Facultative Methylotroph Research Articles

PqqE from Methylobacterium extorquens AM1: a radical S-adenosyl-l-methionine enzyme with an unusual tolerance to oxygen.

Methylobacterium extorquens AM1 is an aerobic facultative methylotroph known to secrete pyrroloquinoline quinone (PQQ), a cofactor of a number of bacterial dehydrogenases, into the culture medium. To elucidate the molecular mechanism of PQQ biosynthesis, we are focusing on PqqE which is believed to be the enzyme catalysing the first reaction of the pathway. PqqE belongs to the radical S-adenosyl-l-methionine (SAM) superfamily, in which most, if not all, enzymes are very sensitive to dissolved oxygen and rapidly inactivated under aerobic conditions. We here report that PqqE from M. extorquens AM1 is markedly oxygen-tolerant; it was efficiently expressed in Escherichia coli cells grown aerobically and affinity-purified to near homogeneity. The purified and reconstituted PqqE contained multiple (likely three) iron-sulphur clusters and showed the reductive SAM cleavage activity that was ascribed to the consensus [4Fe-4S](2+) cluster bound at the N-terminus region. Mössbauer spectrometric analyses of the as-purified and reconstituted enzymes revealed the presence of [4Fe-4S](2+) and [2Fe-2S](2+) clusters as the major forms with the former being predominant in the reconstituted enzyme. PqqE from M.extorquens AM1 may serve as a convenient tool for studying the molecular mechanism of PQQ biosynthesis, avoiding the necessity of establishing strictly anaerobic conditions.

Methylopila turkiensis sp. nov., a new aerobic facultatively methylotrophic phytosymbiont

A new facultative methylotroph, strain Side1T, was isolated from the phyllosphere of Bougainvillea sp. L. The isolate is represented by rod-shaped, aerobic gram-negative asporogenous bacteria which divide by binary fission. Methanol and mono- and trimethylamine were utilized, as well as a limited spectrum of polycarbon substrates, while methane and dichloromethane were not used. Growth occurred at pH 6.0–9.0 with the optimum at pH 7.0 within the temperature range from 20 to 40°C (optimum at 28–30°C) and 0–2.5% NaCl in the medium. The predominant fatty acids were cis-11-octadecenoic (C18:1ω7c), 11-methyl-octadecenoic (C18:ω7c11Me), and stearic (C18:0) acids. Phosphatidylethanolamine, phosphatidylcholine, phosphatidylglycerol, and diphosphatidylglycerol were the dominant phospholipids. Q10 was the dominant ubiquinone. The isolate oxidized methanol and methylamine by the appropriate dehydrogenases. The isocitrate lyase-negative variant of the serine pathway was used. Ammonium assimilation involved glutamate dehydrogenase and the glutamate cycle (glutamate synthase and glutamine synthetase). The strain synthesized indole and siderophores; it solubilized insoluble phosphates. The DNA G+C content (Tm) was 65.4 mol %. While the nucleotide sequence of the 16S rRNA gene of strain Side1 exhibited high similarity to those of Methylopila species (M. musalis MUSAT and M. capsulata IM1T), DNA-DNA homology with these cultures was 32–37%. The results obtained supported classification of strain Side1T as a new species Methylopila turkiensis sp. nov. (VKM B-2748T= DSM 27566T).

Advenella kashmirensis subsp. methylica PK1, a facultative methylotroph from carex rhizosphere

A strain (PK1) of facultative methylobacteria growing on methanol as a carbon and energy source was isolated from carex rhizosphere (Pamukkale National Park, Turkey). The cells were nonmotile gram-negative rods propagating by binary fission. The organism was a strict anaerobe, oxidase- and catalase-positive. Optimal growth occurred at 29°C, pH 8.0-8.5, and 0.5% NaCl; no growth occurred at 2% NaCl. The organism used the ribulose bisphosphate pathway of C1 assimilation. Predominant fatty acids were 11-octodecenoic (18:1ω7) and cis-hexadecenoic (16:1ω7c). Phosphatidylethanolamine and diphosphatidylglycerol were the dominant phospholipids. Q8 was the main ubiquinone. DNA G+C content was 55.4 mol % (mp). Sequencing of the 16S rRNA gene revealed that strain PK1 belonged to the genus Advenella with 98.8 and 99.2% similarity to the type strains A. incenata CCUG 45225T and A. kashmirensis WT001T, respectively. DNA-DNA homology of strain PK1 and A. kashmirensis WT001T was 70%. While MALDI analysis confirmed their close clusterization, RAPD analysis revealed the differences between strain PKI and other Advenella strains. Based on its geno- and phenotypic properties, the isolate PK1 was classified as A. kashmirensis subsp. methylica PK1 (VKM-B 2850 = DSM 27514), the first known methylotroph of the genus Advenella.

Optimizing Plant Geometry and Nutrient Management for Grain Yield and Economics in Irrigated Greengram

Greengram (Vigna radiata L.) is important pulse crop in India. The yield and economics were in-creased by optimization of plant geometry and nutrient management under irrigated condition. The field experiment was conducted during Rabi season of November 2013 to January 2014 at Tamil Nadu Agricultural University, Agricultural College and Research Institute, Killikulam. An experiment was laid out in randomised block design and replicated thrice and the test variety of the crop greengram (CO 6) was used. The plant geometry of 30 × 30 cm, 25 × 25 cm and 30 × 10 cm was adopted. The Soil Test Crop Response (STCR) based fertilizer application, RDF, FYM and ZnSO4 was applied in soil as basal. The foliar spray of Pulse Wonder and Pink-pigmented facultative methylotrophs (PPFM) spray was done at one week after flowering and 1% KNO3 at 50 per cent flowering. Adoption of planting geometry of 30 × 30 cm, application of RDF, 12.5 t of FYM and 25 kg ZnSO4 as basal and foliar spraying of 1% KNO3 at 50 per cent flowering recorded higher dry matter production of 2865 kg·ha-1 and yield attributes viz., number of pod clusters plant-1 (10.34), number of pods plant-1 (53.40), number of seeds pod-1 (13.23), pod length (8.77 cm) and seed test weight (3.42 g). Higher grain yield of 1775 kg·ha-1, haulm yield (2920 kg·ha-1), harvest index (0.38), net return (57,806 Rs·ha-1) and B:C ratio (2.43) were associated with the treatment comprising of 30 × 30 cm spacing, application of RDF, 12.5 t of FYM and 25 kg ZnSO4 as basal and foliar spraying of 1% KNO3 at 50 per cent flowering.

Requirement of carbon dioxide for initial growth of facultative methylotroph, Acidomonas methanolica MB58

The facultative methylotrophic bacterium Acidomonas methanolica MB58 can utilize C1 compounds via the ribulose monophosphate pathway. A large gene cluster comprising three components related to C1 metabolism was found in the genome. From upstream, the first was an mxa cluster encoding proteins for oxidation of methanol to formaldehyde; the second was the rmp cluster encoding enzymes for formaldehyde fixation; and the third was the cbb gene cluster encoding proteins for carbon dioxide (CO2) fixation. Examination of CO2 requirements for growth of A.methanolica MB58 cells demonstrated that it did not grow on any carbon source under CO2-free conditions. Measurement of ribulose-1,5-bisphosphate carboxylase activity and RT-PCR analysis demonstrated enzymatic activity was detected in A.methanolica MB58 at growth phase, regardless of carbon sources. However, methanol dehydrogenase and 3-hexlose-6-phosphate synthase expression was regulated by methanol or formaldehyde; it were detected during growth and apparently differed from ribulose-1,5-bisphosphate carboxylase expression. These results suggested that A.methanolica MB58 may be initially dependent on autotrophic growth and that carbon assimilation was subsequently coupled with the ribulose monophosphate pathway at early- to mid-log phases during methylotrophic growth.

Genome information of Methylobacterium oryzae, a plant-probiotic methylotroph in the phyllosphere.

Pink-pigmented facultative methylotrophs in the Rhizobiales are widespread in the environment, and many Methylobacterium species associated with plants produce plant growth-promoting substances. To gain insights into the life style at the phyllosphere and the genetic bases of plant growth promotion, we determined and analyzed the complete genome sequence of Methylobacterium oryzae CBMB20T, a strain isolated from rice stem. The genome consists of a 6.29-Mb chromosome and four plasmids, designated as pMOC1 to pMOC4. Among the 6,274 coding sequences in the chromosome, the bacterium has, besides most of the genes for the central metabolism, all of the essential genes for the assimilation and dissimilation of methanol that are either located in methylotrophy islands or dispersed. M. oryzae is equipped with several kinds of genes for adaptation to plant surfaces such as defense against UV radiation, oxidative stress, desiccation, or nutrient deficiency, as well as high proportion of genes related to motility and signaling. Moreover, it has an array of genes involved in metabolic pathways that may contribute to promotion of plant growth; they include auxin biosynthesis, cytokine biosynthesis, vitamin B12 biosynthesis, urea metabolism, biosorption of heavy metals or decrease of metal toxicity, pyrroloquinoline quinone biosynthesis, 1-aminocyclopropane-1-carboxylate deamination, phosphate solubilization, and thiosulfate oxidation. Through the genome analysis of M. oryzae, we provide information on the full gene complement of M. oryzae that resides in the aerial parts of plants and enhances plant growth. The plant-associated lifestyle of M. oryzae pertaining to methylotrophy and plant growth promotion, and its potential as a candidate for a bioinoculant targeted to the phyllosphere and focused on phytostimulation are illuminated.

Bacterial metabolism of methylated amines and identification of novel methylotrophs in Movile Cave.

Movile Cave, Romania, is an unusual underground ecosystem that has been sealed off from the outside world for several million years and is sustained by non-phototrophic carbon fixation. Methane and sulfur-oxidising bacteria are the main primary producers, supporting a complex food web that includes bacteria, fungi and cave-adapted invertebrates. A range of methylotrophic bacteria in Movile Cave grow on one-carbon compounds including methylated amines, which are produced via decomposition of organic-rich microbial mats. The role of methylated amines as a carbon and nitrogen source for bacteria in Movile Cave was investigated using a combination of cultivation studies and DNA stable isotope probing (DNA-SIP) using (13)C-monomethylamine (MMA). Two newly developed primer sets targeting the gene for gamma-glutamylmethylamide synthetase (gmaS), the first enzyme of the recently-discovered indirect MMA-oxidation pathway, were applied in functional gene probing. SIP experiments revealed that the obligate methylotroph Methylotenera mobilis is one of the dominant MMA utilisers in the cave. DNA-SIP experiments also showed that a new facultative methylotroph isolated in this study, Catellibacterium sp. LW-1 is probably one of the most active MMA utilisers in Movile Cave. Methylated amines were also used as a nitrogen source by a wide range of non-methylotrophic bacteria in Movile Cave. PCR-based screening of bacterial isolates suggested that the indirect MMA-oxidation pathway involving GMA and N-methylglutamate is widespread among both methylotrophic and non-methylotrophic MMA utilisers from the cave.

Drip fertigation effects on yield, nutrient uptake and soil fertility of Bt Cotton in semi arid tropics

Field experiment was conducted for 2 seasons to study the influence of drip fertigation in combination with or without bio fertilizers on yield, plant uptake and soil fertility of Bt cotton. The treatments comprised of four levels of drip fertigation viz., 75, 100, 125 and 150 per cent of recommended dose of fertilizers (RDF, NPK) combined with and without bio fertilizers, drip irrigation with soil surface application of 100 per cent RDF and surface irrigation with soil surface application of 100 per cent RDF as control. Biofertilizers used for fertigation is azophosmet containing Azospirillum, phosphobacterium and pink pigmented facultative methylotroph. Most of the yield attributes viz., number of sympodial branches per plant, number of fruiting points, bolls per plant, plant uptake and available soil N, P and K of Bt cotton were significantly increased by the drip fertigation treatments. Application of 150 per cent RDF as drip fertigation combined with biofertigation of liquid formulation of azophosmet @ 250 ml (10 12 cells ml -1 ) ha -1 registered the highest seed cotton yield of 3395 kg ha -1 and was significantly superior over control. Biofertigation significantly increased seed cotton yield and a progressive increase in seed yield was noticed with increasing levels of NPK fertilizer application. Application of nutrients through drip fertigation improved seed cotton yield by 43.0 per cent compared with conventional surface irrigation with soil surface application of fertilizers. The nutrient uptake pattern and post harvest soil fertility status also followed similar trend and confirmed the significance of drip fertigation with biofertilizers.

Proteomic analysis of the thermophilic methylotroph Bacillus methanolicusMGA3

Bacillus methanolicus MGA3 is a facultative methylotroph of industrial relevance that is able to grow on methanol as its sole source of carbon and energy. The Gram-positive bacterium possesses a soluble NAD(+) -dependent methanol dehydrogenase and assimilates formaldehyde via the ribulose monophosphate (RuMP) cycle. We used label-free quantitative proteomics to generate reference proteome data for this bacterium and compared the proteome of B. methanolicus MGA3 on two different carbon sources (methanol and mannitol) as well as two different growth temperatures (50°C and 37°C). From a total of approximately 1200 different detected proteins, approximately 1000 of these were used for quantification. While the levels of 213 proteins were significantly different at the two growth temperatures tested, the levels of 109 proteins changed significantly when cells were grown on different carbon sources. The carbon source strongly affected the synthesis of enzymes related to carbon metabolism, and in particular, both dissimilatory and assimilatory RuMP cycle enzyme levels were elevated during growth on methanol compared to mannitol. Our data also indicate that B. methanolicus has a functional tricarboxylic acid cycle, the proteins of which are differentially regulated on mannitol and methanol. Other proteins presumed to be involved in growth on methanol were constitutively expressed under the different growth conditions. All MS data have been deposited in the ProteomeXchange with the identifiers PXD000637 and PXD000638 (http://proteomecentral.proteomexchange.org/dataset/PXD000637, http://proteomecentral.proteomexchange.org/dataset/PXD000638).

Impact of climate change scenario on rice production in two planting methods: a simulation study

In the present study, attempts have been made to simulate the effect of climate change on rice growth and yield, under both control and water-stressed conditions. Between the two planting methods, the system of rice intensification (SRI) practice had an advantage for the elevated CO2 conditions, with an additional yield of 1,325 kg ha−1; while it was only 391 kg ha−1 under traditional system of planting rice (TPR). Similarly, the yield decline due to temperature increment was −2.0, −2.4, −6.2 and −12.8 % under SRI practice as compared to that of −4.0, −9.9, −11.3 and −31.7 % in the TPR system for +1, +2, +3 and +4 °C temperature rise, respectively. Thus, doubling of atmospheric CO2 level will compensate for the detrimental effect of increased temperature up to 2 °C in the SRI method of rice cultivation as compared to TPR system of planting. Thus, SRI practice is the most suitable method of rice cultivation under both elevated CO2 and temperature level. Simulation analysis of the present data using the dynamic model, ORYZA2000, indicated that under future adverse climatic conditions, the grain yield showed little variation (+1.83 %) with doubled CO2 at +2 °C temperature rise especially with the water stress situations. However, this could be further raised (+17.10 %) with the supplementation of pink-pigmented facultative methylotroph bacterium (PPFM) bio-fertilizer in the given scenario. Thus, temperature-induced yield alterations especially under water-stressed environment could be favorably mitigated with the CO2 fertilization along with the supplementation of PPFM bio-fertilizer.

Methyloceanibacter caenitepidi gen. nov., sp. nov., a facultatively methylotrophic bacterium isolated from marine sediments near a hydrothermal vent

A moderately thermophilic, methanol-oxidizing bacterium (strain Gela4(T)) was isolated from methane-utilizing mixed-culture originating from marine sediment near a hydrothermal vent. Phylogenetic analysis of 16S rRNA gene sequences indicated that strain Gela4(T) was closely related to members of the genus 'Methyloligella' (94.7% similarity) within the class Alphaproteobacteria. Strain Gela4(T) was a Gram-staining-negative and aerobic organism. Cells were rod-shaped and non-motile. The temperature range for growth of strain Gela4(T) was 19-43 °C (optimal growth at 35 °C). Strain Gela4(T) tolerated up to 9% NaCl with an optimum at 1%. The organism was a facultative methylotroph that could utilize methanol, methylamine, trimethylamine and a variety of multi-carbon compounds. The major cellular fatty acid and major respiratory quinone were C18 : 1ω7c and ubiquinone-10, respectively. The predominant phospholipids were phosphatidylcholine, phosphatidylglycerol and phosphatidylethanolamine. The DNA G+C content was 63.9 mol%. On the basis of the morphological, physiological, biochemical and genetic information, a novel genus and species, Methyloceanibacter caenitepidi is proposed, with Gela4(T) ( = NBRC 109540(T) = DSM 27242(T)) as the type strain.

Methyloversatilis thermotolerans sp. nov., a novel thermotolerant facultative methylotroph isolated from a hot spring

A newly isolated facultatively methylotrophic bacterium (strain 3t(T)) was investigated. Cells of the isolate were Gram-stain-negative, asporogenous, non-motile rods that multiplied by binary fission. The strain utilized methanol, methylamine and a variety of multicarbon compounds as carbon and energy sources. Growth occurred at pH 6.5-8.5 (optimally at 7.0-7.5) and at 10-45 °C (optimally at 30-37 °C). The major fatty acids of methanol-grown cells were C16 : 1ω7c and C16 : 0. The predominant phospholipids were phosphatidylethanolamine and phosphatidylglycerol. The major ubiquinone was Q-8. Strain 3t(T) possessed pyrroloquinoline quinone (PQQ)-linked methanol dehydrogenase and assimilated C1 units at the level of formaldehyde and CO2 via the serine cycle. The DNA G+C content of the strain was 63.6 mol% (Tm). On the basis of 16S rRNA gene sequence similarity (98.1 %) and rather low DNA-DNA relatedness (30 %) with the type strain of the type species of the genus Methyloversatilis (Methyloversatilis universalis FAM5(T)), and physiological and biochemical characteristics, the isolate was classified as a representative of a new species of the genus and named Methyloversatilis thermotolerans 3t(T) ( = VKM B-2692(T) = CCUG 61694(T) = DSM 25156(T)).

Dominant Colonization and Inheritance ofMethylobacteriumsp. Strain OR01 on Perilla Plants

Pink-pigmented facultative methylotrophs (PPFMs) are major inhabitants of the phyllosphere. In a preceding study, we found that perilla plants harbor a dominant population of PPFMs on their leaves and seeds, and that the closest relative of PPFMs (Methylobacterium sp. strain OR01 as representative strain) isolated from red perilla seeds was M. fujisawaense DSM5686(T). In the present study, the specific interaction between red perilla and Methylobacterium species was investigated. All the PPFMs isolated from red perilla seeds harvested in the Ohara area of Kyoto, Japan in 2009, 2010, and 2011 and the PPFMs isolated from red perilla leaves planted at four geographically different places in Japan had 16S rRNA sequences identical to that of strain OR01. Direct transmission of PPFMs from seeds to leaves and the competitiveness of strain OR01 were confirmed. This report is the first step toward understanding the species-level specificity of the interaction between perilla plants and Methylobacterium species.

Degradation of formaldehyde at high concentrations by phenol-adapted Ralstonia eutropha closely related to pink-pigmented facultative methylotrophs

The ability of the phenol-adapted Ralstonia eutropha to utilize formaldehyde (FD) as the sole source of carbon and energy was studied. Adaptation to FD was accomplished by substituting FD for glucose in a stepwise manner. The bacterium in the liquid test culture could tolerate concentrations of FD up to 900 mg L−1. Degradation of FD was complete in 528 h at 30°C with shaking at 150 rpm (r = 1.67 mg L−1 h−1), q = 0.035 gFD gcell −1 h−1. Substrate inhibition kinetics (Haldane and Luong equations) are used to describe the experimental data. At non-inhibitory concentrations of FD, the Monod equation was used. According to the Luong model, the values of the maximum specific growth rate (μmax), half-saturation coefficient (kS), the maximum allowable formaldehyde concentration (Sm), and the shape factor (n) were 0.117 h−1, 47.6 mg L−1, 900 mg L−1, and 2.2, respectively. The growth response of the test bacterium to consecutive FD feedings was examined, and the FD-adapted R. eutropha cells were able to degrade 1000 mg L−1 FD in 150 h through 4 cycles of FD feeds. During FD degradation, formic acid metabolite was formed. Assimilation of FD, methanol, formic acid, and oxalate by the test bacterium was accompanied by the formation of a pink pigment. The carotenoid nature of the cellular pigment has been confirmed and the test bacterium appeared to be closely related to pink-pigmented facultative methylotrophs (PPFM). The extent of harm to soil exposed to biotreated wastewaters containing FD may be moderated due to the association between methylotrophic/oxalotrophic bacteria and plants.

Distribution of Pink-Pigmented Facultative Methylotrophs on Leaves of Vegetables

The distribution of pink-pigmented facultative methylotrophs (PPFMs) on the leaves of various vegetables was studied. All kinds of vegetable leaves tested gave pink-pigmented colonies on agar plates containing methanol as sole carbon source. The numbers of PPFMs on the leaves, colony-forming units (CFU)/g of fresh leaves, differed among the plants, although they were planted and grown at the same farm. Commercial green perilla, Perilla frutescens viridis (Makino) Makino, gave the highest counts of PPFMs (2.0-4.1×10(7) CFU/g) of all the commercial vegetable leaves tested, amounting to 15% of total microbes on the leaves. The PPFMs isolated from seeds of two varieties of perilla, the red and green varieties, exhibited high sequence similarity as to the 16S rRNA gene to two different Methylobacterium species, M. fujisawaense DSM5686(T) and M. radiotolerans JCM2831(T) respectively, suggesting that there is specific interaction between perilla and the PPFMs.

The abundance of pink-pigmented facultative methylotrophs in the root zone of plant species in invaded coastal sage scrub habitat.

Pink-pigmented facultative methylotrophic bacteria (PPFMs) are associated with the roots, leaves and seeds of most terrestrial plants and utilize volatile C1 compounds such as methanol generated by growing plants during cell division. PPFMs have been well studied in agricultural systems due to their importance in crop seed germination, yield, pathogen resistance and drought stress tolerance. In contrast, little is known about the PPFM abundance and diversity in natural ecosystems, let alone their interactions with non-crop species. Here we surveyed PPFM abundance in the root zone soil of 5 native and 5 invasive plant species along ten invasion gradients in Southern California coastal sage scrub habitat. PPFMs were present in every soil sample and ranged in abundance from 102 to 105 CFU/g dry soil. This abundance varied significantly among plant species. PPFM abundance was 50% higher in the root zones of annual or biennial species (many invasives) than perennial species (all natives). Further, PPFM abundance appears to be influenced by the plant community beyond the root zone; pure stands of either native or invasive species had 50% more PPFMs than mixed species stands. In sum, PPFM abundance in the root zone of coastal sage scrub plants is influenced by both the immediate and surrounding plant communities. The results also suggest that PPFMs are a good target for future work on plant-microorganism feedbacks in natural ecosystems.

Crop Establishment Techniques and Nutrient Management Practices on Yield and Economics of Wet Seeded Lowland Rice

Field experiment was carried out at wetland of Tamil Nadu Agricultural University, Coimbatore during Kharif 2008. The experiment was laid out in a factorial randomized block design with three replications to evaluate the stand establishment techniques and nutrient management practices in wet seeded lowland rice. The treatments consisting of two establishment techniques (Direct planting system and drum seeding) and four levels of nutrient management practices (control, RDF, RDF+FYM and RDF+FYM+Biofertilizers). Direct planting system recorded higher yield components and grain yield compared to drum seeding method of crop establishment. Application of recommended dose of fertilizers (150:50:50 NPK kg ha -1 ), FYM 12.5 t ha -1 and biofertilizers (Azosphosmet @ 2 kg ha -1 ) + PPFM (pink pigmented facultative methylotroph) @ 5 L ha -1 enhanced the grain yield over no manure treatment under Direct Planting System when compared with drum seeding method. The net income and B:C were higher under DPS than drum seeding method due to higher grain yield.

The Ethylmalonyl-CoA Pathway Is Used in Place of the Glyoxylate Cycle by Methylobacterium extorquens AM1 during Growth on Acetate

Acetyl-CoA assimilation was extensively studied in organisms harboring the glyoxylate cycle. In this study, we analyzed the metabolism of the facultative methylotroph Methylobacterium extorquens AM1, which lacks isocitrate lyase, the key enzyme in the glyoxylate cycle, during growth on acetate. MS/MS-based proteomic analysis revealed that the protein repertoire of M. extorquens AM1 grown on acetate is similar to that of cells grown on methanol and includes enzymes of the ethylmalonyl-CoA (EMC) pathway that were recently shown to operate during growth on methanol. Dynamic 13C labeling experiments indicate the presence of distinct entry points for acetate: the EMC pathway and the TCA cycle. 13C steady-state metabolic flux analysis showed that oxidation of acetyl-CoA occurs predominantly via the TCA cycle and that assimilation occurs via the EMC pathway. Furthermore, acetyl-CoA condenses with the EMC pathway product glyoxylate, resulting in malate formation. The latter, also formed by the TCA cycle, is converted to phosphoglycerate by a reaction sequence that is reversed with respect to the serine cycle. Thus, the results obtained in this study reveal the utilization of common pathways during the growth of M. extorquens AM1 on C1 and C2 compounds, but with a major redirection of flux within the central metabolism. Furthermore, our results indicate that the metabolic flux distribution is highly complex in this model methylotroph during growth on acetate and is fundamentally different from organisms using the glyoxylate cycle.

Epiphytic pink-pigmented methylotrophic bacteria enhance germination and seedling growth of wheat (Triticum aestivum) by producing phytohormone

Methylotrophic bacteria were isolated from the phyllosphere of different crop plants such as sugarcane, pigeonpea, mustard, potato and radish. The methylotrophic isolates were differentiated based on growth characteristics and colony morphology on methanol supplemented ammonium mineral salts medium. Amplification of the mxaF gene helped in the identification of the methylotrophic isolates as belonging to the genus Methylobacterium. Cell-free culture filtrates of these strains enhanced seed germination of wheat (Triticum aestivum) with highest values of 98.3% observed using Methylobacterium sp. (NC4). Highest values of seedling length and vigour were recorded with Methylobacterium sp. (NC28). HPLC analysis of production by bacterial strains ranged from 1.09 to 9.89μgml(-1) of cytokinins in the culture filtrate. Such cytokinin producing beneficial methylotrophs can be useful in developing bio-inoculants through co-inoculation of pink-pigmented facultative methylotrophs with other compatible bacterial strains, for improving plant growth and productivity, in an environment-friendly manner.

Genome-scale reconstruction and system level investigation of the metabolic network of Methylobacterium extorquensAM1

BackgroundMethylotrophic microorganisms are playing a key role in biogeochemical processes - especially the global carbon cycle - and have gained interest for biotechnological purposes. Significant progress was made in the recent years in the biochemistry, genetics, genomics, and physiology of methylotrophic bacteria, showing that methylotrophy is much more widespread and versatile than initially assumed. Despite such progress, system-level description of the methylotrophic metabolism is currently lacking, and much remains to understand regarding the network-scale organization and properties of methylotrophy, and how the methylotrophic capacity emerges from this organization, especially in facultative organisms.ResultsIn this work, we report on the integrated, system-level investigation of the metabolic network of the facultative methylotroph Methylobacterium extorquens AM1, a valuable model of methylotrophic bacteria. The genome-scale metabolic network of the bacterium was reconstructed and contains 1139 reactions and 977 metabolites. The sub-network operating upon methylotrophic growth was identified from both in silico and experimental investigations, and 13C-fluxomics was applied to measure the distribution of metabolic fluxes under such conditions. The core metabolism has a highly unusual topology, in which the unique enzymes that catalyse the key steps of C1 assimilation are tightly connected by several, large metabolic cycles (serine cycle, ethylmalonyl-CoA pathway, TCA cycle, anaplerotic processes). The entire set of reactions must operate as a unique process to achieve C1 assimilation, but was shown to be structurally fragile based on network analysis. This observation suggests that in nature a strong pressure of selection must exist to maintain the methylotrophic capability. Nevertheless, substantial substrate cycling could be measured within C2/C3/C4 inter-conversions, indicating that the metabolic network is highly versatile around a flexible backbone of central reactions that allows rapid switching to multi-carbon sources.ConclusionsThis work emphasizes that the metabolism of M. extorquens AM1 is adapted to its lifestyle not only in terms of enzymatic equipment, but also in terms of network-level structure and regulation. It suggests that the metabolism of the bacterium has evolved both structurally and functionally to an efficient but transitory utilization of methanol. Besides, this work provides a basis for metabolic engineering to convert methanol into value-added products.