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Involvement of linear plasmids in aerobic biodegradation of vinyl chloride.

ABSTRACT: 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.).

SUBMITTER: Danko AS

PROVIDER: S-EPMC522125 | biostudies-literature | 2004 Oct

REPOSITORIES: biostudies-literature

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Involvement of linear plasmids in aerobic biodegradation of vinyl chloride.

Danko Anthony S AS Luo Meizhong M Bagwell Christopher E CE Brigmon Robin L RL Freedman David L DL

Applied and environmental microbiology 20041001 10

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-k ...[more]

PMID: 15466555

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Project description:A shotgun metagenome microarray was created and used to investigate gene transcription during vinyl chloride (VC) dechlorination by a microbial enrichment culture called KB1. The array was constructed by spotting genomic fragments amplified from short-insert libraries of KB1 metagenomic DNA. Subsequently, the microarrays were interrogated with RNA extracted from KB1 during VC dechlorination (VC+methanol), and in the absence of VC (methanol-only). The most differentially expressed spots, and spots with the highest intensities, were then chosen to be sequenced. Sequencing revealed that Dehalococcoides (Dhc) genes involved in transcription, translation and energy generation were up-regulated during VC degradation. Furthermore, the results indicated that the reductive dehalogenase homologous (RDH) gene KB1rdhA14 is the only RDH gene up-regulated upon VC degradation, and that multiple RDH genes were more highly transcribed in the absence of VC. Numerous hypothetical genes from Dehalococcoides were also more highly transcribed in methanol only treatments and indicate that many uncharacterized proteins are involved in cell maintenance in the absence of chlorinated substrates. Spots with genes from Spirochaetes, Chloroflexi, Geobacter, Methanogens and phage organisms were differentially expressed and sequencing provided information from these uncultivated organisms that can be used to design primers for more targeted studies. This array format is powerful, as it does not require a priori sequence knowledge. This study provides the first report of such arrays being used to investigate transcription in a mixed community, and shows that this array format can be used to screen metagenomic libraries for functionally important genes. 2 Biological replicate experimens conducted 1 month apart. In the first there were 2 dye-swapped duplicates (total 4) of VC+MeOH versus MeOH only. In the second experiment there was one set of dye swapped arrays. Thus 6 arrays were performed including biological replicates, dye swapped replicates and technical duplicates.

Dataset Information

Involvement of linear plasmids in aerobic biodegradation of vinyl chloride.

Publications

Involvement of linear plasmids in aerobic biodegradation of vinyl chloride.

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