Andrew J. Holmes

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.

Analysis of 16S rRNA and methane monooxygenase gene sequences reveals a novel group of thermotolerant and thermophilic methanotrophs, Methylocaldum gen. nov.

Two methanotrophic bacteria with optimum growth temperatures above 40° C were isolated. Thermotolerant strain LK6 was isolated from agricultural soil, and the moderately thermophilic strain OR2 was isolated from the effluent of an underground hot spring. When compared to the described thermophilic methanotrophs Methylococcus capsulatus and Methylococcus thermophilus, these strains are phenotypically similar to Methylococcus thermophilus. However, their 16S rRNA gene sequences are markedly different from the sequence of Methylococcus thermophilus (∼ 8% divergence) and, together with Methylomonas gracilis, they form a distinct, new genus within the γ-subgroup of the Proteobacteria related to extant Type I methanotrophs. Further phenotypic characterisation showed that the isolates possess particulate methane monooxygenase (pMMO) but do not contain soluble methane monooxygenase. The nucleotide sequence of a gene encoding pMMO (pmoA) was determined for both isolates and for Methylomonas gracilis. PmoA sequence comparisons confirmed the monophyletic nature of this newly recognised group of thermophilic methanotrophs and their relationship to previously described Type I methanotrophs. We propose that strains OR2 and LK6, together with the misclassified thermophilic strains Methylomonas gracilis VKM-14LT and Methylococcus thermophilus IMV-B3122, comprise a new genus of thermophilic methanotrophs, Methylocaldum gen. nov., containing three new species: Methylocaldum szegediense, Methylocaldum tepidum and Methylocaldum gracile.

Methylosulfonomonas methylovora gen. nov., sp. nov., and Marinosulfonomonas methylotropha gen. nov., sp. nov.: novel methylotrophs able to grow on methanesulfonic acid

Abstract Two novel genera of restricted facultative methylotrophs are described; both Methylosulfonomonas and Marinosulfonomonas are unique in being able to grow on methanesulfonic acid as their sole source of carbon and energy. Five identical strains of Methylosulfonomonas were isolated from diverse soil samples in England and were shown to differ in their morphology, physiology, DNA base composition, molecular genetics, and 16S rDNA sequences from the two marine strains of Marinosulfonomonas, which were isolated from British coastal waters. The marine strains were almost indistinguishable from each other and are considered to be strains of one species. Type species of each genus have been identified and named Methylosulfonomonas methylovora (strain M2) and Marinosulfonomonas methylotropha (strain PSCH4). Phylogenetic analysis using 16S rDNA sequencing places both genera in the α-Proteobacteria. Methylosulfonomonas is a discrete lineage within the α-2 subgroup and is not related closely to any other known bacterial genus. The Marinosulfonomonas strains form a monophyletic cluster in the α-3 subgroup of the Proteobacteria with Roseobacter spp. and some other partially characterized marine bacteria, but they are distinct from these at the genus level. This work shows that the isolation of bacteria with a unique biochemical character, the ability to grow on methanesulfonic acid as energy and carbon substrate, has resulted in the identification of two novel genera of methylotrophs that are unrelated to any other extant methylotroph genera.

Molecular analysis of enrichment cultures of marine methane oxidising bacteria

Abstract Methane oxidising bacteria (methanotrophs) are an important group of bacteria in the global cycling of methane and may act as a sink for methane in freshwater, soil and marine environments. Their ecology is fairly well documented in terrestrial and freshwater environments but marine methanotrophs have been less-well characterised, partly due to the difficulties in the isolation, cultivation and identification of these organisms. Molecular ecological techniques are being developed to aid the characterisation and enumeration of marine methane oxidising bacteria and to assess their role in the turn-over of methane in the marine environment. 16S ribosomal RNA gene probes have been developed to detect and identify marine methanotrophs. The use of these probes, coupled with fluorescence microscopy techniques, to aid enrichment, cultivation and subsequent characterisation of marine methanotrophs is described. The use of the polymerase chain reaction to amplify methane monooxygenase genes from enrichments of marine methanotrophs is also described.

Molecular Biology of Particulate Methane Monooxygenase

Methane oxidizing bacteria (methanotrophs) grow on methane as their sole source of carbon and energy by oxidizing this one-carbon (CI) compound, via methanol, formaldehyde and formate, to carbon dioxide. Carbon is assimilated into biomass at the level of formaldehyde by either the serine or the ribulose monophosphate pathway. The first step in the methane oxidation pathway is catalysed by the enzyme methane monooxygenase (MMO). The MMO can exist in two forms, a soluble, cytoplasmic enzyme complex (sMMO) or a membrane-bound, particulate enzyme (pMMO). The sMMO is only found in methanotrophs such as Methylococcus spp., Methylosinus spp., and some Methylocystis and Methylomonas spp. The sMMO has been purified from several methanotrophs and the genes encoding this enzyme complex have been cloned and sequenced from Methylococcus capsulatus (Bath) and Methylosinus trichosporium OB3b. The biochemistry and molecular biology of sMMO has recently been reviewed extensively (Lipscomb 1994; Dalton et al. 1993; Murrell 1992, 1994) and will not be covered here. The pMMO appears to be present in all methanotrophs but is less-well characterised than sMMO. The enzyme has proved difficult to purify in active form, presumably because of instability in the pMMO polypeptides once they are removed from the membranes. In those methanotrophs that possess both sMMO and pMMO, eg Mc. capsulatus (Bath) and Ms. Trichosporium OB3b, the copper-to-biomass ratio of cultures seems to regulate the switch between expression of sMMO or pMMO (Stanley et al. 1983). A high copper-to-biomass ratio favours expression of the pMMO and sMMO is repressed by copper ions (copper also appears to inhibit sMMO activity). Cells grown at low copper-to-biomass ratios express sMMO and there is now good evidence for transcriptional control of sMMO (and pMMO) expression by copper ions (A. Nielsen, J.C. Murrell, unpublished).

Molecular Ecology of Marine Methanotrophs

Methane oxidising bacteria (methanotrophs) are a unique group of methylotrophs (organisms that can utilise one-carbon compounds for growth) that grow using methane as their sole source of carbon and energy. They are generally regarded as obligate organisms and although many methanotrophs can also grow on methanol, they do not utilise multi-carbon compounds. The first report of a methanotroph was by Sohngen who isolated the pink methanotroph Bacillus methanicus (Solingen, 1906) which was subsequently renamed Pseudomonas methanica and then Methylomonas methanica. This was re-isolated many years later by Dworkin and Foster (1956) but there were only a few sporadic reports on methanotrophs until the pioneering work of Whittenbury and colleagues who isolated and characterised over 100 different strains of methane-oxidising bacteria from soils, sediments and freshwater (Whittenbury et al., 1970). These were all strictly aerobic, gram negative bacteria that grew on a simple mineral salts medium and methane. Based on their cell morphology, the resting stages formed, the intracytoplasmic membranes they contained and a few physiological characteristics, these bacteria were divided into five proposed genera, Methylococcus, Methylobacter, Methylomonas, Methylocystis and Methylosinus.

Molecular Ecology of Methanotrophs

Methane oxidizing bacteria (methanotrophs) are unique in growing with methane as their sole source of carbon and energy. They do not grow on multi-carbon compounds, but some can also utilize methanol as a growth substrate. These unique organisms appear to be ubiquitous in the natural environment and have been isolated from a wide variety of soils, sediments and freshwater samples (Whittenbury et al. 1970; Bowman et al. 1993) . There are also marine representatives (reviewed in Murrell and Holmes, 1995). They are all strictly aerobic, gram negative bacteria that grow on a minimal medium and methane and can be classified into two groups on the basis of their intracytoplasmic membranes, pathways of formaldehyde assimilation and 16S rRNA sequence. The five genera Methylomonas, Methylobacter, Methylococcus, Methylocystis and Methylosinus originally proposed by Whittenbury et al. (1970) have largely remained unaltered (Bowman et al. 1993). Type I methanotrophs Methylobacter and Methylomonas are related to bacteria in the γ-subdivision of the Proteobacteria, contain bundles of intracytoplasmic membranes and utilize the ribulose monophosphate (RuMP) pathway for formaldehyde assimilation into biomass.

Working Group Reports

Microbial processes are involved in the exchange of most of the trace gases between the atmosphere and most types of ecosystems. Consequently the regulation of the amounts of gases emitted or taken up are tightly linked to changes in microbial activity.