J. C. Murrell

Molecular Ecology Techniques for the Study of Aerobic Methanotrophs

Methane oxidation can occur in both aerobic and anaerobic environments; however, these are completely different processes involving different groups of prokaryotes. Aerobic methane oxidation is carried out by aerobic methanotrophs, and anaerobic methane oxidizers, discovered recently, thrive under

Metabolic aspects of aerobic obligate methanotrophy

Publisher Summary This chapter discusses the metabolic aspects of aerobic obligate methanotrophy. Aerobic methanotrophs are a unique group of gram-negative bacteria that use methane as carbon and energy source. Methanotrophs have been studied intensively over the past 40 years since these bacteria possess significant metabolic potential for practical use in the biotransformation of a variety of organic substrates, bioremediation of pollutants the production of single-cell protein (SCP), and value-added products. They also play a vital role in the global methane cycle, mitigating the emissions and green-house effects of methane on the Earths climate. Methanotrophs build all of their cell constituents from C 1 compounds by employing special biosynthetic pathways for phosphotrioses, which are different from those of heterotrophic 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.

Stable-isotope probing implicates Methylophaga spp and novel Gammaproteobacteria in marine methanol and methylamine metabolism

The metabolism of one-carbon (C1) compounds in the marine environment affects global warming, seawater ecology and atmospheric chemistry. Despite their global significance, marine microorganisms that consume C1 compounds in situ remain poorly characterized. Stable-isotope probing (SIP) is an ideal tool for linking the function and phylogeny of methylotrophic organisms by the metabolism and incorporation of stable-isotope-labelled substrates into nucleic acids. By combining DNA-SIP and time-series sampling, we characterized the organisms involved in the assimilation of methanol and methylamine in coastal sea water (Plymouth, UK). Labelled nucleic acids were analysed by denaturing gradient gel electrophoresis (DGGE) and clone libraries of 16S rRNA genes. In addition, we characterized the functional gene complement of labelled nucleic acids with an improved primer set targeting methanol dehydrogenase (mxaF) and newly designed primers for methylamine dehydrogenase (mauA). Predominant DGGE phylotypes, 16S rRNA, methanol and methylamine dehydrogenase gene sequences, and cultured isolates all implicated Methylophaga spp, moderately halophilic marine methylotrophs, in the consumption of both methanol and methylamine. Additionally, an mxaF sequence obtained from DNA extracted from sea water clustered with those detected in 13C-DNA, suggesting a predominance of Methylophaga spp among marine methylotrophs. Unexpectedly, most predominant 16S rRNA and functional gene sequences from 13C-DNA were clustered in distinct substrate-specific clades, with 16S rRNA genes clustering with sequences from the Gammaproteobacteria. These clades have no cultured representatives and reveal an ecological adaptation of particular uncultured methylotrophs to specific C1 compounds in the coastal marine environment.

Marine methylotrophs revealed by stable-isotope probing, multiple displacement amplification and metagenomics

The concentrations of one-carbon substrates that fuel methylotrophic microbial communities in the ocean are limited and the specialized guilds of bacteria that use these molecules may exist at low relative abundance. As a result, these organisms are difficult to identify and are often missed with existing cultivation and gene retrieval methods. Here, we demonstrate a novel proof of concept: using environmentally-relevant substrate concentrations in stable-isotope probing (SIP) incubations to yield sufficient DNA for large-insert metagenomic analysis through multiple displacement amplification (MDA). A marine surface-water sample was labelled sufficiently by incubation with near in situ concentrations of methanol. Picogram quantities of labelled (13)C-DNA were purified from caesium chloride gradients, amplified with MDA to produce microgram amounts of high-molecular-weight DNA (<or= 40 kb) and cloned to produce a fosmid library of > 10 000 clones. Denaturing gradient gel electrophoresis (DGGE) demonstrated minimal bias associated with the MDA step and implicated Methylophaga-like phylotypes with the marine metabolism of methanol. Polymerase chain reaction screening of 1500 clones revealed a methanol dehydrogenase (MDH) containing insert and shotgun sequencing of this insert resulted in the assembly of a 9-kb fragment of DNA encoding a cluster of enzymes involved in MDH biosynthesis, regulation and assembly. This novel combination of methodology enables future structure-function studies of microbial communities to achieve the long-desired goal of identifying active microbial populations using in situ conditions and performing a directed metagenomic analysis for these ecologically relevant microorganisms.

Molecular Analysis of the Sulfate Reducing and ArchaealCommunity in a Meromictic Soda Lake (Mono Lake, California) by Targeting 16S rRNA, mcrA , apsA , and dsrAB Genes

Sulfate reduction is the most important process involved in the mineralization of carbon in the anoxic bottom waters of Mono Lake, an alkaline, hypersaline, meromictic Lake in California. Another important biogeochemical process in Mono Lake is thought to be sulfate-dependent methane oxidation (SDMO). However little is known about what types of organisms are involved in these processes in Mono Lake. Therefore, the sulfate-reducing and archaeal microbial community in Mono Lake was analyzed by targeting 16S rRNA, methyl-coenzyme M reductase (mcrA), adenosine-5′-phosphosulfate (apsA), and dissimilatory sulfite reductase (dsrAB) genes to investigate the sulfate-reducing and archaeal community with depth. Most of the 16S rRNA gene sequences retrieved from the samples fell into the δ-subdivision of the Proteobacteria. Phylogenetic analyses suggested that the clones obtained represented sulfate-reducing bacteria, which are probably involved in the mineralization of carbon in Mono Lake, many of them belonging to a novel line of descent in the δ-Proteobacteria. Only 6% of the sequences retrieved from the samples affiliated to the domain Euryarchaeota but did not represent Archaea, which is considered to be responsible for SDMO [Orphan et al. 2001: Appl Environ Microbiol 67:1922–1934; Teske et al.: Appl Environ Microbiol 68:1994–2007]. On the basis of our results and thermodynamic arguments, we proposed that SDMO in hypersaline environments is presumably carried out by SRB alone. Polymerase chain reaction (PCR) amplifications of the mcrA-, apsA-, and dsrAB genes in Mono Lake samples were, in most cases, not successful. Only the PCR amplification of the apsA gene was partially successful. The amplification of these functional genes was not successful because there was either insufficient “target” DNA in the samples, or the microorganisms in Mono Lake have divergent functional genes.

Microbial metabolism of methanesulfonic acid

Abstract Methanesulfonic acid is a very stable strong acid and a key intermediate in the biogeochemical cycling of sulfur. It is formed in megatonne quantities in the atmosphere from the chemical oxidation of atmospheric dimethyl sulfide (most of which is of biogenic origin) and deposited on the Earth in rain and snow, and by dry deposition. Methanesulfonate is used by diverse aerobic bacteria as a source of sulfur for growth, but is not known to be used by anaerobes either as a sulfur source, a fermentation substrate, an electron acceptor, or as a methanogenic substrate. Some specialized methylotrophs (including Methylosulfonomonas, Marinosulfonomonas, and strains of ¶Hyphomicrobium and Methylobacterium) can use it as a carbon and energy substrate to support growth. Methanesulfonate oxidation is initiated by cleavage catalysed by methanesulfonate monooxygenase, the properties and molecular biology of which are discussed.

Hyphomicrobium chloromethanicum sp. nov. and Methylobacterium chloromethanicum sp. nov., chloromethane-utilizing bacteria isolated from a polluted environment.

Two chloromethane-utilizing facultatively methylotrophic bacteria, strains CM2T and CM4T, were isolated from soil at a petrochemical factory. On the basis of their morphological, physiological and genotypical properties, strain CM2T (= VKM B-2176T = NCIMB 13687T) is proposed as a new species of the genus Hyphomicrobium, Hyphomicrobium chloromethanicum, and strain CM4T (= VKM B-2223T = NCIMB 13688T) as a new species of the genus Methylobacterium, Methylobacterium chloromethanicum.

The sea-surface microlayer is a gelatinous biofilm

One of John McN Sieburth’s many contributions to biological oceanography was his proposal of a hydrated gelatinous surface microlayer film at the air–sea interface (Sieburth, 1983). The purpose of this commentary is to highlight John Sieburth’s contribution to surface ocean science and the recent evidence that supports his proposal of a gelatinous microlayer film. His hypothesis was based on many observations made over several years, including those during a Trichodesmium bloom in the Sargasso Sea, when a distinct slick was reported directly above the bloom (Sieburth and Conover, 1965). At the air–sea interface, the sea-surface microlayer is the physical boundary between the ocean and the atmosphere. Roughly considered to be the uppermost 1 mm of the ocean, the sea-surface microlayer is physico-chemically distinct compared with subsurface water and is characteristically enriched with biogenic organic compounds, such as lipids, proteins and polysaccharides (Liss and Duce, 2005). The presence of the surface film and surface tension properties means the sea-surface microlayer is a unique habitat that is often referred to as the neuston. Bacterial communities that are present in the surface microlayer are known as the bacterioneuston (Liss and Duce, 2005). Surface films occur on all water bodies, marine, estuarine and freshwater, sometimes as visible slicks, and are rapidly reformed after mixing by wind or waves (RC UpstillGoddard, personal communication). The widely known effect of pouring oil on troubled waters occurs naturally, with organic films changing the physical properties of surface water, reducing roughness and affecting air–sea gas transfer (Liss and Duce, 2005). The structure of the sea-surface microlayer film, potentially 70% of the Earth’s surface, is of great importance in the exchange of chemicals between the oceans and the atmosphere. During summer blooms of filamentous cyanobacteria, the net oxygen flux is significantly highly between the atmosphere and the surface microlayer compared with the ocean and the surface microlayer, which highlights the importance of autotrophic and heterotrophic organisms in this environment (Ploug, 2008). Also, bacterial activity within the microlayer can mediate the air–sea flux of other gases, such as methane (Upstill-Goddard et al., 2003). Recent work by Wurl and Holmes (2008) has shed new insights into Sieburth’s proposal, with important implications for marine microbial ecology and oceanography. Sea-surface microlayer and subsurface water samples (1-m depth) were collected from around Singapore and subsequently, the concentrations of transparent exopolymer particles (TEPs) were determined (Wurl and Holmes, 2008). They showed an enrichment of TEPs in the sea-surface microlayer. TEPs are generally formed in surface waters from the coagulation of biogenic polysaccharides (Figure 1), particularly those produced by phytoplankton, and are some of the most ubiquitous gel particles in the marine environment (Verdugo et al., 2004). They are critical in the formation of marine aggregates, acting as the binding matrix or ‘glue’ that holds the aggregate together (Verdugo et al., 2004). An abundance of TEPs in the seasurface microlayer supports Sieburth’s proposal made in 1983. The molecular microbial ecology of the seasurface microlayer has only recently been studied. Franklin et al. (2005) showed that the bacterioneuston at a site in the North Sea was distinctly different compared with subsurface water just 0.4 m below the surface. The bacterioneuston was dominated by two genera, Vibrio and Pseudoalteromonas. Both organisms have physiological characteristics that are indicative of adaptations for biofilm survival (Franklin et al., 2005). If the seasurface microlayer is a gelatinous film, then organisms that have biofilm capabilities will have a selective advantage. More recent evidence published in The ISME Journal supports the observations made in the North Sea by Franklin et al. (2005) and shows distinct bacterioneuston communities are also present in estuarine surface microlayers (Cunliffe et al., 2008). Denaturing gradient gel electrophoresis comparison of bacterial and archaeal 16S rRNA genes from surface microlayer and subsurface water samples at The ISME Journal (2009) 3, 1001–1003 & 2009 International Society for Microbial Ecology All rights reserved 1751-7362/09

Methane and methanol utilizers

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