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PLoS Genetics Nov 2019While microbiologists often make the simplifying assumption that genotype determines phenotype in a given environment, it is becoming increasingly apparent that...
Microbial phenotypic heterogeneity in response to a metabolic toxin: Continuous, dynamically shifting distribution of formaldehyde tolerance in Methylobacterium extorquens populations.
While microbiologists often make the simplifying assumption that genotype determines phenotype in a given environment, it is becoming increasingly apparent that phenotypic heterogeneity (in which one genotype generates multiple phenotypes simultaneously even in a uniform environment) is common in many microbial populations. The importance of phenotypic heterogeneity has been demonstrated in a number of model systems involving binary phenotypic states (e.g., growth/non-growth); however, less is known about systems involving phenotype distributions that are continuous across an environmental gradient, and how those distributions change when the environment changes. Here, we describe a novel instance of phenotypic diversity in tolerance to a metabolic toxin within wild-type populations of Methylobacterium extorquens, a ubiquitous phyllosphere methylotroph capable of growing on the methanol periodically released from plant leaves. The first intermediate in methanol metabolism is formaldehyde, a potent cellular toxin that is lethal in high concentrations. We have found that at moderate concentrations, formaldehyde tolerance in M. extorquens is heterogeneous, with a cell's minimum tolerance level ranging between 0 mM and 8 mM. Tolerant cells have a distinct gene expression profile from non-tolerant cells. This form of heterogeneity is continuous in terms of threshold (the formaldehyde concentration where growth ceases), yet binary in outcome (at a given formaldehyde concentration, cells either grow normally or die, with no intermediate phenotype), and it is not associated with any detectable genetic mutations. Moreover, tolerance distributions within the population are dynamic, changing over time in response to growth conditions. We characterized this phenomenon using bulk liquid culture experiments, colony growth tracking, flow cytometry, single-cell time-lapse microscopy, transcriptomics, and genome resequencing. Finally, we used mathematical modeling to better understand the processes by which cells change phenotype, and found evidence for both stochastic, bidirectional phenotypic diversification and responsive, directed phenotypic shifts, depending on the growth substrate and the presence of toxin.
Topics: Drug Tolerance; Formaldehyde; Gene Expression Regulation, Bacterial; Genetic Heterogeneity; Genetic Variation; Genotype; Methanol; Methylobacterium extorquens; Phenotype; Plant Leaves
PubMed: 31710603
DOI: 10.1371/journal.pgen.1008458 -
FEMS Microbiology Ecology Dec 2019Greenland's Dark Zone is the largest contiguous region of bare terrestrial ice in the Northern Hemisphere and microbial processes play an important role in driving its...
Greenland's Dark Zone is the largest contiguous region of bare terrestrial ice in the Northern Hemisphere and microbial processes play an important role in driving its darkening and thereby amplifying melt and runoff from the ice sheet. However, the dynamics of these microbiota have not been fully identified. Here, we present joint 16S rRNA gene and 16S rRNA (cDNA) comparison of input (snow), storage (cryoconite) and output (supraglacial stream water) habitats across the Dark Zone over the melt season. We reveal that all three Dark Zone communities have a preponderance of rare taxa exhibiting high protein synthesis potential (PSP). Furthermore, taxa with high PSP represent highly connected 'bottlenecks' within community structure, consistent with their roles as metabolic hubs. Finally, low abundance-high PSP taxa affiliated with Methylobacterium within snow and stream water suggest a novel role for Methylobacterium in the carbon cycle of Greenlandic snowpacks, and importantly, the export of potentially active methylotrophs to the bed of the Greenland Ice Sheet. By comparing the dynamics of bulk and potentially active microbiota in the Dark Zone of the Greenland Ice Sheet, we provide novel insights into the mechanisms and impacts of the microbial colonization of this critical region of our melting planet.
Topics: Carbon Cycle; Ecosystem; Freezing; Greenland; Ice Cover; Methylobacterium; Microbiota; RNA, Ribosomal, 16S; Seasons; Snow
PubMed: 31697309
DOI: 10.1093/femsec/fiz177 -
Genome Biology and Evolution Oct 2019Phyllosphere is a habitat to a variety of viruses, bacteria, fungi, and other microorganisms, which play a fundamental role in maintaining the health of plants and...
Phyllosphere is a habitat to a variety of viruses, bacteria, fungi, and other microorganisms, which play a fundamental role in maintaining the health of plants and mediating the interaction between plants and ambient environments. A recent addition to this catalogue of microbial diversity was the aerobic anoxygenic phototrophs (AAPs), a group of widespread bacteria that absorb light through bacteriochlorophyll α (BChl a) to produce energy without fixing carbon or producing molecular oxygen. However, culture representatives of AAPs from phyllosphere and their genome information are lacking, limiting our capability to assess their potential ecological roles in this unique niche. In this study, we investigated the presence of AAPs in the phyllosphere of a winter wheat (Triticum aestivum L.) in Denmark by employing bacterial colony based infrared imaging and MALDI-TOF mass spectrometry (MS) techniques. A total of ∼4,480 colonies were screened for the presence of cellular BChl a, resulting in 129 AAP isolates that were further clustered into 21 groups based on MALDI-TOF MS profiling, representatives of which were sequenced using the Illumina NextSeq and Oxford Nanopore MinION platforms. Seventeen draft and four complete genomes of AAPs were assembled belonging in Methylobacterium, Rhizobium, Roseomonas, and a novel Alsobacter. We observed a diverging pattern in the evolutionary rates of photosynthesis genes among the highly homogenous AAP strains of Methylobacterium (Alphaproteobacteria), highlighting an ongoing genomic innovation at the gene cluster level.
Topics: Evolution, Molecular; Genomics; Heterotrophic Processes; Methylobacterium; Photosynthesis; Phototrophic Processes; Phylogeny; Triticum
PubMed: 31626703
DOI: 10.1093/gbe/evz204 -
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 -
ACS Synthetic Biology Nov 2019Genetic tools are a prerequisite to engineer cellular factories for synthetic biology and biotechnology. AM1 is an important platform organism of a future C-bioeconomy....
Genetic tools are a prerequisite to engineer cellular factories for synthetic biology and biotechnology. AM1 is an important platform organism of a future C-bioeconomy. However, its application is currently limited by the availability of genetic tools. Here we systematically tested regions to maintain extrachromosomal DNA in . We used three elements to construct mini-chromosomes that are stably inherited at single copy number and can be shuttled between and . These mini-chromosomes are compatible among each other and with high-copy number plasmids of . We also developed a set of inducible promoters of wide expression range, reaching levels exceeding those currently available, notably the -promoter. In summary, we provide a set of tools to control the dynamic expression and copy number of genetic elements in , which opens new ways to unleash the metabolic and biotechnological potential of this organism for future applications.
Topics: Bacterial Proteins; Biotechnology; DNA Helicases; DNA Replication; Escherichia coli; Extrachromosomal Inheritance; Gene Expression Regulation, Bacterial; Genome, Bacterial; Metabolic Engineering; Methylobacterium extorquens; Plasmids; Promoter Regions, Genetic; Replicon; Synthetic Biology; Trans-Activators
PubMed: 31584803
DOI: 10.1021/acssynbio.9b00220 -
Microbes and Environments Dec 2019Plant-associated bacteria are critical for plant growth and health. However, the effects of plant growth stages on the bacterial community remain unclear. Analyses of... (Comparative Study)
Comparative Study
Plant-associated bacteria are critical for plant growth and health. However, the effects of plant growth stages on the bacterial community remain unclear. Analyses of the microbiome associated with field-grown soybean revealed a marked shift in the bacterial community during the growth stages. The relative abundance of Methylorubrum in the leaf and stem increased from 0.2% to more than 45%, but decreased to approximately 15%, with a peak at the flowering stage at which nitrogen metabolism changed in the soybean plant. These results suggest the significance of a time-series analysis for understanding the relationship between the microbial community and host plant physiology.
Topics: Bacteria; DNA, Bacterial; Methylobacteriaceae; Microbiota; Nitrogen; Plant Leaves; RNA, Ribosomal, 16S; Glycine max
PubMed: 31413227
DOI: 10.1264/jsme2.ME19067 -
PloS One 2019Discoveries of bacterial communities in environments that previously have been described as sterile have in recent years radically challenged the view of these...
Discoveries of bacterial communities in environments that previously have been described as sterile have in recent years radically challenged the view of these environments. In this study we aimed to use 16S rRNA sequencing to describe the composition and temporal stability of the bacterial microbiota in bovine milk from healthy udder quarters, an environment previously believed to be sterile. Sequencing of the 16S rRNA gene is a technique commonly used to describe bacterial composition and diversity in various environments. With the increased use of 16S rRNA gene sequencing, awareness of methodological pitfalls such as biases and contamination has increased although not in equal amount. Evaluation of the composition and temporal stability of the microbiota in 288 milk samples was largely hampered by background contamination, despite careful and aseptic sample processing. Sequencing of no template control samples, positive control samples, with defined levels of bacteria, and 288 milk samples with various levels of bacterial growth, revealed that the data was influenced by contaminating taxa, primarily Methylobacterium. We observed an increasing impact of contamination with decreasing microbial biomass where the contaminating taxa became dominant in samples with less than 104 bacterial cells per mL. By applying a contamination filtration on the sequence data, the amount of sequences was substantially reduced but only a minor impact on number of identified taxa and by culture known endogenous taxa was observed. This suggests that data filtration can be useful for identifying biologically relevant associations in milk microbiota data.
Topics: Animals; Bacteria; DNA Contamination; DNA, Bacterial; Food Contamination; Food Microbiology; High-Throughput Nucleotide Sequencing; Methylobacterium; Microbiota; Milk; RNA, Ribosomal, 16S; Reproducibility of Results; Sequence Analysis, DNA
PubMed: 31194836
DOI: 10.1371/journal.pone.0218257 -
Current Issues in Molecular Biology 2019Experimental evolution has become an increasingly common approach for studying evolutionary phenomena, as well as uncovering physiological connections in a manner... (Review)
Review
Experimental evolution has become an increasingly common approach for studying evolutionary phenomena, as well as uncovering physiological connections in a manner complementary to traditional genetics. Here I describe the development of as a model system for using experimental evolution to study questions at the intersection of metabolism and evolution. Each experiment was initiated to address a particular question inspired by patterns in natural methylotrophs, such as tradeoffs between single-carbon and multi-carbon growth, or the challenges involved in incorporating novel metabolic pathways or genes with poor codon usage that are acquired via horizontal gene transfer. What I could not have appreciated initially, however, was just how many fortuitous surprise findings would emerge. These have ranged from the repeatability of evolution, complex dynamics within populations, epistasis between beneficial mutations, and even the ability to use simple mathematical models to generate testable, quantitative hypotheses about the fitness landscape.
Topics: Directed Molecular Evolution; Epistasis, Genetic; Evolution, Molecular; Gene Expression Regulation, Bacterial; Gene Transfer Techniques; Metabolic Engineering; Metabolic Networks and Pathways; Methylobacterium; Models, Biological; Organisms, Genetically Modified; Research
PubMed: 31166197
DOI: 10.21775/cimb.033.249 -
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 -
Current Issues in Molecular Biology 2019Chloromethane is a halogenated volatile organic compound, produced in large quantities by terrestrial vegetation. After its release to the troposphere and transport to... (Review)
Review
Chloromethane is a halogenated volatile organic compound, produced in large quantities by terrestrial vegetation. After its release to the troposphere and transport to the stratosphere, its photolysis contributes to the degradation of stratospheric ozone. A better knowledge of chloromethane sources (production) and sinks (degradation) is a prerequisite to estimate its atmospheric budget in the context of global warming. The degradation of chloromethane by methylotrophic communities in terrestrial environments is a major underestimated chloromethane sink. Methylotrophs isolated from soils, marine environments and more recently from the phyllosphere have been grown under laboratory conditions using chloromethane as the sole carbon source. In addition to anaerobes that degrade chloromethane, the majority of cultivated strains were isolated in aerobiosis for their ability to use chloromethane as sole carbon and energy source. Among those, the Proteobacterium (recently reclassified as ) harbours the only characterisized 'chloromethane utilization' () pathway, so far. This pathway is not representative of chloromethane-utilizing populations in the environment as genes are rare in metagenomes. Recently, combined 'omics' biological approaches with chloromethane carbon and hydrogen stable isotope fractionation measurements in microcosms, indicated that microorganisms in soils and the phyllosphere (plant aerial parts) represent major sinks of chloromethane in contrast to more recently recognized microbe-inhabited environments, such as clouds. Cultivated chloromethane-degraders lacking the genes display a singular isotope fractionation signature of chloromethane. Moreover, 13CH3Cl labelling of active methylotrophic communities by stable isotope probing in soils identify taxa that differ from the taxa known for chloromethane degradation. These observations suggest that new biomarkers for detecting active microbial chloromethane-utilizers in the environment are needed to assess the contribution of microorganisms to the global chloromethane cycle.
Topics: Bacterial Proteins; Biodegradation, Environmental; Energy Metabolism; Geologic Sediments; Metabolic Networks and Pathways; Methanol; Methyl Chloride; Methylobacterium; Methylophilaceae; Methyltransferases; Proteobacteria; Soil Microbiology
PubMed: 31166190
DOI: 10.21775/cimb.033.149