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 peptideglycan 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.Many cell wall surface proteins of gram-positive bacteria are covalently anchored to the cell wall by a mechanism requiring a C-terminal anchoring motif, which consists of a conserved amino acid sequence, Leu-Pro-X-Thr-Gly (LPXTG, where X is any amino acid), followed by a hydrophobic domain and, in most cases, a tail of positively charged residues (11, 47). A series of elegant studies by Schneewind and his colleagues (32,35,45,46) have determined the function of a membranelocalized cysteine protease, named sortase, using protein A of Staphylococcus aureus as the model. Sortase specifically cleaves the LPXTG sequence in protein A between the threonine and glycine residues and catalyzes the transfer of the processed protein to the free amino group of pentaglycine cross-bridges in the staphylococcal peptidoglycan. Thus, sortase appears to be a multifunctional protease-transpeptidase (58, 59).Computational searches of complete and preliminary genome sequence data have revealed t...

A Comparative Genome Analysis Identifies Distinct Sorting Pathways in Gram-Positive Bacteria

“…The remaining gram-positive subfamilies are numbered 3, 4, and 5, whereas the gram-negative subfamily is also known as subfamily 6. As has been previously noted, the members of a subset of SrtA family sortases (20) are distinguished by their genomic proximity to the gene encoding DNA gyrase subunit A (found in the genera Lactococcus and Streptococcus) (37,54). Table 2 lists how members of each subfamily are distributed in the 49 species of bacteria analyzed in this study.…”

“…Consistent with playing a role in surface protein chemistry, all SrtA homologs contain appropriately positioned active site residues (SrtA residues H120 and C184) (32) and transmembrane segments, and their genes are frequently clustered with genes encoding CWScontaining proteins. Moreover, several homologs have been shown to be directly involved in protein anchoring, since their elimination prevents the display of surface proteins (11,23,26,54). Although the SrtA protein recognizes the sequence LPXTG within its substrates, this motif is widely varied, and a second S. aureus sortase, called SrtB, processes proteins bearing the sequence NPQTN (46).…”

Identification of a Novel Streptococcal Adhesin P (SadP) Protein Recognizing Galactosyl-α1–4-galactose-containing Glycoconjugates

“…3D). Sortases catalyze the covalent linkage of secreted proteins via the LPNTG motif into the peptidoglycan cell wall, and SrtA has been reported to be functionally a major sortase isoform in S. suis (37). As shown in Fig.…”

Sortases and the Art of Anchoring Proteins to the Envelopes of Gram-Positive Bacteria

“…Mrp, a surface protein with a C-terminal sorting signal, is thought to be covalently anchored to the S. suis cell wall envelope, and this has stimulated the study of sortases in this microbe. As S. suis genomic sequences are not yet available, PCR amplification with degenerate primers was used to identify five sortase gene homologs, srtA to -E, in the genome of S. suis serotype 2, the most common disease-associated serotype (146). The srtB to -D genes are clustered with two genes encoding putative surface protein substrates, named orf203 and orf204, specifying IPYTG and LPATG sorting signal motifs, respectively.…”