Moderately haloalkaliphilic aerobic methylobacteria

Aerobic methylobacteria utilizing oxidized and substituted methane derivatives as carbon and energy sources are widespread in nature and involved in the global carbon cycle, being a unique biofilter on the path of these C1 compounds from different ecosystems to the atmosphere. New data on the biological features of moderately halophilic, neutrophilic, and alkaliphilic methylobacteria isolated from biotopes with higher osmolarity (seas, saline and soda lakes, saline soils, and deteriorating marble) are reviewed. Particular attention is paid to the latest advances in the study of the mechanisms of osmoadaptation of aerobic moderately haloalkaliphilic methylobacteria: formation of osmolytes, in particular, molecular and genetic aspects of biosynthesis of the universal bioprotectant ectoine. The prospects for further studies of the physiological and biochemical principles of haloalkalophily and for the application of haloalkaliphilic aerobic methylobacteria in biosynthesis and biodegradation are discussed.     

Phytosymbiosis of aerobic methylobacteria: New facts and views

This review highlightsrecent findings on the phytosymbiosis of aerobic methylobacteria, including their biodiversity, occurrence, and their role in associations with plants, as well as the capacity for biosynthesis of bioactive compounds (auxins, cytokinins, and vitamin B_l2) and nitrogen fixation. Future research directions in phytosymbiosis of aerobic methylobacteria during the postgenomics era are discussed.     

Methylophaga natronica sp. nov., a new alkaliphilic and moderately halophilic, restricted-facultatively methylotrophic bacterium from soda lake of the Southern Transbaikal region

A new, moderately haloalkaliphilic and restricted-facultatively methylotrophic bacterium (strain Bur2T) with the ribulose monophosphate pathway of carbon assimilation is described. The isolate, which utilizes methanol, methylamine and fructose, is an aerobic, Gram-negative, asporogenous, motile short rod multiplying by binary fission. It is auxotrophic for vitamin B12, and requires NaHCO3 or NaCl for growth in alkaline medium. Cellular fatty acids profile consists primarily of straight-chain saturated C16:0, unsaturated C16:1 and C18:1 acids. The major ubiquinone is Q-8. The dominant phospholipids are phosphatidylethanolamine and phosphatidylglycerol. Diphosphatidylglycerol is also present. Optimal growth conditions are 25-29 degrees C, pH 8.5-9.0 and 2-3% (w/v) NaCl. Cells accumulate ectoine and glutamate as the main osmoprotectants. The G + C content of the DNA is 45.0 mol%. Based on 16S rDNA sequence analysis and DNA-DNA relatedness (25-35%) with type strains of marine and soda lake methylobacteria belonging to the genus Methylophaga, the novel isolate was classified as a new species of this genus and named Methylophaga natronica (VKM B-2288T).     

Phosphate-solubilizing activity of aerobic methylobacteria

Phosphate-solubilizing activity was found in 14 strains of plant-associated aerobic methylobacteria belonging to the genera Methylophilus, Methylobacillus, Methylovorus, Methylopila, Methylobacterium, Delftia, and Ancyclobacter. The growth of methylobacteria on medium with methanol as the carbon and energy source and insoluble tricalcium phosphate as the phosphorus source was accompanied by a decrease in pH due to the accumulation of up to 7 mM formic acid as a methanol oxidation intermediate and by release of 120–280 μM phosphate ions, which can be used by both bacteria and plants. Phosphate-solubilizing activity is a newly revealed role of methylobacteria in phytosymbiosis.     

The Biology of Methylobacteria Capable of Degrading Halomethanes

Recent data on the biology of aerobic methylotrophic bacteria capable of utilizing toxic halogenated methane derivatives as sources of carbon and energy are reviewed, with particular emphasis on the taxonomic, physiological, and biochemical diversity of mono- and dihalomethane-degrading methylobacteria and the enzymatic and genetic aspects of their primary metabolism. The initial steps of chloromethane dehalogenation to formate and HCl through a methylated corrinoid and methyltetrahydrofolate are catalyzed by inducible cobalamin methyl transferase, made up of two proteins (CmuA and CmuB) encoded by the cmuA and cmuB genes. At the same time, the primary dehalogenation of dichloromethane to formaldehyde and HCl is catalyzed by cytosolic glutathione transferase with S-chloromethylglutathione as an intermediate. The latter enzyme is encoded by the structural dcmA gene and is under the negative control of the regulatory dcmR gene. In spite of considerable progress in the study of halomethane dehalogenation, some aspects concerning the structural and functional organization of this process and its regulation remain unknown, including the mechanisms of halomethane transport, the release of toxic dehalogenation products (S-chloromethylglutathione, CH₂O, and HCl) from cells, and the maintenance of intracellular pH. Of particular interest is a quantitative evaluation of the ecophysiological role of aerobic methylobacteria in the mineralization of halomethanes and the protection of the biosphere from these toxic pollutants.     

Production and optimization of a commercially viable alkaline protease from a haloalkaliphilic bacterium

Twenty five haloalkaliphilic bacterial strains were isolated from sea water along the Coastal Gujarat (India) and screened for their ability to secret alkaline proteases. Among them, a potent strain S-20-9 (GenBank accession number EU118360), resembling to Halophilic Bacterium MBIC3303 on the basis of 16S rRNA gene sequencing, was selected for the optimization of enzyme production. S-20-9 produced protease optimally, under aerobic conditions during mid-stationary phase over a broad range of salt (5∼25%, w/v) and pH (7∼10). The optimum production was at pH 9 and 15% (w/v) NaCl. The production was suppressed by lactose, maltose, sucrose, and inorganic nitrogen sources, especially ammonium ions. Further, the production was significantly stimulated by KH₂PO₄ and suppressed by glucose. Similarly, the production was also suppressed at higher concentrations of gelatin, yeast extract, peptone, and casamino acids, indicating towards a threshold value for nitrogen requirement. The growth and protease production were enhanced by mono-valent cation (KCl), while the divalent cations acted as inhibitors. The study holds significance as only few reports are available on the alkaline proteases from haloalkaliphilic bacteria, particularly those from moderate saline habitats.     

<Emphasis Type="Italic">Methylophaga murata</Emphasis> sp. nov.: a Haloalkaliphilic Aerobic Methylotroph from Deteriorating Marble

The haloalkaliphilic methylotrophic bacterium (strain Kr3) isolated from material scraped off the deteriorating marble of the Moscow Kremlin masonry has been found to be able to utilize methanol, methylamine, trimethylamine, and fructose as carbon and energy sources. Its cells are gram-negative motile rods multiplying by binary fission. Spores are not produced. The isolate is strictly aerobic and requires vitamin B₁₂ and Na⁺ ions for growth. It is oxidase- and catalase-positive and reduces nitrates to nitrites. Growth occurs at temperatures between 0 and 40°C (with the optimum temperatures being 20–32°C), pH values between 6 and 11 (with the optimum at 8–9), and NaCl concentrations between 0.05 and 3 M (with the optimum at 0.5–1.5 M). The dominant cellular phospholipids are phosphatidylethanolamine, phosphatidylglycerol, and cardiolipin. The major cellular fatty acids are palmitic (C_16:0), palmitoleic (C_16:1), and octadecenoic (C_18:1) acids. The major ubiquinone is Q₈. It accumulates ectoine and glutamate, as well as a certain amount of sucrose, to function as osmoprotectants and synthesizes an exopolysaccharide composed of carbohydrate and protein components. It is resistant to heating at 70°C, freezing, and drying; utilizes methanol, with the resulting production of formic acid, which is responsible for the marble-degrading activity of the isolate; and implements the 2-keto-3-deoxy-6-phosphogluconate variant of the ribulose monophosphate pathway. The G+C content of its DNA is 44.6 mol %. Based on 16S rRNA gene sequencing and DNA-DNA homology levels (23–41%) with neutrophilic and alkaliphilic methylobacteria from the genus Methylophaga, the isolate has been identified as a new species, Methylophaga murata (VKM B-2303^T = NCIMB 13993^T).__________Translated from Mikrobiologiya, Vol. 74, No. 4, 2005, pp. 511–519.Original Russian Text Copyright © 2005 by Doronina, Lee, Ivanova, Trotsenko.     

Biotechnological Potential of Aerobic Methylotrophic Bacteria: A Review of Current State and Future Prospects

Major results of the authors' findings on the implementation of the biotechnological potential of aerobic methylobacteria and methanotrophs for obtaining forage proteins, biopolymers (polybutyrate and polysaccharides), enzymes (oxidoreductases), and bioprotectors (ectoine), as well as for degrading toxic C₁ and C_n compounds, have been reviewed. Unique features of the structural and functional organization of the metabolism of extremophilic (tolerant) methylotrophs are discussed, with a view for their prospective use in various fields of modern biotechnology, including biocatalysis and nanotechnology.     

Methylophaga murata sp. nov.: a haloalkaliphilic aerobic methylotroph from deteriorating marble

The haloalkaliphilic methylotrophic bacterium (strain Kr3) isolated from material scraped off the deteriorating marble of the Moscow Kremlin masonry has been found to be able to utilize methanol, methylamine, trimethylamine, and fructose as carbon and energy sources. Its cells are gram-negative motile rods multiplying by binary fission. Spores are not produced. The isolate is strictly aerobic and requires vitamin B12 and Na+ ions for growth. It is oxidase- and catalase-positive and reduces nitrates to nitrites. Growth occurs at temperatures between 0 and 42 degrees C (with the optimum temperatures being 20-32 degrees C), pH values between 6 and 11 (with the optimum at 8-9), and NaCl concentrations between 0.05 and 3 M (with the optimum at 0.5-1.5 M). The dominant cellular phospholipids are phosphatidylethanolamine, phosphatidylglycerol, and cardiolipin. The major cellular fatty acids are palmitic (C16:0), palmitoleic (C16:1), and octadecenoic (C18:1) acids. The major ubiquinone is Q8. The isolate accumulates ectoine and glutamate, as well as a certain amount of sucrose, to function as osmoprotectants and synthesizes an exopolysaccharide composed of carbohydrate and protein components. It is resistant to heating at 70 degrees C, freezing, and drying; utilizes methanol, with the resulting production of formic acid, which is responsible for the marble-degrading activity of the isolate; and implements the 2-keto-3-deoxy-6-phosphogluconate variant of the ribulose monophosphate pathway. The G+C content of its DNA is 44.6 mol%. Based on 16S rRNA gene sequencing and DNA-DNA homology levels (23-41%) with neutrophilic and alkaliphilic methylobacteria from the genus Methylophaga, the isolate has been identified as a new species, Methylophaga murata (VKM B-2303T = NCIMB 13993T).     

The biology of aerobic methylobacteria capable of degrading halomethanes

Recent data on the biology of aerobic methylotrophic bacteria capable of utilizing toxic halogenated methane derivatives as sources of carbon and energy are reviewed, with particular emphasis on the taxonomic, physiological, and biochemical diversity of mono- and dihalomethane-degrading methylobacteria and the enzymatic and genetic aspects of their primary metabolism. The initial steps of chloromethane dehalogenation to formate and HCl through a methylated corrinoid and methyletrahydrofolate are catalyzed by inducible cobalamin methyl transferase, made up of two proteins (CmuA and CmuB) encoded by the cmuA and cmuB genes. At the same time, the primary dehalogenation of dichloromethane to formaldehyde and HCl is catalyzed by cytosolic glutathione transferase with S-chloromethylglutathione as an intermediate. The latter enzyme is encoded by the structural dcmA gene and is under the negative control of the regulatory dcmR gene. In spite of considerable progress in the study of halomethane dehalogenation, some aspects concerning the structural and functional organization of this process and its regulation remain unknown, including the mechanisms of halomethane transport, the release of toxic dehalogenation products (S-chloromethylglutathione, CH2O, and HCl) from cells, and the maintenance of intracellular pH. Of particular interest is quantitative evaluation of the ecophysiological role of aerobic methylobacteria in the mineralization of halomethanes and protection of the biosphere from these toxic pollutants.     

Enhanced Resistance of Pea Plants to Oxidative Stress Caused by Paraquat during Colonization by Aerobic Methylobacteria

The influence of colonization of the pea (Pisum sativum L.) by aerobic methylobacteria of five different species (Methylophilus flavus Ship, Methylobacterium extorquens G10, Methylobacillus arboreus Iva, Methylopila musalis MUSA, Methylopila turkiensis Side1) on plant resistance to paraquat-induced stresses has been studied. The normal conditions of pea colonization by methylobacteria were characterized by a decrease in the activity of antioxidant enzymes (superoxide dismutase, catalase, and peroxidases) and in the concentrations of endogenous H₂O₂, proline, and malonic dialdehyde, which is a product of lipid peroxidation and indicator of damage to plant cell membranes, and an increase in the activity of the photosynthetic apparatus (the content of chlorophylls а, b and carotenoids). In the presence of paraquat, the colonized plants had higher activities of antioxidant enzymes, stable photosynthetic indices, and a less intensive accumulation of the products of lipid peroxidation as compared to noncolonized plants. Thus, colonization by methylobacteria considerably increased the adaptive protection of pea plants to the paraquat-induced oxidative stress.     

Oxidative formation of lactic acid by a fungus

Experimental evidence has been presented for an aerobic biosynthesis of lactate from a C₂-precursor. The data indicate that the C₄-dicarboxylic acids function as intermediates; oxygen is essential. The aerobic process occurs concomitantly with glycolytic lactate synthesis and accounts for the extra lactate yield under vigorously aerobic conditions. A scheme is presented which accounts for the observed facts and it is evident that the oxidative synthesis of lactate in reality involves an oxidative synthesis of pyruvate. This latter synthesis is discussed from the point of view of its importance to autotrophic organisms, which must of necessity synthesize the important intermediate, pyruvate, from carbon fragments consisting of two carbons or less.     

Methylotrophic producers of bioplastics (Review)

This paper summarizes modern views on the physiological and biochemical properties of aerobic methylobacteria as producers of bioplastics and the prospects for their use in various areas of modern biotechnology.     

Biosynthesis of the bioprotectant ectoin by aerobic methylotrophic bacteria from methanol

It is shown that neutrophilic methylobacteria Methylophaga thalassica and M. marina have higher rates of growth and ectoin accumulation compared to the haloalkaliphilic species M. alcalica and M. natronica and methanotrophs Methylomicrobium alcaliphilum and M. kenyense. The conditions of M. thalassica cultivation in methanol-containing medium were optimized. The yield of this process reached 60 g/l of absolutely dry biomass containing 15–19% (9–11 g/l) ectoine. The scheme of ectoin isolation from the biomass by extraction and subsequent purification, which allowed obtaining preparations of different degree of purity, was developed.     

Comparative characteristics of biosynthesis of polyhydroxybutyrate from methanol by Methylobacteria extorquens G10 and Methyloligella halotolerans C2

The biosynthesis of polyhydroxybutyrate by Methylobacteria extorquens G10 and Methyloligella halotolerans C2 via the serine pathway of C1 metabolism was comparatively studied. Nitrogen limitation stimulated synthesis of the biopolymer in both cultures. It was shown that, despite the similarity of the pathways of methanol metabolism and those of polyhydroxybutyrate biosynthesis, the methylobacteria synthesized polymers of different molecular weights. In the case of M. extorquens G10, an increase in the content of the residual nitrogen in the culture medium was found to result in a reduction of the molecular weight of the polymer from 250 to 85 kDa, whereas M. halotolerans C2 synthesized a polymer of high molecular weight (approximately 3000 kDa) regardless of the residual content of the nitrogen source. It was established that the examined methylobacteria can utilize not only pure methanol but also a crude one, a feature that made it possible to significantly reduce the cost of the resulting polyhydroxybutyrate.     

Chemolithotrophic haloalkaliphiles from soda lakes

This paper summarizes recent data on the occurrence and properties of lithotrophic prokaryotes found in extremely alkaline, saline (soda) lakes. Among the chemolithotrophs found in these lakes the obligately autotrophic sulfur-oxidizing bacteria were the dominant, most diverse group, best adapted to haloalkaline conditions. The culturable forms are represented by three new genera, Thioalkalimicrobium, Thioalkalivibrio and Thioalkalispira in the Gammaproteobacteria. Among them, the genus Thioalkalivibrio was most metabolically diverse, including denitrifying, thiocyanate-oxidizing and facultatively alkaliphilic species. Culturable methane-oxidizing populations in the soda lakes belong to the type I methanotroph group in the Gammaproteobacteria, mostly in the genus Methylomicrobium. The nitrifying bacteria in hyposaline soda lakes were represented by a new species Nitrobacter alkalicus (Alphaproteobacteria), and by an alkaliphilic subspecies of Nitrosomonas halophila (Betaproteobacteria). Both belonged to the low salt-tolerant alkaliphiles. The facultatively autotrophic haloalkaliphilic isolates able to grow with hydrogen as electron donor were identified as representatives of the alpha-3 subclass of the Proteobacteria (aerobic) and of the Natronolimnicola - Alkalispirillum group in the gammaproteobacteria (nitrate-reducing). While all chemolithotrophic isolates from soda lakes belong to the alkaliphiles with a pH optimum for growth around 10, only the sulfur-oxidizing group included species able to grow under hypersaline conditions. This indicates that carbon and nitrogen cycles in the hypersaline alkaline lakes might not be closed.     

Carbon balance and water relations of sorghum exposed to salt and water stress

The daily (24 hour) changes in carbon balance, water loss, and leaf area of whole sorghum plants (Sorghum bicolor L. Moench, cv BTX616) were measured under controlled environment conditions typical of warm, humid, sunny days. Plants were either (a) irrigated frequently with nutrient solution (osmotic potential −0.08 kilojoules per kilogram = −0.8 bar), (b) not irrigated for 15 days, (c) irrigated frequently with moderately saline nutrient (80 millimoles NaCl + 20 millimoles CaCl₂·2H₂O per kilogram water, osmotic potential −0.56 kilojoules per kilogram), or (d) preirrigated with saline nutrient and then not irrigated for 22 days.

Under frequent irrigation, salt reduced leaf expansion and carbon gain, but water use efficiency was increased since the water loss rate was reduced more than the carbon gain. Water stress developed more slowly in the salinized plants and they were able to adjust osmotically by a greater amount. Leaf expansion and carbon gain continued down to lower leaf water potentials.

Some additional metabolic cost associated with salt stress was detected, but under water stress this was balanced by the reduced cost of storing photosynthate rather than converting it to new biomass. Reirrigation produced a burst of respiration associated with renewed synthesis of biomass from stored photosynthate.

It is concluded that although irrigation of sorghum with moderately saline water inhibits plant growth in comparison with irrigation with nonsaline water, it also inhibits water loss and allows a greater degree of osmotic adjustment, so that the plants are able to continue growing longer and reach lower leaf water potentials between irrigations.

     

The Biology and Osmoadaptation of Haloalkaliphilic Methanotrophs

There is increasing evidence for the presence and activity of methanotrophic bacteria in saline and alkaline aquatic environments located in different ecogeographical regions. Alkalitolerant halophilic and alkaliphilic halotolerant methanotrophs of type I were found to be able to utilize methane and methanol, to oxidize ammonium ions, and to transform various organic compounds in a wide range of water salinities (up to 12% NaCl) and pH values (from 5 to 11). The ecophysiological importance of methanotrophs in microbial communities inhabiting saline and alkaline aquatic environments is due to their involvement in the global cycles of methane and major bioelements (C, N, and S). Specific cyto- and biochemical properties of haloalkaliphilic methanotrophs—the synthesis of osmoprotectants (ectoine, 5-oxoproline, and sucrose), the accumulation of potassium ions, the formation of glycoprotein S-layers on the outer surface of their cell walls, and the modification of the chemical composition of their membranes—allow them to adapt to highly saline and alkaline habitats. Due to their specific properties, haloalkaliphilic methanotrophs may be of use in modern biotechnology.     

Changes of chlorine isotope composition characterize bacterial dehalogenation of dichloromethane

Fractionation of dichloromethane (DCM) molecules with different chlorine isotopes by aerobic methylobacteria Methylobacterium dichloromethanicum DM4 and Albibacter nethylovorans DM10; cell-free extract of strain DM4; and transconjugant Methylobacterium evtorquens Al1/pME 8220, expressing the dcmA gene for DCM dehalogenase but unable to grow on DCM, was studied. Kinetic indices of DCM isotopomers for chlorine during bacterial dehalogenation and diffusion were compared. A two-step model is proposed, which suggests diffusional DCM transport to bacterial cells.     

The rate of permafrost carbon release under aerobic and anaerobic conditions and its potential effects on climate

Recent observations suggest that permafrost thaw may create two completely different soil environments: aerobic in relatively well‐drained uplands and anaerobic in poorly drained wetlands. The soil oxygen availability will dictate the rate of permafrost carbon release as carbon dioxide (CO₂) and as methane (CH₄), and the overall effects of these emitted greenhouse gases on climate. The objective of this study was to quantify CO₂ and CH₄ release over a 500‐day period from permafrost soil under aerobic and anaerobic conditions in the laboratory and to compare the potential effects of these emissions on future climate by estimating their relative climate forcing. We used permafrost soils collected from Alaska and Siberia with varying organic matter characteristics and simultaneously incubated them under aerobic and anaerobic conditions to determine rates of CO₂ and CH₄ production. Over 500 days of soil incubation at 15 °C, we observed that carbon released under aerobic conditions was 3.9–10.0 times greater than anaerobic conditions. When scaled by greenhouse warming potential to account for differences between CO₂ and CH₄, relative climate forcing ranged between 1.5 and 7.1. Carbon release in organic soils was nearly 20 times greater than mineral soils on a per gram soil basis, but when compared on a per gram carbon basis, deep permafrost mineral soils showed carbon release rates similar to organic soils for some soil types. This suggests that permafrost carbon may be very labile, but that there are significant differences across soil types depending on the processes that controlled initial permafrost carbon accumulation within a particular landscape. Overall, our study showed that, independent of soil type, permafrost carbon in a relatively aerobic upland ecosystems may have a greater effect on climate when compared with a similar amount of permafrost carbon thawing in an anaerobic environment, despite the release of CH₄ that occurs in anaerobic conditions.