Feedback inhibition in the PhoQ/PhoP signaling system by a membrane peptide

Abstract

The PhoQ/PhoP signaling system responds to low magnesium and the presence of certain cationic antimicrobial peptides. It regulates genes important for growth under these conditions, as well as additional genes important for virulence in many gram-negative pathogens. PhoQ is a sensor kinase that phosphorylates and activates the transcription factor PhoP. Since feedback inhibition is a common theme in stress-response circuits, we hypothesized that some members of the PhoP regulon may play such a role in the PhoQ/PhoP pathway. We therefore screened for PhoP-regulated genes that mediate feedback in this system. We found that deletion of mgrB (yobG), which encodes a 47 amino acid peptide, results in a potent increase in PhoP-regulated transcription. In addition, over-expression of mgrB decreased transcription at both high and low concentrations of magnesium. Localization and bacterial two-hybrid studies suggest that MgrB resides in the inner-membrane and interacts directly with PhoQ. We further show that MgrB homologs from Salmonella typhimurium and Yersinia pestis also repress PhoP-regulated transcription in these organisms. In cell regulatory circuits, feedback has been associated with modulating the induction kinetics and/or the cell-to-cell variability in response to stimulus. Interestingly, we found that elimination of MgrB-mediated feedback did not have a significant effect on the kinetics of reporter protein production and did not decrease the variability in expression among cells. Our results indicate MgrB is a broadly conserved membrane peptide that is a critical mediator of negative feedback in the PhoQ/PhoP circuit. This new regulator may function as a point of control that integrates additional input signals to modulate the activity of this important signaling system.

Figure 1. Deletion of mgrB leads to up-regulation of multiple genes in the PhoQ/PhoP Regulon.

(A) YFP (left) and CFP (right) fluorescence images of a transcriptional reporter strain with deletions of various PhoP-regulated genes, growing on LB agar. The strain contains a chromosomal copy of the PhoP-regulated mgrB promoter (P_mgrB) driving yfp expression and a constitutive promoter driving cfp expression as an internal reference. The specific strains are, clockwise starting with WT, TIM92, TIM100, AML4, AML6, AML8, AML16, AML10, AML12, and AML14. (B) Fluorescence of several strains (as in A) grown in liquid. (C) Fluorescence of phoPQ transcriptional reporters grown in liquid. The strains are, from left to right, TIM148, TIM229, AML22, and AML23. (D) Fluorescence of mgtA transcriptional reporters grown in liquid. The strains are, from left to right TIM91, TIM99, AML24, AML25. For liquid cultures, cells were grown in minimal glucose medium with 100 µM (white) or 10 mM (black) MgSO₄ and cellular fluorescence was measured by microscopy and image analysis as described in Materials and Methods. The error bars represent the range of means for two independent cultures.

Figure 2. Expression of mgrB from a plasmid complements a chromosomal deletion.

Reporter strains for mgrB transcription are either wild-type for the chromosomal mgrB (TIM92) or contain a deletion (AML20) and either a control plasmid (pEB52) or a plasmid bearing mgrB (pAL8). Cells were grown in minimal glucose medium with 100 µM (white) or 10 mM (black) MgSO₄. Cellular fluorescence was measured by microscopy and image analysis as described in Materials and Methods. The error bars represent the range of means for two independent cultures.

Figure 3. MgrB localizes to the inner membrane in vivo.

(A) The amino acid sequence of MgrB. The underlined region is a potential transmembrane domain . The arrows labeled I and II denote potential type I ([34] ) and type II signal sequence cleavage sites, respectively. (B) Western blot of the total cell lysate, envelope fraction (pellet), or the soluble protein fraction (sup), of an mgrB⁻ strain expressing cytoplasmic YFP and CFP (AML20) and containing either a control plasmid (pEB52) or an mgrB expression plasmid (pAL8). Both plasmids also express beta-lactamase, an enzyme that resides in the periplasm. (C) Phase contrast and GFP fluorescence micrographs of AML67 (mgrB ⁻) expressing either GFP (pAL39, top) or a fusion of GFP to the N-terminus of MgrB (pAL38, bottom). Cells were grown in minimal glucose medium with 1 mM MgSO₄.

Figure 4. Alteration of the PhoQ periplasmic domain blocks the repressive effect of MgrB.

Fluorescence of an mgrB transcriptional reporter, AML21 (mgrB ⁻ phoQ ⁻), which was transformed with a control plasmid (pEB52) or a plasmid containing mgrB (pAL8) and with a compatible plasmid expressing wild-type PhoQ (pLPQ2), a chimera in which the periplasmic domain of E coli PhoQ was replaced with the corresponding domain from P. aeruginosa PhoQ (pLPQ*2), or a control plasmid (pGB2). Fluorescence was measured from cells grown at 30°C in minimal glucose medium with 100 µM MgSO₄ as described in Materials and Methods. Cells were grown at 30°C for consistency with bacterial two-hybrid experiments—Figure 5 and Figure S3. Similar results were observed for cells grown at 37°C (data not shown). The error bars represent the range of means of two independent cultures. (WT: wild-type, chim: chimera).

Figure 5. MgrB interacts with PhoQ and with itself.

MgrB interacts with E. coli PhoQ and with itself but not with a PhoQ chimera containing a modified periplasmic domain. Cells expressed protein fusions to the T18 and T25 subunits of B. pertussis adenylyl cyclase. Reconstitution of adenylyl cyclase activity was inferred from beta-galactosidase activity. The cyaA ⁻ strain BTH101 contained plasmids expressing subunits alone (pKT25 or pUT18C) or subunit fusions to the N-terminus of MgrB (pAL25 and pAL33), E. coli PhoQ (pAL27), or a PhoQ chimera (PhoQ_chim—pAL36). For each strain, the mean and standard deviation for three independent measurements are shown.

Figure 6. MgrB homologs identified from genome sequences of various genera of Enterobacteriaceae.

The first 11 sequences were aligned using ClustalW . Genome sequences were from Escherichia coli MG1655, Shigella flexneri 2457T, Citrobacter koseri ATCC BAA-895, Salmonella typhimurium LT2, Enterobacter sakazakii ATCC BAA-894, Klebsiella pneumoniae 342, Yersinia pestis KIM, Photorhabdus luminescens subsp. laumondii TTO1, Serratia proteamaculans 568, Providencia stuartii ATCC 25827, Sodalis glossinidius str. ‘morsitans’, and Proteus penneri ATCC 35198.

Figure 7. Inhibition of PhoP-regulated transcription by MgrB in Salmonella typhimurium and Yersinia pestis.

(A) Salmonella enterica serovar Typhimurium strain 14028s carrying the P_phoN-lacZ reporter plasmid pNL2 and either a plasmid expressing Salmonella mgrB (pAL43) or a control plasmid (pEB52) were grown in LB with antibiotics and 1 mM IPTG. The amino acid sequence of MgrB from strain 14028s (data not shown) is identical to the sequence from strain LT2 (shown in Figure 6). (B) Y. pestis KIM6 carrying pNL2 and either a plasmid expressing the Yersinia pestis mgrB (pAL42) or a control plasmid (pEB52) were grown in BHI with antibiotics and 1mM IPTG. For each strain, the mean and standard deviation for at least three independent cultures are shown.

Figure 8. MgrB mediates negative feedback in the PhoQ/PhoP system.

PhoQ stimulation by low extracellular magnesium or cationic antimicrobial peptides (CAMPs) leads to increased levels of phosphorylated PhoP, which in turn results in increased transcription of mgrB. MgrB inserts in the inner membrane, with its N-terminus in the cytoplasm and C-terminus in the periplasm, and represses PhoQ, resulting in decreased PhoP phosphorylation. The question marks illustrate potential points of control where additional signals could modulate PhoQ/PhoP signaling by regulating MgrB activity or expression.