Genetic organization of pha gene locus affects phaC expression, poly(hydroxyalkanoate) composition and granule morphology in Pseudomonas corrugata

The complete sequence of the pha locus responsible for the biosynthesis of poly(hydroxyalkanoates) (PHAs) in Pseudomonas corrugata 388 was determined. As with the other known pseudomonad pha gene loci, the one in P. corrugata 388 also consists of phaC1 (1,680 bps; PHA synthase 1), phaZ (858 bp; PHA depolymerase) and phaC2 (1,683 bp; PHA synthase 2) genes. A BLAST search showed that the nucleotide sequences of these genes and the amino-acid sequences of their respective gene products are homologous to those of P. corrugata CFBP5454 and P. mediterranea CFBP5447. A putative intrinsic transcription terminator consisting of a dyad symmetry (24 bp; Delta G = -41.8 kcals) that precedes a stretch of dA residues was located in the phaC1-phaZ intergenic region. P. corrugata mutant-clones XI 32-1 and XI 32-4 were constructed in which this intergenic region was replaced with a selectable kanamycin-resistance gene. These mutant clones when grown on oleic acid for 48 h showed 4.7-to 7.0-fold increases of phaC1 and phaC2 relative expression in comparison to the initial inoculants, whereas the parental strain showed only 1.2- to 1.4-fold increases. Furthermore, in comparison to parental P. corrugata with only a few large PHA inclusion bodies, the mutants grown on oleic acid produce numerous smaller PHA granules that line the periphery of the cells. With glucose as a substrate, XI 32-1 and XI 32-4 clones produce mcl-PHA with a high content (26-31 mol%) of the mono-unsaturated 3-hydroxydodecenoate as a repeat-unit monomer. Our results show for the first time the effects of the phaC1-phaZ intergenic region on the substrate-dependent temporal expression of phaC1 and phaC2 genes, the repeat-unit composition of mcl-PHA, and the morphology of the PHA granules

Neofusicoccum parvum causes stem canker of thornless blackberry in Italy

Important damage caused by the fungus Neofusicoccum parvum in thornless blackberry in southern Italy is reported for the first time. The most noticeable symptoms were stem cankers and yellowing of the foliage. Cankers on stems were initially elongated, and infected tissue was darkly pigmented. The fungus was identified based on morphological characteristics and by sequencing the ITS1-5.8S-ITS2 region and part of the translation elongation factor 1-alpha (EF1-a) gene. Pathogenicity tests confirmed that N. parvum caused the disease on blackberry, inducing symptoms similar to those occurring under natural conditions. This is potentially a serious disease of blackberry in southern Italy, where cultivation is expanding

The social representation of sexual violence between "background and surface attitudes" a research with university students

The problem of "sexual violence", a central challenge of our society, keeps existing in spite of condamning stances from the institutions and the cultural world. It's a controvertial problem, especially regarding the rapists' specific behaviours and its quantitative consistence. The present paper was made with a sample of university students in order to verify the influence of gender, level and type of studies, and of psychosocial variables on the represantion of the phenomenon. The first results seem to confirm the hypotheses

Transcriptional analysis of pha genes in Pseudomonas mediterranea CFBP 5447 grown on glycerol

We analysed the draft genome sequence of Pseudomonas mediterranea CFBP 5447 in order to identify firstly the central metabolic pathways that convert fatty acids or carbohydrate intermediates into mcl-PHA and secondly the genes involved in glycerol metabolism (glpF, glpK, glpD, glpR). Absence of the glpF gene, which codifies for the “glycerol uptake facilitator protein”, was highlighted. In order to understand the expression of the pha gene cluster, we investigated the promoter activity of phaC1, phaC2, phaZ, phaD and phaI genes. When glycerol was present as the carbon source, PI was found to be the most active promoter. Expression analysis of the knock-out mutant of the phaD gene, which is a transcriptional regulator belonging to the TetR family, showed that PhaD acts as an activator of the phaI promoter which, in turn, triggers the transcription of the phaIF operon. The activation of PC1, which controls the phaC1ZC2D, by PhaD, was less efficient than PI

Memory effects in annealed hybrid gold nanoparticles/block copolymer bilayers

We report on the use of the self-organization process of sputtered gold nanoparticles on a self-assembled block copolymer film deposited by horizontal precipitation Langmuir-Blodgett (HP-LB) method. The morphology and the phase-separation of a film of poly-n-butylacrylate-block-polyacrylic acid (PnBuA-b-PAA) were studied at the nanometric scale by using atomic force microscopy (AFM) and Time of Flight Secondary Ion Mass Spectrometry (TOF-SIMS). The templating capability of the PnBuA-b-PAA phase-separated film was studied by sputtering gold nanoparticles (NPs), forming a film of nanometric thickness. The effect of the polymer chain mobility onto the organization of gold nanoparticle layer was assessed by heating the obtained hybrid PnBuA-b-PAA/Au NPs bilayer at T >Tg. The nanoparticles' distribution onto the different copolymer domains was found strongly affected by the annealing treatment, showing a peculiar memory effect, which modifies the AFM phase response of the Au NPs layer onto the polar domains, without affecting their surfacial composition. The effect is discussed in terms of the peculiar morphological features induced by enhanced mobility of polymer chains on the Au NPs layer

Production of Polyhydroxyalkanoates and Extracellular Products Using Pseudomonas Corrugata and P. Mediterranea: A Review

Some strains of Pseudomonas corrugata (Pco) and P. mediterranea (Pme) efficiently synthesize medium-chain-length polyhydroxyalkanoates elastomers (mcl-PHA) and extracellular products on related and unrelated carbon sources. Yield and composition are dependent on the strain, carbon source, fermentation process, and any additives. Selected Pco strains produce amorphous and sticky mcl-PHA, whereas strains of Pme produce, on high grade and partially refined biodiesel glycerol, a distinctive filmable PHA, very different from the conventional microbial mcl-PHA, suitable for making blends with polylactide acid. However, the yields still need to be improved and production costs lowered. An integrated process has been developed to recover intracellular mcl-PHA and extracellular bioactive molecules. Transcriptional regulation studies during PHA production contribute to understanding the metabolic potential of Pco and Pme strains. Data available suggest that pha biosynthesis genes and their regulations will be helpful to develop new, integrated strategies for cost-effective production

A real-time PCR quantitative detection assay for Pseudomonas savastanoi pv. nerii in Nerium oleander

A real-time PCR assay based on TaqMan chemistry was developed for the detection of Pseudomonas savastanoi pathovars that cause bacterial knot disease on different plant species. Primers and probe sequences were based on the iaaL gene coding for (indole-3-acetyl)-L-lysine synthetase and previously used in conventional PCR tests. Assay specificity was tested with an extended range of strains of P. savastanoi from eight hosts, with 13 other Pseudomonas spp., and with other microorganisms naturally occurring on or in oleander plants. A pure culture cell suspension was quantified over a seven log concentration range (108 to 102 cfu ml-1 ). Different protocols were developed for the detection and quantification of P. savastanoi pv. nerii from symptomatic and asymptomatic oleander plants. A 24-h bacterial enrichment step either on PVF-1 or OKA-M broth improved the sensitivity of the assay, making it suitable to screen planting material for latent infections