Transcriptional and Post‐Transcriptional Regulation of mtlA in Vibrio cholerae

Vibrio cholerae , the bacterium that causes cholera, performs a complex regulation of genes encoding carbon‐source transporters in order to survive in both aquatic reservoirs and the human small intestine. One such transporter is MtlA, which operates within the phosphotransferase system (PTS), a highly‐conserved system for carbohydrate uptake across bacteria. MtlA concomitantly phosphorylates and transports mannitol, the six‐carbon sugar alcohol produced by algae, into the cell. In addition to mtlA, the mtl locus contains mtlS and mtlR , which encode two major regulators of MtlA. mtlS encodes a 120‐nucleotide small regulatory RNA (sRNA) that posttranscriptionally regulates MtlA synthesis by binding to the mtlA mRNA 5′ untranslated region. Our objective is to identify regulators of mtlS expression. We have constructed a V. cholerae strain harboring a chromosomal mtlS‐lacZ transcriptional fusion. Using a V. cholerae genomic library, we identified putative regulators of mtlS expression. The second regulator of mtlA , encoded by mtlR , showed repressive effects in Escherichia coli , but had not been well characterized in V. cholerae . We studied how MtlR regulates MtlA activity in V. cholerae grown in a variety of carbon sources. Biofilm assays demonstrated that mtlR overexpression results in a greater than 50% decrease in biofilm formation ( p <0.001 by unpaired two‐tailed t ‐test), an activity known to be positively affected by MtlA. qRT‐PCR showed that a ΔmtlR mutant has higher mtlA RNA levels than a wild‐type control when grown in glucose‐supplemented minimal medium ( p <0.0001 by unpaired two‐tailed t‐ test), and even higher mtlA RNA levels when grown in maltose‐ and mannose‐ supplemented minimal medium ( p <0.01 by unpaired two‐tailed t‐ test). Western blotting analysis reflected these increases; MtlA protein levels were more elevated in ΔmtlR compared to wild‐type when grown in glucose, maltose, and mannose. The ΔmtlR mutant was partially complemented, as demonstrated by qRT‐PCR ( p <0.01 by unpaired two‐tailed t test) and western blotting analysis. This shows that the observed increases in MtlA were a direct effect of the gene product of mtlR. These results contribute to a model in which MtlR acts as a transcriptional repressor of mtlA in non‐mannitol conditions, working synergistically with MtlS to regulate mtlA. Support or Funding Information NIH R15 AI090606 This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .