Figure 2 The effects of a pstS mutation on secondary metabolism see more and QS are occurring via PhoBR. (A) Pig, (B) Car and (C) AHL production were measured from WT, pstS mutant (ROP2), phoR mutant (BR1), phoB mutant (BR9), pstS, phoR double mutant (PCF60) and pstS, phoB double mutant (PCF59) cells. Production was assayed from cells grown to early stationary phase in LB. Insertions within phoBR abolish transcriptional upregulation of pigA and smaI in the pstS mutant Phenotypic analysis showed that PhoBR are required for the upregulation of secondary metabolism and QS in response to mutation of the pstSCAB-phoU operon (Ganetespib described above). To confirm that these effects are exerted at the

transcriptional level, primer extension analysis was used to AZD0156 in vivo assess levels of the pigA and smaI transcripts throughout growth in WT, pstS mutant and pstS, phoB double mutant strains. The abundance of pigA mRNA in the pstS, phoB double mutant was restored to levels similar

to those displayed in WT Serratia 39006 (Fig. 3A). A chromosomal pigA::lacZ transcriptional fusion was used to confirm this result; a 3-fold increase in pigA transcription was observed in a pstS mutant, this was restored to WT levels following a secondary mutation in phoB or phoR (Fig. 3B). The upregulation of smaI transcription in the pstS mutant was also abolished by a phoB mutation (Fig. 3C). This is consistent with the hypothesis that PhoB, either directly or indirectly, activates expression of pigA and smaI in response to constitutive phosphorylation by PhoR as a result of the pstS insertion. Figure 3 A pstS mutation effects transcription of pigA and smaI via PhoBR. Primer extension analysis was used to measure the level of (A) pigA or (C) smaI transcripts in WT, pstS mutant (ROP2), or pstS, phoB (RBR9) double mutant strains throughout growth in LB. (B) β-Galactosidase Ribociclib price activity was measured from a chromosomal pigA::lacZ fusion in an otherwise WT background (NW60), or in pstS (PCF76), phoR (PCF75), phoB (PCF74), pstS, phoR double (PCF78) or pstS, phoB double (PCF77) mutant backgrounds. Activity was assayed from cells grown to early stationary

phase in LB. Insertions within pstSCAB-phoU result in increased transcription of rap A complex network of regulators controls secondary metabolism in Serratia 39006 [27]. Therefore, it was possible that the effects on Pig and AHL production, in response to a pst mutation, were mediated via one or more of these regulators. To test if the effect on smaI and pigA transcription was mediated through any of the known secondary metabolite regulators, the expression of chromosomal lacZ transcriptional fusions in pigP, pigQ, pigR, pigS, pigT, pigV, pigW, pigX, pigZ, rap and carR was assessed throughout growth in the presence or absence of a pstS::mini-Tn5Sm/Sp insertion (data not shown). No effect was seen on any of the regulatory genes except for rap. The expression of rap was increased by 1.4-fold in the pstS mutant (Fig. 4A).