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Virulence of Metallo-ß-Lactamase-Producing Pseudomonas aeruginosa In Vitro and In Vivo. S. Aoki, 2004.We evaluated the virulence of Pseudomonas aeruginosa carrying blaIMP, a metallo-ß-lactamase gene, and the efficacy of ceftazidime, imipenem-cilastatin, and ciprofloxacin in the endogenous bacteremia model . The presence of blaIMP did not practically change the virulence of the parent strain, and ciprofloxacin was effective against infection with P . aeruginosa carrying blaIMP . Detection and Enumeration of Vibrio vulnificus in Oysters from Two Estuaries along the Southwest Coast of India, Using Molecular Methods. Ammini Parvathi, 2004. The MexJK Efflux Pump of Pseudomonas aeruginosa Requires OprM for Antibiotic Efflux but Not for Efflux of Triclosan. Rungtip Chuanchuen, 2002.Using the biocide triclosan as a selective agent, several triclosan-resistant mutants of a susceptible Pseudomonas aeruginosa strain were isolated . Cloning and characterization of a DNA fragment conferring triclosan resistance from one of these mutants revealed a hitherto uncharacterized efflux system of the resistance nodulation cell division (RND) family, which was named MexJK and which is encoded by the mexJK operon . Expression of this operon is negatively regulated by the product of mexL, a gene located upstream of and transcribed divergently from mexJK . The triclosan-resistant mutant contained a single nucleotide change in mexL, which caused an amino acid change in the putative helix-turn-helix domain of MexL . The MexL protein belongs to the TetR family of repressor proteins . The MexJK system effluxed tetracycline and erythromycin but only in the presence of the outer membrane protein channel OprM; OprJ and OprN did not function with MexJK . Triclosan efflux required neither of the outer membrane protein channels tested but necessitated the MexJ membrane fusion protein and the MexK inner membrane RND transporter . The results presented in this study suggest that MexJK may function as a two-component RND pump for triclosan efflux but must associate with OprM to form a tripartite antibiotic efflux system . Furthermore, the results confirm that triclosan is an excellent tool for the study of RND multidrug efflux systems and that this popular biocide therefore readily selects mutants which are cross-resistant with antibiotics . Identification and Characterization of a New Enoyl Coenzyme A Hydratase Involved in Biosynthesis of Medium-Chain-Length Polyhydroxyalkanoates in Recombinant Escherichia coli. Si Jae Park, 2003.The biosynthetic pathway of medium-chain-length (MCL) polyhydroxyalkanoates (PHAs) from fatty acids has been established in fadB mutant Escherichia coli strain by expressing the MCL-PHA synthase gene . However, the enzymes that are responsible for the generation of (R)-3-hydroxyacyl coenzyme A (R3HA-CoAs), the substrates for PHA synthase, have not been thoroughly elucidated . Escherichia coli MaoC, which is homologous to Pseudomonas aeruginosa (R)-specific enoyl-CoA hydratase (PhaJ1), was identified and found to be important for PHA biosynthesis in a fadB mutant E . coli strain . When the MCL-PHA synthase gene was introduced, the fadB maoC double-mutant E . coli WB108, which is a derivative of E . coli W3110, accumulated 43% less amount of MCL-PHA from fatty acid compared with the fadB mutant E . coli WB101 . The PHA biosynthetic capacity could be restored by plasmid-based expression of the maoCEc gene in E . coli WB108 . Also, E . coli W3110 possessing fully functional ß-oxidation pathway could produce MCL-PHA from fatty acid by the coexpression of the maoCEc gene and the MCL-PHA synthase gene . For the enzymatic analysis, MaoC fused with His6-Tag at its C-terminal was expressed in E . coli and purified . Enzymatic analysis of tagged MaoC showed that MaoC has enoyl-CoA hydratase activity toward crotonyl-CoA . These results suggest that MaoC is a new enoyl-CoA hydratase involved in supplying (R)-3-hydroxyacyl-CoA from the ß-oxidation pathway to PHA biosynthetic pathway in the fadB mutant E . coli strain . Arthrobacter Strain VAI-A Utilizes Acyl-Homoserine Lactone Inactivation Products and Stimulates Quorum Signal Biodegradation by Variovorax paradoxus. Suvi Flagan, 2003.Many Proteobacteria produce acyl-homoserine lactones (acyl-HSLs) and employ them as dedicated cell-to-cell signals in a process known as quorum sensing . Previously, Variovorax paradoxus VAI-C was shown to utilize diverse acyl-HSLs as sole sources of energy and nitrogen . We describe here the properties of a second isolate, Arthrobacter strain VAI-A, obtained from the same enrichment culture that yielded V . paradoxus VAI-C . Although strain VAI-A grew rapidly and exponentially on a number of substrates, it grew only slowly and aberrantly (i.e., linearly) in media amended with oxohexanoyl-HSL as the sole energy source . Increasing the culture pH markedly improved the growth rate in media containing this substrate but did not abolish the aberrant kinetics . The observed growth was remarkably similar to the known kinetics of the pH-influenced half-life of acyl-HSLs, which decay chemically to yield the corresponding acyl-homoserines . Strain VAI-A grew rapidly and exponentially when provided with an acyl-homoserine as the sole energy or nitrogen source . The isolate was also able to utilize HSL as a sole source of nitrogen but not as energy for growth . V . paradoxus, known to release HSL as a product of quorum signal degradation, was examined for the ability to support the growth of Arthrobacter strain VAI-A in defined cocultures . It did . Moreover, the acyl-HSL-dependent growth rate and yield of the coculture were dramatically superior to those of the monocultures . This suggested that the original coenrichment of these two organisms from the same soil sample was not coincidental and that consortia may play a role in quorum signal turnover and mineralization . The fact that Arthrobacter strain VAI-A utilizes the two known nitrogenous degradation products of acyl-HSLs, acyl-homoserine and HSL, begins to explain why none of the three compounds are known to accumulate in the environment . A Modified Saccharomyces cerevisiae Strain That Consumes L-Arabinose and Produces Ethanol. Jessica Becker, 2003.Metabolic engineering is a powerful method to improve, redirect, or generate new metabolic reactions or whole pathways in microorganisms . Here we describe the engineering of a Saccharomyces cerevisiae strain able to utilize the pentose sugar L-arabinose for growth and to ferment it to ethanol . Expanding the substrate fermentation range of S . cerevisiae to include pentoses is important for the utilization of this yeast in economically feasible biomass-to-ethanol fermentation processes . After overexpression of a bacterial L-arabinose utilization pathway consisting of Bacillus subtilis AraA and Escherichia coli AraB and AraD and simultaneous overexpression of the L-arabinose-transporting yeast galactose permease, we were able to select an L-arabinose-utilizing yeast strain by sequential transfer in L-arabinose media . Molecular analysis of this strain, including DNA microarrays, revealed that the crucial prerequisite for efficient utilization of L-arabinose is a lowered activity of L-ribulokinase . Moreover, high L-arabinose uptake rates and enhanced transaldolase activities favor utilization of L-arabinose . With a doubling time of about 7.9 h in a medium with L-arabinose as the sole carbon source, an ethanol production rate of 0.06 to 0.08 g of ethanol per g (dry weight) · h-1 under oxygen-limiting conditions, and high ethanol yields, this yeast strain should be useful for efficient fermentation of hexoses and pentoses in cellulosic biomass hydrolysates .
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