Identification and characterization of a Streptococcus pyogenes ABC transporter with multiple specificity for metal cations

Metal ions are crucial trace elements for bacteria infecting the human host . The Lral (lipoprotein receptor-associated antigen I) transporter in Strep tococcus spp. belongs to the superfamily of ABC transporters. The transport er consists of a lipoprotein, an ATP-binding protein and a hydrophobic inte gral membrane protein. Here, we describe a new member of the Lral family in the important human pathogen Streptococcus pyogenes. The system was identi fied in silico by analysis of the S. pyogenes Genome Sequencing Project. Th e S, pyogenes operon exhibits an atypical organization compared with equiva lents in other Streptococcus spp. The presence and atypical organization of the operon was verified in a number of S, pyogenes strains of different se rotypes. Transcriptional analysis of the Lral operon demonstrates a polycis tronic transcription attenuated by a stable stem-loop structure, which allo ws the lipoprotein to be expressed in larger quantities than the other two components. The localization of the native lipoprotein at the bacterial sur face was shown by proteolytic digestion of S. pyogenes bacteria and NH2-ter minal sequencing of a released lipoprotein fragment. Recombinant lipoprotei n was expressed as a GST fusion protein, and studies of molecular interacti ons with metal radioisotopes demonstrated that the protein has affinity for Zn(II), Fe(III) and Cu(II). Zn(II) and Cu(II) were found to compete for th e same binding site, whereas Fe(III) uses a second site. Also, proton-induc ed X-ray analysis of lipoprotein samples identified iron, copper and zinc. Finally, a mutant strain lacking a functional mtsABC operon was generated a nd showed reduced uptake of Fe-55 and Zn-65 compared with the wild-type str ain. The operon encoding this novel ABC transporter with multiple specifici ty for metal cations is designated mtsABC, for metal transporter of Strepto coccus.