Methylotrophic Bacteria Research Articles

Hansschlegelia beijingensis sp. nov., an aerobic, pink-pigmented, facultatively methylotrophic bacterium isolated from watermelon rhizosphere soil

A novel Gram-stain-negative, aerobic, rod-shaped strain designated PG04(T) was isolated from the rhizosphere of watermelon plants cultivated in Beijing, China. A polyphasic taxonomic study was performed on the new isolate. On the basis of 16S rRNA gene sequence similarity studies, isolate PG04(T) belonged clearly to the genus Hansschlegelia and was most closely related to Hansschlegelia zhihuaiae (97.3 % similarity to the type strain). The predominant respiratory quinone was ubiquinone 10 (Q-10) and the polar lipid profile was composed of the major lipids diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine and phosphatidylcholine. The major fatty acids were C18 : 1ω7c (41.3 %), C19 : 0 cyclo ω8c (30.6 %) and C16 : 0 (19.1 %). The G+C content of the DNA was about 64.4 mol%. DNA-DNA hybridization experiments showed 34.4 % relatedness between strain PG04(T) and H. zhihuaiae DSM 18984(T). The results of physiological and biochemical tests and differences in fatty acid profiles allowed clear phenotypic differentiation of strain PG04(T) from the most closely related species in the genus, H. zhihuaiae. Strain PG04(T) therefore represents a novel species within the genus Hansschlegelia, for which the name Hansschlegelia beijingensis sp. nov. is proposed, with the type strain PG04(T) ( = DSM 25481(T) = ACCC 05759(T)).

The 380 kb pCMU01 Plasmid Encodes Chloromethane Utilization Genes and Redundant Genes for Vitamin B12- and Tetrahydrofolate-Dependent Chloromethane Metabolism in Methylobacterium extorquens CM4: A Proteomic and Bioinformatics Study

Chloromethane (CH3Cl) is the most abundant volatile halocarbon in the atmosphere and contributes to the destruction of stratospheric ozone. The only known pathway for bacterial chloromethane utilization (cmu) was characterized in Methylobacterium extorquens CM4, a methylotrophic bacterium able to utilize compounds without carbon-carbon bonds such as methanol and chloromethane as the sole carbon source for growth. Previous work demonstrated that tetrahydrofolate and vitamin B12 are essential cofactors of cmuA- and cmuB-encoded methyltransferases of chloromethane dehalogenase, and that the pathway for chloromethane utilization is distinct from that for methanol. This work reports genomic and proteomic data demonstrating that cognate cmu genes are located on the 380 kb pCMU01 plasmid, which drives the previously defined pathway for tetrahydrofolate-mediated chloromethane dehalogenation. Comparison of complete genome sequences of strain CM4 and that of four other M. extorquens strains unable to grow with chloromethane showed that plasmid pCMU01 harbors unique genes without homologs in the compared genomes (bluB2, btuB, cobA, cbiD), as well as 13 duplicated genes with homologs of chromosome-borne genes involved in vitamin B12-associated biosynthesis and transport, or in tetrahydrofolate-dependent metabolism (folC2). In addition, the presence of both chromosomal and plasmid-borne genes for corrinoid salvaging pathways may ensure corrinoid coenzyme supply in challenging environments. Proteomes of M. extorquens CM4 grown with one-carbon substrates chloromethane and methanol were compared. Of the 49 proteins with differential abundance identified, only five (CmuA, CmuB, PurU, CobH2 and a PaaE-like uncharacterized putative oxidoreductase) are encoded by the pCMU01 plasmid. The mainly chromosome-encoded response to chloromethane involves gene clusters associated with oxidative stress, production of reducing equivalents (PntAA, Nuo complex), conversion of tetrahydrofolate-bound one-carbon units, and central metabolism. The mosaic organization of plasmid pCMU01 and the clustering of genes coding for dehalogenase enzymes and for biosynthesis of associated cofactors suggests a history of gene acquisition related to chloromethane utilization.

Ethylene emission and PR protein synthesis in ACC deaminase producing Methylobacterium spp. inoculated tomato plants (Lycopersicon esculentum Mill.) challenged with Ralstonia solanacearum under greenhouse conditions

Bacteria of genus Methylobacterium have been found to promote plant growth and regulate the level of ethylene in crop plants. This work is aimed to test the induction of defense responses in tomato against bacterial wilt by stress ethylene level reduction mediated by the ACC deaminase activity of Methylobacterium strains. Under greenhouse conditions, the disease index value in Methylobacterium sp. inoculated tomato plants was lower than control plants. Plants treated with Methylobacterium sp. challenge inoculated with Ralstonia solanacearum (RS) showed significantly reduced disease symptoms and lowered ethylene emission under greenhouse condition. The ACC and ACO (1-aminocyclopropane-1-carboxylate oxidase) accumulation in tomato leaves were significantly reduced with Methylobacterium strains inoculation. While ACC oxidase gene expression was found higher in plants treated with R. solanacearum than Methylobacterium sp. treatment, PR proteins related to induced systemic resistance like β-1,3-glucanase, PAL, PO and PPO were increased in Methylobacterium sp. inoculated plants. A significant increase in β-1,3-glucanase and PAL gene expression was found in all the Methylobacterium spp. treatments compared to the R. solanacearum treatment. This study confirms the activity of Methylobacterium sp. in increasing the defense enzymes by modulating the ethylene biosynthesis pathway and suggests the use of methylotrophic bacteria as potential biocontrol agents in tomato cultivation.

Methylobacterium haplocladii sp. nov. and Methylobacterium brachythecii sp. nov., isolated from bryophytes

Pink-pigmented, facultatively methylotrophic bacteria, strains 87e(T) and 99b(T), were isolated from the bryophytes Haplocladium microphyllum and Brachythecium plumosum, respectively. The cells of both strains were Gram-reaction-negative, motile, non-spore-forming rods. On the basis of 16S rRNA gene sequence similarity, strains 87e(T) and 99b(T) were found to be related to Methylobacterium organophilum ATCC 27886(T) (97.1% and 97.7%, respectively). Strains 87e(T) and 99b(T) showed highest 16S rRNA gene similarity to Methylobacterium gnaphalii 23e(T) (98.3 and 99.0%, respectively). The phylogenetic similarities to all other species of the genus Methylobacterium with validly published names were less than 97%. Major cellular fatty acids of both strains were C(18:1)ω7c and C(18:0). The results of DNA-DNA hybridization, phylogenetic analyses based on 16S rRNA and cpn60 gene sequences, fatty acid profiles, whole-cell matrix-assisted, laser-desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS) analysis, and physiological and biochemical tests allowed genotypic and phenotypic differentiation of strains 87e(T) and 99b(T) from their phylogenetically closest relatives. We propose that strains 87e(T) and 99b(T) represent novel species within the genus Methylobacterium, for which the names Methylobacterium haplocladii sp. nov. (type strain 87e(T) =DSM 24195(T) =NBRC 107714(T)) and Methylobacterium brachythecii sp. nov. (type strain 99b(T) =DSM 24105(T) =NBRC 107710(T)) are proposed.

Bacteriohopanoid inventory of Geobacter sulfurreducens and Geobacter metallireducens

Geobacter spp. are ubiquitous prokaryotes in diverse sedimentary environments, coupling the oxidation of various carbon compounds with the reduction of metals, such as ferric iron. One specific attribute of such species, namely Geobacter metallireducens and Geobacter sulfurreducens, is their ability to synthesize hopanoids. Hopanoids are among the most specific and abundant molecular fossils in the sedimentary record and are commonly used to trace bacterial activity in recent to sub-recent sediments. The production of polyfunctionalized bacteriohopanepolyols (BHPs) by G. sulfurreducens and G. metallireducens is known, but the intact BHPs were not identified. In this study, the complete hopanoid inventory of both species was investigated. Among the hopanoids, bacteriohopanetetrol cyclitol ether was the major compound in both, while guanidine-substituted bacteriohopanetetrol cyclitol ether and bacteriohopanetetrol glucosamine were also found in high abundance in G. sulfurreducens, but only in trace amounts in G. metallireducens. Interestingly, the BHP inventories are similar to those of some aerobic or facultative anaerobic methylotrophic bacteria, but have been identified in anaerobic cultures of Geobacter for the first time. The ubiquity of Geobacter spp. in anoxic sediments provokes us to speculate that such species may be among the predominant sources of composite BHPs in many sedimentary environments.

ISOLATION AND IDENTIFICATION OF SOME PPFMs BACTERIAL ISOLATES AND THEIR POTENTIALITY AS BIOFERTILIZERS AND BIOCONTROL AGENTS TO Rhizoctonia solani

A total of 12 isolates of pink-pigmented, facultative methylotrophic bacteria (PPFMs) were isolated on selective AMS medium from clay soil. Samples were collected from two locations (Sidi Salem and Agricultural Farm, Sakha Agri. Res. Station). There after, all the classical recommended methods used for identification of all the obtained isolates were carried out including cultural, morphological, microscopical as well as biochemical characteristics. The results revealed that all the isolates were gram negative, rod shaped, motile, colonies were light pink or pink or orange/pink in colour due to pigmentation. Biochemical characteristics revealed that all the isolates showed positive results for oxidase, catalase, urease and citirate test as well as indole production. None of the isolates showed positive results for casein hydrolysis. The isolates of ML4, ML8 and ML10 gave a positive reaction for starch hydrolysis test, while the others showed negative results. Regarding, cellulose degradation, the isolates ML2, ML7,ML8 and ML12 gave a positive reaction. The obtained results highly recommended that all the obtained isolates could be classified as follows: ML1and ML6 Methylobacterium radiotolerans; ML2 and ML5 Methylobacterium suomiense; ML3 Methylobacterium aminovorans; ML4 and ML9 Methylobacterium thiocyanatum; ML7, ML8 andML10 Methylobacterium rhodesianum; ML11 Methylobacterium fujisawaens and ML12 Methylobacterium rhodinum. Screening the isolates for beneficial growth characters and biocontrol agent to R. solani. In general, Methylobacterium rhodinum.and Methylobacterium aminovorans recorded the highest values of indole acetic acid production, 28.15 and 27.50 μg ml-1, and 70.41 and 67.46 μg ml-1 for GA production, and 0.580, 0.560 μ moles of siderophores production, and 1.217 and 1.193 mg N/g for fixed nitrogen respectively. Also, the two species exhibiting obvious antibiosis against R. solani as recorded 72.61 and 71.20% for inhibition of R. solani on PDA medium, and 69.30 and 66.80% for inhibition of R. solani on AMS medium respectively.

Detection of Metabolic Key Enzymes of Methane Turnover Processes in Cold Seep Microbial Biofilms

In anoxic environments, methane oxidation is conducted in a syntrophic process between methanotrophic archaea (ANME) and sulfate reducing bacteria (SRB). Microbial mats consisting of ANME, SRB and other microorganisms form methane seep-related carbonate buildups in the anoxic bottom waters of the Black Sea Crimean shelf. To shed light on the localization of the biochemical processes at the level of single cells in the Black Sea microbial mats, we applied antibody-based markers for key enzymes of the relevant metabolic pathways. The dissimilatory adenosine-5′-phosphosulfate (APS) reductase, methyl-coenzyme M reductase (MCR) and methanol dehydrogenase (MDH) were selected to localize sulfate respiration, reverse methanogenesis and aerobic methane oxidation, respectively. The key enzymes could be localized by double immunofluorescence and immunocytochemistry at light- and electron microscopic levels. In this study we show that sulfate reduction is conducted synchronized and in direct proximity to reverse methanogenesis of ANME archaea. Microcolonies in interspaces between ANME/SRB express methanol dehydrogenase, which is indicative for oxidation of C1 compounds by methylotrophic or methanotrophic bacteria. Thus, in addition to syntrophic AOM, oxygen-dependent processes are also conducted by a small proportion of the microbial population.

Methyloligella halotolerans gen. nov., sp. nov. and Methyloligella solikamskensis sp. nov., two non-pigmented halotolerant obligately methylotrophic bacteria isolated from the Ural saline environments

Two newly isolated halotolerant obligately methylotrophic bacteria (strains C2T and SK12T) with the serine pathway of C1 assimilation are described. The isolates are strictly aerobic, Gram negative, asporogenous, non-motile rods, forming rosettes, multiplying by binary fission. Mesophilic and neutrophilic, accumulate intracellularly compatible solute ectoine and poly-β-hydroxybutyrate. The novel strains are able to grow at 0 up to 16% NaCl (w/v), optimally at 3–5% NaCl. The major cellular fatty acids are C18:1ω7c and C19:0cyc and the prevailing quinone is Q-10. The predominant phospholipids are phosphatidylcholine, phosphatidylglycerol, phosphatidyldimethylethanolamine and phosphatidylethanolamine. Assimilate NH4+ by glutamate dehydrogenase and via the glutamate cycle (glutamine synthetase and glutamate synthase). The DNA G+C contents of strains C2T and SK12T are 60.9 and 60.5mol% (Tm), respectively. 16S rRNA gene sequence similarity between the two new isolates are 99% but below 94% with other members of the Alphaproteobacteria thus indicating that they can be assigned to a novel genus Methyloligella. Rather low level of DNA–DNA relatedness (53%) between the strains C2T and SK12T indicates that they represent two separate species of the new genus, for which the names Methyloligella halotolerans gen. nov., sp. nov. and Methyloligella solikamskensis sp. nov. are proposed. The type strain of Methyloligella halotolerans is C2T (=VKM B-2706T=CCUG 61687T=DSM 25045T) and the type strain of Methyloligella solikamskensis is SK12T (=VKM B-2707T=CCUG 61697T=DSM 25212T).

Phylogeny of bacterial methylotrophy genes reveals robustness in Methylobacterium mxaF sequences and mxa operon construction

mxaF, the structural gene for the methanol dehydrogenase (MDH enzyme), encodes for alpha subunit of the MDH enzyme. This member of the mxa family is required for production of a functional MDH enzyme. Methylobacterium species, pink-pigmented facultatively methylotrophic bacteria (PPFMs), are plant colonizing, methylotrophic bacteria that utilize methanol as a carbon and energy source during phyllosphere and rhizosphere growth. In a previous study that examined the phylogeny of soil and blueberry skin biofilm isolates, mxaF sequence was found to be a more useful indicator of species distinction within the Methylobacterium genus than 16S rRNA gene sequence and that it was not influenced by several environmental properties, including pH. Here, the phylogeny of methylotrophy genes including mxaF is examined to assess the potential for use of mxaF sequence as a biomarker for the Methylobacterium genus. A phylogenetic comparison of 311 archived PPFM and non-PPFM GenBank bacterial sequences of mxaF, xoxF, and generically annotated PQQ-dependent dehydrogenase genes in the methanol/ethanol family was conducted, including 270 mxaF, mxaF′ and mxaF-like sequences. In addition, construction of operons within the mxa gene family was compared using GenBank completed genome sequences to investigate potential horizontal gene transfer (HGT) events within the mxa methylotrophy system. mxaF sequence and mxa gene family operon construction was found to be highly conserved within the genus Methylobacterium as compared to other bacterial genera. The data reveal evidence of HGT events between Methylobacterium species and other bacterial genera as well as the potential for mxaF sequence to be used as a taxonomic indicator for environmental Methylobacterium strains.

Application of mxaF functional gene sequence to determine genetic relatedness among environmental Methylobacterium strains (PPFMs)

Pink-pigmented, facultatively methylotrophic bacteria (PPFMs) of the Methylobacterium genus utilize plant-derived methanol as a carbon and energy source when growing on plant roots. To accomplish this, they employ the methanol dehydrogenase (MDH) enzyme, for which mxaF is the structural gene. Changes in mxaF sequence can result in decreased enzyme efficiency. Environmental conditions such as soil pH can alter MDH efficiency and impact bacterial proliferation in soil by affecting the solubility and chemical speciation of metals and nutrients. Soil pH can be influenced by location, soil type, ecosystem type and plant cover. Phenotypic analyses as well as 16S rRNA and mxaF functional gene phylogeny and denaturing gradient gel electrophoresis (DGGE) were performed on nine New Jersey agricultural soil PPFM isolates to compare gene characteristics and to determine the feasibility of using mxaF as a genetic tool to distinguish between PPFM species. 16S rRNA and mxaF functional gene phylogeny of 114 New Jersey, South Carolina, and California forest and agricultural soil Methylobacterium isolates were examined in order to apply mxaF sequence as a tool for describing genetic relatedness among newly acquired soil Methylobacterium strains. The pH of soil sample source was considered as a potential influencing factor for PPFM species distribution and thus mxaF sequence in soil environments. Nucleotide sequences of PPFM 16S rRNA gene fragments were too similar to be useful for species characterization. DGGE separation of mxaF fragments revealed species-specific banding patterns for PPFM strains whereas 16S rRNA patterns were nearly identical for multiple strains. pH, location and plant type were found not to be a selecting factor for mxaF sequence and PPFM species distribution in soil, suggesting the robust and versatile nature of the mxaF gene in Methylobacterium.

Микробиологический синтез [2H]инозина высокого уровня дейтерированности грамположительной хемогетеротрофной бактериейBacillus subtilis

A 2H-labeled purine ribonucleoside inosine was microbiologically synthesized (yield, 3.9 g/L of culture liquid) using a deuterium-adapted strain ofthe gram-positive chemoheterotrophic bacterium Bacillus subtilis, cultivated in a heavy water medium with a high degree of deuteration (99.8 at % 2H) containing 2% hydrolysate of deuterated biomass of the methylotrophic bacterium Brevibacterium methylicum as a source of 2H-labeled growth substrate produced in an M9 minimal medium with 98% 2H20 and 2% [2H]-methanol. The inosine extracted from the culture liquid of the producer strain was fractionated by adsorption (desorption) on an activated carbon surface, extraction with 0.3 M ammonium-formate buffer (pH 8.9), subsequent crystallization in 80% ethanol, and ion exchange chromatography on a column with AG50WX 4 cation exchange resin equilibrated with 0.3 M ammonium-formate buffer containing 0.045 M NH4Cl. Fast atom bombardment (FAB) mass spectrometry demonstrated incorporation of five deuterium atoms in the inosine molecule (62.5% 2H), three of which were contained in the ribose moiety and two in the hypoxanthine moiety.

A Study of Antibacterial Activity of Fatty Acids Extracted from Pseudomonas sp. LP1 by using Chromatography technique

A bacterium that was capable of metabolizing C1- compounds was isolated and identified in Moscow State Academy of Fine Chemical Technology (Russia), as Pseudomonas sp. LP1 based on detailed taxonomic studies. This organism is a new strain of obligate methylotrophic bacteria, which was cultured on the methanol-containing medium (1%) as its sole carbon source. This study included culture of Pseudomonas sp. LP1 in M9 modified medium. The lipids were extracted from biomass of bacteria by organic solvents where a mixture of fatty acid methyl esters (FAMEs) was obtained. Antibacterial activity of total fatty acids was tested against some standard strains of both gram positive and gram negative bacteria by using wells in agar plates. Fatty acids mixture were found to have antibacterial effects. Thin-layer chromatography was used to tentatively identified the mixture components of fatty acids. A gas-liquid chromatographic system referred that Palmitic (16:0), Palmitoleic (16:1) and Oleic (18:1) acids were present.

A Catalytic Role of XoxF1 as La3+-Dependent Methanol Dehydrogenase in Methylobacterium extorquens Strain AM1

In the methylotrophic bacterium Methylobacterium extorquens strain AM1, MxaF, a Ca2+-dependent methanol dehydrogenase (MDH), is the main enzyme catalyzing methanol oxidation during growth on methanol. The genome of strain AM1 contains another MDH gene homologue, xoxF1, whose function in methanol metabolism has remained unclear. In this work, we show that XoxF1 also functions as an MDH and is La3+-dependent. Despite the absence of Ca2+ in the medium strain AM1 was able to grow on methanol in the presence of La3+. Addition of La3+ increased MDH activity but the addition had no effect on mxaF or xoxF1 expression level. We purified MDH from strain AM1 grown on methanol in the presence of La3+, and its N-terminal amino acid sequence corresponded to that of XoxF1. The enzyme contained La3+ as a cofactor. The ΔmxaF mutant strain could not grow on methanol in the presence of Ca2+, but was able to grow after supplementation with La3+. Taken together, these results show that XoxF1 participates in methanol metabolism as a La3+-dependent MDH in strain AM1.

Atmospheric methane removal by boreal plants

Several studies have proposed aerobic methane (CH4) emissions by plants. If confirmed, these findings would further increase the imbalance in the global CH4 budget which today underestimates CH4sinks. Oxidation by OH‐radicals in the troposphere is the major identified sink followed by smaller contribution from stratospheric loss and oxidation by methano‐ and methylotrophic bacteria in soils. This study directly investigated CH4 exchange by plants in their natural environment. At a forest site in central Sweden, in situ branch chamber measurements were used to study plant ambient CH4 exchange by spruce (Picea abies), birch (Betula pubescens), rowan (Sorbus aucuparia) and pine (Pinus sylvestris). The results show a net uptake of CH4 by all the studied plants, which might be of importance for the methane budget.

Methylobacillus rhizosphaerae sp. nov., a novel plant-associated methylotrophic bacterium isolated from rhizosphere of red pepper

A novel plant-associated obligate methylotrophic bacterium, designated strain Ca-68(T), was isolated from the rhizosphere soil of field-grown red pepper from India. The isolates are strictly aerobic, Gram negative, motile rods multiplying by binary fission and formaldehyde is assimilated via the ribulose monophosphate pathway. A comparative 16S rRNA gene sequence-based phylogenetic analysis placed the strain in a clade with the species Methylobacillus flagellatus, Methylobacillus glycogens and Methylobacillus pratensis, with which it showed pairwise similarity of 97.8, 97.4 and 96.2 %, respectively. The major fatty acids are C(16:0), C(10:0) 3OH and C(16:1) ω7c. The G+C content of the genomic DNA is 59.7 mol%. The major ubiquinone is Q-8. Dominant phospholipids are phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol. Based on 16S rRNA gene sequence analysis and DNA-DNA relatedness (14-19 %) with type strains of the genus Methylobacillus, the novel isolate was classified as a new species of this genus and named Methylobacillus rhizosphaerae Ca-68(T) (=KCTC 22383(T) = NCIMB 14472(T)).

Deep Sequencing of Myxilla (Ectyomyxilla) methanophila, an Epibiotic Sponge on Cold-Seep Tubeworms, Reveals Methylotrophic, Thiotrophic, and Putative Hydrocarbon-Degrading Microbial Associations

The encrusting sponge Myxilla (Ectyomyxilla) methanophila (Poecilosclerida: Myxillidae) is an epibiont on vestimentiferan tubeworms at hydrocarbon seeps on the upper Louisiana slope of the Gulf of Mexico. It has long been suggested that this sponge harbors methylotrophic bacteria due to its low δ(13)C value and high methanol dehydrogenase activity, yet the full community of microbial associations in M. methanophila remained uncharacterized. In this study, we sequenced 16S rRNA genes representing the microbial community in M. methanophila collected from two hydrocarbon-seep sites (GC234 and Bush Hill) using both Sanger sequencing and next-generation 454 pyrosequencing technologies. Additionally, we compared the microbial community in M. methanophila to that of the biofilm collected from the associated tubeworm. Our results revealed that the microbial diversity in the sponges from both sites was low but the community structure was largely similar, showing a high proportion of methylotrophic bacteria of the genus Methylohalomonas and polycyclic aromatic hydrocarbon (PAH)-degrading bacteria of the genera Cycloclasticus and Neptunomonas. Furthermore, the sponge microbial clone library revealed the dominance of thioautotrophic gammaproteobacterial symbionts in M. methanophila. In contrast, the biofilm communities on the tubeworms were more diverse and dominated by the chemoorganotrophic Moritella at GC234 and methylotrophic Methylomonas and Methylohalomonas at Bush Hill. Overall, our study provides evidence to support previous suggestion that M. methanophila harbors methylotrophic symbionts and also reveals the association of PAH-degrading and thioautotrophic microbes in the sponge.

Structure and Catalytic Mechanism of N5,N10-Methenyl-tetrahydromethanopterin Cyclohydrolase

Methenyltetrahydromethanopterin (methenyl-H(4)MPT(+)) cyclohydrolase (Mch) catalyzes the interconversion of methenyl-H(4)MPT(+) and formyl-H(4)MPT in the one-carbon energy metabolism of methanogenic, methanotrophic, and sulfate-reducing archaea and of methylotrophic bacteria. To understand the catalytic mechanism of this reaction, we kinetically characterized site-specific variants of Mch from Archaeoglobus fulgidus (aMch) and determined the X-ray structures of the substrate-free aMch(E186Q), the aMch:H(4)MPT complex, and the aMch(E186Q):formyl-H(4)MPT complex. (Formyl-)H(4)MPT is embedded inside a largely preformed, interdomain pocket of the homotrimeric enzyme with the pterin and benzyl rings being oriented nearly perpendicular to each other. The active site is primarily built up by the segment 93:95, Arg183 and Glu186 that either interact with the catalytic water attacking methenyl-H(4)MPT(+) or with the formyl oxygen of formyl-H(4)MPT. The catalytic function of the strictly conserved Arg183 and Glu186 was substantiated by the low enzymatic activities of the E186A, E186D, E186N, E186Q, R183A, R183Q, R183E, R183K, and R183E-E186Q variants. Glu186 most likely acts as a general base. Arg183 decisively influences the pK(a) value of Glu186 and the proposed catalytic water mainly by its positive charge. In addition, Glu186 appears to be also responsible for product specificity by donating a proton to the directly neighbored N(10) tertiary amine of H(4)MPT. Thus, N(10) becomes a better leaving group than N(5) which implies the generation of N(5)-formyl-H(4)MPT. For comparison, methenyltetrahydrofolate (H(4)F) cyclohydrolase produces N(10)-formyl-H(4)F in an analogous reaction. An enzymatic mechanism of Mch is postulated and compared with that of other cyclohydrolases.

Methylopila musalis sp. nov., an aerobic, facultatively methylotrophic bacterium isolated from banana fruit

A newly isolated, facultatively methylotrophic bacterium (strain MUSA(T)) was investigated. The isolate was strictly aerobic, Gram-stain-negative, asporogenous, motile, rod-shaped and multiplied by binary fission. The strain utilized methanol, methylamine and an apparently narrow range of multi-carbon compounds, but not methane, dichloromethane or CO2/H2, as the carbon and energy sources. Growth occurred at pH 5.5-9.5 (optimum, pH 7.0) and 16-40 °C (optimum, 28-30 °C). The major fatty acids of methanol-grown cells were C18 : 1ω7c, C18 : 0 and 11-methyl-C18 : 1ω7c . The predominant phospholipids were phosphatidylethanolamine, phosphatidylcholine, phosphatidylglycerol and phosphatidylmonomethylethanolamine. The major ubiquinone was Q-10. The strain had methanol and methylamine dehydrogenases as well as the enzymes of the N-methylglutamate pathway (lyases of γ-glutamylmethylamide and N-methylglutamate). C1 assimilation occurs via the isocitrate lyase-negative serine pathway. Ammonium was assimilated by glutamate dehydrogenase and the glutamate cycle (glutamate synthase/glutamine synthetase). The DNA G+C content of the strain was 64.5 mol% (determined from the melting temperature). Based on 16S rRNA gene sequence similarity (97.0-98.9 %) and DNA-DNA relatedness (36-38 %) with representatives of the genus Methylopila (Methylopila capsulata IM1(T) and Methylopila jiangsuensis JZL-4(T)) the isolate was classified as a novel species of the genus Methylopila, for which the name Methylopila musalis sp. nov. is proposed. The type strain is MUSA(T) ( = VKM B-2646(T) = DSM 24986(T) = CCUG 61696(T)).

Comparative proteomic analysis reveals insights into anoxic growth of Methyloversatilis universalis FAM5 on methanol and ethanol

Methyloversatilis universalis FAM5 is a facultative methylotrophic bacterium that has been found in a variety of natural and engineered ecosystems. The goal of this study was to investigate M. universalis FAM5 responses to different electron/carbon donors, e.g. methanol or ethanol, during anoxic growth in chemostats with nitrate as the electron acceptor. During steady-state anoxic growth on either methanol or ethanol, over 90% of the influent nitrate was reduced primarily to nitrite. The cell yield on methanol was lower, possibly due to high energy requirements for C(1) assimilation. Label-free proteomics further revealed that methanol-grown cells displayed elevated concentrations of the enzymes involved in C(1) metabolism (H(4)MPT/H(4) F pathways, formate oxidation and serine cycle). In contrast, C(2) metabolism (glyoxylate shunt and tri-carboxylic acid cycle) and polyhydroxy-β-butyrate (PHB) synthesis related proteins were overrepresented during subsequent growth on ethanol. Notably, the expression of respiratory nitrate reductase was not affected by the carbon sources applied. Furthermore, the changes in the proteome upon switching back to methanol were mostly reversible. Therefore, M. universalis displays wide-ranging responses to adapt between growth on methanol and ethanol. Such metabolic versatility could be particularly useful in wastewater treatment systems, which need to switch between different electron donors, while still reliably meeting effluent nitrogen discharge goals.

Role of EctR as transcriptional regulator of ectoine biosynthesis genes in Methylophaga thalassica

In the halophilic aerobic methylotrophic bacterium Methylophaga thalassica, the genes encoding the enzymes for biosynthesis of the osmoprotectant ectoine were shown to be located in operon ectABC-ask. Transcription of the ect-operon was started from the two promoters homologous to the σ(70)-dependent promoter of Escherichia coli and regulated by protein EctR, whose encoding gene, ectR, is transcribed from three promoters. Genes homologous to ectR of methylotrophs were found in clusters of ectoine biosynthesis genes in some non-methylotrophic halophilic bacteria. EctR proteins of methylotrophic and heterotrophic halophiles belong to the MarR-family of transcriptional regulators but form a separate branch on the phylogenetic tree of the MarR proteins.