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Background: Methanotrophs is a promising biocatalyst in biotechnological applications with their ability to utilize single carbon (C1) feedstock to produce high-value compounds. Understanding the behavior of biological networks of methanotrophic bacteria in different parameters is vital to systems biology and metabolic engineering. Interestingly, methanotrophic bacteria possess the pyrophosphate-dependent 6-phosphofructokinase (PPi-PFK) instead of the ATP-dependent 6-phosphofructokinase, indicating their potentials to serve as promising model for investigation the role of inorganic pyrophosphate (PPi) and PPi-dependent glycolysis in bacteria. Gene knockout experiments along with global-omics approaches can be used for studying gene functions as well as unraveling regulatory networks that rely on the gene product., Results: In this study, we performed gene knockout and RNA-seq experiments in Methylotuvimicrobium alcaliphilum 20Z to investigate the functional roles of PPi-PFK in C1 metabolism when cells were grown on methane and methanol, highlighting its metabolic importance in C1 assimilation in M. alcaliphilum 20Z. We further conducted adaptive laboratory evolution (ALE) to investigate regulatory architecture in pfk knockout strain. Whole-genome resequencing and RNA-seq approaches were performed to characterize the genetic and metabolic responses of adaptation to pfk knockout. A number of mutations, as well as gene expression profiles, were identified in pfk ALE strain to overcome insufficient C1 assimilation pathway which limits the growth in the unevolved strain., Conclusions: This study first revealed the regulatory roles of PPi-PFK on C1 metabolism and then provided novel insights into mechanism of adaptation to the loss of this major metabolic enzyme as well as an improved basis for future strain design in type I methanotrophs.

The bacteria utilizing methane as a growth substrate (methanotrophs) are important constituents of the biosphere. Methanotrophs mitigate the emission of anthropogenic and natural greenhouse gas methane to the environment and are the promising agents for future biotechnologies. Many aspects of CH4 bioconversion by methanotrophs require further clarification. This study was aimed at characterizing the biochemical properties of the malic enzyme (Mae) from the halotolerant obligate methanotroph Methylotuvimicrobium alcaliphilum 20Z. The His6-tagged Mae was obtained by heterologous expression in Escherichia coli BL21 (DE3) and purified by affinity metal chelating chromatography. As determined by gel filtration and non-denaturating gradient gel electrophoresis, the molecular mass of the native enzyme is 260 kDa. The homotetrameric Mae (65x4 kDa) catalyzed an irreversible NAD+-dependent reaction of L-malate decarboxylation into pyruvate with a specific activity of 32 ± 2 units mg-1 and Km value of 5.5 ± 0.8 mM for malate and 57 ± 5 μM for NAD+. The disruption of the mae gene by insertion mutagenesis resulted in a 20-fold increase in intracellular malate level in the mutant compared to the wild type strain. Based on both enzyme and mutant properties, we conclude that the malic enzyme is involved in the control of intracellular L-malate level in Mtm. alcaliphilum 20Z. Genomic analysis has revealed that Maes present in methanotrophs fall into two different clades in the amino acid-based phylogenetic tree, but no correlation of the division with taxonomic affiliations of the host bacteria was observed., Competing Interests: The authors have declared that no competing interests exist.

Nitrite-dependent methane-oxidizing bacteria couple the reduction of nitrite to the oxidation of methane via a unique oxygen-producing pathway. This process is carried out by members of the genus Methylomirabilis that belong to the NC10 phylum. Contrary to other known anaerobic methane oxidizers, they do not employ the reverse methanogenesis pathway for methane activation but instead a canonical particulate methane monooxygenase similar to those used by aerobic methanotrophs. Methylomirabilis -like bacteria are detected in many natural and manmade ecosystems, but their physiology is not well understood. Here, using continuous cultivation techniques, batch activity assays, and state-of-the-art membrane-inlet mass spectrometry, we determined growth rate, doubling time, and methane and nitrite affinities of the nitrite-dependent methane-oxidizing bacterium " Candidatus Methylomirabilis lanthanidiphila." Our results provide insight into understanding the interactions of these microorganisms with methanotrophs and other nitrite-reducing microorganisms, such as anaerobic ammonium-oxidizing bacteria. Furthermore, our data can be used in modeling studies as well as wastewater treatment plant design. IMPORTANCE Methane is an important greenhouse gas with a radiative forcing 28 times that of carbon dioxide over a 100-year time scale. The emission of methane to the atmosphere is controlled by aerobic and anaerobic methanotrophs, which are microorganisms that are able to oxidize methane to conserve energy. While aerobic methanotrophs have been studied for over a century, knowledge on the physiological characteristics of anaerobic methanotrophs is scarce. Here, we describe kinetic properties of " Candidatus Methylomirabilis lanthanidiphila," a nitrite-dependent methane-oxidizing microorganism, which is ecologically important and can be applied in wastewater treatment., (Copyright © 2019 American Society for Microbiology.)

Biological methane utilization, one of the main sinks of the greenhouse gas in nature, represents an attractive platform for production of fuels and value-added chemicals. Despite the progress made in our understanding of the individual parts of methane utilization, our knowledge of how the whole-cell metabolic network is organized and coordinated is limited. Attractive growth and methane-conversion rates, a complete and expert-annotated genome sequence, as well as large enzymatic, 13 C-labeling, and transcriptomic datasets make Methylomicrobium alcaliphilum 20Z R an exceptional model system for investigating methane utilization networks. Here we present a comprehensive metabolic framework of methane and methanol utilization in M. alcaliphilum 20Z R . A set of novel metabolic reactions governing carbon distribution across central pathways in methanotrophic bacteria was predicted by in-silico simulations and confirmed by global non-targeted metabolomics and enzymatic evidences. Our data highlight the importance of substitution of ATP-linked steps with PPi-dependent reactions and support the presence of a carbon shunt from acetyl-CoA to the pentose-phosphate pathway and highly branched TCA cycle. The diverged TCA reactions promote balance between anabolic reactions and redox demands. The computational framework of C 1 -metabolism in methanotrophic bacteria can represent an efficient tool for metabolic engineering or ecosystem modeling.

Microbial methane oxidizers play a crucial role in the oxidation of methane in marine ecosystems, as such preventing the escape of excessive methane to the atmosphere. Despite the important role of methanotrophs in marine ecosystems, only a limited number of isolates are described, with only four genomes available. Here, we report on two genomes of gammaproteobacterial methanotroph cultures, affiliated with the deep-sea cluster 2, obtained from North Sea sediment. Initial enrichments using methane as sole source of carbon and energy and mimicking the in situ conditions followed by serial subcultivations and multiple extinction culturing events over a period of 3 years resulted in a highly enriched culture. The draft genomes of the methane oxidizer in both cultures showed the presence of genes typically found in type I methanotrophs, including genes encoding particulate methane monooxygenase (pmoCAB), genes for tetrahydromethanopterin (H4MPT)- and tetrahydrofolate (H4F)-dependent C1-transfer pathways, and genes of the ribulose monophosphate (RuMP) pathway. The most distinctive feature, when compared to other available gammaproteobacterial genomes, is the absence of a calcium-dependent methanol dehydrogenase. Both genomes reported here only have a xoxF gene encoding a lanthanide-dependent XoxF5-type methanol dehydrogenase. Thus, these genomes offer novel insight in the genomic landscape of uncultured diversity of marine methanotrophs.

Unlabelled: Many methylotrophic taxa harbor two distinct methanol dehydrogenase (MDH) systems for oxidizing methanol to formaldehyde: the well-studied calcium-dependent MxaFI type and the more recently discovered lanthanide-containing XoxF type. MxaFI has traditionally been accepted as the major functional MDH in bacteria that contain both enzymes. However, in this study, we present evidence that, in a type I methanotroph, Methylomicrobium buryatense, XoxF is likely the primary functional MDH in the environment. The addition of lanthanides increases xoxF expression and greatly reduces mxa expression, even under conditions in which calcium concentrations are almost 100-fold higher than lanthanide concentrations. Mutations in genes encoding the MDH enzymes validate our finding that XoxF is the major functional MDH, as XoxF mutants grow more poorly than MxaFI mutants under unfavorable culturing conditions. In addition, mutant and transcriptional analyses demonstrate that the lanthanide-dependent MDH switch operating in methanotrophs is mediated in part by the orphan response regulator MxaB, whose gene transcription is itself lanthanide responsive., Importance: Aerobic methanotrophs, bacteria that oxidize methane for carbon and energy, require a methanol dehydrogenase enzyme to convert methanol into formaldehyde. The calcium-dependent enzyme MxaFI has been thought to primarily carry out methanol oxidation in methanotrophs. Recently, it was discovered that XoxF, a lanthanide-containing enzyme present in most methanotrophs, can also oxidize methanol. In a methanotroph with both MxaFI and XoxF, we demonstrate that lanthanides transcriptionally control genes encoding the two methanol dehydrogenases, in part by controlling expression of the response regulator MxaB. Lanthanides are abundant in the Earth's crust, and we demonstrate that micromolar amounts of lanthanides are sufficient to suppress MxaFI expression. Thus, we present evidence that XoxF acts as the predominant methanol dehydrogenase in a methanotroph., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

The aim of this study was first to identify lysozymes paralogs in the deep sea mussel Bathymodiolus azoricus then to measure their relative expression or activity in different tissue or conditions. B. azoricus is a bivalve that lives close to hydrothermal chimney in the Mid-Atlantic Ridge (MAR). They harbour in specialized gill cells two types of endosymbiont (gram-bacteria): sulphide oxidizing bacteria (SOX) and methanotrophic bacteria (MOX). This association is thought to be ruled by specific mechanism or actors of regulation to deal with the presence of symbiont but these mechanisms are still poorly understood. Here, we focused on the implication of lysozyme, a bactericidal enzyme, in this endosymbiosis. The relative expression of Ba-lysozymes paralogs and the global anti-microbial activity, were measured in natural population (Lucky Strike--1700 m, Mid-Atlantic Ridge), and in in situ experimental conditions. B. azoricus individuals were moved away from the hydrothermal fluid to induce a loss of symbiont. Then after 6 days some mussels were brought back to the mussel bed to induce a re-acquisition of symbiotic bacteria. Results show the presence of 6 paralogs in B. azoricus. In absence of symbionts, 3 paralogs are up-regulated while others are not differentially expressed. Moreover the global activity of lysozyme is increasing with the loss of symbiont. All together these results suggest that lysozyme may play a crucial role in symbiont regulation.

A novel exonuclease, designated as MszExo I, was cloned from Methylocaldum szegediense, a moderately thermophilic methanotroph. It specifically digests single-stranded DNA in the 3' to 5' direction. The protein is composed of 479 amino acids, and it shares 47% sequence identity with E. coli Exo I. The crystal structure of MszExo I was determined to a resolution of 2.2 Å and it aligns well with that of E. coli Exo I. Comparative studies revealed that MszExo I and E. coli Exo I have similar metal ion binding affinity and similar activity at mesophilic temperatures (25-47°C). However, the optimum working temperature of MszExo I is 10°C higher, and the melting temperature is more than 4°C higher as evaluated by both thermal inactivation assays and DSC measurements. More importantly, two thermal transitions during unfolding of MszExo I were monitored by DSC while only one transition was found in E. coli Exo I. Further analyses showed that magnesium ions not only confer structural stability, but also affect the unfolding of MszExo I. MszExo I is the first reported enzyme in the DNA repair systems of moderately thermophilic bacteria, which are predicted to have more efficient DNA repair systems than mesophilic ones.

Sterols are essential biological molecules in the majority of life forms. Sterol reductases including Δ(14)-sterol reductase (C14SR, also known as TM7SF2), 7-dehydrocholesterol reductase (DHCR7) and 24-dehydrocholesterol reductase (DHCR24) reduce specific carbon-carbon double bonds of the sterol moiety using a reducing cofactor during sterol biosynthesis. Lamin B receptor (LBR), an integral inner nuclear membrane protein, also contains a functional C14SR domain. Here we report the crystal structure of a Δ(14)-sterol reductase (MaSR1) from the methanotrophic bacterium Methylomicrobium alcaliphilum 20Z (a homologue of human C14SR, LBR and DHCR7) with the cofactor NADPH. The enzyme contains ten transmembrane segments (TM1-10). Its catalytic domain comprises the carboxy-terminal half (containing TM6-10) and envelops two interconnected pockets, one of which faces the cytoplasm and houses NADPH, while the other one is accessible from the lipid bilayer. Comparison with a soluble steroid 5β-reductase structure suggests that the reducing end of NADPH meets the sterol substrate at the juncture of the two pockets. A sterol reductase activity assay proves that MaSR1 can reduce the double bond of a cholesterol biosynthetic intermediate, demonstrating functional conservation to human C14SR. Therefore, our structure as a prototype of integral membrane sterol reductases provides molecular insight into mutations in DHCR7 and LBR for inborn human diseases.

The melting of permafrost and the associated potential for methane emissions to the atmosphere are major concerns in the context of global warming. However, soils can also represent a significant sink for methane through the activity of methane-oxidizing bacteria (MOB). In this study, we looked at the activity, diversity, and community structure of MOB at two sampling depths within the active layer in three soils from the Canadian high Arctic. These soils had the capacity to oxidize methane at low (15 ppm) and high (1000 ppm) methane concentrations, but rates differed greatly depending on the sampling date, depth, and site. The pmoA gene sequences related to two genotypes of uncultured MOB involved in atmospheric methane oxidation, the 'upland soil cluster gamma' and the 'upland soil cluster alpha', were detected in soils with near neutral and acidic pH, respectively. Other groups of MOB, including Type I methanotrophs and the 'Cluster 1' genotype, were also detected, indicating a broader diversity of MOB than previously reported for Arctic soils. Overall, the results reported here showed that methane oxidation at both low and high methane concentrations occurs in high Arctic soils and revealed that different groups of atmospheric MOB inhabit these soils., (© 2014 National Research Council Canada FEMS Microbiology Ecology © 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd.)

The Zoige wetland of the Tibetan Plateau is a high-altitude tundra wetland and one of the biggest methane emission centers in China. In this study, methanotrophs with respect to community structure, abundance, and activity were investigated in peat soils collected in the vicinity of different marshland plants that dominate different regions of the wetland, including Polygonum amphibium, Carex muliensis, and Eleocharis valleculosa (EV). 16S rRNA gene and particulate methane monooxygenase gene (pmoA) clone library sequence data indicated the presence of methanotrophs with two genera, Methylobacter and Methylocystis. Methylococcus, like pmoA gene sequences, were also retrieved and showed low similarity to those from Methylococcus spp. and thus indicates the existence of novel methanotrophs in the Zoige wetland. Quantitative polymerase chain reaction (qPCR) assays were used to measure the abundance of methantrophs and detected 10(7) to 10(8) of total pmoA gene copies per gram dry weight of soil in the three marshes. Group-specific qPCR and reverse transcriptase qPCR results found that the Methylobacter genus dominates the wetland, and Methylocystis methanotrophs were less abundant, although this group of methanotrophs was estimated to be more active according to mRNA/DNA ratio. Furthermore, EV marsh demonstrated the highest methanotrophs abundance and activity among the three marshes investigated. Our study suggests that both type I and type II methanotrophs contribute to the methane oxidation in the Zoige wetland.

Little is known about population dynamics and contribution of specific taxa to methane oxidation in flooded rice paddies. In this article we investigate the succession of methanotrophs in oxygen-methane counter-gradients. We used a gradient microcosm system that simulates oxic-anoxic interfaces of a water-saturated paddy soils, and measured pmoA-based (gene encoding particulate methane monooxygenase) terminal restriction fragment length polymorphism (T-RFLP) profiles at both the transcription (mRNA) and the population (DNA) levels. The DNA T-RFLP profiles indicated that the methanotrophic community present clearly differed from the active methanotrophic community. We observed a succession of the methanotrophic community over time without any direct effect of pore water chemistry on the community structure. Both the total population and the active subpopulation changed with time, whereas methane oxidation rates remained nearly constant. Hence, we suggest that a diverse microbial seed bank of methanotrophs is important in maintaining the function in a dynamic ecosystem.

Pyrophosphate-dependent 6-phosphofructokinase (PPi-PFK) was obtained as His₆-tagged protein by cloning of the pfp gene from the aerobic obligate methanotroph Methylomicrobium alcaliphilum 20Z and characterized. The recombinant PPi-PFK (4×45 kDa) was highly active, non-allosteric and stringently specific to pyrophosphate as the phosphoryl donor. The enzyme was more specific for the reverse reaction substrate fructose-1,6-bisphosphate (K(m) 0.095 mM, V(max) 805 U/mg of protein) than for the forward reaction substrate fructose-6-phosphate (K(m) 0.64 mM, V(max) 577 U/mg of protein). It also phosphorylated sedoheptulose-7-phosphate with much lower efficiency (K(m) 1.01 mM, V(max) 0.118 U/mg of protein). The kinetic properties of the M. alcaliphilum PP(i)-PFK were analyzed and compared with those of PP(i)-PFKs from other methanotrophs. The PP(i)-PFK from M. alcaliphilum shows highest sequence identity to PPi-PFK from obligate mesophilic methanotroph Methylomonas methanica (89%), and only low identity to the enzyme from thermotolerant Methylococcus capsulatus Bath (16%). This extensive sequence divergence of PPi-PFKs correlated with differential ability to phosphorylate sedoheptulose-7-phosphate and with the metabolic patterns of these bacteria assimilating C₁ substrate either via the ribulose monophoshate (RuMP) cycle or simultaneously via the RuMP and the Calvin cycles. Based on enzymic and genomic data, the involvement of PPi-PFK in pyrophosphate-dependent glycolysis in M. alcaliphilum 20Z was fist proposed., (Copyright © 2010 Institut Pasteur. Published by Elsevier SAS. All rights reserved.)

The nucleotide sequence of an open reading frame (corB) downstream of the copper-repressible CorA-encoding gene of the methanotrophic bacterium Methylomicrobium album BG8 was obtained by restriction enzyme digestion and inverse PCR. The amino acid sequence deduced from this gene showed significant sequence similarity to the surface-associated di-haem cytochrome c peroxidase (SACCP) previously isolated from Methylococcus capsulatus (Bath), including both c-type haem-binding motifs. Homology analysis placed this protein, phylogenetically, within the subfamily containing the M. capsulatus SACCP of the bacterial di-haem cytochrome c peroxidase (BCCP) family of proteins. Immunospecific recognition confirmed synthesis of the M. album CorB as a protein non-covalently associated with the outer membrane and exposed to the periplasm. corB expression is regulated by the availability of copper ions during growth and the protein is most abundant in M. album when grown at a low copper-to-biomass ratio, indicating an important physiological role of CorB under these growth conditions. corB was co-transcribed with the gene encoding CorA, constituting a copper-responding operon, which appears to be under the control of a sigma(54)-dependent promoter. M. album CorB is the second isolated member of the recently described subfamily of the BCCP family of proteins. So far, these proteins have only been described in methanotrophic bacteria.

Methane vents are of significant geochemical and ecological importance. Notable progress has been made toward understanding anaerobic methane oxidation in marine sediments; however, the diversity and distribution of aerobic methanotrophs in the water column are poorly characterized. Both environments play an essential role in regulating methane release from the oceans to the atmosphere. In this study, the diversity of particulate methane monooxygenase (pmoA) and 16S rRNA genes from two methane vent environments along the California continental margin was characterized. The pmoA phylotypes recovered from methane-rich sediments and the overlying water column differed. Sediments harbored the greatest number of unique pmoA phylotypes broadly affiliated with the Methylococcaceae family, whereas planktonic pmoA phylotypes formed three clades that were distinct from the sediment-hosted methanotrophs and distantly related to established methanotrophic clades. Water column-associated phylotypes were highly similar between field sites, suggesting that planktonic methanotroph diversity is controlled primarily by environmental factors rather than geographical proximity. Analysis of 16S rRNA genes from methane-rich waters did not readily recover known methanotrophic lineages, with only a few phylotypes demonstrating distant relatedness to Methylococcus. The development of new pmo primers increased the recovery of monooxygenase genes from the water column and led to the discovery of a highly diverged monooxygenase sequence which is phylogenetically intermediate to Amo and pMMO. This sequence potentiates insight into the amo/pmo superfamily. Together, these findings lend perspective into the diversity and segregation of aerobic methanotrophs within different methane-rich habitats in the marine environment.

135 years ago Ferdinand Cohn, the founder of bacteriology, microscopically observed a conspicuous filamentous bacterium with a complex life cycle and described it as Crenothrix polyspora. This uncultured bacterium is infamous for mass developments in drinking water systems, but its phylogeny and physiology remained unknown. We show that C. polyspora is a gammaproteobacterium closely related to methanotrophs and capable of oxidizing methane. We discovered that C. polyspora encodes a phylogenetically very unusual particulate methane monooxygenase whose expression is strongly increased in the presence of methane. Our findings demonstrate a previously unrecognized complexity of the evolutionary history and cell biology of methane-oxidizing bacteria.

Mono Lake is an alkaline hypersaline lake that supports high methane oxidation rates. Retrieved pmoA sequences showed a broad diversity of aerobic methane oxidizers including the type I methanotrophs Methylobacter (the dominant genus), Methylomicrobium, and Methylothermus, and the type II methanotroph Methylocystis. Stratification of Mono Lake resulted in variation of aerobic methane oxidation rates with depth. Methanotroph diversity as determined by analysis of pmoA using new denaturing gradient gel electrophoresis primers suggested that variations in methane oxidation activity may correlate with changes in methanotroph community composition.

All aerobic methanotrophic bacteria described to date are unable to grow on substrates containing carbon-carbon bonds. Here we demonstrate that members of the recently discovered genus Methylocella are an exception to this. These bacteria are able to use as their sole energy source the one-carbon compounds methane and methanol, as well as the multicarbon compounds acetate, pyruvate, succinate, malate, and ethanol. To conclusively verify facultative growth, acetate and methane were used as model substrates in growth experiments with the type strain Methylocella silvestris BL2. Quantitative real-time PCR targeting the mmoX gene, which encodes a subunit of soluble methane monooxygenase, showed that copies of this gene increased in parallel with cell counts during growth on either acetate or methane as the sole substrate. This verified that cells possessing the genetic basis of methane oxidation grew on acetate as well as methane. Cloning of 16S rRNA genes and fluorescence in situ hybridization with strain-specific and genus-specific oligonucleotide probes detected no contaminants in cultures. The growth rate and carbon conversion efficiency were higher on acetate than on methane, and when both substrates were provided in excess, acetate was preferably used and methane oxidation was shut down. Our data demonstrate that not all methanotrophic bacteria are limited to growing on one-carbon compounds. This could have major implications for understanding the factors controlling methane fluxes in the environment.

PCR amplification, restriction fragment length polymorphism, and phylogenetic analysis of oxygenase genes were used for the characterization of in situ methane- and ammonia-oxidizing bacteria from free-living and attached communities in the Eastern Snake River Plain aquifer. The following three methane monooxygenase (MMO) PCR primer sets were used: A189-A682, which amplifies an internal region of both the pmoA gene of the MMO particulate form and the amoA gene of ammonia monooxygenase; A189-mb661, which specifically targets the pmoA gene; and mmoXA-mmoXB, which amplifies the mmoX gene of the MMO soluble form (sMMO). Whole-genome amplification (WGA) was used to amplify metagenomic DNA from each community to assess its applicability for generating unbiased metagenomic template DNA. The majority of sequences in each archive were related to oxygenases of type II-like methanotrophs of the genus Methylocystis. A small subset of type I sequences found only in free-living communities possessed oxygenase genes that grouped nearest to Methylobacter and Methylomonas spp. Sequences similar to that of the amoA gene associated with ammonia-oxidizing bacteria (AOB) most closely matched a sequence from the uncultured bacterium BS870 but showed no substantial alignment to known cultured AOB. Based on these functional gene analyses, bacteria related to the type II methanotroph Methylocystis sp. were found to dominate both free-living and attached communities. Metagenomic DNA amplified by WGA showed characteristics similar to those of unamplified samples. Overall, numerous sMMO-like gene sequences that have been previously associated with high rates of trichloroethylene cometabolism were observed in both free-living and attached communities in this basaltic aquifer.

Diversity and community structure of aerobic methane-oxidizing bacteria in the littoral sediment of Lake Constance was investigated by cloning analysis and terminal restriction fragment length polymorphism (T-RFLP) fingerprinting of the pmoA gene. Phylogenetic analysis revealed a high diversity of type I and type II methanotrophs in the oxygenated uppermost centimeter of the sediment. T-RFLP profiles indicated a high similarity between the active methanotrophic community in the oxic layer and the inactive community in an anoxic sediment layer at a 10-cm depth. There were also no major changes in community structure between littoral sediment cores sampled in summer and winter. By contrast, the fingerprint patterns showed substantial differences between the methanotrophic communities of littoral and profundal sediments.

Methane oxidation in soils is mostly accomplished by methanotrophic bacteria. Little is known about the abundance of methanotrophs in soils, since quantification by cultivation and microscopic techniques is cumbersome. Comparison of 16S ribosomal DNA and pmoA (alpha subunit of the particulate methane monooxygenase) phylogenetic trees showed good correlation and revealed five distinct groups of methanotrophs within the alpha and gamma subclasses of Proteobacteria: the Methylococcus group, the Methylobacter/Methylosarcina group, the Methylosinus group, the Methylocapsa group, and the forest clones group (a cluster of pmoA sequences retrieved from forest soils). We developed quantitative real-time PCR assays with SybrGreen for each of these five groups and for all methanotrophic bacteria by targeting the pmoA gene. Detection limits were between 10(1) and 10(2) target molecules per reaction for all assays. Real-time PCR analysis of soil samples spiked with cells of Methylococcus capsulatus, Methylomicrobium album, and Methylosinus trichosporium recovered almost all the added bacteria. Only the Methylosinus-specific assay recovered only 20% of added cells, possibly due to a lower lysis efficiency of type II methanotrophs. Analysis of the methanotrophic community structure in a flooded rice field soil showed (5.0 +/- 1.4) x 10(6) pmoA molecules g(-1) for all methanotrophs. The Methylosinus group was predominant (2.7 x 10(6) +/- 1.1 x 10(6) target molecules g(-1)). In addition, bacteria of the Methylobacter/Methylosarcina group were abundant (2.0 x 10(6) +/- 0.9 x 10(6) target molecules g of soil(-1)). On the other hand, pmoA affiliated with the forest clones and the Methylocapsa group was below the detection limit of 1.9 x 10(4) target molecules g of soil(-1). Our results showed that pmoA-targeted real-time PCR allowed fast and sensitive quantification of the five major groups of methanotrophs in soil. This approach will thus be useful for quantitative analysis of the community structure of methanotrophs in nature.

X-band (9.1 GHz) and S-band (3.4 GHz) electron paramagnetic resonance (EPR) spectra for particulate methane monooxygenase (pMMO) in whole cells from Methylococcus capsulatus (Bath) grown on (63)Cu and (15)N were obtained and compared with previously reported spectra for pMMO from Methylomicrobium album BG8. For both M. capsulatus (Bath) and M. album BG8, two nearly identical Cu(2+) EPR signals with resolved hyperfine coupling to four nitrogens are observed. The EPR parameters for pMMO from M. capsulatus (Bath) (g( parallel) = 2.244, A( parallel) = 185 G, and A(N) = 19 G for signal one; g( parallel) = 2.246, A( parallel) = 180 G, and A(N) = 19 G for signal two) and for pMMO from M. album BG8 (g( parallel) = 2.243, A( parallel) = 180 G, and A(N) = 18 G for signal one; g( parallel) = 2. 251, A( parallel) = 180 G, and A(N) = 18 G for signal two) are very similar and are characteristic of type 2 Cu(2+) in a square planar or square pyramidal geometry. In three-pulse electron spin echo envelope modulation (ESEEM) data for natural-abundance samples, nitrogen quadrupolar frequencies due to the distant nitrogens of coordinated histidine imidazoles were observed. The intensities of the quadrupolar combination bands indicate that there are three or four coordinated imidazoles, which implies that most, if not all, of the coordinated nitrogens detected in the continuous wave spectra are from histidine imidazoles.

The soluble methane monooxygenase (sMMO; EC 1.14.13.25) from the pseudothermophile Methylococcus capsulatus (Bath) is a three-component enzyme system that catalyzes the selective oxidation of methane to methanol. We have used NMR spectroscopy to produce a highly refined structure of MMOB, the 16-kDa regulatory protein of this system. This structure has a unique and intricate fold containing seven beta-strands forming two beta-sheets oriented perpendicular to each other and bridged by three alpha-helices. The rate and efficiency of the methane hydroxylation by sMMO depend on dynamic binding interactions of the hydroxylase with the reductase and regulatory protein components during catalysis. We have monitored by NMR the binding of MMOB to the hydroxylase in the presence and absence of the reductase. The results of these studies provide structural insight into how the regulatory protein interacts with the hydroxylase.

Background: The soluble methane monooxygenase (sMMO) system in methanotrophic bacteria uses three protein components to catalyze the selective oxidation of methane to methanol. The coupling protein B (MMOB) both activates the carboxylate-bridged diiron center in the hydroxylase (MMOH) for substrate oxidation and couples the reaction to electron transfer from NADH through the sMMO reductase. Although the X-ray structure of the hydroxylase is known, little structural information is available regarding protein B., Results: Wild-type protein B from Methylococcus capsulatus (Bath) is very susceptible to degradation. The triple mutant protein B, Gly10-->Ala, Gly13-->Gln, Gly16-->Ala is resistant to degradation. Analyzing wild-type and mutant forms of protein B using size exclusion chromatography and circular dichroism spectroscopy suggests that the amino terminus of MMOB (Ser1-Ala25) is responsible for the proteolytic sensitivity and unusual mobility of the protein. We used the stable triple glycine protein B mutant to generate an affinity column for the hydroxylase and investigated the interaction between MMOH and MMOB. These results suggest the interaction is dominated by hydrophobic contacts., Conclusions: A structural model is presented for protein B that explains both its proclivity for degradation and its anomalous behavior during size exclusion chromatography. The model is consistent with previously published biophysical data, including the NMR structure of the phenol hydroxylase regulatory protein P2. Furthermore, this model allows for detailed and testable predictions about the structure of protein B and the role of proposed recognition sites for the hydroxylase.

Genes for the subunits of particulate methane monooxygenase, PmoABC, have been sequenced from the gamma-proteobacterial methanotroph Methylococcus capsulatus Bath. M. capsulatus Bath contains two complete copies of pmoCAB, as well as a third copy of pmoC. The two pmoCAB regions were almost identical at the nucleotide sequence level, differing in only 13 positions in 3183 bp. At the amino acid level, each translated gene product contained only one differing residue in each copy. However, the pmoC3 sequence was more divergent from the two other pmoC copies at both the far N-terminus and far C-terminus. Chromosomal insertion mutations were generated in all seven genes. Null mutants could not be obtained for pmoC3, suggesting that it may play an essential role in growth on methane. Null mutants were obtained for pmoC1, pmoC2, pmoA1, pmoA2, pmoB1 and pmoB2. All of these mutants grew on methane, demonstrating that both gene copies were functional. Copy 1 mutants showed about two-thirds of the wild-type whole-cell methane oxidation rate, while copy 2 mutants showed only about one-third of the wild-type rate, indicating that both gene copies were necessary for wild-type particulate methane monooxygenase activity. It was not possible to obtain double null mutants that were defective in both pmo copies, which may indicate that some expression of pMMO is important for growth.

Radical clock substrate probes were used to assess the viability of a discrete substrate radical species in the mechanism of hydrocarbon oxidation by the soluble methane monooxygenase (sMMO) from Methylococcus capsulatus (Bath). New substituted cyclopropane probes were used with very fast ring-opening rate constants and other desirable attributes, such as the ability to discriminate between radical and cationic intermediates. Oxidation of these substrates by a reconstituted sMMO system resulted in no rearranged products, allowing an upper limit of 150 fs to be placed on the lifetime of a putative radical species. This limit strongly suggests that there is no such substrate radical intermediate. The two enantiomers of trans-1-methyl-2-phenyl-cyclopropane were prepared, and the regioselectivity of their oxidation to the corresponding cyclopropylmethanol and cyclopropylphenol products was determined. The results are consistent with selective orientation of the two enantiomeric substrates in the hydrophobic cavity at the active site of sMMO, specific models for which were examined by molecular modeling.

EPR spectra were obtained for the type 2 Cu2+ site in particulate methane monooxygenase (pMMO) from Methylomicrobium album BG8 grown on K15NO3 and 63Cu(NO3)2. The concentration of the type 2 Cu2+ signal was approximately 200 microM per 25 mg/ml protein in packed cells and membrane fractions, a concentration that is consistent with its attribution to pMMO, and the EPR parameters were consistent with electron paramagnetic resonance (EPR) parameters previously assigned to pMMO. The superhyperfine structure due to nitrogen is better resolved because I = 1/2 for 15N whereas I = 1 for 14N and A(15N)/A(14N) = 1.4. Under these conditions, superhyperfine structure is resolved in the g region of the X-band spectrum. At low microwave frequency (S-band) the resolution of the nitrogen superhyperfine structure improves. Signals are attributed to type 2 Cu2+ in which cupric ion is bound to four (less likely three) nitrogen donor atoms.

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.

We attempted to mimic in small upflow anaerobic sludge bed (UASB) bioreactors the metabolic association found in nature between methanogens and methanotrophs. UASB bioreactors were inoculated with pure cultures of methanotrophs, and the bioreactors were operated by using continuous low-level oxygenation in order to favor growth and/or survival of methanotrophs. Unlike the reactors in other similar studies, the hybrid anaerobic-aerobic bioreactors which we used were operated synchronously, not sequentially. Here, emphasis was placed on monitoring various methanotrophic populations by using classical methods and also a PCR amplification assay based on the mmoX gene fragment of the soluble methane monooxygenase (sMMO). The following results were obtained: (i) under the conditions used, Methylosinus sporium appeared to survive better than Methylosinus trichosporium; (ii) the PCR method which we used could detect as few as about 2,000 sMMO gene-containing methanotrophs per g (wet weight) of granular sludge; (iii) inoculation of the bioreactors with pure cultures of methanotrophs contributed greatly to increases in the sMMO-containing population (although the sMMO-containing population decreased gradually with time, at the end of an experiment it was always at least 2 logs larger than the initial population before inoculation); (iv) in general, there was a good correlation between populations with the sMMO gene and populations that exhibited sMMO activity; and (v) inoculation with sMMO-positive cultures helped increase significantly the proportion of sMMO-positive methanotrophs in reactors, even after several weeks of operation under various regimes. At some point, anaerobic-aerobic bioreactors like those described here might be used for biodegradation of various chlorinated pollutants.

An 8.6 kDa protein, which the authors call a modifin, has been purified from Methylococcus capsulatus (Bath) and has been shown to alter the substrate specificity and kinetics of NAD+-linked formaldehyde dehydrogenase (FDH) isolated from the same organism. Purification methods for both the modifin and FDH are presented which reliably produced pure protein for further analysis. Analysis of the molecular mass and N-terminal sequence of both FDH and the modifin indicate that they are unique proteins and show no similarity to alcohol or aldehyde dehydrogenase enzymes isolated from methylotrophic bacteria. Substrate specificity studies demonstrated that FDH oxidized formaldehyde exclusively in the presence of the modifin; a diverse range of aldehydes and alcohols were oxidized by FDH in the absence of the modifin. No formaldehyde oxidation was detected in the absence of the modifin. Attempts to replace the modifin with glutathione or high concentrations of methanol to stimulate formaldehyde oxidation failed. With acetaldehyde as substrate, FDH showed standard Michaelis-Menten kinetics; interaction of FDH with the modifin using formaldehyde as substrate altered the kinetics of the reaction to sigmoidal. Kinetic analysis during turnover experiments indicated that the FDH may be associated with bound formaldehyde following enzyme isolation and that NAD may also be associated with the enzyme but in a form that is less tightly bound than found with the methanol dehydrogenase from Bacillus methanolicus. Data are presented which indicate that the modifin may play an important role in regulating formaldehyde concentration in vivo.

The particulate methane monooxygenase (pMMO) is known to be very difficult to study mainly due to its unusual activity instability in vitro. By cultivating Methylococcus capsulatus (Bath) under methane stress conditions and high copper levels in the growth medium, membranes highly enriched in the pMMO with exceptionally stable activity can be isolated from these cells. Purified and active pMMO can be subsequently obtained from these membrane preparations using protocols in which an excess of reductants and anaerobic conditions were maintained during membrane solubilization by dodecyl beta-D-maltoside and purification by chromatography. The pMMO was found to be the major constituent in these membranes, constituting 60-80% of total membrane proteins. The dominant species of the pMMO was found to consist of three subunits, alpha, beta, and gamma, with an apparent molecular mass of 45, 26, and 23 kDa, respectively. A second species of the pMMO, a proteolytically processed version of the enzyme, was found to be composed of three subunits, alpha', beta, and gamma, with an apparent molecular mass of 35, 26, and 23 kDa, respectively. The alpha and alpha' subunits from these two forms of the pMMO contain identical N-terminal sequences. The gamma subunit, however, exhibits variation in its N-terminal sequence. The pMMO is a copper-containing protein only and shows a requirement for Cu(I) ions. Approximately 12-15 Cu ions per 94-kDa monomeric unit were observed. The pMMO is sensitive to dioxygen tension. On the basis of dioxygen sensitivity, three kinetically distinct forms of the enzyme can be distinguished. A slow but air-stable form, which is converted into a "pulsed" state upon direct exposure to atmospheric oxygen pressure, is considered as type I pMMO. This form was the subject of our pMMO isolation effort. Other forms (types II and III) are deactivated to various extents upon exposure to atmospheric dioxygen pressure. Under inactivating conditions, these unstable forms release protons to the buffer (approximately 10 H+/94-kDa monomeric unit) and eventually become completely inactive.

The ab initio structures of 2,7,9-tricarboxypyrroloquinoline quinone (PQQ), semiquinone (PQQH), and dihydroquinone (PQQH2) have been determined and compared with ab initio structures of the (PQQ)Ca2+, (PQQH)Ca2+, and (PQQH2)Ca2+ complexes as well as the x-ray structure of (PQQ)Ca2+ bound at the active site of the methanol dehydrogenase (MDH) of methyltropic bacteria. Plausible mechanisms for the MDH oxidation of methanol involving the (PQQ)Ca2+ complex are explored via ab initio computations and discussed. Considering the reaction of methanol with PQQ in the absence of Ca2+, nucleophilic addition of methanol to the PQQ C-5 carbonyl followed by a retro-ene elimination is deemed unlikely due to large energy barrier. A much more favorable disposition of the methanol C-5 adduct to provide formaldehyde involves proton ionization of the intermediate followed by elimination of methoxide concerted with hydride transfer to the oxygen of the C-4 carbonyl. Much the same transition state is reached if one searches for the transition state beginning with Asp-303-CO2-general-base removal of the methanol proton of the (PQQ)Ca2+O(H)CH3 complex concerted with hydride transfer to the oxygen at C-4. For such a mechanism the role of the Ca2+ moiety would be to (i) contribute to the formation of the ES complex (ii) provide a modest decrease in the pKa of methanol substrate,; and (iii) polarize the oxygen at C-5.

The regulatory protein B of soluble methane monooxygenase (sMMO) from Methylococcus capsulatus (Bath), exists as a mixture of the full-length active form and truncated forms, B' and B". Electrospray ionisation mass spectrometry (ESI-MS) was used to identify a cleavage site between Met12 and Gly13, such that 12 amino acids were lost from the N-terminus of protein B. This truncate was designated B' and molecular masses were assigned to proteins B and B' of 15,852.6+/-0.4 Da and 14,629.5+/-0.3 Da, respectively. A cleavage site between Gln29 and Val30 was also identified such that 29 amino acids were lost from the N-terminus of protein B. This truncate was designated B" and had a molecular mass of 12,709.93+/-0.02 Da. Proteins B' and B" were found to be inactive in the sMMO system. Addition of protease inhibitors or the heterologous expression of protein B in various strains of lon-deficient or ompT-deficient Escherichia coli, did not inhibit B' formation. Expression of protein B as a glutathione S-transferase fusion protein and subsequent purification of protein B from E. coli using affinity chromatography resulted in preparations of protein B with higher enzyme activities than that of wild-type protein B. However, ESI-MS confirmed that protein B' was still present. Alteration of the Met12-Gly13 cleavage site to Met12-Gln13 revealed that the stability of G13Q at 20 degrees C and 37 degrees C was higher than that of wild-type preparations. ESI-MS indicated that protein B' was absent and could only be identified after prolonged incubation at room temperature. The amount of active protein B present in the cell may be controlled by protein B cleavage, thereby regulating electron transfer. Alternatively, it may allow protein B to maintain a certain conformation necessary for enzyme activity and this may control the activity of sMMO in response to the supply of methane to the cell.

Transformation yields for the aerobic cometabolic degradation of five chlorinated ethenes were determined by using a methanotrophic mixed culture expressing particulate methane monooxygenase (pMMO). Transformation yields (expressed as moles of chlorinated ethene degraded per mole of methane consumed) were 0.57, 0.25, 0.058, 0.0019, and 0.00022 for trans-1,2-dichloroethylene (t-DCE), vinyl chloride (VC), cis-1,2-dichloroethylene (c-DCE), trichloroethylene (TCE), and 1,1-dichloroethylene (1,1-DCE), respectively. Degradation of t-DCE and VC was observed only in the presence of formate or methane, sources of reducing energy necessary for cometabolism. The t-DCE and VC transformation yields represented 35 and 15%, respectively, of the theoretical maximum yields, based on reducing-energy availability from methane dissimilation to carbon dioxide, exclusive of all other processes that require reducing energy. The yields for t-DCE and VC were 20 times greater than the yields reported by others for cells expressing soluble methane monooxygenase (sMMO). Transformation yields for c-DCE, TCE, and 1,1-DCE were similar to or less than those for cultures expressing sMMO. Although methanotrophic biotreatment systems have typically been designed to incorporate cultures expressing sMMO, these results suggest that pMMO expression may be highly advantageous for degradation of t-DCE or VC. It may also be much easier to maintain pMMO expression in treatment systems, because pMMO is expressed by all methanotrophs whereas sMMO is expressed only by type II methanotrophs under copper-limited conditions.

The redox properties of the hydroxylase component of soluble methane monooxygenase from Methylococcus capsulatus (Bath) have been thoroughly investigated. Previous studies used redox indicator titrations and spectroscopic methods for the determination of the concentrations of reduced species. Herein we report, for the first time, direct electrochemistry (i.e. without the use of mediators) of the diiron centers of the hydroxylase from M. capsulatus (Bath) at a modified gold electrode giving rise to two waves at 4(+/- 10) mV and -386(+/- 14) mV versus saturated calomel electrode (SCE). In addition, the effects of proteins B and B' on the redox reactions were determined. The redox potentials of the complex with protein B are -25(+/- 14) mV and -433(+/- 8) mV versus SCE whereas protein B' had no effect though it did alter the effect of protein B on the redox potentials.

Cytochrome c' was isolated from the obligate methylotroph Methylococcus capsulatus Bath. The native and subunit molecular masses of the cytochrome were 34.9 kDa and 16.2 kDa, respectively, with an isoelectric pH of 7.0. The amino acid composition and N-terminal amino acid sequence were consistent with identification of the protein as a cytochrome c'. The electron paramagnetic resonance spectrum of the monoheme cytochrome indicated the presence of a high spin, S = 5/2, heme center that is diagnostic of cytochromes c'. The optical absorption spectra of ferric or ferrous cytochrome c' were also characteristic of cytochromes c'. The ferrocytochrome bound carbon monoxide and nitric oxide, but not isocyanide, cyanide, or azide. Changes in physical properties due to binding of CO or NO to some other c'-type cytochromes have been interpreted as an indication of dimer dissociation. In the case of cytochrome c' from M. capsulatus Bath, analytical ultracentrifugation of the ferricytochrome, the ferrocytochrome, and the ferrocytochrome-CO complex indicate that the changes induced by binding of CO are conformational and are not consistent with dimer dissociation. EPR spectra show that cytochrome c' was reduced in the presence of hydroxylamine only when in a complex with cytochrome P-460. The value of the midpoint potential, Em 7.0, was -250 mV for cytochrome c' from M. capsulatus Bath, which is well below the range of values reported for other cytochromes c'. The values of midpoint potentials for cytochrome P-460 (Em 7.0 = -300 mV to -380 mV) and cytochrome C555 (Em 7.0 = +175 mV to +195 mV) are less than and greater than, respectively, the value for cytochrome c' and suggest the possibility that the latter may function as an electron shuttle between cytochrome P-460 and cytochrome C555.

Methane is oxidized to methanol by the enzyme methane mono-oxygenase (MMO) in methanotrophic bacteria. In previous work, this multicomponent enzyme system has been extensively characterized at the biochemical and molecular level. Copper ions have been shown to irreversibly inhibit MMO activity in vivo and in vitro, but the effect of copper ions on transcription of the genes encoding the soluble (cytoplasmic) MMO (sMMO) has not previously been investigated. To examine more closely the regulation of bacterial methane oxidation and to determine the role of copper in this process, we have investigated transcriptional regulation of the sMMO gene cluster in the methanotrophic bacterium Methylococcus capsulatus (Bath). Using Northern blot analysis and primer extension experiments, it was shown that the six ORFs of the sMMO gene cluster are organized as an operon and the transcripts produced upon expression of this operon have been identified. The synthesis of these transcripts was under control of a single copper-regulated promoter, which is as yet not precisely defined.

A method for reducing RNA and DNA in the bacterium Methylococcus capsulatus (Bath) has been developed. Endogenous RNase and DNase were activated by a 10 s heat shock at 90 degrees C. Cells were then incubated at 60 degrees C for 20 min to allow degradation of the nucleic acids. The optimum pH for the process was 7.0. The protein loss was less than 10% and occurred during the initial heat shock. No protein loss was found during incubation. The total dry-weight loss in connection with an 80% reduction of the nucleic acid content was 20%-25%, giving a final product with a raw protein content of approximately 75%. Reduction of both RNA and DNA was inhibited by CuSO4 and ZnSO4. DNA reduction was stimulated by other minerals. Optimal stimulation was found at 1 mM FeSO4. Reduction of RNA was not increased by any of the minerals tested.

An active preparation of the membrane-associated methane monooxygenase (pMMO) from Methylococcus capsulatus Bath was isolated by ion-exchange and hydrophobic interaction chromatography using dodecyl beta-D-maltoside as the detergent. The active preparation consisted of three major polypeptides with molecular masses of 47,000, 27,000, and 25,000 Da. Two of the three polypeptides (those with molecular masses of 47,000 and 27,000 Da) were identified as the polypeptides induced when cells expressing the soluble MMO are switched to culture medium in which the pMMO is expressed. The 27,000-Da polypeptide was identified as the acetylene-binding protein. The active enzyme complex contained 2.5 iron atoms and 14.5 copper atoms per 99,000 Da. The electron paramagnetic resonance spectrum of the enzyme showed evidence for a type 2 copper center (g perpendicular = 2.057, g parallel = 2.24, and magnitude of A parallel = 172 G), a weak high-spin iron signal (g = 6.0), and a broad low-field (g = 12.5) signal. Treatment of the pMMO with nitric oxide produced the ferrous-nitric oxide derivative observed in the membrane fraction of cells expressing the pMMO. When duroquinol was used as a reductant, the specific activity of the purified enzyme was 11.1 nmol of propylene oxidized.min-1.mg of protein-1, which accounted for approximately 30% of the cell-free propylene oxidation activity. The activity was stimulated by ferric and cupric metal ions in addition to the cytochrome b-specific inhibitors myxothiazol and 2-heptyl-4-hydroxyquinoline-N-oxide.

A fluorescence method is described for direct measurement of the interaction between methanol dehydrogenase (MDH) and its electron acceptor cytochrome cL. This has permitted a distinction to be made between factors affecting electron transfer and those affecting the initial binding or docking process. It was confirmed that the initial interaction is electrostatic, but previous conclusions with respect to the mechanism of EDTA inhibition have been modified. It is proposed that the initial 'docking' of MDH and cytochrome cL is by way of ionic interactions between lysyl residues on its surface and carboxylate groups on the surface of cytochrome cL. This interaction is not inhibited by EDTA, which we suggest acts by binding to nearby lysyl residues, thus preventing movement of the 'docked' cytochrome to its optimal position for electron transfer, which probably involves interaction with the hydrophobic funnel in the surface of MDH.

Methanotrophic bacteria were isolated from a natural environment potentially favorable to heat-tolerant methanotrophs. An improved colony plate assay was developed and used to identify putative methanotrophic colonies with high confidence. Fourteen new isolates were purified and partially characterized. These new isolates exhibit a DNA sequence homology of up to 97% with the conserved regions in the mmoX and mmoC genes of the soluble methane monooxygenase (MMO)-coding gene cluster of Methylococcus capsulatus Bath. The copper regulation of soluble MMO expression in the same isolates, however, differs from that of M. capsulatus Bath, as the new isolates can tolerate up to 0.8 microM copper without loss of MMO activity while a drastic reduction of MMO activity occurs already at 0.1 microM copper in M. capsulatus Bath. The isolates can be cultivated and utilized at elevated temperatures, and their copper- and heat-tolerant MMO activity makes these bacteria ideal candidates for future biotechnological use.

A 45-kDa membrane polypeptide that is associated with activity of the particulate methane monooxygenase (pMMO) has been purified from three methanotrophic bacteria, and the N-terminal amino acid sequence was found to be identical in 17 of 20 positions for all three polypeptides and identical in 14 of 20 positions for the N terminus of AmoB, the 43-kDa subunit of ammonia monooxygenase. DNA from a variety of methanotrophs was screened with two probes, an oligonucleotide designed from the N-terminal sequence of the 45-kDa polypeptide from Methylococcus capsulatus Bath and an internal fragment of amoA, which encodes the 27-kDa subunit of ammonia monooxygenase. In most cases, two hybridizing fragments were identified with each probe. Three overlapping DNA fragments containing one of the copies of the gene encoding the 45-kDa pMMO polypeptide (pmoB) were cloned from Methylococcus capsulatus Bath. A 2.1-kb region was sequenced and found to contain both pmoB and a second gene, pmoA. The predicted amino acid sequences of these genes revealed high identity with those of the gene products of amoB and amoA, respectively. Further hybridization experiments with DNA from Methylococcus capsulatus Bath and Methylobacter albus BG8 confirmed the presence of two copies of pmoB in both strains. These results suggest that the 45- and 27-kDa pMMO-associated polypeptides of methanotrophs are subunits of the pMMO and are present in duplicate gene copies in methanotrophs.

We designed PCR primers by using the DNA sequences of the soluble methane monooxygenase gene clusters of Methylosinus trichosporium OB3b and Methylococcus capsulatus (Bath), and these primers were found to be specific for four of the five structural genes in the soluble methane monooxygenase gene clusters of several methanotrophs. We also designed primers for the gram-negative methylotroph-specific methanol dehydrogenase gene moxF. The specificity of these primers was confirmed by hybridizing and sequencing the PCR products obtained. The primers were then used to amplify methanotroph DNAs in samples obtained from various aquatic and terrestrial environments. Our sequencing data suggest that a large number of different methanotrophs are present in peat samples and also that there is a high level of variability in the mmoC gene, which codes for the reductase component of the soluble methane monooxygenase, while the mmoX gene, which codes for the alpha subunit of the hydroxylase component of this enzyme complex, appears to be highly conserved in methanotrophs.

The data from the deuterium isotope experiments in this study show that the primary kinetic isotope effect for methane oxidation catalysed by soluble methane monooxygenase from Methylococcus capsulatus (Bath) is very small, < 2. In contrast, the primary kinetic isotope effect for -CH3 group oxidation in toluene is large, > 7. A mechanistic pathway in which a substrate radical is formed from hydrogen atom abstraction by a ferryl species is believed to operate for CH4, the toluene -CH3 group and similar alkanes. Direct oxygen atom addition, rather than H atom abstraction, is indicated for aromatic ring oxidations in benzene and toluene and for styrene oxide formation from styrene. Thus, more than one mechanistic pathway appears to operate in soluble methane-monooxygenase-catalysed reactions and, in some cases, the pathway chosen may be dictated by the substrate. In the soluble methane-monooxygenase-catalysed oxidation of toluene the rates of: (a) substrate dissociation from the enzyme-substrate complex, (b) product formation and (c) product release (benzyl alcohol and p-cresol) from the enzyme-product complex are comparable in magnitude. Therefore all three of these steps are partially rate-determining in the soluble methane monooxygenase catalytic cycle for toluene oxidation.

An enzyme capable of the oxidation of hydroxylamine to nitrite was isolated from the obligate methylotroph Methylococcus capsulatus Bath. The absorption spectra in cell extracts, electron paramagnetic resonance spectra, molecular weight, covalent attachment of heme group to polypeptide, and enzymatic activities suggest that the enzyme is similar to cytochrome P-460, a novel iron-containing protein previously observed only in Nitrosomonas europaea. The native and subunit molecular masses of the M. capsulatus Bath protein were 38,900 and 16,390 Da, respectively; the isoelectric point was 6.98. The enzyme has approximately one iron and one copper atom per subunit. The electron paramagnetic resonance spectrum of the protein showed evidence for a high-spin ferric heme. In contrast to the enzyme from N. europaea, a 13-nm blue shift in the soret band of the ferrocytochrome (463 nm in cell extracts to 450 nm in the final sample) occurred during purification. The amino acid composition and N-terminal amino acid sequence of the enzyme from M. capsulatus Bath was similar but not identical to those of cytochrome P-460 of N. europaea. In cell extracts, the identity of the biological electron acceptor is as yet unestablished. Cytochrome c-555 is able to accept electrons from cytochrome P-460, although the purified enzyme required phenazine methosulfate for maximum hydroxylamine oxidation activity (specific activity, 366 mol of O2 per s per mol of enzyme). Hydroxylamine oxidation rates were stimulated approximately 2-fold by 1 mM cyanide and 1.5-fold by 0.1 mM 8-hydroxyquinoline.

Soluble methane monooxygenase (sMMO) from Methylosinus trichosporium OB3b can degrade many halogenated aliphatic compounds that are found in contaminated soil and groundwater. This enzyme oxidizes the most frequently detected pollutant, trichloroethylene (TCE), at least 50 times faster than other enzymes. However, slow growth of the strain, strong competition between TCE and methane for sMMO, and repression of the smmo locus by low concentrations of copper ions limit the use of this bacterium. To overcome these obstacles, the 5.5-kb smmo locus of M. trichosporium OB3b was cloned into a wide-host-range vector (to form pSMMO20), and this plasmid was electroporated into five Pseudomonas strains. The best TCE degradation results were obtained with Pseudomonas putida F1/pSMMO20. The plasmid was maintained stably, and all five of the sMMO proteins (alpha, beta, and gamma hydroxylase proteins, reductase, and component B) were observed clearly by both sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western immunoblotting. TCE degradation rates were quantified for P. putida F1/pSMMO20 with a gas chromatograph (Vmax = 5 nmol per min per mg of protein), and the recombinant strain mineralized 55% of the TCE (10 microM) as indicated by measuring chloride ion concentrations with a chloride ion-specific electrode. The maximum TCE degradation rate obtained with the recombinant strain was lower than that of M. trichosporium OB3b but greater than other TCE-degrading recombinants and most well-studied pseudomonads. In addition, this recombinant strain mineralizes chloroform (a specific substrate for sMMO), grows much faster than M. trichosporium OB3b, and degrades TCE without competitive inhibition from the growth substrate.

It is shown that the particulate methane monooxygenase (pMMO) has an obligate requirement for copper. The MMO activity in the particulate fractions obtained from Methylococcus capsulatus (Bath) cells is found to increase with increasing copper content of the membranes. The enzyme activity from membranes obtained from cells grown at low copper levels can be stimulated further by the addition of Cu(II) ions to the assay medium. The membrane-bound copper ions can exist in both Cu(II) and Cu(I) forms. EPR and magnetic susceptibility characterizations of the membranes indicate the presence of an exchange-coupled trinuclear Cu(II) cluster when the bulk of the copper ions is oxidized. However, the functional form of the enzyme is the reduced or partially reduced form. The copper ions in the membrane fractions as isolated often exhibit a high level of reduction. An EPR spectrum with one unpaired electron spin delocalized over three copper nuclei has been observed for the two-electron reduced trinuclear copper cluster. The high correlation between the copper level in the membranes and enzymatic activity as well as the high reactivity of the reduced copper clusters toward dioxygen strongly indicate that the membrane-bound copper ions constitute the active sites of the pMMO.

We have cloned the homoserine dehydrogenase genes (hom) from the gram-negative obligate methylotrophs Methylobacillus glycogenes ATCC 21276 and ATCC 21371 by complementation of an Escherichia coli homoserine dehydrogenase-deficient mutant. The 4.15-kb DNA fragment cloned from M. glycogenes ATCC 21371 also complemented an E. coli threonine synthase-deficient mutant, suggesting the DNA fragment contained the thrC gene in addition to the hom gene. The homoserine dehydrogenases expressed in the E. coli recombinants were hardly inhibited by L-threonine, L-phenylalanine, or L-methionine. However, they became sensitive to the amino acids after storage at 4 degrees C for 4 days as in M. glycogenes. The structures of the homoserine dehydrogenases overexpressed in E. coli were thought to be different from those in M. glycogenes, probably in subunit numbers of the enzyme, and were thought to have converted to the correct structures during the storage. The nucleotide sequences of the hom and thrC genes were determined. The hom genes of M. glycogenes ATCC 21276 and ATCC 21371 encode peptides with M(r)s of 48,225 and 44,815, respectively. The thrC genes were located 50 bp downstream of the hom genes. The thrC gene of ATCC 21371 encodes a peptide with an M(r) of 52,111, and the gene product of ATCC 21276 was truncated. Northern (RNA) blot analysis suggests that the hom and thrC genes are organized in an operon. Significant homology between the predicted amino acid sequences of the hom and thrC genes and those from other microorganisms was found.