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.

Project description:A microarray targeting four sequenced strains in the Dehalococcoides (Dhc) genus was used to analyze gene expression in a robust long-term trichloroethene (TCE)-degrading microbial community (designated ANAS) during feeding cycles that involve conditions of periodic substrate supply. The Dhc transcriptome was examined at three time-points throughout a batch feeding cycle: T1 (27 h) when TCE, dichloroethene (DCE), and vinyl chloride (VC) were present; T2 (54 h) when only VC remained; and T3 (13 d) when Dhc had been starved of substrate for nine days. 90% of the Dhc ORFs that were detected in the ANAS DNA were found to be expressed as RNA sometime during the time course, demonstrating extraordinary utilization of the streamlined genome. 97% of these transcripts were differentially expressed during the time course, indicating efficiency of transcription through regulation in Dhc. Most Dhc genes were significantly down-regulated at T3, responding to a lack of substrate as would be expected. The tceA and vcrA genes, which code for proteins with known chlorinated ethene reduction functions, were highly expressed at both T1 and T2, whereas two other putative reductive dehalogenase genes (DET0173 and DET1545) were most highly expressed at T2, likely in response to the presence of VC. Hydrogenases were most highly expressed at T1, reflecting their important role in accumulating electrons used to initiate reductive dechlorination and other biosynthesis pathways. Cobalamin transport genes were preferentially expressed at T2, reflecting an increase in corrinoid transport as chloroethenes were degraded and a decrease in activity of the transport system after dehalogenation was complete. This is the first application of a microarray targeting a known genus, including both core genomes and identified strain-specific genes, applied to improve our understanding of transcriptional dynamics within an undefined microbial community. Replicate samples were independently collected, and simultaneously but individually extracted, fragmented, labeled, and hybridized to arrays. Three DNA samples (one from each of the three replicate cycles) and nine RNA samples (one from each of the three time-points in each of the three replicate cycles) were prepared for microarray analysis.

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Dechlorinating enrichment culture KB-1

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