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

Abstract

analysis has demonstrated that sapA2 was not expressed in the C. fetus strain from which it was cloned. Further Southern analyses revealed increasing sapA diversity as probes increasingly 3 within the ORF were used. Pulsed-field gel electrophoresis and then Southern blotting with the conserved N-terminal region of the sapA homologs as a probe showed that these genes were tightly clustered on the chromosome. Deletion mutagenesis revealed that the S-layer protein bound serospecifically to the C. fetus lipopolysaccharide via its conserved N-terminal region. These data indicated that the S-layer proteins shared functional activity in the conserved N terminus but diverged in a semiconservative manner for the remainder of the molecule. Variation in S-layer protein expression may involve rearrangement of complete gene copies from a single large locus containing multiple sapA homologs.

S-Layer Proteins

“…In type A cells, the S-layer protein SapA and its homologs are bound via a conserved N-terminal region of 184 amino acids to the type A LPS. The S-layer protein SapB and its homologs possess a different Nterminal region and recognize cells with type B LPS as a binding site (31,32).…”

Molecular mechanisms of Campylobacter fetus surface layer protein expression

“…As SLPs of the same strain type share the first N-terminal 184 amino acids and then diverge, and because sapA and sapB are identical except for that region, we hypothesized that the binding domain would reside within the N-terminus. Subsequent reattachment studies demonstrating LPS serotype-specific binding with recombinant truncated type A polypeptides (Dworkin et al, 1995b) confirmed the hypothesis.…”

“…Southern hybridization using the 5Ј conserved region to probe genomic DNA from type A C. fetus strains indicates that eight or nine SLP gene cassettes are present in wildtype strains (Tummuru and Blaser, 1992;Fujita et al, 1995). Pulsed-field gel electrophoresis indicates that these cassettes are tightly clustered on a region of less than 93 kb, representing less than 8% of the genome (Dworkin et al, 1995b;Fujita et al, 1995). Beyond the 5Ј conserved region, each SLP gene cassette possesses a variable region that becomes progressively more divergent as the 3Ј end is approached; thus, each homologue encodes a unique SLP due in part to a unique C-terminus (Dworkin et al, 1995b).…”

“…Third, mutagenesis studies (Blaser et al, 1994) indicated that SLP expression variation is associated with exchange of cassettes 3Ј to the sapA promoter. Fourth, spontaneous S ¹ mutant strains that have a 9 kb deletion including this promoter (Tummuru and Blaser, 1992;Fujita and Amako, 1994;Dworkin et al, 1995b), are unable to produce an S-layer under any circumstance tested, including strong in vivo and in vitro selection for the S þ phenotype (Dworkin and Blaser, 1996).…”

“…Second, programmed gene rearrangements can use elements of homologous recombination; our findings provide further evidence that the previous distinction between homologous and site-specific events is insufficient, as earlier identified by studies with transposon-initiated homologous recombination (Hagemann and Craig, 1993 . DNA inversion between two oppositely oriented cassettes flanking a 6.2 kb segment follows DNA strand exchange within the 5Ј conserved region (small red box) (Dworkin et al, 1995b). The intervening segment is topologically reversed (rotated 180Њ) leading to ordered rearrangement of the SLP gene cassettes.…”