He. Valentin et al., INVESTIGATION OF THE FUNCTION OF PROTEINS ASSOCIATED TO POLYHYDROXYALKANOATE INCLUSIONS IN PSEUDOMONAS-PUTIDA BMO1, Journal of biotechnology, 64(2-3), 1998, pp. 145-157
Polyhydroxyalkanoate (PHA) granule associated proteins from Pseudomona
s oleovorans were purified and the N-terminal sequences of two major p
roteins 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 DMA fragments from P. ol
eovorans and Pseudomonas putida encoding GA1 and GA2. DNA sequence ana
lysis of the fragments obtained from P. putida revealed that the genes
encoding these proteins were adjacent to phaC2 and ORF3, the PHA synt
hase II gene and an open reading frame of unknown function, respective
ly, found at the P. oleovorans and P. aeruginosa PHA synthase gene loc
us. The open reading frames encoding GA1, GA2 and ORF3 or smaller frag
ments beginning at GA1 were inactivated by chromosomal insertion of th
e Tn5 kanamycin resistance gene block (neo). When these mutants were g
rown on mineral salts agar media under nitrogen limitation, containing
gluconate or decanoate as carbon sources, they appeared more transluc
ent than the wild-type grown under similar conditions. Gas-chromatogra
phic analysis of the cellular dry mass revealed that the mutant strain
s accumulated 30-50% less PHA than the P. putida wild type. (C) 1998 E
lsevier Science B.V. All rights reserved.