Identification of DysI, the immunity factor of the streptococcal bacteriocin dysgalacticin

Abstract

DysI is identified as the protein that confers specific immunity to dysgalacticin, a plasmid-encoded streptococcal bacteriocin. dysI is transcribed as part of the copG-repB-dysI replication-associated operon. DysI appears to function at the membrane level to prevent the inhibitory effects of dysgalacticin on glucose transport, membrane integrity, and intracellular ATP content.

FIG. 1.

(A) Organization and nucleotide sequence of the copG-repB-ORF1 (dysI) region in pW2580. The genes shown are not drawn to scale. The putative ribosome binding site for dysI (AGAGG; nt 889 to 903) and the stop codon for repB (TAG; nt 907 to 909) are underlined. The hydrophobic amino acid residues predicted to form transmembrane helices are in bold text and underlined. The unique HindIII site that was digested and end filled to generate the frameshift mutation in ORF1 is in italics and boxed. The arrows indicate the positions and orientations of the various primers (P1 to P4) used in RT-PCR experiments. (B) RT-PCR amplicons generated from total RNA extracted from S. dysgalactiae W2580 grown to mid-logarithmic phase. Lanes: M, DNA size marker (Fermentas, Maryland); 2, P3 + P4 (dysI only; 174 bp); 4, P2 + P4 (repB-dysI; 792 bp); 6, P1 + P4 (copG-dysI; 989 bp); 1, 3, and 5, negative-control reactions for each of the above lanes in which reverse transcriptase was not added. (C) ORF1 (dysI) confers immunity to dysgalacticin. Twenty-microgram amounts of recombinant dysgalacticin were applied to lawns of S. dysgalactiae W2580C and its dysI-related derivatives in order to determine bacteriocin sensitivity. The indicator strains were as follows: a, wild-type S. dysgalactiae W2580 (immune control); b, strain W2580C (sensitive control); c, DWPS2 (W2580C carrying pWPS2, ΔdysI); d, DWPS1 (W2580C carrying pWPS1, dysI⁺).

FIG. 2.

Western blot analysis of DysI-His₆ localization. Cell lysate (Cl), cytoplasmic (Cyt), and membrane (Mem) fractions of DysI-His₆-expressing S. dysgalactiae strain DWPS3.

FIG. 3.

Protective effect of DysI in S. pyogenes strain PWPS1 (FF22 carrying pWPS1) with respect to [³H]2DG uptake (A), glucose fermentation (B), membrane permeabilization (C), or intracellular ATP content (D). Cells were energized with glucose in all experiments except that analyzed in panel A and a glucose-free control (B). Untreated controls of wild-type S. pyogenes FF22 are shown as filled squares, and cells treated with 11 μg/ml of dysgalacticin are represented by filled triangles. “I⁺” represents dysI⁺ S. pyogenes strain PWPS1, which was treated with dysgalacticin (open triangles), untreated controls (open squares), or untreated controls without glucose (open circles). The arrow indicates the time point at which purified recombinant dysgalacticin (9) was added to the cells at a final concentration of 11 μg/ml. Nisin, a known pore-forming bacteriocin, was included as a positive control (open diamonds) in the membrane permeabilization experiments in order to maintain consistency with previous studies (15) and was added at a concentration of 25 μg/ml. The MIC of nisin for S. pyogenes is 0.78 μg/ml.