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Microbial Biotechnology Nov 2022L-5-Methyltetrahydrofolate (L-5-MTHF) is the only biologically active form of folate in the human body. Production of L-5-MTHF by using microbes is an emerging...
L-5-Methyltetrahydrofolate (L-5-MTHF) is the only biologically active form of folate in the human body. Production of L-5-MTHF by using microbes is an emerging consideration for green synthesis. However, microbes naturally produce only a small amount of L-5-MTHF. Here, Escherichia coli BL21(DE3) was engineered to increase the production of L-5-MTHF by overexpressing the intrinsic genes of dihydrofolate reductase and methylenetetrahydrofolate (methylene-THF) reductase, introducing the genes encoding formate-THF ligase, formyl-THF cyclohydrolase and methylene-THF dehydrogenase from the one-carbon metabolic pathway of Methylobacterium extorquens or Clostridium autoethanogenum and disrupting the gene of methionine synthase involved in the consumption and synthesis inhibition of the target product. Thus, upon its native pathway, an additional pathway for L-5-MTHF synthesis was developed in E. coli, which was further analysed and confirmed by qRT-PCR, enzyme assays and metabolite determination. After optimizing the conditions of induction time, temperature, cell density and concentration of IPTG and supplementing exogenous substances (folic acid, sodium formate and glucose) to the culture, the highest yield of 527.84 μg g of dry cell weight for L-5-MTHF was obtained, which was about 11.8 folds of that of the original strain. This study paves the way for further metabolic engineering to improve the biosynthesis of L-5-MTHF in E. coli.
Topics: Humans; Escherichia coli; Tetrahydrofolates; Folic Acid; Escherichia coli Infections
PubMed: 36070350
DOI: 10.1111/1751-7915.14139 -
PloS One 2015Methylobacterium species frequently inhabit plant surfaces and are able to utilize the methanol emitted from plants as carbon and energy sources. As some of the...
Methylobacterium Species Promoting Rice and Barley Growth and Interaction Specificity Revealed with Whole-Cell Matrix-Assisted Laser Desorption/Ionization-Time-of-Flight Mass Spectrometry (MALDI-TOF/MS) Analysis.
Methylobacterium species frequently inhabit plant surfaces and are able to utilize the methanol emitted from plants as carbon and energy sources. As some of the Methylobacterium species are known to promote plant growth, significant attention has been paid to the mechanism of growth promotion and the specificity of plant-microbe interactions. By screening our Methylobacterium isolate collection for the high growth promotion effect in vitro, we selected some candidates for field and pot growth tests for rice and barley, respectively. We found that inoculation resulted in better ripening of rice seeds, and increased the size of barley grains but not the total yield. In addition, using whole-cell matrix-assister laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF/MS) analysis, we identified and classified Methylobacterium isolates from Methylobacterium-inoculated rice plants. The inoculated species could not be recovered from the rice plants, and in some cases, the Methylobacterium community structure was affected by the inoculation, but not with predomination of the inoculated species. The isolates from non-inoculated barley of various cultivars grown in the same field fell into just two species. These results suggest that there is a strong selection pressure at the species level of Methylobacterium residing on a given plant species, and that selection of appropriate species that can persist on the plant is important to achieve growth promotion.
Topics: Hordeum; Likelihood Functions; Methylobacterium; Molecular Sequence Data; Oryza; Phylogeny; Plant Leaves; RNA, Ribosomal, 16S; Seeds; Species Specificity; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
PubMed: 26053875
DOI: 10.1371/journal.pone.0129509 -
Microbiome Jun 2023Several investigations on the microbial diversity and functional properties of the International Space Station (ISS) environment were carried out to understand the...
BACKGROUND
Several investigations on the microbial diversity and functional properties of the International Space Station (ISS) environment were carried out to understand the influence of spaceflight conditions on the microbial population. However, metagenome-assembled genomes (MAGs) of ISS samples are yet to be generated and subjected to various genomic analyses, including phylogenetic affiliation, predicted functional pathways, antimicrobial resistance, and virulence characteristics.
RESULTS
In total, 46 MAGs were assembled from 21 ISS environmental metagenomes, in which metaSPAdes yielded 20 MAGs and metaWRAP generated 26 MAGs. Among 46 MAGs retrieved, 18 bacterial species were identified, including one novel genus/species combination (Kalamiella piersonii) and one novel bacterial species (Methylobacterium ajmalii). In addition, four bins exhibited fungal genomes; this is the first-time fungal genomes were assembled from ISS metagenomes. Phylogenetic analyses of five bacterial species showed ISS-specific evolution. The genes pertaining to cell membranes, such as transmembrane transport, cell wall organization, and regulation of cell shape, were enriched. Variations in the antimicrobial-resistant (AMR) and virulence genes of the selected 20 MAGs were characterized to predict the ecology and evolution of biosafety level (BSL) 2 microorganisms in space. Since microbial virulence increases in microgravity, AMR gene sequences of MAGs were compared with genomes of respective ISS isolates and corresponding type strains. Among these 20 MAGs characterized, AMR genes were more prevalent in the Enterobacter bugandensis MAG, which has been predominantly isolated from clinical samples. MAGs were further used to analyze if genes involved in AMR and biofilm formation of viable microbes in ISS have variation due to generational evolution in microgravity and radiation pressure.
CONCLUSIONS
Comparative analyses of MAGs and whole-genome sequences of related ISS isolates and their type strains were characterized to understand the variation related to the microbial evolution under microgravity. The Pantoea/Kalamiella strains have the maximum single-nucleotide polymorphisms found within the ISS strains examined. This may suggest that Pantoea/Kalamiella strains are much more subjective to microgravity changes. The reconstructed genomes will enable researchers to study the evolution of genomes under microgravity and low-dose irradiation compared to the evolution of microbes here on Earth. Video Abstract.
Topics: Metagenome; Phylogeny; Bacteria; Space Flight; Gammaproteobacteria; Anti-Infective Agents; Metagenomics
PubMed: 37264385
DOI: 10.1186/s40168-023-01545-7 -
FEMS Microbiology Letters Jan 2023Methylobacterium species are abundant colonizers of the phyllosphere due to the availability of methanol, a waste product of pectin metabolism during plant cell...
Methylobacterium species are abundant colonizers of the phyllosphere due to the availability of methanol, a waste product of pectin metabolism during plant cell division. The phyllosphere is an extreme environment, with a landscape that is heterogeneous and continuously changing as the plant grows and is exposed to high levels of ultraviolet irradiation. Geographically, New Zealand (NZ) has been isolated for over a million years, has a biologically diverse flora, and is considered a biodiversity hotspot, with most native plants being endemic. We therefore hypothesize that the phyllosphere of NZ native plants harbor diverse groups of Methylobacterium species. Leaf imprinting using methanol-supplemented agar medium was used to isolate bacteria, and diversity was determined using ARDRA and 16S rRNA gene sequencing. Methylobacterium species were successfully isolated from the phyllosphere of 18 of the 20 native NZ plant species in this study, and six different species were identified: M. marchantiae, M. mesophilicum, M. adhaesivum, M. komagatae, M. extorquens, and M. phyllosphaerae. Other α, β, and γ-Proteobacteria, Actinomycetes, Bacteroidetes, and Firmicutes were also isolated, highlighting the presence of other potentially novel methanol utilizers within this ecosystem. This study identified that Methylobacterium are abundant members of the NZ phyllosphere, with species diversity and composition dependent on plant species.
Topics: Methylobacterium; Ecosystem; RNA, Ribosomal, 16S; Methanol; New Zealand; Plants; Plant Leaves
PubMed: 37985695
DOI: 10.1093/femsle/fnad124 -
Current Issues in Molecular Biology 2019Methanol, commercially generated from methane, is a renewable chemical feedstock that is highly soluble, relatively inexpensive, and easy to handle. The concept of... (Review)
Review
Methanol, commercially generated from methane, is a renewable chemical feedstock that is highly soluble, relatively inexpensive, and easy to handle. The concept of native methylotrophic bacteria serving as whole cell catalysts for production of chemicals and materials using methanol as a feedstock is highly attractive. In recent years, the available omics data for methylotrophic bacteria, especially for , the most well-characterized model methylotroph, have provided a solid platform for rational engineering of methylotrophic bacteria for industrial production. In addition, there is a strong interest in converting the more traditional heterotrophic production platforms toward the use of single carbon substrates, including methanol, through metabolic engineering. In this chapter, we review the recent progress toward achieving the desired growth and production yields from methanol, by genetically engineered native methylotrophic strains and by the engineered synthetic methylotrophs.
Topics: Biological Products; Biotransformation; Metabolic Engineering; Metabolic Networks and Pathways; Methane; Methanol; Methylobacterium extorquens; Organisms, Genetically Modified; Synthetic Biology
PubMed: 31166195
DOI: 10.21775/cimb.033.225 -
The ISME Journal Nov 2018Microbes frequently rely on metabolites excreted by other bacterial species, but little is known about how this cross-feeding influences the effect of antibiotics. We...
Microbes frequently rely on metabolites excreted by other bacterial species, but little is known about how this cross-feeding influences the effect of antibiotics. We hypothesized that when species rely on each other for essential metabolites, the minimum inhibitory concentration (MIC) for all species will drop to that of the "weakest link"-the species least resistant in monoculture. We tested this hypothesis in an obligate cross-feeding system that was engineered between Escherichia coli, Salmonella enterica, and Methylobacterium extorquens. The effect of tetracycline and ampicillin were tested on both liquid and solid media. In all cases, resistant species were inhibited at significantly lower antibiotic concentrations in the cross-feeding community than in monoculture or a competitive community. However, deviation from the "weakest link" hypothesis was also observed in cross-feeding communities apparently as result of changes in the timing of growth and cross-protection. Comparable results were also observed in a clinically relevant system involving facultative cross-feeding between Pseudomonas aeruginosa and an anaerobic consortium found in the lungs of cystic fibrosis patients. P. aeruginosa was inhibited by lower concentrations of ampicillin when cross-feeding than when grown in isolation. These results suggest that cross-feeding significantly alters tolerance to antibiotics in a variety of systems.
Topics: Ampicillin; Anti-Bacterial Agents; Cystic Fibrosis; Drug Resistance, Bacterial; Escherichia coli; Humans; Methylobacterium extorquens; Pseudomonas aeruginosa; Salmonella enterica; Tetracycline
PubMed: 29991761
DOI: 10.1038/s41396-018-0212-z -
Environmental Science and Ecotechnology Sep 2024The pathogen , responsible for a variety of diseases, poses a considerable threat to global crop yields. Emerging biocontrol strategies employ antagonistic...
The pathogen , responsible for a variety of diseases, poses a considerable threat to global crop yields. Emerging biocontrol strategies employ antagonistic microorganisms, utilizing phyllosphere microecology and systemic resistance to combat this disease. However, the interactions between phyllosphere microbial dynamics and the activation of the plant defense system remain poorly understood. Here we show significant alterations in phyllosphere microbiota structure and plant gene expression following the application of biocontrol agents. We reveal enhanced collaboration and integration of and within the microbial co-occurrence network. Notably, inhibits by disrupting pathogen chemotaxis and virulence. Additionally, both and activate plant defenses by upregulating pathogenesis-related gene expression through abscisic acid, ethylene, jasmonate acid, and salicylic acid signaling pathways. Our results highlighted that biocontrol agents promote plant health, from reconstructing beneficial microbial consortia to enhancing plant immunity. The findings enrich our comprehension of the synergistic interplays between phyllosphere microbiota and plant immunity, offering potential enhancements in biocontrol efficacy for crop protection.
PubMed: 38883559
DOI: 10.1016/j.ese.2024.100431 -
The New Phytologist Jan 2022Living trees in forests emit methane (CH ) from their stems. However, the magnitudes, patterns, drivers, origins, and biogeochemical pathways of these emissions remain...
Living trees in forests emit methane (CH ) from their stems. However, the magnitudes, patterns, drivers, origins, and biogeochemical pathways of these emissions remain poorly understood. We measured in situ CH fluxes in poplar stems and soils using static chambers and investigated the microbial communities of heartwood and sapwood by sequencing bacterial 16S, archaeal 16S, and fungal ITS rRNA genes. Methane emissions from poplar stems occurred throughout the sampling period. The mean CH emission rate was 2.7 mg m stem d . Stem CH emission rate increased significantly with air temperature, humidity, soil water content, and soil CH fluxes, but decreased with increasing sampling height. The CO reduction and methylotrophic methanogenesis were the major methanogenic pathways in wood tissues. The dominant methanogen groups detected in stem tissues were Methanobacterium, Methanobrevibacter, Rice Cluster I, Methanosarcina, Methanomassiliicoccus, Methanoculleus, and Methanomethylophilaceae. In addition, three methanotrophic genera were identified in the heartwood and sapwood - Methylocystis, Methylobacterium, and Paracoccus. Overall, stem CH emissions can originate directly from the internal tissues or co-occur from soils and stems. The co-existence of methanogens and methanotrophs within heartwood and sapwood highlights a need for future research in the microbial mechanisms underlying stem CH exchange with the atmosphere.
Topics: Archaea; Methane; Populus; Soil; Trees
PubMed: 34617594
DOI: 10.1111/nph.17778 -
Molecular Microbiology May 2019Recently, rare-earth elements lanthanides (Ln ) have emerged as enzyme cofactors of methanol dehydrogenases of the XoxF type. It is now understood that XoxF enzymes can...
Recently, rare-earth elements lanthanides (Ln ) have emerged as enzyme cofactors of methanol dehydrogenases of the XoxF type. It is now understood that XoxF enzymes can functionally replace the alternative, calcium-dependent, MxaFI-type methanol dehydrogenases, when Ln are available. These rare-earth metals are not only essential for XoxF activity, but they also regulate gene expression, in a reverse fashion, activating the expression of XoxF and repressing the expression of MxaFI. This type of regulation has created multiple conundrums, including the details of the solubility, transport, sensing and selection mechanisms for Ln by the bacterial cells, as well as the questions relevant to the evolution of the alternative enzymes and their potentially different redox properties. Overall, the newly discovered biological activity of Ln presents a big puzzle. Ochsner et al. add several pieces to this puzzle, utilizing a model phyllosphere colonizer Methylobacterium extorquens PA1. They determine that Ln sensing by this organism can take place via both XoxF-dependent and XoxF-independent mechanisms. They also identify genes for a TonB-dependent transporter and an ABC-type transporter and demonstrate that both are essential for Ln -dependent methanol metabolism. The puzzle still requires multiple additional pieces for completion, but great strides have been made toward the goal of solving it.
PubMed: 30673122
DOI: 10.1111/mmi.14210 -
Journal of Microorganism Control 2023Bedside dialysis monitoring equipment for hemodialysis are located in the bioburden section upstream of the endotoxin-retentive filter for dialysis fluid sterilization....
Bedside dialysis monitoring equipment for hemodialysis are located in the bioburden section upstream of the endotoxin-retentive filter for dialysis fluid sterilization. We observed 26 equipment at our institution for bacterial contamination at least once every 4 weeks for 5 years with another ultrafiltration membrane upstream to prevent bacterial contamination. Bacterial contamination levels were highest and most diverse at the time of the first flush. During subsequent initial cleanng, the contamination level decreased, and bacterial species converged almost exclusively to one genus, namely Methylobacterium spp. During clinical use, the equipment were cleaned and disinfected daily after dialysis, and daily operations and maintenance were performed using aseptic techniques. Although the frequency of bacterial detection decreased annually, the same bacterial genotypes observed at the first flush were isolated even after long time periods and were thought to persist in the equipment possibly by forming biofilm. Pseudomonas aeruginosa was newly detected after the replacement of parts during breakdown maintenance, indicating the need to sterilize replacement parts. Thus, the bioburden should be assessed regularly as part of the management of in-house-produced dialysis fluid.
Topics: Renal Dialysis; Bacteria; Dialysis Solutions; Ultrafiltration; Endotoxins
PubMed: 37866898
DOI: 10.4265/jmc.28.3_69