Adaptation of aerobic methylobacteria to dichloromethane degradation

A shortening of the lag phase in dichloromethane (DCM) consumption was observed in the methylobacteria Methylopila helvetica DM6 and Albibacter methylovorans DM10 after prior growth on methanol with the presence of 1.5% NaCl. Neither heat nor acid stress accelerated methylobacterium adaptation to DCM consumption. Sodium azide (1 mM) and potassium cyanide (1 mM) inhibited consumption of DCM by these degraders but not by transconjugants Methylobacterium extorquens AM1, expressing DCM dehalogenase but unable to grow on DCM. This indicates that the degrader strains possess energy-dependent systems of transport of DCM or chloride anions produced during DCM dehalogenation. Inducible proteins were found in the membrane fraction of A. methylovorans DM10 cells adapted to DCM and elevated NaCl concentration.     

Effect of DNA-damaging agents on the aerobic methylobacteria capable and incapable of utilizing dichloromethane

Methylobacterium dichloromethanicum DM4, a degrader of dichloromethane (DCM), was more tolerant to the effect of H2O2 and UV irradiation than Methylobacterium extorquens AM1, which does not consume DCM. Addition of CH2Cl2 to methylobacteria with active serine, ribulose monophosphate, and ribulose bisphosphate pathways of C1 metabolism, grown on methanol, resulted in a 1.1- to 2.5-fold increase in the incorporation of [alpha-32P]dATP into DNA Klenow fragment (exo-). As DCM dehalogenase was not induced in this process, the increase in total lengths of DNA gaps resulted from the action of DCM rather than S-chloromethylglutathione (intermediate of primary dehalogenation). The degree of DNA damage in the presence of CH2Cl2 was lower in DCM degraders than methylobacteria incapable of degrading this pollutant. This suggests that DCM degraders possess a more efficient mechanism of DNA repair.     

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.     

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.     

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.     

Physiological and biochemical analysis of the transformants of aerobic methylobacteria expressing the dcm A gene of dichloromethane dehydrogenase

The transformants of Methylobacterium dichloromethanicum DM4 (DM4-2cr-/pME8220 and DM4-2cr-/pME8221) and of Methylobacterium extorquens AM1 (AM1/pME8220 and AM1/pME8221) that express the dcm A gene of dichloromethane dehalogenase undergo lysis when incubated in the presence of dichloromethane and are sensitive to acidic shock. The lysis of the transformants was found to be related neither to the accumulation of Cl- ions, CH2O, and HCOOH, nor to the impairment of glutathione synthesis or to the maintenance of intracellular pH. The (exo-) Klenow fragment-mediated incorporation of [alpha-32P]dATP into the DNA of the transformants DM4-2cr-/pME8220 and AM1/pME8220 was considerably greater when the transformed cells were incubated with CH2Cl2 than when they were incubated with CH3OH, indicating the occurrence of a significant increase in the total length of gaps. At the same time, the strain AM1 (which lacks dichloromethane dehalogenase) and the dichloromethane-degrading strain DM4 incubated with CH2Cl2 showed an insignificant increase in the total length of the gaps. The transformed cells are likely to lyse due to the relatively inefficient repair of DNA lesions that are induced in response to the alkylating action of S-chloromethylglutathione, an intermediate product of CH2Cl2 degradation. The data obtained suggest that the bacterial mineralization of dichloromethane requires an efficient DNA repair system.     

The aerobic degradation of dichloromethane: Structural-functional aspects (a review)

This review summarizes current notions on the mechanisms of transport and degradation of dichloromethane (DCM) by aerobic methylotrophic bacteria as well as enzymological and genetic aspects of DCM dehalogenation, including probable pathways used by cells to overcome accompanying stresses (acid, osmotic, and oxidative). The topicality of the problem of the degradation of this genotoxic solvent is associated with the search for and creation of new DCM-destroying strains, which would provide for more efficient bioremediation of industrial sewage and ecosystems with extreme pH and salinity and could be used for the development of modern techniques for DCM degradation on the basis of existing strains. Special attention is given to the consideration of methodological approaches to the interpretation of physiological—biochemical and molecular bases of adaptation of bacteria to the utilization of DCM and other halogenated pollutants.     

Aerobic methylobacteria as the basis for a biosensor for dichloromethane detection

Cells of dichloromethane (DChM) bacteria-destructors were immobilized by sorption on different types of membranes, which were fixed on the measuring surface of a pH-sensitive field transistor. The presence of DChM in the medium (0.6–8.8 mM) led to a change in the transistor’s output signal, which was determined by the appearance of H⁺ ions in the medium due to DChM utilization by methylobateria. Among four strains of methylobacteria—Methylobacterium dichloromethanicum DM4, Methylobacterium extorquens DM17, Methylopila helvetica DM6, and Ancylobacter dichloromethanicus DM16—the highest and most stable activity toward DChM degradation was observed in the strain M. dichloromethanicum DM4. Among 11 types of membranes for cell immobilization, Millipore nitrocellulose membranes and chromatographic fiber paper GF/A, which allow one to obtain stable biosensor signals for 2 weeks without a bioreceptor change, were chosen as optimal carriers.     

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.     

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.     

Halophilic and halotolerant aerobic methylobacteria from the technogenic Solikamsk biotopes

Seven strains of moderately halophilic and halotolerant aerobic methylobacteria from the technogenic Solikamsk biotopes (Perm krai, Russia) were isolated in pure cultures and characterized. The isolates were represented by gram-negative and gram-positive (strain 2395B) cells. All the cells were shown to multiply by binary fission without formation of spores or prosthecae. All isolates except strain 2395B were able to oxidize methanol by a classical methanol dehydrogenase. The ribulose monophosphate (RMP) (strain LS), serine (strains S12, S3, 2395A), or ribulose bisphosphate (strains SK15 and S3270) pathways of C₁-assimilation were used. In strain 2395B, the key enzymes of the RMP and serine metabolic pathways were determined. Using polyphasic taxonomy, three strains were identified as representatives of the known species: Arthrobacter protophormiae 2395B, Methylophaga thalassica LS, and Ancylobacter rudongensis S3270. Three more strains were identified as members of new species: Methylopila oligotropha sp. nov. (strain 2395AT; VKM B-2788^T = CCUG 63805^T), Ancylobacter defluvii sp. nov. (strain SK15^T; VKM B-2789^T = CCUG 63806^T), and Paracoccus communis sp. nov. (strain S3^T; VKM B-2787^T = CCUG 63804^T). According to the results of 16S rRNA gene sequencing, the obligately methylotrophic strain S12 had less than 94% similarity with the known genera of the Proteobacteria and was probably a representative of a novel genus.     

Cloning and characterization of dichloromethane dehalogenase from Methylobacterium rhodesianum for dichloromethane degradation

The gene encoding dichloromethane dehalogenase from Methylobacterium rhodesianum was cloned. Bioinformatic analysis showed that dichloromethane dehalogenase gene sequence from M. rhodesianum is almost identical to the one from Methylobacterium extorquens, with only one base difference. Dichloromethane dehalogenase was subsequently expressed in Escherichia coli BL21 (DE3) and purified. It was found that enzyme activity in recombinant cells was 3 times higher than that in the wild-type M. rhodesianum. Further investigation showed that recombinant dichloromethane dehalogenase was most active at 40°C at pH 7–8, and its K_M was 10.96 mM when treated with dichloromethane as substrate. The fitted curve of dichloromethane degradation gave a V_max of 0.43 mM/h of in 0.01 M phosphate buffer. Degradation efficiency of dichloromethane reached 86.11% within 20 h. In addition, it was found that degradation efficiency of dichloromethane was highly associated with glutathione concentration, supporting the reports that glutathione functions as coenzyme of dichloromethane dehalogenase for dichloromethane degradation.     

Biodiversity of aerobic methylobacteria associated with the phyllosphere of the southern Moscow region

During the summer period (15–25°C), 34 strains of methylotrophic bacteria associated with different species of herbs, shrub, and trees in Pushchino (Moscow oblast, Russia) were isolated on the medium with methanol. Predominance of pink-colored Methylobacterium strains in the phyllosphere of many plants was confirmed by microscopy, enumeration of the colonies from grass leaves, and sequencing of the 16S rRNA genes. Colorless and yellow-pigmented methylotrophs belonged to the genera Methylophilus, Methylobacillus, Hansschlegelia, Methylopila, Xanthobacter, and Paracoccus. All isolates were able to synthesize plant hormones auxins from L-tryptophan (5?50 μg/mL) and are probably plant symbionts.     

Phylogenetic analysis of aerobic methylotrophic bacteria, using dichloromethane

The phylogenetic relationships of 12 aerobic dichloromethane-degrading bacteria that implement different C1-assimilation pathways was determined based on 16S ribosomal RNA sequences and DNA-DNA hybridization data. The restricted facultative methylotroph "Methylophilus leisingerii" DM11 with the ribulose monophosphate pathway was found to belong to the genus Methylophilus cluster of the beta subdivision of the phylogenetic kingdom Proteobacteria. The facultative methylotroph Methylorhabdus multivorans DM13 was assigned to a separate branch of the alpha-2 group of Proteobacteria. Paracoccus methylutens DM12, which utilizes C1-compounds via the Calvin cycle was found to belong to the alpha-3 group of the Proteobacteria (more precisely, to the genus Paracoccus cluster). Thus, phylogenetic analysis confirmed the taxonomic status of these recently characterized bacteria. According to the degree of DNA homology, several novel strains of methylotrophic bacteria were divided into three genotypic groups within the alpha-2 group of the Proteobacteria. Genotypic group 1, comprising strains DM1, DM3, and DM5 through DM9, and genotypic group 3, comprising strain DM10, were phylogenetically close to the methylotrophic bacteria of the genus Methylopila, whereas genotypic group 2 (strain DM4) was close to bacteria of the genus Methylobacterium. The genotypic groups obviously represent distinct taxa of methylotrophic bacteria, whose status should be confirmed by phenotypic analysis.     

Bacterial degradation of dichloromethane in cultures and natural environments

Dichloromethane (DCM) is a toxic pollutant showing prolonged persistence in water. DCM biodegradation is usually determined from increases in Cl ions, gas chromatography, or by using radioisotopes. Herein, we present an original and easy spectrophotometric method to estimate DCM concentrations in cultures and environmental samples during DCM biodegradation experiments.     

Analysis of the key functional genes in new aerobic degraders of dichloromethane

The genes of dichloromethane (CH₂C1₂, DCM) degradation have been characterized in the aerobic degraders “Gottschalkia methylica” DM15, “Ancylobacter dichloromethanicus” DM16, and Methylobac- terium extorquens DM17, isolated from different regions of Russia. The sequencing of the structural gene dcmA of DCM dehalogenase, followed by phylogenetic analysis, showed that the new degraders possess A-type dehalogenases. The DcmAs of the strains DM15 and DM17 were identical to the known orthologous proteins of Methylorhabdus multivorans DM 13 and Methylobacterium dichloromethanicum DM4, respectively. DcmA of the degrader DM16 differed by three amino acid substitutions from DcmA of strain DM4. In agreement with the organization of the cluster of DCM degradation genes in M. dichloromethanicum DM4, the regulatory gene dcmR and the open reading frame orf353, flanking dcmA, were identified in the new degraders. The similarity of DCM degradation genes in aerobic degraders of different taxonomic position and geographical origin suggests their distribution among methylotrophic bacteria by means of horizontal transfer.     

Stable carbon isotope fractionation during degradation of dichloromethane by methylotrophic bacteria

Stable carbon isotope fractionation during dichloromethane (DCM) degradation by methylotrophic bacteria was investigated under aerobic and nitrate-reducing conditions. The strains studied comprise several Hyphomicrobium strains, Methylobacterium, Methylopila, Methylophilus and Methylorhabdus spp. that are considered to degrade DCM by a glutathione (GSH)-dependent dehalogenase enzyme system in the initial step. The stable carbon isotope fractionation factors (alphaC) of the strains varied under aerobic conditions between 1.043 and 1.071 and under nitrate-reducing conditions between 1.048 and 1.065. Comparison of isotope fractionation under aerobic and nitrate-reducing conditions by individual strains revealed only minor to no differences. The variability in isotope fractionation among strains was found to be related to the polymorphism of the functional genes encoding the DCM dehalogenase.