Modularity of methylotrophy, revisited

Methylotrophy is a metabolic capability possessed by microorganisms that allows them to build biomass and to obtain energy from organic substrates containing no carbon-carbon bonds (C1 compounds, such as methane, methanol, etc.). This phenomenon in microbial physiology has been a subject of study for over 100 years, elucidating a set of well-defined enzymatic systems and pathways enabling this capability. The knowledge gained from the early genetic and genomic approaches to understanding methylotrophy pointed towards the existence of alternative enzymes/pathways for the specific metabolic goals. Different combinations of these systems in different organisms suggested that methylotrophy must be modular in its nature. More recent insights from genomic analyses, including the genomes representing novel types of methylotrophs, seem to reinforce this notion. This review integrates the new findings with the previously developed concept of modularity of methylotrophy.     

Multiphyletic origins of methylotrophy in Alphaproteobacteria,exemplified by comparative genomics of Lake Washington isolates

We sequenced the genomes of 19 methylotrophic isolates from Lake Washington, which belong to nine genera within eight families of the Alphaproteobacteria, two of the families being the newly proposed families. Comparative genomic analysis with a focus on methylotrophy metabolism classifies these strains into heterotrophic and obligately or facultatively autotrophic methylotrophs. The most persistent metabolic modules enabling methylotrophy within this group are the N‐methylglutamate pathway, the two types of methanol dehydrogenase (MxaFI and XoxF), the tetrahydromethanopterin pathway for formaldehyde oxidation, the serine cycle and the ethylmalonyl‐CoA pathway. At the same time, a great potential for metabolic flexibility within this group is uncovered, with different combinations of these modules present. Phylogenetic analysis of key methylotrophy functions reveals that the serine cycle must have evolved independently in at least four lineages of Alphaproteobacteria and that all methylotrophy modules seem to be prone to lateral transfers as well as deletions.     

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.     

Characterization of a novel methanol dehydrogenase in representatives of Burkholderiales: implications for environmental detection of methylotrophy and evidence for convergent evolution

Some members of Burkholderiales are able to grow on methanol but lack the genes (mxaFI) responsible for the well-characterized two-subunit pyrroloquinoline quinone-dependent quinoprotein methanol dehydrogenase that is widespread in methylotrophic Proteobacteria. Here, we characterized novel, mono-subunit enzymes responsible for methanol oxidation in four strains, Methyloversatilis universalis FAM5, Methylibium petroleiphilum PM1, and unclassified Burkholderiales strains RZ18-153 and FAM1. The enzyme from M. universalis FAM5 was partially purified and subjected to matrix-assisted laser desorption ionization-time of fight peptide mass fingerprinting. The resulting peptide spectrum was used to identify a gene candidate in the genome of M. petroleiphilum PM1 (mdh2) predicted to encode a type I alcohol dehydrogenase related to the characterized methanol dehydrogenase large subunits but at less than 35% amino acid identity. Homologs of mdh2 were amplified from M. universalis FAM5 and strains RZ18-153 and FAM1, and mutants lacking mdh2 were generated in three of the organisms. These mutants lost their ability to grow on methanol and ethanol, demonstrating that mdh2 is responsible for oxidation of both substrates. Our findings have implications for environmental detection of methylotrophy and indicate that this ability is widespread beyond populations possessing mxaF, the gene traditionally used as a genetic marker for environmental detection of methanol-oxidizing capability. Our findings also have implications for understanding the evolution of methanol oxidation, suggesting a convergence toward the enzymatic function for methanol oxidation in MxaF and Mdh2-type proteins.     

Isolation and genomic characterization of Novimethylophilus kurashikiensis gen. nov. sp. nov., a new lanthanide‐dependent methylotrophic species of Methylophilaceae

Recently, it has been found that two types of methanol dehydrogenases (MDHs) exist in Gram‐negative bacterial methylotrophs, calcium‐dependent MxaFI‐MDH and lanthanide‐dependent XoxF‐MDH and the latter is more widespread in bacterial genomes. We aimed to isolate and characterize lanthanide‐dependent methylotrophs. The growth of strain La2‐4^T on methanol, which was isolated from rice rhizosphere soil, was strictly lanthanide dependent. Its 16S rRNA gene sequence showed only 93.4% identity to that of Methylophilus luteus Mim^T, and the name Novimethylophilus kurashikiensis gen. nov. sp. nov. is proposed. Its draft genome (ca. 3.69 Mbp, G + C content 56.1 mol%) encodes 3579 putative CDSs and 84 tRNAs. The genome harbors five xoxFs but no mxaFI. XoxF4 was the major MDH in the cells grown on methanol and methylamine, evidenced by protein identification and quantitative PCR analysis. Methylamine dehydrogenase gene was absent in the La2‐4^T genome, while genes for the glutamate‐mediated methylamine utilization pathway were detected. The genome also harbors those for the tetrahydromethanopterin and ribulose monophosphate pathways. Additionally, as known species, isolates of Burkholderia ambifaria, Cupriavidus necator and Dyadobacter endophyticus exhibited lanthanide dependent growth on methanol. Thus, lanthanide can be used as an essential growth factor for methylotrophic bacteria that do not harbor MxaFI‐MDH.     

Plasmid-dependent methylotrophy in thermotolerant Bacillus methanolicus

Bacillus methanolicus can efficiently utilize methanol as a sole carbon source and has an optimum growth temperature of 50 degrees C. With the exception of mannitol, no sugars have been reported to support rapid growth of this organism, which is classified as a restrictive methylotroph. Here we describe the DNA sequence and characterization of a 19,167-bp circular plasmid, designated pBM19, isolated from B. methanolicus MGA3. Sequence analysis of pBM19 demonstrated the presence of the methanol dehydrogenase gene, mdh, which is crucial for methanol consumption in this bacterium. In addition, five genes (pfk, encoding phosphofructokinase; rpe, encoding ribulose-5-phosphate 3-epimerase; tkt, encoding transketolase; glpX, encoding fructose-1,6-bisphosphatase; and fba, encoding fructose-1,6-bisphosphate aldolase) with deduced roles in methanol assimilation via the ribulose monophosphate pathway are encoded by pBM19. A shuttle vector, pTB1.9, harboring the pBM19 minimal replicon (repB and ori) was constructed and used to transform MGA3. Analysis of the resulting recombinant strain demonstrated that it was cured of pBM19 and was not able to grow on methanol. A pTB1.9 derivative harboring the complete mdh gene could not restore growth on methanol when it was introduced into the pBM19-cured strain, suggesting that additional pBM19 genes are required for consumption of this carbon source. Screening of 13 thermotolerant B. methanolicus wild-type strains showed that they all harbor plasmids similar to pBM19, and this is the first report describing plasmid-linked methylotrophy in any microorganism. Our findings should have an effect on future genetic manipulations of this organism, and they contribute to a new understanding of the biology of methylotrophs.     

Isolation, sequencing, and mutagenesis of the gene encoding cytochrome c553i of Paracoccus denitrificans and characterization of the mutant strain.

The periplasmically located cytochrome c553i of Paracoccus denitrificans was purified from cells grown aerobically on choline as the carbon source. The purified protein was digested with trypsin to obtain several protein fragments. The N-terminal regions of these fragments were sequenced. On the basis of one of these sequences, a mix of 17-mer oligonucleotides was synthesized. By using this mix as a probe, the structural gene encoding cytochrome c553i (cycB) was isolated. The nucleotide sequence of this gene was determined from a genomic bank. The N-terminal region of the deduced amino acid sequence showed characteristics of a signal sequence. Based on the deduced amino acid sequence of the mature protein, the calculated molecular weight is 22,427. The gene encoding cytochrome c553i was mutated by insertion of a kanamycin resistance gene. As a consequence of the mutation, cytochrome c553i was absent from the periplasmic protein fraction. The mutation in cycB resulted in a decreased maximum specific growth rate on methanol, while the molecular growth yield was not affected. Growth on methylamine or succinate was not affected at all. Upstream of cycB the 3' part of an open reading frame (ORF1) was identified. The deduced amino acid sequence of this part of ORF1 showed homology with methanol dehydrogenases from P. denitrificans and Methylobacterium extorquens AM1. In addition, it showed homology with other quinoproteins like alcohol dehydrogenase from Acetobacter aceti and glucose dehydrogenase from both Acinetobacter calcoaceticus and Escherichia coli. Immediately downstream from cycB, the 5' part of another open reading frame (ORF2) was found. The deduced amino acid sequence of this part of ORF2 showed homology with the moxJ gene products from P. denitrificans and M. extorquens AM1.     

Purification and characterization of azurin from the methylamine-utilizing obligate methylotroph Methylobacillus flagellatus KT

Methylamine dehydrogenase (MADH) and azurin were purified from the periplasmic fraction of the methylamine-grown obligate methylotroph Methylobacillus flagellatus KT. The molecular mass of the purified azurin was 16.3 kDa, as measured by SDS-PAGE, or 13 920 Da as determined by MALDI-TOF mass spectrometry. Azurin of M. flagellatus KT contained 1 copper atom per molecule and had an absorption maximum at 620 nm in the oxidized state. The redox potential of azurin measured at pH 7.0 by square-wave voltammetry was +275 mV versus normal hydrogen electrode. MADH reduced azurin in the presence of methylamine, indicating that this cupredoxin is likely to be the physiological electron acceptor for MADH in the electron transport chain of the methylotroph. A scheme of electron transport functioning in M. flagellatus KТ during methylamine oxidation is proposed.     

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.     

Expressed Genome of Methylobacillus flagellatus as Defined through Comprehensive Proteomics and New Insights into Methylotrophy

In recent years, techniques have been developed and perfected for high-throughput identification of proteins and their accurate partial sequencing by shotgun nano-liquid chromatography-tandem mass spectrometry (nano-LC-MS/MS), making it feasible to assess global protein expression profiles in organisms with sequenced genomes. We implemented comprehensive proteomics to assess the expressed portion of the genome of Methylobacillus flagellatus during methylotrophic growth. We detected a total of 1,671 proteins (64% of the inferred proteome), including all the predicted essential proteins. Nonrandom patterns observed with the nondetectable proteins appeared to correspond to silent genomic islands, as inferred through functional profiling and genome localization. The protein contents in methylamine- and methanol-grown cells showed a significant overlap, confirming the commonality of methylotrophic metabolism downstream of the primary oxidation reactions. The new insights into methylotrophy include detection of proteins for the N-methylglutamate methylamine oxidation pathway that appears to be auxiliary and detection of two alternative enzymes for both the 6-phosphogluconate dehydrogenase reaction (GndA and GndB) and the formate dehydrogenase reaction (FDH1 and FDH4). Mutant analysis revealed that GndA and FDH4 are crucial for the organism''s fitness, while GndB and FDH1 are auxiliary.Methylotrophy is the ability of some microorganisms to grow on compounds containing no carbon-carbon bonds (C₁ compounds) as sole sources of carbon and energy. This type of metabolism requires special enzyme systems and pathways that accomplish three principal metabolic goals: (i) energy-producing primary oxidation of a C₁ substrate to formaldehyde or methyl transfer to produce methyl-tetrahydrofolate (H₄F), (ii) energy-producing oxidation of formaldehyde or methyl-H₄F to CO₂, and (iii) energy-consuming assimilation of formaldehyde, methylene-H₄F, and/or CO₂ into biomass (1, 22). In the course of microbial evolution, multiple pathways for each step have been assembled, and, theoretically, any combination of these should enable methylotrophy (9). Moreover, some organisms possess multiple pathways for certain metabolic tasks. For example, methane can be oxidized to methanol by either soluble or particulate methane monooxygenase, and many methanotrophs possess both enzymes (25). The presence of multiple formate dehydrogenases is typical among methylotrophs (10). Some methylotrophs, such as Methylococcus capsulatus, encode multiple pathways for C₁ assimilation (37). Methylotrophs employing the ribulose monophosphate (RuMP) cycle for formaldehyde assimilation can also oxidize formaldehyde via this pathway by employing a single additional reaction, catalyzed by 6-phosphogluconate dehydrogenase (Gnd) (1, 22). In addition, most RuMP cycle methylotrophs also possess a linear pathway for formaldehyde oxidation employing tetrahydromethanopterin (H₄MPT) as a cofactor (35).Methylobacillus flagellatus is a typical representative of RuMP cycle methylotrophs. It has a very limited substrate repertoire, growing robustly only on methanol or methylamine (16). Genomic analysis has revealed specific lesions in pathways for utilization of multicarbon compounds, confirming that, indeed, this organism must rely exclusively on methylotrophy to sustain its growth (11). At the same time, redundant methylotrophy pathways have been deduced from the genome. For example, in addition to the bona fide methanol dehydrogenase, four homologs of the large subunit are encoded (11). Besides the well-characterized methylamine dehydrogenase, an alternative system for methylamine oxidation is encoded, consisting of N-methylglutamate synthase and N-methylglutamate dehydrogenase (N-methylglutamate pathway) (21). Both cyclic and linear pathways for formaldehyde oxidation are encoded, along with two formate dehydrogenases. In addition, multiple terminal cytochrome oxidases are encoded (11).It has been assumed that metabolism of both methanol and methylamine, with the exception of the respective specific primary oxidation step, is carried out by M. flagellatus (and other methylotrophs) in exactly the same fashion (1, 22). However, this assumption has not been experimentally tested. The relative contributions of each of the redundant pathways encoded in the genome (for example, linear versus cyclic oxidation of formaldehyde) also remained unclear (7). These questions can be assessed through analysis of the expressed portion of the genome under specific growth conditions and especially precisely through a comprehensive analysis of the protein content in the cell (proteome) (3).In recent years, techniques have been developed and perfected for high-throughput identification of proteins and their accurate partial sequencing by shotgun nano-liquid chromatography-tandem mass spectrometry (nanoLC-MS/MS), making it feasible to assess relatively complete global protein expression profiles in prokaryotic organisms with sequenced genomes (2, 17). In this study, we employed comprehensive proteomics of M. flagellatus to determine what portion of the encoded proteome is detectable during methylotrophic growth under defined laboratory conditions and to obtain new insights into the physiology of the organism through this analysis.     

Characterization of a new marine methylotroph

Abstract A methanol-oxidizing bacterium from a marine environment has been isolated and characterized. The bacterium was a Gram-negative rod, capable of growth on methanol and methylamine, but not on multicarbon compounds. It showed a temperature optimum of 30°C, a salt optimum of 0.4% (w/v) and the mol % G + C of its DNA was 46%. Carbon was assimilated via the ribulose monophosphate pathway for formaldehyde fixation during growth on methanol. This bacterium superficially resembled other obligate methylotrophs requiring NaCl reported previously which were designated Methylomonas thalassica . It also appeared similar to many strains of obligate freshwater methylotrophs, except for its NaCl requirement and its lower mol % G + C.     

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.     

一株新的产吡咯喹啉醌甲基营养菌全基因组测序和比较基因组学分析

【背景】由于甲基营养菌被发现的时间较短,而且可以生产吡咯喹啉醌(pyrroloquinoline quinone,PQQ)的甲基杆菌属细菌只有少数菌株的全基因组序列被公布,增加了该类细菌基因组学和生物代谢途径研究的难度。【目的】将本实验室筛选的PQQ生产菌经多种诱变方式处理,用于提高PQQ的发酵产量。对高产突变菌株进行全基因组解析,以探究甲基杆菌PQQ合成的分子机制,为后续分子育种提供序列背景信息。【方法】将野生型PQQ生产菌株进行紫外诱变、亚硝基胍诱变、甲基磺酸乙酯诱变、硫酸二乙酯诱变和紫外-氯化锂复合诱变。将突变菌株利用PromethION三代测序平台和MGISEQ-2000二代测序平台测序,然后进行组装和功能注释。组装得到的全基因组序列与模式菌株扭脱甲基杆菌AM1 (Methylobacterium extorquens AM1)进行比较基因组学分析。【结果】经11轮诱变获得一株突变菌株NI91,其PQQ产量为19.49mg/L,相较原始菌株提高44.91%。突变菌株NI91的基因组由一个5 409 262 bp的染色体组成,共编码4 957个蛋白,与模式菌株M. extorqu...     

Taxonomic studies on methylotrophic bacteria by 5S ribosomal RNA sequencing

Nucleotide sequences of 5S ribosomal RNA (rRNA) isolated from 19 strains of Gram-negative methylotrophic bacteria were determined. Comparison of these sequences allowed construction of a tentative phylogenetic tree and showed that the bacteria analysed belong to the Proteobacteria and fell into several clusters, including obligate methanotrophs, obligate methylotrophs and several groups of facultative methylotrophs. Taxonomic relations between methylotrophic and non-methylotrophic bacteria are discussed, and the polyphyletic nature of methylotrophy as a taxonomic feature is highlighted.     

Nature, nomenclature and taxonomy of obligate methanol utilizing strains

In a screening program, a number of different bacterial strains with the ability to utilize methanol as a sole carbon and energy source were isolated and described. They are well known methanol utilizing genera Pseudomonas, Klebsiella, Micrococcus, Methylomonas or, on the contrary, the new, unknown genera and species of methylotrophic bacteria. In the last category, Acinetobacter and Alcaligenes are the new reported genera of organisms able to use methanol as a sole carbon and energy source. The present paper reports the very complex physiological and biochemical modifications when very versatile bacteria such as Pseudomonas aeruginosa and Acinetobacter calcoaceticus are cultured on methanol and when the obligate methylotrophic state is compared with the facultative methylotrophic state of the same bacterial strain. Based on experiments and comparisons with literature data, it seems that Methylomonas methanica is the obligate methylotrophic state of Pseudomonas aeruginosa and that Acinetobacter calcoaceticus is the facultative methylotrophic state of Methylococcus capsulatus, an obligate methylotroph. The relationship of the obligate to the facultative and of the facultative to the obligate methylotrophy were established. These new methylotrophic genera and species, the profound physiological and biochemical modifications as well as the new data concerning nature, nomenclature and taxonomy of methanol utilizing bateria were reported for the first time in 1983.     

Development of a formaldehyde biosensor with application to synthetic methylotrophy

Formaldehyde is a prevalent environmental toxin and a key intermediate in single carbon metabolism. The ability to monitor formaldehyde concentration is, therefore, of interest for both environmental monitoring and for metabolic engineering of native and synthetic methylotrophs, but current methods suffer from low sensitivity, complex workflows, or require expensive analytical equipment. Here we develop a formaldehyde biosensor based on the FrmR repressor protein and cognate promoter of Escherichia coli. Optimization of the native repressor binding site and regulatory architecture enabled detection at levels as low as 1 µM. We then used the sensor to benchmark the in vivo activity of several NAD‐dependent methanol dehydrogenase (Mdh) variants, the rate‐limiting enzyme that catalyzes the first step of methanol assimilation. In order to use this biosensor to distinguish individuals in a mixed population of Mdh variants, we developed a strategy to prevent cross‐talk by using glutathione as a formaldehyde sink to minimize intercellular formaldehyde diffusion. Finally, we applied this biosensor to balance expression of mdh and the formaldehyde assimilation enzymes hps and phi in an engineered E. coli strain to minimize formaldehyde build‐up while also reducing the burden of heterologous expression. This biosensor offers a quick and simple method for sensitively detecting formaldehyde, and has the potential to be used as the basis for directed evolution of Mdh and dynamic formaldehyde control strategies for establishing synthetic methylotrophy.     

An Escherichia coli S1-like ribosomal protein is immunologically conserved in Gram-negative bacteria, but not in Gram-positive bacteria

Abstract A study was made of the enzymology of primary and intermediary pathways of C₁ metabolism in three strains of non-motile obligately methylotrophic bacteria. Each uses a variant of the ribulosemonophosphate (RMP) cycle of formaldehyde fixation which involves the Entner-Doudoroff route for hexose-phosphate cleavage and transaldolase/transketolase mode of rearrangement. The organisms possess high levels of hexulose-phosphate synthase and NAD(P)-linked glucose-6-phosphate and 6-phosphogluconate dehydrogenases. In addition they contain small activities of dye-linked methanol and methylamine dehydrogenases, PMS- and NAD-linked formaldehyde and formate dehydrogenases. This indicates cyclic rather than direct oxidation of formaldehyde derived from methanol or methylamine. The tricarboxylic acid cycle is defective in 2-ketoglutarate dehydrogenase and the glyoxylate shunt is not operating because of the absence of malate synthase. Oxaloacetate is regenerated by (phosphoenol) pyruvate carboxylases. NH⁺ ₄ is assimilated mainly by glutamate dehydrogenase. The results show metabolic similarities between motile and non-motile obligate methanol and methylamine utilizers.     

Multiple formate dehydrogenase enzymes in the facultative methylotroph Methylobacterium extorquens AM1 are dispensable for growth on methanol

Formate dehydrogenase has traditionally been assumed to play an essential role in energy generation during growth on C(1) compounds. However, this assumption has not yet been experimentally tested in methylotrophic bacteria. In this study, a whole-genome analysis approach was used to identify three different formate dehydrogenase systems in the facultative methylotroph Methylobacterium extorquens AM1 whose expression is affected by either molybdenum or tungsten. A complete set of single, double, and triple mutants was generated, and their phenotypes were analyzed. The growth phenotypes of the mutants suggest that any one of the three formate dehydrogenases is sufficient to sustain growth of M. extorquens AM1 on formate, while surprisingly, none is required for growth on methanol or methylamine. Nuclear magnetic resonance analysis of the fate of [(13)C]methanol revealed that while cells of wild-type M. extorquens AM1 as well as cells of all the single and the double mutants continuously produced [(13)C]bicarbonate and (13)CO(2), cells of the triple mutant accumulated [(13)C]formate instead. Further studies of the triple mutant showed that formate was not produced quantitatively and was consumed later in growth. These results demonstrated that all three formate dehydrogenase systems must be inactivated in order to disrupt the formate-oxidizing capacity of the organism but that an alternative formate-consuming capacity exists in the triple mutant.     

Organization of the methylamine utilization (mau) genes in Methylophilus methylotrophus W3A1-NS.

The organization of genes involved in utilization of methylamine (mau genes) was studied in Methylophilus methylotrophus W3A1. The strain used was a nonmucoid variant termed NS (nonslimy). The original mucoid strain was shown to be identical to the NS strains on the basis of chromosomal digest and hybridization patterns. An 8-kb PstI fragment of the chromosome from M. methylotrophus W3A1-NS encoding the mau genes was cloned and a 6,533-bp region was sequenced. Eight open reading frames were found inside the sequenced area. On the basis of a high level of sequence identity with the Mau polypeptides from Methylobacterium extorquens AM1, the eight open reading frames were identified as mauFBEDAGLM. The mau gene cluster from M. methylotrophus W3A1 is missing two genes, mauC (amicyanin) and mauJ (whose function is unknown), which have been found between mauA and mauG in all studied mau gene clusters. Mau polypeptides sequenced so far from five different bacteria show considerable identity. A mauA mutant of M. methylotrophus W3A1-NS that was constructed lost the ability to grow on all amines as sources of nitrogen but still retained the ability to grow on trimethylamine as a source of carbon. Thus, unlike M. extorquens AM1 and Methylobacillus flagellatum KT, M. methylotrophus W3A1-NS does not have an additional methylamine dehydrogenase system for amine oxidation. Using a promoter-probe vector, we identified a promoter upstream of mauF and used it to construct a potential expression vector, pAYC229.