The polyhydroxyalkanoate (PHA) synthase 1 of Pseudomonas sp. H9 synthesized a 3-hydroxybutyrate-dominant hybrid of short- and medium-chain-length PHA.

Pseudomonas sp. H9 (H9), an environmental strain isolated from a riverbank soil sample collected in southern Taiwan, is Gram-negative and shares a 99 % sequence identity to Pseudomonas putida KT2440 based on 16S rRNA gene analysis. H9 produced novel polyhydroxyalkanoates (PHA) including a hybrid PHA, comprised of 3-hydroxyvalerate (37 mol%) and medium-chain-length (MCL) monomers from valerate, as well as a 3-hydroxyhexanoate-dominant (93 mol%) MCL-PHA from hexanoate. Next-generation sequencing analysis showed H9 had a typical class II PHA operon, consisted of phaC1H9-phaZH9-phaC2H9, in which phaC1H9 was the sole active PHA synthase in H9. Deletion of phaC1H9 gene led to a complete loss of its PHA accumulation capability. Knockout of phaC2H9 gene, in contrast, affected neither bacterial growth nor PHA accumulation. When co-expressed with the phaAB genes of Ralstonia eutropha H16 in the PHA mutant strain Pseudomonas sp. H9ΔC1, phaC1H9 synthesized a hybrid PHA consisted of 3-hydroxybutyrate (3HB) (75 mol%) and MCL-monomers, confirmed in analyses using hot-acetone fractionation and 13C-NMR spectroscopy. As a novel PHA synthase, PhaC1H9 possesses a broad substrate specificity to synthesize a hybrid of SCL- and MCL-PHA, known to have many mechanical properties for potential applications.

Cultivation temperature modulated the monomer composition and polymer properties of polyhydroxyalkanoate synthesized by Cupriavidus sp. L7L from levulinate as sole carbon source.

Cupriavidus sp. L7L, a newly isolated wild-type soil bacterium, was found to synthesize polyhydroxyalkanoate (PHA) terpolymers from levulinic acid (LA), a top bio-based platform chemical, as the sole carbon source. NMR spectra showed that the terpolymers consisted mainly of 3-hydroxybutyrate (3HB), 3-hydroxyvalerate (3HV), and a small amount (2.1-4.8 mol%) of 4-hydroxyvalerate (4HV). The combination of monomers together with high molecular weights improved markedly the thermal and mechanical properties of the terpolymers. Increasing cultivation temperature of Cupriavidus sp. L7L increased the proportions of 3HB and decreased the proportions of 3HV. A fed-batch fermentation using LA as the sole carbon source without pH control produced a biomass of 15.8 dry weight g L-1 that contained 81 dry weight% of a terpolymer P(3HB-co-33.7 mol% 3HV-co-2.1 mol% 4HV), equaling a productivity of 0.213 g PHA L-1 h-1. The terpolymer showed a melting point of 92 °C and elongation at break of 630%, compared to 84.6 °C and 462%, respectively, for the copolymer P(3HB-co-35.9 mol% 3HV) that contained no 4HV. This study showed that Cupriavidus sp. L7L exhibited a great potential for producing PHA polymers with excellent mechanical property that could be modulated by cultivation temperature when cultivated exclusively in LA.

Isolation and characterization of the unicellular diazotrophic cyanobacterium Group C TW3 from the tropical western Pacific Ocean.

A unicellular diazotrophic cyanobacterium strain of Group C, designated TW3, was isolated from the oligotrophic Kuroshio Current of the western Pacific Ocean. To our knowledge, this represents the first successful laboratory culture of a Group C unicellular diazotroph from oceanic water. TW3 cells are green rods, 2.5-3.0 µm in width and 4.0-6.0 µm in length. Phylogenetic analyses of both 16S rRNA and nifH gene fragments indicated that the TW3 sequences were over 98% identical to those of the previously isolated Cyanothece sp. ATCC51142 and Gloeocapsa sp., suggesting that TW3 is a member of the Group C unicellular diazotrophs. In addition, both TW3 and Cyanothece sp. ATCC51142 share morphological characteristics; both strains are sheathless and rod-shaped, display binary fission in a single plane, and possess dispersed thylakoids. TW3 grows aerobically in nitrogen-deficient artificial seawater, and exhibited the highest observed growth rate of 0.035 h(-1) when cultured at 30°C and 140 µmol m(-2) s(-1) of light intensity. The nitrogen fixation rate, when grown optimally using a 12 h/12 h light-dark cycle, was 7.31 × 10(-15) mol N cell(-1) day(-1) . Immunocytochemical staining using Trichodesmium sp. NIBB1067 nitrogenase antiserum revealed the existence of diazotrophic cells sharing morphological characteristics of TW3 in the Kuroshio water from which TW3 was isolated.

Cloning of peroxisome proliferators activated receptors in the cobia (Rachycentron canadum) and their expression at different life-cycle stages under cage aquaculture.

We present the cDNA sequences and tissue mRNA expression of peroxisome proliferator-activated receptor (PPAR) alpha, beta and gamma isotypes in the cobia (Rachycentron canadum), a warm water pelagic fish that is becoming a fish of choice for offshore cage farming. RT-PCR and real-time PCR showed that PPARalpha mRNA predominated in red muscle, heart and liver whereas PPARbeta was expressed mainly in liver and pyloric caeca. In contrast, PPARgamma transcripts were detected in all of the tissues examined, with the highest level occurring in visceral fat depot. Our 52-wk time-series investigation showed that while the mRNA expression of PPARgamma in the cobia was positively (P < 0.05) related to its body lipid deposition, a negative (P < 0.05) relationship was found between PPARalpha expression in the liver and body lipid deposition. There was a significant increase in body lipid deposition and hepatic PPARgamma expression as the fish grew. The hepatic PPARgamma expression could be a sufficient parameter describing the bodily expression of PPARgamma because of its positive correlation with PPARgamma expressions in all other tissues. These results showed that PPARgamma and alpha played a pivotal role in the control of lipid metabolic and storage functions in the liver, muscle and visceral fat depot of the cobia.

Differential time-series expression of immune-related genes of Pacific white shrimp Litopenaeus vannamei in response to dietary inclusion of beta-1,3-glucan.

Time-series changes in transcript abundance of nine genes encoding important immune proteins in haemocytes or hepatopancreas of Pacific white shrimp Litopenaeus vannamei fed daily in a 1-week feeding trial diets containing three levels (0%, 0.2% or 1%) of beta-1,3-glucan from Schizophyllum commune were quantified by real-time PCR. As a whole, the immune modulation elicited by beta-glucan is bimodal, one swift reaction of up- or down-regulation occurred within 24h and a delayed regulation was commenced as late as 3-7days. Haemocyanin, crustin, prophenoloxidase (proPO) and transglutaminase (TGase) did not respond to the glucan treatment. While penaeidin 3 (Litvan PEN3) was swiftly down-regulated (0-24h), lysozyme and cytosolic manganese superoxide dismutase (cMnSOD) were swiftly up-regulated (0-24h). In contrast, the two pattern recognition proteins (PRPs), beta-glucan binding protein-high density lipoprotein (BGBP-HDL) and lipopolysaccharide/beta-glucan binding protein (LGBP), showed a delayed up-regulation. Their expressions were not maximized until as late as 72h or 7days, respectively, which coincide with the initiation of reported immune enhancement (6-24days) of PO and SOD activity, phagocytosis and superoxide anion production in penaeid shrimp receiving glucan-containing diet. These immune responses could be the downstream effects of the two PRP gene up-regulation that predispose the shrimp to a state of high immune responsiveness. Increased dosage of beta-glucan from 2 to 10gkg(-1) diet did not affect the expressions of the genes, indicating the sufficiency of beta-glucan supplementation at 2gkg(-1) diet.

Tissue expressions of nine genes important to immune defence of the Pacific white shrimp Litopenaeus vannamei.

The tissue expressions of nine immune related genes in apparently healthy Pacific white shrimp Litopenaeus vannamei were analyzed by conventional RT-PCR, quantitative real time PCR (qPCR) and in situ hybridisation. The nine genes were beta-glucan binding protein-high density lipoprotein (BGBP-HDL), lipopolysaccharide-beta-glucan binding protein (LGBP), haemocyanin, prophenoloxidase (proPO), transglutaminase (TGase), crustins, penaeidins (PEN), cytosolic manganese superoxide dismutase (cMnSOD), and lysozyme. Transcripts of all nine genes were detected in all tissues with differential expression levels when examined by RT-PCR and qPCR. BGBP-HDL, LGBP and haemocyanin were mainly expressed in the hepatopancreas and their expressions levels were about 1/10-1/3 those of beta-actin. Their expressions in other tissues were relatively limited. ProPO, TGase, crustins, PEN-3, and lysozyme showed the highest levels of expression in haemocytes and the lowest in hepatopancreas. Their expression levels in the haemocytes were 3 (PEN-3) to 10(-2) (proPO) times those of beta-actin. In contrast to the other genes, cMnSOD showed higher expression levels in haemolymph related organ, stomach and muscle; and lower expression levels in haemocyte, migut, neural ganglion and hepatopancreas. When examined by in situ hybridisation, hepatopancreatic F cells were found to be the major cell type that produced transcripts of BGBP-HDL, LGBP and haemocyanin. On the other hand, circulatory haemocytes and haemocytes infiltrated in various tissues contributed to the expressions of proPO, TGase, crustins, PEN-3 and lysozyme. Both hepatopancreatic F cell and haemocyte generated cMnSOD transcripts. Using in situ hybridisation, the present study is the first to show the tissue distributions of BGBP-HDL, LGBP, haemocyanin, TGase, crustins and cMnSOD in healthy white shrimp. The present results provide a baseline data of physiological expressions for the genes that are important in immune activation and modulation in Pacific white shrimp and a guideline of tissue or organ sampling for effective gene expression analyses for future immunological studies.

Tissue distribution of prophenoloxidase transcript in the Pacific white shrimp Litopenaeus vannamei.

The expression of prophenoloxidase (proPO) transcript in the tissues of the Pacific white shrimp Litopenaeus vannamei was studied by Northern blot, RT-PCR and in situ hybridisation. Northern blot analysis reveals that proPO was constitutive mainly in shrimp haemocytes. Its transcripts, when detected with RT-PCR, were widely distributed in haemocytes, gill, heart, lymphoid organ, stomach, midgut, anterior midgut caecum and ganglion. A lower expression was found in hepatopancreas, muscle and cuticular epidermis. The results of in situ hybridisations showed that gill, heart, muscle, haematopoitic tissue and hepatopancreas that were infiltrated by haemocytes also presented positive signals. ProPO transcripts, however, were detected in non-haemocyte cells, including F and E cells of the hepatopancreas, epithelium in stomach, anterior midgut caecum, glia cell in nervous cord and neurosecretory cells in ganglions.

Dietary beta-1,3-glucan effectively improves immunity and survival of Penaeus monodon challenged with white spot syndrome virus.

The effectiveness of dietary beta-1,3-glucan (BG), derived from Schizophyllum commune, in modulating the non-specific immunity of the grass prawn Penaeus monodon and its resistance to white spot syndrome virus (WSSV) were investigated. Juvenile P. monodon (6.5+/-0.4 g) were fed for 20 days on a series of test diets containing graded levels of BG (0, 1, 2, 10, 20 g kg(-1)diet) and were then challenged by injection of WSSV. The haemolymph total haemocyte count (THC), phagocytosis (PI), phenoloxidase (PO), superoxide anion (O(2)(-)) and superoxide dismutase (SOD) production were measured at days 0, 1, 3, 6, 9, 12 and 24 after challenge, and shrimp survival rate was also recorded. All the shrimps fed on diets containing BG no more than 1 g kg(-1)died by day 12. Conversely, the survival rate of shrimp fed with the diet containing 10 g kg(-1)BG was significantly higher (P<0.05) by day 9 than that of the other groups. When screened by the WSSV PCR diagnostic procedure, the percentages of surviving juveniles of the BG 2, 10, 20 g kg(-1)groups that were 2-step WSSV negative, were 55, 65 and 65%, respectively. The haemolymph THC, PO, O(2)(-)and SOD production of the 2, 10 and 20 g kg(-1)BG diet groups dropped drastically immediately after the WSSV challenge but subsequently returned to normal. Therefore, oral administration of BG at an optimal level of 10 g kg(-1)diet for 20 days effectively enhanced the immune system and improved the survival of WSSV-infected P. monodon.

Docosahexaenoic acid is superior to eicosapentaenoic acid as the essential fatty acid for growth of grouper, Epinephelus malabaricus.

Juvenile grouper (Epinephelus malabaricus) were fed seven experimental diets, one control diet and one reference diet for 12 wk to determine the dietary requirement of grouper for docosahexaenoic (DHA) and eicosapentaenoic (EPA) acids. Each of the seven diets contained 1 g/100 g DHA and EPA in various combinations and 9 g/100 g tristearin. The control diet contained 1 g/100 g trilinolenin and trilinolein (3:1, wt/wt), and no supplemental EPA or DHA. The reference diet contained only natural oils from a mixture of cod liver oil, linseed oil and safflower oil at a ratio of 2:1:1 (wt/wt/wt). Significant differences (P < 0.05) in growth were observed among the dietary treatments but not in survival rate or relative liver weight. Only the diet with the highest DHA/EPA ratio (3:1) promoted significantly greater growth than the control diet. Purified EPA and DHA did not perform better in promoting growth than did the impure EPA and DHA oils. Enhanced growth was observed when the dietary DHA/EPA ratio was greater than 1, indicating that DHA was superior to EPA in promoting fish growth. Neutral lipid (NL) was the predominant lipid fraction (>70%) in both liver and muscle. Tissue NL/polar lipid did not differ among groups except the reference diet group that had a higher ratio (P < 0.05). DHA and EPA levels in the grouper tissues, especially muscle, were highly reflective of dietary levels of DHA and EPA, indicating that direct incorporation was likely. In addition, the 20:1(n-9), concentration in NL fractions seems to be an appropriate indicator of dietary essential fatty acid deficiency in grouper.