Biomedical subjects

J Colin Murrell

Publications and source records attributed to J Colin Murrell.

44 records · Page 3Linked to original sources

PubMed2002

Identification of active methylotroph populations in an acidic forest soil by stable-isotope probing.

Stable-isotope probing (SIP) is a culture-independent technique that enables the isolation of DNA from micro-organisms that are actively involved in a specific metabolic process. In this study, SIP was used to characterize the active methylotroph populations in forest soil (pH 3.5) microcosms that were exposed to (13)CH(3)OH or (13)CH(4). Distinct (13)C-labelled DNA ((13)C-DNA) fractions were resolved from total community DNA by CsCl density-gradient centrifugation. Analysis of 16S rDNA sequences amplified from the (13)C-DNA revealed that bacteria related to the genera Methylocella, Methylocapsa, Methylocystis and Rhodoblastus had assimilated the (13)C-labelled substrates, which suggested that moderately acidophilic methylotroph populations were active in the microcosms. Enrichments targeted towards the active proteobacterial CH(3)OH utilizers were successful, although none of these bacteria were isolated into pure culture. A parallel analysis of genes encoding the key enzymes methanol dehydrogenase and particulate methane monooxygenase reflected the 16S rDNA analysis, but unexpectedly revealed sequences related to the ammonia monooxygenase of ammonia-oxidizing bacteria (AOB) from the beta-subclass of the PROTEOBACTERIA: Analysis of AOB-selective 16S rDNA amplification products identified Nitrosomonas and Nitrosospira sequences in the (13)C-DNA fractions, suggesting certain AOB assimilated a significant proportion of (13)CO(2), possibly through a close physical and/or nutritional association with the active methylotrophs. Other sequences retrieved from the (13)C-DNA were related to the 16S rDNA sequences of members of the Acidobacterium division, the beta-Proteobacteria and the order Cytophagales, which implicated these bacteria in the assimilation of reduced one-carbon compounds or in the assimilation of the by-products of methylotrophic carbon metabolism. Results from the (13)CH(3)OH and (13)CH(4) SIP experiments thus provide a rational basis for further investigations into the ecology of methylotroph populations in situ.

Ammonia↗

PubMed2002

Duplicate copies of genes encoding methanesulfonate monooxygenase in Marinosulfonomonas methylotropha strain TR3 and detection of methanesulfonate utilizers in the environment.

Marinosulfonomonas methylotropha strain TR3 is a marine methylotroph that uses methanesulfonic acid (MSA) as a sole carbon and energy source. The genes from M. methylotropha strain TR3 encoding methanesulfonate monooxygenase, the enzyme responsible for the initial oxidation of MSA to formaldehyde and sulfite, were cloned and sequenced. They were located on two gene clusters on the chromosome of this bacterium. A 5.0-kbp HindIII fragment contained msmA, msmB, and msmC, encoding the large and small subunits of the hydroxylase component and the ferredoxin component, respectively, of the methanesulfonate monooxygenase, while a 6.5-kbp HindIII fragment contained duplicate copies of msmA and msmB, as well as msmD, encoding the reductase component of methanesulfonate. Both sets of msmA and msmB genes were virtually identical, and the derived msmA and msmB sequences of M. methylotropha strain TR3, compared with the corresponding hydroxylase from the terrestrial MSA utilizer Methylosulfonomonas methylovora strain M2 were found to be 82 and 69% identical. The msmA gene was investigated as a functional gene probe for detection of MSA-utilizing bacteria. PCR primers spanning a region of msmA which encoded a unique Rieske [2Fe-2S] binding region were designed. These primers were used to amplify the corresponding msmA genes from newly isolated Hyphomicrobium, Methylobacterium, and Pedomicrobium species that utilized MSA, from MSA enrichment cultures, and from DNA samples extracted directly from the environment. The high degree of identity of these msmA gene fragments, compared to msmA sequences from extant MSA utilizers, indicated the effectiveness of these PCR primers in molecular microbial ecology.

Alphaproteobacteria↗

PubMed2002

Improved system for protein engineering of the hydroxylase component of soluble methane monooxygenase.

Soluble methane monooxygenase (sMMO) of Methylosinus trichosporium OB3b is a three-component oxygenase that catalyses the O(2)- and NAD(P)H-dependent oxygenation of methane and numerous other substrates. Despite substantial interest in the use of genetic techniques to study the mechanism of sMMO and manipulate its substrate specificity, directed mutagenesis of active-site residues was previously impossible because no suitable heterologous expression system had been found for expression in a highly active form of the hydroxylase component, which is an (alphabetagamma)(2) complex containing the binuclear iron active site. A homologous expression system that enabled the expression of recombinant wild-type sMMO in a derivative of M. trichosporium OB3b from which the chromosomal copy of the sMMO-encoding operon had been partially deleted was previously reported. Here we report substantial development of this method to produce a system for the facile construction and expression of mutants of the hydroxylase component of sMMO. This new system has been used to investigate the functions of Cys 151 and Thr 213 of the alpha subunit, which are the only nonligating protonated side chains in the hydrophobic active site. Both residues were found to be critical for the stability and/or activity of sMMO, but neither was essential for oxygenation reactions. The T213S mutant was purified to >98% homogeneity. It had the same iron content as the wild type and had 72% wild-type activity toward toluene but only 17% wild-type activity toward propene; thus, its substrate profile was significantly altered. With these results, we have demonstrated proof of the principle for protein engineering of this uniquely versatile enzyme.

Amino Acid Substitution↗

PubMed2002

Identification of the functionally active methanotroph population in a peat soil microcosm by stable-isotope probing.

The active population of low-affinity methanotrophs in a peat soil microcosm was characterized by stable-isotope probing. "Heavy" (13)C-labeled DNA, produced after microbial growth on (13)CH(4), was separated from naturally abundant (12)C-DNA by cesium chloride density gradient centrifugation and used as a template for the PCR. Amplification products of 16S rRNA genes and pmoA, mxaF, and mmoX, which encode key enzymes in the CH(4) oxidation pathway, were analyzed. Sequences related to extant type I and type II methanotrophs were identified, indicating that these methanotrophs were active in peat exposed to 8% (vol/vol) CH(4). The (13)C-DNA libraries also contained clones that were related to beta-subclass Proteobacteria, suggesting that novel groups of bacteria may also be involved in CH(4) cycling in this soil.

Alcohol Oxidoreductases↗

PubMed2001

Detection and localization of two hydrogenases in Methylococcus capsulatus (Bath) and their potential role in methane metabolism.

Methylococcus capsulatus (Bath) was shown to contain two distinct hydrogenases, a soluble hydrogenase and a membrane-bound hydrogenase. This is the first report of a membrane-bound hydrogenase in methanotrophs. Both enzymes were expressed apparently constitutively under normal growth conditions. The soluble hydrogenase was capable of reducing NAD(+) with molecular hydrogen. The activities of both soluble and particulate methane monooxygenases could be driven by molecular hydrogen. This confirmed that molecular hydrogen could be used as a source of reducing power for methane oxidation. Hydrogen-driven methane monooxygenase activities tolerated elevated temperatures and moderate oxygen concentrations. The significance of these findings for biotechnological applications of methanotrophs is discussed.

Hydrogen↗

PubMed1999

Homologous expression of soluble methane monooxygenase genes in Methylosinus trichosporium OB3b.

An homologous expression system has been developed for soluble methane monooxygenase (sMMO) genes from Methylosinus trichosporium OB3b. sMMO-minus mutants were previously obtained after marker-exchange mutagenesis, by the insertion of a kanamycin-resistance cassette into the mmoX gene of the sMMO operon. Complementation of the sMMO-minus genotype was achieved by conjugation with broad-host-range plasmids containing the native promoter and sMMO operon from Ms. trichosporium OB3b (pVK100Sc and pHM2). In wild-type methanotrophs, copper ions present in the growth medium at concentrations greater than 0.25 microM inhibit transcription of sMMO genes. The stable maintenance of pVK100Sc resulted in transconjugant methanotrophs with a decreased sensitivity to copper, since expression of sMMO occurred at copper sulphate concentrations of 7.5 microM. sMMO activity was only detected in soluble extracts after the addition of purified sMMO reductase component, which is inhibited by copper ions in vitro. This phenomenon could have arisen due to the increased number of sMMO gene copies (derived from pVK100Sc) in the cell. Transconjugants obtained from conjugations with pHM2 expressed sMMO at copper concentrations of 0-2.5 microM only and sMMO activity was not restored by the addition of purified reductase component at copper concentrations higher than 2.5 microM. Southern hybridization showed that the plasmid had integrated into the chromosome, probably by a single homologous recombination event. This is the first report of homologous sMMO expression in a methanotroph with enzyme activities that are comparable to the activity reported in wild-type strains. This expression system will be useful for site-directed mutagenesis of active-site residues of sMMO from Ms. trichosporium OB3b.

Blotting, Southern↗

PubMed1996

Metabolism of methanesulfonic acid involves a multicomponent monooxygenase enzyme.

A novel methylotroph, strain M2, capable of utilizing methanesulfonic acid (MSA) as a sole source of carbon and energy was the subject of these investigations. The initial step in the biodegradative pathway of MSA in strain M2 involved an inducible NADH-specific monooxygenase enzyme (MSAMO). Partial purification of MSAMO from cell-free extracts by ion-exchange chromatography led to the loss of MSAMO activity. Activity was restored by the mixing of three distinct protein fractions designated A, B and C. The reconstituted enzyme had a narrow substrate specificity relative to crude cell-free extracts. Addition of FAD and ferrous ions to the reconstituted enzyme complex resulted in a fivefold increase in enzyme activity, suggesting the loss of FAD and ferrous ion from the multicomponent enzyme on purification. Analysis of mutants of strain M2 defective in the metabolism of C1 compounds indicated that methanol was not an intermediate in the degradative pathway of MSA and also confirmed the involvement of a multicomponent enzyme in the degradation of MSA by methylotroph strain M2.

Bacteria↗

PubMed1995

Detection of novel marine methanotrophs using phylogenetic and functional gene probes after methane enrichment.

A major limitation of rRNA-targeted group-specific probes is that they may cross-react with organisms of other physiological, or even phylogenetic groups when applied to environmental samples containing unknown sequences. We have exploited the restricted physiology of methane-oxidizing bacteria to assess the specificity and efficiency of probes for this physiological type which target the 16S rRNA or genes involved in methanotroph physiology. Seawater samples were enriched for methanotrophs by addition of methane and essential nutrients. The changes in composition of the bacterial population were monitored by analysis of 16S rRNA gene libraries. Methanotroph group-specific probes failed to give a signal with samples from these enrichments even though a methanol dehydrogenase structural gene was detected. A 16S rDNA sequence that was abundant only after methane addition was recovered and found to show a close phylogenetic relationship to Methylomonas. Organisms containing this sequence were observed in enrichments by in situ hybridization. The combination of enrichment on methane and screening with the broad specificity methanol dehydrogenase probe allowed detection of novel methanotrophs that were not detected with the original suite of methanotroph group-specific probes.

Alcohol Oxidoreductases↗