Cloning and molecular analysis of the poly(3‐hydroxyalkanoic acid) gene locus of Pseudomonas aeruginosa PAO1

Abstract

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.

Screening of different contaminated environments for polyhydroxyalkanoates-producing bacterial strains

“…Five out of nine PHA producers showed positive results on amplification of gene fragment of PHA polymerase. The other four strains were also PHA producers but not amplified on PCR that may be due to the non-complementation of their PHA synthase to primers, or these strains may not belong to the PHA synthase class II (Timm & Steinbüchel 1992;Shamala et al 2003). Solaiman et al (2000) used the PCR-based strategy to confirm the PHA producers from different Pseudomonas sp.…”

“…The class II PHA synthase genes PhaC1 and PhaC2 that favour mcl 3HA have been identified and characterized in Pseudomonas sp. including Pseudomonas aeruginosa (Timm & Steinbüchel 1992), Pseudomonas oleovorans (Huisman et al 1991), Pseudomonas sp. 61-3 (Matsusaki et al 1998), and Pseudomonas mendocina (Hein et al 2002).…”

Rapid detection of polyhydroxyalkanoate-accumulating bacteria isolated from the environment by colony PCR

“…(GenBank accession no. U78047), Methylobacterium extorquens (Valentin & Steinbu$ chel, 1993), Paracoccus denitrificans (Ueda et al, 1996), Pseudomonas aeruginosa (Timm & Steinbu$ chel, 1992) (containing two phaC genes), P. oleovorans (Huisman et al, 1991) (containing two phaC genes), Rhizobium etli (Cevallos et al, 1996), Rhizobium meliloti (Tombolini et al, 1995) and Zoogloea ramigera (GenBank accession no. U66242).…”

Physiological and biochemical changes accompanying the loss of mucoidy by Pseudomonas aeruginosa

“…These intracellular granules increased from < 0.1 YO of the cell volume at the start of the experiment to 8.9% at the end of the experiment, i.e. from a concentration of approximately 0 to 0.05 g 1-', confirming that substantially more PHA was made by non-mucoid cells than by mucoid cells (see also Timm 8i Steinbuchel, 1990Steinbuchel, , 1992. Overall, therefore, the decrease in the concentration of glucose in the effluent medium during the loss of mucoidy (A glucose: -3-80 g 1-' ) was closely matched by the net change in the total concentration of the products (alginate, gluconate, 2-ketogluconate and P HA ;…”