Salivaricin G32, a Homolog of the Prototype Streptococcus pyogenes Nisin-Like Lantibiotic SA-FF22, Produced by the Commensal Species Streptococcus salivarius

Abstract

Salivaricin G32, a 2667 Da novel member of the SA-FF22 cluster of lantibiotics, has been purified and characterized from Streptococcus salivarius strain G32. The inhibitory peptide differs from the Streptococcus pyogenes-produced SA-FF22 in the absence of lysine in position 2. The salivaricin G32 locus was widely distributed in BLIS-producing S. salivarius, with 6 (23%) of 26 strains PCR-positive for the structural gene, slnA. As for most other lantibiotics produced by S. salivarius, the salivaricin G32 locus can be megaplasmid encoded. Another member of the SA-FF22 family was detected in two Streptococcus dysgalactiae of bovine origin, an observation supportive of widespread distribution of this lantibiotic within the genus Streptococcus. Since the inhibitory spectrum of salivaricin G32 includes Streptococcus pyogenes, its production by S. salivarius, either as a member of the normal oral microflora or as a commercial probiotic, could serve to enhance protection of the human host against S. pyogenes infection.

Figure 1

Reversed phase fractionation of salivaricin G32 preparations. (a) Representative C₈ reversed phase fractionation of concentrated XAD-2 fractions containing inhibitory activity against indicator organism Micrococcus luteus. Elution of the column was with a gradient of 18–30% acetonitrile at a flow rate of 1 mL·min⁻¹ with detection of absorbance at 214 nm. (b) C₁₈ reversed phase fractionation of pooled C₈ reversed phase fractionated samples having inhibitory activity as indicated in panel A by the solid bar labelled 1. Elution was with isocratic 45% acetonitrile (containing 0.1% TFA) over 60 minutes at a flow rate of 0.7 mL·min⁻¹. Active fractions (solid bar 2) from multiple runs were pooled then analysed by MALDI-TOF MS as indicated by the grey insert.

Figure 2

(1) Alignment of (a) the leader sequences and (b) the propeptide sequences of salivaricin G32, SA-FF22, and macedocin (- indicates a spacer introduced to aid the alignment). The N-terminal sequence derived from the purified G32 preparation is shown below, where X indicates a blank cycle in the Edman reaction. (2) Alignment of (a) the predicted translated leader sequences and (b) the predicted translated propeptide sequences for slnA1, scnA1, and mcdA1. The number of amino acids in each sequence is indicated in front of each sequence name.

Figure 3

Graphical representation of the SA-FF22 locus illustrating the section of the salivaricin G32 locus that has been sequenced. Black-filled arrows indicate genes that have been completely sequenced. Unfilled arrows indicate genes for which the complete sequence has not yet been determined.

Figure 4

PFGE Analysis of megaplasmid content of total DNA of S. salivarius strains G32 (lane 1), 20P3 (lane 2), S. dysgalactiae strains 67 (lane 3), and 61 (lane 4), and S. pyogenes strain FF22 (lane 5). Lane 6 was left blank and lane 7 contained low range PFG marker (New England Biolabs). Megaplasmid bands are indicated with white arrows and marker sizes are indicated to the right of the gel.