Biology subjects

Podar, M.

Publications and source records attributed to Podar, M..

9 recordsLinked to original sources

bioRxiv2025

BONCAT-Live for isolation and cultivation of active environmental bacteria

In diverse environments, microbes drive a myriad of processes, from geochemical and nutrient cycling to interspecies interactions, including in association with plants and animals. Their physiological state is dynamic and impacted by abiotic and biotic conditions, responding to environmental fluctuations by changes in cellular metabolism, according to their genetic potential. Molecular, cellular and genomic approaches can identify and measure microbial responses and adaptation to environmental changes in native communities. However, isolating the individual microbes that respond to specific changes has been difficult. To address that, we implemented BONCAT-Live, by integrating bio-orthogonal non-canonical amino acid tagging (BONCAT) in diverse native communities, with isolation and cultivation of cells responding to specific stimuli, at different time scales. In frozen Arctic permafrost samples, we identified and isolated dormant bacteria that become active after thawing under native or nutrient enriched conditions. From a Populus tree rhizosphere, we isolated strains that thrive under high concentrations of root exudates that act as defense compounds and nutrients. In the human oral microbiome, we identified and isolated bacteria that rapidly proliferated when exposed to metabolites provided by the host or other co-occurring microbes. Further characterization of isolated bacterial strains will provide opportunities for in depth determination of how these microbes adapt to changes in their environments, individually and as part of model communities. ImportanceDynamic microbial activity transforms environments and impacts health and disease in associations with plants and animals, including humans. Identifying the contribution of individual microbes to those processes in real time has not been generally compatible with their selective cultivation. BONCAT-Live tracks which microbes in environmental samples are translationally active and couples it with single cell isolation and cultivation. By studying the response of individual community member to specific natural or induced physical or chemical changes in the environment and culturing those organisms, BONCAT-Live enables further insights into microbial metabolic strategies, community dynamics and environmental adaptations.

microbiology↗

bioRxiv2022

Transcriptional control of hgcAB by an ArsR-like regulator in Pseudodesulfovibrio mercurii ND132

The hgcAB gene pair encodes mercury (Hg) methylation capability in a diverse group of microorganisms, but its evolution and transcriptional regulation remain unknown. Working from the possibility that the evolutionary function of HgcAB may not be Hg methylation, we test a possible link to arsenic resistance. Using model Hg-methylator Pseudodesulfovibrio mercurii ND132, we specifically evaluated transcriptional control of hgcAB by a putative ArsR encoded upstream and co-transcribed with hgcAB. This regulator shares homology with ArsR repressors of arsenic resistance and S-adenosyl-homocysteine (SAH) responsive regulators of methionine biosynthesis but is distinct from other ArsR/SahR in Pseudodesulfovibrio mercurii ND132. Using qPCR and RNA-seq analyses we confirmed this ArsR regulates hgcAB transcription, and is responsive to arsenic and SAH. Additionally, RNA-seq indicated a possible link between hgcAB activity and arsenic transformations by Pseudodesulfovibrio mercurii ND132, with significant up-regulation of other ArsR-regulated arsenic resistance operons alongside hgcAB. Interestingly, wild-type ND132 was less sensitive to AsV (but not AsIII) than an hgcAB knockout strain, supporting the idea that hgcAB may be linked to arsenic resistance. Arsenic significantly impacted Hg-methylation rates by ND132, however, responses varied with culture conditions. Differences in growth and overall metabolic activity did not account for arsenic impacts on methylation. One goal of this research is to better predict MeHg production in nature. However, we found that hgcAB gene and transcript abundance was not a good predictor of Hg-methylation rates. Our finding that hgcAB activity is linked to arsenic may hold clues to the possible environmental drivers of horizontal transfer of hgcAB. IMPORTANCEThis work reveals a link between microbial mercury methylation and arsenic resistance and may hold clues to the evolution of mercury methylation genes (hgcAB). Microbes with hgcAB produce methylmercury, a strong neurotoxin that readily accumulates in the food web. This study addresses a critical gap in our understanding about the environmental factors that control hgcAB expression. We show that hgcAB expression is controlled by an ArsR-like regulator responsive to both arsenic and S-adenosyl-homocysteine in our model organism, Pseudodesulfovibrio mercurii ND132. Exposure to arsenic also significantly impacted Pseudodesulfovibrio mercurii ND132 mercury methylation rates. However, expression of hgcAB was not always a good predictor of Hg methylation rates, highlighting the roles of Hg bioavailability and other biochemical mechanisms in methylmercury production. This study improves our understanding of the controls on hgcAB expression which is needed to better predict environmental methylmercury production.

microbiology↗

bioRxiv2022

A consensus protocol for the recovery of mercury methylation genes from metagenomes

Mercury methylation genes (hgcAB) mediate the formation of the toxic methylmercury and have been identified from diverse environments, including freshwater and marine ecosystems, Arctic permafrost, forest and paddy soils, coal-ash amended sediments, chlor-alkali plants discharges and geothermal springs. Here we present the first attempt at a standardized protocol for the detection, identification and quantification of hgc genes from metagenomes. Our Hg-MATE (Hg-cycling Microorganisms in Aquatic and Terrestrial Ecosystems) database, a catalogue of hgc genes, provides the most accurate information to date on the taxonomic identity and functional/metabolic attributes of microorganisms responsible for Hg methylation in the environment. Furthermore, we introduce "marky-coco", a ready-to-use bioinformatic pipeline based on de novo single-metagenome assembly, for easy and accurate characterization of hgc genes from environmental samples. We compared the recovery of hgc genes from environmental metagenomes using the marky-coco pipeline with an approach based on co-assembly of multiple metagenomes. Our data show similar efficiency in both approaches for most environments except those with high diversity (i.e., paddy soils) for which a co-assembly approach was preferred. Finally, we discuss the definition of true hgc genes and methods to normalize hgc gene counts from metagenomes.

microbiology↗

bioRxiv2021

Microbial diversity and sulfur cycling in an early earth analogue: From ancient novelty to modern commonality

Life emerged and diversified in the absence of molecular oxygen. The prevailing anoxia and unique sulfur chemistry in the Paleo-, Meso- and Neoarchean, and early Proterozoic eons may have supported microbial communities that are drastically different than those currently thriving on the earths surface. Zodletone spring in southwestern Oklahoma represents a unique habitat where spatial sampling could substitute for geological eons: from the anoxic, surficial light exposed sediments simulating a preoxygenated earth, to overlaid water column where air exposure simulates the relentless oxygen intrusion during the Neo Proterozoic. We document a remarkably diverse microbial community in the anoxic spring sediments, with 340/516 (65.89%) of genomes recovered in a metagenomic survey belonging to 200 bacterial and archaeal families that were either previously undescribed or that exhibit an extremely rare distribution on the current earth. Such diversity is underpinned by the widespread occurrence of sulfite-, thiosulfate, tetrathionate-, and sulfur-reduction, and paucity of sulfate-reduction machineries in these taxa; hence greatly expanding lineages mediating reductive sulfur cycling processes in the tree of life. Analysis of the overlaying water community demonstrated that oxygen intrusion lead to the development of a significantly less diverse community dominated by well-characterized lineages and a prevalence of oxidative sulfur cycling processes. Such transition from ancient novelty to modern commonality underscores the profound impact of the great oxygenation event on the earths surficial anoxic community. It also suggests that novel and rare lineages encountered in current anaerobic habitats could represent taxa once thriving in an anoxic earth, but have failed to adapt to earths progressive oxygenation.

microbiology↗

bioRxiv2020

An improved hgcAB primer set and direct high throughput sequencing expand Hg-methylator diversity in nature

The gene pair hgcAB is essential for microbial mercury methylation. Our understanding of its abundance and diversity in nature is rapidly evolving. In this study we developed a new broad-range primer set for hgcAB, plus an expanded hgcAB reference library, and used these to characterize Hg-methylating communities from diverse environments. We applied this new Hg-methylator database to assign taxonomy to hgcA sequences from clone, amplicon, and metagenomic datasets. We evaluated potential biases introduced in primer design, sequence length, and classification, and suggest best practices for studying Hg-methylator diversity. Our study confirms the emerging picture of an expanded diversity of HgcAB-encoding microbes in many types of ecosystems, with abundant putative mercury methylators Nitrospirae and Chloroflexi in several new environments including salt marsh and peat soils. Other common microbes encoding HgcAB included Phycisphaerae, Aminicenantes, Spirochaetes, and Elusimicrobia. Gene abundance data also corroborate the important role of two "classic" groups of methylators (Deltaproteobacteria and Methanomicrobia) in many environments, but generally show a scarcity of hgcAB+ Firmicutes. The new primer set was developed to specifically target hgcAB sequences found in nature, reducing degeneracy and providing increased sensitivity while maintaining broad diversity capture. We evaluated mock communities to confirm primer improvements, including culture spikes to environmental samples with variable DNA extraction and PCR amplification efficiencies. For select sites, this new workflow was combined with direct high-throughput hgcAB sequencing. The hgcAB diversity generated by direct amplicon sequencing confirmed the potential for novel Hg-methylators previously identified using metagenomic screens. A new phylogenetic analysis using sequences from freshwater, saline, and terrestrial environments showed Deltaproteobacteria HgcA sequences generally clustered among themselves, while metagenome-resolved HgcA sequences in other phyla tended to cluster by environment, suggesting horizontal gene transfer into many clades. HgcA from marine metagenomes often formed distinct subtrees from those sequenced from freshwater ecosystems. Overall the majority of HgcA sequences branch from a cluster of HgcAB fused proteins related to Thermococci, Atribacteria (candidate division OP9), Aminicenantes (OP8), and Chloroflexi. The improved primer set and library, combined with direct amplicon sequencing, provide a significantly improved assessment of the abundance and diversity of hgcAB+ microbes in nature. Contribution to the Field StatementThe gene pair hgcAB is essential for microbial production of the neurotoxin methylmercury. In recent years these genes have been used as biomarkers to determine the potential of a microbiome to generate methylmercury via PCR amplification using degenerate primers from several research groups. However, improved techniques for capturing hgcAB diversity are necessary for identifying the major environmental producers of the neurotoxin as well as the expanding diversity of novel putative methylators, and the genes evolutionary history. The work described herein advances hgcAB detection in environmental samples through an updated primer set coupled with a direct high-throughput sequencing method that enables broader diversity capture. We provide an expanded hgcAB sequence reference library that allows for more sensitive and robust estimations of Hg-methylator diversity and potential for MeHg generation in the environment. The hgcAB diversity generated by high-throughput sequencing confirms the potential for novel Hg-methylators previously only identified using metagenomic screens. This study provides a significantly improved assessment of the abundance and diversity of hgcAB+ microbes in nature. By expanding our understanding of the microbial metabolic clades associated with mercury methylation, this work improves our ability to predict environmental conditions that drive production and accumulation of the neurotoxin in aquatic ecosystems.

molecular biology↗

bioRxiv2019

Comparative microbial diversity across temperature gradients in hot springs from Yellowstone and Iceland

Geothermal hot springs are a natural setting to study microbial adaptation to a wide range of temperatures reaching up to boiling. Temperature gradients lead to distinct microbial communities that inhabit their optimum niches. We sampled three distant but chemically similar hot springs in Yellowstone and Iceland that had outflows and presented a wide range of temperatures. The microbial composition at different niches was determined by deep DNA sequencing of rRNA gene amplicons. Over three dozen phyla of Archaea and Bacteria were identified, representing over 1700 unique organisms. We observed a significant reduction in the number of microbial taxa as the temperature increased from warm (38{degrees}C) to boiling. The community structure was primarily driven by temperature and, at high taxonomic levels, was similar between Yellowstone and Iceland, suggesting that the environment rather than biogeographic location play the largest role in the microbial ecology of hot springs. We observed significant endemism at genus level, especially in thermophilic phototrophs, potentially driven by distinct environmental conditions and dispersal limitations.

microbiology↗

bioRxiv2019

Integrated characterization of subsurface media from locations up- and down-gradient of a uranium-contaminated aquifer

The processing of sediment to accurately characterize the spatially-resolved depth profiles of geophysical and geochemical properties along with signatures of microbial density and activity remains a challenge especially in complex contaminated environments. To provide site assessment for a larger study, we processed cores from two sediment boreholes from background and contaminated core sediments and surrounding groundwater from the ENIGMA Field Research Site at the United States Department of Energy (DOE) Oak Ridge Reservation (ORR). We compared fresh core sediments by depth to capture the changes in sediment structure, sediment minerals, biomass, and pore water geochemistry in terms of major and trace elements including contaminants, cations, anions, and organic acids. Soil porewater samples were matched to groundwater level, flow rate, and preferential flows and compared to homogenized groundwater-only samples from neighboring monitoring wells. This environmental systems approach provided detailed site-specific biogeochemical information from the various properties of subsurface media to reveal the influences of solid, liquid, and gas phases. Groundwater analysis of nearby wells only revealed high sulfate and nitrate concentrations while the same analysis using sediment pore water samples with depth was able to suggest areas high in sulfate- and nitrate- reducing bacteria based on their decreased concentration and production of reduced by-products that could not be seen in the groundwater samples. Positive correlations among porewater content, total organic carbon, trace metals and clay minerals revealed a more complicated relationship among contaminant, sediment texture, groundwater table, and biomass. This suggested that groundwater predominantly flowed through preferential paths with high flux and little mixing with water in the interstices of sediment particles, which could impact microbial activity. The abundant clay minerals with high surface area and high water-holding capacity of micro-pores of the fine clay rich layer suggest suppression of nutrient supply to microbes from the surface. The fluctuating capillary interface had high concentrations of Fe and Mn-oxides combined with trace elements including U, Th, Sr, Ba, Cu, and Co. This suggests the mobility of highly toxic elements, sediment structure, and biogeochemical factors are all linked together to impact microbial communities, emphasizing that solid interfaces play an important role in determining the abundance of bacteria in the sediments.

systems biology↗

bioRxiv2019

Comparative genomics guides elucidation of vitamin B12 biosynthesis in novel human associated Akkermansia

BackgroundAkkermansia muciniphila is a mucin-degrading bacterium found in the gut of most healthy humans and is considered a next-generation probiotic. However, knowledge of the genomic and physiological diversity of human associated Akkermansia is limited, as only one species has been formally described. ResultsTo begin to fill this knowledge gap, we reconstructed 35 high-quality metagenome assembled genomes from children and combined them with 40 other publicly available genomes from adults and mice for comparative genomic analysis. We identified at least four species-level phylogroups (AmI-AmIV) with distinct functional potentials. Most notably, we identified the presence of putative cobalamin (vitamin B12) biosynthesis genes within the AmII (n=26/28) and AmIII (n=2/2) phylogroups. To test these predictions, 10 novel strains of Akkermansia were isolated from adults and screened for essential vitamin B12 biosynthesis genes via PCR. Two strains of the AmII phylogroup were positive for the presence of vitamin B12 biosynthesis genes, while all AmI strains, including the type strain A. muciniphila MucT, were negative. To demonstrate vitamin B12 biosynthesis, we measured the production of acetate, succinate, and propionate in the presence and absence of vitamin supplementation in representative strains of the AmI and AmII phylogroups since cobalamin is a cofactor in propionate metabolism. Results show that the Akkermansia AmII strain produced acetate and propionate in the absence of supplementation, which is indicative of de novo vitamin B12 biosynthesis. In contrast, acetate and succinate were the main fermentation products for the AmI strains when vitamin B12 was not supplied in the culture medium. ConclusionsWe identified Akkermansia strains as potentially important vitamin B12 biosynthetic bacteria in the human gut. This novel physiological trait of human associated Akkermansia may impact how these bacteria interact with the human host and other members of the human gut microbiome.

genomics↗

bioRxiv2018

Systems genetic discovery of host-microbiome interactions reveals mechanisms of microbial involvement in disease

The role of the microbiome in health and disease involves complex networks of host genetics, genomics, microbes and environment. Identifying the mechanisms of these interactions has remained challenging. Systems genetics in the laboratory mouse enables data-driven discovery of network components and mechanisms of host-microbial interactions underlying multiple disease phenotypes. To examine the interplay among the whole host genome, transcriptome and microbiome, we mapped quantitative trait loci and correlated the abundance of cecal mRNA, luminal microflora, physiology and behavior in incipient strains of the highly diverse Collaborative Cross mouse population. The relationships that are extracted can be tested experimentally to ascribe causality among host and microbe in behavior and physiology, providing insight into disease. Application of this strategy in the Collaborative Cross population revealed experimentally validated mechanisms of microbial involvement in models of autism, inflammatory bowel disease and sleep disorder.\n\neTOC BlurbHost genetic diversity provides a variable selection environment and physiological context for microbiota and their interaction with host physiology. Using a highly diverse mouse population Bubier et al. identified a variety of host, microbe and potentially disease interactions.\n\nHighlights* 18 significant species-specific QTL regulating microbial abundance were identified\n* Cis and trans eQTL for 1,600 cecal transcripts were mapped in the Collaborative Cross\n* Sleep phenotypes were highly correlated with the abundance of B.P. Odoribacter\n* Elimination of sleep-associated microbes restored normal sleep patterns in mice.

genetics↗