Cometabolic biodegradation of quizalofop-p-ethyl by Methylobacterium populi YC-XJ1 and identification of QPEH1 esterase

BACKGROUND: Quizalofop-p-ethyl (QPE), a unitary R configuration aromatic oxyphenoxypropionic acid ester (AOPP) herbicide, was widely used and had led to detrimental environmental effects. For finding the QPEdegrading bacteria and promoting the biodegradation of QPE, a series of studies were carried out. RESULTS: A QPE-degrading bacterial strain YC-XJ1 was isolated from desert soil and identified as Methylobacterium populi, which could degrade QPE with methanol by cometabolism. Ninety-seven percent of QPE (50 mg/L) could be degraded within 72 h under optimum biodegradation condition of 35°C and pH 8.0. The maximum degradation rate of QPE was 1.4 mg/L/h, and the strain YC-XJ1 exhibited some certain salinity tolerance. Two novel metabolites, 2-hydroxy-6-chloroquinoxaline and quinoxaline, were found by high-performance liquid chromatography/mass spectroscopy analysis. The metabolic pathway of QPE was predicted. The catalytic efficiency of strain YC-XJ1 toward different AOPPs herbicides in descending order was as follows: haloxyfop-pmethyl ≈ diclofop-methyl ≈ fluazifop-p-butyl N clodinafop-propargyl N cyhalofop-butyl N quizalofop-p-ethyl N fenoxaprop-p-ethyl N propaquizafop N quizalofop-p-tefuryl. The genome of strain YC-XJ1 was sequenced using a combination of PacBio RS II and Illumina platforms. According to the annotation result, one α/ß hydrolase gene was selected and named qpeh1, for which QPE-degrading function has obtained validation. Based on the phylogenetic analysis and multiple sequence alignment with other QPE-degrading esterases reported previously, the QPEH1 was clustered with esterase family V. CONCLUSION: M. populi YC-XJ1 could degrade QPE with a novel pathway, and the qpeh1 gene was identified as one of QPE-degrading esterase gene.

High Production of Ergothioneine in Escherichia coli using the Sulfoxide Synthase from Methylobacterium strains.

We previously constructed a heterologous production system for ergothioneine (ERG) in Escherichia coli using five ERG biosynthesis genes (egtABCDE) from Mycobacterium smegmatis. However, significant amounts of hercynine (HER), an intermediate of ERG, as ERG were accumulated, suggesting that the reaction of EgtB catalyzing the attachment of γ-glutamylcysteine (γGC) to HER to yield hercynyl-γ-glutamylcysteine sulfoxide was a bottleneck. In this study, we searched for other EgtBs and found many egtB orthologs in diverse microorganisms. Among these, Methylobacterium strains possessed EgtBs that catalyze the direct conversion of HER into hercynylcysteine sulfoxide with l-cysteine (l-Cys) as a sulfur donor, in a manner similar to those of acidobacterial CthEgtB and fungal Egt1. An in vitro study with recombinant EgtBs from Methylobacterium brachiatum and Methylobacterium pseudosasicola clearly showed that both enzymes accepted l-Cys but not γGC. We reconstituted the ERG production system in E. coli with egtB from M. pseudosasicola; ERG productivity reached 657 mg L-1.

Purification and characterization of formaldehyde dismutases of Methylobacterium sp. FD1.

In the present study, we purified and characterized three formaldehyde dismutases (Fdms) (EC 1.2.98.1) (Fdm1, Fdm2, and Fdm3) of Methylobacterium sp. FD1. These Fdms (with His-tag) were produced in the recombinant E. coli and purified by immobilized metal affinity chromatography from the E. coli extracts. In each of the three Fdms, the enzyme-bound coenzyme was nicotinamide adenine dinucleotide (NAD(H)) and the enzyme-bound metal was zinc. The quaternary structures of these Fdms were estimated as homotetrameric. The optimal pHs and temperatures of Fdm1, Fdm2, and Fdm3 were approximately 6.5, 6.0, and 6.0, and 35°C, 25°C, and 30°C, respectively. The Km values of Fdm1, Fdm2, and Fdm3 were 621, 865, and 414 mM, respectively. These results were similar to the properties of already-known Fdms. However, each of the Fdms of FD1 had methanol:p-nitroso-N,N-dimethylaniline oxidoreductase activity that is not found in already-known Fdms.

Crossing the Border: From Keto- to Imine Reduction in Short-Chain Dehydrogenases/Reductases.

The family of NAD(P)H-dependent short-chain dehydrogenases/reductases (SDRs) comprises numerous biocatalysts capable of C=O or C=C reduction. The highly homologous noroxomaritidine reductase (NR) from Narcissus sp. aff. pseudonarcissus and Zt_SDR from Zephyranthes treatiae, however, are SDRs with an extended imine substrate scope. Comparison with a similar SDR from Asparagus officinalis (Ao_SDR) exhibiting keto-reducing activity, yet negligible imine-reducing capability, and mining the Short-Chain Dehydrogenase/Reductase Engineering Database indicated that NR and Zt_SDR possess a unique active-site composition among SDRs. Adapting the active site of Ao_SDR accordingly improved its imine-reducing capability. By applying the same strategy, an unrelated SDR from Methylobacterium sp. 77 (M77_SDR) with distinct keto-reducing activity was engineered into a promiscuous enzyme with imine-reducing activity, thereby confirming that the ability to reduce imines can be rationally introduced into members of the "classical" SDR enzyme family. Thus, members of the SDR family could be a promising starting point for protein approaches to generate new imine-reducing enzymes.

Efficient methanol-degrading aerobic bacteria isolated from a wetland ecosystem.

Methylotrophs present in the soil play an important role in the regulation of one carbon compounds in the environment, and thereby aid in mitigating global warming. The study envisages the isolation and characterization of methanol-degrading bacteria from Kuttanad wetland ecosystem, India. Three methylotrophs, viz. Achromobacter spanius KUT14, Acinetobacter sp. KUT26 and Methylobacterium radiotolerans KUT39 were isolated and their phylogenetic positions were determined by constructing a phylogenetic tree based on 16S rDNA sequences. In vitro activity of methanol dehydrogenase enzyme, responsible for methanol oxidation was evaluated and the genes involved in methanol metabolism, mxaF and xoxF were partially amplified and sequenced. The specific activity of methanol dehydrogenase (451.9 nmol min-1 mg-1) observed in KUT39 is the highest, reported ever to our knowledge from a soil bacterium. KUT14 recorded the least activity of 50.15 nmol min-1 mg-1 and is the first report on methylotrophy in A. spanius.

Distribution of 1-aminocyclopropane-1-carboxylate deaminase and D-cysteine desulfhydrase genes among type species of the genus Methylobacterium.

The presence of 1-aminocyclopropane-1-carboxylate (ACC) deaminase determines the ability of bacteria to increase the resistance of plants to various types of stress. The genes of ACC deaminase (acdS) and the closely related enzyme D-cysteine desulfhydrase (dcyD) were searched in type strains of various representatives of the genus Methylobacterium. Using PCR screening and in silico searching in the available complete genome sequences of type strains, the genes were found in 28 of 48 species of the genus. Phylogenetic analysis of amino acid sequences of proteins revealed two large groups of sequences of the AcdS protein and one of the DcyD protein. The distribution of these groups correlates well with the phylogenetic tree based on the sequences of the 16S rRNA genes, which apparently indicates a different evolutionary adaptation to association with plants in the representatives of these groups. For the first time for aerobic methylotrophs it was demonstrated that the gene dcyD encodes D-cysteine desulfhydrase by cloning and recombinant protein characterization.

Toblerols: Cyclopropanol-Containing Polyketide Modulators of Antibiosis in Methylobacteria.

Trans-AT polyketide synthases (PKSs) are a family of biosynthetically versatile modular type I PKSs that generate bioactive polyketides of impressive structural diversity. In this study, we detected, in the genome of several bacteria a cryptic, architecturally unusual trans-AT PKS gene cluster which eluded automated PKS prediction. Genomic mining of one of these strains, the model methylotroph Methylobacterium extorquens AM1, revealed unique epoxide- and cyclopropanol-containing polyketides named toblerols. Relative and absolute stereochemistry were determined by NMR experiments, chemical derivatization, and the comparison of CD data between the derivatized natural product and a synthesized model compound. Biosynthetic data suggest that the cyclopropanol moiety is generated by carbon-carbon shortening of a more extended precursor. Surprisingly, a knock-out strain impaired in polyketide production showed strong inhibitory activity against other methylobacteria in contrast to the wild-type producer. The activity was inhibited by complementation with toblerols, thus suggesting that these compounds modulate an as-yet unknown methylobacterial antibiotic.

Construction of a novel anaerobic pathway in Escherichia coli for propionate production.

BACKGROUND: Propionate is widely used as an important preservative and important chemical intermediate for synthesis of cellulose fibers, herbicides, perfumes and pharmaceuticals. Biosynthetic propionate has mainly been produced by Propionibacterium, which has various limitations for industrial application. RESULTS: In this study, we engineered E. coli by combining reduced TCA cycle with the native sleeping beauty mutase (Sbm) cycle to construct a redox balanced and energy viable fermentation pathway for anaerobic propionate production. As the cryptic Sbm operon was over-expressed in E. coli MG1655, propionate titer reached 0.24 g/L. To increase precursor supply for the Sbm cycle, genetic modification was made to convert mixed fermentation products to succinate, which slightly increased propionate production. For optimal expression of Sbm operon, different types of promoters were examined. A strong constitutive promoter Pbba led to the highest titer of 2.34 g/L. Methylmalonyl CoA mutase from Methylobacterium extorquens AM1 was added to strain T110(pbba-Sbm) to enhance this rate limiting step. With optimized expression of this additional Methylmalonyl CoA mutase, the highest production strain was obtained with a titer of 4.95 g/L and a yield of 0.49 mol/mol glucose. CONCLUSIONS: With various metabolic engineering strategies, the propionate titer from fermentation achieved 4.95 g/L. This is the reported highest anaerobic production of propionate by heterologous host. Due to host advantages, such as non-strict anaerobic condition, mature engineering and fermentation techniques, and low cost minimal media, our work has built the basis for industrial propionate production with E. coli chassis.

Mixed regiospecificity compromises alkene synthesis by a cytochrome P450 peroxygenase from Methylobacterium populi.

Intensive interest has focused on enzymes that are capable of synthesizing hydrocarbons, alkenes and alkanes, for sustainable fuel production. A recently described cytochrome P450 (OleTJE) from the CYP152 family catalyzes an unusual carbon-carbon scission reaction, transforming Cn fatty acids to Cn-1 1-alkenes. Here, we show that a second CYP152, CYP-MP from Methylobacterium populi ATCC BAA 705, also catalyzes oxidative substrate decarboxylation. Alkene production is accompanied with the production of fatty alcohol products, underscoring the mechanistic similarity of the decarboxylation reaction with canonical P450 monooxygenation chemistry. The branchpoint of these two chemistries, and regiospecificity of oxidation products, is strongly chain length dependent, suggesting an importance of substrate coordination for regulating alkene production.

Characterization of a cypermethrin-degrading Methylobacterium sp. strain A-1 and molecular cloning of its carboxylesterase gene.

A novel mesophilic bacterial strain, designated A-1, was isolated from microbially contaminated biopolymer microcapsules. The bacterium was able to withstand and grow in liquid cultures supplemented with the pyrethroid cypermethrin in concentrations up to 400 mg L(-1) . Furthermore, strain A-1 could use cypermethrin as sole carbon source and could degrade >50% of it in 12 h. Based on phenotypic and chemotaxonomic characterization, and phylogenetic analysis of 16S rRNA gene sequence, the strain A-1 was identified as Methylobacterium sp., which is the first reported cypermethrin degrader of methylotrophic bacteria. A role for esterase activity in cypermethrin biodegradation was presumed. Therefore, the carboxylesterase gene mse1 was amplified from the Methylobacterium sp. strain A-1 genome and the resulting 1 kb amplicon cloned into E. coli. Sequence analysis of the mse1-DNA insert revealed an open reading frame of 633 bp encoding for a putative carboxylesterase of 210 amino acid residues with a predicted molecular mass of 22 kDa. The amino acid sequence of the deduced enzyme MsE1 with the catalytic triad Ser106 , Asp156 , and His187 was found to be similar to that of α/ß-hydrolase fold proteins. The active site Ser106 residue is located in the consensus pentapeptide motif Gly-X-Ser-X-Gly that is typical of esterases.

[Functionality of the xoxF Gene in Methylobacterium dichloromethanicum DM4].

Activation of expression of the xoxFgene encoding PQQ-dependent methanol/ethanol dehydrogenase (METDI2492) in dichloromethane- (DCM) -grown Methylobacterium dichloromethanicum DM4 was first demonstrated. The sequence of the only XoxF homolog found in the genome of strain DM4 exhibited 50% identity to that of the protein (MxaF) of the large subunit of methanol dehydrogenase (MDH). A knockout mutant with the inactivate xoxF gene (ΔxoxF) was found to be unable to grow on methanol due to the absence of the expression of the gene cluster of the classical MDH, as was confirmed by the GFP test. When grown of succinate, the ΔxoxF mutant exhibited a lower growth rate on DCM than the original strain and was more sensitive to various stress factors (oxidative, osmotic, and heat shock). Based on these data, the xoxF gene was hypothesized to belong to a group of genes affecting expression of the proteins of general stress response.

Real time expression of ACC oxidase and PR-protein genes mediated by Methylobacterium spp. in tomato plants challenged with Xanthomonas campestris pv. vesicatoria.

Biotic stress like pathogenic infection increases ethylene biosynthesis in plants and ethylene inhibitors are known to alleviate the severity of plant disease incidence. This study aimed to reduce the bacterial spot disease incidence in tomato plants caused by Xanthomonas campestris pv. vesicatoria (XCV) by modulating stress ethylene with 1-aminocyclopropane-1-carboxylate (ACC) deaminase activity of Methylobacterium strains. Under greenhouse condition, Methylobacterium strains inoculated and pathogen challenged tomato plants had low ethylene emission compared to pathogen infected ones. ACC accumulation and ACC oxidase (ACO) activity with ACO related gene expression increased in XCV infected tomato plants over Methylobacterium strains inoculated plants. Among the Methylobacterium spp., CBMB12 resulted lowest ACO related gene expression (1.46 Normalized Fold Expression), whereas CBMB20 had high gene expression (3.42 Normalized Fold Expression) in pathogen challenged tomato. But a significant increase in ACO gene expression (7.09 Normalized Fold Expression) was observed in the bacterial pathogen infected plants. In contrast, Methylobacterium strains enhanced ß-1,3-glucanase and phenylalanine ammonia-lyase (PAL) enzyme activities in pathogen challenged tomato plants. The respective increase in ß-1,3-glucanase related gene expressions due to CBMB12, CBMB15, and CBMB20 strains were 66.3, 25.5 and 10.4% higher over pathogen infected plants. Similarly, PAL gene expression was high with 0.67 and 0.30 Normalized Fold Expression, in pathogen challenged tomato plants inoculated with CBMB12 and CBMB15 strains. The results suggest that ethylene is a crucial factor in bacterial spot disease incidence and that methylobacteria with ACC deaminase activity can reduce the disease severity with ultimate pathogenesis-related protein increase in tomato.

Changes in methane oxidation activity and methanotrophic community composition in saline alkaline soils.

The soil of the former Lake Texcoco is a saline alkaline environment where anthropogenic drainage in some areas has reduced salt content and pH. Potential methane (CH4) consumption rates were measured in three soils of the former Lake Texcoco with different electrolytic conductivity (EC) and pH, i.e. Tex-S1 a >18 years drained soil (EC 0.7 dS m(-1), pH 8.5), Tex-S2 drained for ~10 years (EC 9.0 dS m(-1), pH 10.3) and the undrained Tex-S3 (EC 84.8 dS m(-1), pH 10.3). An arable soil from Alcholoya (EC 0.7 dS m(-1), pH 6.7), located nearby Lake Texcoco was used as control. Methane oxidation in the soil Tex-S1 (lowest EC and pH) was similar to that in the arable soil from Alcholoya (32.5 and 34.7 mg CH4 kg(-1) dry soil day(-1), respectively). Meanwhile, in soils Tex-S2 and Tex-S3, the potential CH4 oxidation rates were only 15.0 and 12.8 mg CH4 kg(-1) dry soil day(-1), respectively. Differences in CH4 oxidation were also related to changes in the methane-oxidizing communities in these soils. Sequence analysis of pmoA gene showed that soils differed in the identity and number of methanotrophic phylotypes. The Alcholoya soil and Tex-S1 contained phylotypes grouped within the upland soil cluster gamma and the Jasper Ridge, California JR-2 clade. In soil Tex-S3, a phylotype related to Methylomicrobium alcaliphilum was detected.

Development of alginate-based aggregate inoculants of Methylobacterium sp. and Azospirillum brasilense tested under in vitro conditions to promote plant growth.

AIM: To develop co-aggregated bacterial inoculant comprising of Methylobacterium oryzae CBMB20/Methylobacterium suomiense CBMB120 strains with Azospirillum brasilense (CW903) strain and testing their efficiency as inoculants for plant growth promotion (PGP). METHODS AND RESULTS: Biofilm formation and co-aggregation efficiency was studied between A. brasilense CW903 and methylobacterial strains M. oryzae CBMB20 and M. suomiense CBMB120. Survival and release of these co-aggregated bacterial strains entrapped in alginate beads were assessed. PGP attributes of the co-aggregated bacterial inoculant were tested in tomato plants under water-stressed conditions. Results suggest that the biofilm formation efficiency of the CBMB20 and CBMB120 strains increased by 15 and 34%, respectively, when co-cultivated with CW903. Co-aggregation with CW903 enhanced the survivability of CBMB20 strain in alginate beads. Water stress index score showed least stress index in plants inoculated with CW903 and CBMB20 strains maintained as a co-aggregated inoculant. CONCLUSIONS: This study reports the development of co-aggregated cell inoculants containing M. oryzae CBMB20 and A. brasilense CW903 strains conferred better shelf life and stress abatement in inoculated tomato plants. SIGNIFICANCE AND IMPACT OF THE STUDY: These findings could be extended to other PGP bacterial species to develop multigeneric bioinoculants with multiple benefits for various crops.

Rare earth metals are essential for methanotrophic life in volcanic mudpots.

Growth of Methylacidiphilum fumariolicum SolV, an extremely acidophilic methanotrophic microbe isolated from an Italian volcanic mudpot, is shown to be strictly dependent on the presence of lanthanides, a group of rare earth elements (REEs) such as lanthanum (Ln), cerium (Ce), praseodymium (Pr) and neodymium (Nd). After fractionation of the bacterial cells and crystallization of the methanol dehydrogenase (MDH), it was shown that lanthanides were essential as cofactor in a homodimeric MDH comparable with one of the MDHs of Methylobacterium extorquens AM1. We hypothesize that the lanthanides provide superior catalytic properties to pyrroloquinoline quinone (PQQ)-dependent MDH, which is a key enzyme for both methanotrophs and methylotrophs. Thus far, all isolated MxaF-type MDHs contain calcium as a catalytic cofactor. The gene encoding the MDH of strain SolV was identified to be a xoxF-ortholog, phylogenetically closely related to mxaF. Analysis of the protein structure and alignment of amino acids showed potential REE-binding motifs in XoxF enzymes of many methylotrophs, suggesting that these may also be lanthanide-dependent MDHs. Our findings will have major environmental implications as metagenome studies showed (lanthanide-containing) XoxF-type MDH is much more prominent in nature than MxaF-type enzymes.

1-Aminocyclopropane-1-carboxylate (ACC) deaminases from Methylobacterium radiotolerans and Methylobacterium nodulans with higher specificity for ACC.

The 1-aminocyclopropane-1-carboxylate (ACC) deaminases (EC 3.4.99.7), the key enzymes of degradation of the precursor of the phytohormone ethylene, have not been well studied despite their great importance for plant-bacterial interactions. Using blast, the open reading frames encoding ACC deaminases were found in the genomes of epiphytic methylotroph Methylobacterium radiotolerans JCM2831 and nodule-forming endosymbiont Methylobacterium nodulans ORS2060. These genes were named acdS and cloned; recombinant proteins were expressed and purified from Escherichia coli. The enzyme from M. nodulans displayed the highest substrate specificity among all of the characterized ACC deaminases (Km 0.80 ± 0.04 mM), whereas the enzyme from M. radiotolerans had Km 1.8 ± 0.3 mM. The kcat values were 111.8 ± 0.2 and 65.8 ± 2.8 min(-1) for the enzymes of M. nodulans and M. radiotolerans, respectively. Both enzymes are homotetramers with a molecular mass of 144 kDa, as was demonstrated by size exclusion chromatography and native PAGE. The purified enzymes displayed the maximum activity at 45-50 °C and pH 8.0. Thus, the priority data have been obtained, extending the knowledge of biochemical properties of bacterial ACC deaminases.

[Properties of modified amperometric biosensors based on methanol dehydrogenase and Methylobacterium nodulans cells].

The properties of amperometric biosensors based on methanol dehydrogenase (MDH), Methylobacterium nodulans cells, and the ferrocene-modified carbon paste electrode were investigated. It was shown that the addition ofhydroxyapatite (HA) to a carbon paste increased the sensitivity and operating stability of MDH biosensors. The linear range of the electrode was 0.0135-0.5 and 0.032-1.5 mM for methanol and formaldehyde, respectively. The detection limit of methanol and formaldehyde was 4.5 and 11.0 microM, respectively. The loss of activity of the electrode within 10 days of storage in the presence of 2.0 mM KCN did not exceed 12%. Cyanide (10 mM) completely inhibited the sensor responses to formaldehyde (1.0 mM), which allowed for the selective determination of methanol in the presence of formaldehyde. The biosensor based on cells exhibited lower stability and sensitivity toward methanol and formaldehyde; the sensitivity coefficients were 980 and 21 nA/mM, respectively.

Characterization of recombinant PPi-dependent 6-phosphofructokinases from Methylosinus trichosporium OB3b and Methylobacterium nodulans ORS 2060.

The properties of the purified recombinant PPi-dependent 6-phosphofructokinases (PPi-PFKs) from the methanotroph Methylosinus trichosporium OB3b and rhizospheric phytosymbiont Methylobacterium nodulans ORS 2060 were determined. The dependence of activities of PPi-PFK-His(6)-tag from Ms. trichosporium OB3b (6 × 45 kDa) and PPi-PFK from Mb. nodulans ORS 2060 (4 × 43 kDa) on the concentrations of substrates of forward and reverse reactions conformed to Michaelis-Menten kinetics. Besides fructose-6-phosphate, the enzymes also phosphorylated sedoheptulose-7-phosphate. ADP or AMP (1 mM each) inhibited activity of the Ms. trichosporium PPi-PFK but did not affect the activity of the Mb. nodulans enzyme. Preference of PPi-PFKs to fructose-1,6-bisphosphate implied a predominant function of the enzymes in hexose phosphate synthesis in these bacteria. PPi-PFKs from the methylotrophs have low similarity of translated amino acid sequences (17% identity) and belong to different phylogenetic subgroups of type II 6-phosphofructokinases. The relationship of PPi-PFKs with microaerophilic character of Ms. trichosporium OB3b and adaptation of Mb. nodulans ORS 2060 to anaerobic phase of phytosymbiosis are discussed.

[Purification and characteristics of methanol dehydrogenase of Methylobacterium nodulans rhizosphere phytosymbionts].

Methanol dehydrogenase (MDG) of the facultative methylotrophic phytosymbiont Methylobacterium nodulans has been purified for the first time to an electrophoretically homogeneous state and characterized. The native protein with a molecular mass of 70 kDa consists of large (60 kDa) and small (6 kDa) subunits. The purified protein displayed a specter identical to that of pyrochinolinchinon (PCC)-containing MDGs (pI 8.7, pH optimum in the range 9-10). The enzyme was inactive in the absence of ammonium or methylamine and exhibited a wide substrate specificity with regard to C1-C2 alcohols with the highest affinity to methanol (K(M) = 70 mM), but it did not oxidize benzyl and secondary alcohols. The apparent values of K(M) to primary alcohols increased with the length of the carbonic chain. The enzyme was characterized by a high stability level even in the absence of a substrate. An immobilized enzyme was used for amperometric methanol detection.

Molecular structure of La3+-induced methanol dehydrogenase-like protein in Methylobacterium radiotolerans.

La(3+) and not Ca(2+) increases methanol dehydrogenase (MDH) activity in Methylobacterium radiotolerans NBRC15690. La(3+)- and Ca(2+)-MDH-like proteins were found to be homodimeric (α(2)) and heterotetrameric (α(2)ß(2)), respectively. N-terminal amino acid sequences of these proteins revealed that La(3+)- and Ca(2+)-MDH-like proteins were encoded by xoxF and mxaFI, respectively.