Methylobacillus methanolivorans sp. nov., a novel non-pigmented obligately methylotrophic bacterium.

Three strains of obligately methylotrophic Betaproteobacteria (ZT, SP and M3) with the ribulose monophosphate pathway of C1 assimilation are described. The isolates were strictly aerobic, Gram-stain-negative, asporogenous, motile (strains ZT and M3) or non-motile (strain SP) rods that multiplied by binary fisson, and were mesophilic and neutrophilic. All three strains utilized methanol but only strains SP and M3 utilized methylamine as carbon and energy sources. The prevailing cellular fatty acids were straight-chain saturated C16 : 0 and unsaturated C16 : 1ω7c acids. The major ubiquinone was Q-8. The predominant phospholipids were phosphatidylethanolamine, phosphatidylglycerol and cardiolipin. Ammonia was assimilated by glutamate dehydrogenase. The DNA G+C contents of strains ZT, SP and M3 were 51.0, 52.0 and 52.0 mol% (Tm), respectively. Levels of 16S rRNA gene sequence similarity between the three strains were very high (99.9-100 %), and they shared high levels of DNA-DNA relatedness (88-98 %). Based on 16S rRNA gene sequence analysis and DNA-DNA relatedness (19-30 %) with the type strains of the genus Methylobacillus, the novel isolates ZT, SP and M3 are classified as representing a novel species of this genus, for which the name Methylobacillus methanolivorans sp. nov. is proposed. The type strain is ZT (=VKM B-3037T=JCM 31401T=CCUG 68999T).

Novel and efficient screening of PQQ high-yielding strains and subsequent cultivation optimization.

A novel and efficient screening method for pyrroloquinoline quinone (PQQ) high-yielding methylotrophic strains was developed by using glucose dehydrogenase apoenzyme (GDHA) which depended on PQQ as the cofactor. Using this high-throughput method, PQQ high-yielding strains were rapidly screened out from thousands of methylotrophic colonies at a time. The comprehensive phylogenetic analysis revealed that the highest PQQ-producing strain zju323 (CCTCC M 2016079) could be assigned to a novel species in the genus Methylobacillus of the Betaproteobacteria. After systematic optimization of different medium components and cultivation conditions, about 33.4 mg/L of PQQ was obtained after 48 h of cultivation with Methylobacillus sp. zju323 at the shake flask scale. Further cultivations of Methylobacillus sp. zju323 were carried out to investigate the biosynthesis of PQQ in 10-L bench-top fermenters. In the batch operation, the PQQ accumulation reached 78 mg/L in the broth after 53 h of cultivation. By adopting methanol feeding strategy, the highest PQQ concentration was improved up to 162.2 mg/L after 75 h of cultivation. This work developed a high-throughput strategy of screening PQQ-producing strains from soil samples and also demonstrated one potential bioprocess for large-scale PQQ production with the isolated PQQ strain.

Bifunctional sucrose phosphate synthase/phosphatase is involved in the sucrose biosynthesis by Methylobacillus flagellatus KT.

The aerobic obligate methylotroph Methylobacillus flagellatus KT was shown to synthesize sucrose in the presence of 0.5-2% NaCl in the growth medium. In the genome of this bacterium, an open reading frame (ORF) encoding a predicted 84-kD polypeptide homologous to the plant and cyanobacterial sucrose phosphate synthases (SPSs) was found. Using heterologous expression of the putative sps gene in Escherichia coli, followed by affinity chromatography, pure recombinant protein SPS-His6 was obtained. The enzyme catalyzed two reactions: conversion of fructose 6-phosphate and UDP-glucose into sucrose 6-phosphate and hydrolysis of sucrose 6-phosphate to sucrose. The bifunctional sucrose phosphate synthase/phosphatase (SPS/SPP) was a 340 kDa homotetrameric Mg(2+) -dependent enzyme activated by fructose 1,6-phosphate2 and ATP but inhibited by glucose 6-phosphate, fructose 1-phosphate, AMP and inorganic phosphate. The amino acid sequence of the protein had a C-terminal domain homologous to SPPs. This correlated with the absence of the spp gene in the M. flagellatus chromosome. The ORFs homologous to the M. flagellatus SPS were found in the genomes of another obligate methylotroph Methylovorus glucosetrophus as well as the lithoautotrophic bacteria Acidithiobacillus ferrooxidans, Nitrosomonas europaea and Nitrosospira multiformis whose genomes lacked the spp genes. Thus, data extending the knowledge of biochemical properties of bacterial SPSs have been obtained.

Methylobacillus rhizosphaerae sp. nov., a novel plant-associated methylotrophic bacterium isolated from rhizosphere of red pepper.

A novel plant-associated obligate methylotrophic bacterium, designated strain Ca-68(T), was isolated from the rhizosphere soil of field-grown red pepper from India. The isolates are strictly aerobic, Gram negative, motile rods multiplying by binary fission and formaldehyde is assimilated via the ribulose monophosphate pathway. A comparative 16S rRNA gene sequence-based phylogenetic analysis placed the strain in a clade with the species Methylobacillus flagellatus, Methylobacillus glycogens and Methylobacillus pratensis, with which it showed pairwise similarity of 97.8, 97.4 and 96.2 %, respectively. The major fatty acids are C(16:0), C(10:0) 3OH and C(16:1) ω7c. The G+C content of the genomic DNA is 59.7 mol%. The major ubiquinone is Q-8. Dominant phospholipids are phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol. Based on 16S rRNA gene sequence analysis and DNA-DNA relatedness (14-19 %) with type strains of the genus Methylobacillus, the novel isolate was classified as a new species of this genus and named Methylobacillus rhizosphaerae Ca-68(T) (=KCTC 22383(T) = NCIMB 14472(T)).

Membrane topology and functional analysis of Methylobacillus sp. 12S genes epsF and epsG, encoding polysaccharide chain-length determining proteins.

The EpsF and EpsG of the methanol-assimilating bacterium Methylobacillus sp. 12S are involved in the synthesis of a high molecular weight exopolysaccharide, methanolan. These proteins share homology with chain-length determiners in other polysaccharide-producing bacteria. The N- and C-termini of EpsF were found to locate to the cytoplasm, and EpsF was predicted to have two transmembrane regions. EpsG showed both ATPase and autophosphorylation activities.

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.

Methylobacillus arboreus sp. nov., and Methylobacillus gramineus sp. nov., novel non-pigmented obligately methylotrophic bacteria associated with plants.

Two newly isolated obligate methanol-utilizing bacteria (strains Iva(T) and Lap(T)) with the ribulose monophosphate pathway of C(1) assimilation are described. The isolates are strictly aerobic, Gram negative, asporogenous, motile rods multiplying by binary fission, mesophilic and neutrophilic, synthesize indole-3-acetate. The prevailing cellular fatty acids are straight-chain saturated C(16:0) and unsaturated C(16:1) acids. The major ubiquinone is Q-8. The predominant phospholipids are phosphatidylethanolamine, phosphatidylglycerol and cardiolipin. Ammonia is assimilated by glutamate dehydrogenase. The DNA G+C contents of strains Iva(T) and Lap(T) are 54.0 and 50.5mol% (T(m)), respectively. Based on 16S rRNA gene sequence analysis and DNA-DNA relatedness (38-45%) with type strains of the genus Methylobacillus, the novel isolates are classified as the new species of this genus and named Methylobacillus arboreus Iva(T) (VKM B-2590(T), CCUG 59684(T), DSM 23628(T)) and Methylobacillus gramineus Lap(T) (VKM B-2591(T), CCUG 59687(T), DSM 23629(T)). The GenBank accession numbers for the 16S rRNA gene and mxaF gene sequences of the strains Iva(T) and Lap(T) are GU937479, GU937478 and HM030736, HM030735, respectively.

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.

Genome of Methylobacillus flagellatus, molecular basis for obligate methylotrophy, and polyphyletic origin of methylotrophy.

Along with methane, methanol and methylated amines represent important biogenic atmospheric constituents; thus, not only methanotrophs but also nonmethanotrophic methylotrophs play a significant role in global carbon cycling. The complete genome of a model obligate methanol and methylamine utilizer, Methylobacillus flagellatus (strain KT) was sequenced. The genome is represented by a single circular chromosome of approximately 3 Mbp, potentially encoding a total of 2,766 proteins. Based on genome analysis as well as the results from previous genetic and mutational analyses, methylotrophy is enabled by methanol and methylamine dehydrogenases and their specific electron transport chain components, the tetrahydromethanopterin-linked formaldehyde oxidation pathway and the assimilatory and dissimilatory ribulose monophosphate cycles, and by a formate dehydrogenase. Some of the methylotrophy genes are present in more than one (identical or nonidentical) copy. The obligate dependence on single-carbon compounds appears to be due to the incomplete tricarboxylic acid cycle, as no genes potentially encoding alpha-ketoglutarate, malate, or succinate dehydrogenases are identifiable. The genome of M. flagellatus was compared in terms of methylotrophy functions to the previously sequenced genomes of three methylotrophs, Methylobacterium extorquens (an alphaproteobacterium, 7 Mbp), Methylibium petroleiphilum (a betaproteobacterium, 4 Mbp), and Methylococcus capsulatus (a gammaproteobacterium, 3.3 Mbp). Strikingly, metabolically and/or phylogenetically, the methylotrophy functions in M. flagellatus were more similar to those in M. capsulatus and M. extorquens than to the ones in the more closely related M. petroleiphilum species, providing the first genomic evidence for the polyphyletic origin of methylotrophy in Betaproteobacteria.

Expression and purification of HtpX-like small heat shock integral membrane protease of an unknown organism related to Methylobacillus flagellatus.

The M48 conserved family of peptidases contains a single catalytic zinc ion tetrahedrally co-ordinated by two histidines within an HEXXH motif. The proteases of this class are generally toxic to the cell and thus difficult to express and purify. Here, we report the expression and purification of the small HtpX-like heat shock metalloprotease from an unknown organism related to the obligate methylotrophic anaerobic bacterium, Methylobacillus flagellatus. The protease was expressed in the Escherichia coli vector - pT7. Optimization of expression was done to increase the yield and solubility of the expressed protein. Improved refolding procedures from inclusion bodies of pT7 E. coli system were devised to get the protease in an active and stable form. The protease was purified to near homogeneity in its active form from the refolded proteins of the inclusion bodies by a two-step (cation exchange followed by gel filtration) high performance liquid chromatography (HPLC). The purified protease was active on zymography and casein hydrolysis assays. The activity of the protease was found to be optimum at pH 7.4 and at a temperature of 37 degrees C but significant activity was also retained at higher temperatures of 45-50 degrees C. Centrifugal fractionation showed that it is a membrane localized endopeptidase. The methods described here can serve as guidelines to express and purify other homologues of M48 family of proteases for functional and structural studies.

Cloning and sequencing of the aldehyde oxidase gene from Methylobacillus sp. KY4400.

The aldehyde oxidase genes (aods) from Methylobacillus sp. KY4400 were cloned, and sequenced. The sequences for small (aodS, 489 bp), medium (aodM, 993 bp), and large (aodL, 2,328 bp) subunit genes were determined. At least one additional ORF was indispensable for the expression of enzyme activity. The structural genes contained two [2Fe-2S] centers, an FAD binding site, and a molybdenum cofactor binding site.

Genes involved in the synthesis of the exopolysaccharide methanolan by the obligate methylotroph Methylobacillus sp strain 12S.

Methylobacillus sp. strain 12S produces an exopolysaccharide (EPS), methanolan, composed of glucose, mannose and galactose. Twenty-four ORFs flanking a Tn5 insertion site in an EPS-deficient mutant were identified, and 21 genes (epsCBAKLDEFGHIJMNOPQRSTU) were predicted to participate in methanolan synthesis on the basis of the features of the primary sequence. Gene disruption analyses revealed that epsABCEFGIJNOP and epsR are required for methanolan synthesis, whereas epsKD and epsH are not essential. EpsFG and EpsE showed homology with Wzc (chain length regulator) and Wza (export protein) of group 1 capsule-producing Escherichia coli, suggesting that methanolan was synthesized via a Wzy-like biosynthesis system. This possibility was supported by the fact that the putative hydropathy profiles of EpsH and EpsM were similar to those of Wzx and Wzy, which are also involved in the flipping of the repeating unit in the cytoplasmic membrane and the polymerization of the capsule in the Wzy-dependent system. EpsBJNOP and EpsR are probably glycosyltransferases involved in the synthesis of the repeating unit onto the lipid carrier. In particular, EpsB appeared to catalyse the initial transfer of the glucose moiety. On the basis of their predicted location in the cells, it is proposed that EpsI and EpsL are involved in methanolan export to the cell surface. E. coli strains expressing EpsQ, EpsS and EpsT showed enhanced activities of GDP-mannose pyrophosphorylase, UDP-galactose 4-epimerase and UDP-glucose pyrophosphorylase, respectively, revealing that they were responsible for the production of the activated compositional sugars of methanolan. EpsU contains a conserved a lytic transglycosylase motif, indicating that it could participate in the degradation of polysaccharides. EpsA and EpsK, which have conserved DNA-binding and cAMP-binding motifs, respectively, were deduced to be transcriptional regulators. In particular, EpsA seems to positively regulate the transcription of methanolan synthesis genes, since the constitutive expression of epsA in strain 12S increased the EPS production. Interestingly, EpsD showed homology with peptidyl prolyl cis-trans isomerases that catalyse the folding of proteins following translocation across the cytoplasmic membrane.

Overproduction of L-Lysine from methanol by Methylobacillus glycogenes derivatives carrying a plasmid with a mutated dapA gene.

The dapA gene, encoding dihydrodipicolinate synthase (DDPS) partially desensitized to inhibition by L-lysine, was cloned from an L-threonine- and L-lysine-coproducing mutant of the obligate methylotroph Methylobacillus glycogenes DHL122 by complementation of the nutritional requirement of an Escherichia coli dapA mutant. Introduction of the dapA gene into DHL122 and AL119, which is the parent of DHL122 and an L-threonine producing mutant, elevated the specific activity of DDPS 20-fold and L-lysine production 2- to 3-fold with concomitant reduction of L-threonine in test tube cultures. AL119 containing the dapA gene produced 8 g of L-lysine per liter in a 5-liter jar fermentor from methanol as a substrate. Analysis of the nucleotide sequence of the dapA gene shows that it encodes a peptide with an M(r) of 30,664 and that the encoded amino acid sequence is extensively homologous to those of other organisms. In order to study the mutation that occurred in DHL122, the dapA genes of the wild type and AL119 were cloned and sequenced. Comparison of the nucleotide sequences of the dapA genes revealed that the amino acid at residue 88 was F in DHL122 whereas it was L in the wild type and AL119, suggesting that this amino acid alteration that occurred in DHL122 caused the partial desensitization of DDPS to the inhibition by L-lysine. The similarity in the amino acid sequences of DDPS in M. glycogenes and other organisms suggests that the mutation of the dapA gene in DHL122 is located in the region concerned with interaction of the allosteric effector, L-lysine.

Saccharide production from methanol by transposon 5 mutants derived from the extracellular polysaccharide-producing bacterium Methylobacillus sp. strain 12S.

A CH3OH-utilizing bacterium that has the ability to produce extracellular polysaccharide (EPS) was isolated from a soil sample, and was identified as the obligate methylotroph Methylobacillus sp. strain 12S on the basis of its 16S rDNA sequence and growth-substrate specificity. The EPS produced by strain 12S was purified and the sugar composition was analysed by GC-MS and HPLC to reveal that the EPS was a heteropolymer composed of glucosyl, galactosyl, and mannosyl residues in the molar ratio 3:1:1. In order to produce mono- and/or oligosaccharides by single-step fermentation from CH3OH, stain 12S was mutagenized by transposon 5. Among eleven EPS-deficient mutants, three strains were found to accumulate significant amounts of reducing sugars in the media. The amounts of the reducing sugars produced by the mutants ( > ca. 700 mg glucose equivalent/l) were > 11-22 times higher than those produced by the wild-type strain (

Analysis of two formaldehyde oxidation pathways in Methylobacillus flagellatus KT, a ribulose monophosphate cycle methylotroph.

The roles of cyclic formaldehyde oxidation via 6-phosphogluconate dehydrogenase and linear oxidation via the tetrahydromethanopterin (H4MPT)-linked pathway were assessed in an obligate methylotroph, Methylobacillus flagellatus KT, by cloning, sequencing and mutating two chromosomal regions containing genes encoding enzymes specifically involved in these pathways: 6-phosphogluconate dehydrogenase, glucose-6-phosphate dehydrogenase and methenyl H4MPT cyclohydrolase (gndA, zwf and mch). No null mutants were obtained in gndA or zwf, implying that the cyclic oxidation of formaldehyde is required for C1 metabolism in this obligate methylotroph, probably as the main energy-generating pathway. In contrast, null mutants were generated in mch, indicating that the H4MPT-linked pathway is dispensable. These mutants showed enhanced sensitivity to formaldehyde, suggesting that this pathway plays a secondary physiological role in this methylotroph. This function is in contrast to Methylobacterium extorquens AM1, in which the H4MPT-linked pathway is essential.

Organization of threonine biosynthesis genes from the obligate methylotroph Methylobacillus flagellatus.

The genes encoding aspartate kinase (ask), homoserine dehydrogenase (hom), homoserine kinase (thrB) and threonine synthase (thrC) from the obligate methylotroph Methylobacillus flagellatus were cloned. In maxicells hom and thrC directed synthesis of 51 and 48 kDa polypeptides, respectively. The hom, thrB and thrC genes and adjacent DNA areas were sequenced. Of the threonine biosynthesis genes, only hom and thrC were tightly linked in the order hom-thrC. The gene for thymidylate synthase (thyA) followed thrC and the gene for aspartate aminotransferase (aspC) preceded hom. All four genes (aspC-hom-thrC-thyA) were transcribed in the same direction. mRNA analysis indicated that hom-thrC are apparently transcribed in one 7.5 kb transcript in M. flagellatus. Promoter analysis showed the presence of a functional promoter between aspC and hom. No functional promoter was found to be associated with the DNA stretch between hom and thrC. The thrB gene encoded an unusual type of homoserine kinase and was not linked to other threonine biosynthesis genes.

[Use of the recombinant bacterial strain to control blood-sucking mosquito larvae]. / Vozmozhnost' primeneniia rekombinantnogo shtamma bakterii dlia bor'by s lichinkami krovososushchikh komarov.

The recombinant strain of Methylobacillus flagellatum with the cloned synthesis gene Cry 4B of the toxic Bac. thuringiensis var. israelensis protein proved to be effective against larvae of the Anopheles stephensi, An. atroparvus, An. pulcherrimus, An. superpictus, and An. sacharovi cultured in the laboratory. The use of M. flagellatum in combination with T. pyriformis may greatly expand the scope of use of the recombinant strain to control malaria mosquito larvae. Their combined use shows a 6-fold increase in the rate of strain action and a 4-fold decrease in the concentration of the agent. The optimum effects are shown following 24-hour combined intubation of M. flagellatum and Tetrahymena pyriformis.

Isolation and characterization of a mutant defective in the production of methanol dehydrogenase from a new restricted facultative methanol-oxidizing bacterium.

A new restricted facultative methanol-oxidizing bacterium Methylobacillus sp. strain SK1 (DSM 8269) was subjected to insertion mutagenesis studies with the transposon Tn5 to generate mutants deficient in the production of methanol dehydrogenase (MDH). The transposon was conjugally transferred into the methylotroph by using the triparental mating procedure. The mutants induced by Tn5 were selected directly from a plate containing succinate by using the allyl alcohol selection procedure. The transposition of Tn5 to the bacterium showed a moderate transposition frequency (10(-5) - 10(-6)). Southern hybridization analysis confirmed that the transposon Tn5 was inserted into the chromosomal DNA of the mutants. In addition, the mutants had no RNA transcripts produced from the mdh gene, as judged by Northern blot analysis. The mutants could neither grow on methanol nor produce MDH protein, as determined by Western blot analysis with anti-MDH antibody. These results suggest that Tn5 mutagenesis maybe a useful tool for the molecular analysis of a facultative methanol-oxidizing bacterium.