Two polyhydroxyalkanoate synthase genes, phaC1 from Pseudomonas pseudoalcaligenes HBQ06 and phaC2 from Pseudomonas nitroreducens 0802, were cloned using a PCR cloning strategy based on the type II pha loci property of Pseudomonas strains. The complete open reading frames (ORFs) of phaC1 (P. nitroreducens HBQ06) and phaC2 (P. nitroreducens 0802) were identified from the PCR products. Using the sequence information, the complete PHA synthase genes were PCR cloned directly from the genomic DNA and expressed in Escherichia coli as confirmed by Fourier transform-infrared spectroscopy and gas chromatography. The differences between PhaC1 and PhaC2 were analyzed and the two proteins were suggested to contain different functions and evolution history.

SUMMARY Juvenile gilthead sea bream were exposed to diluted seawater (2.5‰salinity; DSW) for 3 h or, in a second experiment, acclimated to DSW and fed a control or calcium-deficient diet for 30 days. Branchial Ca2+influx, drinking rate and plasma calcium levels were assessed. Sea bream plasma parathyroid hormone related protein (sPTHrP) was measured, and mRNAs of pthrp, its main receptor, pth1r, and the calcium-sensing receptor (casr) were quantified in osmoregulatory tissues and the pituitary gland. When calcium is limited in water or diet, sea bream maintain calcium balance; however, both plasma Ca2+ and plasma sPTHrP concentrations were lower when calcium was restricted in both water and diet. Positive correlations between plasma sPTHrP and plasma Ca2+(R2=0.30, N=39, P<0.05), and plasma sPTHrP and body mass of the fish (R2=0.37, N=148, P<0.001) were found. Immunoreactive sPTHrP was demonstrated in pituitary gland pars intermedia cells that border the pars nervosa and co-localises with somatolactin. In the pituitary gland, pthrp, pth1r and casr mRNAs were downregulated after both short-and long-term exposure to DSW. A correlation between pituitary gland pthrp mRNA expression and plasma Ca2+(R2=0.71, N=7, P<0.01) was observed. In gill tissue, pthrp and pth1r mRNAs were significantly upregulated after 30 days exposure to DSW, whereas no effect was found for casr mRNA expression. We conclude that in water of low salinity,declining pituitary gland pthrp mRNA expression accompanied by constant plasma sPTHrP levels points to a reduced sPTHrP turnover and that sPTHrP, through paracrine interaction, is involved in the regulation of branchial calcium handling, independently of endocrine pituitary gland sPTHrP.

The PCR cloning strategy for type II polyhydroxyalkanoate (PHA) biosynthesis genes established previously for Pseudomonas was successfully applied to Burkholderia caryophylli strain AS 1.2741. The whole pha locus containing PHA synthase genes phaC1, phaC2 and PHA depolymerase gene phaZ was cloned. The complete open reading frames of phaC1(Bc), phaC2(Bc) and phaZ(Bc) were identified. Sequence analyses of the phaC1(Bc), phaZ(Bc) and phaC2(Bc) showed more than 77.7%, 73.7% and 68.5% identities compared with the corresponding pha loci of the known Pseudomonas strains, respectively. The functional expression of the phaC1(Bc) or phaC2(Bc) in Escherichia coli strain KM32B (fadB deleted mutant) showed the abilities of PHA production by the estimated PHA synthase genes. Over 1% PHA consisting of 3-hydroxyhexanoate (3HHx), 3-hydroxyoctanoate (3HO) and 3-hydroxydecanoate (3HD) was detected from cells of recombinant E. coli KM32B (pHXM11) harboring phaC1(Bc), grown on octanoate. At the same time over 3% of PHA consisting of 3HO and 3HD was produced from cells of recombinant E. coli KM32B (pHXM21) harboring phaC2(BC), grown on decanoate. Results showed the PCR cloning strategy developed previously can be applied to non-Pseudomonas strains such as Burkholderia in this case. This result also provided evidence for the presumption that the Burkholderia strain possesses not only polyhydroxybutyrate synthase genes, but also synthase for medium-chain-length polyhydroxyalkanoates consisting of 3HHx, 3HO and 3HD.