©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Segmental Conservation of sapA Sequences in Type B Campylobacter fetus Cells (*)

Joel Dworkin (1) (2), Murali K. R. Tummuru (1), Martin J. Blaser (1) (2) (3)(§)

From the (1)Division of Infectious Diseases, Department of Medicine and the (2)Department of Microbiology and Immunology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-2605 and (3)Medical Service, Department of Veterans Affairs Medical Center, Nashville, Tennessee 37212

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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.


INTRODUCTION

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) 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).

C. fetus strains may stably exist as either of two (type A or B) defined serogroups based on their lipopolysaccharide (LPS)()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) .

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.


EXPERIMENTAL PROCEDURES

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) .


RESULTS

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.


DISCUSSION

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 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.

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) ,()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.

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.

  
Table: Campylobacter fetus subspecies fetus strains used in this study



FOOTNOTES

*
This work was supported in part by National Institutes of Health Grant RO1-AI24145 and by the Medical Research Service of the Department of Veterans Affairs. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Vanderbilt University School of Medicine, Division of Infectious Diseases, A-3310 Medical Center North, Nashville, TN 37232-2605. Tel.: 615-322-2035; Fax: 615-343-6160.

The abbreviations used are: LPS, lipopolysaccharide; kb, kilobase(s); bp, base pair(s); ORF, open reading frame; PCR, polymerase chain reaction.

J. Dworkin, O. Shedd, and M. J. Blaser, manuscript in preparation.

J. Dworkin, M. K. R. Tummuru, and M. J. Blaser, unpublished data.

J. Dworkin, M. K. R. Tummuru, E. Wang, and M. J. Blaser, unpublished observations.


ACKNOWLEDGEMENTS

We thank Drs. Stuart Thompson and Richard Breyer for helpful discussions and technical assistance.


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