Project description:Members of the bacterial phylum Spirochaetes are primarily studied for their commensal and pathogenic roles in animal hosts. However, Spirochaetes are also frequently detected in anoxic hydrocarbon-contaminated environments but their ecological role in such ecosystems has so far remained unclear. Here we provide a functional trait to these frequently detected organisms with an example of a sulfate-reducing, naphthalene-degrading enrichment culture consisting of a sulfate-reducing deltaproteobacterium Desulfobacterium naphthalenivorans and a novel spirochete Rectinema cohabitans. Using a combination of genomic, proteomic, and physiological studies we show that R. cohabitans grows by fermentation of organic compounds derived from biomass from dead cells (necromass). It recycles the derived electrons in the form of H2 to the sulfate-reducing D. naphthalenivorans, thereby supporting naphthalene degradation and forming a simple microbial loop. We provide metagenomic evidence that equivalent associations between Spirochaetes and hydrocarbon-degrading microorganisms are of general importance in hydrocarbon- and organohalide-contaminated ecosystems. We propose that environmental Spirochaetes form a critical component of a microbial loop central to nutrient cycling in subsurface environments. This emphasizes the importance of necromass and H2-cycling in highly toxic contaminated subsurface habitats such as hydrocarbon-polluted aquifers.
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:Isoprene-metabolizing bacteria represent a global regulator for atmospheric isoprene concentrations. Under anoxic conditions, isoprene can be used as an electron acceptor reducing it to methylbutene. This study describes the proteogenomic profiling of an isoprene reducing enrichment culture to identify organisms and genes responsible for the isoprene hydrogenation reaction. A metagenome assembled genome (MAG) of the most abundant (88 % rel. abundance) lineage in the enrichment, Acetobacterium wieringae, was obtained. Comparative proteogenomics and RT-PCR identified a five-gene operon from the A. wieringae MAG upregulated during isoprene reduction. The operon encodes a putative oxidoreductase, three pleiotropic nickel chaperones (HypA, HypA, HypB) and one 4Fe-4S ferredoxin. The oxidoreductase is proposed as the putative isoprene reductase with a binding site for NADH, FAD as well as two pairs of [4Fe-4S]-clusters. Other Acetobacterium strains (A. woodii DSM 1030, A. wieringae DSM 1911, A. malicum DSM 4132 and A. dehalogenans DSM 11527) do not encode the isoprene reduction operon and could not reduce isoprene. Uncharacterized homologs of the putative isoprene reductase are observed across the Firmicutes, Spirochaetes, Tenericutes, Actinobacteria, Chloroflexi, Bacteroidetes and Proteobacteria, suggesting the ability of biohydrogenation of non-functionalized conjugated doubled bonds in other unsaturated hydrocarbons.
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.