J. Colin Murrell

Evidence that participate methane monooxygenase and ammonia monooxygenase may be evolutionarily related

Genes encoding particulate methane monooxygenase and ammonia monooxygenase share high sequence identity. Degenerate oligonucleotide primers were designed, based on regions of shared amino acid sequence between the 27-kDa polypeptides, which are believed to contain the active sites, of particulate methane monooxygenase and ammonia monooxygenase. A 525-bp internal DNA fragment of the genes encoding these polypeptides (pmoA and amoA) from a variety of methanotrophic and nitrifying bacteria was amplified by PCR, cloned and sequenced. Representatives of each of the phylogenetic groups of both methanotrophs (alpha- and gamma-Proteobacteria) and ammonia-oxidizing nitrifying bacteria (beta- and gamma-Proteobacteria) were included. Analysis of the predicted amino acid sequences of these genes revealed strong conservation of both primary and secondary structure. Nitrosococcus oceanus AmoA showed higher identity to PmoA sequences from other members of the gamma-Proteobacteria than to AmoA sequences. These results suggest that the particulate methane monooxygenase and ammonia monooxygenase are evolutionarily related enzymes despite their different physiological roles in these bacteria.

Stable isotope probing - linking microbial identity to function.

Stable isotope probing (SIP) is a technique that is used to identify the microorganisms in environmental samples that use a particular growth substrate. The method relies on the incorporation of a substrate that is highly enriched in a stable isotope, such as 13C, and the identification of active microorganisms by the selective recovery and analysis of isotope-enriched cellular components. DNA and rRNA are the most informative taxonomic biomarkers and 13C-labelled molecules can be purified from unlabelled nucleic acid by density-gradient centrifugation. The future holds great promise for SIP, particularly when combined with other emerging technologies such as microarrays and metagenomics.

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.

Stable-isotope probing of nucleic acids: a window to the function of uncultured microorganisms

Phylogeny based on ribosomal RNA sequences alone is rarely a reliable indicator of microbial function. To circumvent this problem, nucleic acid based techniques have been developed that exploit the physical properties of stable isotopes to study microbially mediated processes within complex environmental samples. Investigations using labelled substrates, or which detect variations in the natural abundance of isotopes, have thus revealed the metabolic function of microorganisms without the need to isolate them in culture.

Regulation of expression of methane monooxygenases by copper ions

Many methanotrophs contain both a soluble and a particulate methane monooxygenase. A unique metabolic switch, mediated by copper ions, regulates the expression of these enzymes. When the copper-to-biomass ratio of the cell is low, the soluble enzyme is expressed, and when the copper-to-biomass ratio is high, the particulate enzyme is expressed. A model for the mechanism of this switch is proposed.

Molecular biology and regulation of methane monooxygenase

Abstract. Methanotrophs are ubiquitous in the environment and play an important role in mitigating global warming due to methane. They are also potentially interesting for industrial applications such as production of bulk chemicals or bioremediation. The first step in the oxidation of methane is the conversion to methanol by methane monooxygenase, the key enzyme, which exists in two forms: the cytoplasmic, soluble methane monooxygenase (sMMO) and the membrane-bound, particulate methane monooxygenase (pMMO). This paper reviews the biochemistry and molecular biology of both forms of MMO. In the past few years there have been many exciting new findings. sMMO components have been expressed in heterologous and homologous hosts. The pMMO has been purified and biochemically studied in some detail and the genes encoding the pMMO have been sequenced. Copper ions have been shown to play a key role in regulating the expression of both MMO enzyme complexes. We also present a model for copper regulation based on results from Northern analysis, primer-extensions and new sequence data, and raise a number of unanswered questions for future studies.

Methodological Considerations for the Use of Stable Isotope Probing in Microbial Ecology

Stable isotope probing (SIP) is a method used for labeling uncultivated microorganisms in environmental samples or directly in field studies using substrate enriched with stable isotope (e.g., 13C). After consumption of the substrate, the cells of microorganisms that consumed the substrate become enriched in the isotope. Labeled biomarkers, such as phospholipid-derived fatty acid (PLFA), ribosomal RNA, and DNA can be analyzed with a range of molecular and analytical techniques, and used to identify and characterize the organisms that incorporated the substrate. The advantages and disadvantages of PLFA-SIP, RNA-SIP, and DNA-SIP are presented. Using examples from our laboratory and from the literature, we discuss important methodological considerations for a successful SIP experiment.

Copper-dependent reciprocal transcriptional regulation of methane monooxygenase genes in Methylococcus capsulatus and Methylosinus trichosporium.

The methanotrophic bacteria Methylococcus capsulatus (Bath) and Methylosinus trichosporium OB3b convert methane to methanol using the enzyme, methane monooxygenase (MMO). These bacteria are able to express two distinct MMOs: a cytoplasmic or soluble form (sMMO) and a membrane‐bound or particulate form (pMMO). Differential expression of sMMO and pMMO is regulated by the amount of copper ions available to the cells; sMMO is expressed at low copper–biomass ratios, whereas pMMO is expressed at high copper–biomass ratios. In both methanotrophs, transcription of the sMMO gene cluster is negatively regulated by copper ions. Data suggest that transcription of the M. trichosporium OB3b sMMO gene cluster is directed from a σ54‐like and a σ70‐like promoter. The pMMO (pmo ) genes of M. capsulatus (Bath) are transcribed into a polycistronic mRNA of 3.3 kb. The synthesis of this mRNA was activated by copper ions. Activation of pmo transcription by copper ions was concomitant with repression of sMMO gene transcription in both methanotrophs. This suggests that a common regulatory pathway may be involved in the transcriptional switch between sMMO and pMMO gene expression.

Regulation of methane oxidation in the facultative methanotroph Methylocella silvestris BL2

The molecular regulation of methane oxidation in the first fully authenticated facultative methanotroph Methylocella silvestris BL2 was assessed during growth on methane and acetate. Problems of poor growth of Methylocella spp. in small‐scale batch culture were overcome by growth in fermentor culture. The genes encoding soluble methane monooxygenase were cloned and sequenced, which revealed that the structural genes for soluble methane monooxygenase, mmoXYBZDC, were adjacent to two genes, mmoR and mmoG, encoding a σ54 transcriptional activator and a putative GroEL‐like chaperone, located downstream (3′) of mmoC. Transcriptional analysis revealed that the genes were all cotranscribed from a σ54‐dependent promoter located upstream (5′) of mmo X. The transcriptional start site was mapped. Transcriptional analysis of soluble methane monooxygenase genes and expression studies on fermentor grown cultures showed that acetate repressed transcription of sMMO in M. silvestris BL2. The possibility of the presence of a particulate, membrane‐bound methane monooxygenase enzyme in M. silvestris BL2 and the copper‐mediated regulation of soluble methane monooxygenase was investigated. Both were shown to be absent. A promoter probe vector was constructed and used to assay transcription of the promoter of the soluble methane monoxygenase genes of M. silvestris BL2 grown under various conditions and with different substrates. These data represent the first insights into the molecular physiology of a facultative methanotroph.

When metagenomics meets stable-isotope probing: progress and perspectives

The application of metagenomics, the culture-independent capture and subsequent analysis of genomic DNA from the environment, has greatly expanded our knowledge of the diversity of microbes and microbial protein families; however, the metabolic functions of many microorganisms remain largely unknown. DNA stable-isotope probing (DNA-SIP) is a recently developed method in which the incorporation of stable isotope from a labelled substrate is used to identify the function of microorganisms in the environment. The technique has now been used in conjunction with metagenomics to establish links between microbial identity and particular metabolic functions. The combination of DNA-SIP and metagenomics not only permits the detection of rare low-abundance species from metagenomic libraries but also facilitates the detection of novel enzymes and bioactive compounds.