From the
Campylobacter fetus cells may exist as either of two
defined serogroups (type A or B) based on their lipopolysaccharide
(LPS) composition. Wild-type strains contain surface array proteins
(S-layer proteins) that have partial antigenic cross-reactivity but
bind exclusively to LPS from homologous (type A or B) cells. Type A
cells possess 8 homologs of sapA, which encodes a 97-kDa
S-layer protein; the gene products of these homologs have a conserved N
terminus of 184 amino acids. To further explore the structural
relationships between the C. fetus S-layer proteins and their
encoding genes, we sought to clone and express an S-layer protein from
type B strain 84-91. The cloned type B gene (sapB) was
similar in structure to the previously cloned type A gene (sapA) and encoded a full-length 936-amino acid (97-kDa)
S-layer protein. Sequence analysis of sapB indicated that the
conserved N-terminal encoding region in sapA was absent but
that the remainder of the ORF (encoding 751 amino acids) was identical
to that of sapA in spite of the nonconserved nature of this
region among sapA homologs. Noncoding sequences both 300 base
pairs 5` and 1000 base pairs 3` to the sapB and sapA
ORFs, including the sapA promoter and transcriptional
terminator sequences, were essentially identical. Southern analyses
revealed that the sapB N-terminal encoding region was
conserved in multiple copies in type B strains but was absent in type A
strains. Recombinant sapA and sapB products bound to
a substantially greater degree to cells of the homologous LPS type
compared with the heterologous LPS type, indicating that the conserved sapA- and sapB-encoded N termini are critical for LPS
binding specificity. The parallel genetic organization and identity at
the nucleotide level in both coding and noncoding regions for sap homologs in types A and B cells indicates the necessity of both
homolog conservation and high fidelity DNA replication in the biology
of sap diversity.
Campylobacter fetus are spiral, microaerophilic
Gram-negative bacteria that cause infertility and infectious abortion
in sheep and cattle and extraintestinal infections in humans
(1-4). As with many other bacteria(5) , wild type C.
fetus strains possess an outermost crystalline surface layer of
regular closely packed protein subunits (S-layer proteins). The C.
fetus S-layer protein has been shown to be a critical virulence
factor in resistance to host immune
defenses(6, 7, 8, 9, 10) . C. fetus strains possessing S-layer proteins
(S
C. fetus strains may stably exist as either of two (type A
or B) defined serogroups based on their lipopolysaccharide (LPS)
In the present
study, we sought to clone and express an S-layer protein from a type B
strain to understand the extent of conservation of the sap homologs across serotype and define the determinants in the type B
protein needed for binding to the type B cell surface. We now report an
unexpected and remarkable homolog conservation across serotype; sapB homologs are naturally occurring chimeras of sapA homologs differing significantly only within their
encoded N termini. This conservation, at the nucleotide level, also
involves noncoding regions surrounding the homologs.
These studies have broadened our understanding of the nature
of the genes encoding C. fetus S-layer proteins from both type
A and type B strains and have further characterized the functional
domains of the proteins. Southern hybridization analyses demonstrate
that the organization of the sap homologs in type B strains is
parallel to that described for type A strains. Both type A and B
strains possess 8 sap homologs with a conserved region
encoding the N terminus and exhibit increasing diversity for the
remainder of the ORFs in a 5` to 3` manner. That all strains possess a
like number of homologs in their genome indicates that recombinatorial
events that accompany changes in S-layer expression (11) must be
highly precise and do not result in homolog duplication or deletion,
consistent with our earlier findings(17, 19) .
The C. fetus S-layer proteins must share relatively conserved
features, as dictated by requirements for crystalline
structure(29, 30) , binding to divalent
cations(14) , and complement resistance(7, 8) .
In addition, S-layer proteins bind specifically to the LPS layer from
homologous (type A or B) but not heterologous cells (this paper and
Refs. 14 and 16). Our data indicate that the N terminus of each of the sapA or sapB products, found exclusively within the
homologous type A or type B strain, respectively, is critical for the
LPS binding specificity. Since C. fetus S-layer proteins are
not glycosylated or lipidated (21), interaction with the LPS must be
directly mediated by that portion of the polypeptide. Although the
N-terminal regions of each homolog differ substantially, it is
uncertain whether the entire region or a limited number of residues is
necessary for LPS binding specificity. The predicted secondary
structures of these homologs are strikingly similar for the first 40
amino acid residues, which may be particularly important in mediating
binding.
The remainder of the sapA and sapB coding
region, which is dispensable for LPS-binding(16) , is conserved
among unrelated type A and type B strains, as are the remainder of the sapA1/sapB1 and sapA2/sapB2 coding
regions. Conservation of these 3` variable regions is present at the
nucleotide level, including flanking noncoding regions (Fig. 2),
and indicates a stringent preservation of these genes. Global
conservation of the sap products may be imposed by the
functional requirements for export, self-assembly, mediation of
complement resistance, and formation of crystalline structures among
all the C. fetus S-layer proteins. Preservation of individual
S-layer proteins across serotype (e.g.sapA and sapB products) also may be necessary since individual S-layer
proteins may be uniquely adapted for the survival of C. fetus within differing environments during its life cycle in
vivo(3, 31) .
The genes encoding S-layer
proteins are able to rearrange with a frequency approximately
10
A characteristic of the C. fetus surface layer is that a single bacterial strain may
express one of three different S-layer proteins ranging from 97 to 149
kDa with one form predominating in mixed
cultures(19, 21, 29, 33) . Since both sapA and sapB were expressed copies while other
cloned homologs were silent(15) ,
That the N-terminal region should be conserved, in a
type-specific manner, was not unexpected. Previous N-terminal amino
acid sequencing demonstrated identity among homologous type SLPs (21, 33) and Southern hybridization in type A strains
using a probe to the N-terminal encoding regions indicated the presence
of multiple (eight) sapA homologs(15) . However, that
the homolog-specific 3` segments also were maintained with essentially
complete fidelity even among divergent type A and B strains was
unexpected. The C. fetus S-layer genes are novel in that they
present a biological system in which the encoded proteins are naturally
chimeric across serotypes. The LPS molecules that define C. fetus serotype (13) differ in carbohydrate composition; compared with
type A LPS, type B LPS contained a novel sugar, L-acofriose,
higher molar ratios of L-rhamnose, and lower molar ratios of D-mannose(12) . It is possible that SLP divergence
between the two serotypes occurred following a change in the LPS
characteristics of the organism, and selection for changes in the
LPS-binding domain of the sap homolog that permitted binding
to the new protein followed. By gene duplication, such changes could
later have been amplified in each of the sap homologs. Why the
differences found in the LPS-binding domain are so extensive given
apparently limited differences in LPS carbohydrate composition (12) must await further analysis.
We thank Drs. Stuart Thompson and Richard Breyer for
helpful discussions and technical assistance.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
) but not spontaneous mutants or chemically-treated
strains lacking S-layers (S
) are resistant to C3b
binding and phagocytosis by polymorphonuclear
leukocytes(7, 8) . The in vivo ability of C. fetus to vary the S-layer protein expressed also
facilitates resistance to antibody-mediated phagocytosis (11).
(
)composition(12, 13) . S-layer proteins
bind specifically to LPS molecules from homologous (type A or B) but
not heterologous cells(14) . Type A cells possess eight sapA homologs that encode 97-kDa or larger S-layer proteins
(15); these homologs are tightly clustered on a 93-kb region
representing less than 10% of the bacterial chromosome(16) .
Cloning of three of these homologs (sapA, sapA1, and sapA2) has demonstrated two regions of identity. The first (5`
conserved region) begins 74 base pairs (bp) upstream of the open
reading frame (ORF), proceeding 552 bp into the ORF to encode the
N-terminal 184 amino acids, and the second (3` conserved region) begins
downstream of the ORF(16, 17, 18) . Beyond the
5` conserved region, the sapA homologs possess a variable
region that diverges 5` to 3` in a semiconservative manner(16) .
Despite this diversity and evidence that sapA homologs undergo
high frequency rearrangement associated with SLP
variation(17, 19) , Southern analyses indicate that sapA, sapA1, and sapA2 are conserved in all
type A strains(16) . Deletion mutagenesis revealed that the type
A S-layer protein binds serospecifically to the C. fetus LPS
via its conserved N-terminal region(16) .
Bacterial Strains and Culture
Conditions
Six C. fetus subspecies fetus strains, of either S or S
phenotype possessing LPS of type A or B, were used in this study (). Stock cultures were stored and grown as described
elsewhere(16) . Escherichia coli strains used in this
study, including DH5
and XL1-blue (Stratagene, La Jolla, CA), were
grown in L broth or on L plates(20) .
Chemicals and
Enzymes
Isopropyl--D-thiogalactopyranoside and
5-bromo-4-chloro-3-indolyl
-D-galactoside were purchased
from Jersey Lab Supply (Livingston, NJ) and were used at 50 and 30
µg/ml, respectively. Restriction enzymes, T4 DNA ligase, and E.
coli DNA polymerase large (Klenow) fragment were from Promega and
U.S. Biochemical Corp. [
-
P]dATP (650
Ci/mmol) was from ICN Radiochemicals (Irvine, CA).
Production of Antiserum to C. fetus S-layer
Proteins
Antiserum to the 97-kDa S-layer protein of type A
strain 82-40 LP was raised in adult New Zealand White female
rabbits and was absorbed by passage through an affinity column of
Zap II-E. coli lysate linked to glass beads. This
antiserum shows broad recognition of C. fetus S-layer proteins
as described elsewhere(21, 22) .
Detection and Purification of Recombinant
Clones
Partial Sau3A digests of chromosomal DNA from
type B strain 84-91 containing fragments of 20-40 kb were
ligated to the XbaI-digested, dephosphorylated, and BamHI-digested ScosI vector, packaged, and transduced into the E. coli strain DH5. Cloning of the gene (sapB)
encoding an S-layer protein from a type B strain, was accomplished by
screening the cosmid bank library with a 1.6-kb PstI sapA-specific fragment derived from pBG1 (18) spanning the
first half of the sapA ORF. A cosmid subclone recognized by
the antiserum to the 97-kDa protein, and possessing a 12-kb insert was
identified, and a 5.4-kb EcoRI fragment encoding a full-length
recombinant S-layer protein (as detected by immunoblot of recombinant E. coli lysate) was subcloned into the pBluescript vector to
produce pBJM120 (Fig. 1).
Figure 1:
Physical map
comparisons of sapA- and sapB-containing plasmids. Arrows beneath pBJM120, pBG1, and pMJ1 represent the locations
of genes and directions of transcription. Openbox,
type B upstream and N-terminal encoding conserved regions; shadedboxes, type A upstream and N-terminal encoding conserved
regions; blackboxes, downstream conserved regions.
The locations of DNA probes used for Southern hybridizations are
indicated by numbers1-8. 1, 0.41-kb
PCR fragment of the sapB conserved region; 2, 0.5-kb ClaI-NdeI fragment of the sapA promoter; 3, 0.42-kb PCR fragment of the sapA conserved region; 4, 0.61-kb PCR fragment of the sapA middle region; 5, 0.38-kb PCR fragment of the sapA C-terminal
region; 6, 0.36-kb PCR fragment of the sapA2 region
proximal to the N terminus; 7, 0.45-kb HindIII
fragment of the sapA2 middle region; 8, 0.33-kb PstI fragment of the sapA2 C terminus. Restriction
endonuclease cleavage sites were as follows: AccI (A), BglII (B), BstI (B1), ClaI (C), EcoRI (E), HincII (H), HindIII (H3), NdeI (N), PstI (P), PvuII (P2), SspI (S), and XbaI (X).
Genetic Techniques and DNA Sequence
Analyses
Chromosomal DNA was prepared as described
previously(23) . Plasmids were isolated by the procedure
described by Birnboim and Doly(24) . All other standard
molecular genetic techniques were used, as described
elsewhere(20) . The nucleotide sequence was determined for both
strands using the dideoxy chain termination reaction (25) and
deposited in GenBank with accession number U25133.
Southern Hybridizations
C. fetus chromosomal DNA was digested with HaeIII, HincII, or HindIII, electrophoresed, transferred to
nylon membranes, and hybridized exactly as described
elsewhere(15) . The probes were gel-purified DNA fragments from
pBG1 (18) or polymerase chain reaction (PCR) products specific
for sapA, sapB, or sapA2 homologs (Fig. 1) and were radiolabeled by primer extension with random
hexameric oligonucleotides(26) . The filters were washed
successively three times for 30 min each in 2 SSC (1
SSC is 0.15 M NaCl plus 0.015 M sodium citrate), 1
SSC, and O.5
SSC at 65 °C and were exposed to XAR-2
x-ray film.
PCR
Chromosomal DNA from each strain was prepared
and used as a template for PCR. Oligonucleotide primers were
5`-GATAGTCCAGGGGCGGCT (ACF), 5`-ATTTTATTAAGGAGTTCG (BCF),
5`-AACCTTATCAAGATCACTAGC (ACR), 5`-AGCTATAGTATCAGCAAC (BCR),
5`-CTACGTAATCATACTGCTACC (A1R), and 5`-AGCTACTGTGATTGTATTAGC (A2R) (see Fig. 9). Samples were processed through 30 cycles of
amplification, denaturing at 95 °C, annealing at 41 °C or 48
°C, and then they were extended at 72 °C. Amplifications from
type A or type B templates were done separately to avoid possible
contamination.
Figure 9:
Reattachment of recombinant S-layer
protein from E. coli lysates to C. fetus S-cells, as
shown by immunoblot with antiserum to the 97-kDa S-layer protein. LanesA represent type A S cells,
and lanesB represent type B S
cells. Full-length recombinant SapA product (encoded on pBG1)
from type A strain and full-length SapB product (encoded on pBJM120)
from type B strain reattached to the homologous type S
cells but not to the heterologous type S
cells. Arrow, position of full-length 97-kDa recombinant
protein.
In Vitro Reattachment of Recombinant S-layer Protein to
the C. fetus Cell Surface
Reattachment of recombinant proteins
was performed exactly as described (16) and then assessed by
immunoblot analysis with anti-97-kDa SLP following SDS-polyacrylamide
gel electrophoresis, as described previously(14) .
Sequence of sapB, Encoding a C. fetus Type B S-layer
Protein
To explore the relationship between the S-layer proteins
and their binding specificities to C. fetus cells of type A or
B LPS, we cloned sapB from C. fetus type B strain
84-91 in pBJM120 (Fig. 1). Physical map comparison
demonstrated identity of restriction sites between pBJM120 (encoding sapB) and pBG1 (encoding sapA; Fig. 1) with the
exception of the region encoding the N termini of SapA and SapB. The
sequence of the sapB insert was determined (Fig. 2A), and only a single ORF greater than 500 bp was
found in any of the six reading frames. This ORF encoded a polypeptide
of 936 amino acids (Fig. 2B) with a predicted molecular
mass of 96.2 kDa and an apparent molecular mass of 97 kDa as estimated
by SDS-polyacrylamide gel electrophoresis (see Fig. 8). Searches
of the protein and gene banks failed to reveal any significant
similarity to other known proteins except other S-layer proteins of C. fetus(16, 17, 18) . Alignment with
the sapA homolog cloned from C. fetus type A strain
23D (18) demonstrated that the two genes (sapA and sapB) were nearly identical except for the 5` (549-bp) region
encoding the N terminus conserved among type A strains (Fig. 2, A-C). The region encoding the N-terminal 183 amino acids
was distinct for type A and type B strains, with apparent conservation
in secondary structure limited to the domain's extremities.
Hydrophilicity and Chou-Fasman analyses predicted a hydrophobic
-sheet turn-hydrophilic
-sheet turn structure with low
surface probability for both sapA and sapB homologs
within their first 40 amino acid residues (Fig. 3, A-C). Other algorithms, including those for flexibility,
amphiphilic sheet, amphiphilic helix, and antigenic index, indicated
substantial structural similarity between the two N-terminal regions
(not shown), despite their significant differences in primary
structure.
Figure 2:
Comparison of sapA and sapB homologs. A, nucleotide sequence comparison.
Promoter elements (-35 and -10), the ribosome-binding site
(Shine-Delgarno sequence, S.D.), and the position of the start
codon (arrow) are indicated. Capital letters, ORF.
Palindromic sequences are indicated by opposing arrows. B,
amino acid sequence comparison. Vertical dash, sequence
identity; colon, conserved substitution; period,
semi-conservative substitution. C, schematic representation of
the sapB and sapA homologs. Arrows beneath sap homologs represent the locations of genes and the
directions of transcription. Shaded boxes represent the
upstream and N terminal encoding conserved regions for the sapA homologs; black boxes, downstream conserved
region; hatched boxes, sapA and sapB region
of coding identity; bold lines, regions of noncoding sequence
identity; small arrow with P, a functional promoter site.
Figure 8:
Panel A,
schematic localization of PCR primers in relation to sapA1 and sapA2 and putative genes sapB1 and sapB2. Panel B, PCR products from sapA homologs present in
type A and type B C. fetus strains. When the forward primer is
based on the conserved region of the sapA homologs (ACF),
amplification occurs only in type A strain (23D). Similarly, when the
forward primer is based on the conserved region of the sapB
homologs (BCF), amplification occurs only for the type B strain
(84-107). Use of sapA1 and sapA2-specific
primers indicates that the variable portion of these genes exists in
both the type A and the type B strains.
Figure 3:
Structural analyses of the deduced
N-terminal 184 amino acids encoded by sapA (black
lines/boxes) and sapB (gray area/boxes). The
hydrophilicity profiles of the two proteins are based on the
Kyte-Doolittle algorithm (34), using a window of 7, as shown in A; the Chou-Fasman algorithm (35) for protein secondary
structure is shown in B; the surface probability profiles,
based on the algorithm of Emini et al. (36), are shown in C. Analyses were performed using the MacVector sequence
analysis programs (IBI, New Haven, CT).
Sequence analyses also indicated that the noncoding
sequences surrounding sapB and sapA were nearly
identical at least 300 bp 5` and 1000 bp 3` to the ORF, including the sapA promoter, translation initiation (Shine-Delgarno)
sequence, transcriptional terminator sequences, and the conserved type
A 3` box (Fig. 2, A and C). However, the
Chi-like sequence (GCTGGTGA) (27, 28) present upstream
of all type A homologs (17) was absent in sapB (Fig. 2B). The present data demonstrate that sapB differs significantly from sapA only in the 5`
region encoding the first 183 amino acids; the homologies and
divergence among the cloned sap family members from type A and
B strains are illustrated in Fig. 2, C and D.
The sapB Homolog Is Conserved among C. fetus Type B
Strains
In type A C. fetus strains, sapA
homologs are conserved, and unrelated strains share similar genomic
organization of these homologs(15, 16, 17) . To
investigate whether the sapB homolog was conserved among type
A and type B strains, a series of Southern hybridizations were
performed. Genomic DNA from five C. fetus strains were
digested with either HindIII or HincII and hybridized
under high stringency conditions with probes 1-5 (Fig. 1).
Probe 3, encoding the type A (sapA) 5` conserved region,
hybridized to multiple (eight) bands in C. fetus strains of
the homologous type A but not the heterologous type B (Fig. 4, leftpanel). In contrast, probe 1, based on the sapB 5` region encoding the N terminus, hybridized to multiple
(eight) bands in C. fetus strains of the homologous type B but
not the heterologous type A (Fig. 4, rightpanel). These results demonstrate that there are multiple sapB homologs in type B strains that possess a conserved 5`
region encoding the N terminus, parallel in organization to that
observed for sapA homologs in type A strains (Fig. 4)(16) . That two unrelated type B strains showed
identical hybridization patterns, indicates the substantial
conservation present in this system. Southern analyses using probes to
the sapA promoter region (Fig. 5, probe2) and middle region of the sapA or sapB ORFs (Fig. 5, probe4) hybridized
to the identical 5.2-kb restriction fragment in both type A and type B
strains, suggesting that both the promoter and coding sequence 3` to
the N-terminal encoding region were conserved in continuity in both
type A and type B strains. An additional hybridizing fragment also was
seen in type A strains (Fig. 5, probes2 and 4) and indicated the presence of an additional homolog in type
A strains. To examine the continuity of the entire sapA and sapB ORFs, probes increasingly 3` were used (Fig. 6, probes4 and 5). These probes, to the
late-middle and C-terminal encoding regions, hybridized to the same
restriction fragment for the C. fetus type B strains and to a
different fragment for the C. fetus type A strains (Fig. 6). In total, these results indicated that sapA
and sapB are conserved in their entirety among type A and type
B wild type C. fetus strains, respectively.
Figure 4:
Southern hybridization of HindIII-digested genomic DNAs from C. fetus type B
strains 84-91 and 84-107 and C. fetus type A
strains 23D, 23B, and LP3, using conserved region probe 3 from sapA (left panel) or probe 1 from sapB (right panel), as indicated in Fig.
1.
Figure 5:
Southern
hybridization of HindIII-digested genomic DNAs from C.
fetus type B strains 84-91 and 84-107 and C. fetus type A strains 23D, 23B, and LP3, using sapA probe 2 (left panel) and 4 (right panel), as indicated in
Fig. 1.
Figure 6:
Southern hybridization of HincII-digested genomic DNAs from C. fetus type B
strains 84-91 and 84-107 and C. fetus type A
strains 23D, 23B, and LP3, using sapA probe 4 (left
panel) or 5 (right panel), as indicated in Fig.
1.
Conservation of sapA Homologs in Type B Strains
To
determine whether the conservation between type A and type B strains of C. fetus can be generalized to other sapA homologs,
we performed Southern hybridizations with probes from regions of the sapA2 homolog (Fig. 7). We previously have shown that
the sapA2 homolog exists as a complete gene copy within type A
strains, and the homology between sapA2 and other sapA homologs diminishes toward the 3` end of the
gene(16) . As expected for type A strains, sapA2 probe
6 hybridized to multiple fragments, whereas the more 3` probe 8
hybridized to only one fragment (Fig. 7). Similarly, for type B
strains, multiple (four) hybridizing bands were observed with probe 6
and fewer (two) were detected with probe 8 (Fig. 7). Continuity
of the sapA2 ORF was demonstrated further by Southern
hybridization using probe 7 (data not shown) and by PCR analyses using sapA2-specific primers located 3` of the N-terminal conserved
region and at the C terminus and indicated that sapA2 exists
in its entirety in type B strains (Fig. 8). Similarly, PCR
analyses indicated that the sapA homolog sapA1 also
exists in its entirety in type B strains (Fig. 8). The Southern
hybridization patterns and PCR analyses (Fig. 5-8 and data
not shown), demonstrate that the organization of the sap homologs was identical among two unrelated wild-type B strains
and, with the exception of minor differences with conserved region
probe 3, was identical among two wild-type A strains. The remarkable
conservation of the 3` variable regions across serotype indicates that
the sapB homologs are naturally occurring chimeras of sapA homologs, differing significantly only within the 5`
region encoding the N terminus. Preservation of the variable regions
among strains of either serotype at the nucleotide level further
indicates that these particular sequences may have important functional
properties.
Figure 7:
Southern
hybridization of HaeIII-digested genomic DNAs from C.
fetus type B strains 84-91 and 84-107 and C. fetus type A strains 23D, 23B, and LP3, using sapA2 probe 6 (left panel) or 8 (right panel), as indicated in Fig.
1.
Recombinant sapB Adheres Specifically to Type B
LPS
C. fetus S-layer proteins bind to the cell surface
in an LPS type-specific manner(14) . Deletion mutagenesis
revealed that the type A S-layer protein binds specifically to the type
A (but not type B) C. fetus LPS via its conserved N-terminal
region(16) . The cloned type B homolog, encoded by sapB, differed from the coding region encoded by sapA
solely within the N-terminal region (Fig. 2B).
Therefore, we anticipated that the binding domain for type B LPS must
reside within its N terminus. To verify this hypothesis, we utilized
the fact that the sapA and sapB products are
naturally occurring chimeras differing only in the N-terminal 183 amino
acids. The full-length recombinant sapA or sapB
products are recognized by antiserum to the 97-kDa SLP, and the lysates
of the host E. coli strains contain equivalent amounts of
these products (data not shown). When we evaluated reattachment to
SC. fetus cells, as expected, the
recombinant sapA and sapB products bound to cells of
the homologous LPS type to a substantially greater extent than to cells
of the heterologous LPS type (Fig. 9). In conjunction with the
previous studies(16) , the present data indicate that the
conserved N terminus of each of the sapA and sapB
gene products is critical for its unique LPS binding specificity.
to
10
/generation(19) .
(
)The conservation at the nucleotide level indicates
that the recombination events accompanying changes in S-layer gene
expression (11) are highly precise so as to preserve the
antecedent sequences, and/or are constrained by particular sequence
requirements. Such requirements may include specific targeting signals
or the presence of substantial identity among sap homologs
allowing for homologous pairing during strand exchange that may
accompany recombination events. The strict conservation of noncoding
flanking sequences is consistent with these hypotheses, and in type A
strains, palindromic sequences flanking the homologs within the
conserved 5` and 3` noncoding regions may serve as the recombination
targeting signals(16) . Recent observations also demonstrate
that recombination among sapA homologs is a recA-dependent process
and suggest the involvement
of homologous sequences in sap rearrangement (32). That both
the coding and noncoding divergent regions should be conserved in
continuity, despite recom-bination, also suggests that the structure of
each sap homolog is never changed.
(
)and
laboratory-passaged type A strains tend toward expression of sapA,
(
)we hypothesize that sapA and sapB genes represent the ``basal''
S-layer genes that are preferentially expressed in vitro. A
smaller S-layer represents less of a metabolic burden to the bacterial
cell and may explain why a 97-kDa S-layer is preferred in
vitro, but it does not explain why other 97-kDa S-layers are not
also favored.
Table: Campylobacter fetus subspecies fetus strains
used in this study
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.