Lanpepsy is a novel lanthanide-binding protein involved in the lanthanide response of the obligate methylotroph Methylobacillus flagellatus.

Lanthanides were recently discovered as metals required in the active site of certain methanol dehydrogenases. Since then, the characterization of the lanthanome, that is, proteins involved in sensing, uptake, and utilization of lanthanides, has become an active field of research. Initial exploration of the response to lanthanides in methylotrophs has revealed that the lanthanome is not conserved and that multiple mechanisms for lanthanide utilization must exist. Here, we investigated the lanthanome in the obligate model methylotroph Methylobacillus flagellatus. We used a proteomic approach to analyze differentially regulated proteins in the presence of lanthanum. While multiple known proteins showed induction upon growth in the presence of lanthanum (Xox proteins, TonB-dependent receptor), we also identified several novel proteins not previously associated with lanthanide utilization. Among these was Mfla_0908, a periplasmic 19 kDa protein without functional annotation. The protein comprises two characteristic PepSY domains, which is why we termed the protein lanpepsy (LanP). Based on bioinformatic analysis, we speculated that LanP could be involved in lanthanide binding. Using dye competition assays, quantification of protein-bound lanthanides by inductively coupled plasma mass spectrometry, as well as isothermal titration calorimetry, we demonstrated the presence of multiple lanthanide binding sites that showed selectivity over the chemically similar calcium ion. LanP thus represents the first member of the PepSY family that binds lanthanides. Although the physiological role of LanP is still unclear, its identification is of interest for applications toward the sustainable purification and separation of rare-earth elements.

Crystal structure of an aldehyde oxidase from Methylobacillus sp. KY4400.

Hetero-trimeric aldehyde oxidases of bacterial origin, which use O2 to catalyse the oxidation of various aldehydes but not those of aromatic N-heterocycles, belong to the xanthine oxidase family. In the present study, the crystal structure of a recombinant aldehyde oxidase from Methylobacillus sp. KY4400 (Mb-AOX) was determined at 2.5 Å resolution. The structures of its subunits resemble those of the corresponding subunits or domains of other structurally characterised enzymes belonging to the family, and include a [4Fe-4 S] cluster in the medium subunit like that found in Escherichia coli periplasmic aldehyde oxidoreductase (EP-AOR). A funnel leading to the si-face of the isoalloxazine ring of FAD, which is narrower than those in mouse liver AOX3 and human AOX1, is also present and it is even narrower than that in EP-AOR. The environment surrounding the ring in Mb-AOX and EP-AOR is subtly different, which might account for their different abilities to use O2. A remarkable characteristic of the Mo catalytic centre in Mb-AOX is a tryptophan situated near the centre instead of the alanine present in other xanthine oxidase family members. The tryptophan residue together with other residue differences might play an important role in binding to aldehydes such as n-heptylaldehyde in Mb-AOX.

Biochemical Properties and Phylogeny of Hydroxypyruvate Reductases from Methanotrophic Bacteria with Different C1-Assimilation Pathways.

In the aerobic methanotrophic bacteria Methylomicrobium alcaliphilum 20Z, Methylococcus capsulatus Bath, and Methylosinus trichosporium OB3b, the biochemical properties of hydroxypyruvate reductase (Hpr), an indicator enzyme of the serine pathway for assimilation of reduced C1-compounds, were comparatively analyzed. The recombinant Hpr obtained by cloning and heterologous expression of the hpr gene in Escherichia coli catalyzed NAD(P)H-dependent reduction of hydroxypyruvate or glyoxylate, but did not catalyze the reverse reactions of D-glycerate or glycolate oxidation. The absence of the glycerate dehydrogenase activity in the methanotrophic Hpr confirmed a key role of the enzyme in utilization of C1-compounds via the serine cycle. The enzyme from Ms. trichosporium OB3b realizing the serine cycle as a sole assimilation pathway had much higher special activity and affinity in comparison to Hpr from Mm. alcaliphilum 20Z and Mc. capsulatus Bath assimilating carbon predominantly via the ribulose monophosphate (RuMP) cycle. The hpr gene was found as part of gene clusters coding the serine cycle enzymes in all sequenced methanotrophic genomes except the representatives of the Verrucomicrobia phylum. Phylogenetic analyses revealed two types of Hpr: (i) Hpr of methanotrophs belonging to the Gammaproteobacteria class, which use the serine cycle along with the RuMP cycle, as well as of non-methylotrophic bacteria belonging to the Alphaproteobacteria class; (ii) Hpr of methylotrophs from Alpha- and Betaproteobacteria classes that use only the serine cycle and of non-methylotrophic representatives of Betaproteobacteria. The putative role and origin of hydroxypyruvate reductase in methanotrophs are discussed.

Screening and Degradation Mechanism of a Cold-Resistant Nitrobenzene-Degrading Microorganism.

A cold-resistant nitrobenzene-degrading strain was screened from river sediment. The strain was identified as Methylobacillus glycogens, which has never been reported to be capable of degrading nitrobenzene. The degradation rates of 900 µg/L nitrobenzene reached respectively 99.3% and 88.6% in 144 h under both aerobic and anaerobic environments (30 mL inoculation volume at 12 ± 0.5 °C and pH7.0 ± 0.1). When aerobically degraded, nitrobenzene was firstly oxidized into o-nitrophenol, which was further oxidized into 1,2-benzenediol, meanwhile releasing NO2-. Then the 1,2-benzenediol was metabolized through either the ortho-cleavage into succinic acid and acetyl-CoA, or meta-cleavage into pyruvic acid and acetaldehyde, as well as other small molecule substances of non-toxicity or low-toxicity, which were finally decomposed into CO2 and H2O. When anaerobically degraded, nitrobenzene was firstly degraded into aniline (C6H5NH2), which was further degraded into 4-amino benzoic acid. The benzoic acid was degraded into benzoyl, which was finally metabolized and decomposed.

Medium optimization for pyrroloquinoline quinone (PQQ) production by Methylobacillus sp. zju323 using response surface methodology and artificial neural network-genetic algorithm.

Methylobacillus sp. zju323 was adopted to improve the biosynthesis of pyrroloquinoline quinone (PQQ) by systematic optimization of the fermentation medium. The Plackett-Burman design was implemented to screen for the key medium components for the PQQ production. CoCl2 · 6H2O, ρ-amino benzoic acid, and MgSO4 · 7H2O were found capable of enhancing the PQQ production most significantly. A five-level three-factor central composite design was used to investigate the direct and interactive effects of these variables. Both response surface methodology (RSM) and artificial neural network-genetic algorithm (ANN-GA) were used to predict the PQQ production and to optimize the medium composition. The results showed that the medium optimized by ANN-GA was better than that by RSM in maximizing PQQ production and the experimental PQQ concentration in the ANN-GA-optimized medium was improved by 44.3% compared with that in the unoptimized medium. Further study showed that this ANN-GA-optimized medium was also effective in improving PQQ production by fed-batch mode, reaching the highest PQQ accumulation of 232.0 mg/L, which was about 47.6% increase relative to that in the original medium. The present work provided an optimized medium and developed a fed-batch strategy which might be potentially applicable in industrial PQQ production.

Enhanced fed-batch production of pyrroloquinoline quinine in Methylobacillus sp. CCTCC M2016079 with a two-stage pH control strategy.

The effects of pH control strategy and fermentative operation modes on the biosynthesis of pyrroloquinoline quinine (PQQ) were investigated systematically with Methylobacillus sp. CCTCC M2016079 in the present work. Firstly, the shake-flask cultivations and benchtop fermentations at various pH values ranging from 5.3 to 7.8 were studied. Following a kinetic analysis of specific cell growth rate (µ x ) and specific PQQ formation rate (µ p ), the discrepancy in optimal pH values between cell growth and PQQ biosynthesis was observed, which stimulated us to develop a novel two-stage pH control strategy. During this pH-shifted process, the pH in the broth was controlled at 6.8 to promote the cell growth for the first 48 h and then shifted to 5.8 to enhance the PQQ synthesis until the end of fermentation. By applying this pH-shifted control strategy, the maximum PQQ production was improved to 158.61 mg/L in the benchtop fermenter, about 44.9% higher than that under the most suitable constant pH fermentation. Further fed-batch study showed that PQQ production could be improved from 183.38 to 272.21 mg/L by feeding of methanol at the rate of 11.5 mL/h in this two-stage pH process. Meanwhile, the productivity was also increased from 2.02 to 2.84 mg/L/h. In order to support cell growth during the shifted pH stage, the combined feeding of methanol and yeast extract was carried out, which brought about the highest concentration (353.28 mg/L) and productivity (3.27 mg/L/h) of PQQ. This work has revealed the potential of our developed simple and economical strategy for the large-scale production of PQQ.

Biochemical and molecular characterization of the isocitrate dehydrogenase with dual coenzyme specificity from the obligate methylotroph Methylobacillus Flagellatus.

The isocitrate dehydrogenase (MfIDH) with unique double coenzyme specificity from Methylobacillus flagellatus was purified and characterized, and its gene was cloned and overexpressed in E. coli as a fused protein. This enzyme is homodimeric,-with a subunit molecular mass of 45 kDa and a specific activity of 182 U mg -1 with NAD+ and 63 U mg -1 with NADP+. The MfIDH activity was dependent on divalent cations and Mn2+ enhanced the activity the most effectively. MfIDH exhibited a cofactor-dependent pH-activity profile. The optimum pH values were 8.5 (NAD+) and 6.0 (NADP+).The Km values for NAD+ and NADP+ were 113 µM and 184 µM respectively, while the Km values for DL-isocitrate were 9.0 µM (NAD+), 8.0 µM (NADP+). The MfIDH specificity (kcat/Km) was only 5-times higher for NAD+ than for NADP+. The purified MfIDH displayed maximal activity at 60°C. Heat-inactivation studies showed that the MfIDH was remarkably thermostable, retaining full activity at 50°C and losting ca. 50% of its activity after one hour of incubation at 75°C. The enzyme was insensitive to the presence of intermediate metabolites, with the exception of 2 mM ATP, which caused 50% inhibition of NADP+-linked activity. The indispensability of the N6 amino group of NAD(P)+ in its binding to MfIDH was demonstrated. MfIDH showed high sequence similarity with bacterial NAD(P)+-dependent type I isocitrate dehydrogenases (IDHs) rather than with eukaryotic NAD+-dependent IDHs. The unique double coenzyme specificity of MfIDH potentially resulted from the Lys340, Ile341 and Ala347 residues in the coenzyme-binding site of the enzyme. The discovery of a type I IDH with double coenzyme specificity elucidates the evolution of this subfamily IDHs and may provide fundamental information for engineering enzymes with desired properties.

Biodiversity of aerobic methylobacteria associated with the phyllosphere of the southern Moscow Oblast.

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

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).

A distinct holoenzyme organization for two-subunit pyruvate carboxylase.

Pyruvate carboxylase (PC) has important roles in metabolism and is crucial for virulence for some pathogenic bacteria. PC contains biotin carboxylase (BC), carboxyltransferase (CT) and biotin carboxyl carrier protein (BCCP) components. It is a single-chain enzyme in eukaryotes and most bacteria, and functions as a 500 kD homo-tetramer. In contrast, PC is a two-subunit enzyme in a collection of Gram-negative bacteria, with the α subunit containing the BC and the ß subunit the CT and BCCP domains, and it is believed that the holoenzyme has α4ß4 stoichiometry. We report here the crystal structures of a two-subunit PC from Methylobacillus flagellatus. Surprisingly, our structures reveal an α2ß4 stoichiometry, and the overall architecture of the holoenzyme is strikingly different from that of the homo-tetrameric PCs. Biochemical and mutagenesis studies confirm the stoichiometry and other structural observations. Our functional studies in Pseudomonas aeruginosa show that its two-subunit PC is important for colony morphogenesis.

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.

Biosynthesis of glucosyl glycerol, a compatible solute, using intermolecular transglycosylation activity of amylosucrase from Methylobacillus flagellatus KT.

A putative α-amylase gene (accession number, CP000284) of Methylobacillus flagellatus KT ATCC51484 was cloned in Escherichia coli, and its gene product was expressed and characterized. The purified recombinant enzyme (MFAS) displayed a typical amylosucrase (ASase) activity by the demonstration of multiple activities of hydrolysis, isomerization, and polymerization although it was designated as an α-amylase. The optimal reaction temperature and pH for the sucrose hydrolysis activity of MFAS were determined to be 45 °C and pH 8.5, respectively. MFAS has relatively high thermostable characteristics compared with other ASases, as demonstrated by a half-life of 19.3 min at 50 °C. MFAS also showed polymerization activity using sucrose as a sole substrate. Glycerol was transglycosylated by the intermolecular transglycosylation activity of MFAS. Two major products were observed by thin-layer chromatography and isolated by paper chromatography and recycling HPLC. Using (1)H and (13)C NMR, their chemical structures were determined to be (2S)-1-O-α-D-glucosyl-glycerol or (2R)-1-O-α-D-glucosyl-glycerol and 2-O-α-D-glucosyl-glycerol, in which a glucose molecule is linked to glycerol via an α-glycosidic linkage.

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.

Isolation of a Methylobacillus sp. that degrades microcystin toxins associated with cyanobacteria.

Sludge from cyanobacteria-salvaged yard in Meiliang Bay, Lake Taihu in Wuxi, China was cultured and acclimated by inoculating microcystins (MCs) extract. Strain J10 was isolated by degrading the MC-RR and MC-LR and was identified as Methylobacillus sp. Further research showed that both MC-LR and MC-RR could be completely degraded at 17h after inoculation of J10, and the degradation probably was mediated by oxygen. Different enzymes, oxygen-dependent as well as oxygen-independent, with MC-degrading activity were found in the different fractions of J10 culture. However, the enzymes mainly responsible for MC degradation by J10 were oxygen-dependent and were probably bound to cell wall or outside the cytoplasmic membrane.