1株拮抗植物病原真菌的细菌筛选及其抑菌机理的初步研究

从上海市郊农作物根际土壤中共分离纯化得到276株细菌,利用平板对峙法筛选出1株对多种植物病原真菌具有较强拮抗作用的菌株。经过形态学观察、生理生化特征以及16S rDNA的同源性分析,初步鉴定该菌株为桔黄假单胞菌(Pseudomonas aurantiaca),该菌株的16S rDNA序列已在GenBank中注册,登录号为GQ358919。通过显微镜观察,发现JD37菌株的主要抑菌机理是通过产生拮抗物质造成病原真菌菌丝体断裂、扭曲、畸形等异常生长现象。该菌产生的拮抗物质对酸、碱较为稳定,但对高温敏感。     

The lid is a structural and functional determinant of lipase activity and selectivity

In several lipases access to the enzyme active site is regulated by the position of a mobile structure named the lid. The role of this region in modulating lipase function is reviewed in this paper analysing the results obtained with three different recombinant lipases modified in the lid sequence: Candida rugosa lipase isoform 1 (CRL1), Pseudomonas fragi lipase (PFL) and Bacillus subtilis lipase A (BSLA). A CRL chimera enzyme obtained by replacing its lid with that of another C. rugosa lipase isoform (CRL1LID3) was found to be affected in both activity and enantioselectivity in organic solvent. Variants of the PFL protein in which three polar lid residues were replaced with amino acids strictly conserved in homologous lipases displayed altered chain length preference profile and increased thermostability. On the other hand, insertion of lid structures from structurally homologous enzymes into BSLA, a lipase that naturally does not possess such a lid structure, caused a reduction in the enzyme activity and an altered substrate specificity. These results strongly support the concept that the lid plays an important role in modulating not only activity but also specifity, enantioselectivity and stability of lipase enzymes.     

Characterization of 1,3-regiospecific lipases from new Pseudomonas and Bacillus isolates

Lipase producing ability of 120 bacterial isolates was examined qualitatively, resulting in 32 lipase producers, which were further screened for 1,3-regiospecificity. Three Bacillus (GK-8, GK-31 and GK-42) and one Pseudomonas (GK-80) were found to produce 1,3-regiospecific lipases. These lipases were alkaline in nature as they showed pH optima of 9.0 and high stability in the alkaline pH range of 8.0–11.0. The lipases from three Bacillus isolates, viz. GK-8, GK-31 and GK-42 showed temperature optima of 37 °C, whereas the Pseudomonas (GK-80) lipase showed optimum activity at 50 °C. The lipase of GK-8 was highly stable and showed enhanced activity in different organic solvents like petroleum ether (172%), diethyl ether (143%) and acetone (135%).     

The effect of bio-converted polyunsaturated fatty acids on the oxidation of TAG containing highly unsaturated fatty acids

Microbial modification of vegetable fatty acids can often lead to special changes in their structure and in biological function. A bacterial strain, Pseudomonas aeruginosa PR3, is known to carry out multiple hydroxylations on polyunsaturated fatty acids containing 1,4-cis, cis diene structural units, resulting in antibacterial activity. In this paper, in an effort to understand the overall mechanism involved in the varied biological functions of the complicated metabolites of bio-converted polyunsaturated fatty acids, we performed bioconversion of several polyunsaturated fatty acids using PR3, and determined their oxidative activities against fish oil. Bio-converted linoleic acid, eicosapentanoic acid, and docosahexanoic acid promoted effectively oxidation of fish oil. It is assumed that this oxidative effect could plausibly play an important role in the antimicrobial function of these bio-converted fatty acids.     

Chitinase-mediated destructive antagonistic potential of Pseudomonas aeruginosa GRC1 against Sclerotinia sclerotiorum causing stem rot of peanut

Pseudomonas aeruginosa GRC₁ exhibited strong antagonistic activity against Sclerotinia sclerotiorum, in vitro and in vivo. Scanning electron microscopic (SEM) studies showed morphological abnormalities such as perforation, lysis and fragmentation of hyphae of S. sclerotiorum caused by P. aeruginosa GRC₁. This strain produced extracellular chitinase enzyme, the role of which was clearly demonstrated through Tn5 mutagenesis. Bacterization of peanut seeds with GRC₁ resulted in increased seed germination and reduced stem-rot of peanut in S. sclerotiorum-infested soil by 97%. Other vegetative and yield plant parameters such as nodules per plant, pods and grain yield per plant were enhanced with a statistical significance in comparison to control. Neomycin resistant (GRC₁^neo+) bacterium was a good root colonizer and frequently isolated from rhizosphere of peanut plants. These findings showed P. aeruginosa GRC₁ as a potential biocontrol agent against S. sclerotiorum.     

Interference in adhesion of bacteria and yeasts isolated from explanted voice prostheses to silicone rubber by rhamnolipid biosurfactants

AIMS: The effects and extent of adhesion of four different bacterial and two yeast strains isolated from explanted voice prostheses to silicone rubber with and without an adsorbed rhamnolipid biosurfactant layer obtained from Pseudomonasaeruginosa DS10-129 was studied. METHODS AND RESULTS: The ability of rhamnolipid biosurfactant to inhibit adhesion of micro-organisms to silicone rubber was investigated in a parallel-plate flow chamber. The anti-adhesive activity of the biosurfactant at different concentrations was significant against all the strains and depended on the micro-organism tested. The results showed an effective reduction in the initial deposition rates, and the number of bacterial cells adhering after 4 h, for all micro-organisms tested at the 4 g l(-1) undiluted rhamnolipid solution. Maximum initial reduction of adhesion rate (an average of 66%) occurred for Streptococcus salivarius GB 24/9 and Candida tropicalis GB 9/9. The number of cells adhering after 4 h on silicone rubber conditioned with biosurfactant was reduced to 48% for Staphylococcus epidermidis GB 9/6, Strep. salivarius GB 24/9, Staphylococcus aureus GB 2/1 and C. tropicalis GB 9/9 in comparison to controls. Perfusing the flow chamber with biosurfactant containing solution followed by the passage of a liquid-air interface, to investigate detachment of micro-organisms adhering to silicone rubber, produced high detachment (96%) of adhered cells for all micro-organisms studied, except for Staph. aureus GB 2/1 (67%). SIGNIFICANCE AND IMPACT OF THE STUDY: It is concluded that biosurfactant represent suitable compounds that should be considered in developing future strategies to prevent the microbial colonization of silicone rubber voice prostheses.     

Characterization and gene cloning of a novel serine protease with nematicidal activity from Trichoderma pseudokoningii SMF2

Pseudomonas aeruginosa is a pathogenic bacterium widely investigated for its high incidence in clinical environments and its ability to form strong biofilms. During biofilm development, sessile cells acquire physiological characteristics differentiating them from planktonic cells. But after treatment with disinfectants, or to ensure survival of the species in hostile environments, biofilm cells can detach. This complicates disinfection procedures. This study aimed to physiologically characterize cells detached from a P. aeruginosa biofilm and to compare them with their sessile and planktonic counterparts. We first tested planktonic growth kinetics and capacities to form new biofilms. Then we investigated cell-surface properties. And finally, we tested in vitro susceptibility to antibiotics. The results first indicated that sessile and detached cells have similar planktonic growth kinetics and cell-surface properties, distinguishable from those of planktonic cells. Interestingly, the three populations exhibited different biofilm-forming capacities, suggesting that there is a transitional phenotype between sessile and planktonic states, at least during the first hours following cell detachment. It is important to consider this observation when developing treatments to optimize disinfection processes. Surprisingly, the three populations showed the same antibiotic susceptibility profile.     

Comparative genomics-guided loop-mediated isothermal amplification for characterization of Pseudomonas syringae pv. phaseolicola

Aims:  To design and evaluate a loop-mediated isothermal amplification (LAMP) protocol by combining comparative genomics and bioinformatics for characterization of Pseudomonas syringae pv. phaseolicola (PSP), the causal agent of halo blight disease of bean ( Phaseolus vulgaris L.).
Methods and Results:  Genomic sequences of Pseudomonas syringae pathovars, P. fluorescens and P. aeruginosa were analysed using multiple sequence alignment. A pathovar-specific region encoding pathogenicity-related secondary metabolites in the PSP genome was targeted for developing a LAMP assay. The final assay targeted a polyketide synthase gene, and readily differentiated PSP strains from other Pseudomonas syringae pathovars and other Pseudomonas species, as well as other plant pathogenic bacteria, e.g. species of Pectobacterium , Erwinia and Pantoea .
Conclusion:  A LAMP assay has been developed for rapid and specific characterization and identification of PSP from other pathovars of P. syringae and other plant-associated bacteria .
Significance and Impact of the Study:  This paper describes an approach combining a bioinformatic data mining strategy and comparative genomics with the LAMP technology for characterization and identification of a plant pathogenic bacterium. The LAMP assay could serve as a rapid protocol for microbial identification and detection with significant applications in agriculture and environmental sciences.     

Immunoelectron microscopy of Bacillus subtilis cells secreting human interferon α1 or staphylokinase

Uptake of 14C-labelled chlorhexidine diacetate (14C-CHA) by wild-type and envelope mutant strains of Escherichia coli and Pseudomonas aeruginosa was very rapid. Maximum uptake was observed within a contact time of 20 s with no additional binding on increased contact, and was concentration-dependent. In contrast to this rapid binding of 14C-CHA, bactericidal studies revealed that the lethal activity of low concentrations of unlabelled CHA was slow, although higher concentrations had a rapid effect. Comparison of a wild-type strain with its envelope mutants indicated that there was little difference in 14C-CHA uptake, in minimal inhibitory concentrations or in bactericidal activity. Azolectin was found to be an effective neutralising agent of biguanide action, but in in vitro agar tests and in reducing or removing the amount of 14C-CHA taken up by the cells.     

Effect of the proton motive force inhibitor carbonyl cyanide‐m‐chlorophenylhydrazone (CCCP) on Pseudomonas aeruginosa biofilm development

Aims: Proton motive force (PMF) inhibition enhances the intracellular accumulation of autoinducers possibly interfering with biofilm formation. We evaluated the effect of the PMF inhibitor carbonyl cyanide‐m‐chlorophenylhydrazone (CCCP) on Pseudomonas aeruginosa biofilm development. Methods and Results: Four epidemiologically unrelated P. aeruginosa isolates were studied. A MexAB‐oprM overproducing strain was used as control. Expression of gene mexB was examined and biofilm formation after incubation with 0, 12·5 and 25 μmol l^?1 of CCCP was investigated. Mean values of optical density were analysed with one‐way analysis of variance and t‐test. Two isolates subexpressed mexB gene and only 25 μmol l^?1 of CCCP affected biofilm formation. Biofilms of the other two isolates and control strain PA140 exhibited significantly lower absorbance (P ranging from <0·01 to <0·05) with either 12·5 or 25 μmol l^?1 of CCCP. Conclusions: The PMF inhibitor CCCP effect was correlated with the expression of MexAB‐OprM efflux system and found to compromise biofilm formation in P. aeruginosa. Significance and Impact of the Study: These data suggest that inhibition of PMF‐dependent trasporters might decrease biofilm formation in P. aeruginosa.