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Distribution and Organization of Auxotrophic Genes on the Multichromosomal Genome of Burkholderia multivorans ATCC 17616. Harunobu Komatsu, 2003.The Burkholderia multivorans strain ATCC 17616 carries three circular chromosomes with sizes of 3.4, 2.5, and 0.9 Mb . To determine the distribution and organization of the amino acid biosynthetic genes on the genome of this ß-proteobacterium, various auxotrophic mutations were isolated using a Tn5 derivative that was convenient not only for the determination of its insertion site on the genome map but also for the structural analysis of the flanking regions . Analysis by pulsed-field gel electrophoresis revealed that 20 out of 23 insertion mutations were distributed on the 3.4-Mb chromosome . More detailed analysis of the his, trp, arg, and lys mutations and their flanking regions revealed the following properties of these auxotrophic genes: (i) all nine his genes were clustered on the 3.4-Mb chromosome; (ii) seven trp genes were organized within two distinct regions, i.e., a trpEGDC cluster on the 3.4-Mb chromosome and a trpFBA cluster on the 2.5-Mb chromosome; (iii) the leu gene cluster, leuCDB, was also located close to the trpFBA cluster; and (iv) lysA and argG genes were located on the 2.5-Mb chromosome, in contrast to the argH gene, which was located on the 3.4-Mb chromosome . Southern hybridization analysis, allelic exchange mutagenesis of ATCC 17616, and complementation tests demonstrated that all of the genes examined were functional and existed as a single copy within the genome . The present findings also indicated that the 2.5-Mb chromosome carried various auxotrophic genes with no structural or functional counterparts on the remaining two chromosomes . Analysis of Structural and Physiological Profiles To Assess the Effects of Cu on Biofilm Microbial Communities. B. Massieux, 2004.We investigated the effects of copper on the structure and physiology of freshwater biofilm microbial communities . For this purpose, biofilms that were grown during 4 weeks in a shallow, slightly polluted ditch were exposed, in aquaria in our laboratory, to a range of copper concentrations (0, 1, 3, and 10 µM) . Denaturing gradient gel electrophoresis (DGGE) revealed changes in the bacterial community in all aquaria . The extent of change was related to the concentration of copper applied, indicating that copper directly or indirectly caused the effects . Concomitantly with these changes in structure, changes in the metabolic potential of the heterotrophic bacterial community were apparent from changes in substrate use profiles as assessed on Biolog plates . The structure of the phototrophic community also changed during the experiment, as observed by microscopic analysis in combination with DGGE analysis of eukaryotic microorganisms and cyanobacteria . However, the extent of community change, as observed by DGGE, was not significantly greater in the copper treatments than in the control . Yet microscopic analysis showed a development toward a greater proportion of cyanobacteria in the treatments with the highest copper concentrations . Furthermore, copper did affect the physiology of the phototrophic community, as evidenced by the fact that a decrease in photosynthetic capacity was detected in the treatment with the highest copper concentration . Therefore, we conclude that copper affected the physiology of the biofilm and had an effect on the structure of the communities composing this biofilm . Importance of Different tfd Genes for Degradation of Chloroaromatics by Ralstonia eutropha JMP134. Iris Plumeier, 2002.The tfdCIDIEIFI, and tfdDIICIIEIIFII gene modules of plasmid pJP4 of Ralstonia eutropha JMP134 encode complete sets of functional enzymes for the transformation of chlorocatechols into 3-oxoadipate, which are all expressed during growth on 2,4-dichlorophenoxyacetate (2,4-D) . However, activity of tfdI-encoded enzymes was usually higher than that of tfdII-encoded enzymes, both in the wild-type strain grown on 2,4-D and in 3-chlorobenzoate-grown derivatives harboring only one tfd gene module . The tfdDII-encoded chloromuconate cycloisomerase exhibited special kinetic properties, with high activity against 3-chloromuconate and poor activity against 2-chloromuconate and unsubstituted muconate, thus explaining the different phenotypic behaviors of R . eutropha strains containing different tfd gene modules . The enzyme catalyzes the formation of an equilibrium between 2-chloromuconate and 5-chloro- and 2-chloromuconolactone and very inefficiently catalyzes dehalogenation to form trans-dienelactone as the major product, thus differing from all (chloro)muconate cycloisomerases described thus far . Changes in Microbial Community Composition and Function during a Polyaromatic Hydrocarbon Phytoremediation Field Trial. Steven D. Siciliano, 2003.The purpose of this study was to investigate the mechanism by which phytoremediation systems promote hydrocarbon degradation in soil . The composition and degradation capacity of the bulk soil microbial community during the phytoremediation of soil contaminated with aged hydrocarbons was assessed . In the bulk soil, the level of catabolic genes involved in hydrocarbon degradation (ndoB, alkB, and xylE) as well as the mineralization of hexadecane and phenanthrene was higher in planted treatment cells than in treatment cells with no plants . There was no detectable shift in the 16S ribosomal DNA (rDNA) composition of the bulk soil community between treatments, but there were plant-specific and -selective effects on specific catabolic gene prevalence . Tall Fescue (Festuca arundinacea) increased the prevalence of ndoB, alkB, and xylE as well as naphthalene mineralization in rhizosphere soil compared to that in bulk soil . In contrast, Rose Clover (Trifolium hirtum) decreased catabolic gene prevalence and naphthalene mineralization in rhizosphere soil . The results demonstrated that phytoremediation systems increase the catabolic potential of rhizosphere soil by altering the functional composition of the microbial community . This change in composition was not detectable by 16S rDNA but was linked to specific functional genotypes with relevance to petroleum hydrocarbon degradation . Assessment of Bioavailability of Soil-Sorbed Atrazine. Jeong-Hun Park, 2003.Bioavailability of pesticides sorbed to soils is an important determinant of their environmental fate and impact . Mineralization of sorbed atrazine was studied in soil and clay slurries, and a desorption-biodegradation-mineralization (DBM) model was developed to quantitatively evaluate the bioavailability of sorbed atrazine . Three atrazine-degrading bacteria that utilized atrazine as a sole N source (Pseudomonas sp . strain ADP, Agrobacterium radiobacter strain J14a, and Ralstonia sp . strain M91-3) were used in the bioavailability assays . Assays involved establishing sorption equilibrium in sterile soil slurries, inoculating the system with organisms, and measuring the CO2 production over time . Sorption and desorption isotherm analyses were performed to evaluate distribution coefficients and desorption parameters, which consisted of three desorption site fractions and desorption rate coefficients . Atrazine sorption isotherms were linear for mineral and organic soils but displayed some nonlinearity for K-saturated montmorillonite . The desorption profiles were well described by the three-site desorption model . In many instances, the mineralization of atrazine was accurately predicted by the DBM model, which accounts for the extents and rates of sorption/desorption processes and assumes biodegradation of liquid-phase, but not sorbed, atrazine . However, for the Houghton muck soil, which manifested the highest sorbed atrazine concentrations, enhanced mineralization rates, i.e., greater than those expected on the basis of aqueous-phase atrazine concentration, were observed . Even the assumption of instantaneous desorption could not account for the elevated rates . A plausible explanation for enhanced bioavailability is that bacteria access the localized regions where atrazine is sorbed and that the concentrations found support higher mineralization rates than predicted on the basis of aqueous-phase concentrations . Characteristics of high sorbed-phase concentration, chemotaxis, and attachment of cells to soil particles seem to contribute to the bioavailability of soil-sorbed atrazine .
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