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Microbiology (Reading, England) Jun 2009Heterogeneity of cells within exponentially growing populations was addressed in a bacterium, the facultative methylotroph Methylobacterium extorquens AM1. A...
Heterogeneity of cells within exponentially growing populations was addressed in a bacterium, the facultative methylotroph Methylobacterium extorquens AM1. A transcriptional fusion between a well-characterized methanol-inducible promoter (P(mxaF)) and gfp(uv) was used with flow cytometry to analyse the distribution of gene expression in populations grown on either succinate or methanol, correlated with forward scatter as a measure of cell size. These cell populations were found to consist of three major subpopulations defined by cells that were actively growing and dividing, newly divided, and non-dividing. Through the use of flow cytometry, it was demonstrated that a significant percentage of the total population did not respond to carbon shift. In addition, these experiments demonstrated that a small subset of the total population was significantly brighter than the rest of the population and dominated fluorimetry data. These results were corroborated with a continuous flow-through system and laser scanning microscopy, confirming that subpopulations, not discernible in the population average, dominate population response. These results demonstrate that the combination of flow cytometry and microscopic single-cell analysis can be effectively used to determine the dynamics of subpopulations in population response. In addition, they support the concept that physiological diversity in isogenic populations can poise some proportion of the population to respond appropriately to changing conditions.
Topics: Cell Division; Flow Cytometry; Gene Expression Regulation, Bacterial; Genes, Reporter; Green Fluorescent Proteins; Methanol; Methylobacterium extorquens; Microscopy, Confocal; Recombinant Fusion Proteins; Succinic Acid
PubMed: 19383691
DOI: 10.1099/mic.0.025890-0 -
Biocontrol Science 2015The killing effect of peppermint vapor (PMV) against pink-slime forming microorganisms, Methylobacterium mesophilicum as a bacterium and Rhodotorula mucilaginosa as a...
The killing effect of peppermint vapor (PMV) against pink-slime forming microorganisms, Methylobacterium mesophilicum as a bacterium and Rhodotorula mucilaginosa as a yeast, was investigated by the agar vapor assay. In this method, microbial cells were spread over the agar surface exposed to PMV in a petri dish, and then transferred into a recovery liquid. When 60μl of the peppermint liquid was added to a paper disc, a marked killing effect of PMV was observed after 48h against M. mesophilicum and after 168h against R. mucilaginosa. M. mesophilicum and R. mucilaginosa were found to be more resistant to PMV than Escherichia coli and Candida albicans, used as reference microorganisms, respectively. With the addition of 0.03% sodium pyruvate as a hydrogen peroxide scavenger in agar, the killing effect of PMV against E. coli and C. albicans was decreased, whereas it was little changed against M. mesophilicum and R. mucilaginosa. In fact, the properties of the killing effect of hydrogen peroxide solution at 0.2-1.0mM was in accord with those of PMV. M. mesophilicum and R. mucilaginosa were more resistant to the oxidant than E. coli and C. albicans, respectively. Results obtained suggested that reactive oxygen species (ROS) may be involved in the killing action of PMV and therefore pink-slime formers are more resistant to PMV than non-pink-slime formers because of the presence of carotenoids as an antioxidant in cells. We also suggest that the use of PMV appeared to be a potential tool for the control of pink-slime forming microorganisms occurring in wet areas of houses such as the bathroom and washing room.
Topics: Anti-Infective Agents; Biofilms; Candida albicans; Escherichia coli; Mentha piperita; Methylobacterium; Microbial Viability; Rhodotorula; Volatile Organic Compounds
PubMed: 26133506
DOI: 10.4265/bio.20.91 -
Science Translational Medicine Sep 2020Dysbiosis of the skin microbiota is increasingly implicated as a contributor to the pathogenesis of atopic dermatitis (AD). We previously reported first-in-human safety... (Randomized Controlled Trial)
Randomized Controlled Trial
Dysbiosis of the skin microbiota is increasingly implicated as a contributor to the pathogenesis of atopic dermatitis (AD). We previously reported first-in-human safety and clinical activity results from topical application of the commensal skin bacterium for the treatment of AD in 10 adults and 5 children older than 9 years of age. Here, we examined the potential mechanism of action of treatment and its impact on children with AD less than 7 years of age, the most common age group for children with AD. In 15 children with AD, treatment was associated with amelioration of disease severity, improvement in epithelial barrier function, reduced burden on the skin, and a reduction in topical steroid requirements without severe adverse events. Our observed response rates to treatment were greater than those seen in historical placebo control groups in prior AD studies. Skin improvements and colonization by persisted for up to 8 months after cessation of treatment. Analyses of cellular scratch assays and the MC903 mouse model of AD suggested that production of sphingolipids by , cholinergic signaling, and flagellin expression may have contributed to therapeutic impact through induction of a TNFR2-mediated epithelial-to-mesenchymal transition. These results suggest that a randomized, placebo-controlled trial of treatment in individuals with AD is warranted and implicate commensals in the maintenance of the skin epithelial barrier.
Topics: Adult; Child; Dermatitis, Atopic; Eczema; Humans; Lipids; Methylobacteriaceae; Skin
PubMed: 32908007
DOI: 10.1126/scitranslmed.aaz8631 -
International Journal of Molecular... Jan 2018Metal contaminated soils are increasing worldwide. Metal-tolerant plants growing on metalliferous soils are fascinating genetic and microbial resources. Seeds can...
Metal contaminated soils are increasing worldwide. Metal-tolerant plants growing on metalliferous soils are fascinating genetic and microbial resources. Seeds can vertically transmit endophytic microorganisms that can assist next generations to cope with environmental stresses, through yet poorly understood mechanisms. The aims of this study were to identify the core seed endophyte microbiome of the pioneer metallophyte throughout three generations, and to better understand the plant colonisation of the seed endophyte sp. Cp3. Strain Cp3 was detected in seeds across three successive generations and showed the most dominant community member. When inoculated in the soil at the time of flowering, strain Cp3 migrated from soil to seeds. Using confocal microscopy, Cp3-mCherry was demonstrated to colonise the root cortex cells and xylem vessels of the stem under metal stress. Moreover, strain Cp3 showed genetic and potential to promote seed germination and seedling development. We revealed, for the first time, that the seed microbiome of a pioneer plant growing in its natural environment, and the colonisation behaviour of an important plant growth promoting systemic seed endophyte. Future characterization of seed microbiota will lead to a better understanding of their functional contribution and the potential use for seed-fortification applications.
Topics: Crotalaria; Endophytes; Environmental Pollution; Metals; Methylobacterium; Microbiota; Plant Development; Plant Roots; Seeds; Soil Microbiology; Soil Pollutants; Symbiosis
PubMed: 29351192
DOI: 10.3390/ijms19010291 -
Bioscience, Biotechnology, and... Mar 2019A number of pink-pigmented facultative methylotrophs (PPFMs) belonging to Methylobacterium spp. isolated from living plant samples were found to require B vitamins for...
A number of pink-pigmented facultative methylotrophs (PPFMs) belonging to Methylobacterium spp. isolated from living plant samples were found to require B vitamins for their growth in minimal medium, and most B vitamin-auxotrophic PPFMs required pantothenate (vitamin B). Further investigation of pantothenate auxotrophy using the representative strain Methylobacterium sp. OR01 demonstrated that this strain cannot synthesize β-alanine, one of the precursors of pantothenate. β-alanine and several precursors of pantothenate restored the growth of Methylobacterium sp. OR01 in minimal medium. Furthermore, this strain could colonize leaves of Arabidopsis thaliana cultivated in medium without pantothenate or its precursors. Pantothenate, β-alanine and several precursors were detected in the suspension of A. thaliana leaves. These results suggest that pantothenate-auxotrophic PPFMs can symbiotically colonize the surface of plant leaves by acquiring β-alanine and other precursors, in addition to pantothenate. Finally, the fitness advantage of B vitamin auxotrophy of PPFMs in the phyllosphere environment is discussed.
Topics: Arabidopsis; Autotrophic Processes; Methylobacterium; Pantothenic Acid; Plant Leaves; beta-Alanine
PubMed: 30475153
DOI: 10.1080/09168451.2018.1549935 -
MSphere Jan 2020Microbial communities in the evaporator core (EC) of automobile air-conditioning systems have a large impact on indoor air quality, such as malodor and allergenicity....
Microbial communities in the evaporator core (EC) of automobile air-conditioning systems have a large impact on indoor air quality, such as malodor and allergenicity. DNA-based microbial population analysis of the ECs collected from South Korea, China, the United States, India, and the United Arab Emirates revealed the extraordinary dominance of species in EC biofilms. Mixed-volatile organic compound (VOC) utilization and biofilm-forming capabilities were evaluated to explain the dominance of species in the ECs. The superior growth of all species could be possible under mixed-VOC conditions. Interestingly, two lifestyle groups of species could be categorized as the aggregator group, which sticks together but forms a small amount of biofilm, and the biofilm-forming group, which forms a large amount of biofilm, and their genomes along with phenotypic assays were analyzed. Pili are some of the major contributors to the aggregator lifestyle, and succinoglycan exopolysaccharide production may be responsible for the biofilm formation. However, the coexistence of these two lifestyle groups enhanced their biofilm formation compared to that with each single culture. Air-conditioning systems (ACS) are indispensable for human daily life; however, microbial community analysis in automobile ACS has yet to be comprehensively investigated. A bacterial community analysis of 24 heat exchanger fins from five countries (South Korea, China, the United States, India, and the United Arab Emirates [UAE]) revealed that species are some of the dominant bacteria in automobile ACS. Furthermore, we suggested that the predominance of species in automobile ACS is due to the utilization of mixed volatile organic compounds and their great ability for aggregation and biofilm formation.
Topics: Air Conditioning; Automobiles; Biofilms; China; India; Methylobacterium; Microbiota; Republic of Korea; United Arab Emirates; United States; Volatile Organic Compounds
PubMed: 31941811
DOI: 10.1128/mSphere.00761-19 -
Sheng Wu Gong Cheng Xue Bao = Chinese... May 2018Pyrroloquinoline quinone (PQQ) is a bacterial dehydrogenase coenzyme. PQQ can promote body growth and regulate the function of free radical level of the body. It could...
Pyrroloquinoline quinone (PQQ) is a bacterial dehydrogenase coenzyme. PQQ can promote body growth and regulate the function of free radical level of the body. It could be applied in food, medicine and other fields. Due to the extremely high cost of chemical synthesis, the production of PQQ by microbial fermentation attracted more and more attention. At present, the production titer of PQQ by fermentation method is too low to achieve industrial application. Due to the lack of a thorough understanding of the PQQ biosynthesis and its regulation mechanisms, and the lack of necessary genetic engineering modification methods for wild type strains, metabolic engineering of microorganisms to enhance PQQ production still lacks essential requirements. In this study, a PQQ-producing bacterium, Methylobacterium extorquens I-F2, was employed as a model strain. By integration of Atmospheric and room temperature plasma (ARTP) mutagenesis, flow cytometry sorting and high-throughput screening strategies, optimization of sample preparation and flow sorting process, a high-titer PQQ mutant strain was obtained. The titer of PQQ was increased by 98.02% compared with that of M. extorqunens I-F2. The process described here showed that the combination of the flow cytometry with high-throughput screening method can be used to obtain high-titer mutants more simply and rapidly, compared with genetic engineering and traditional screening methods.
Topics: High-Throughput Screening Assays; Industrial Microbiology; Methylobacterium extorquens; Mutagenesis; PQQ Cofactor
PubMed: 29893087
DOI: 10.13345/j.cjb.170440 -
Journal of Bacteriology Feb 2001Several DNA regions containing genes involved in poly-beta-hydroxybutyrate (PHB) biosynthesis and degradation and also in fatty acid degradation were identified from...
Several DNA regions containing genes involved in poly-beta-hydroxybutyrate (PHB) biosynthesis and degradation and also in fatty acid degradation were identified from genomic sequence data and have been characterized in the serine cycle facultative methylotroph Methylobacterium extorquens AM1. Genes involved in PHB biosynthesis include those encoding beta-ketothiolase (phaA), NADPH-linked acetoacetyl coenzyme A (acetyl-CoA) reductase (phaB), and PHB synthase (phaC). phaA and phaB are closely linked on the chromosome together with a third gene with identity to a regulator of PHB granule-associated protein, referred to as orf3. phaC was unlinked to phaA and phaB. Genes involved in PHB degradation include two unlinked genes predicted to encode intracellular PHB depolymerases (depA and depB). These genes show a high level of identity with each other at both DNA and amino acid levels. In addition, a gene encoding beta-hydroxybutyrate dehydrogenase (hbd) was identified. Insertion mutations were introduced into depA, depB, phaA, phaB, phaC, and hbd and also in a gene predicted to encode crotonase (croA), which is involved in fatty acid degradation, to investigate their role in PHB cycling. Mutants in depA, depB, hbd, and croA all produced normal levels of PHB, and the only growth phenotype observed was the inability of the hbd mutant to grow on beta-hydroxybutyrate. However, the phaA, phaB, and phaC mutants all showed defects in PHB synthesis. Surprisingly, these mutants also showed defects in growth on C(1) and C(2) compounds and, for phaB, these defects were rescued by glyoxylate supplementation. These results suggest that beta-hydroxybutyryl-CoA is an intermediate in the unknown pathway that converts acetyl-CoA to glyoxylate in methylotrophs and Streptomyces spp.
Topics: Acetyl-CoA C-Acyltransferase; Acyltransferases; Alcohol Oxidoreductases; Carboxylic Ester Hydrolases; Cloning, Molecular; Escherichia coli; Ethanol; Fatty Acids; Genes, Bacterial; Genome, Bacterial; Hydroxybutyrates; Isocitrate Lyase; Methanol; Methylobacterium extorquens; Models, Biological; Molecular Sequence Data; Mutagenesis, Insertional; NADP Transhydrogenases; Phenotype; Polyesters; Recombinant Proteins; Sequence Analysis, DNA; Serine
PubMed: 11208803
DOI: 10.1128/JB.183.3.1038-1046.2001 -
Scientific Reports Apr 2017The aim of the present study was to evaluate the microbiota of children with severe or complicated acute viral gastroenteritis (AGE). To that end, next-generation...
The aim of the present study was to evaluate the microbiota of children with severe or complicated acute viral gastroenteritis (AGE). To that end, next-generation sequencing (NGS) technology was used to sequence the 16S ribosomal RNA (16S rRNA) gene in 20 hospitalized pediatric patients with severe or complicated AGE and a further 20 otherwise healthy children; the fecal microbiome was then assessed. Comparative metagenomics data were analyzed by a Wilcoxon rank-sum test and hierarchical clustering analysis of bacterial reads. The statistical analyses showed a significantly decreased Shannon diversity index (entropy score) of the intestinal microbiota in patients with severe AGE compared with normal controls (P = 0.017) and patients with mild-to-moderate AGE (P = 0.011). The intestinal microbiota score of the 5 patients with rotavirus AGE was significantly lower than that of those with norovirus infection (P = 0.048). Greater richness in Campylobacteraceae (P = 0.0003), Neisseriaceae (P = 0.0115), Methylobacteriaceae (P = 0.0004), Sphingomonadaceae (P = 0.0221), and Enterobacteriaceae (P = 0.0451) was found in patients with complicated AGE compared with normal controls. The data suggest a significant reduction in intestinal microbial diversity in patients with severe AGE, particularly those with rotavirus infection.
Topics: Acute Disease; Biodiversity; Caliciviridae Infections; Case-Control Studies; Child; Gastroenteritis; Gastrointestinal Microbiome; Humans; RNA, Ribosomal, 16S; Rotavirus Infections; Sequence Analysis, RNA; Severity of Illness Index
PubMed: 28397879
DOI: 10.1038/srep46130 -
The Journal of Biological Chemistry 2021Methylofuran (MYFR) is a formyl-carrying coenzyme essential for the oxidation of formaldehyde in most methylotrophic bacteria. In Methylorubrum extorquens, MYFR contains...
Methylofuran (MYFR) is a formyl-carrying coenzyme essential for the oxidation of formaldehyde in most methylotrophic bacteria. In Methylorubrum extorquens, MYFR contains a large and branched polyglutamate side chain of up to 24 glutamates. These glutamates play an essential role in interfacing the coenzyme with the formyltransferase/hydrolase complex, an enzyme that generates formate. To date, MYFR has not been identified in other methylotrophs, and it is unknown whether its structural features are conserved. Here, we examined nine bacterial strains for the presence and structure of MYFR using high-resolution liquid chromatography-mass spectrometry (LC-MS). Two of the strains produced MYFR as present in M. extorquens, while a modified MYFR containing tyramine instead of tyrosine in its core structure was detected in six strains. When M. extorquens was grown in the presence of tyramine, the compound was readily incorporated into MYFR, indicating that the biosynthetic enzymes are unable to discriminate tyrosine from tyramine. Using gene deletions in combination with LC-MS analyses, we identified three genes, orf5, orfY, and orf17 that are essential for MYFR biosynthesis. Notably, the orfY and orf5 mutants accumulated short MYFR intermediates with only one and two glutamates, respectively, suggesting that these enzymes catalyze glutamate addition. Upon homologous overexpression of orf5, a drastic increase in the number of glutamates in MYFR was observed (up to 40 glutamates), further corroborating the function of Orf5 as a glutamate ligase. We thus renamed OrfY and Orf5 to MyfA and MyfB to highlight that these enzymes are specifically involved in MYFR biosynthesis.
Topics: Coenzymes; Formaldehyde; Furans; Glutamic Acid; Hydrolases; Hydroxymethyl and Formyl Transferases; Methylobacterium extorquens; Polyglutamic Acid
PubMed: 33894199
DOI: 10.1016/j.jbc.2021.100682