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We will investigate connection between the phenol and benzoate catabolism and physiology in P. putida strain carrying the pheBA genes. Mechanisms how the presence of other nutrients in growth media of bacteria would influence catabolism of phenol will be investigated. Transcription of the phenol degadation genes is activated by CatR protein in the presence of inducer molecule cis,cis-muconate (CCM), but this transcription is repressed in the presence of amino acids in growth media of bacteria. We plan to study molecular mechanisms which control transcription from the CatR-regulated promoters under conditions when amino acids are added into growth media. We will examine the possibility whether the silencing of transcription from CatR-regulated promoters would occur due to binding of repressor protein to these promoters. We will also study regulation of transcription of ben operon encoding benzoate catabolism and transport in P. putida grown under different environmental conditions. The promoter of the ben genes will be isolated and cloned upstream of the luxAB reporter system and lacZ reporter system. The effects of growth media composition and presence of alternative sigma factors on transcription from this promoter will be assayed either by measuring luciferase activities or ß-galactosidase, respectively. We plan to investigate a possible role of BenKEF transport system on benzoate and phenol uptake in P. putida. At first, we will construct a knockout mutant of this system. Growth of a benKEF mutant and the wild type strain on phenol and benzoate will be compared by measuring doubling times of cells grown on these substrates. Also, uptake assays will be performed by adding [14C]benzoate and [14C]phenol into growth media of the wild type and mutant cells. |