Unknown,Transcriptomics,Genomics,Proteomics

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Vinyl Chloride Degradation by Mixed Microbial Community KB1

ABSTRACT: 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.

ORGANISM(S): dechlorinating enrichment culture KB-1

SUBMITTER: Alison Waller

PROVIDER: E-GEOD-20041 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:The genomes of three newly isolated Dehalococcoides strains (11a, 11a5 and MB) were compared against known genomes in the Dehalococcoides genus via a microarray targeting four sequenced Dehalococcoides strains (195, CBDB1, BAV1, and VS). All three strains exhibit different dechlorination patterns, with strains 11a dechlorinating TCE to ethene, 11a5 dechlorinating TCE to VC and MB dechlorinating PCE only to isomers of DCE. Hybridization of their respective genomic DNA to the microarrays showed that the genomes of strains 11a and 11a5 show great similarity to each other and to strains CBDB1 and BAV1 of the Pinellas subgroup, while strain MB shows strong genome similarity to members of the Cornell subgroup. All genes within the respective subgroups that were not detected by microarray are within the respective high plasticity regions or integrated elements of the sequenced strains. A large number of reductive dehalogenase (RDase)-encoding genes are present within each genome, and the presence of the vcrA and tceA genes in strains 11a and 11a5 respectively, and the absence of any of the four functionally-characterized chlorinated ethene RDases (pceA, tceA, vcrA, bvcA) within strain MB appear to dictate chlorinated ethene usages regardless of the respective core genome phylogeny of the three strains. Considering the current data set together with previous comparative genomics results from application of the Dehalococcoides genus microarray to two other un-sequenced strains, the observed incongruence between the core genome phylogeny and chlorinated ethene usage of Dehalococcoides strains is likely driven by horizontal gene transfer of functional RDases. The other genomic features that are repeatedly observed in the microarray analyses of all five un-sequenced Dehalococcoides strains as well as the environmental implications on this work are presented in this study. The genomic DNA (gDNA) of each culture was analyzed in triplicate. gDNA from the two newly isolated Dehalococcoides strains 11a and 11a5 were analyzed.

Project description:Tetrachloroethene (PCE) and trichloroethene (TCE) are prevalent groundwater contaminants that can be completely reductively dehalogenated by Dehalococcoides organisms. A Dehalococcoides-containing microbial consortium (ANAS) with the ability to degrade TCE to ethene, an innocuous end-product, was previously enriched from contaminated soil. A whole-genome photolithographic microarray was developed based on the genome of Dehalococcoides ethenogenes 195 (strain 195). This microarray contains probes designed to hybridize to >99% of the predicted protein-coding sequences in the strain 195 genome. DNA from ANAS was hybridized to the microarray to characterize the genomic content of the ANAS enrichment. The microarray revealed that the genes associated with central metabolism including an apparently incomplete carbon fixation pathway, cobalamin salvaging system, nitrogen fixation pathway, and five hydrogenase complexes are present in both strain 195 and ANAS. Although the gene encoding the TCE reductase tceA was detected, 13 of the 19 reductive dehalogenase genes present in strain 195 were not detected in ANAS. Additionally, 88% of the genes in predicted integrated genetic elements in strain 195 were not detected in ANAS, consistent with these elements being genetically mobile. Sections of the tryptophan operon and an operon encoding an ABC transporter in strain 195 were also not detected in ANAS. These insights into the diversity of Dehalococcoides genomes will improve our understanding of the physiology and evolution of these bacteria which is essential in developing effective strategies for bioremediation of PCE and TCE in the environment. Keywords: comparative genomic hybridization Genomic DNA from each culture was divided into replicate samples which were independently fragmented, labeled, and hybridized to arrays. Two microarrays were processed for the positive control (strain 195), two for the negative control (D. restrictus), and five for the ANAS enrichment culture(two analyses from one biological sample followed one year later by three analyses of a second biological sample).

Project description:The chlorinated ethene-respiring bacteria of the genus Dehalococcoides are important for bioremediation. A microarray targeting genes from all available sequenced genomes of the Dehalococcoides genus was designed with 4305 probe sets to target 98.6% of all genes from strains 195, CBDB1, BAV1, and VS. The microarrays were validated with genomic DNA (gDNA) of strains 195 and BAV1 and satisfactory analytical reproducibility, quantitative response and gene detection accuracy were obtained. These microarrays were applied to query the genomes of two recently isolated Dehalococcoides strains, ANAS1 and ANAS2. Strains ANAS1 and ANAS2 can both couple the reduction of TCE, cDCE and 1,1-DCE but not PCE and tDCE with growth while only strain ANAS2 couples VC reduction to growth. Analysis of the respective gDNA using the microarrays showed that the genomes of both strains are similar to each other and to strain 195, except for genes that are within the previously defined integrated elements (IEs) or high plasticity regions (HPRs). Similar results to the combined isolates were obtained when gDNA of ANAS, the enrichment culture from which the two Dehalococcoides isolates originated, was applied to the microarrays. The genome similarities, together with the distinct chlorinated ethene usage of strains ANAS1, ANAS2 and 195 demonstrate that closely phylogenetically related strains can be physiologically different. This incongruence between physiology and core genome phylogeny appears to be driven by the presence of distinct reductive dehalogenase (RDase)-encoding genes with characterized chlorinated ethene functions (pceA, tceA in strain 195; tceA in strain ANAS1; vcrA in strain ANAS2). Genes encoding central metabolic functions of strain 195 were all detected in strains ANAS1 and ANAS2, while interestingly, the tryptophan operon of these strains is similar to that of strain VS. Overall, the microarrays are a valuable high-throughput tool for comparative genomics of un-sequenced Dehalococcoides-containing samples. The genomic DNA (gDNA) of each culture was analyzed in triplicate. gDNA from strain 195 and strain BAV1 was used as positive control, while gDNA from Anaeromyxobacter dehalogenans strain 2CP-C served as negative control in validating the microarray. gDNA from the two newly isolated strains ANAS1 and ANAS2 as well as the enrichment culture (ANAS) where the two strains originated was also analyzed.

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Vinyl Chloride Degradation by Mixed Microbial Community KB1

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