Dysgalacticin: a novel, plasmid-encoded antimicrobial protein (bacteriocin) produced by Streptococcus dysgalactiae subsp. equisimilis

Abstract

Dysgalacticin is a novel bacteriocin produced by Streptococcus dysgalactiae subsp. equisimilis strain W2580 that has a narrow spectrum of antimicrobial activity directed primarily against the principal human streptococcal pathogen Streptococcus pyogenes. Unlike many previously described bacteriocins of Gram-positive bacteria, dysgalacticin is a heat-labile 21?5 kDa anionic protein that kills its target without inducing lysis. The N-terminal amino acid sequence of dysgalacticin [Asn-Glu-Thr-Asn-Asn-Phe-Ala-Glu-Thr-Gln-Lys-Glu-Ile-Thr-Thr-Asn-(Asn)-Glu-Ala] has no known homologue in publicly available sequence databases. The dysgalacticin structural gene, dysA, is located on the indigenous plasmid pW2580 of strain W2580 and encodes a 220 aa preprotein which is probably exported via a Sec-dependent transport system. Natural dysA variants containing conservative amino acid substitutions were also detected by sequence analyses of dysA elements from S. dysgalactiae strains displaying W2580-like inhibitory profiles. Production of recombinant dysgalacticin by Escherichia coli confirmed that this protein is solely responsible for the inhibitory activity exhibited by strain W2580. A combination of in silico secondary structure prediction and reductive alkylation was employed to demonstrate that dysgalacticin has a novel structure containing a disulphide bond essential for its biological activity. Moreover, dysgalacticin displays similarity in predicted secondary structure (but not primary amino acid sequence or inhibitory spectrum) with another plasmid-encoded streptococcal bacteriocin, streptococcin A-M57 from S. pyogenes, indicating that dysgalacticin represents a prototype of a new class of antimicrobial proteins. INTRODUCTIONNumerous genera of Eubacteria and Archaea produce proteinaceous antimicrobial substances known as bacteriocins that target related species, presumably to provide the producing organism with an ecological advantage in its microenvironment (Riley & Gordon, 1999;Riley & Wertz, 2002). Broadly speaking, bacteriocins fall into one of four main categories, depending on whether they are produced by (and therefore act primarily on) Gram-positive or Gram-negative bacteria, and whether they are of high (>10 kDa) or relatively low molecular mass (<10 kDa). There is a wealth of scientific literature concerning bacteriocins from three of these categories: (i) the >20 kDa bacteriocins of Gramnegative bacteria, epitomized by the plasmid-encoded colicins from Escherichia coli (Gillor et al., 2004;; the <10 kDa antimicrobial peptides or microcins produced by Gram-negative species (Gillor et al., 2004); and (iii) the low-molecular-mass bacteriocins of Gram-positive bacteria (reviewed by Jack et al., 1998; Eijsink et al., 2002;Fimland et al., 2005;Cotter et al., 2005). However, comparatively little is known about the colicin equivalents of Gram-positive bacteria, since reports of high-molecularmass bacteriocins from such bacteria are scarce, the most extensively studied being the peptidoglycan-hydrolysing enzyme lyso...

ThefsrQuorum-Sensing System and Cognate Gelatinase Orchestrate the Expression and Processing of Proprotein EF_1097 into the Mature Antimicrobial Peptide Enterocin O16

“…To investigate the genetic basis of enterocin O16 biosynthesis, both bioinformatics and functional genetic analyses were performed. EF_1097 is a member of a family of antimicrobial proteins (15). The ef1097 locus ( Fig.…”

“…The fsr quorum-sensing system, via the activity of GelE, contributes to several important aspects of E. faecalis physiology, including fratricide, cell surface protein presentation, and protein turnover, and thus is important for virulence (13,15,33). It has also been shown that the promoter regions of three operons have binding sites recognized by the phosphorylated FsrA transcription factor (12).…”

“…The fsr regulatory system thus controls the expression of the GelE and SprE proteases, which are implicated in several aspects of E. faecalis physiology, including protein turnover, biofilm formation, and virulence (12)(13)(14). The last gene, ef1097, which constitutes part of the E. faecalis core genome, is physically located 800 kb apart from the other fsr-controlled loci, and it is a member of a gene family encoding antimicrobial proteins (15).…”

Identification of DysI, the Immunity Factor of the Streptococcal Bacteriocin Dysgalacticin

“…The dysgalacticin structural gene dysA is located on a 3,043-bp plasmid, pW2580 (9). However, strain W2580C, a plasmid-cured derivative of strain W2580, not only is defective in dysgalacticin production but also is sensitive to exogenously added recombinant dysgalacticin (9).…”

“…equisimilis strain W2580 which exhibits a narrow spectrum of inhibitory activity directed mainly against the human pathogen Streptococcus pyogenes (9). We have previously reported that the mode of action of dysgalacticin is nonlytic and is likely to involve receptor binding to the membrane-bound glucose and/or mannose phosphotransferase system (PTS), followed by disruption of cytoplasmic membrane integrity and then subsequent K ϩ efflux and dissipation of the membrane potential in S. pyogenes (9,15).…”