Acetyl phosphate‐sensitive regulation of flagellar biogenesis and capsular biosynthesis depends on the Rcs phosphorelay

Abstract

SummaryAs part of our attempt to map the impact of acetyl phosphate (acetyl~P) on the entire network of twocomponent signal transduction pathways in Escherichia coli, we asked whether the influence of acetyl~P on capsular biosynthesis and flagellar biogenesis depends on the Rcs phosphorelay. To do so, we performed a series of epistasis experiments: mutations in the components of the pathway that controls acetyl~P levels were combined with mutations in components of the Rcs phosphorelay. Cells that did not synthesize acetyl~P produced no capsule under normally permissive conditions, while those that accumulated acetyl~P synthesized capsule under conditions previously considered to be nonpermissive. Acetyl~P-dependent capsular biosynthesis required both RcsB and RcsA, while the lack of RcsC restored capsular biosynthesis to acetyl~P-deficient cells. Similarly, acetyl~P-sensitive repression of flagellar biogenesis was suppressed by the loss of RcsB (but not of RcsA), while it was enhanced by the lack of RcsC. Taken together, these results show that both acetyl~P-sensitive activation of capsular biosynthesis and acetyl~P-sensitive repression of flagellar biogenesis require the Rcs phosphorelay. Moreover, they provide strong genetic support for the hypothesis that RcsC can function as either a kinase or a phosphatase dependent on environmental conditions. Finally, we learned that RcsB and RcsC inversely regulated the timing of flagellar biogenesis: rcsB mutants elaborated flagella prematurely, while rcsC mutants delayed their display of flagella. Temporal control of flagella biogenesis implicates the Rcs phosphorelay (and, by extension, acetyl~P) in the transition of motile, planktonic individuals into sessile biofilm communities.

The glutamate-dependent acid resistance system in Escherichia coli: essential and dual role of the His–Asp phosphorelay RcsCDB/AF

“…However, in the ackA pta strain, where there is no production of acetyl phosphate, or in the rcsC ack pta triple mutant, acid resistance is maintained (data not shown). If the basal RcsB activity depends on the phosphorylated form of the protein, then a third phosphorylation pathway (parallel to those of RcsC and acetyl phosphate) must exist as already suggested by Majdalani et al (2005) and Fredericks et al (2006). Alternatively, the unphosphorylated form of RcsB might have a regulatory activity on its own.…”

“…However, OmpR still needs to be activated, probably by receiving a phosphoryl group from acetyl phosphate (Bang et al, 2000). Evidence exists that RcsB can also be activated through phosphorylation by acetyl phosphate, notably when the phosphatase activity of the sensor RcsC is missing (Fredericks et al, 2006;Majdalani et al, 2005;our unpublished data). However, in the ackA pta strain, where there is no production of acetyl phosphate, or in the rcsC ack pta triple mutant, acid resistance is maintained (data not shown).…”

Deletion analysis of RcsC reveals a novel signalling pathway controlling poly-N-acetylglucosamine synthesis and biofilm formation in Escherichia coli

“…Other plasmids used to generate mutants were pKD46 and pKD4 (Datsenko & Wanner, 2000). E. coli AJW678 and the AJW1939 (ackA) and AJW2013 (pta-ackA) mutant strains were previously described (Fredericks et al, 2006;Kumari et al, 2000). For the complete list of strains used in this study see Table S1 (available in the online Supplementary Material).…”

“…It is important to mention that several studies with RcsC have been done by cloning rcsC on multicopy plasmids under the control of inducible promoters to enhance RcsCassociated phenotypes (Fredericks et al, 2006;Huang et al, 2006Huang et al, , 2009, which was the approach followed here; further efforts to study the phenotype evidenced in this report, under more physiological conditions, are under way.…”

“…When unstimulated, RcsC functions as a net phosphatase (Fredericks et al, 2006). Furthermore, RcsA is a substrate of the Lon protease (Brill et al, 1988;Chen et al, 2001;Fredericks et al, 2006;Gottesman et al, 1985;Salscheider et al, 2014;Takeda et al, 2001;Torres-Cabassa & Gottesman, 1987); for a review see Majdalani & Gottesman (2005).…”