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[Phylogenetic analysis of facultative methylotrophic bacteria of the genus Methylobacterium].

Phylogenetic analysis of Methylobacterium genus species using analysis of nucleotide sequence of 16S rRNA genes has shown that Methylobacterium zatmanii and M. rhodesianum possess high-level similarity with M. extorquens (99.4-98.8%), and M. fujisawaense and M. radiotolerans--with M. mesophilicum (98.9-97.8%). These species are also similar as to their phenotypical properties and their total DNAs have also close similarity. Thus, the right of M. zatmanii, M. rhodesianum, M. fujisawaense and M. radiotolerans species for existence needs confirmation. Some phenotypical properties which were assummed as a basis for formation of certain species of the genus Methylobacterium (radioresistance, e.g.) cannot serve as the differentiating characteristic of this genus species. High resistance to gamma- and UV-radiation is a specific sign of representatives of Methylobacterium genus.

Base Sequence↗

PubMed1988

Plasmid analysis and cloning of the dichloromethane-utilization genes of Methylobacterium sp. DM4.

The dichloromethane (DCM)-utilizing facultative methylotroph Methylobacterium sp. DM4 was shown to contain three plasmids with approximate size of 120 kb, 40 kb and 8 kb. Curing experiments suggested that the DCM-utilization character was correlated with the possession of an intact 120 kb plasmid. The DCM-utilization genes were cloned on the broad-host-range vector pVK100. Plasmid pME1510, a recombinant plasmid carrying a 21 kb HindIII fragment complemented DCM-utilization-negative derivatives of Methylobacterium sp. DM4 and conferred the DCM-utilization-positive phenotype to a number of Gram-negative methylotrophic bacteria. In Southern hybridization experiments with pMe1510 as a probe, chromosomal DNA from Methylobacterium sp. DM4 gave definite signals while purified plasmid DNA did not. Plasmid pME1510 did not hybridize with total DNA from a cured DCM-non-utilizing derivative of Methylobacterium sp. DM4. It is concluded that the DCM-utilization genes are located on the chromosome or on a megaplasmid. Curing procedures thus led to the formation of a chromosomal or megaplasmid deletion larger than 21 kb and covering the DCM-utilization genes or to the loss of an undetected megaplasmid.

Chromosomes, Bacterial↗

PubMed2006

Regulation of ethylene levels in canola (Brassica campestris) by 1-aminocyclopropane-1-carboxylate deaminase-containing Methylobacterium fujisawaense.

We report the presence of ACC deaminase in Methylobacterium fujisawaense and its lowering of ethylene levels and promotion of root elongation in canola seedlings under gnotobiotic conditions. To test a part of the previous model proposed for ACC deaminase producing bacteria with Methylobacterium, ACC levels and various enzyme activities were monitored in canola. Lower amounts of ACC were present in the tissues of seeds treated with M. fujisawaense strains than in control seeds treated with MgSO(4). Though the increased activities of ACC synthase in the tissue extracts of the treated seedlings might be due to bacterial indole-3-acetic acid, the amount of ACC was reduced due to bacterial ACC deaminase activity. The activities of ACC oxidase, the enzyme catalyzing conversion of ACC to ethylene remained lower in M. fujisawaense treated seedlings. This consequently lowered the ethylene in plants and prevented ethylene inhibition of root elongation. Our results collectively suggest that Methylobacterium commonly found in soils, as well as on the surfaces of leaves, seeds, and in the rhizosphere of a wide variety of plants could be better exploited to promote plant growth.

Brassica↗

PubMed2006

Characterizing NAD-dependent alcohol dehydrogenase enzymes of Methylobacterium extorquens and strawberry (Fragaria x ananassa cv. Elsanta).

The methylotroph Methylobacterium extorquens (strain with CABI registration number IMI 369321), which has been isolated from strawberry (Fragaria x ananassa cv. Elsanta) callus cultures, was grown on a mixture of methanol (0.25% v/v) and 1,2-propanediol (0.75% v/v). The microbial biotransformation of 1,2-propanediol to 2-hydroxypropanal (lactaldehyde) was studied. The bacterial alcohol dehydrogenase (ADH) enzymatic activities were assessed, and the optimum pH for ADH activity was found to be pH 6.0. Enzyme assays were carried out for both the bacterial and the strawberry extracts to define the best substrate specificity. For Methylobacterium extorquens, the best substrates were found to be methanol (Km = 0.78 mM) and 1,2-propanediol (Km = 15.84 mM), whereas for strawberries, 1-propanol (Km = 3.54 mM) and ethanol (Km = 6.66 mM) were the best substrates. A wide variety of metals as well as EDTA were shown to decrease the enzymatic activity. Furthermore, SDS-PAGE experiments showed molecular weights of 45.0 and 24.6 kDa for the alcohol dehydrogenases of Methylobacterium extorquens and Fragaria x ananassa, respectively.

Alcohol Dehydrogenase↗

PubMed2002

A Methylobacterium-like organism from algal crusts covering silicone rubber electric insulators in Africa.

AIMS: The primary goals of this study were to isolate, identify and characterize culturable bacteria living in a close association with microalgae within green crusts covering silicone rubber electric insulators in Tanzania. METHODS AND RESULTS: Twenty-four bacterial colonies were isolated from an Apatococcus crust. Characterization by statistical analyses of total cellular protein profiles demonstrated that they were highly similar to one another. Final identification was achieved using 16S rDNA sequencing and fatty acid methyl ester profiling. These analyses revealed the presence of microbes with high similarity to Methylobacterium radiotolerans. The selected isolate, A1, displayed strong inhibitory activity against Rhizoctonia solani and was found to be resistant to relatively high concentrations of zinc in the growth medium. CONCLUSIONS: This study revealed the presence of M. radiotolerans bacteria in a novel environment--within algal crusts formed on electrical insulators in Africa. Moreover, this bacterium was found to be a predominant culturable species within those complex algal-microbial associations. The isolate also shared some traits of biotechnological importance with other members of the Methylobacterium genus. SIGNIFICANCE AND IMPACT OF THE STUDY: The data presented provide a valuable contribution concerning the formation and function of associations between green microalgae and bacteria. This study also provides some information about the utility of bacteria from the genus Methylobacterium in biotechnological applications, such as biocontrol of rhizoctoniosis and bioremediation of heavy metal-contaminated soils.

Chlorophyta↗

PubMed2005

Methylotrophic metabolism is advantageous for Methylobacterium extorquens during colonization of Medicago truncatula under competitive conditions.

Facultative methylotrophic bacteria of the genus Methylobacterium are commonly found in association with plants. Inoculation experiments were performed to study the importance of methylotrophic metabolism for colonization of the model legume Medicago truncatula. Competition experiments with Methylobacterium extorquens wild-type strain AM1 and methylotrophy mutants revealed that the ability to use methanol as a carbon and energy source provides a selective advantage during colonization of M. truncatula. Differences in the fitness of mutants defective in different stages of methylotrophic metabolism were found; whereas approximately 25% of the mutant incapable of oxidizing methanol to formaldehyde (deficient in methanol dehydrogenase) was recovered, 10% or less of the mutants incapable of oxidizing formaldehyde to CO2 (defective in biosynthesis of the cofactor tetrahydromethanopterin) was recovered. Interestingly, impaired fitness of the mutant strains compared with the wild type was found on leaves and roots. Single-inoculation experiments showed, however, that mutants with defects in methylotrophy were capable of plant colonization at the wild-type level, indicating that methanol is not the only carbon source that is accessible to Methylobacterium while it is associated with plants. Fluorescence microscopy with a green fluorescent protein-labeled derivative of M. extorquens AM1 revealed that the majority of the bacterial cells on leaves were on the surface and that the cells were most abundant on the lower, abaxial side. However, bacterial cells were also found in the intercellular spaces inside the leaves, especially in the epidermal cell layer and immediately underneath this layer.

Alcohol Oxidoreductases↗

PubMed2002

Chloromethane-induced genes define a third C1 utilization pathway in Methylobacterium chloromethanicum CM4.

Methylobacterium chloromethanicum CM4 is an aerobic alpha-proteobacterium capable of growth with chloromethane as the sole carbon and energy source. Two proteins, CmuA and CmuB, were previously purified and shown to catalyze the dehalogenation of chloromethane and the vitamin B12-mediated transfer of the methyl group of chloromethane to tetrahydrofolate. Three genes located near cmuA and cmuB, designated metF, folD and purU and encoding homologs of methylene tetrahydrofolate (methylene-H4folate) reductase, methylene-H4folate dehydrogenase-methenyl-H4folate cyclohydrolase and formyl-H4folate hydrolase, respectively, suggested the existence of a chloromethane-specific oxidation pathway from methyl-tetrahydrofolate to formate in strain CM4. Hybridization and PCR analysis indicated that these genes were absent in Methylobacterium extorquens AM1, which is unable to grow with chloromethane. Studies with transcriptional xylE fusions demonstrated the chloromethane-dependent expression of these genes. Transcriptional start sites were mapped by primer extension and allowed to define three transcriptional units, each likely comprising several genes, that were specifically expressed during growth of strain CM4 with chloromethane. The DNA sequences of the deduced promoters display a high degree of sequence conservation but differ from the Methylobacterium promoters described thus far. As shown previously for purU, inactivation of the metF gene resulted in a CM4 mutant unable to grow with chloromethane. Methylene-H4folate reductase activity was detected in a cell extract of strain CM4 only in the presence of chloromethane but not in the metF mutant. Taken together, these data provide evidence that M. chloromethanicum CM4 requires a specific set of tetrahydrofolate-dependent enzymes for growth with chloromethane.

Amidohydrolases↗

PubMed1995

A comparison of organochlorine removal from bleached kraft pulp and paper-mill effluents by dehalogenating Pseudomonas, Ancylobacter and Methylobacterium strains.

The relative importance of each of three dechlorinating species to overall organochlorine removal from bleached kraft-mill effluents (BKME) was assessed. Ancylobacter aquaticus A7, Pseudomonas P1, and Methylobacterium CP13, strains indigenous to a BKME treatment system, were tested for growth on chlorinated acetic acids and alcohols, and for adsorbable organic halogen (AOX) reduction in batch cultures of sterile BKME from three sources. A. aquaticus A7 exhibited the broadest substrate range, but could only affect significant AOX reduction in softwood effluents. Methylobacterium CP13 exhibited a limited substrate range, but was capable of removing significant amounts of AOX from both hardwood and softwood effluents. By contrast, Pseudomonas sp. P1 exhibited a limited substrate range and poor to negligible reductions in AOX levels from both effluent types. Mixed inocula of all three species combined and inocula of sludge from mill treatment systems removed as much AOX from softwood effluents as did pure populations of Methylobacterium CP13. When BKME was hydrolysed prior to AOX analysis, the subsequent estimates of recalcitrant, or non-hydrolysable, AOX levels were far less variable than their counterpart total AOX measures. It is suggested that this is a relevant and useful measure of AOX for biodegradation studies.

Biodegradation, Environmental↗

PubMed1993

Identification of methanol-regulated promoter sequences from the facultative methylotrophic bacterium Methylobacterium organophilum XX.

A promoter-probe vector (pHX200) was constructed using the broad-host-range cosmid pLA2917 and a promoterless xylE gene of Pseudomonas as the reporter gene. Insertion of the cloned promoter fragment of the methanol dehydrogenase large subunit gene moxF (methanol oxidation) in front of the xylE gene in pHX200V-47 resulted in high-level expression of the xylE gene product--catechol 2,3-dioxygenase--in Methylobacterium organophilum XX. The specific activity of the enzyme was four times higher in methanol-grown M. organophilum XX culture than in succinate-grown culture. Interestingly, the insertion of the same fragment in the opposite orientation in front of the xylE gene (pHX200V-74) also led to elevated catechol 2,3-dioxygenase activity. This promoter activity was also methanol regulated. A total of 21 methanol-regulated promoter clones were identified that originate from three gene clusters (groups V, VI and VII) on the M. organophilum XX chromosome involved in methanol oxidation. Vector pHX200 and its derivatives were successfully mobilized into cells of three phylogenetically diverse methylotrophic bacteria: Methylophilus methylotrophus AS1, Methylobacterium extorquens AM1 and Methylobacterium sp. DM4. The reporter gene (xylE) was functionally expressed in all three bacteria with the aid of a proper promoter. Transcriptional fusions of methanol-regulated promoters with the xylE gene were mobilized into Mox- mutants of M. organophilum XX and M. extorquens AM1 to study the roles of methanol oxidation genes, especially regulatory genes. It appeared that vector pHX200 is an efficient promoter probe with wide host-range and an excellent tool for studies of structure and function of promoters/regulators.

Base Sequence↗

PubMed1992

Susceptibility testing of clinical isolates of Methylobacterium species.

Methylobacterium species represent a relatively new genus which is being increasingly isolated from cases of opportunistic infections. This study reports on 3 reference strains and 15 clinical isolates of Methylobacterium species. Susceptibility tests were performed by the agar dilution and commercial broth microdilution methods at both 30 and 35 degrees C. Readings were made at 24, 48, and 72 h. Incubation conditions of 48 h and 30 degrees C were found to be optimum. Both the agar dilution and broth microdilution methods gave equivalent results. Drugs tested and their MICs for 90% of isolates (in micrograms per milliliter) were as follows: amikacin, less than or equal to 1; gentamicin, 1; ciprofloxacin, 1; trimethoprim-sulfamethoxazole, 2/38; ceftriaxone, 16; and ceftizoxime, 16. The majority of our isolates were resistant to six other beta-lactam drugs tested. Nine of the 15 Methylobacterium isolates were beta-lactamase positive.

Anti-Bacterial Agents↗

PubMed1986

Isolation and complementation analysis of 10 methanol oxidation mutant classes and identification of the methanol dehydrogenase structural gene of Methylobacterium sp. strain AM1.

A method has been developed for the direct selection of methanol oxidation mutants of the facultative methylotroph Methylobacterium sp. strain AM1 (formerly Pseudomonas sp. strain AM1). Using this direct selection technique, we have isolated mutants of Methylobacterium sp. strain AM1 that are no longer capable of growth on methanol but retain the ability to grow on methylamine. These methanol oxidation (Mox) mutants were complemented with a genomic clone bank of this organism constructed in the broad-host-range cosmid pVK100, and subcloning and Tn5 mutagenesis experiments have assigned the Mox mutants to 10 distinct complementation groups. Using an open reading frame beta-galactosidase fusion vector and antibodies specific for Methylobacterium sp. strain AM1 methanol dehydrogenase, we have identified the methanol dehydrogenase structural gene and determined the direction of transcription. The results suggest that the synthesis and utilization of an active methanol dehydrogenase in this organism requires at least 10 different gene functions.

1-Propanol↗

PubMed1993

[Isolation of Methylobacterium spp. from drinking tank-water and resistance of isolates to chlorine].

On bacteriological examination of 100 samples of drinking tank water, standard plate count bacteria (36 degrees C, 24 h) and coliforms were not detected, conforming to the water quality criteria under the Water Supply Law. Ninety-five percent of test samples had concentrations of residual chlorine exceeding 0.1 mg/l. However, one characteristic heterotroph was isolated from 70% of these water samples. The bacteria showed glucose non-fermentative Gram-negative rods and formed pink colonies when cultured on standard agar medium at 30 degrees C for 7 days. It was oxidase-positive, catalase-positive and motile. Furthermore, utilization of methyl alcohol was a characteristic. From these characteristics, it was identified as genus Methylobacterium. The isolated Methylobacterium of 118 strains were divided into 60 types using 20 biochemical tests. Thirty Methylobacterium strains were examined for tolerance to chloride by contact with free residual chlorine of 0.1 mg/l concentration for 5 min. Considerable resistance to residual chlorine was evident. The TW-7 strain showed especially high tolerance, even surviving contact at 1.0 mg/l concentration of free residual chlorine for 10 minutes.

Chlorine↗

PubMed2004

Development of an insertional expression vector system for Methylobacterium extorquens AM1 and generation of null mutants lacking mtdA and/or fch.

Over the past few years, the genetic 'toolkit' available for use with Methylobacterium extorquens AM1 has expanded significantly. Here a further advance is presented and demonstrated, an insertional expression system that allows expression of genes from a stable, unmarked chromosomal locus. This system has been used to better understand the role of the tetrahydrofolate (H4F) pathway in methylotrophy. Previously, it has not been possible to generate null mutants lacking either mtdA (encoding an NADP-dependent methylene-H4F/methylene-tetrahydromethanopterin dehydrogenase) or fch (encoding methenyl-H4F cyclohydrolase). An unmarked strain was generated that expressed the analogous folD gene (encoding a bifunctional NADP-dependent methylene-H4F dehydrogenase/methenyl-H4F cyclohydrolase) from Methylobacterium chloromethanicum CM4T. In this strain, null mutants could be obtained that grew normally on multicarbon substrates but were defective for growth on C1 substrates. Additionally, null mutants of mtdA and/or fch could also be generated in the wild-type by supplementing the succinate medium with formate. These strains were unable to grow on C1 compounds but were not methanol-sensitive. These approaches have demonstrated that the apparent essentiality of mtdA and fch is due to the need for formyl-H4F for biosynthesis of purines and other compounds, and have provided clear genetic evidence that the H4F pathway is required for methylotrophy.

Formates↗

PubMed2006

Production of polyhydroxyalkanoates from methanol by a new methylotrophic bacterium Methylobacterium sp. GW2.

A new bacterial strain, isolated from groundwater contaminated with explosives, was characterized as a pink-pigmented facultative methylotroph, affiliated to the genus Methylobacterium. The bacterial isolate designated as strain GW2 was found capable of producing the homopolymer poly-3-hydroxybutyrate (PHB) from various carbon sources such as methanol, ethanol, and succinate. Methanol acted as the best substrate for the production of PHB reaching 40 % w/w dry biomass. PHB accumulation was observed to be a growth-associated process, so that there was no need for two-step fermentation. Optimal growth occurred at 0.5 % (v/v) methanol concentration, and growth was strongly inhibited at alpha concentration above 2 % (v/v). Methylobacterium sp. strain GW2 was also able to accumulate the copolyester poly-3-hydroxybutyrate-poly-3-hydroxyvalerate (PHB/HV) when valeric acid was supplied as an auxiliary carbon source to methanol. After 66 h, a copolymer content of 30 % (w/w) was achieved with a PHB to PHV ratio of 1:2. Biopolymers produced by strain GW2 had an average molecular weight ranging from 229,350 to 233,050 Da for homopolymer PHB and from 362,430 to 411,300 Da for the copolymer PHB/HV.

Biopolymers↗

PubMed2007

Intercellular colonization and growth promoting effects of Methylobacterium sp. with plant-growth regulators on rice (Oryza sativa L. Cv CO-43).

The Methylobacterium sp. strain NPFM-SB3, isolated from Sesbania rostrata stem nodules possessed nitrogenase activity and nodA genes. Pure culture of NPFM-SB3 strain produced indole-3-acetic acid, cytokinins and on inoculation to rice plants resulted in numerous lateral roots. Inoculation of synthetic auxins 2,4-dichlorophenoxy acetic acid, naphthalene acetic acid or flavonoids naringenin and dihydroxy-4-methoxyisoflavone individually or to bacterial inoculated rice seedlings improved the plant growth and lateral root formation under hydroponic condition. The formation of nodule-like structure and nitrogenase activity which is purely auxin dependent was observed in 2,4-dichlorophenoxy acetic acid treatments to Methylobacterium sp. NPFM-SB3 inoculated rice plants. The rhizobia entered through fissures formed due to lateral root emergence and spread intercellularly in the nodular structures concluded that the effect of 2,4-dichlorophenoxy acetic acid treatment for rice seedlings grown under gnotobiotic conditions is to create a niche in which these bacteria can grow.

Bacterial Proteins↗

PubMed2005

Rapid, specific and quantitative assays for the detection of the endophytic bacterium Methylobacterium mesophilicum in plants.

Xylella fastidiosa is a xylem-limited bacterium that causes citrus variegated chlorosis disease in sweet orange. There is evidence that X. fastidiosa interacts with endophytic bacteria present in the xylem of sweet orange, and that these interactions, particularly with Methylobacterium mesophilicum, may affect disease progress. However, these interactions cannot be evaluated in detail until efficient methods for detection and enumeration of these bacteria in planta are developed. We have previously developed standard and quantitative PCR-based assays specific for X. fastidiosa using the LightCycler system [Li, W.B., Pria Jr., L.P.M.W.D., X. Qin, and J.S. Hartung, 2003. Presence of Xylella fastidiosa in sweet orange fruit and seeds and its transmission to seedlings. Phytopathology 93:953-958.], and now report the development of both standard and quantitative PCR assays for M. mesophilicum. The assays are specific for M. mesophilicum and do not amplify DNA from other species of Methylobacterium or other bacteria commonly associated with citrus or plant tissue. Other bacteria tested included Curtobacterium flaccumfaciens, Pantoea agglomerans, Enterobacter cloacae, Bacillus sp., X. fastidiosa, Xanthomonas axonopodis pv. citri, and Candidatus Liberibacter asiaticus. We have demonstrated that with these methods we can quantitatively monitor the colonization of xylem by M. mesophilicum during the course of disease development in plants artificially inoculated with both bacteria.

Citrus sinensis↗

PubMed2000

Methylopila helvetica sp. nov. and Methylobacterium dichloromethanicum sp. nov.--novel aerobic facultatively methylotrophic bacteria utilizing dichloromethane.

Eight strains of Gram-negative, aerobic, asporogenous, neutrophilic, mesophilic, facultatively methylotrophic bacteria are taxonomically described. These icl- serine pathway methylobacteria utilize dichloromethane, methanol and methylamine as well as a variety of polycarbon compounds as the carbon and energy source. The major cellular fatty acids of the non-pigmented strains DM1, DM3, and DM5 to DM9 are C18:1, C16:0, C18:0, Ccy19:0 and that of the pink-pigmented strain DM4 is C18:1. The main quinone of all the strains is Q-10. The non-pigmented strains have similar phenotypic properties and a high level of DNA-DNA relatedness (81-98%) as determined by hybridization. All strains belong to the alpha-subgroup of the alpha-Proteobacteria. 16S rDNA sequence analysis led to the classification of these dichloromethane-utilizers in the genus Methylopila as a new species - Methylopila helvetica sp.nov. with the type strain DM9 (=VKM B-2189). The pink-pigmented strain DM4 belongs to the genus Methylobacterium but differs from the known members of this genus by some phenotypic properties, DNA-DNA relatedness (14-57%) and 16S rDNA sequence. Strain DM4 is named Methylobacterium dichloromethanicum sp. nov. (VKM B-2191 = DSMZ 6343).

Bacterial Typing Techniques↗

PubMed2001

Chloromethane: tetrahydrofolate methyl transfer by two proteins from Methylobacterium chloromethanicum strain CM4.

The cmuA and cmuB genes are required for growth of Methylobacterium chloromethanicum strain CM4 with chloromethane as the sole carbon source. While CmuB was previously shown to possess methylcobalamin:tetrahydrofolate methyltransferase activity, sequence analysis indicated that CmuA represented a novel and so far unique two-domain methyltransferase/corrinoid-binding protein involved in methyl transfer from chloromethane to a corrin moiety. CmuA was purified from wild-type M. chloromethanicum strain CM4 and characterized as a monomeric, cobalt-containing and zinc-containing enzyme of molecular mass 67 kDa with a bound vitamin B12 cofactor. In combination, CmuA and CmuB proteins catalyze the in vitro transfer of the methyl group of chloromethane to tetrahydrofolate, thus affording a direct link between chloromethane dehalogenation and core C1 metabolism of Methylobacterium. Chloromethane dehalogenase activity in vitro is limited by CmuB, as formation of methyltetrahydrofolate from chloromethane displays apparent Michaelis-Menten kinetics with respect to methylated CmuA, with an apparent Km of 0.27 microM and a Vmax of 0.45 U x mg(-1). This contrasts with sequence-related systems for methyl transfer from methanogens, which involve methyltransferase and corrinoid protein components in well-defined stoichiometric ratios.

Bacterial Proteins↗