From genomic libraries, the polyhydroxyalkanoate gene locus of Pseudomonas aeruginosa PAOl was cloned and characterised at the molecular level. Two genes coding for polyhydroxyalkanoate synthases, phaCl,, and p h~C 2~, , a polyhydroxyalkanoate depolymerase gene, phaDp,, and four adjacent open reading frames (ORFl,ORF2,ORF3 and ORF4) were identified from the nucleotide sequence. Two transcriptional start sites, which were preceded by sequences resembling the Escherichia coli consensus sequences for C T~~ and g 7 0 promoters, were identified experimentally upstream of phaCl,,, which was shown by Northern blot analysis to constitute an operon together with phaDp,. A third putative promoter resembling the E. coli consensus sequence for ~'O-dependent promoters was proposed upstream of p h~C 2~, , which is in a bicistronic operon with ORF3. Investigations of rpollrnegative mutants of related strains revealed that polyhydroxyalkanoate accumulation from gluconate required an intact rpoN locus in P. aeruginosa. Complementation experiments revealed multiple evidence that either polyhydroxyalkanoate synthase is involved in polyhydroylkanoate accumulation from gluconate as well as from octanoate. The P. aeruginosa PAOl polyhydroxyalkanoate gene locus was expressed in the polyhydroxyalkanoate-negative mutant Alcaligenes eutrophus PHB -4 and in the poly(3-hydroxybutyrate)-accumulating strain P. oleovorans DSM1045. It conferred on the latter the ability to synthesize and accumulate polyhydroxyalkanoates consisting of medium-chain-length 3-hydroxyalkanoic acids from unrelated substrates in addition to poly(3-hydroxybutyrate). The sequence of the putative translational product of ORFl was similar to those of the leukotoxin repressor of Pmteurella haemolytica and to the ORF9 product of Azotobacter vinelandii, and that of ORF4 was similar to the algP product of P. aeruginosa and to eukaryotic histone H1 proteins. The proteins of ORF2 and ORF3 appear to be previously unidentified.
Pseudomonas aeruginosa PAO and 15 other strains of this species synthesized a polyester with 3hydroxydecanoate as the main constituent (55 to 76 mol%) if the cells were cultivated in the presence of gluconate and if the nitrogen source was exhausted; 3-hydroxyhexanoate, 3-hydroxyoctanoate, and 3-hydroxydodecanoate were minor constituents of the polymer. The polymer was deposited in granules within the cell and amounted to 70% of the cell dry matter in some strains. Among 55 different strains of 41 Pseudomonas species tested, P. aureofaciens (21.6% of cellular dry matter), P. citronellolis (78.0%), P. chlororaphis (8.5%), P. marginalis (11.4%), P. mendocina (50.7%), P. putida (33.5%), and Pseudomonas sp. strain DSM 1650 (54.6%) accumulated this type of polymer at significant levels (>5%) during cultivation on gluconate. In two strains of P. facilis and P. fluorescens, as well as in one strain of P. syringae, this polymer was detected as a minor constituent (<5%). All other strains accumulated either poly(3-hydroxybutyrate) or a polymer consisting mainly of 3-hydroxyoctanoate with octanoate but no polyester with gluconate as the carbon source. Only a few species (e.g., P. stutzeri) were unable to accumulate poly(hydroxyalkanoic acids) (PHA) at all. These results indicated that the formation of PHA depends on a pathway which is distinct from all other known PHAbiosynthetic pathways. The polyesters accumulated by gluconateor octanoate-grown cells of recombinant strains of P. aeruginosa and P. putida, which harbored the Alcaligenes eutrophus poly(3-hydroxybutyrate)biosynthetic genes, contained 3-hydroxybutyrate as an additional constituent.
Recombinant strains of Pseudomonas oleovorans, which harbour the poly(3-hydroxybutyrate)-biosynthetic genes of Alcaligenes eutrophus, accumulated poly(hydroxyalkanoates), composed of 3-hydroxybutyrate(3HB), 3-hydroxyhexanoate (3HHx) and 3-hydroxyoctanoate (3HO), up to 70% of the cell dry weight if the cells were cultivated with sodium octanoate as the carbon source. Physiological and chemical analysis revealed multiple evidence that this polymer is a blend of the homopolyester poly(3HB) and of the copolyester poly(3HHx-co-3HO) rather than a random or a block copolyester of 3HB, 3HHx and 3HO. The molar ratio between poly(3HHx-co-3HO) and poly(3HB) varied drastically during the process of fermentation. Whereas synthesis of poly(3HHx-co-3HO) started immediately after ammonium was exhausted in the medium, synthesis of poly(3HB) occurred only after a lag-phase. From freeze-dried cells poly(3HHx-co-3HO) was much more readily extracted with chloroform than was poly(3HB). The blend was fractionated into petrol-ether-insoluble poly(3HB) and petrol-ether-soluble poly(3HHx-co-3HO). The molecular weight values of these polyesters measured by gel permeation chromatography were 2.96 × 10 6 and 0.35 × 10 6 and were similar of those polymers accumulated by A. eutrophus or by wild-type P. oleovorans, respectively.
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