Regulation of the pha genes in Pseudomonas putida KT2440: Involvement of PhaD and the stationary phase sigma factor

LAURA J. RAIGER- IUSTMAN; PAULA M. TRIBELLI; MARTINIANO M. RICARDI; JIMENA A. RUIZ

Villa Carlos Paz- Cordoba

Congreso; VI Congreso Argentino de Microbiología General; 2009

SAMIGE

Pseudopmonas putida KT2440 synthesizes and accumulates medium-chain-length polyhydroxyalkanoates (PHAs) under conditions of nutrient limitation and carbon excess. The pha locus of P. putida KT2440 consists of seven genes: two PHA polymerases: phaC1 and phaC2, a PHA depolymerase: phaZ, a putative transcriptional regulator of the TetR family: phaD and two PHA granule- associated proteins: phaI and phaF. Previous result from our group (Raiger and Ruiz, 2008) demonstrated that the global regulator S negatively affects the expression of the promoter that drives the expression of the genes phaC1 and phaZ (pC1) during the stationary phase. Since RpoS is a sigma factor, and a negative regulation by a direct action of a sigma factor is not a common mechanism, we suggested that there must be other regulator acting on the pC1 promoter. The putative TetR-type transcriptional regulator, PhaD, was a good candidate, because a mutation in the gene that codes for PhaD had the same effect on PHA metabolism (Kinkle et al, 2000) that a mutation in rpoS. In this work we showed, by means of Real time PCR experiments, that in the rpoS mutant the expression of phaD is greatly reduced (about 11 fold), indicating that S has a positive effect on the expression of phaD. On the other hand, the over-expression of phaD in P. putida KT2440 pC1::lacZ, significantly reduced the expression driven by the pC1 promoter. These results would indicate that S and the still not characterized regulator PhaD form part of a cascade that regulates pha genes expression in P. putida. The role of PhaD as a specific- negative regulator of the pha genes is currently under research.