The Listeria monocytogenes Sortase-B Recognizes Varied Amino Acids at Position 2 of the Sorting Motif

Javier F Mariscotti,Francisco García-Del Portillo,M.Graciela Pucciarelli

doi:10.1074/jbc.m807989200

Abstract

Sortases are bacterial enzymes that anchor surface proteins covalently to the peptidoglycan upon cleavage of a motif located at their C-terminal end. Motifs recognized by sortases of the class-B (SrtB) are defined by the consensus sequence NP(Q/K)(T/S)(N/G/S)(D/A). Evidence supporting this consensus is limited to IsdC of Staphylococcus aureus and Bacillus anthracis, cleaved at motifs NPQTN and NPKTG, respectively. In Listeria monocytogenes, StrB has two substrates, Lmo2185 and Lmo2186, containing NAKTN and NKVTN (or the overlapping sequence NPKSS) as putative sorting motifs. Some of these motifs do not match the consensus, because they lack either proline (P) at position 2 or glutamine/lysine (Q/K) at position 3. Here, we identified NPKSS as a sorting motif of Lmo2186 by monitoring anchoring to peptidoglycan of chimeras lacking each of its two predicted motifs. Motif-swapping experiments confirmed that NPKSS, but not NKVTN, could replace NAKTN for anchoring of an Lmo2185 chimera. Residue substitutions in the NPKSS sequence revealed the essentiality of proline at position 2 for recognition of this particular motif. Lysine at position 3 was however dispensable. Deletion of NAKTN, on the other hand, abrogated SrtB-mediated anchoring of the Lmo2185 chimera. NAKTN, therefore, represents an exception to the rule of a conserved proline in position 2 of the sorting motif. Taken together, our data indicate that proline is not absolutely required for substrate recognition by sortases of the class-B. In addition, they prove the capacity of a single sortase, as SrtB of L. monocytogenes, to recognize varied amino acids at position 2 of the sorting motif.

Highlights

NPKSS Is a Motif Recognized by Sortase-B (SrtB) of L. monocytogenes in Lmo2186—The surface protein Lmo2186 (207 amino acids) contains a C-terminal sorting region predicted to initiate with the motif NKVTNPKSS, followed by a stretch of 22 hydrophobic amino acids and a positively charged tail (Fig. 1)
New InlBCter-Lmo2186 chimeras were constructed bearing either NKVTN or NPKSS as the only motif (Fig. 2A). These variants were expressed in wild-type (EGD-e) and isogenic ⌬srtA or ⌬srtB bacteria, which were grown to stationary phase in BHI nutrient medium
Our results showed that the variants containing either NKVTNPKSS or NPKSS were efficiently anchored to the peptidoglycan (Fig. 2B)

Summary

The abbreviations used are

Proteomic analyses of cell wall material isolated from L monocytogenes srtA and srtB mutants revealed that SrtA anchors proteins bearing LPXTG motifs, while two surface proteins, Lmo2185 and Lmo2186, are anchored to the peptidoglycan in a StrB-dependent manner [12, 24] Both lmo2185 and lmo2186 genes are clustered in the same operon containing srtB [21]. Proteomic analyses reported the presence of a Lmo2186 peptide with the sequence SDSSNKVTNPK in peptidoglycan material of L. monocytogenes containing strongly associated mature proteins [24] This finding provided an indirect evidence for NPKSS as the motif recognized and cleaved by SrtB. We provide the identity of these motifs and uncover the capacity of this sortase for naturally recognizing motifs with varied amino acid at position 2 of the sorting motif

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION