Table 1.
Peptide pheromones known or tested as substrates of PptAB.
Fig 1.
Transposon mutagenesis identifies novel components of the Rgg-SHP quorum sensing circuit.
(A) Sixteen loci were identified according to our criteria (see text). Gene names and chromosomal location are indicated, with the replication origin (*) at twelve o’clock. Components of the QS circuit previously identified are indicated (†). (B) Fourteen unique insertions (arrowheads) mapped to pptAB, a predicted ABC transporter. pptAB were replaced with a cassette containing aphA3, which confers resistance to kanamycin.
Fig 2.
Rgg-SHP signaling in a S. pyogenes pptAB mutant.
(A) Luciferase expression from Pshp3 reporters integrated into wild-type (WT; NZ131), Δrgg3 (JCC131), and Δrgg3ΔpptAB (JCC209) GAS strains with (closed symbols) and without (open symbols) the addition of 100nM synthetic SHP3-C8 (C8) peptide. Data shown are representative of experiments performed at least three times. (B) Maximum Pshp3-lux reporter activity in WT, Δrgg3, or Δrgg3ΔpptAB strains carrying a plasmid encoding pptAB (pJC252) or empty vector (pLZ12-Sp). (C) Maximum Pshp3-lux reporter-inducing activity in conditioned supernatants prepared from WT, Δrgg3, or Δrgg3ΔpptAB donor strains expressing pptAB (pJC252) or empty vector (pLZ12-Sp). Donor cultures were grown to OD ~0.5, cells were removed by centrifugation and filtration, and Pshp3-lux activity of a Δrgg3 shpGGG reporter strain (BNL204) was measured. For B and C, data shown are the mean and SD from at least three experiments.
Fig 3.
PptAB exports SHP pheromones in GAS and S. mutans.
(A) Maximum Pshp3-lux reporter-inducing activity in culture supernatants prepared from (A) GAS and (B) S. mutans WT and ΔpptAB donor strains expressing PrecA-shp2 (pJC350) or PrecA-shp3 (pJC352). Both GAS donor strains are deleted for chromosomal copies of shp2 and shp3 (shpGGG; BNL170 and JCC218, respectively). Donor cultures were grown to OD ~0.5, cells were removed by centrifugation, supernatants were filtered or chemically sterilized, and Pshp3-lux activity of a Δrgg3 shpGGG reporter strain (BNL204) was measured. Data shown are the mean and SD from at least three experiments.
Fig 4.
ComRS signaling in a S. mutans pptAB mutant.
Maximum luciferase activity of WT (UA159), ΔcomR (MW02), ΔcomS (MW05) and ΔpptAB (JCC263) mutants carrying a multi-copy PsigX-lux reporter (pWAR304). Cells were grown to mid-log phase in CDM then diluted to an OD600 ~0.05 in a 96-well plate, and growth and luciferase activity were measured every 20 minutes in a Synergy 2 plate reader (Biotek). Data shown are the mean and SD from three experiments.