Pyocin S2 (Sa) kills Pseudomonas aeruginosa strains via the FpvA type I ferripyoverdine receptor

Abstract

Soluble (S-type) pyocins are Pseudomonas aeruginosa bacteriocins that kill nonimmune P. aeruginosa strains via a specific receptor. The genes coding for pyocin Sa (consisting of a killing protein and an immunity protein) were cloned and expressed in Escherichia coli. Sequence analysis revealed that Sa is identical to pyocin S2. Seventy-nine strains of P. aeruginosa were tested for their sensitivity to pyocins S1, S2, and S3, and their ferripyoverdine receptors were typed by multiplex PCR. No strain was found to be sensitive to both S2 and S3, suggesting that the receptors for these two pyocins cannot coexist in one strain. As expected, all S3-sensitive strains had the type II ferripyoverdine receptor fpvA gene, confirming our previous reports. S1 killed strains irrespective of the type of ferripyoverdine receptor they produced. All S2-sensitive strains had the type I fpvA gene, and the inactivation of type I fpvA in an S2-sensitive strain conferred resistance to the S2 pyocin. Accordingly, complementation with type I fpvA in trans restored sensitivity to S2. Some S2-resistant type I fpvA-positive strains were detected, the majority (all but five) of which had the S1-S2 immunity gene. Comparison of type I fpvA sequences from immunity gene-negative S2-sensitive and S2-resistant strains revealed only a valine-to-isoleucine substitution at position 46 of type I FpvA. However, both type I fpvA genes conferred the capacity for type I pyoverdine utilization and sensitivity to S2. When these two type I fpvA genes were introduced into strain 7NSK2 carrying mutations in type II fpvA (encoding the type II pyoverdine receptor) and fpvB (encoding the alternative type I receptor), growth in the presence of type I pyoverdine was observed and the strain became sensitive to S2. We also found that type I pyoverdine could signal type II pyoverdine production via the type I FpvA receptor in 7NSK2.

FIG. 1.

pys2-PA1153 locus. The open reading frames in dark gray are those annotated in the PAO1 genome (http://www.pseudomonas.com), while those in white are predicted based on a BLASTX analysis. The first one (orfA) corresponds to part of a conserved hypothetical protein gene of the Tn3 family of transposons. The two other open reading frames (orfB and orfC) encode a putative colicin immunity protein and a hypothetical protein, respectively. The region between imm2 and PA1153 is shown below in more detail. The genes are not to scale, but the numbers represent the nucleotide coordinates in the P. aeruginosa PAO genome.

FIG. 2.

Effect of the inactivation of type I fpvA in the pyocin S2-sensitive strain W15Dec1 and complementation in trans with the two type I fpvA genes (encoding V46 and I46). (A) Killing activity of pyocin S2 on wild-type W15Dec1 (top left panel), W15Dec1fpvA⁻ (top right panel), W15Dec1fpvA⁻ complemented with type I fpvA(V46) (bottom left panel), and W15Dec1fpvA⁻ complemented with type I fpvA(I46) (bottom right panel). (B) SDS-PAGE analysis of outer membrane proteins from CAA-grown wild-type W15Dec1 (lane 2), the W15Dec1 type I fpvA mutant (lane 3), the W15Dec1 type I fpvA mutant complemented with type I fpvA(V46) (lane 4), and the W15Dec1 type I fpvA mutant complemented with type I fpvA(I46) (lane 5). The band below the 85-kDa marker is FpvA. Lane 1, molecular mass markers.

FIG. 3.

Complementation of the type II pyoverdine 7NSK2fpvA⁻fpvB⁻ mutant strain. (A) Growth stimulation of 7NSK2fpvA⁻fpvB⁻ on CAA plates containing EDDHA around a type I pyoverdine-impregnated disk (panel 1) and the same mutant complemented with type I fpvA(V46) (panel 2) and type I fpvA(I46) (panel 3). Panels 4 and 5 are the plates from panels 2 and 3 shown under UV illumination to demonstrate the fluorescence due to type II pyoverdine production. (B) Results from an S2 pyocin killing assay of 7NSK2fpvA⁻fpvB⁻ (resistant; panel 1) and the same mutant complemented with type I fpvA(V46) (sensitive; panel 2) and type I fpvA(I46) (sensitive; panel 3). Panel 4 shows the killing of the sensitive indicator strain O:9 as a positive control.