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MBio Mar 2020Population-level analyses are rapidly becoming inadequate to answer many of biomedical science and microbial ecology's most pressing questions. The role of microbial...
Population-level analyses are rapidly becoming inadequate to answer many of biomedical science and microbial ecology's most pressing questions. The role of microbial populations within ecosystems and the evolutionary selective pressure on individuals depend fundamentally on the metabolic activity of single cells. Yet, many existing single-cell technologies provide only indirect evidence of metabolic specialization because they rely on correlations between transcription and phenotype established at the level of the population to infer activity. In this study, we take a top-down approach using isotope labels and secondary ion mass spectrometry to track the uptake of carbon and nitrogen atoms from different sources into biomass and directly observe dynamic changes in anabolic specialization at the level of single cells. We investigate the classic microbiological phenomenon of diauxic growth at the single-cell level in the model methylotroph In nature, this organism inhabits the phyllosphere, where it experiences diurnal changes in the available carbon substrates, necessitating an overhaul of central carbon metabolism. We show that the population exhibits a unimodal response to the changing availability of viable substrates, a conclusion that supports the canonical model but has thus far been supported by only indirect evidence. We anticipate that the ability to monitor the dynamics of anabolism in individual cells directly will have important applications across the fields of ecology, medicine, and biogeochemistry, especially where regulation downstream of transcription has the potential to manifest as heterogeneity that would be undetectable with other existing single-cell approaches. Understanding how genetic information is realized as the behavior of individual cells is a long-term goal of biology but represents a significant technological challenge. In clonal microbial populations, variation in gene regulation is often interpreted as metabolic heterogeneity. This follows the central dogma of biology, in which information flows from DNA to RNA to protein and ultimately manifests as activity. At present, DNA and RNA can be characterized in single cells, but the abundance and activity of proteins cannot. Inferences about metabolic activity usually therefore rely on the assumption that transcription reflects activity. By tracking the atoms from which they build their biomass, we make direct observations of growth rate and substrate specialization in individual cells throughout a period of growth in a changing environment. This approach allows the flow of information from DNA to be constrained from the distal end of the regulatory cascade and will become an essential tool in the rapidly advancing field of single-cell metabolism.
Topics: Biomass; Carbon; Isotope Labeling; Methylobacterium extorquens; Nitrogen; Phenotype; Single-Cell Analysis; Spectrometry, Mass, Secondary Ion
PubMed: 32127448
DOI: 10.1128/mBio.01519-19 -
PloS One 2020The ecology and distribution of many bacteria is strongly associated with specific eukaryotic hosts. However, the impact of such host association on bacterial ecology...
The ecology and distribution of many bacteria is strongly associated with specific eukaryotic hosts. However, the impact of such host association on bacterial ecology and evolution is not well understood. Bacteria from the genus Methylobacterium consume plant-derived methanol, and are some of the most abundant and widespread plant-associated bacteria. In addition, many of these species impact plant fitness. To determine the ecology and distribution of Methylobacterium in nature, we sampled bacteria from 36 distinct rice landraces, traditionally grown in geographically isolated locations in North-East (NE) India. These landraces have been selected for diverse phenotypic traits by local communities, and we expected that the divergent selection on hosts may have also generated divergence in associated Methylobacterium strains. We determined the ability of 91 distinct rice-associated Methylobacterium isolates to use a panel of carbon sources, finding substantial variability in carbon use profiles. Consistent with our expectation, across spatial scales this phenotypic variation was largely explained by host landrace identity rather than geographical factors or bacterial taxonomy. However, variation in carbon utilisation was not correlated with sugar exudates on leaf surfaces, suggesting that bacterial carbon use profiles do not directly determine bacterial colonization across landraces. Finally, experiments showed that at least some rice landraces gain an early growth advantage from their specific phyllosphere-colonizing Methylobacterium strains. Together, our results suggest that landrace-specific host-microbial relationships may contribute to spatial structure in rice-associated Methylobacterium in a natural ecosystem. In turn, association with specific bacteria may provide new ways to preserve and understand diversity in one of the most important food crops of the world.
Topics: Carbon; Crops, Agricultural; Ecosystem; Genetic Variation; Host-Pathogen Interactions; India; Methylobacterium; Oryza; Phenotype; Phylogeny; Plant Leaves
PubMed: 32092057
DOI: 10.1371/journal.pone.0228550 -
Journal of Biological Inorganic... Mar 2020Methanol dehydrogenases (MDH) have recently taken the spotlight with the discovery that a large portion of these enzymes in nature utilize lanthanides in their active...
Methanol dehydrogenases (MDH) have recently taken the spotlight with the discovery that a large portion of these enzymes in nature utilize lanthanides in their active sites. The kinetic parameters of these enzymes are determined with a spectrophotometric assay first described by Anthony and Zatman 55 years ago. This artificial assay uses alkylated phenazines, such as phenazine ethosulfate (PES) or phenazine methosulfate (PMS), as primary electron acceptors (EAs) and the electron transfer is further coupled to a dye. However, many groups have reported problems concerning the bleaching of the assay mixture in the absence of MDH and the reproducibility of those assays. Hence, the comparison of kinetic data among MDH enzymes of different species is often cumbersome. Using mass spectrometry, UV-Vis and electron paramagnetic resonance (EPR) spectroscopy, we show that the side reactions of the assay mixture are mainly due to the degradation of assay components. Light-induced demethylation (yielding formaldehyde and phenazine in the case of PMS) or oxidation of PES or PMS as well as a reaction with assay components (ammonia, cyanide) can occur. We suggest here a protocol to avoid these side reactions. Further, we describe a modified synthesis protocol for obtaining the alternative electron acceptor, Wurster's blue (WB), which serves both as EA and dye. The investigation of two lanthanide-dependent methanol dehydrogenases from Methylorubrum extorquens AM1 and Methylacidiphilum fumariolicum SolV with WB, along with handling recommendations, is presented. Lanthanide-dependent methanol dehydrogenases. Understanding the chemistry of artificial electron acceptors and redox dyes can yield more reproducible results.
Topics: 2,6-Dichloroindophenol; Alcohol Oxidoreductases; Electrons; Methylobacterium extorquens; Methylphenazonium Methosulfate; Molecular Structure; Phenazines; Tetramethylphenylenediamine; Verrucomicrobia
PubMed: 32060650
DOI: 10.1007/s00775-020-01752-9 -
Scientific Reports Jan 2020A mud volcano (MV) is a naturally hydrocarbon-spiked environment, as indicated by the presence of various quantities of PAHs and aromatic isotopic shifts in its...
A mud volcano (MV) is a naturally hydrocarbon-spiked environment, as indicated by the presence of various quantities of PAHs and aromatic isotopic shifts in its sediments. Recurrent expulsion of various hydrocarbons consolidates the growth of hydrocarbonoclastic bacterial communities in the areas around MVs. In addition to the widely-known availability of biologically malleable alkanes, MVs can represent hotbeds of polyaromatic hydrocarbons (PAHs), as well - an aspect that has not been previously explored. This study measured the availability of highly recalcitrant PAHs and the isotopic signature of MV sediments both by GC-MS and δC analyses. Subsequently, this study highlighted both the occurrence and distribution of putative PAH-degrading bacterial OTUs using a metabarcoding technique. The putative hydrocarbonoclastic taxa incidence are the following: Enterobacteriaceae (31.5%), Methylobacteriaceae (19.9%), Bradyrhizobiaceae (16.9%), Oxalobacteraceae (10.2%), Comamonadaceae (7.6%) and Sphingomonadaceae (5.5%). Cumulatively, the results of this study indicate that MVs represent polyaromatic hydrocarbonoclastic hotbeds, as defined by both natural PAH input and high incidence of putative PAH-degrading bacterial OTUs.
PubMed: 31988316
DOI: 10.1038/s41598-020-58282-2 -
Microbes and Environments 2020The Vietnamese Mekong delta is one of the largest rice-producing areas globally. Methylobacterium spp. are persistent colonizers of the rice plant and exert beneficial...
The Vietnamese Mekong delta is one of the largest rice-producing areas globally. Methylobacterium spp. are persistent colonizers of the rice plant and exert beneficial effects on plant growth and health. Sixty-one Methylobacterium strains belonging to seven species were predominantly isolated from the phyllosphere of rice cultivated in six Mekong delta provinces. Inoculation tests revealed that some strains exhibited plant growth-promoting activity. Moreover, three strains possessed the novel characteristics of inducing leaf bleaching and killing rice seedlings. These results revealed the complex diversity of Methylobacterium in Mekong delta rice and that healthy and productive rice cultivation requires a proper balance of Methylobacterium.
Topics: Genetic Variation; Lactuca; Methylobacterium; Oryza; Plant Components, Aerial; Plant Growth Regulators; Vietnam
PubMed: 31969531
DOI: 10.1264/jsme2.ME19111 -
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 -
ELife Jan 2020Rank orders have been studied in evolutionary biology for almost a hundred years. Constraints on the order in which mutations accumulate are known from cancer drug...
Rank orders have been studied in evolutionary biology for almost a hundred years. Constraints on the order in which mutations accumulate are known from cancer drug treatment, and order constraints for species invasions are important in ecology. However, current theory on rank orders in biology is somewhat fragmented. Here, we show how our previous work on inferring genetic interactions from comparative fitness data (Crona et al., 2017) is related to an influential approach to rank orders based on sign epistasis. Our approach depends on order perturbations that indicate interactions. We apply our results to malaria parasites and find that order perturbations beyond sign epistasis are prevalent in the antimalarial drug-resistance landscape. This finding agrees with the observation that reversed evolution back to the ancestral type is difficult. Another application concerns the adaptation of bacteria to a methanol environment.
Topics: Adaptation, Physiological; Alleles; Antimalarials; Computer Simulation; Epistasis, Genetic; Evolution, Molecular; Genetic Fitness; Genotype; Methylobacterium extorquens; Models, Genetic; Mutation; Normal Distribution; Plasmodium vivax; Selection, Genetic
PubMed: 31934856
DOI: 10.7554/eLife.51004 -
Angewandte Chemie (International Ed. in... Mar 2020The synthesis of complex molecules from simple, renewable carbon units is the goal of a sustainable economy. Here we explored the biocatalytic potential of the...
The synthesis of complex molecules from simple, renewable carbon units is the goal of a sustainable economy. Here we explored the biocatalytic potential of the thiamine-diphosphate-dependent (ThDP) oxalyl-CoA decarboxylase (OXC)/2-hydroxyacyl-CoA lyase (HACL) superfamily that naturally catalyzes the shortening of acyl-CoA thioester substrates through the release of the C -unit formyl-CoA. We show that the OXC/HACL superfamily contains promiscuous members that can be reversed to perform nucleophilic C -extensions of various aldehydes to yield the corresponding 2-hydroxyacyl-CoA thioesters. We improved the catalytic properties of Methylorubrum extorquens OXC by rational enzyme engineering and combined it with two newly described enzymes-a specific oxalyl-CoA synthetase and a 2-hydroxyacyl-CoA thioesterase. This enzymatic cascade enabled continuous conversion of oxalate and aromatic aldehydes into valuable (S)-α-hydroxy acids with enantiomeric excess up to 99 %.
Topics: Aldehydes; Biocatalysis; Carboxy-Lyases; Humans; Hydroxy Acids; Kinetics; Methylobacteriaceae; Mutagenesis, Site-Directed; Recombinant Proteins; Stereoisomerism; Substrate Specificity; Thiamine Pyrophosphate
PubMed: 31894608
DOI: 10.1002/anie.201915155 -
Scientific Reports Nov 2019Phlebotomine sand flies are remarkable vectors of several etiologic agents (virus, bacterial, trypanosomatid Leishmania), posing a heavy health burden for human...
Wild specimens of sand fly phlebotomine Lutzomyia evansi, vector of leishmaniasis, show high abundance of Methylobacterium and natural carriage of Wolbachia and Cardinium types in the midgut microbiome.
Phlebotomine sand flies are remarkable vectors of several etiologic agents (virus, bacterial, trypanosomatid Leishmania), posing a heavy health burden for human populations mainly located at developing countries. Their intestinal microbiota is involved in a wide range of biological and physiological processes, and could exclude or facilitate such transmission of pathogens. In this study, we investigated the Eubacterial microbiome from digestive tracts of Lu. evansi adults structure using 16S rRNA gene sequence amplicon high throughput sequencing (Illumina MiSeq) obtained from digestive tracts of Lu. evansi adults. The samples were collected at two locations with high incidence of the disease in humans: peri-urban and forest ecosystems from the department of Sucre, Colombia. 289,068 quality-filtered reads of V4 region of 16S rRNA gene were obtained and clustered into 1,762 operational taxonomic units (OTUs) with 97% similarity. Regarding eubacterial diversity, 14 bacterial phyla and 2 new candidate phyla were found to be consistently associated with the gut microbiome content. Proteobacteria, Firmicutes, and Bacteroidetes were the most abundant phyla in all the samples and the core microbiome was particularly dominated by Methylobacterium genus. Methylobacterium species, are known to have mutualistic relationships with some plants and are involved in shaping the microbial community in the phyllosphere. As a remarkable feature, OTUs classified as Wolbachia spp. were found abundant on peri-urban ecosystem samples, in adult male (OTUs n = 776) and unfed female (OTUs n = 324). Furthermore, our results provide evidence of OTUs classified as Cardinium endosymbiont in relative abundance, notably higher with respect to Wolbachia. The variation in insect gut microbiota may be determined by the environment as also for the type of feeding. Our findings increase the richness of the microbiota associated with Lu. evansi. In this study, OTUs of Methylobacterium found in Lu. evansi was higher in engorged females, suggesting that there are interactions between microbes from plant sources, blood nutrients and the parasites they transmit during the blood intake.
Topics: Animals; Bacteroidetes; Female; Gastrointestinal Microbiome; Humans; Insect Vectors; Leishmaniasis; Male; Methylobacterium; Psychodidae; RNA, Ribosomal, 16S; Wolbachia
PubMed: 31780680
DOI: 10.1038/s41598-019-53769-z -
Proceedings of the National Academy of... Dec 2019Methylotrophy, the ability of microorganisms to grow on reduced one-carbon substrates such as methane or methanol, is a feature of various bacterial species. The...
Methylotrophy, the ability of microorganisms to grow on reduced one-carbon substrates such as methane or methanol, is a feature of various bacterial species. The prevailing oxidation pathway depends on tetrahydromethanopterin (HMPT) and methylofuran (MYFR), an analog of methanofuran from methanogenic archaea. Formyltransferase/hydrolase complex (Fhc) generates formate from formyl-HMPT in two consecutive reactions where MYFR acts as a carrier of one-carbon units. Recently, we chemically characterized MYFR from the model methylotroph and identified an unusually long polyglutamate side chain of up to 24 glutamates. Here, we report on the crystal structure of Fhc to investigate the function of the polyglutamate side chain in MYFR and the relatedness of the enzyme complex with the orthologous enzymes in archaea. We identified MYFR as a prosthetic group that is tightly, but noncovalently, bound to Fhc. Surprisingly, the structure of Fhc together with MYFR revealed that the polyglutamate side chain of MYFR is branched and contains glutamates with amide bonds at both their α- and γ-carboxyl groups. This negatively charged and branched polyglutamate side chain interacts with a cluster of conserved positively charged residues of Fhc, allowing for strong interactions. The MYFR binding site is located equidistantly from the active site of the formyltransferase (FhcD) and metallo-hydrolase (FhcA). The polyglutamate serves therefore an additional function as a swinging linker to shuttle the one-carbon carrying amine between the two active sites, thereby likely increasing overall catalysis while decreasing the need for high intracellular MYFR concentrations.
Topics: Bacterial Proteins; Coenzymes; Crystallography; Formates; Furans; Hydroxymethyl and Formyl Transferases; Methane; Methanol; Methylobacterium extorquens; Polyglutamic Acid
PubMed: 31776258
DOI: 10.1073/pnas.1911595116