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Microbiological Research 2009The nitrogen fixing methylotrophic bacteria were isolated from the nodules of tropical legumes. Two isolates CMCJ317 and CMSA322 isolated from Crotalaria juncea and...
The nitrogen fixing methylotrophic bacteria were isolated from the nodules of tropical legumes. Two isolates CMCJ317 and CMSA322 isolated from Crotalaria juncea and Sesbania aculeata possessing high nitrogenase activities under pure culture conditions and able to form nodules under inoculated conditions were further characterized. The biochemical characteristics revealed their close relationship with Methylobacterium nodulans type strain ORS2060. The PCR amplification of nodA and mxaF genes showed the expected 584 and 555 bp products, respectively, similar to M. nodulans ORS2060 and digestion with restriction enzymes revealed that the two isolates differed. The strains showed significantly higher nitrogenase activity and also improved nodulation and shoot nitrogen of the plants when inoculated to Macroptilum atropurpureum. CMCJ317 and CMSA322 formed nodules on C. juncea and M. atropurpureum under green house conditions and also significantly increased the nitrogen concentration in shoots. These findings show that the ability to establish symbiosis with legumes is more widespread in Methylobacterium.
Topics: Bacterial Proteins; Fabaceae; Methanol; Methylobacterium; Nitrogen Fixation; Nitrogenase; Plant Root Nodulation; Root Nodules, Plant; Symbiosis; Tropical Climate
PubMed: 17074473
DOI: 10.1016/j.micres.2006.08.009 -
Microbial Cell Factories Dec 2018Butadiene is a platform chemical used as an industrial feedstock for the manufacture of automobile tires, synthetic resins, latex and engineering plastics. Currently,...
BACKGROUND
Butadiene is a platform chemical used as an industrial feedstock for the manufacture of automobile tires, synthetic resins, latex and engineering plastics. Currently, butadiene is predominantly synthesized as a byproduct of ethylene production from non-renewable petroleum resources. Although the idea of biological synthesis of butadiene from sugars has been discussed in the literature, success for that goal has so far not been reported. As a model system for methanol assimilation, Methylobacterium extorquens AM1 can produce several unique metabolic intermediates for the production of value-added chemicals, including crotonyl-CoA as a potential precursor for butadiene synthesis.
RESULTS
In this work, we focused on constructing a metabolic pathway to convert crotonyl-CoA into crotyl diphosphate, a direct precursor of butadiene. The engineered pathway consists of three identified enzymes, a hydroxyethylthiazole kinase (THK) from Escherichia coli, an isopentenyl phosphate kinase (IPK) from Methanothermobacter thermautotrophicus and an aldehyde/alcohol dehydrogenase (ADHE2) from Clostridium acetobutylicum. The K and k of THK, IPK and ADHE2 were determined as 8.35 mM and 1.24 s, 1.28 mM and 153.14 s, and 2.34 mM and 1.15 s towards crotonol, crotyl monophosphate and crotonyl-CoA, respectively. Then, the activity of one of rate-limiting enzymes, THK, was optimized by random mutagenesis coupled with a developed high-throughput screening colorimetric assay. The resulting variant (THK) isolated from over 3000 colonies showed 8.6-fold higher activity than wild-type, which helped increase the titer of crotyl diphosphate to 0.76 mM, corresponding to a 7.6% conversion from crotonol in the one-pot in vitro reaction. Overexpression of native ADHE2, IPK with THK under a strong promoter mxaF in M. extorquens AM1 did not produce crotyl diphosphate from crotonyl-CoA, but the engineered strain did generate 0.60 μg/mL of intracellular crotyl diphosphate from exogenously supplied crotonol at mid-exponential phase.
CONCLUSIONS
These results represent the first step in producing a butadiene precursor in recombinant M. extorquens AM1. It not only demonstrates the feasibility of converting crotonol to key intermediates for butadiene biosynthesis, it also suggests future directions for improving catalytic efficiency of aldehyde/alcohol dehydrogenase to produce butadiene precursor from methanol.
Topics: Butadienes; High-Throughput Screening Assays; Metabolic Engineering; Metabolic Networks and Pathways; Methylobacterium extorquens
PubMed: 30572892
DOI: 10.1186/s12934-018-1042-4 -
The Journal of Biological Chemistry Jun 2020The lanthanide elements (Ln), those with atomic numbers 57-63 (excluding promethium, Pm), form a cofactor complex with pyrroloquinoline quinone (PQQ) in bacterial XoxF...
The lanthanide elements (Ln), those with atomic numbers 57-63 (excluding promethium, Pm), form a cofactor complex with pyrroloquinoline quinone (PQQ) in bacterial XoxF methanol dehydrogenases (MDHs) and ExaF ethanol dehydrogenases (EDHs), expanding the range of biological elements and opening novel areas of metabolism and ecology. Other MDHs, known as MxaFIs, are related in sequence and structure to these proteins, yet they instead possess a Ca-PQQ cofactor. An important missing piece of the Ln puzzle is defining what features distinguish enzymes that use Ln-PQQ cofactors from those that do not. Here, using XoxF1 MDH from the model methylotrophic bacterium AM1, we investigated the functional importance of a proposed lanthanide-coordinating aspartate residue. We report two crystal structures of XoxF1, one with and another without PQQ, both with La bound in the active-site region and coordinated by Asp Using constructs to produce either recombinant XoxF1 or its D320A variant, we show that Asp is needed for catalytic function, activity, and La coordination. XoxF1 and XoxF1 D320A, when produced in the absence of La, coordinated Ca but exhibited little or no catalytic activity. We also generated the parallel substitution in ExaF to produce ExaF D319S and found that this variant loses the capacity for efficient ethanol oxidation with La These results provide evidence that a Ln-coordinating aspartate is essential for the enzymatic functions of XoxF MDHs and ExaF EDHs, supporting the notion that sequences of these enzymes, and the genes that encode them, are markers for Ln metabolism.
Topics: Alcohol Oxidoreductases; Amino Acid Substitution; Aspartic Acid; Bacterial Proteins; Biocatalysis; Calcium; Crystallography, X-Ray; Lanthanoid Series Elements; Methanol; Methylobacterium extorquens; Oxidation-Reduction; Structure-Activity Relationship
PubMed: 32366463
DOI: 10.1074/jbc.RA120.013227 -
PloS One 2014Pink-pigmented facultative methylotrophs in the Rhizobiales are widespread in the environment, and many Methylobacterium species associated with plants produce plant...
Pink-pigmented facultative methylotrophs in the Rhizobiales are widespread in the environment, and many Methylobacterium species associated with plants produce plant growth-promoting substances. To gain insights into the life style at the phyllosphere and the genetic bases of plant growth promotion, we determined and analyzed the complete genome sequence of Methylobacterium oryzae CBMB20T, a strain isolated from rice stem. The genome consists of a 6.29-Mb chromosome and four plasmids, designated as pMOC1 to pMOC4. Among the 6,274 coding sequences in the chromosome, the bacterium has, besides most of the genes for the central metabolism, all of the essential genes for the assimilation and dissimilation of methanol that are either located in methylotrophy islands or dispersed. M. oryzae is equipped with several kinds of genes for adaptation to plant surfaces such as defense against UV radiation, oxidative stress, desiccation, or nutrient deficiency, as well as high proportion of genes related to motility and signaling. Moreover, it has an array of genes involved in metabolic pathways that may contribute to promotion of plant growth; they include auxin biosynthesis, cytokine biosynthesis, vitamin B12 biosynthesis, urea metabolism, biosorption of heavy metals or decrease of metal toxicity, pyrroloquinoline quinone biosynthesis, 1-aminocyclopropane-1-carboxylate deamination, phosphate solubilization, and thiosulfate oxidation. Through the genome analysis of M. oryzae, we provide information on the full gene complement of M. oryzae that resides in the aerial parts of plants and enhances plant growth. The plant-associated lifestyle of M. oryzae pertaining to methylotrophy and plant growth promotion, and its potential as a candidate for a bioinoculant targeted to the phyllosphere and focused on phytostimulation are illuminated.
Topics: Chromosomes; DNA, Bacterial; Genome, Bacterial; Methylobacterium; Molecular Sequence Annotation; Oxidative Stress; Phylogeny; Plant Stems; Probiotics; Sequence Analysis, DNA
PubMed: 25211235
DOI: 10.1371/journal.pone.0106704 -
Annals of Botany Jul 2013The legume clade Lotononis sensu lato (s.l.; tribe Crotalarieae) comprises three genera: Listia, Leobordea and Lotononis sensu stricto (s.s.). Listia species are...
Nodule morphology, symbiotic specificity and association with unusual rhizobia are distinguishing features of the genus Listia within the Southern African crotalarioid clade Lotononis s.l.
BACKGROUND AND AIMS
The legume clade Lotononis sensu lato (s.l.; tribe Crotalarieae) comprises three genera: Listia, Leobordea and Lotononis sensu stricto (s.s.). Listia species are symbiotically specific and form lupinoid nodules with rhizobial species of Methylobacterium and Microvirga. This work investigated whether these symbiotic traits were confined to Listia by determining the ability of rhizobial strains isolated from species of Lotononis s.l. to nodulate Listia, Leobordea and Lotononis s.s. hosts and by examining the morphology and structure of the resulting nodules.
METHODS
Rhizobia were characterized by sequencing their 16S rRNA and nodA genes. Nodulation and N2 fixation on eight taxonomically diverse Lotononis s.l. species were determined in glasshouse trials. Nodules of all hosts, and the process of infection and nodule initiation in Listia angolensis and Listia bainesii, were examined by light microscopy.
KEY RESULTS
Rhizobia associated with Lotononis s.l. were phylogenetically diverse. Leobordea and Lotononis s.s. isolates were most closely related to Bradyrhizobium spp., Ensifer meliloti, Mesorhizobium tianshanense and Methylobacterium nodulans. Listia angolensis formed effective nodules only with species of Microvirga. Listia bainesii nodulated only with pigmented Methylobacterium. Five lineages of nodA were found. Listia angolensis and L. bainesii formed lupinoid nodules, whereas nodules of Leobordea and Lotononis s.s. species were indeterminate. All effective nodules contained uniformly infected central tissue. Listia angolensis and L. bainesii nodule initials occurred on the border of the hypocotyl and along the tap root, and nodule primordia developed in the outer cortical layer. Neither root hair curling nor infection threads were seen.
CONCLUSIONS
Two specificity groups occur within Lotononis s.l.: Listia species are symbiotically specific, while species of Leobordea and Lotononis s.s. are generally promiscuous and interact with rhizobia of diverse chromosomal and symbiotic lineages. The seasonally waterlogged habitat of Listia species may favour the development of symbiotic specificity.
Topics: Africa, Southern; Bradyrhizobium; Fabaceae; Genes, Bacterial; Methylobacteriaceae; Methylobacterium; Nitrogen Fixation; Phylogeny; Plant Roots; RNA, Ribosomal, 16S; Rhizobium; Sequence Analysis, DNA; Species Specificity; Symbiosis
PubMed: 23712451
DOI: 10.1093/aob/mct095 -
Antonie Van Leeuwenhoek Dec 2023Methylorubrum extorquens is an important model methylotroph and has enormous potential for the development of C1-based microbial cell factories. During strain...
Methylorubrum extorquens is an important model methylotroph and has enormous potential for the development of C1-based microbial cell factories. During strain construction, regulated promoters with a low background expression level are important genetic tools for expression of potentially toxic genes. Here we present an accordingly optimised promoter, which can be used for that purpose. During construction and testing of terpene production strains harbouring a recombinant mevalonate pathway, strong growth defects were observed which made strain development impossible. After isolation and characterisation of suppressor mutants, we discovered a variant of the cumate-inducible promoter P used in this approach. Deletion of 28 nucleotides resulted in an extremely low background expression level, but also reduced the maximal expression strength to about 30% of the original promoter. This tightly repressed promoter version is a powerful module for controlled expression of potentially toxic genes in M. extorquens.
Topics: Promoter Regions, Genetic; Methylobacterium extorquens; Methanol
PubMed: 37751033
DOI: 10.1007/s10482-023-01880-7 -
Microbes and Environments Sep 2016Methylobacterium inhabits the phyllosphere of a large number of plants. We herein report the results of comparative metagenome analyses on methylobacterial communities...
Methylobacterium inhabits the phyllosphere of a large number of plants. We herein report the results of comparative metagenome analyses on methylobacterial communities of soybean plants grown in an experimental field in Tohoku University (Kashimadai, Miyagi, Japan). Methylobacterium was identified as the most dominant genus (33%) among bacteria inhabiting soybean stems. We classified plant-derived Methylobacterium species into Groups I, II, and III based on 16S rRNA gene sequences, and found that Group I members (phylogenetically close to M. extorquens) were dominant in soybean-associated Methylobacterium. By comparing 29 genomes, we found that all Group I members possessed a complete set of genes for the N-methylglutamate pathway for methylamine utilization, and genes for urea degradation (urea carboxylase, urea amidolyase, and conventional urease). Only Group I members and soybean methylobacterial isolates grew in a culture supplemented with methylamine as the sole carbon source. They utilized urea or allantoin (a urea-related compound in legumes) as the sole nitrogen source; however, group III also utilized these compounds. The utilization of allantoin may be crucial in soybean-bacterial interactions because allantoin is a transported form of fixed nitrogen in legume plants. Soybean-derived Group I strain AMS5 colonized the model legume Lotus japonicus well. A comparison among the 29 genomes of plant-derived and other strains suggested that several candidate genes are involved in plant colonization such as csgG (curli fimbriae). Genes for the N-methylglutamate pathway and curli fimbriae were more abundant in soybean microbiomes than in rice microbiomes in the field. Based on these results, we discuss the lifestyle of Methylobacterium in the legume phyllosphere.
Topics: Allantoin; Cluster Analysis; DNA, Bacterial; DNA, Ribosomal; Genotype; Japan; Metagenome; Metagenomics; Methylamines; Methylobacterium; Phylogeny; Plant Stems; RNA, Ribosomal, 16S; Sequence Analysis, DNA; Glycine max; Urea
PubMed: 27431374
DOI: 10.1264/jsme2.ME16035 -
Journal of Bacteriology Nov 2002Methylobacterium extorquens AM1, a serine cycle facultative methylotroph, accumulates poly-beta-hydroxybutyrate (PHB) as a carbon and energy reserve material during...
Methylobacterium extorquens AM1, a serine cycle facultative methylotroph, accumulates poly-beta-hydroxybutyrate (PHB) as a carbon and energy reserve material during growth on both multicarbon- and single-carbon substrates. Recently, the identification and mutation of the genes involved in the biosynthesis and degradation of PHB have been described for this bacterium, demonstrating that two of the genes of the PHB cycle (phaA and phaB) are also involved in C(1) and C(2) metabolism, as part of a novel pathway for glyoxylate regeneration in the serine cycle (N. Korotkova and M. E. Lidstrom, J. Bacteriol. 183:1038-1046, 2001; N. Korotkova, L. Chistoserdova, V. Kuksa, and M. E. Lidstrom, J. Bacteriol. 184:1750-1758, 2002). In this work, three new genes involved in PHB biosynthesis in this bacterium have been investigated via mutation and phenotypic analysis: gap11, gap20, and phaR. We demonstrate that gap11 and gap20 encode two major granule-associated proteins (phasins) and that mutants with mutations in these genes are defective in PHB production and also in growth on C(2) compounds, while they show wild-type growth characteristics on C(1) or multicarbon compounds. The phaR mutant shows defects in both PHB accumulation and growth characteristics when grown on C(1) compounds and has defects in PHB accumulation but grows normally on C(3) and C(4) compounds, while both PHB accumulation and growth rate are at wild-type levels during growth on C(2) compounds. Our results suggest that this phenotype is due to altered fluxes of acetyl coenzyme A (CoA), a major intermediate in C(1), C(2), and heterotrophic metabolism in M. extorquens AM1, as well as the entry metabolite for the PHB cycle. Therefore, it seems likely that PhaR acts to control acetyl-CoA flux to PHB in this methylotrophic bacterium.
Topics: Acetyl Coenzyme A; Bacterial Proteins; Culture Media; DNA, Bacterial; DNA-Binding Proteins; Gene Expression Regulation, Bacterial; Hydroxybutyrates; Methanol; Methylobacterium extorquens; Molecular Sequence Data; Mutagenesis, Site-Directed; Mutation; Polyesters; Repressor Proteins; Sequence Analysis, DNA; Succinic Acid; Transcription, Genetic
PubMed: 12399487
DOI: 10.1128/JB.184.22.6174-6181.2002 -
Scientific Reports Oct 2019In vitro and animal studies have demonstrated that topical application and oral consumption of pomegranate reduces UVB-induced skin damage. We therefore investigated if... (Randomized Controlled Trial)
Randomized Controlled Trial
Pomegranate Juice and Extract Consumption Increases the Resistance to UVB-induced Erythema and Changes the Skin Microbiome in Healthy Women: a Randomized Controlled Trial.
In vitro and animal studies have demonstrated that topical application and oral consumption of pomegranate reduces UVB-induced skin damage. We therefore investigated if oral pomegranate consumption will reduce photodamage from UVB irradiation and alter the composition of the skin microbiota in a randomized controlled, parallel, three-arm, open label study. Seventy-four female participants (30-45 years) with Fitzpatrick skin type II-IV were randomly assigned (1:1:1) to 1000 mg of pomegranate extract (PomX), 8 oz of pomegranate juice (PomJ) or placebo for 12 weeks. Minimal erythema dose (MED) and melanin index were determined using a cutometer (mexameter probe). Skin microbiota was determined using 16S rRNA sequencing. The MED was significantly increased in the PomX and PomJ group compared to placebo. There was no significant difference on phylum, but on family and genus level bacterial composition of skin samples collected at baseline and after 12 week intervention showed significant differences between PomJ, PomX and placebo. Members of the Methylobacteriaceae family contain pigments absorbing UV irradiation and might contribute to UVB skin protection. However, we were not able to establish a direct correlation between increased MED and bacterial abundance. In summary daily oral pomegranate consumption may lead to enhanced protection from UV photodamage.
Topics: Adult; Erythema; Female; Fruit and Vegetable Juices; Humans; Inflammation; Microbiota; Middle Aged; Plant Extracts; Pomegranate; RNA, Ribosomal, 16S; Skin; Ultraviolet Rays
PubMed: 31601842
DOI: 10.1038/s41598-019-50926-2 -
Journal of the American Chemical Society Jul 2020Understanding the relationship between the metallocofactor and its protein environment is the key to uncovering the mechanism of metalloenzymes. PqqE, a radical...
Understanding the relationship between the metallocofactor and its protein environment is the key to uncovering the mechanism of metalloenzymes. PqqE, a radical adenosylmethionine enzyme in pyrroloquinoline quinone (PQQ) biosynthesis, contains three iron-sulfur cluster binding sites. Two auxiliary iron-sulfur cluster binding sites, designated as AuxI and AuxII, use distinctive ligands compared to other proteins in the family while their functions remain unclear. Here, we investigate the electronic properties of these iron-sulfur clusters and compare the catalytic efficiency of wild-type (WT) AM1 PqqE to a range of mutated constructs. Using native mass spectrometry, protein film electrochemistry, and electron paramagnetic resonance spectroscopy, we confirm the previously proposed incorporation of a mixture of [2Fe-2S] and [4Fe-4S] clusters at the AuxI site and are able to assign redox potentials to each of the three iron-sulfur clusters. Significantly, a conservative mutation at AuxI, C268H, shown to selectively incorporate a [4Fe-4S] cluster, catalyzes an enhancement of uncoupled adenosylmethionine cleavage relative to WT, together with the elimination of detectable peptide cross-linked product. While a [4Fe-4S] cluster can be tolerated at the AuxI site, the aggregate findings suggest a functional [2Fe-2S] configuration within the AuxI site. PqqE variants with nondestructive ligand replacements at AuxII also show that the reduction potential at this site can be manipulated by changing the electronegativity of the unique aspartate ligand. A number of novel mechanistic features are proposed based on the kinetic and spectroscopic data. Additionally, bioinformatic analyses suggest that the unique ligand environment of PqqE may be relevant to its role in PQQ biosynthesis within an oxygen-dependent biosynthetic pathway.
Topics: Bacterial Proteins; Biocatalysis; Crystallography, X-Ray; Endopeptidases; Iron; Methylobacterium extorquens; Models, Molecular; Molecular Structure; Sulfur
PubMed: 32643933
DOI: 10.1021/jacs.0c02044