Yuri A. Trotsenko

Methylocella palustris gen. nov., sp. nov., a new methane-oxidizing acidophilic bacterium from peat bogs, representing a novel subtype of serine-pathway methanotrophs.

A new genus, Methylocella, and a new species, Methylocella palustris, are proposed for three strains of methane-oxidizing bacteria isolated from acidic Sphagnum peat bogs. These bacteria are aerobic, Gram-negative, colourless, non-motile, straight and curved rods that utilize the serine pathway for carbon assimilation, multiply by normal cell division and contain intracellular poly-beta-hydroxybutyrate granules (one at each pole). These strains use methane and methanol as sole sources of carbon and energy and are moderately acidophilic organisms with growth between pH 4.5 and pH 7.0, the optimum being at pH 5.0-5.5. The temperature range for growth is 10-28 degrees C with the optimum at 15-20 degrees C. The intracytoplasmic membrane system is different from those of type I and II methanotrophs. Cells contain an extensive periplasmic space and a vesicular membrane system connected to the cytoplasmic membrane. The strains grew only on media with a low salt content (0.2-0.5 g l(-1)). All three strains were found to possess soluble methane monooxygenase and are able to fix atmospheric nitrogen via an oxygen-sensitive nitrogenase. No products were observed in a PCR with particulate methane monooxygenase-targeted primers; hybridization with a pmoA probe was also negative. The major phospholipid fatty acids are 18:1 acids. The G+C content of the DNA is 61.2 mol%. The three strains share identical 16S rRNA gene sequences and represent a novel lineage of methane-oxidizing bacteria within the alpha-subclass of the class Proteobacteria and are only moderately related to type II methanotrophs of the Methylocystis-Methylosinus group. The three strains are most closely related to the acidophilic heterotrophic bacterium Beijerinckia indica subsp. indica (96.5% 16S rDNA sequence similarity). Collectively, these strains comprise a new species and genus Methylocella palustris gen. nov., sp. nov.; strain KT (= ATCC 700799T) is the type strain.

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.

Methylocapsa acidiphila gen. nov., sp. nov., a novel methane-oxidizing and dinitrogen-fixing acidophilic bacterium from Sphagnum bog

A novel genus and species, Methylocapsa acidiphila gen. nov., sp. nov., are proposed for a methane-oxidizing bacterium isolated from an acidic Sphagnum peat bog. This bacterium, designated strain B2T, represents aerobic, gram-negative, colourless, non-motile, curved coccoids that form conglomerates covered by an extracellular polysaccharide matrix. The cells use methane and methanol as sole sources of carbon and energy and utilize the serine pathway for carbon assimilation. Strain B2T is a moderately acidophilic organism with growth between pH 4.2 and 7.2 and at temperatures from 10 to 30 degrees C. The cells possess a well-developed system of intracytoplasmic membranes (ICM) packed in parallel on only one side of the cell membrane. This type of ICM structure represents a novel arrangement, which was termed type III. The resting cells are Azotobacter-type cysts. Strain B2T is capable of atmospheric nitrogen fixation; it possesses particulate methane monooxygenase and does not express soluble methane monooxygenase. The major phospholipid fatty acid is 18:1omega7c and the major phospholipids are phosphatidylglycerols. The G+C content of the DNA is 63.1 mol%. This bacterium belongs to the alpha-subclass of the Proteobacteria and is most closely related to the acidophilic methanotroph Methylocella palustris KT (97.3% 16S rDNA sequence similarity). However, the DNA-DNA hybridization value between strain B2T and Methylocella palustris K(T) is only 7%. Thus, strain B2T is proposed to comprise a novel genus and species, Methylocapsa acidiphila gen. nov., sp. nov. Strain B2T (= DSM 13967T = NCIMB 13765T) is the type strain.

Highly efficient methane biocatalysis revealed in a methanotrophic bacterium

Methane is an essential component of the global carbon cycle and one of the most powerful greenhouse gases, yet it is also a promising alternative source of carbon for the biological production of value-added chemicals. Aerobic methane-consuming bacteria (methanotrophs) represent a potential biological platform for methane-based biocatalysis. Here we use a multi-pronged systems-level approach to reassess the metabolic functions for methane utilization in a promising bacterial biocatalyst. We demonstrate that methane assimilation is coupled with a highly efficient pyrophosphate-mediated glycolytic pathway, which under oxygen limitation participates in a novel form of fermentation-based methanotrophy. This surprising discovery suggests a novel mode of methane utilization in oxygen-limited environments, and opens new opportunities for a modular approach towards producing a variety of excreted chemical products using methane as a feedstock.

Biology of extremophilic and extremotolerant methanotrophs

Abstract. This review summarizes recent findings on the biology of obligate methanotrophic bacteria living in various extreme environments. By using molecular ecology techniques, it has become clear that obligate methanotrophs are ubiquitous in nature and well adapted to high or low temperature, pH and salinity. The isolation and characterization of pure cultures has led to the discovery of several new genera and species of extremophilic/tolerant methanotrophs. Their major physiological role is participation in the methane cycle and supplying C1 intermediates and various metabolites to other members of microbial communities in extreme ecosystems. To survive under extreme conditions, methanotrophs have developed diverse structure-function adaptive mechanisms including cell-surface layer formation, changes in cellular phospholipid composition and de novo synthesis of organic osmolytes such as ectoine, 5-oxoproline and sucrose. However, despite the above advances, basic knowledge of other stress protectants, as well as bioenergetic and genetic aspects of methanotroph adaptation, is still lacking. This information is necessary for better understanding the molecular mechanisms underlying the versatility of methanotrophs and for the development of novel biotechnological processes.

Osmoadaptation in halophilic and alkaliphilic methanotrophs

Abstract By using 1H- and 13C-NMR spectroscopy, an accumulation of sucrose and two cyclic amino acids [ectoine (1,4,5,6-tetrahydro-2-methyl-4-pyrimidine carboxylic acid) and 5-oxoproline (pyrrolidone carboxylic acid)] was detected in the halotolerant methanotrophs Methylobacter alcaliphilus 20Z and Methylobacter modestohalophilus 10S. The organic solute pool was found to increase upon raising the NaCl concentration. In M. alcaliphilus 20Z, the intracellular level of the total solutes was shown to be sufficient to balance the osmotic pressure of the medium, whereas in M. modestohalophilus 10S their content was several times lower. Additionally, phosphatidylglycerol and phosphatidylcholine were predominant cell phospholipids in salt-adapted M. alcaliphilus 20Z. However, no phosphatidylcholine was found in M. modestohalophilus 10S, and the portion of phosphatidylglycerol increased while phosphatidylethanolamine decreased upon elevated external NaCl concentrations. Regularly arranged glycoprotein surface layers (S-layers) of hexagonal and linear (p2) symmetry were observed on the outer cell walls of M. alcaliphilus 20Z and M. modestohalophilus 10S. The S-layer in M alcaliphilus 20Z consisting of tightly packed, cup-shaped subunits was lost during growth at pH 7.2 (the lowest possible pH) in the absence of NaCl. Hence, osmoadaptation in the methanotrophs studied involves structure/function alterations of cell envelopes and changes in the chemical composition of membranes as well as de novo synthesis of compatible solutes.

Isolation and characterization of halotolerant alkaliphilic methanotrophic bacteria from Tuva soda lakes

Abstract. Two strains (5Z and 20Z) of halotolerant alkaliphilic obligate methanotrophic bacteria were first isolated from moderately saline soda lakes in Tuva (Central Asia). The strains grow fastest at pH 9.0–9.5 and much more slowly at pH 7.0. No growth occurred at pH ≤ 6.8. They require NaHCO3 or NaCl for growth in alkaline medium. Gram-negative, motile rods with ordered cup-shaped cell wall structures and Type I intracytoplasmic membranes assimilate methane and methanol via the ribulose monophosphate pathway. The G + C content of strains 5Z and 20Z are 47.6 and 47.9 mol%, respectively. Based on their alkaliphilic physiology, both strains were referred to as Methylobacter alcaliphilus sp. nov. The changes in cell phospholipids, fatty acids, and amino acids have been observed upon varying salinity and pH of the medium, thus suggesting structure-function osmoadaptation of the strains studied. Whole-cell experiments revealed the salt- and pH-dependence of CH4 oxidation and assimilation rates. Cell motility was also Na+ dependent and sensitive to some energy uncouplers and ionophores.

Taxonomic Characterization of New Alkaliphilic and Alkalitolerant Methanotrophs from Soda Lakes of the Southeastern Transbaikal Region and description of Methylomicrobium buryatense sp.nov.

Five strains of obligate methanotrophic bacteria (4G, 5G, 6G, 7G and 5B) isolated from bottom sediments of Southeastern Transbaikal soda lakes (pH 9.5-10.5) are taxonomically described. These bacteria are aerobic, Gram-negative monotrichous rods having tightly packed cup-shaped structures on the outer cell wall surface (S-layers) and Type I intracytoplasmic membranes. All the isolates possess particulate methane monooxygenase (pMMO) and one strain (5G) also contains soluble methane monooxygenase (sMMO). They assimilate methane and methanol via the ribulose monophosphate pathway (RuMP). The isolates are alkalitolerant or facultatively alkaliphilic, able to grow at pH 10.5-11.0 and optimally at pH 8.5-9.5. These organisms are obligately dependent on the presence of sodium ions in the growth medium and tolerate up to 0.9-1.4 M NaCl or 1 M NaHCO3. Although being mesophilic, all the isolates are resistant to heating (80 degrees C, 20 min), freezing and drying. Their cellular fatty acids profiles primarily consist of C(16:1). The major phospholipids are phosphatidylethanolamine and phosphatidylglycerol. The main quinone is Q-8. The DNA G+C content ranges from 49.2-51.5 mol %. Comparative 16S rDNA sequencing showed that the newly isolated methanotrophs are related to membres of the Methylomicrobium genus. However, they differ from the known members of this genus by DNA-DNA relatedness. Based on pheno- and genotypic characteristics, we propose a new species of the genus Methylomicrobium Methylomicrobium buryatense sp. nov.

Characterization of the ectoine biosynthesis genes of haloalkalotolerant obligate methanotroph "Methylomicrobium alcaliphilum 20Z".

The genes involved in biosynthesis of the major compatible solute ectoine (1,4,5,6-tetrahydro-2-methylpyrimidine carboxylic acid) in halotolerant obligate methanotroph “Methylomicrobium alcaliphilum 20Z” were studied. The complete nucleotide sequences of the structural genes encoding l-aspartokinase (Ask), l-2,4-diaminobutyric acid transaminase (EctB), l-2,4-diaminobutyric acid acetyltransferase (EctA), and l-ectoine synthase (EctC) were defined and shown to be transcribed as a single operon ectABCask. Phylogenetic analysis revealed high sequence identities (34–63%) of the Ect proteins to those from halophilic heterotrophs with the highest amino acid identities being to Vibrio cholerae enzymes. The chromosomal DNA fragment from “M. alcaliphilum 20Z” containing ectABC genes and putative promoter region was expressed in Escherichia coli. Recombinant cells could grow in the presence of 4% NaCl and synthesize ectoine. The data obtained suggested that despite the ectoine biosynthesis pathway being evolutionary well conserved with respect to the genes and enzymes involved, some differences in their organization and regulation could occur in various halophilic bacteria.

Methylomonas scandinavica sp. nov., a new methanotrophic psychrotrophic bacterium isolated from deep igneous rock ground water of Sweden

Methane-utilizing bacteria were enriched from deep igneous rock environments and affiliated by amplification of functional and phylogenetic gene probes. Type I methanotrophs belonging to the genera Methylomonas and Methylobacter dominated in enrichment cultures from depths below 400 m. A pure culture of an obligate methanotroph (strain SR5) was isolated and characterized. Pink-pigmented motile rods of the new isolate contained intracytoplasmic membranes as stacks of vesicles, assimilated methane via the ribulose monophosphate pathway and had an incomplete tricarboxylic acid cycle. Phosphatidyl glycerol, methylene ubiquinone and cytochrome c552 were prevailing. The DNA G+C content is 53.3 mol %. Strain SR5 grew at temperatures between 5 and 30 degrees C with optimum at 15 degrees C, close to its in situ temperature. Analyses of 16S rRNA gene, whole cell protein, enzymatic and physiological analyses of strain SR-5 revealed significant differences compared to the other representatives of Type I methanotrophs. Based on pheno- and genotypic characteristics we propose to refer the strain SR5 as to a new species, Methylomonas scandinavica.