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Involvement of Linear Plasmids in Aerobic Biodegradation of Vinyl Chloride. Anthony S. Danko, 2004.Pseudomonas putida strain AJ and Ochrobactrum strain TD were isolated from hazardous waste sites based on their ability to use vinyl chloride (VC) as the sole source of carbon and energy under aerobic conditions . Strains AJ and TD also use ethene and ethylene oxide as growth substrates . Strain AJ contained a linear megaplasmid (approximately 260 kb) when grown on VC or ethene, but it contained no circular plasmids . While strain AJ was growing on ethylene oxide, it was observed to contain a 100-kb linear plasmid, and its ability to use VC as a substrate was retained . The linear plasmids in strain AJ were cured, and the ability of strain AJ to consume VC, ethene, and ethylene oxide was lost following growth on a rich substrate (Luria-Bertani broth) through at least three transfers . Strain TD contained three linear plasmids, ranging in size from approximately 90 kb to 320 kb, when growing on VC or ethene . As with strain AJ, the linear plasmids in strain TD were cured following growth on Luria-Bertani broth and its ability to consume VC and ethene was lost . Further analysis of these linear plasmids may help reveal the pathway for VC biodegradation in strains AJ and TD and explain why this process occurs at many but not all sites where groundwater is contaminated with chloroethenes . Metabolism of VC and ethene by strains AJ and TD is initiated by an alkene monooxygenase . Their yields during growth on VC (0.15 to 0.20 mg of total suspended solids per mg of VC) are similar to the yields reported for other isolates (i.e., Mycobacterium sp., Nocardioides sp., and Pseudomonas sp.) . Comparison of Diversities and Compositions of Bacterial Populations Inhabiting Natural Forest Soils. Evelyn Hackl, 2004.The diversity and composition of soil bacterial communities were compared among six Austrian natural forests, including oak-hornbeam, spruce-fir-beech, and Austrian pine forests, using terminal restriction fragment length polymorphism (T-RFLP, or TRF) analysis and sequence analysis of 16S rRNA genes . The forests studied differ greatly in soil chemical characteristics, microbial biomass, and nutrient turnover rates . The aim of this study was to relate these differences to the composition of the bacterial communities inhabiting the individual forest soils . Both TRF profiling and clone sequence analysis revealed that the bacterial communities in soils under Austrian pine forests, representing azonal forest types, were distinct from those in soils under zonal oak-hornbeam and spruce-fir-beech forests, which were more similar in community composition . Clones derived from an Austrian pine forest soil were mostly affiliated with high-G+C gram-positive bacteria (49%), followed by members of the  -Proteobacteria (20%) and the Holophaga/Acidobacterium group (12%) . Clones in libraries from oak-hornbeam and spruce-fir-beech forest soils were mainly related to the Holophaga/Acidobacterium group (28 and 35%), followed by members of the Verrucomicrobia (24%) and the
-Proteobacteria (27%), respectively . The soil bacterial communities in forests with distinct vegetational and soil chemical properties appeared to be well differentiated based on 16S rRNA gene phylogeny . In particular, the outstanding position of the Austrian pine forests, which are determined by specific soil conditions, was reflected in the bacterial community composition .
Freshwater Bacteria Can Methylate Selenium through the Thiopurine Methyltransferase Pathway. Lionel Ranjard, 2003.Involvement of the bacterial thiopurine methyltransferase (bTPMT) in natural selenium methylation by freshwater was investigated . A freshwater environment that had no known selenium contamination but exhibited reproducible emission of dimethyl selenide (DMSe) or dimethyl diselenide (DMDSe) when it was supplemented with an organic form of selenium [(methyl)selenocysteine] or an inorganic form of selenium (sodium selenite) was used . The distribution of the bTPMT gene (tpm) in the microflora was studied . Freshwater bacteria growing on 10 µM sodium selenite and 10 µM sodium selenate were isolated, and 4.5 and 10% of the strains, respectively, were shown by colony blot hybridization to hybridize with a Pseudomonas syringae tpm DNA probe . Ribotyping showed that these strains are closely related . The complete rrs sequence of one of the strains, designated Hsa.28, was obtained and analyzed . Its closest phyletic neighbor was found to be the Pseudomonas anguilliseptica rrs sequence . The Hsa.28 strain grown with sodium selenite or (methyl)selenocysteine produced significant amounts of DMSe and DMDSe . The Hsa.28 tpm gene was isolated by genomic DNA library screening and sequencing . BLASTP comparisons of the deduced Hsa.28 bTPMT sequence with P . syringae, Pseudomonas aeruginosa, Vibrio cholerae, rat, and human thiopurine methyltransferase sequences revealed that the levels of similarity were 52 to 71% . PCR-generated Escherichia coli subclones containing the Hsa.28 tpm open reading frame were constructed . E . coli cells harboring the constructs and grown with sodium selenite or (methyl)selenocysteine produced significant levels of DMSe and DMDSe, confirming that the gene plays a role in selenium methylation . The effect of strain Hsa.28 population levels on freshwater DMSe and DMDSe emission was investigated . An increase in the size of the Hsa.28 population was found to enhance significantly the emission of methyl selenides by freshwater samples supplemented with sodium selenite or (methyl)selenocysteine . These data suggest that bTPMT can play a role in natural freshwater selenium methylation processes . Sampling Natural Viral Communities from Soil for Culture-Independent Analyses. Kurt E. Williamson, 2003.An essential first step in investigations of viruses in soil is the evaluation of viral recovery methods suitable for subsequent culture-independent analyses . In this study, four elution buffers (10% beef extract, 250 mM glycine buffer, 10 mM sodium pyrophosphate, and 1% potassium citrate) and three enumeration techniques (plaque assay, epifluorescence microscopy [EFM], and transmission electron microscopy [TEM]) were compared to determine the best method of extracting autochthonous bacteriophages from two Delaware agricultural soils . Beef extract and glycine buffer were the most effective in eluting viable phages inoculated into soils (up to 29% recovery); however, extraction efficiency varied significantly with phage strain . Potassium citrate eluted the highest numbers of virus-like particles from both soils based on enumerations by EFM (mean, 5.3 x 108 g of dry soil-1), but specific soil-eluant combinations posed significant problems to enumeration by EFM . Observations of virus-like particles under TEM gave confidence that the particles were, in fact, phages, but TEM enumerations yielded measurements of phage abundance (mean, 1.5x108 g of dry soil-1) that were about five times lower . Clearly, the measurement of phage abundance in soils varies with both the extraction and enumeration methodology; thus, it is important to assess multiple extraction and enumeration approaches prior to undertaking ecological studies of phages in a particular soil .
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