Localization of methanol dehydrogenase in two strains of methylotrophic bacteria detected by immunogold labeling.

Antibodies to methanol dehydrogenase purified from Methylobacterium sp. strain AM1 and Methylomonas sp. strain A4 were raised. The antibody preparations were used in indirect immunogold labeling studies. With this approach, methanol dehydrogenase was found to be preferentially localized to the periplasmic region of the methylotroph Methylobacterium sp. strain AM1 and to the intracytoplasmic membrane of the methanotroph Methylomonas sp. strain A4. Antibody cross-reactivity to other methylotrophic bacteria was detected.     

Identification of putative methanol dehydrogenase (moxF) structural genes in methylotrophs and cloning of moxF genes from Methylococcus capsulatus bath and Methylomonas albus BG8.

An open-reading-frame fragment of a Methylobacterium sp. strain AM1 gene (moxF) encoding a portion of the methanol dehydrogenase structural protein has been used as a hybridization probe to detect similar sequences in a variety of methylotrophic bacteria. This hybridization was used to isolate clones containing putative moxF genes from two obligate methanotrophic bacteria, Methylococcus capsulatus Bath and Methylomonas albus BG8. The identity of these genes was confirmed in two ways. A T7 expression vector was used to produce methanol dehydrogenase protein in Escherichia coli from the cloned genes, and in each case the protein was identified by immunoblotting with antiserum against the Methylomonas albus methanol dehydrogenase. In addition, a moxF mutant of Methylobacterium strain AM1 was complemented to a methanol-positive phenotype that partially restored methanol dehydrogenase activity, using broad-host-range plasmids containing the moxF genes from each methanotroph. The partial complementation of a moxF mutant in a facultative serine pathway methanol utilizer by moxF genes from type I and type X obligate methane utilizers suggests broad functional conservation of the methanol oxidation system among gram-negative methylotrophs.     

Isolation and nucleotide sequence of the methanol dehydrogenase structural gene from Paracoccus denitrificans.

A genomic clone bank of Paracoccus denitrificans DNA has been constructed in the expression vector set pEX1, pEX2, and pEX3. Screening of this clone bank with antibodies raised against P. denitrificans methanol dehydrogenase resulted in the isolation of a clone, pNH3, that synthesized methanol dehydrogenase cross-reactive proteins. The nucleotide sequence of the P. denitrificans DNA fragment inserted in this clone has been determined and shown to contain the full methanol dehydrogenase structural gene. DNA cross-hybridization was found with DNA fragments which have been reported to contain the methanol dehydrogenase structural genes from Methylobacterium sp. strain AM1 and Methylobacterium organophilum.     

Phenotypic characterization of 10 methanol oxidation mutant classes in Methylobacterium sp. strain AM1

Twenty-five methanol oxidation mutants of the facultative methylotroph Methylobacterium sp. strain AM1 have been characterized by complementation analysis and assigned to 10 complementation groups, Mox A1, A2, A3, and B through H (D. N. Nunn and M. E. Lidstrom, J. Bacteriol. 166:582-591, 1986). In this study we have characterized each of the mutants belonging to the 10 Mox complementation groups for the following criteria: phenazine methosulfate-dichlorophenolindophenol dye-linked methanol dehydrogenase activity; methanol-dependent whole-cell oxygen consumption; the presence or absence of methanol dehydrogenase protein by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting; the absorption spectra of purified mutant methanol dehydrogenase proteins; and the presence or absence of the soluble cytochrome c proteins of Methylobacterium sp. strain AM1, as determined by reduced-oxidized difference spectra and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. With this information, we have proposed functions for each of the genes deficient in the mutants of the 10 Mox complementation groups. These proposed gene functions include two linked genes that encode the methanol dehydrogenase structural protein and the soluble cytochrome cL, a gene encoding a secretion function essential for the synthesis and export of methanol dehydrogenase and cytochrome cL, three gene functions responsible for the proper association of the pyrrolo-quinoline quinone prosthetic group with the methanol dehydrogenase apoprotein, and four positive regulatory gene functions controlling the expression of the ability to oxidize methanol.     

Organization of Genes Required for the Oxidation of Methanol to Formaldehyde in Three Type II Methylotrophs

Restriction maps of genes required for the synthesis of active methanol dehydrogenase in Methylobacterium organophilum XX and Methylobacterium sp. strain AM1 have been completed and compared. In these two species of pink-pigmented, type II methylotrophs, 15 genes were identified that were required for the expression of methanol dehydrogenase activity. None of these genes were required for the synthesis of the prosthetic group of methanol dehydrogenase, pyrroloquinoline quinone. The structural gene required for the synthesis of cytochrome c(L), an electron acceptor uniquely required for methanol dehydrogenase, and the genes encoding small basic peptides that copurified with methanol dehydrogenases were closely linked to the methanol dehydrogenase structural genes. A cloned 22-kilobase DNA insert from Methylsporovibrio methanica 81Z, an obligate type II methanotroph, complemented mutants that contained lesions in four genes closely linked to the methanol dehydrogenase structural genes. The methanol dehydrogenase and cytochrome c(L) structural genes were found to be transcribed independently in M. organophilum XX. Only two of the genes required for methanol dehydrogenase synthesis in this bacterium were found to be cotranscribed.     

16S ribosomal RNA sequence analysis for determination of phylogenetic relationship among methylotrophs.

16S ribosomal RNAs (rRNA) of 12 methylotrophic bacteria have been almost completely sequenced to establish their phylogenetic relationships. Methylotrophs that are physiologically related are phylogenetically diverse and are scattered among the purple eubacteria (class Proteobacteria). Group I methylotrophs can be classified in the beta- and the gamma-subdivisions and group II methylotrophs in the alpha-subdivision of the purple eubacteria, respectively. Pink-pigmented facultative and non-pigmented obligate group II methylotrophs form two distinctly separate branches within the alpha-subdivision. The secondary structures of the 16S rRNA sequences of 'Methylocystis parvus' strain OBBP, 'Methylosinus trichosporium' strain OB3b, 'Methylosporovibrio methanica' strain 81Z and Hyphomicrobium sp. strain DM2 are similar, and these non-pigmented obligate group II methylotrophs form one tight cluster in the alpha-subdivision. The pink-pigmented facultative methylotrophs, Methylobacterium extorquens strain AM1, Methylobacterium sp. strain DM4 and Methylobacterium organophilum strain XX form another cluster within the alpha-subdivision. Although similar in phenotypic characteristics, Methylobacterium organophilum strain XX and Methylobacterium extorquens strain AM1 are clearly distinguishable by their 16S rRNA sequences. The group I methylotrophs, Methylophilus methylotrophus strain AS1 and methylotrophic species DM11, which do not utilize methane, are similar in 16S rRNA sequence to bacteria in the beta-subdivision. The methane-utilizing, obligate group I methanotrophs, Methylococcus capsulatus strain BATH and Methylomonas methanica, are placed in the gamma-subdivision. The results demonstrate that it is possible to distinguish and classify the methylotrophic bacteria using 16S rRNA sequence analysis.     

Genetic and physical analyses of Methylobacterium organophilum XX genes encoding methanol oxidation.

When allyl alcohol was used as a suicide substrate, spontaneous mutants and UV light- and nitrous acid-generated mutants of Methylobacterium organophilum XX were selected which grew on methylamine but not on methanol. There was no detectable methanol dehydrogenase (MDH) activity in crude extracts of these mutants, yet Western blots revealed that some mutants still produced MDH protein. Complementation of 50 mutants by a cosmid gene bank of M. organophilum XX demonstrated that three major regions of the genome, each of which was separated by a minimum of 40 kilobases, were required for expression of active MDH. By subcloning and Tn5 insertion mutagenesis of subcloned fragments, at least 11 genes clustered within these three regions were subsequently identified. The identity of the MDH structural gene, which was initially determined by hybridization to the structural gene of Methylobacterium sp. strain AM1, was confirmed by Western blot analysis of an MDH-beta-galactosidase fusion protein.     

Soluble cytochromes from the marine methanotroph Methylomonas sp. strain A4.

Soluble c-type cytochromes are central to metabolism of C1 compounds in methylotrophic bacteria. In order to characterize the role of c-type cytochromes in methane-utilizing bacteria (methanotrophs), we have purified four different cytochromes, cytochromes c-554, c-553, c-552, and c-551, from the marine methanotroph Methylomonas sp. strain A4. The two major species, cytochromes c-554 and c-552, were monoheme cytochromes and accounted for 57 and 26%, respectively, of the soluble c-heme. The approximate molecular masses were 8,500 daltons (Da) (cytochrome c-554) and 14,000 Da (cytochrome c-552), and the isoelectric points were pH 6.4 and 4.7, respectively. Two possible diheme c-type cytochromes were also isolated in lesser amounts from Methylomonas sp. strain A4, cytochromes c-551 and c-553. These were 16,500 and 34,000 Da, respectively, and had isoelectric points at pH 4.75 and 4.8, respectively. Cytochrome c-551 accounted for 9% of the soluble c-heme, and cytochrome c-553 accounted for 8%. All four cytochromes differed in their oxidized versus reduced absorption maxima and their extinction coefficients. In addition, cytochromes c-554, c-552, and c-551 were shown to have different electron paramagnetic spectra and N-terminal amino acid sequences. None of the cytochromes showed significant activity with purified methanol dehydrogenase in vitro, but our data suggested that cytochrome c-552 is probably the in vivo electron acceptor for the methanol dehydrogenase.     

Molecular detection and isolation from antarctica of methylotrophic bacteria able to grow with methylated sulfur compounds

This study is the first demonstration that a diverse facultatively methylotrophic microbiota exists in some Antarctic locations. PCR amplification of genes diagnostic for methylotrophs was carried out with bacterial DNA isolated from 14 soil and sediment samples from ten locations on Signy Island, South Orkney Islands, Antarctica. Genes encoding the mxaF of methanol dehydrogenase, the fdxA for Afipia ferredoxin, the msmA of methanesulfonate monooxygenase, and the 16S rRNA gene of Methylobacterium were detected in all samples tested. The mxaF gene sequences corresponded to those of Hyphomicrobium, Methylobacterium, and Methylomonas. Over 30 pure cultures of methylotrophs were isolated on methanesulfonate, dimethylsulfone, or dimethylsulfide from ten Signy Island lakes. Some were identified from 16S rRNA gene sequences (and morphology) as Hyphomicrobium species, strains of Afipia felis, and a methylotrophic Flavobacterium strain. Antarctic environments thus contain diverse methylotrophic bacteria, growing on various C1-substrates, including C1-sulfur compounds.     

Genetics of carbon metabolism in methylotrophic bacteria

Abstract The application of genetic techniques to the methylotrophic bacteria has greatly enhanced studies of these important organisms. Two methylotrophic systems have been studied in some detail, the serine cycle for formaldehyde assimilation and the methanol oxidation system. In both cases, genes have been cloned and mapped in Methylobacterium species (facultative serine cycle methanol-utilizers). In addition, methanol oxidation genes have been studied in an autotrophic methanol-utilizer ( Paracoccus denitrificans ) and three methanotrophs ( Methylosporovibrio methanica, Methylomonas albus and Methylomonas sp. A4). Although much remains to be learned in these systems, it is becoming clear that the order of C₁ genes has been conserved to some extent in methylotrophic bacteria, and that many C₁ genes are loosely clustered on the chromosome. Operons appear to be rare, but some examples have been observed. The extension of genetic approaches to both the obligate and facultative methylotrophs holds much promise for the future in understanding and manipulating the activities of these bacteria.     

The moxFG region encodes four polypeptides in the methanol-oxidizing bacterium Methylobacterium sp. strain AM1.

The polypeptides encoded by a putative methanol oxidation (mox) operon of Methylobacterium sp. strain AM1 were expressed in Escherichia coli, using a coupled in vivo T7 RNA polymerase/promoter gene expression system. Two mox genes had been previously mapped to this region: moxF, the gene encoding the methanol dehydrogenase (MeDH) polypeptide; and moxG, a gene believed to encode a soluble type c cytochrome, cytochrome cL. In this study, four polypeptides of Mr 60,000, 30,000, 20,000, and 12,000 were found to be encoded by the moxFG region and were tentatively designated moxF, -J, -G, and -I, respectively. The arrangement of the genes (5' to 3') was found to be moxFJGI. The identities of three of the four polypeptides were determined by protein immunoblot analysis. The product of moxF, the Mr-60,000 polypeptide, was confirmed to be the MeDH polypeptide. The product of moxG, the Mr-20,000 polypeptide, was identified as mature cytochrome cL, and the product of moxI, the Mr-12,000 polypeptide, was identified as a MeDH-associated polypeptide that copurifies with the holoenzyme. The identity of the Mr-30,000 polypeptide (the moxJ gene product) could not be determined. The function of the Mr-12,000 MeDH-associated polypeptide is not yet clear. However, it is not present in mutants that lack the Mr-60,000 MeDH subunit, and it appears that the stability of the MeDH-associated polypeptide is dependent on the presence of the Mr-60,000 MeDH polypeptide. Our data suggest that both the Mr-30,000 and -12,000 polypeptides are involved in methanol oxidation, which would bring to 12 the number of mox genes in Methylobacterium sp. strain AM1.     

The second subunit of methanol dehydrogenase of Methylobacterium extorquens AM1.

The nucleotide and deduced amino acid sequence of a novel small (beta) subunit of methanol dehydrogenase of Methylobacterium extorquens AM1 (previously Pseudomonas AM1) has been determined. Work with the whole protein has shown that is has an alpha 2 beta 2 configuration.     

1株甲基杆菌Methylobacterium sp.WGM16的鉴定及降解甲醇的最佳培养条件

从水稻根部土壤中筛选到1株粉红色、需氧的兼性甲基营养型菌株WGM16,该菌为革兰阴性杆菌。根据菌株16S rRNA基因序列比对分析及结合菌株常规形态特征、生理生化性状的鉴定,将该菌初步鉴定为Methylobacterium sp.PCR扩增到菌株WGM16编码甲醇脱氢酶α-亚基的mxaF基因,表明菌株WGM16中存在甲基营养代谢途径。在培养温度为32℃、以1%的甲醇作为碳源、pH值为8.0的培养条件下,其甲醇降解率可达75%。     

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.     

Isolation and characterization of a blue copper protein from Thiobacillus versutus

The blue copper protein induced during growth of Thiobacillus versutus on methylamine was purified and characterized. It is an acidic protein (isoelectric point 4.7), contains one Cu2+ ion/enzyme molecule, is a monomeric protein (molecular mass about 14 kDa), has a maximum in its absorption spectrum at 596 nm (molar absorption coefficient 3.9 X 10(3) M-1 cm-1), shows an axial type-I electron paramagnetic resonance spectrum (g parallel = 2.239, g perpendicular = 2.046 and A parallel = 5.6 mT) and has a redox potential (Eo) of + 260 mV. In view of these properties and in view of the fact that the protein is active as an electron carrier between methylamine dehydrogenase and cytochrome c, it is concluded that it is similar to the amicyanins isolated from Methylomonas sp. strain J and Pseudomonas sp. strain AM 1.     

The methanol-oxidizing system of Methylobacterium extorquens AM1 reconstituted with purified constituents

The electron transport system (with cytochrome aa3) coupled to the oxidation of methanol in Methylobacterium extorquens AM1 (former Pseudomonas AM1) was reconstituted with highly purified constituents of the system. A mixture of 2.7 microM methanol dehydrogenase, 3.2 microM cytochrome cH, and 71 nM cytochrome c oxidase (= cytochrome aa3) consumed oxygen at a lower rate in the presence of methanol, while its activity was enhanced 3-fold by the addition of 1.4 microM cytochrome cL (74 mol of O2 consumed/mol of heme a of cytochrome c oxidase per min). Further addition of amicyanin to the above mixture did not affect the activity. Although ammonium ion greatly activated the activity of methanol dehydrogenase, the ion had little effect on the oxygen consumption activity of the above mixture. On the basis of the results obtained in the present study, an electron transport system is proposed for the oxidation of methanol in M. extorquens AM1.     

Tricarboxylic acid-cycle enzymes and ATP pool in facultative and obligate methylotrophs: Pseudomonas J26 and Methylomonas Pl1.

1. No essential differences were found in the activities of tricarboxylic acid-cycle enzymes in the newly isolated facultative methylotroph Pseudomonas J26 and obligate methylotroph Methylomonas Pl1. 2-Oxoglutarate dehydrogenase and succinate dehydrogenase were absent in Methylomonas Pl1; in Pseudomonas J26 the functioning of the cycle was imparied only on the methanol medium. Citrate synthase of both organisms showed low sensitivity to 2-oxoglutarate, NADH and ATP. 2. In both methylotrophs, methanol dehydrogenase was inhibited non-competitively by ATP: the activity was reduced by half by ATP at a concentration of 5 mM. 3. Concentration of ATP in the log-phase cultures of Methylomonas Pl1 was about twice as high as in Pseudomonas J26 (4.7 and 1.7 mumol/g dry wt., respectively). 4. Differences between the energy state of Methylomonas Pl1 and Pseudomonas J26 might be due to the higher ability of the former to oxidize methanol and/or lower energy requirement for C1 assimilation by the hexulose pathway in the obligate methylotroph.     

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

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