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.
Highlights
Bacteria use two-component signal transduction systems (TCSs) to respond to changing extracellular conditions and intracellular physiological cues, enabling them to initiate an appropriate pattern of gene expression or protein activity
We hypothesized that the homologs of this TCS in P. aeruginosa and P. entomophila, as well as those in non-O1/non-O139 V. cholerae strains, would regulate acetyl-CoA synthase (acsA) expression
To confirm that the reduction in acsA expression was linked to interruptions in this pathway, both deletion mutant strains were transformed with a plasmid that overexpressed the response regulator of this TCS
Summary
Bacteria use two-component signal transduction systems (TCSs) to respond to changing extracellular conditions and intracellular physiological cues, enabling them to initiate an appropriate pattern of gene expression or protein activity. Acs transcription is regulated by the CrbS/R TCS, and expression of acsA, crbR, and crbS are all required for V. cholerae virulence towards Drosophila [7]. This mechanism was discovered and characterized in a pandemic strain of V. cholerae of the O139 serotype that carries both the cholera toxin and toxincoregulated pilus genes required for causing cholera. This TCS is well conserved in sequenced V. cholerae strains, including environmental, non-toxigenic V. cholerae isolates. P. entomophila is a natural pathogen of insects [13], and P. aeruginosa can infect humans as well as a variety of other hosts in the environment [6]
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