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Characterization and Functional Analysis of the poxB Gene, Which Encodes Pyruvate Oxidase in Lactobacillus plantarum. Frédérique Lorquet, 2004.The pyruvate oxidase gene (poxB) from Lactobacillus plantarum Lp80 was cloned and characterized . Northern blot and primer extension analyses revealed that transcription of poxB is monocistronic and under the control of a vegetative promoter . poxB mRNA expression was strongly induced by aeration and was repressed by glucose . Moreover, Northern blotting performed at different stages of growth showed that poxB expression is maximal in the early stationary phase when glucose is exhausted . Primer extension and in vivo footprint analyses revealed that glucose repression of poxB is mediated by CcpA binding to the cre site identified in the promoter region . The functional role of the PoxB enzyme was studied by using gene overexpression and knockout in order to evaluate its implications for acetate production . Constitutive overproduction of PoxB in L . plantarum revealed the predominant role of pyruvate oxidase in the control of acetate production under aerobic conditions . The  poxB
mutant strain exhibited a moderate (20 to 25%) decrease in acetate
production when it was grown on glucose as the carbon source, and
residual pyruvate oxidase activity that was between 20 and 85% of the
wild-type activity was observed with glucose limitation (0.2%
glucose) . In contrast, when the organism was grown on maltose, the
poxB mutation resulted in a large (60 to 80%) decrease in acetate
production . In agreement with the latter observation, the level
of residual pyruvate oxidase activity with maltose limitation (0.2%
maltose) was less than 10% of the wild-type level of activity .
Change in Bacterial Community Structure during In Situ Biostimulation of Subsurface Sediment Cocontaminated with Uranium and Nitrate. Nadia N. North, 2004.Previous studies have demonstrated that metal-reducing microorganisms can effectively promote the precipitation and removal of uranium from contaminated groundwater . Microbial communities were stimulated in the acidic subsurface by pH neutralization and addition of an electron donor to wells . In single-well push-pull tests at a number of treated sites, nitrate, Fe(III), and uranium were extensively reduced and electron donors (glucose, ethanol) were consumed . Examination of sediment chemistry in cores sampled immediately adjacent to treated wells 3.5 months after treatment revealed that sediment pH increased substantially (by 1 to 2 pH units) while nitrate was largely depleted . A large diversity of 16S rRNA gene sequences were retrieved from subsurface sediments, including species from the  , ß,
 , and
 subdivisions of the class Proteobacteria, as well as low- and high-G+C gram-positive species . Following in situ biostimulation of microbial communities within contaminated sediments, sequences related to previously cultured metal-reducing
-Proteobacteria increased from 5% to nearly 40% of the clone libraries . Quantitative PCR revealed that Geobacter-type 16S rRNA gene sequences increased in biostimulated sediments by 1 to 2 orders of magnitude at two of the four sites tested . Evidence from the quantitative PCR analysis corroborated information obtained from 16S rRNA gene clone libraries, indicating that members of the
-Proteobacteria subdivision, including Anaeromyxobacter dehalogenans-related and Geobacter-related sequences, are important metal-reducing organisms in acidic subsurface sediments . This study provides the first cultivation-independent analysis of the change in metal-reducing microbial communities in subsurface sediments during an in situ bioremediation experiment .
PorA Represents the Major Cell Wall Channel of the Gram-Positive Bacterium Corynebacterium glutamicum. Noelia Costa-Riu, 2003.The cell wall of the gram-positive bacterium Corynebacterium glutamicum contains a channel (porin) for the passage of hydrophilic solutes . The channel-forming polypeptide PorA is a 45-amino-acid acidic polypeptide with an excess of four negatively charged amino acids, which is encoded by the 138-bp gene porA . porA was deleted from the chromosome of C.glutamicum wild-type strain ATCC 13032 to obtain mutant ATCC 13032  porA . Southern blot analysis demonstrated that porA was deleted . Lipid bilayer experiments revealed that PorA was not present in the cell wall of the mutant strain . Searches within the known chromosome of C . glutamicum by using National Center for Biotechnology Information BLAST and reverse transcription-PCR showed that no other PorA-like protein is encoded on the chromosome or is expressed in the deletion strain . The porA deletion strain exhibited slower growth and longer growth times than the C . glutamicum wild-type strain . Experiments with different antibiotics revealed that the susceptibility of the mutant strain was much lower than that of the wild-type C . glutamicum strain . The results presented here suggest that PorA represents a major hydrophilic pathway through the cell wall and that C . glutamicum contains cell wall channels which are not related to PorA .
Seasonal Changes in Fungal Production and Biomass on Standing Dead Scirpus lacustris Litter in a Northern Prairie Wetland. Brij Verma, 2003.Decaying macrophytes are an important source of carbon and nutrients in fungal and bacterial communities of northern prairie wetlands . Dead macrophytes do not collapse into the water column immediately after death, and decomposition by fungi and bacteria begins while the plants are standing . The seasonal variations in fungal biomass and production on Scirpus lacustris stems, both above and below water, were measured to assess which environmental factors were dominant in affecting these variations in a typical prairie wetland . Fungal biomass and production were measured from early May to November, just prior to freeze-up . Fungal decomposition began and was greatest in the spring despite low water temperatures . The fungal production, as measured by the incorporation of [1-14C]acetate into ergosterol, ranged from 1.8 to 376 µg of C g of ash-free dry mass (AFDM)-1 day-1, and the biomass, as estimated by using ergosterol, ranged from nondetectable to 5.8 mg of C g of AFDM-1 . There was no significant difference in biomass or production between aerial and submerged portions of Scirpus stems . The water temperature was correlated with fungal production (r = 0.7, P < 0.005) for aerial stem pieces but not for submerged pieces . However, in laboratory experiments water temperature had a measurable effect on both biomass and production in submerged stem pieces . Changes in fungal biomass and productivity on freshly cut green Scirpus stems decaying in the water either exposed to natural solar radiation or protected from UV radiation were monitored over the summer . There was no significant difference in either fungal biomass (P = 0.76) or production (P = 0.96) between the two light treatments . The fungal biomass and rates of production were within the lower range of the values reported elsewhere, probably as a result of the colder climate and perhaps the lower lability of Scirpus stems compared to the labilities of the leaves and different macrophytes examined in other studies performed at lower latitudes . Modification of the Monomer Composition of Polyhydroxyalkanoate Synthesized in Saccharomyces cerevisiae Expressing Variants of the ß-Oxidation-Associated Multifunctional Enzyme. Silvia Marchesini, 2003.Expression by Saccharomyces cerevisiae of a polyhydroxyalkanoate (PHA) synthase modified at the carboxy end by the addition of a peroxisome targeting signal derived from the last 34 amino acids of the Brassica napus isocitrate lyase (ICL) and containing the terminal tripeptide Ser-Arg-Met resulted in the synthesis of PHA . The ability of the terminal peptide Ser-Arg-Met and of the 34-amino-acid peptide from the B . napus ICL to target foreign proteins to the peroxisome of S . cerevisiae was demonstrated with green fluorescent protein fusions . PHA synthesis was found to be dependent on the presence of both the enzymes generating the ß-oxidation intermediate 3-hydroxyacyl-coenzyme A (3-hydroxyacyl-[CoA]) and the peroxin-encoding PEX5 gene, demonstrating the requirement for a functional peroxisome and a ß-oxidation cycle for PHA synthesis . Using a variant of the S . cerevisiae ß-oxidation multifunctional enzyme with a mutation inactivating the B domain of the R-3-hydroxyacyl-CoA dehydrogenase, it was possible to modify the PHA monomer composition through an increase in the proportion of the short-chain monomers of five and six carbons .
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