Browsing by Author "Khadka, Roshan"

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    Diversity of methane and short chain hydrocarbon degrading bacteria with an emphasis on methane biofilter systems
    (2018-10-03) Khadka, Roshan; Dunfield, Peter F.; Voordouw, Gerrit; Hubert, Casey R. J.
    Methanotrophs house enzymes capable of methane oxidation, act as a sink for atmospheric methane and play a key role in the global carbon cycle. This study conducted multiple studies on methanotrophs, including: examination of the evolutionary history of copper membrane monooxygenases (CuMMOs), application of methanotrophic communities in protocol design for monitoring methane biofilter systems, and the analyses of single cell genomes containing new CuMMO-encoding genes. CuMMOs are encoded by three genes, usually in an operon of xmoCAB, and oxidize ammonia, methane, and short chain alkanes and alkenes. To examine the evolutionary history of CuMMOs, phylogenetic inferences and compositional genome analyses were applied to a set of 66 genomes. Individual phylogeny of all genes xmoA, xmoB, and xmoC closely matched in almost all genomes, indicating this operon evolved as a unit. However in Verrucomicrobia pmoB has a distinct phylogeny from pmoA and pmoC. The gammaproteobacteria AMO (Nitrosococcus spp.), the gammaproteobacterial Pxm, the thaumarcheotal AMO and the NC10 pMMO showed little or no compositional bias in the xmo operon indicating similar compositional biases to its genome. Based on the analysis, possible lateral gene transfer events of xmoCAB genes were predicted. The nitrifying bacterium Nitrosococcus postulated as the donor of pmoCAB to both the alpha- and gammaproteobacterial methanotrophs. To design a monitoring protocol that would allow a simple, cost effective and accurate estimation of whether a methane biofilter is operating efficiently, microcosms using compost as a biofilter material were tested via growth and starvation experiments for long periods. Analysis of 16S rRNA gene sequences suggested that non-methanotrophic methylotrophic bacteria belonging the family Methylophilaceae showed a rapid response to biofilter methane oxidation activity and may be a good monitoring target. A monitoring system based on these “methanotroph-associated methylotrophs” is proposed and a ratio of Methylophilaceae to Methylococcaceae of 0.35 was related to high methane activity and 0.1 to low activity. Novel copper membrane monooxygenase encoding operons (xmoCAB) were detected while screening metagenomes obtained from oil sands environments. Quantitative PCR assays were developed for detection of xmoCAB genes in methane, ethane and propane enrichment cultures from environmental samples. Single cell genomes were sequenced from the xmoCAB positive sorted cells of a propane enrichment culture. Screening the genomes identified Polaromonas and Rhodoferax as containing multiple xmoCAB operons. Potential propane oxidation pathways were predicted based on enzymes present in single cell genomes of these two genera.
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    Novel copper-containing membrane monooxygenases (CuMMOs) encoded by alkane-utilizing Betaproteobacteria
    (Nature Publishing Group, 2019-07-01) Rochman, Fauziah F.; Kwon, Miye; Khadka, Roshan; Tamas, Ivica; Lopez-Jauregui, Abraham; Sheremet, Andriy; Smirnova, Angela V.; Malmstrom, Rex; Yoon, Sukhwan; Woyke, Tanja; Dunfield, Peter F.; Verbeke, Tobin J.
    Copper-containing membrane monooxygenases (CuMMOs) are encoded by xmoCAB(D) gene clusters and catalyze the oxidation of methane, ammonia, or some short chain alkanes and alkenes. In a metagenome constructed from an oilsands tailings pond we detected an xmoCABD gene cluster with <59% derived amino acid identity to genes from known bacteria. Stable isotope probing experiments combined with a specific xmoA qPCR assay demonstrated that the bacteria possessing these genes were incapable of methane assimilation, but did grow on ethane and propane. Single-cell genomes (SAGs) from propane-enriched samples were therefore constructed and screened with the specific PCR assay to identify bacteria possessing the target gene cluster. Multiple SAGs of Betaproteobacteria belonging to the genera Rhodoferax and Polaromonas possessed close homologues of the metagenomic xmoCABD gene cluster. Unexpectedly, each of these two genera also possessed other xmoCABD paralogs, representing two additional lineages in phylogenetic analyses. Metabolic reconstructions from SAGs predicted that neither bacterium was capable of catabolic methane or ammonia oxidation, but that both were capable of higher n-alkane degradation. The involvement of the encoded CuMMOs in alkane oxidation was further suggested by reverse transcription PCR analyses, which detected elevated transcription of the xmoA genes upon enrichment of water samples with propane as the sole energy source. Enrichments, isotope incorporation studies, genome reconstructions, and gene expression studies therefore all agreed that the unknown xmoCABD operons did not encode methane or ammonia monooxygenases, but rather n-alkane monooxygenases. This study broadens the known diversity of CuMMOs and identifies non-nitrifying Betaproteobacteria as possessing these enzymes.