Characterization of the Campylobacter fetus sapA promoter: evidence that the sapA promoter is deleted in spontaneous mutant strains

Abstract

Wild-type Campylobacterfetus cells possess S-layer proteins (S+ phenotype), whereas after laboratory passage, spontaneous stable mutants that do not express these proteins (S-phenotype) arise. To determine the molecular mechanisms by which C.fetus changes to the S-phenotype, we studied wild-type strain 23D, from which the sapA gene encoding the 97-kDa S-layer protein has been cloned, and strain 23B, a spontaneous S-mutant. We compared these strains with another pair of strains, LP (S+) and HP (S5 Northern RNA blot analysis showed no sapA mRNA in strain 23B. These data indicate that the lack of S-layer protein expression in spontaneous mutant strains is caused by the deletion of promoter sequences.Many microorganisms possess regular surface layers (Slayers) that are noncovalently attached to membrane structures (35). The superficial location of these proteins suggests that these layers are important mediators of interactions with hosts (4,5,21,24). Although the morphological properties of S-layers have been extensively studied for a wide range of microorganisms (22,34,35), relatively little is known about the regulatory mechanisms involved in the biosynthesis, transport, and assembly of S-layer proteins.Campylobacterfetus is an important veterinary pathogen that causes infertility and infectious abortion in sheep and cattle (36) and extraintestinal infections (16) and a thrombotic diathesis (8) in humans. The ability of C. fetus to produce these diseases is associated with the presence of surface array proteins (4,5,11,21,24). The C. fetus S-layer is composed of high-molecular-mass protein subunits that migrate at approximately 97, 127, and 149 kDa; usually a subunit of a single size predominates for a given strain (10, 25). C. fetus strains may contain either type A or type B lipopolysaccharide, and their S-layer proteins are lipopolysaccharide type specific (38). The type A S-layer proteins are antigenically related and have identical N-terminal amino acid sequences (25) but differ from the type B proteins (10, 38).S-layer protein expression in C. fetus is subject to antigenic variation (10,25,(30)(31)(32). It is possible to isolate C. fetus variants that express from zero to up to three different S-layer proteins (10, 25), but the determinants of which protein is predominantly expressed are not known. carrying S-layer proteins (S+ phenotype), but not spontaneous mutants lacking them (S-phenotype), resist complement-mediated killing by normal or immune serum and resist phagocytosis (4,5,21). Strain 23B is an S-spontaneous mutant of the wild-type S+ strain 23D (21).The mechanisms by which pathogens alter their surface antigens are varied but often involve programmed DNA rearrangements (7). As a first step in elucidating the molecular mechanisms involved in the antigenic variation of the C. fetus S-layer proteins, we recently cloned and sequenced the strain 23D sapA gene, which encodes a polypeptide of 97 kDa (3). The aim of our present studies is to assess the molecular principles of C. fetus S-layer protein pro...

Protein shift and antigenic variation in the S-layer of Campylobacter fetus subsp. venerealis during bovine infection accompanied by genomic rearrangement of sapA homologs

“…Furthermore, we provide evidence that each of the shifts in S-layer protein expression and antigenicity was associated with genomic rearrangement. This finding is consistent with the multiplicity of sapA homologs (25) and their organization, with both conserved and variable regions (26). Our combined observations indicate the dynamic changes in C. fetus S-layer protein antigen expression during vaginal colonization and show that this in vivo antigenic variation is accompanied by genomic rearrangement and possibly amplification of the sapA homologs, as indicated by the increase in copy numbers in week 2 to 9 isolates.…”

“…Antigenic variation may be achieved by shifts in the expression of S-layer proteins which result in different immunodominant epitopes during in vivo persistence in the bovine genital tract (28). A gene (sapA) encoding the 97-kDa S-layer protein has been cloned (1), and it now is clear that both the wild type and spontaneous mutants lacking the S-layer proteins possess multiple sapA homologs (25). In a single strain, in vitro antigenic shift was associated with rearrangement of sapA homologs (26).…”

Transcriptional analysis of the Aeromonas salmonicida S-layer protein gene vapA

“…In keeping with other S-layer subunit proteins (18,29,43,45), the vapA transcript expressed from the P1 promoter was found to be monocistronic. Indeed, only the Bacillus S-layers have been shown to be polycistronic (1).…”

The surface (S)-layer gene cspB of Corynebacterium glutamicum is transcriptionally activated by a LuxR-type regulator and located on a 6 kb genomic island absent from the type strain ATCC 13032

“…fetus TK and Camp. fetus 23B, the promoter region of the S-layer gene sapA was found to be truncated (Fujita et al, 1997;Tummuru & Blaser, 1992). A Chi-like sequence located upstream of the S-layer gene, and most likely recognized by the RecBCD system of the cell, might be responsible for inactivation of the S-layer gene (Tummuru & Blaser, 1993).…”