Dysgalacticin is a large, 21.5 kDa bacteriocin that belongs to subgroup B of the class III bacteriocins. It is ribosomally produced by Streptococcus dysgalactiae subsp. equisimilis strain W2580 and exerts inhibitory activity mainly against the medically important pathogen Streptococcus pyogenes by a "non-lytic" mechanism. Despite numerous studies of the mechanisms of action of a wide variety of bacteriocins and of the basis of their producer strain self-immunity, relatively little is known about the "non-lytic" class of bacteriocins. The structural gene encoding for dysgalacticin (dysA) was known to be carried on a small, rolling circle plasmid pW2580 (3.04 kb) (Heng et al., 2006). However, the dysgalacticin immunity gene (dysI) had not been identified prior to the present study. The aims of this research were to elucidate the mechanism of action of dysgalacticin against S. pyogenes and to identify the genetic basis and the mechanism of producer strain self-immunity. Recombinantly-produced dysgalacticin was used to determine the mode of action against S. pyogenes. Dysgalacticin was bactericidal for S. pyogenes, increasing the permeability of the cytoplasmic membrane and ultimately leading to leakage of intracellular potassium ions. Moreover, dysgalacticin dissipated the membrane potential and inhibited [�⁴C]serine uptake, a membrane potential-dependent process in S. pyogenes. Interestingly, dysgalacticin inhibited glucose fermentation by non-growing cell suspensions and blocked transport of both glucose and the nonmetabolisable analogue 2-deoxyglucose. This finding indicates that dysgalacticin may target the phosphophenolpyruvate (PEP)-dependent glucose and mannose phosphotransferase system (PTS) of S. pyogenes. Taken together, these data suggest that dysgalacticin targets the glucose-PTS and/or mannose-PTS as a receptor, leading to inhibition of sugar uptake, and a subsequent dissipation of the membrane potential leading to cell death. Complementation studies demonstrated that dysI is located on pW2580. RNA analysis showed that dysI is co-transcribed with genes encoding for the plasmid copy control protein, copG and replication initiation protein, repB. S. pyogenes transformed with a plasmid containing dysI displayed a markedly higher dysgalacticin MIC (1024 nM) than the corresponding dysgalacticin-sensitive, plasmid-negative strain (8 nM). Further studies of this DysI-expressing S. pyogenes showed that membrane integrity, glucose fermentation and [�H]2DG uptake were not affected by dysgalacticin treatment. These findings are consistant with a mechanism whereby the immunity peptide binds to the target-binding site of dysgalacticin, effectively blocking access by the bacteriocin. H₆DysI was found to localise to the cytoplasmic membrane, further indicating that DysI may bind to the proposed target of dysgalacticin, i.e., the membrane-bound glucose-PTS and mannose-PTS. Thus both the mode of action and the producer strain self-immunity of dysgalacticin are likely to be cytoplasmic-membrane based. Homology searching revealed that the bacteriocin SA-M7 produced by M-type 57 S. pyogenes has structural similarities to dysgalacticin, as do two hypothetical proteins, EF1097 and YpkK, of Enterococcus faecalis and Corynebacterium jeikeium, respectively (Heng et al., 2004, 2006). These proteins were all predicted to contain relatively unstructured N-termini and helix-loop-helix structured C-termini. In each case the C-termini contain two conserved cysteine residues that are predicted to form a disulphide bridge. Heterologous expression of SA-M57, EF1097 and YpkK in Escherchia coli demonstrated that all three proteins have antimicrobial activity, but of differeing activity spectra. Reductive-alkylation of SA-M57, EF1097 and YpkK confirmed that their predicted disulphide bonds were essential for biological activity. These proteins were later renamed streptococcin A-M57, enterococcin V583 and corynicin JK respectively. The outcome of preliminary domain-swapping experiments supported the existence of functional domain-type segments in streptococcin A-M57, enterococcin V583, corynicin JK and dysgalacticin. The N-terminal domain of each of these proteins and also the C-terminal domain of corynicin JK were successfully expressed in E. coli. The failure to express the C-termini of the remaining proteins was thought possibly due to toxicity of thses pepetides for the E. coli host. Nevertheless, the C-terminus of corynicin JK displayed an inhibitory spectrum apparently identical to that of the full-length corynicin, indicating that the N-terminus may not always be required for target binding of this class of antimicrobials. Preliminary mode of action studies revealed that streptococcin A-M57, enterococcin V583 and corynicin JK all resemble dysgalacticin in that they exert inhibitory activity by non-lytic means. These results, in combination with the protein structural predictions indicate that dysgalacticin, streptococcin A-M57, enterococcin V583 and corynicin JK are all members of the same basic class of "non-lytic" bactericoicns.

Dysgalacticin is a large, 21.5 kDa bacteriocin that belongs to subgroup B of the class III bacteriocins. It is ribosomally produced by Streptococcus dysgalactiae subsp. equisimilis strain W2580 and exerts inhibitory activity mainly against the medically important pathogen Streptococcus pyogenes by a "non-lytic" mechanism. Despite numerous studies of the mechanisms of action of a wide variety of bacteriocins and of the basis of their producer strain self-immunity, relatively little is known about the "non-lytic" class of bacteriocins. The structural gene encoding for dysgalacticin (dysA) was known to be carried on a small, rolling circle plasmid pW2580 (3.04 kb) (Heng et al., 2006). However, the dysgalacticin immunity gene (dysI) had not been identified prior to the present study. The aims of this research were to elucidate the mechanism of action of dysgalacticin against S. pyogenes and to identify the genetic basis and the mechanism of producer strain self-immunity. Recombinantly-produced dysgalacticin was used to determine the mode of action against S. pyogenes. Dysgalacticin was bactericidal for S. pyogenes, increasing the permeability of the cytoplasmic membrane and ultimately leading to leakage of intracellular potassium ions. Moreover, dysgalacticin dissipated the membrane potential and inhibited [�⁴C]serine uptake, a membrane potential-dependent process in S. pyogenes. Interestingly, dysgalacticin inhibited glucose fermentation by non-growing cell suspensions and blocked transport of both glucose and the nonmetabolisable analogue 2-deoxyglucose. This finding indicates that dysgalacticin may target the phosphophenolpyruvate (PEP)-dependent glucose and mannose phosphotransferase system (PTS) of S. pyogenes. Taken together, these data suggest that dysgalacticin targets the glucose-PTS and/or mannose-PTS as a receptor, leading to inhibition of sugar uptake, and a subsequent dissipation of the membrane potential leading to cell death. Complementation studies demonstrated that dysI is located on pW2580. RNA analysis showed that dysI is co-transcribed with genes encoding for the plasmid copy control protein, copG and replication initiation protein, repB. S. pyogenes transformed with a plasmid containing dysI displayed a markedly higher dysgalacticin MIC (1024 nM) than the corresponding dysgalacticin-sensitive, plasmid-negative strain (8 nM). Further studies of this DysI-expressing S. pyogenes showed that membrane integrity, glucose fermentation and [�H]2DG uptake were not affected by dysgalacticin treatment. These findings are consistant with a mechanism whereby the immunity peptide binds to the target-binding site of dysgalacticin, effectively blocking access by the bacteriocin. H₆DysI was found to localise to the cytoplasmic membrane, further indicating that DysI may bind to the proposed target of dysgalacticin, i.e., the membrane-bound glucose-PTS and mannose-PTS. Thus both the mode of action and the producer strain self-immunity of dysgalacticin are likely to be cytoplasmic-membrane based. Homology searching revealed that the bacteriocin SA-M7 produced by M-type 57 S. pyogenes has structural similarities to dysgalacticin, as do two hypothetical proteins, EF1097 and YpkK, of Enterococcus faecalis and Corynebacterium jeikeium, respectively (Heng et al., 2004, 2006). These proteins were all predicted to contain relatively unstructured N-termini and helix-loop-helix structured C-termini. In each case the C-termini contain two conserved cysteine residues that are predicted to form a disulphide bridge. Heterologous expression of SA-M57, EF1097 and YpkK in Escherchia coli demonstrated that all three proteins have antimicrobial activity, but of differeing activity spectra. Reductive-alkylation of SA-M57, EF1097 and YpkK confirmed that their predicted disulphide bonds were essential for biological activity. These proteins were later renamed streptococcin A-M57, enterococcin V583 and corynicin JK respectively. The outcome of preliminary domain-swapping experiments supported the existence of functional domain-type segments in streptococcin A-M57, enterococcin V583, corynicin JK and dysgalacticin. The N-terminal domain of each of these proteins and also the C-terminal domain of corynicin JK were successfully expressed in E. coli. The failure to express the C-termini of the remaining proteins was thought possibly due to toxicity of thses pepetides for the E. coli host. Nevertheless, the C-terminus of corynicin JK displayed an inhibitory spectrum apparently identical to that of the full-length corynicin, indicating that the N-terminus may not always be required for target binding of this class of antimicrobials. Preliminary mode of action studies revealed that streptococcin A-M57, enterococcin V583 and corynicin JK all resemble dysgalacticin in that they exert inhibitory activity by non-lytic means. These results, in combination with the protein structural predictions indicate that dysgalacticin, streptococcin A-M57, enterococcin V583 and corynicin JK are all members of the same basic class of "non-lytic" bactericoicns.