. rectus. RNA end mapping and sequence analyses were used to define
the crs promoter; there is an exact match to the
Escherichia coli 
10 promoter consensus sequence but only
a weak match to the 35 consensus element. Southern blots of DNA from
another strain of C. rectus, ATCC 33238, demonstrated that
the crs gene is also present in that strain but that there are numerous restriction fragment length polymorphisms in the second
half of the gene. This finding suggests that the carboxy halves of the
S-layer proteins from strains 314 and 33238 differ. It remains to be
determined whether the diversities in sequence are reflected in
functional or antigenic differences important for the pathogenesis of
different C. rectus isolates.
| |
INTRODUCTION |
Campylobacter rectus
(formerly Wolinella recta), a gram-negative, anaerobic
bacterium, has been strongly implicated in the etiology of adult
periodontitis (31), rapidly progressive periodontitis (11), and periodontitis associated with certain diseases
such as AIDS and diabetes (40, 41). However, the pathogenic
mechanisms of C. rectus are poorly characterized. One strong
candidate for a C. rectus virulence determinant is the
paracrystalline cell surface layer (S-layer), which appears to be
composed of a single protein (21, 26). Although the first 15 amino acids of the S-layer proteins are identical in several strains of
C. rectus, the molecular mass of the S-layer protein varies
from 150 to 166 kDa among strains (30). Nitta et al.
(30) have also shown, by peptide analysis, that there is
internal sequence heterogeneity between the S-layer proteins from
strains 314 and ATCC 33238.
Evidence that the C. rectus S-layer is a virulence factor
stems from studies of a strain of C. rectus that lost its
S-layer during long-term in vitro subculture (7). The
S-layer negative cells were more adherent to human gingival fibroblasts
than were other strains of C. rectus which had their
S-layers (7). In addition, strains which had been passaged
15 to 17 times in vitro formed smaller lesions in a mouse abscess model
for soft tissue destruction than did low-passage C. rectus
strains (19). These studies have led to the proposal that
the C. rectus S-layer helps the organism evade host defense
mechanisms. However, the results need to be interpreted cautiously
since comparisons were being made between unrelated strains and because
the levels of proteins other than the S-layer protein are also
different between low-passage and high-passage C. rectus
cells (7).
A role for the S-layer in pathogenesis has been shown for other
bacteria. For example, the S-layer of the fish pathogen Aeromonas salmonicida protects the bacterial cells against proteolysis and complement lysis and is required for macrophage resistance (14, 15, 29, 36). Similarly, Campylobacter fetus, which
causes bovine infertility, has an S-layer that makes the organism
resistant to phagocytic uptake and to the bactericidal activity of
serum (3, 5, 27). Although the S-layer is involved in
evading host defense mechanisms in these two bacteria, the precise
roles of the two S-layers are quite distinct. This is not necessarily surprising given that the S-layer proteins from C. fetus and
A. salmonicida are not homologous (25).
To begin to determine the role of the C. rectus S-layer in
pathogenesis, the gene encoding this putative virulence factor has been
cloned, sequenced, and characterized. The C. rectus crs gene
from strain 314 encodes an S-layer protein with limited amino acid
sequence similarity to S-layer proteins from other bacteria. The
S-layer gene is single copy and part of a single-gene operon. Although
the gene is present in another strain, 33238, there are many
restriction site polymorphisms between the two strains. The polymorphisms are limited to the second half of the crs
gene, and the 33238 restriction map is consistent with that reported previously by Miyamoto et al. (28). These results suggest
that the precise function of the C. rectus S-layer in
pathogenesis may differ from the molecular roles for the S-layers from
other organisms.
| |
MATERIALS AND METHODS |
Bacterial strains and culture conditions.
C. rectus
314 and ATCC 33238 were originally clinical isolates (7,
35). C. rectus ATCC 33238 (S
) is a
variant of the ATCC 33238 strain whose S-layer was lost spontaneously
during in vitro passage (7). C. rectus cells were
grown in mycoplasma-formate-fumarate (MFF) broth (17), supplemented with 5 µg of hemin per ml and 10% horse serum, in a Coy
anaerobic growth chamber (5% CO2, 10% H2,
85% N2) at 37°C. The strains were maintained by transfer
on MFF agar containing 5 µg of hemin per ml and 10% horse serum
twice a week. Plasmid pUC19 was used as a vector for cloning.
Recombinant constructs were propagated in Escherichia coli
TB-1 in Luria broth medium after transformation by the
CaCl2 procedure or by electroporation (2).
Isolation and labeling of hybridization probes.
DNA
fragments used as hybridization probes were isolated from agarose gels
by a freeze-thaw-phenol extraction procedure described previously
(34). DNA probes were labeled with
[
-32P]dATP by using a nick translation labeling kit or
a random primer DNA labeling system from Life Technologies
(Gaithersburg, Md.). For oligonucleotide probes, the 5' end was labeled
with T4 polynucleotide kinase and [
-32P]ATP
(2).
DNA isolations and Southern blots.
Chromosomal DNA was
isolated from C. rectus 314 by a detergent-proteinase K
lysis procedure that included treatment with cetyltrimethyl ammonium
bromide to remove polysaccharides and cell debris (2). Plasmid DNAs from E. coli were prepared by a miniprep method
involving alkaline lysis and boiling (2).
Southern blot hybridizations were done as described previously
(23). Hybridizations normally were carried out overnight at
65°C for DNA fragment probes and at 42°C for oligonucleotide probes
(22). The filters were washed three times in 2× SSC (1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate) at 65°C after DNA
fragment probe hybridizations and in 6× SSC at 42°C after oligonucleotide probe hybridizations. For low-stringency experiments with the DNA fragment probe, the hybridization and washes were done at
55°C in 2× SSC.
Cloning the crs gene.
Oligonucleotides CR102 and
CR103 (5'-GCICCYTCIGGIACRTCICCRAA-3' and
5'-GCIYTIACICARACICAAGT-3', where I = inosine, Y = C or T,
and R = A or G) were designed from the previously determined sequence of the amino-terminal 22 amino acids of the purified S-layer
protein (30). The primers, 2.5 µg of each, were used in a
PCR with 0.5 µg of genomic DNA from C. rectus 314 as a
template. After an initial 7-min denaturation step at 93°C, the
reaction was amplified for 39 cycles (2 min at 93°C, 1 min at 45°C,
and 1 min at 72°C) in a model PTC-100 thermocycler from MJ Research. The 65-bp PCR product generated was cloned into pUC19 and sequenced. A
22-base non-primer-derived sequence from the middle of the PCR fragment
was used to design another oligonucleotide, which was used as a
hybridization probe to clone a 3.0-kb SacI DNA fragment from
a limited genomic library (23) of SacI-digested,
size-separated C. rectus 314 DNA in pUC19. DNA sequence
analysis of part of the clone, pDK572 (Fig.
1), indicated that it only contained the
beginning of the crs gene.
View larger version (9K):
[in this window]
[in a new window]
|
FIG. 1.
Restriction endonuclease maps of the S-layer gene region
from C. rectus 314 and ATCC 33238. Key restriction
endonuclease sites are marked, but all restriction endonuclease sites
are not shown. Restriction endonuclease sites that differ between the
two strains are underlined. The extent of the coding region of the
crs gene, determined by sequencing, is delimited by vertical
lines. The direction of transcription of the S-layer RNA is indicated
by the large arrow, as is the position of the RNA start site. The
positions of the probes used in Southern and Northern blot analyses are
indicated by the lines labeled I, II, and III. The size of the DNA
fragment used in the S1 nuclease analysis of the RNA start site is
shown by the small arrow labeled S1 probe. B, BstEII; Bs,
BsaI; C, ClaI; H, HindIII; P,
PstI; S, SacI; Sc, ScaI.
|
|
To clone more of the coding region of the
crs gene, a 170-bp
PstI/
EcoRI fragment from clone pDK572 was used as
a hybridization
probe of a
ClaI-digested, size-separated
limited genomic DNA library.
Clone pDK580 (Fig.
1) contained the first
half of the
crs gene
on a 1.9-kb
ClaI fragment in
the
AccI site of pUC19. Finally,
the second half of the gene
was cloned from a
SacI-limited genomic
DNA library, on a
7.3-kb
SacI fragment, using a 150-bp
PstI
fragment
from the most
crs promoter-distal region of the
insert in pDK580
as probe. This clone was designated pDK594 (Fig.
1).
DNA sequencing and computer analysis.
The nucleotide
sequence of the S-layer gene was determined by the dideoxy-chain
termination method. Portions of some clones were sequenced by using a
Sequenase sequencing kit (United States Biochemical), but the majority
of the DNA was sequenced in the Center for Advanced Technology at the
University of Texas Health Science Center at San Antonio, using an
Applied Biosystems model 373A sequencing system. All sequences were
determined independently from both strands. The deduced amino acid
sequence of the C. rectus S-layer protein was compared to
the nonredundant protein database of the National Center for
Biotechnology Information, using the BLAST searching algorithm
(1). The Multalin 4.0 software (Cherwell Scientific) was
used to find the best alignment between the C. rectus and
C. fetus S-layer proteins.
RNA isolation and analyses.
C. rectus RNA was prepared
by a sodium dodecyl sulfate lysis-CsCl cushion procedure previously
used for isolation of RNA from Actinobacillus
actinomycetemcomitans (24). RNAs were isolated from
50-ml samples of C. rectus cells in the mid-logarithmic
phase of growth (optical density of 0.2 to 0.4 at 660 nm). Northern blots were performed as previously described (12).
S1 nuclease analyses were done as described previously (
8,
24), with minor modifications. The DNA probe was end labeled
with
T4 polynucleotide kinase and [
-
32P]ATP, and about 20 ng of probe was mixed with 5 to 45 µg of RNA
in 15 µl of
hybridization buffer [0.83 M NaCl, 1.7 M
piperazine-
N,
N'-bis(2-ethanesulfonic
acid)
(PIPES; pH 6.5)]. After being boiled for 3 min, the samples
were
incubated at 67°C for 60 min, and 300 µl of iced S1 nuclease
buffer
and 983 U of S1 nuclease were added. The samples were incubated
at
37°C for 30 min and then extracted with phenol and precipitated
with
ethanol. The pellets were dissolved in 3 µl of TE buffer
(10 mM Tris
[pH 7.4], 1 mM EDTA), 2 µl of sequencing sample buffer
was added,
and the samples were electrophoresed on 6% polyacrylamide-urea
gels
after being heated at 75°C for 2 min.
The protocol of Kolodrubetz et al. (
24), with minor changes,
was used for primer extension assays. About 0.7 pmol of end-labeled
oligonucleotide primer was mixed with 1 to 45 µg of RNA in 30
µl of
reverse transcriptase buffer (50 mM Tris [pH 8.3], 75 mM
KCl, 3 mM
MgCl
2). The mixture was heated to 85°C for 3 min and
then
allowed to anneal at 37°C for 90 min. One microliter of Moloney
murine leukemia virus reverse transcriptase (200 U/µl; Life
Technologies)
and 2 µl of 0.1 M dithiothreitol were added to the
samples along
with each deoxynucleoside triphosphate to 1.25 mM. The
total volume
of the reaction was brought to 40 µl by the addition of
2 µl of
5× reverse transcriptase buffer and 3 µl of diethyl
pyrocarbonate-treated
water. After 1 h of incubation at 37°C,
each sample was precipitated
with ethanol and electrophoresed as
described above.
Nucleotide sequence accession number.
The nucleotide
sequence of the crs gene has been deposited in GenBank with
accession no. AF010143.
| |
RESULTS AND DISCUSSION |
The crs gene from C. rectus encodes a novel
member of the S-layer protein family.
The sequence of the
amino-terminal 22 amino acids of the purified S-layer protein
(30) was used to design two degenerate oligonucleotides for
use in PCR with genomic DNA from C. rectus 314 as the
template. A 65-bp PCR product encoding the amino terminus of the
S-layer protein was generated, as expected. Because the oligonucleotides used to make the PCR product were degenerate, a
22-base oligonucleotide whose sequence was derived from the middle of
the PCR fragment was used as a hybridization probe to clone a 3.0-kb
SacI DNA fragment from a limited genomic library. Partial
DNA sequencing of this clone, pDK572 (Fig. 1), indicated that it
contained part of the crs gene, since near one end of the
fragment, there was a partial open reading frame whose amino-terminal sequence matched the previously determined amino acid sequence of the
protein. In addition to proving that the correct gene had been cloned,
this result indicates that the C. rectus S-layer protein is
placed on the outside of the cell without the cleavage of a signal
peptide. So far, the S-layer proteins from Caulobacter crescentus and Campylobacter spp. are the only ones
which are synthesized without a cleavable signal peptide
(6).
The rest of the S-layer gene was then cloned, by hybridization, as two
overlapping DNA fragments: a 1.9-kb
ClaI fragment
containing
the first half of the gene (pDK580), and a 7.3-kb
SacI fragment
containing the second half of the gene as
well as over 4.5 kb
of 3' flanking sequence (pDK594) (Fig.
1). The
nucleotide sequence
of the
crs gene was determined
independently from both DNA strands.
Analysis of the DNA sequence
revealed a 4.1-kb open reading frame
encoding a protein of 1,361 amino
acids with a calculated size
of 144 kDa (Fig.
1 and
2). When the deduced amino acid sequence
was used to search the protein databases with the BLAST program,
the
four sequenced S-layer proteins (SapA, SapA1, SapA2, and SapB)
from
C. fetus (
4,
9,
10,
38) gave the most significant
matches. However, the matches were limited to several short
(<30-amino-acid
long) regions. The program Multalin was used to find
the best
alignment of the
C. rectus S-layer protein with
each
C. fetus protein; SapA2 gave the alignment with the
most identities, 15.8%
(Fig.
2). There were two small regions with
more striking identity.
The amino termini of the two proteins were
identical in 15 of
28 amino acids, and there was a stretch, centered at
position
495, in which 9 of 11 amino acids were identical between the
two
proteins. This latter segment of identity was not present in the
other three Sap proteins from
C. fetus, and the SapB protein
of
C. fetus matched the amino terminus of the
C. rectus Crs protein
at only 10 of 28 amino acids. Overall, since
the S-layer protein
from
C. rectus shows less than 16%
identity with any other S-layer
protein, we conclude that the
C. rectus crs gene encodes a novel
S-layer protein.
View larger version (70K):
[in this window]
[in a new window]
|
FIG. 2.
Deduced amino acid sequence of the C. rectus
S-layer protein from strain 314 aligned with the S-layer protein A2
from C. fetus (9). The residues that are
identical between the two sequences are marked by dots. The two
sequences were aligned by using the Multalin 4.0 alignment program
(Cherwell Scientific). The alignment shown is the one in which gaps
were minimized.
|
|
Finally, it is interesting that the first 16 amino acids of the
C. rectus S-layer protein are identical to those of a
104-kDa
cytotoxin secreted by
C. rectus 33238, the only
strain in which
it was looked for (
18). The amino-terminal
amino acid sequence
for this cytotoxin was derived from protein
sequencing, and so
the extent of its identity with the S-layer protein
is unknown.
Having the cloned
crs gene will facilitate
future genetic analyses
to determine whether the cytotoxin and S-layer
protein are made
from the same gene.
crs from C. rectus is a single-copy
monocistronic gene.
In C. fetus, there are multiple
S-layer genes which are involved in the S-layer antigenic variation
found in that organism (38). To determine whether the
crs gene is present in multiple copies in C. rectus, DNA from strain 314 was analyzed by Southern blotting
using three nonoverlapping probes (Fig. 1): probe I, a 1.9-kb
ClaI fragment which contains the first half of the
crs gene; probe II, a 0.7-kb
PstI/AflII fragment from the center of the gene;
and probe III, a 1.5-kb AflII fragment encompassing the last
third of the gene. At normal stringency, the only significant hybridizations with each of the probes were to the DNA fragments of the
sizes expected from the sequence of the crs region (Fig. 3A). These data suggest that
crs is a single-copy gene in the chromosome of C. rectus, the same conclusion that Miyamoto et al. (28)
reached in their Southern blot analysis of the crs gene from
another strain, 33238. To extend this conclusion, the Southern blot
analyses were repeated under lower-stringency conditions using two DNA
fragments which together contain the entire crs gene as
hybridization probes. The hybridization and washes were done at a
temperature and salt concentration which we have shown previously to
generate a strong hybridization signal with as little as 75% identity
over 200 bp between the probe and the target DNAs (22). A
comparison of the hybridization signals found at high stringency (Fig.
3A) and low stringency (Fig. 3B) shows that there are no new signals
found under low stringency. This observation confirms the conclusion
that the S-layer gene is a single-copy gene in C. rectus.
These results are also consistent with the lack of any reports of
antigenic variation of the S-layer within strain 314. Although the
antigenicity of the C. rectus S-layer has been reported to
differ between strains (20, 30), no antigenic differences
are found when a given strain is passaged multiple times in vitro
(7). This differs from what is found with C. fetus, where long-term passage does result in a shift to
antigenically different S-layer proteins (39).
View larger version (59K):
[in this window]
[in a new window]
|
FIG. 3.
Hybridization of S-layer gene probes to genomic DNA from
C. rectus 314. DNA (8 µg) from strain 314 was digested
with the indicated restriction endonucleases (C, ClaI; E,
EcoRI; H, HindIII; P, PstI),
electrophoresed on a 0.75% agarose gel, and analyzed by Southern blot
hybridization. (A) Each blot was hybridized with one of
32P-labeled probes I, II, and III, which are from different
segments of the crs gene (Fig. 1). The filters were
hybridized and washed under normal-stringency conditions (65°C). (B)
The blot was hybridized with two 32P-labeled probes at the
same time; the probes were a 1.94-kb BstEII/ClaI
DNA fragment containing the first half of the crs gene and a
2.54-kb SacI/BsaI DNA fragment containing the
second half of the gene. The hybridization and washings were done at a
lower stringency (55°C). The faint hybridization bands are due to
incomplete digestions with some restriction endonucleases. The
positions of the molecular size standards are indicated.
|
|
Bacterial genes are often organized in operons containing other genes
that are involved in the same metabolic or regulatory
pathway. To
determine whether the
crs gene is cotranscribed with
other
genes, RNA from
C. rectus was analyzed by Northern blotting.
When RNA from strain 314 was hybridized with a
crs probe, a
4.3-kb
hybridization signal was seen (Fig.
4A). No larger band was found
even after
a long exposure (data not shown). Since the transcript
is only 200 bases longer than the
crs coding region, we conclude
that
the
crs gene in
C. rectus is monocistronic. This
is similar
to what has been found for the S-layer genes in other
bacteria
where it has been examined (
25).
View larger version (44K):
[in this window]
[in a new window]
|
FIG. 4.
Expression of S-layer RNA in various strains of C. rectus. RNAs isolated from C. rectus 314, ATCC 33238 (S+), and ATCC 33238 (S) and from A. actinomycetemcomitans Y4 (37) were used in Northern
blot analyses. The two S lanes are RNAs prepared from the
same strain at different times. (A) The blot was first hybridized with
32P-labeled probe I, corresponding to a segment from the
first half of the crs gene (Fig. 1). (B) The blot in panel A
was stripped and then hybridized to a 32P-labeled DNA
fragment from the glyA gene of A. actinomycetemcomitans.
|
|
To identify potential promoter sequences for the
crs gene,
the position of the 5' start site of the
crs mRNA was
determined.
In S1 nuclease protection assays with a 543-bp end-labeled
HindIII/
PstI
fragment encompassing the region
from +112 to
431 (Fig.
1), two
RNA-dependent bands were protected
from S1 nuclease (Fig.
5A).
The major
protected band is approximately 250 bp in size, which
indicates that
the major RNA start site is around position
140
relative to the
crs coding region (Fig.
5C). The minor protected
band, with
a size of 195 bp, suggests that there might be a second
RNA start site
at position
86. To confirm these results and to
map the
crs RNA start site(s) more accurately, primer extension
was
performed with a primer that is 73 bp downstream of the minor
band. One
RNA-dependent band was found (Fig.
5B); its size indicated
that the
crs RNA initiated at position
140, consistent with the
position found for the major RNA start site by S1 nuclease analysis.
There was no primer extension product at the position expected
for the
minor band found in the S1 analysis. The minor band could
be an S1
artifact or could represent the 3' end of an upstream
RNA since the
HindIII end of the S1 probe was also labeled. We
conclude that the
crs RNA initiates at one position 140 bp
before
the
crs coding region.
View larger version (69K):
[in this window]
[in a new window]
|
FIG. 5.
Determination of the 5' end of the S-layer RNA. (A) S1
nuclease protection experiment using an end-labeled 543-bp
PstI/HindIII fragment (probe) encompassing
the promoter region of the crs gene (Fig. 1). In reaction c,
the probe was hybridized to RNA from strain 314 and then subjected to
S1 nuclease treatment as described in Materials and Methods. The lane
marked stds contains a radiolabeled 123-bp ladder DNA (Life
Technologies). (B) Primer extension reaction products obtained with the
5'-end-labeled primer CR120, which is the reverse complement of the
sequence at the start of the crs coding region (C), and
increasing amounts of RNA (1, 3, 15, and 45 µg) from C. rectus 314. The samples (lanes a to d) were electrophoresed on a
high-resolution sequencing gel alongside the products of a dideoxy
sequencing reaction (seq. rxn.) of 314 DNA with primer CR120. (C)
Sequence of the region upstream of the S-layer coding sequence. The
position of the codon for the first amino acid is designated +1. The
dot marks the S-layer RNA start site. The putative 10 and 35
promoter elements are indicated by lines. The position of
oligonucleotide CR120, which is the reverse complement of the sequence
shown, is marked by the arrow.
|
|
The DNA upstream of the
crs RNA initiation site was examined
for potential promoter elements. A hexamer sequence that is identical
to the
E. coli 10 consensus promoter element was found 10 bp
in front of the
crs RNA start site (Fig.
5C). When the
region
16 to 20 bp further upstream was examined, the best match to the
E. coli 35 consensus sequence was identical only at three
of
six bases (Fig.
5C). Interestingly, the 20 bp immediately upstream
of this putative
35 element are very A/T rich. This resembles
the UP
element that has been identified at the same position in
the promoters
for some strongly expressed genes in
E. coli (
32,
33) and other organisms (
16). Proof that this sequence
has
a similar function in the
C. rectus crs promoter awaits
further
experimentation.
As has also been found with S-layer genes in other bacteria
(
25), the leader sequence for the
C. rectus crs
RNA is rather
long, 140 bp. Interestingly, even though the promoter
regions
of the
crs gene and the
sapA S-layer gene
from
C. fetus show no
significant similarities, the first 27 bases of the
C. rectus S-layer RNA are almost identical (26 of 27 bases) to the 5' end
of the
sapA S-layer RNA from
C. fetus (
38). Contained in this
conserved region
is a 12-base palindrome 5'-ACTATCGATAGT-3' whose
function is
unknown, although such sequences in the leader segments
of RNA may play
a role in RNA stability (
13).
The second half of the S-layer gene from strain ATCC 33238 shows
restriction fragment length polymorphisms.
Nitta et al.
(30) used peptide analysis to show that the S-layer proteins
from strains 314 and ATCC 33238 differed. Although the amino-terminal
sequences and the sequences of one peptide were identical between the
two strains, another peptide from 33238 showed no homology to any
peptide from strain 314. This finding suggests that the sequences of
the crs genes from 314 and ATCC 33238 should show some
polymorphisms. To test this possibility, we looked for restriction site
differences between the crs genes of the two strains by
Southern blot analysis. The hybridization patterns of DNA from strains
314 and ATCC 33238 were compared after digestion with one of several
different restriction endonucleases and subsequent hybridization with
probes from three regions of the crs gene. With all three
probes, there were several restriction fragment length polymorphisms
(compare Fig. 3 and 6). The restriction map generated from the Southern
blot data for strain 33238 is consistent with that of Miyamoto et al.
(28), and comparison to the map for strain 314 suggests that
the sequences between strains 314 and 33238 are homologous for the
first half of the crs gene but diverge in the second half
(Fig. 1). This result is also supported by the previous peptide
analysis of the S-layer proteins from the two strains (30).
The peptides whose sequences were identical between 314 and 33238 are
identical to deduced sequences from the beginning of the gene, whereas
the peptide from strain 33238 that is different has homology with the
deduced sequence from the second half of the crs gene. It is
yet to be determined whether the differences in the carboxy portion of
the S-layer proteins from the two C. rectus strains impart a
difference in function or simply reflect the antigenic diversity of
proteins with the same function.
A previously identified S-layer-negative isolate of C. rectus has the crs gene but not the full-length
crs RNA.
ATCC 33238 (S) is a strain
whose S-layer was lost spontaneously during long term in vitro passage
of strain 33238. The reason for the loss of the S-layer is unknown, as
is the possible occurrence of other mutations in this strain. To begin
to determine the molecular basis for the loss of the S-layer in this
strain, Southern and Northern blot analyses were performed. On Southern
blots, the hybridization patterns were identical for DNA from the
CRS+ and CRS strains of ATCC 33238 (Fig.
6). This finding indicates that the crs gene is present in the spontaneous CRS
isolate and that there are no insertions or deletions larger than 100 bp in the crs gene from the spontaneous CRS
isolate. Miyamoto et al. (28) reached the same conclusion in their analysis of strain 33384 but did not delve further into the
reason for the lack of an S-layer in the spontaneous mutant.
View larger version (75K):
[in this window]
[in a new window]
|
FIG. 6.
Hybridization of S-layer gene probes to genomic DNA from
various C. rectus strains. DNA (8 µg) from ATCC 33238 (S+) and ATCC 33238 (S) was digested with the
indicated restriction endonucleases (C, ClaI; E,
EcoRI; H, HindIII; P, PstI),
electrophoresed on a 0.75% agarose gel, and analyzed by Southern blot
hybridization. Each blot was hybridized with one of the
32P-labeled probes I, II, or III, which are from different
segments of the crs gene (Fig. 1). The filters were
hybridized and washed under normal-stringency conditions. The positions
of the molecular size standards are indicated.
|
|
Although the
crs gene is present in the spontaneous
CRS
isolate, the intact 4.4-kb
crs RNA was not
found by Northern blot
analysis (Fig.
4A). Instead, a broad
hybridization signal ranging
from 1 to 2 kb in size was found. This
signal was seen only when
probe I, which recognizes the first half of
the
crs RNA, was used
in the hybridization (data for probes
II and III are not shown).
All three probes hybridized to a 4.3-kb RNA
in the parental strain,
CRS
+ 33238, indicating that the
hybridization to a smaller RNA in
the CRS
33238 isolate
is not due to strain differences with strain 314.
Finally, the smaller
crs RNA in the CRS
isolate is not due to
general RNA degradation in that sample
since another hybridization
probe, the
glyA gene from
A. actinomycetemcomitans,
gave a similar signal on Northern blots
with RNA from all three
strains (Fig.
4B). These results suggest that
the initiation of
crs RNA synthesis may be normal in the
spontaneous CRS
isolate of 33238 but that the RNA either
terminates early in
the gene or is rapidly degraded from the 3' end.
The availability of the cloned S-layer gene from
C. rectus
will allow molecular genetic approaches to be used in the analysis
of
the function of the S-layer protein in pathogenesis. In particular,
the
tools are being developed to create isogenic
crs and
crs+ strains of
C. rectus in order to
elucidate directly the importance
of the S-layer as a virulence factor
in in vivo and in vitro studies.
| |
ACKNOWLEDGMENTS |
We thank L. Phillips for cloning the initial PCR product. We
appreciate the helpful discussions with S. Holt and J. Ebersole.
This work was supported by Public Health Service grant DE-10960 from
the National Institutes of Health.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Microbiology, University of Texas Health Science Center, 7703 Floyd
Curl Dr., San Antonio, TX 78284. Phone: (210) 567-3967. Fax: (210) 567-6612. E-mail: djk{at}giskard.uthscsa.edu.
Editor: J. G. Cannon
| |
REFERENCES |
| 1.
|
Altschul, S. F.,
W. Gish,
W. Miller,
E. W. Myers, and D. J. Lipman.
1990.
Basic local alignment search tool.
J. Mol. Biol.
215:403-410[Medline].
|
| 2.
|
Ausubel, F. M.,
R. Brent,
R. E. Kingston,
D. D. Moore,
J. G. Seidman,
J. A. Smith, and K. Struhl (ed.).
1994.
.
Current protocols in molecular biology.
Wiley Interscience, New York, N.Y.
|
| 3.
|
Blaser, M. J.
1994.
Role of the S-layer proteins of Campylobacter fetus in serum-resistance and antigenic variation: a model of bacterial pathogenesis.
Am. J. Med. Sci.
306:325-329.
|
| 4.
|
Blaser, M. J., and E. C. Gotschlich.
1990.
Surface array protein of Campylobacter fetus. Cloning and gene structure.
J. Biol. Chem.
265:14529-14535[Abstract/Free Full Text].
|
| 5.
|
Blaser, M. J.,
P. F. Smith,
J. E. Repine, and K. A. Joiner.
1988.
Pathogenesis of Campylobacter fetus infections. Failure of encapsulated Campylobacter fetus to bind C3b explains serum and phagocytosis resistance.
J. Clin. Invest.
81:1434-1444.
|
| 6.
|
Boot, H. J., and P. H. Pouwels.
1996.
Expression, secretion and antigenic variation of bacterial S-layer proteins.
Mol. Microbiol.
21:1117-1123[Medline].
|
| 7.
|
Borinski, R., and S. C. Holt.
1990.
Surface characteristics of Wolinella recta ATCC 33238 and human clinical isolates: correlation of structure with function.
Infect. Immun.
58:2770-2776[Abstract/Free Full Text].
|
| 8.
|
Deretic, V., and W. M. Konyecsni.
1989.
Control of mucoidy in Pseudomonas aeruginosa: transcriptional regulation of algR and identification of the second regulatory gene, algQ.
J. Bacteriol.
171:3680-3688[Abstract/Free Full Text].
|
| 9.
|
Dworkin, J.,
M. K. Tummuru, and M. J. Blaser.
1995.
A lipopolysaccharide-binding domain of the Campylobacter fetus S-layer protein resides within the conserved N terminus of a family of silent and divergent homologs.
J. Bacteriol.
177:1734-1741[Abstract/Free Full Text].
|
| 10.
|
Dworkin, J.,
M. K. Tummuru, and M. J. Blaser.
1995.
Segmental conservation of sapA sequences in type B Campylobacter fetus cells.
J. Biol. Chem.
270:15093-15101[Abstract/Free Full Text].
|
| 11.
|
Dzink, J. L.,
S. S. Socransky, and A. D. Haffajee.
1988.
The predominant cultivable microbiota of active and inactive lesions of destructive periodontal diseases.
J. Clin. Periodontol.
15:316-323[Medline].
|
| 12.
| Ebersole, J. L., E. Kraig, G. Bauman, J. K. Spitznagel, and D. Kolodrubetz. 1990. Molecular approaches to
leucotoxin as a virulence component in Actinobacillus
actinomycetemcomitans. Arch. Oral Biol.
35(Suppl.):69S-78S.
|
| 13.
|
Emory, S. A.,
P. Bouvet, and J. G. Belasco.
1992.
A 5'-terminal stem-loop structure can stabilize mRNA in Escherichia coli.
Genes Dev.
6:135-148[Abstract/Free Full Text].
|
| 14.
|
Evenberg, D., and B. Lugtenberg.
1982.
Cell surface of the fish pathogenic bacterium Aeromonas salmonicida. II. Purification and characterization of a major cell envelope protein related to autoagglutination, adhesion and virulence.
Biochim. Biophys. Acta
684:249-254[Medline].
|
| 15.
|
Evenberg, D.,
R. Versluis, and B. Lugtenberg.
1985.
Biochemical and immunological characterization of the cell surface of the fish pathogenic bacterium Aeromonas salmonicida.
Biochim. Biophys. Acta
815:233-244[Medline].
|
| 16.
|
Fredrick, K.,
T. Caramori,
Y. F. Chen,
A. Galizzi, and J. D. Helmann.
1995.
Promoter architecture in the flagellar regulon of Bacillus subtilis: high-level expression of flagellin by the sigma D RNA polymerase requires an upstream promoter element.
Proc. Natl. Acad. Sci. USA
92:2582-2586[Abstract/Free Full Text].
|
| 17.
|
Gillespie, J., and S. C. Holt.
1987.
Growth studies of Wolinella recta, a gram-negative periodontopathogen.
Oral Microbiol. Immunol.
2:105-111[Medline].
|
| 18.
|
Gillespie, M. J.,
J. Smutko,
G. G. Haraszthy, and J. J. Zambon.
1993.
Isolation and partial characterization of the Campylobacter rectus cytotoxin.
Microb. Pathog.
14:203-215[Medline].
|
| 19.
|
Kesavalu, L.,
S. C. Holt,
R. R. Crawley,
R. Borinski, and J. L. Ebersole.
1991.
Virulence of Wolinella recta in a murine abscess model.
Infect. Immun.
59:2806-2817[Abstract/Free Full Text].
|
| 20.
|
Kobayashi, Y.,
H. Ohta,
S. Kokeguchi,
Y. Murayama,
K. Kato,
H. Kurihara, and K. Fukui.
1993.
Antigenic properties of Campylobacter rectus (Wolinella recta) major S-layer proteins.
FEMS Microbiol. Lett.
108:275-280[Medline].
|
| 21.
|
Kokeguchi, S.,
K. Kato,
H. Kurihara, and Y. Murayama.
1989.
Cell surface protein antigen from Wolinella recta ATCC 33238.
J. Clin. Microbiol.
27:1210-1217[Abstract/Free Full Text].
|
| 22.
|
Kolodrubetz, D., and A. Burgum.
1990.
Duplicated NHP6 genes of Saccharomyces cerevisiae encode proteins homologous to bovine high mobility group protein 1.
J. Biol. Chem.
265:3234-3239[Abstract/Free Full Text].
|
| 23.
|
Kolodrubetz, D.,
T. Dailey,
J. Ebersole, and E. Kraig.
1989.
Cloning and expression of the leukotoxin gene from Actinobacillus actinomycetemcomitans.
Infect. Immun.
57:1465-1469[Abstract/Free Full Text].
|
| 24.
|
Kolodrubetz, D.,
J. Spitznagel, Jr.,
B. Wang,
L. H. Phillips,
C. Jacobs, and E. Kraig.
1996.
cis elements and trans factors are both important in strain-specific regulation of the leukotoxin gene in Actinobacillus actinomycetemcomitans.
Infect. Immun.
64:3451-3460[Abstract].
|
| 25.
|
Kuen, B., and W. Lubitz.
1996.
Analysis of S-layer proteins and genes, p. 77-102. In
U. B. Sleytr, P. Messner, D. Pum, and M. Sara (ed.), Crystalline bacterial cell surface proteins.
Academic Press, San Diego, Calif.
|
| 26.
|
Lai, C. H.,
M. A. Listgarten,
A. C. R. Tanner, and S. S. Socransky.
1981.
Ultrastructure of Bacteroides gracilis, Campylobacter concisus, Wolinella recta and Eikenella corrodens, all from humans with periodontal disease.
Int. J. Syst. Bacteriol.
31:465-475.
|
| 27.
|
McCoy, E. C.,
D. Doyle,
K. Burda,
L. B. Corbeil, and A. J. Winter.
1975.
Superficial antigens of Campylobacter (Vibrio) fetus: characterization of antiphagocytic component.
Infect. Immun.
11:517-525[Abstract/Free Full Text].
|
| 28.
|
Miyamoto, M.,
Y. Kobayashi,
S. Kokeguchi,
H. Ohta,
H. Kurihara,
K. Fukui, and Y. Murayama.
1994.
Molecular cloning of the S-layer protein gene of Campylobacter rectus ATCC 33238.
FEMS Microbiol. Lett.
116:13-18.
|
| 29.
|
Munn, C. B.,
E. E. Ishiguro,
W. W. Kay, and T. J. Trust.
1982.
Role of surface components in serum resistance of virulent Aeromonas salmonicida.
Infect. Immun.
36:1069-1075[Abstract/Free Full Text].
|
| 30.
|
Nitta, H.,
S. C. Holt, and J. L. Ebersole.
1997.
Purification and characterization of Campylobacter rectus surface layer proteins.
Infect. Immun.
65:478-483[Abstract].
|
| 31.
|
Rams, T. E.,
D. Feik, and J. Slots.
1993.
Campylobacter rectus in human periodontitis.
Oral Microbiol. Immunol.
8:230-235[Medline].
|
| 32.
|
Rao, L.,
W. Ross,
J. A. Appleman,
T. Gaal,
S. Leirmo,
P. J. Schlax,
M. T. Record, Jr., and R. L. Gourse.
1994.
Factor independent activation of rrnB P1. An "extended" promoter with an upstream element that dramatically increases promoter strength.
J. Mol. Biol.
235:1421-1435[Medline].
|
| 33.
|
Ross, W.,
K. K. Gosink,
J. Salomon,
K. Igarashi,
C. Zou,
A. Ishihama,
K. Severinov, and R. L. Gourse.
1993.
A third recognition element in bacterial promoters: DNA binding by the alpha subunit of RNA polymerase.
Science
262:1407-1413[Abstract/Free Full Text].
|
| 34.
|
Silhavy, T. J.,
M. L. Berman, and L. W. Enquist.
1984.
.
Experiments with gene fusions.
Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.
|
| 35.
|
Tanner, A. C. R.,
S. Badger,
C.-H. Lai,
M. A. Listgarten,
R. A. Visconti, and S. S. Socransky.
1981.
Wolinella gen. nov., Wolinella succinogenes (Vibrio succinogenes Wolin et al.) comb. nov., and description of Bacteroides concisus sp. nov., Wolinella recta sp. nov., Campylobacter concisus sp. nov., and Eikenella corrodens from humans with periodontal disease.
Int. J. Syst. Bacteriol.
31:432-445[Abstract/Free Full Text].
|
| 36.
|
Trust, T. J.,
W. W. Kay, and E. E. Ishiguro.
1983.
Cell surface hydrophobicity and macrophage association of Aeromonas salmonicida.
Curr. Microbiol.
9:315-318.
|
| 37.
|
Tsai, C. C.,
W. P. McArthur,
P. C. Baehni,
B. F. Hammond, and N. S. Taichman.
1979.
Extraction and partial characterization of a leukotoxin from a plaque-derived gram-negative microorganism.
Infect. Immun.
25:427-439[Abstract/Free Full Text].
|
| 38.
|
Tummuru, M. K., and M. J. Blaser.
1993.
Rearrangement of sapA homologs with conserved and variable regions in Campylobacter fetus.
Proc. Natl. Acad. Sci. USA
90:7265-7269[Abstract/Free Full Text].
|
| 39.
|
Wang, E.,
M. M. Garcia,
M. S. Blake,
Z. Pei, and M. J. Blaser.
1993.
Shift in S-layer protein expression responsible for antigenic variation in Campylobacter fetus.
J. Bacteriol.
175:4979-4984[Abstract/Free Full Text].
|
| 40.
|
Zambon, J. J.,
H. Reynolds,
J. G. Fisher,
M. Shlossman,
R. Dunford, and R. J. Genco.
1988.
Microbiological and immunological studies of adult periodontitis in patients with noninsulin-dependent diabetes mellitus.
J. Periodontol.
59:23-31[Medline].
|
| 41.
|
Zambon, J. J.,
H. S. Reynolds, and R. J. Genco.
1990.
Studies of the subgingival microflora in patients with acquired immunodeficiency syndrome.
J. Periodontol.
61:699-704[Medline].
|
Infect Immun, April 1998, p. 1521-1526, Vol. 66, No. 4
0019-9567/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
This article has been cited by other articles:
-
Muryoi, N., Sato, M., Kaneko, S., Kawahara, H., Obata, H., Yaish, M. W. F., Griffith, M., Glick, B. R.
(2004). Cloning and Expression of afpA, a Gene Encoding an Antifreeze Protein from the Arctic Plant Growth-Promoting Rhizobacterium Pseudomonas putida GR12-2. J. Bacteriol.
186: 5661-5671
[Abstract]
[Full Text]
-
Wang, B., Kraig, E., Kolodrubetz, D.
(2000). Use of Defined Mutants To Assess the Role of the Campylobacter rectus S-Layer in Bacterium-Epithelial Cell Interactions. Infect. Immun.
68: 1465-1473
[Abstract]
[Full Text]
-
Sára, M., Sleytr, U. B.
(2000). S-Layer Proteins. J. Bacteriol.
182: 859-868
[Full Text]
-
Braun, M., Kuhnert, P., Nicolet, J., Burnens, A. P., Frey, J.
(1999). Cloning and Characterization of Two Bistructural S-Layer-RTX Proteins from Campylobacter rectus. J. Bacteriol.
181: 2501-2506
[Abstract]
[Full Text]
-
Thompson, S. A., Shedd, O. L., Ray, K. C., Beins, M. H., Jorgensen, J. P., Blaser, M. J.
(1998). Campylobacter fetus Surface Layer Proteins Are Transported by a Type I Secretion System. J. Bacteriol.
180: 6450-6458
[Abstract]
[Full Text]
Top
Abstract
Introduction
