FIG 5.

rapP³⁶¹⁰ codes for an N236T substitution mutation compared to the consensus, which renders it insensitive to the PhrP peptide. (A) Sequence alignment of amino acids 226 to 250 from RapP³⁶¹⁰ with the sequences of other RapP homologues and with other Rap proteins found in strain 3610. Asparagine residue 236 is conserved in all Rap proteins except for the product of rapP³⁶¹⁰, where it is replaced with threonine. Residue 236 of RapP is shown in boldface, and the aligned column is boxed for emphasis. (B) The addition of an IPTG-inducible phrP gene suppresses the effect of rapP^T236N but not of rapP³⁶¹⁰ on P_srfA-3×YFP expression. Shown are YFP expression levels of a P_srfA-3×YFP reporter in strain PY79 with different backgrounds, where each genotype was measured with (+) or without (−) a P_hs-phrP construct. Genotypes are as follows: wild-type PY79 (AES1334), P_hs-phrP (AES1477), P_rapP-rapP³⁶¹⁰ (AES1380), P_rapP-rapP³⁶¹⁰ P_hs-phrP (AES1478), P_rapP-rapP^T236N (AES1678), and P_rapP-rapP^T236N P_hs-phrP (AES1709). Expression was measured at an OD₆₀₀ of 1.5, and IPTG was added at 10 μM when needed. (C) P_srfA-3×YFP expression was monitored in a P_rapP-rapP^T236N (AES1678) background as a function of the concentration of the externally added peptides. Three peptides were tested for their ability to repress rapP^T236N activity: the putative signaling peptides of PhrP (DRAAT or ADRAAT) and the hexapeptide signal of PhrH (TDRNTT).