Transcriptional Analysis of Pha Genes in Pseudomonas Mediterranea CFBP 5447 Grown on Glycerol

We analysed the draft genome sequence of Pseudomonas mediterranea CFBP 5447 in order to identify firstly the central metabolic pathways that convert fatty acids or carbohydrate intermediates into mcl-PHA and secondly the genes involved in glycerol metabolism (glpF, glpK, glpD, glpR). Absence of the glpF gene, which codifies for the "glycerol uptake facilitator protein", was highlighted. In order to understand the expression of the pha gene cluster, we investigated the promoter activity of phaC1, phaC2, phaZ, phaD and phal genes. When glycerol was present as the carbon source, Pi was found to be the most active promoter. Expression analysis of the knock-out mutant of the phaD gene, which is a transcriptional regulator belonging to the TetR family, showed that PhaD acts as an activator of the phal promoter which, in turn, triggers the transcription of the phalF operon. The activation of P-C1, which controls the phaC1ZC2D, by PhaD, was less efficient than P-l.