Surface layers of methanotrophic bacteria

Structural and functional characteristics of the regular glycoprotein layers in prokaryotes are analyzed with a special emphasis on aerobic methanotrophic bacteria. S-Layers are present at the surfaces of Methylococcus, Methylothermus, and Methylomicrobium cells. Different Methylomicrobium species either synthesize S-layers with planar (p2, p4) symmetry or form cup-shaped or conical structures with hexagonal (p6) symmetry. A unique, copper-binding polypeptide ‘CorA’/MopE (27/45 kDa), which is coexpressed with the diheme periplasmic cytochrome c peroxidase ‘CorB’/Mca (80 kDa) was found in Methylomicrobium album BG8, Methylomicrobium alcaliphilum 20Z, and Methylococcus capsulatus Bath. This tandem of the surface proteins is functionally analogous to a new siderophore: methanobactin. Importantly, no ‘CorA’/MopE homologue was found in methanotrophs not forming S-layers. The role of surface proteins in copper metabolism and initial methane oxidation is discussed.     

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-14L^T 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. Received: 2 April 1997 / Accepted: 23 July 1997     

Methanotrophic communities in the soils of Russian northern taiga and subarctic tundra

The PCR analysis of DNA extracted from soil samples taken in Russian northern taiga and subarctic tundra showed that the DNA extracts contain genes specific to methanotrophic bacteria, i.e., the mmoX gene encoding the conserved alpha-subunit of the hydroxylase component of soluble methane monooxygenase, the pmoA gene encoding the alpha-subunit of particulate methane monooxygenase, and the mxaF gene encoding the alpha-subunit of methanol dehydrogenase. PCR analysis with group-specific primers also showed that methanotrophic bacteria in the northern taiga and subarctic tundra soils are essentially represented by the type I genera Methylobacter, Methylomonas, Methylosphaera, and Methylomicrobium and that some soil samples contain type II methanotrophs close to members of the genera Methylosinus and Methylocystis. The electron microscopic examination of enrichment cultures obtained from the soil samples confirmed the presence of methanotrophic bacteria in the ecosystems studied and showed that the methanotrophs contain only small amounts of intracytoplasmic membranes.     

Biosynthesis of ectoine by the methanotrophic bacterial consortium isolated from Bogdanka coalmine (Poland)

Ectoine belongs to the family of compatible solutes, which are known to contribute mainly to the adaptation of the cell to osmotic stress by mediation of a constant turgor. In addition, the cell’s essential functions are maintained under difficult conditions like high salinity, heat, or aridity stress. Biosynthesis of ectoine has been found in halophilic and halotolerant microorganisms. We showed that the methanotrophic bacterial consortium (MBC) isolated from coalbed rocks from coalmine Bogdanka (Poland) and resistant to extreme environmental conditions (low content of moisture) was able to synthesize ectoine. MBC was cultured in mineral nitrate mineral salts medium supplied with NaCl at atmospheric air enriched with 10% of methane. The levels of methanotrophic activity were determined by the gas chromatography technique (943.05 ± 30.73 ? 94.14 ± 0.85 μM CH₄ gDW^?1 day^?1) and the biomass concentration of MBC was evaluated based on OD₆₀₀, as well as biosynthesis of ectoine in relation to the salinity (0–5% NaCl) of the medium. The levels of ectoine tested by NIR measurements ranged from 1.33 ± 0.10 mg gDW^?1 to 0.42 ± 0.08 mg gDW^?1 depending on the salinity of the solution. In addition, we identified MBC based on the pmoA gene.     

Synthesis of osmoprotectants by halophilic and alkaliphilic methanotrophs

The¹H-NMR analysis of methanol extracts of halophilic and halotolerant alkaliphilic methanotrophs isolated from the soda lakes of Southern Transbaikal and Tuva showed that bacterial cells grown at an optimum salinity accumulated mainly sucrose and 5-oxo-1-proline, whereas cells adapted to 0.5–1.0 M NaCl additionally synthesized ectoine. A more detailed study showed that nitrogen deficiency in the growth medium ofMethylobacter alcaliphilus 20Z decreased the synthesis of nitrogen-contaihing osmoprotectants, ectoine and 5-oxo-1-proline.M. alcaliphilus 20Z cells exhibited activities of UDP-glucose pyrophosphorylase and sucrose-phosphate synthase involved in sucrose synthesis. Glutamine synthetase in vitro did not require NH ₄ ⁺ ions, which implies that this enzyme is involved in 5-oxo-1-proline synthesis. Cells grown at high salinity exhibited elevated levels of aspartate kinase, aspartate-semialdehyde dehydrogenase, and ectoine synthase. This suggests that ectoine is synthesized via aspartate and aspartate-semialdehyde, i.e., via the route earlier established for extremely halophilic bacteria.     

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.Dedicated to the 70th birthday of Professor Gerhard Gottschalk who inspired our studies on methylotrophic haloalkaliphiles.     

Methanotrophic community structure and activity under warming and grazing of alpine meadow on the Tibetan Plateau

Knowledge about methanotrophs and their activities is important to understand the microbial mediation of the greenhouse gas CH₄ under climate change and human activities in terrestrial ecosystems. The effects of simulated warming and sheep grazing on methanotrophic abundance, community composition, and activity were studied in an alpine meadow soil on the Tibetan Plateau. There was high abundance of methanotrophs (1.2–3.4 × 10⁸ pmoA gene copies per gram of dry weight soil) assessed by real-time PCR, and warming significantly increased the abundance regardless of grazing. A total of 64 methanotrophic operational taxonomic units (OTUs) were obtained from 1,439 clone sequences, of these OTUs; 63 OTUs (98.4%) belonged to type I methanotrophs, and only one OTU was Methylocystis of type II methanotrophs. The methanotroph community composition and diversity were not apparently affected by the treatments. Warming and grazing significantly enhanced the potential CH₄ oxidation activity. There were significantly negative correlations between methanotrophic abundance and soil moisture and between methanotrophic abundance and NH₄–N content. The study suggests that type I methanotrophs, as the dominance, may play a key role in CH₄ oxidation, and the alpine meadow has great potential to consume more CH₄ under future warmer and grazing conditions on the Tibetan Plateau.     

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 °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.     

Measurement of the content of the osmoprotectant ectoine in methylotrophic bacteria by means of normal-phase high performance liquid chromatography

Detection and quantitative analysis of ectoine in bacterial biomass were performed by normal-phase high performance liquid chromatography with ultraviolet detection at 230 nm. Quantitative analysis was not hindered by glutamate and sucrose accumulation in bacteria. Measurement of ectoine concentration in haloalkaliphilic methanotrophs Methylobacter marinus 7C and Methylomicrobium alcaliphilum 5S showed that ectoine accumulation reached maximum (5 and 12% of dry cell weight) in the presence of NaCl at concentrations of 4 and 6%, respectively.     

Role of Nγ-Acetyldiaminobutyrate as an Enzyme Stabilizer and an Intermediate in the Biosynthesis of Hydroxyectoine

Strain CHR63 is a salt-sensitive mutant of the moderately halophilic wild-type strain Halomonas elongata DSM 3043 that is affected in the ectoine synthase gene (ectC). This strain accumulates large amounts of Nγ-acetyldiaminobutyrate (NADA), the precursor of ectoine (D. Cánovas, C. Vargas, F. Iglesias-Guerra, L. N. Csonka, D. Rhodes, A. Ventosa, and J. J. Nieto, J. Biol. Chem. 272:25794–25801, 1997). Hydroxyectoine, ectoine, and glucosylglycerate were also identified by nuclear magnetic resonance (NMR) as cytoplasmic organic solutes in this mutant. Accumulation of NADA, hydroxyectoine, and ectoine was osmoregulated, whereas the levels of glucosylglycerate decreased at higher salinities. The effect of the growth stage on the accumulation of solutes was also investigated. NADA was purified from strain CHR63 and was shown to protect the thermolabile enzyme rabbit muscle lactate dehydrogenase against thermal inactivation. The stabilizing effect of NADA was greater than the stabilizing effect of ectoine or potassium diaminobutyrate. A ¹H NMR analysis of the solutes accumulated by the wild-type strain and mutants CHR62 (ectA::Tn1732) and CHR63 (ectC::Tn1732) indicated that H. elongata can synthesize hydroxyectoine by two different pathways—directly from ectoine or via an alternative pathway that converts NADA into hydroxyectoine without the involvement of ectoine.     

Family- and genus-level 16S rRNA-targeted oligonucleotide probes for ecological studies of methanotrophic bacteria

Methanotrophic bacteria play a major role in the global carbon cycle, degrade xenobiotic pollutants, and have the potential for a variety of biotechnological applications. To facilitate ecological studies of these important organisms, we developed a suite of oligonucleotide probes for quantitative analysis of methanotroph-specific 16S rRNA from environmental samples. Two probes target methanotrophs in the family Methylocystaceae (type II methanotrophs) as a group. No oligonucleotide signatures that distinguish between the two genera in this family, Methylocystis and Methylosinus, were identified. Two other probes target, as a single group, a majority of the known methanotrophs belonging to the family Methylococcaceae (type I/X methanotrophs). The remaining probes target members of individual genera of the Methylococcaceae, including Methylobacter, Methylomonas, Methylomicrobium, Methylococcus, and Methylocaldum. One of the family-level probes also covers all methanotrophic endosymbionts of marine mollusks for which 16S rRNA sequences have been published. The two known species of the newly described genus Methylosarcina gen. nov. are covered by a probe that otherwise targets only members of the closely related genus Methylomicrobium. None of the probes covers strains of the newly proposed genera Methylocella and "Methylothermus," which are polyphyletic with respect to the recognized methanotrophic families. Empirically determined midpoint dissociation temperatures were 49 to 57 degrees C for all probes. In dot blot screening against RNA from positive- and negative-control strains, the probes were specific to their intended targets. The broad coverage and high degree of specificity of this new suite of probes will provide more detailed, quantitative information about the community structure of methanotrophs in environmental samples than was previously available.     

Methanotrophic Communities in the Soils of the Russian Northern Taiga and Subarctic Tundra

The PCR analysis of DNA extracted from soil samples taken in the Russian northern taiga and subarctic tundra showed that the DNA extracts contain genes specific to methanotrophic bacteria, i.e., the mmoX gene encoding the conserved -subunit of the hydroxylase component of soluble methane monooxygenase, the pmoA gene encoding the -subunit of particulate methane monooxygenase, and the mxaFgene encoding the -subunit of methanol dehydrogenase. PCR analysis with group-specific primers also showed that methanotrophic bacteria in the northern taiga and subarctic tundra soils are essentially represented by the type I genera Methylobacter, Methylomonas, Methylosphaera, and Methylomicrobium and that some soil samples contain type II methanotrophs close to members of the genera Methylosinus and Methylocystis. The electron microscopic examination of enrichment cultures obtained from the soil samples confirmed the presence of methanotrophic bacteria in the ecosystems studied and showed that the methanotrophs contain only small amounts of intracytoplasmic membranes.     

Characterization and phylogenetic analysis of ectoine biosynthesis genes from <Emphasis Type="Italic">Bacillus halodurans</Emphasis>

Ectoine, a cyclic tetrahydropyrimidine (2-methyl-1,4,5,6-tetrahydropyrimidine-4-carboxylic acid), is a natural compound, which serves as a protective substance in many bacterial cells. In this study, the putative ectABC gene cluster from Bacillus halodurans was heterologously expressed in E. coli and the production of ectoine was confirmed by HPLC analysis. The activity of the enzymes coded by the ectA, B and C genes were found to be higher in induced transgenic cells compared to the uninduced cells. Phylogenetic analysis revealed sequence identities ranging from 36–73% for ectA gene, 55–81% for ectB gene and 55–80% for ectC gene indicating that the enzymes are evolutionarily well conserved.     

Electroporation-Based Genetic Manipulation in Type I Methanotrophs

Methane is becoming a major candidate for a prominent carbon feedstock in the future, and the bioconversion of methane into valuable products has drawn increasing attention. To facilitate the use of methanotrophic organisms as industrial strains and accelerate our ability to metabolically engineer methanotrophs, simple and rapid genetic tools are needed. Electroporation is one such enabling tool, but to date it has not been successful in a group of methanotrophs of interest for the production of chemicals and fuels, the gammaproteobacterial (type I) methanotrophs. In this study, we developed electroporation techniques with a high transformation efficiency for three different type I methanotrophs: Methylomicrobium buryatense 5GB1C, Methylomonas sp. strain LW13, and Methylobacter tundripaludum 21/22. We further developed this technique in M. buryatense, a haloalkaliphilic aerobic methanotroph that demonstrates robust growth with a high carbon conversion efficiency and is well suited for industrial use for the bioconversion of methane. On the basis of the high transformation efficiency of M. buryatense, gene knockouts or integration of a foreign fragment into the chromosome can be easily achieved by direct electroporation of PCR-generated deletion or integration constructs. Moreover, site-specific recombination (FLP-FRT [FLP recombination target] recombination) and sacB counterselection systems were employed to perform marker-free manipulation, and two new antibiotics, zeocin and hygromycin, were validated to be antibiotic markers in this strain. Together, these tools facilitate the rapid genetic manipulation of M. buryatense and other type I methanotrophs, promoting the ability to perform fundamental research and industrial process development with these strains.     

Methanotrophic diversity in an agricultural soil as evaluated by denaturing gradient gel electrophoresis profiles of pmoA, mxaF and 16S rDNA sequences

Molecular methods were used to characterize the diversity of a methanotrophic population in an agricultural soil. For this purpose we have used DGGE analysis of functional and phylogenetic markers. Functional markers utilised comprised the pmoA-gene coding for the -subunit of the particulate methane monooxygenase (pMMO) present in all known methanotrophs and the mxaF-gene coding for the -subunit of methanol dehydrogenase (MDH) present in all Gram-negative methylotrophs. In addition, we have used 16S rDNA as a phylogenetic marker. DGGE patterns of an enrichment culture, and sequencing of major DGGE bands obtained with the bacterial specific primers showed that the community structure was dominated by methanotrophic populations related to Methylobacter sp. and Methylomicrobium sp. The PCR products amplified with the functional primer sets were related to both type I and type II methanotrophs. We also designed a new pmoA-targeting primer set which could be used in a nested protocol to amplify PCR-products from DNA extracted directly from the soil.     

Primary characterization of dominant cell surface proteins of halotolerant methanotroph <Emphasis Type="Italic">Methylomicrobium alcaliphilum</Emphasis> 20Z

Cell surface-associated proteins with molecular masses of 27 and 80 kDa found in Methylomicrobium alcaliphilum were studied. The MEALZv2_1030034 and MEALZv2_1030035 genes encoding these proteins were identified in the partially annotated genome of the methanotroph. Polypeptides MEALZv2_1030034 and MEALZv2_1030035 showed high homology (>50% identity) with the surface proteins CorA and CorB of Methylomicrobium album BG8 expressed in conditions of low copper content in the growth medium. Electron microscopic analysis with antibodies revealed localization of the 27-kDa protein in the base of the cup-shaped structures of S-layers. The mutant with an insertion in the MEALZv2_1030034 gene lost the ability to grow on the medium with methane, but grew in the presence of 0.2% methanol. In this case, the cup-shaped structures of S-layers were not bound to the cell wall but occurred as regular aggregates in the intercellular space. The 80-kDa protein was found mainly in the periplasm, had a peroxide-degrading activity, and was classified as a di-heme cytochrome c peroxidase. The mutant deficient in the MEALZv2_1030035 gene grew on methane at a high rate and had higher activities of C₁ compound oxidation enzymes than did the parent strain. The role of the proteins MEALZv2_1030034 and MEALZv2_1030035 and distribution of their homologues in other methanotrophs are discussed.     

Comparison of Aerobic Methanotrophic Communities in Littoral and Profundal Sediments of Lake Constance by a Molecular Approach

Analysis of pmoA and 16S rRNA gene clone libraries of methanotrophic bacteria in Lake Constance revealed an overall dominance of type I methanotrophs in both littoral and profundal sediments. The sediments exhibited minor differences in their methanotrophic community structures. Type X methanotrophs made up a significant part of the clone libraries only in the profundal sediment and were also found only there as a prominent peak by T-RFLP analyses.