Cat and ben gene regulation
Tumen-Velasquez, Melissa Patricia
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BenM and CatM are LysR-type transcriptional regulators (LTTR) from Acinetobacter baylyi ADP1 that control a complex regulon involved in benzoate catabolism. Although previous studies demonstrate similarities in the structure and function of these paralogs, there are key differences that distinguish their regulatory roles. As described in this thesis, mutants were isolated and characterized that grow on benzoate without BenM. In the wild-type strain, benzoate consumption is prevented by the failure of CatM to activate high-level transcription of the benABCDE operon. At this locus, CatM typically activates low-level transcription in response to, cis,cis-muconate, a catabolite of benzoate. The goal of the current research was to alter CatM to make it function more like BenM. This approach was used to reveal the molecular basis of transcriptional control by these two regulators and, by extrapolation, other members of the important LTTR family. Amino acid changes that enable CatM variants to substitute for the loss of BenM were identified in all regions of the protein: the N-terminal DNA-binding domain, the linker helix, and the C-terminal effector-binding domain. In no previous study had mutations been isolated that affect the DNA-binding domain or linker-helix region of these proteins. The novel CatM variants increased ben-gene expression via elevated transcriptional regulation in response to muconate. However, these changes did not confer a distinct regulatory feature of BenM, the ability to respond to two effector compounds and activate transcription of the benABCDE operon synergistically. A different approach was successful in generating this feature. CatM variants that were engineered by rational design acquired the synergistic response to two effectors when changes were made in both the N- and C-terminal domains. Engineered regulatory variants were also examined for their transcriptional effects at two other promoter regions (catA and catBCIJFD). These studies show that the N-terminal domain is not solely responsible for promoter specificity. Similarly, the C-terminal domain does not solely control the response to effectors. Rather, there appears to be subtle interplay between the domains that can affect transcription. These studies expand our understanding of LTTRs and microbial aromatic metabolism. They also facilitate the development of new biotechnology applications.