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.

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.

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.

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.

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.

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.

[Establishment of the screening method and isolation of PQQ producing strains].

Pyrroloquinoline quinone (PQQ) is a cofactor of some oxido-reductases with many important physiological effects and potential pharmaceutical applications. The glucose dehydrogenase of Escherichia coli, being a candidate for enzymic detection of PQQ, is known to be a quinoprotein which is obligately dependant on PQQ as cofactor. The gdh gene of E. coli was amplified and cloned into plasmid pET28a. The recombinant GDH was overexpressed in soluble form in E. coli BL21 (DE3). A bioassay method was established for determination of PQQ by the purified GDH. A screening model was set up for the enrichment of methylotrophic bacteria. Together with the above bioassay method, over 2000 soil samples were screened for the isolation of high-yielding PQQ producing strains. A methylotrophic strain, named MP606, was thus isolated. The PQQ production of MP606 is determined to be 113mg/L without conditional optimization and genetic breeding. The PQQ crystal was obtained from the culture supernatant which has been identified by HPLC, absorption spectra assay, and enzymatic analysis. The 16S rDNA of MP606 was amplified and sequenced. According to the comparison of 16S rDNA sequences, overall similarity value between strain MP606 and 12 typical methylotrophic bacteria is above 95% . The highest value is with two strains of Methylovorus, which reached at 99%.

Purification and some properties of a blue copper protein from Methylobacillus sp. strain SK1 DSM 8269.

A blue protein was purified from the Methylobacillus sp. strain SK1 that is grown on methanol in the presence of copper ion. This protein was found to be a monomer with a molecular weight of 13,500. The Isoelectric point of the protein was estimated to be 8.8. The spectrum of the protein that was treated with ferricyanide showed a broad peak around 620 nm, but that of the dithionite-treated protein revealed no peaks. It contained 0.83 mol of EDTA-stable copper per mol protein. Under air, the protein accelerated the inactivation of methanol dehydrogenase (MDH). The protein was reducible by phenazine methosulfate or by active MDH that was prepared from cells that were grown in the absence of added copper, but not by methanol, dichlorophenol indophenol, or inactive MDH that was prepared from cells that were grown in the presence of added copper. It was also reducible by active MDH in the presence of methanol. The absorption peak at 340 nm of the active MDH disappeared after the enzyme was treated with ferricyanide, hydrogen peroxide, or the purified blue protein. The inactive MDH also showed no peak at 340 nm. The 340-nm peak was not recovered after incubation of the inactive MDH and blue protein-treated active MDH with dithionite or methanol. The inactive MDH and blue protein-treated active MDH co-migrated with the active MDH preparation on nondenaturing polyacrylamide gel, and contained two non-identical subunits with molecular weights that were identical to those of the active MDH. The N-terminal amino acid sequence of the protein was Ala-Gly-Cys-Ser-Val-Asp-Val-Glu-Ala-Asn-Asp-Ala-Met-Gln-Phe. An analysis of the amino acid composition revealed that the protein contained no tryptophan. It contained three cysteines per mol protein. The blue protein in Methylobacillus sp. strain SK1 was produced only in the cells that were grown in the copper-supplemented medium.

[Use of methylotrophic bacteria Methylobacillus flagellatum KT for isolation of deuterated exogenous carbohydrates]. / Ispol'zovanie metilotrofnykh bakterii Methylobacillus flagellatum KT dlia polucheniia deiterirovannykh ékzogennykh uglevodov.

The cultivation conditions optimal for biosynthesis of exogenous polysaccharides by methylotrophic bacteria Methylobacillus flagellatum were evaluated. The mutant strain most active in accumulating exogenous polysaccharides was selected. Gradual adaptation of this strain to the deuterated medium containing 1 vol % CD3OD in deuterium oxide intensified biosynthesis of exogenous polysaccharides and inhibited bacterial growth (compared to the standard medium). The monomeric composition of exogenous polysaccharides obtained during cultivation on standard and deuterated media was estimated by high-performance liquid chromatography and nuclear magnetic resonance spectroscopy. Nondeuterated exogenous polysaccharides contained only fructose, while deuterated exogenous polysaccharides included 98% fructose and 2% ribose. Paramagnetic resonance spectroscopy showed that the degree of deuterium incorporation into molecules of biosynthetic carbohydrates was 89%.

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.

Effect of growth conditions on the synthesis of terminal oxidases in Methylobacillus flagellatus KT.

Membranes of the obligate methylotroph Methylobacillus flagellatus KT contained hemes B, O, and C and cytochromes b, o, and c both in batch and in continuous cultures. Neither heme A nor heme D was detected in the membranes. The cytochromes o and bb were the main components reversibly binding carbon monoxide (CO) in the terminal part of the respiratory chain. The alpha-region and especially the alpha-peaks at 568 and 573 nm and the alpha-troughs at 586 and 592 on the CO-difference spectra were diagnostic for the cytochromes o and bb, respectively. The cytochrome o content increased up to 1.8 times upon increasing the dilution rate of the culture from 0.15 to 0.55 h(-1) under methanol limitation. By contrast, the level of the CO-binding cytochrome bb was not affected by methanol concentration but its content increased up to 1.9 times when the level of oxygen decreased from 95 to 21 microM under the constant dilution rate (mu = 0.55 h(-1)). The maximum ratio between the cytochromes o and bb reached 2 during continuous cultivation under methanol-limited conditions (mu = 0.55 h(-1)), whereas the minimum ratio between them was about 0.7 during batch cultivation at stationary phase of growth. The synthesis of the CO-binding cytochrome bb but not of the cytochrome o in M. flagellatus KT was assumed to depend on the ambient redox potential of the medium. The cytochrome o synthesis was supposed to depend on the transmembrane gradient of protons (Delta(mu)H+).

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.

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.

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 (