Regulation of acetyl-CoA synthetase transcription by the CrbS/R two-component system is conserved in genetically diverse environmental pathogens

Abstract

The CrbS/R two-component signal transduction system is a conserved regulatory mechanism through which specific Gram-negative bacteria control acetate flux into primary metabolic pathways. CrbS/R governs expression of acetyl-CoA synthase (acsA), an enzyme that converts acetate to acetyl-CoA, a metabolite at the nexus of the cell’s most important energy-harvesting and biosynthetic reactions. During infection, bacteria can utilize this system to hijack host acetate metabolism and alter the course of colonization and pathogenesis. In toxigenic strains of Vibrio cholerae, CrbS/R-dependent expression of acsA is required for virulence in an arthropod model. Here, we investigate the function of the CrbS/R system in Pseudomonas aeruginosa, Pseudomonas entomophila, and non-toxigenic V. cholerae strains. We demonstrate that its role in acetate metabolism is conserved; this system regulates expression of the acsA gene and is required for growth on acetate as a sole carbon source. As a first step towards describing the mechanism of signaling through this pathway, we identify residues and domains that may be critical for phosphotransfer. We further demonstrate that although CrbS, the putative hybrid sensor kinase, carries both a histidine kinase domain and a receiver domain, the latter is not required for acsA transcription. In order to determine whether our findings are relevant to pathogenesis, we tested our strains in a Drosophila model of oral infection previously employed for the study of acetate-dependent virulence by V. cholerae. We show that non-toxigenic V. cholerae strains lacking CrbS or CrbR are significantly less virulent than are wild-type strains, while P. aeruginosa and P. entomophila lacking CrbS or CrbR are fully pathogenic. Together, the data suggest that the CrbS/R system plays a central role in acetate metabolism in V. cholerae, P. aeruginosa, and P. entomophila. However, each microbe’s unique environmental adaptations and pathogenesis strategies may dictate conditions under which CrbS/R-mediated acs expression is most critical.

Modulation of CrbS-Dependent Activation of the Acetate Switch in Vibrio cholerae

“…Finally, CrbS carries a receiver (REC) domain, with the conserved aspartate residue that receives the phosphoryl group. The presence of a REC domain implies that CrbS functions as a hybrid histidine kinase that initiates a multistep phosphorelay, but our results indicate that the receiver domain is not necessary for signaling, and it may instead act as a negative regulator (21). We have shown, however, that the phosphor-accepting His residue located in the DHp domain is required for acs expression, indicating that phosphorylation is associated with the "on" state of this pathway (21).…”

“…Removal of acetate from the surrounding medium is controlled by the expression and activity of acetyl coenzyme A (acetyl-CoA) synthetase (Acs), which converts acetate to acetyl-CoA (10,20). In V. cholerae and other members of the Vibrionaceae, acs transcription is positively regulated by the CrbS/R two-component system (10,21,22). Due to its role in controlling acs, the CrbS/R pathway is necessary for V. cholerae infection and virulence toward Drosophila; without it, V. cholerae is virtually avirulent (10,21).…”

“…In V. cholerae and other members of the Vibrionaceae, acs transcription is positively regulated by the CrbS/R two-component system (10,21,22). Due to its role in controlling acs, the CrbS/R pathway is necessary for V. cholerae infection and virulence toward Drosophila; without it, V. cholerae is virtually avirulent (10,21). By connecting acetate metabolism to the CrbS/R two-component system, V. cholerae and related pathogens may link this metabolic switch to additional environmental cues.…”

“…Finally, we tested whether this strain reduces acs expression during infection, by performing reverse transcription-quantitative real-time PCR (RT-qPCR) on RNA isolated from flies infected with the WT or crbSΔSTAC V. cholerae strain. As controls, we also tested acs expression in flies infected with the ΔcrbS strain, which should reduce acs expression, as well as a strain carrying a deletion in the crbS REC domain, which we expected to express acs similarly to the wild-type strain (21). We confirmed that the acs expression level is lower in flies carrying the crbS deletion (10), and we observed that acs expression was equivalent to that of the wild-type strain in both the STAC and REC domain deletions (Fig.…”

Contextual flexibility inPseudomonas aeruginosacentral carbon metabolism during growth in single carbon sources

“…Growth on acetate as a sole carbon source elicited a similarly informative set of changes. There was a strong induction of the glyoxylate shunt enzymes (AceA and GlcB), which are known to be essential for growth on acetate [32,33], and of enzymes directly involved in acetate activation (such as AcsA, AckA and Pta) and acetate uptake (PA3234) [34]. The TCA cycle-associated enzymes were almost all up-regulated during growth on acetate, as was the membrane-bound malate-quinone oxidoreductase, MqoB (Figure 2).…”