Characterization of Streptococcus suis genes encoding proteins homologous to sortase of gram-positive bacteria

Abstract

Many surface proteins which are covalently linked to the cell wall of gram-positive bacteria have a consensus C-terminal motif, Leu-Pro-X-Thr-Gly (LPXTG). This sequence is cleaved, and the processed protein is attached to an amino group of a cross-bridge in the peptidoglycan by a specific enzyme called sortase. Using the type strain of Streptococcus suis, NCTC 10234, we found five genes encoding proteins that were homologous to sortases of other bacteria and determined the nucleotide sequences of the genetic regions. One gene, designated srtA, was linked to gyrA, as were the sortase and sortase-like genes of other streptococci. Three genes, designated srtB, srtC, and srtD, were tandemly clustered in a different location, where there were three segments of directly repeated sequences of approximately 110 bp in close vicinity. The remaining gene, designated srtE, was located separately on the chromosome with a pseudogene which may encode a transposase. The deduced amino acid sequences of the five Srt proteins showed 18 to 31% identity with the sortases of Streptococcus gordonii and Staphylococcus aureus, except that SrtA of S. suis had 65% identity with that of S. gordonii. Isogenic mutants deficient for srtA, srtBCD, or srtE were generated by allelic exchanges. The protein fraction which was released from partially purified cell walls by digestion with N-acetylmuramidase was profiled by two-dimensional gel electrophoresis. More than 15 of the protein spots were missing in the profile of the srtA mutant compared with that of the parent strain, and this phenotype was completely complemented by srtA cloned from S. suis. Four genes encoding proteins corresponding to such spots were identified and sequenced. The deduced translational products of the four genes possessed the LPXTG motif in their C-terminal regions. On the other hand, the protein spots that were missing in the srtA mutant appeared in the profiles of the srtBCD and srtE mutants. These results provide evidence that the cell wall sorting system involving srtA is also present in S. suis.

FIG. 1.

Physical and genetic map of srtA (A), srtBCD (B), and srtE (C) loci in NCTC 10234 and construction of the mutant strains. orf204 contains a contiguous region of sntA. The wild-type srtA, srtBCD, and srtE were replaced by the ΔsrtA::cat, ΔsrtBCD::cat, and ΔsrtE::cat alleles carried on the knockout vectors pSAD1, pSBD1, and pSED1, respectively, as described in Materials and Methods, to generate the mutants. Only restriction endonuclease sites used for inverse PCR, construction of the plasmid, or genomic Southern hybridization are indicated. Black arrows, srtA homologs; gray arrows, other genes in the srtA-homologous regions; open boxes, directly repeated segments; tnp, a pseudogene potentially encoding a transposase; gradated arrow, cat gene; B, BamHI; C, ClaI; E, EcoRI; H, HindIII; N, NcoI; RV, EcoRV; S, SpeI; Sal, SalI; T22, EcoT22I; X, XhoI.

FIG. 2.

Sequence alignment of SrtA, SrtB, SrtC, SrtD, and SrtE of S. suis as well as SrtA of S. aureus and S. gordonii. The presumed C-terminal hydrophobic segments of class B enzymes are underlined. Black background, amino acids identical among all the sequences aligned; gray background, amino acids functionally conserved among the sequences aligned; dashes, gaps in the aligned sequences.

FIG. 3.

2D-PAGE profiles of guanidine-insoluble muramidase-released proteins from cell wall materials of mutant and parental strains of S. suis. (A) Parental strain NCTC 10234, (B) SRTΔA1, (C) SRTΔBCD1, (D) SRTΔE1, (E) SRTΔA1(pSAComp1). Equivalent amounts of lyophilized samples resolved in rehydration buffer were loaded on each gel. After electrophoresis, gels were silver stained. Protein spots marked by the rectangles were subjected to N-terminal amino acid sequence determination. The following molecular weight standards were used in the second dimension: myosin (200,000), β-galactosidase (116,300), phosphorylase b (97,400), bovine serum albumin (66,300), glutamate dehydrogenase (55,400), lactate dehydrogenase (36,500), carbonic anhydrase (31,000), and trypsin inhibitor (21,500).