Investigation of the function of proteins associated to polyhydroxyalkanoate inclusions in Pseudomonas putida BMO1

Polyhydroxyalkanoate (PHA) granule associated proteins from Pseudomonas oleovorans were purified and the N-terminal sequences of two major proteins migrating in sodium dodecyl sulfate polyacrylamide gels with a relative molecular mass of 18 and 43 kDa (GA1 and GA2, respectively) were analyzed. Radiolabeled degenerate probes deduced from these amino acid sequences were used to identify genomic DNA fragments from P. oleovorans and Pseudomonas putida encoding GA1 and GA2. DNA sequence analysis of the fragments obtained from P. putida revealed that the genes encoding these proteins were adjacent to phaC2 and ORF3, the PHA synthase II gene and an open reading frame of unknown function, respectively, found at the P. oleovorans and P. aeruginosa PHA synthase gene locus. The open reading frames encoding GA1, GA2 and ORF3 or smaller fragments beginning at GA1 were inactivated by chromosomal insertion of the Tn5 kanamycin resistance gene block (neo). When these mutants were grown on mineral salts agar media under nitrogen limitation, containing gluconate or decanoate as carbon sources, they appeared more translucent than the wild-type grown under similar conditions. Gas-chromatographic analysis of the cellular dry mass revealed that the mutant strains accumulated 30-50% less PHA than the P. putida wild type.