A new member of the S-layer protein family: characterization of the crs gene from Campylobacter rectus

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.

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.

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.

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.

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.

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.

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.