Biology subjects

Chakraborty, R.

Publications and source records attributed to Chakraborty, R..

At least 55 records · Page 3Linked to original sources

bioRxiv2023

Fine scale sampling reveals spatial heterogeneity of rhizosphere microbiome in young Brachypodium plants

For a deeper and comprehensive understanding of the diversity, composition and function of rhizosphere microbiomes, we need to focus at the scale of individual roots in standardized growth containers. Root exudation patterns are known to vary across distinct parts of the root giving rise to spatially distinct microbial niches. To address this, we analyzed microbial community from two spatially distinct zones of the primary root (the tip vs. the base) in Brachypodium distachyon, grown in natural soil using standardized fabricated ecosystems known as EcoFABs as well as in more conventional pot and tubes. 16S rRNA based community analysis showed a stronger rhizosphere effect in the root base vs. bulk soil compared to the root tips vs. bulk soil, resulting in an enrichment of Actinobacteria, Bacteroidetes, Firmicutes and Proteobacteria, few OTUs belonging to less characterized lineages such as Verrucomicrobia and Acidobacteria. While the microbial community distributions are similar across growth containers, the EcoFAB displayed higher replicate reproducibility. Genome-resolved and bulk metagenomics revealed that genes associated with transcriptional regulation, transport of nutrients and catabolic enzymes indicating active metabolism, biofilm formation and root colonization were enriched in root tips. On the other hand, genes associated with nutrient-limitation and environmental stress were prominent in the bulk soil compared to the root tips, implying the presence of easily available, labile carbon and nutrients in the rhizosphere relative to bulk soil. Such insights into the relationships between root structure, exudation and microbial communities are critical for developing understanding of plant-microbe interactions.

ecology↗

bioRxiv2022

Tunneling nanotubes between neuronal and microglial cells allow bi-directional transfer of α-Synuclein and mitochondria

Tunneling Nanotubes (TNTs) facilitate contact-mediated intercellular communication over long distances. Material transfer via TNTs can range from ions and intracellular organelles to protein aggregates and pathogens. Prion-like toxic protein aggregates accumulating in several disorders, such as Alzheimers, Parkinsons, and Huntingtons diseases have been shown to spread via TNTs not only between neurons, but also between neurons-astrocytes, and neurons-pericytes, indicating the importance of TNTs in mediating neuron-glia interactions. TNT-like structures were also reported between microglia, however their roles in neuron-microglia interaction remain elusive. In this work, we quantitatively characterise microglial TNTs and their cytoskeletal composition, and demonstrate that TNTs form between human neuronal and microglial cells. We show that -Synuclein (-Syn) aggregates increase the global TNT-mediated connectivity between cells, along with the number of TNT connections per cell pair. Homotypic TNTs formed between microglial cells, and heterotypic TNTs between neuronal and microglial cells are furthermore shown to be functional, allowing movement of both -Syn and mitochondria. Quantitative analysis shows that -Syn aggregates are transferred predominantly from neuronal to microglial cells, possibly as a mechanism to relieve the burden of accumulated aggregates. By contrast, microglia transfer mitochondria preferably to -Syn burdened neuronal cells over the healthy ones, likely as a potential rescue mechanism. Besides describing novel TNT-mediated communication between neurons and microglia, this work allows us to better understand the cellular mechanisms of spreading of neurodegenerative diseases, shedding light on the role of microglia.

cell biology↗

bioRxiv2022

Thermal endurance by a hot-spring-dwelling phylogenetic relative of the mesophilic Paracoccus

High temperature growth/survival was revealed in a phylogenetic relative (strain SMMA_5) of the mesophilic Paracoccus isolated from the 78-85{degrees}C water of a Trans- Himalayan sulfur-borax spring. After 12 h at 50{degrees}C, or 45 minutes at 70{degrees}C, in mineral salts thiosulfate (MST) medium, SMMA_5 retained [~]2% colony-forming units (CFUs), whereas comparator Paracoccus had 1.5% and 0% CFU left at 50{degrees}C and 70{degrees}C respectively. After 12 h at 50{degrees}C, the thermally-conditioned sibling SMMA_5_TC exhibited [~]1.5 time increase in CFU-count; after 45 minutes at 70{degrees}C, SMMA_5_TC had 7% of the initial CFU-count intact. 1000-times diluted Reasoners 2A medium, and MST supplemented with lithium, boron or glycine-betaine (solutes typical of the SMMA_5 habitat), supported higher CFU-retention/CFU-growth than MST. With or without lithium/boron/glycine-betaine in MST, a higher percentage of cells always remained viable (cytometry data), compared with what percentage remained capable of forming single colonies (CFU data). SMMA_5, compared with other Paracoccus, contained 335 unique genes, mostly for DNA replication/recombination/repair, transcription, secondary metabolites biosynthesis/transport/catabolism, and inorganic ion transport/metabolism. Its also exclusively enriched in cell wall/membrane/envelope biogenesis, and amino acid metabolism, genes. SMMA_5 and SMMA_5_TC mutually possessed 43 nucleotide polymorphisms, of which 18 were in protein-coding genes with 13 nonsynonymous and seven radical amino acid replacements. Such biochemical and biophysical mechanisms could be involved in thermal stress mitigation which streamline the cells energy and resources towards system-maintenance and macromolecule-stabilization, thereby relinquishing cell-division for cell-viability. Thermal conditioning apparently helped memorize those potential metabolic states which are crucial for cell-system maintenance, while environmental solutes augmented the indigenous stability-conferring mechanisms. IMPORTANCEFor a holistic understanding of microbial lifes high-temperature adaptation it is imperative to explore the biology of the phylogenetic relatives of mesophilic bacteria which get stochastically introduced to geographically and geologically diverse hot spring systems by local geodynamic forces. Here, in vitro endurance of high heat up to the extent of growth under special (habitat-inspired) conditions was discovered in a hot- spring-dwelling phylogenetic relative of the mesophilic Paracoccus species. Thermal conditioning, extreme oligotrophy, metabolic deceleration, presence of certain habitat- specific inorganic/organic solutes, and typical genomic specializations were found to be the major enablers of this conditional (acquired) thermophilicity. Feasibility of such phenomena across the taxonomic spectrum can well be paradigm-changing for the established scopes of microbial adaptation to the physicochemical extremes. Applications of conditional thermophilicity in microbial process biotechnology may be far reaching and multi-faceted.

microbiology↗

bioRxiv2022

Bone marrow age dictates clonality of smooth muscle-derived cells in the atherosclerotic plaque

Aging is the predominant risk factor for atherosclerosis, the leading cause of death. Rare smooth muscle cell (SMC) progenitors clonally expand giving rise to up to [~]70% of atherosclerotic plaque cells; however, the effect of age on SMC clonality is not known. Our results indicate that aged bone marrow (BM)-derived cells non-cell autonomously induce SMC polyclonality and worsen atherosclerosis. Indeed, in myeloid cells from aged mice and humans, TET2 levels are reduced which epigenetically silences integrin {beta}3 resulting in increased tumor necrosis factor [TNF]- signaling. In turn, TNF signals through TNF receptor 1 on SMCs to promote proliferation and induces recruitment and expansion of multiple SMC progenitors into the atherosclerotic plaque. Notably, integrin {beta}3 overexpression in aged BM preserves dominance of the lineage of a single SMC progenitor and attenuates plaque burden. Our results demonstrate a molecular mechanism of aged macrophage-induced SMC polyclonality and atherogenesis and suggest novel therapeutic strategies. Graphical abstractAge of BM-derived monocytes/macrophages determines clonality of SMC lineage in the atherosclerotic plaque. Atherogenesis is depicted in a young (a) or aged (b) host. Aged monocytes/macrophages have decreased levels of the epigenetic regulator TET2, leading to reduction of the 5-hydroxymethylcytosine (5hmC) mark on the Itgb3 promoter. The resulting low integrin {beta}3 levels in aged monocytes/macrophages induces high TNF- levels, facilitating recruitment and expansion of multiple SMC progenitors (polyclonality) in the atherosclerotic plaque and worse disease burden. In contrast, the young control is characterized by mono/oligoclonal SMC expansion in a smaller plaque. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=135 SRC="FIGDIR/small/476756v2_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@12f7090org.highwire.dtl.DTLVardef@186ff92org.highwire.dtl.DTLVardef@1f4da69org.highwire.dtl.DTLVardef@2f509c_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2021

TRPA1: A Potential Prognostic Indicator for Oral Squamous Cell Carcinoma

IntroductionTransient receptors are related to oral cancer pain. Previously capsaicin (TRPV1 agonist) was shown to induce cell death in oral cancer cells. We hypothesised that these receptors are present in oral cancer. MethodWe examined the presence of cannabinoid receptors (CB1 and CB2) and targets (TRPV1, TRPA1, CaV 3.1, CaV 3.2, CaV 3.3) via quantitative polymerase chain reaction (qPCR) in oral cancer cells SCC4, SCC9, SCC25, Cal27, and normal oral cell line OKF6. ResultCannabinoid receptors are absent in all the cell lines, while TRPA1 is only present in normal cells, but absent in all the oral cancer cell lines. Voltage-gated calcium channels are present in all the cell lines. Conclusion and Future AspectsTRPA1 could be the possible future prognostic indicator of oral squamous cell carcinoma. Future functionality assays could use precancerous cell lines to follow the loss of TRPA1.

cell biology↗

bioRxiv2021

Pseudomonas syringae pv. tomato DC3000 Effector HopG1 is a multi-faceted protein that Triggers Necrotic Cell Death that is attenuated by the Nonhost Resistance 2B (AtNHR2B) Protein

The plant pathogenic bacterium Pseudomonas syringae pv. tomato DC3000 (Pst DC3000) has become a paradigm in plant-bacteria interactions due to its ability to cause disease in the model plant Arabidopsis thaliana. Pst DC3000 uses the type III secretion system to deliver type III secreted effectors (T3SEs) directly into the plant cytoplasm. Pst DC3000 T3SEs contribute to pathogenicity by suppressing plant defense responses and targeting plants physiological processes. Although the complete repertoire of effectors encoded in the Pst DC3000 genome have been identified, the specific function for most of them remains to be elucidated. The mitochondrial-localized T3E HopG1, suppresses plant defense responses and promotes the development of disease symptoms. Here, we show that HopG1 triggers necrotic cell death that enables the growth of non-adapted pathogens. We further showed that HopG1 interacts with the plant immunity-related protein AtNHR2B and that AtNHR2B attenuates HopG1-virulence functions.

plant biology↗

bioRxiv2021

The mitochondrial gene CMPK2 functions as a rheostat for macrophage homeostasis in inflammation

In addition to their role in cellular energy production, mitochondria are increasingly recognized as regulators of the innate immune response of phagocytes. Here, we demonstrate that altering expression levels of the mitochondria associated enzyme, cytidine monophosphate kinase 2 (CMPK2), disrupts mitochondrial physiology and significantly de-regulates the resting immune homeostasis of macrophages. Both CMPK2 silenced as well as constitutively over expressing macrophage lines portray mitochondrial stress with marked depolarization of their membrane potential, enhanced ROS and disturbed architecture culminating in the enhanced expression of the pro-inflammatory genes-IL1{beta}, TNF and IL8. Interestingly, the long-term modulation of CMPK2 expression resulted in an increased glycolytic flux of macrophages akin to the altered physiological state of activated M1 macrophages. While infection induced inflammation for restricting pathogens is regulated, our observation of a total dysregulation of basal inflammation by bidirectional alteration of CMPK2 expression, only highlights a critical role of this gene in mitochondria mediated control of inflammation.

immunology↗

bioRxiv2021

Biofilm Interaction Mapping and Analysis (BIMA): A tool for deconstructing interspecific interactions in co-culture biofilms

Pseudomonas species are ubiquitous in nature and include numerous medically, agriculturally and technologically beneficial strains of which the interspecific interactions are of great interest for biotechnologies. Specifically, co-cultures containing Pseudomonas stutzeri have been used for bioremediation, biocontrol, aquaculture management and wastewater denitrification. Furthermore, the use of P. stutzeri biofilms, in combination with consortia based approaches, may offer advantages for these processes. Understanding the interspecific interaction within biofilm co-cultures or consortia provides a means for improvement of current technologies. However, the investigation of biofilm based consortia has been limited. We present an adaptable and scalable method for the analysis of macroscopic interactions (colony morphology, inhibition and invasion) between colony forming bacterial strains using an automated printing method followed by analysis of the genes and metabolites involved in the interactions. Using Biofilm Interaction Mapping and Analysis (BIMA), these interactions were investigated between P. stutzeri strain RCH2, a denitrifier isolated from chromium (VI) contaminated soil, and thirteen other species of pseudomonas isolated from non-contaminated soil. The metabolites and genes associated with both active co-culture growth and inhibitory growth were investigated using mass spectrometry based metabolomics and mutant fitness profiling of a DNA-barcoded mutant library. One interaction partner, Pseudomonas fluorescens N1B4 was selected for mutant fitness profiling; with this approach four genes of importance were identified and the effects on interactions were evaluated with deletion mutants and metabolomics. IMPORTANCEThe Biofilm Interaction Mapping and Analysis (BIMA) methodology provides a way to rapidly screen for positive and negative interspecific interactions, followed by an analysis of the genes and metabolites that may be involved. Knowledge of these may offer opportunities for engineered strains with improved function in biotechnology systems. P. stutzeri, an organism with wide-spread utilization in consortia based biotechnologies, was used to demonstrate the utility of this approach. Where little is known about the factors influencing biofilm based interactions, elucidation of the genes and metabolites involved allows for better control of the system for improved function or yield.

microbiology↗

bioRxiv2021

A defined medium based on R2A for cultivation and exometabolite profiling of soil bacteria

Exometabolomics is an approach to assess how microorganisms alter their environments through the depletion and secretion of chemical compounds. Comparisons of inoculated with uninoculated media can be used to provide direct biochemical observations on depleted and secreted metabolites which can be used to predict resource competition, cross-feeding and secondary metabolite production in microbial isolates and communities. This approach is most powerful when used with defined media that enable tracking of all depleted metabolites. However, microbial growth media have traditionally been developed for the isolation and growth of microorganisms but not metabolite utilization profiling through LC-MS/MS. Here, we describe the construction of a defined medium, the Northen Lab Defined Medium (NLDM), that not only supports the growth of diverse bacteria but is defined and therefore suited for exometabolomic experiments. Metabolites included in NLDM were selected based on their presence in R2A medium and soil, elemental stoichiometry requirements, as well as knowledge of metabolite usage by different bacteria. We found that NLDM supported the growth of 53 phylogenetically diverse soil bacterial isolates and all of its metabolites were trackable through LC-MS/MS analysis. These results demonstrate the viability and utility of the constructed NLDM medium for cultivating and characterizing diverse microbial isolates and communities. Originality-Significance StatementWe build a defined medium based on the metabolite composition of R2A medium and soil, elemental stoichiometry requirements, and knowledge of metabolite usage by different bacteria. The newly formulated defined medium was evaluated on its ability to support the growth of soil isolates and its application for metabolite utilization profiling. We found that of 53 phylogenetically diverse soil bacterial isolates grew on the defined medium and all of its metabolites were trackable through LC-MS/MS analysis. This demonstrates the viability and utility of the constructed defined medium for cultivating and characterizing diverse microbial isolates and communities.

microbiology↗

bioRxiv2021

Conservation of isolate substrate preferences in mixed communities revealed through ribosomal marker protein profiling

Assessment of structure-function relationships is a central theme in microbial ecology. However, the degree that isolate metabolic activities are conserved in communities remains unclear. This is because tracking population dynamics and substrate partitioning in microbial communities remains technically challenging. Here, we describe the application of a mass spectrometry-based ribosomal marker protein profiling with stable isotope probing approach that allows for concurrent monitoring of community structure dynamics and resource assimilation within a five-member synthetic soil bacterial community. Using this approach, we find that isolate substrate preferences for glutamine and phenylalanine are largely conserved in the community and can be predicted using a weighted-sum model. However, time-series monitoring revealed a significant delay in phenylalanine incorporation by two of the strains, as well as enhanced growth for Variovorax paradoxus presumably due to interspecies interactions. The unique utility of this approach to temporally probe resource incorporation and community structure enables deciphering the dynamic interactions occurring within the community. Extension of this approach to other communities under various environmental perturbations is needed to reveal the generality of microbial conservation of substrate preferences.

microbiology↗

bioRxiv2021

Measurement of Volatile Compounds for Real-time Analysis of Soil Microbial Metabolic Response to Simulated Snowmelt

Snowmelt dynamics are a significant determinant of microbial metabolism in soil and regulate global biogeochemical cycles of carbon and nutrients by creating seasonal variations in soil redox and nutrient pools. With an increasing concern that climate change accelerates both snowmelt timing and rate, obtaining an accurate characterization of microbial response to snowmelt is important for understanding biogeochemical cycles intertwined with soil. However, observing microbial metabolism and its dynamics non-destructively remains a major challenge for systems such as soil. Microbial volatile compounds (mVCs) emitted from soil represent information-dense signatures and when assayed non-destructively using state-of-the-art instrumentation such as Proton Transfer Reaction-Time of Flight-Mass Spectrometry (PTR-TOF-MS) provide time resolved insights into the metabolism of active microbiomes. In this study, we used PTR-TOF-MS to investigate the metabolic trajectory of microbiomes from a subalpine forest soil, and their response to a simulated wet-up event akin to snowmelt. Using an information theory approach based on the partitioning of mutual information, we identified mVC metabolite pairs with robust interactions, including those that were non-linear and with time lags. The biological context for these mVC interactions was evaluated by projecting the connections onto the Kyoto Encyclopedia of Genes and Genomes (KEGG) network of known metabolic pathways. Simulated snowmelt resulted in a rapid increase in the production of trimethylamine (TMA) suggesting that anaerobic degradation of quaternary amine osmo/cryoprotectants, such as glycine betaine, may be important contributors to this resource pulse. Unique and synergistic connections between intermediates of methylotrophic pathways such as dimethylamine, formaldehyde and methanol were observed upon wet-up and indicate that the initial pulse of TMA was likely transformed into these intermediates by methylotrophs. Increases in ammonia oxidation signatures (transformation of hydroxylamine to nitrite) were observed in parallel, and while the relative role of nitrifiers or methylotrophs cannot be confirmed, the inferred connection to TMA oxidation suggests either a direct or indirect coupling between these processes. Overall, it appears that such mVC time-series from PTR-TOF-MS combined with causal inference represents an attractive approach to non-destructively observe soil microbial metabolism and its response to environmental perturbation.

microbiology↗

bioRxiv2021

Identification and characterization of novel plasma proteins in drug resistant HIV/AIDS patients by SWATH-MS

Background and ObjectivesAcquired immunodeficiency syndrome is one of the most important diseases caused by human immunodeficiency virus. Understanding its molecular pathogenesis is essential to manage the disease at the population level. In this study, a quantitative analysis of plasma proteins was carried out in drug resistant and drug respondent patients using the SWATH-MS. MethodsSequential window acquisition of all theoretical mass spectra (SWATH-MS) is a prime technique to seek the key plasma proteins involved in virus replication and drug metabolism during therapy. ResultsIn total, 204 proteins were identified and quantified, 57 proteins were differentially expressed, 25 proteins were down regulated and 32 proteins were upregulated in drug resistant patients. Six proteins such as complement C4-A, immunoglobulin heavy variable 1-2, carboxylic ester hydrolase, fibulin-1, immunoglobulin lambda constant 7, secreted phosphoprotein 24 were statistically expressed in drug resistant patients compared to the drug respondent patients. Gene ontology study and protein-protein interaction networks were established in six statistically significant differentially expressed proteins of the drug resistant patients. Interpretation and ConclusionOur findings high lights the novel proteins that were differentially expressed in drug resistant patients. A label-free quantitative proteomics method for depleted human plasma samples by SWATH-MS that can be useful in plasma proteomics research in any biological system.

biochemistry↗

bioRxiv2020

Lipid Droplets Promote Phase Separation of Ago2 to Accelerate Dicer1 Loss and Decelerate miRNA Activity in Lipid Exposed Hepatic Cells

miR-122 is a liver specific miRNA that plays an important role in controlling metabolic homeostasis in mammalian liver cells. Interestingly, miR-122 on exposure to lipotoxic stress is reduced in liver cells. To fight stress, miRNA processor Dicer1 is depleted to cause reduced miR-122 production and the lowering of miRNA level ensures a better stress response in hepatocytes under lipotoxic stress. Interestingly, lipid droplets, formed in the liver cells on exposure to high fat, ensure cytoplasmic phase separation of Ago2 and prevent interaction of Ago2 with Dicer1. Lipid droplets bind miRNA and enhance miRNA-Ago2 uncoupling and Ago2 phase separation. Loss of interaction between Ago2 and Dicer1 eventually facilitates export and lowering of cellular Dicer1, a process also dependent on the endosomal maturation controller protein Alix, thereby ceasing pre-miRNA processing by Dicer1 in lipid exposed cells. Depletion of lipid droplets by downregulation of Perilipins with siRNAs resulted in a rescue of cellular Dicer1 level and Ago2-Dicer1 interaction. This is a novel mechanism that liver cells adopt to restrict cellular miRNA levels under stress condition. Thus, lipid droplets prevent cell death upon exposure to high fat by reducing intra and extracellular pool of miR-122 in hepatic tissue.

cell biology↗

bioRxiv2020

No change in oxytocin level before a human intergroup competition: study among Polish players before rugby and handball matches

ObjectivesThe aim of the present study was to assess the changes in urinary oxytocin concentration during the period between five days before, and on the day of match, among rugby and handball players. MethodsThe study used a repeated measures design with the relative oxytocin level as the outcome variable measured at two subsequent points of time, viz., on five days before as well as on the days of matches. Nine male rugby players with a mean age of 27.62 years (SD = 4.21) and 18 male handball players with a mean age of 17.03 years (SD = 0.57) participated. Urinary oxytocin level was measured by ELISA immunoassay as a ratio to the concentration of creatinine [mg/ml] measured through colorimetric detection. Differences in oxytocin level were assessed by ANOVA with repeated measurements. ResultsThe OT/CRE levels significantly differed between the type of player (rugby or handball) but not between times of measurements. Tukeys post-hoc tests revealed that significant differences were only between OT/CRE level in a day of match in rugby players and in 5 days before match in handball players (p<0.05). ConclusionThere was no change in oxytocin levels during the time periods between five days before and on the day of a match, in either of the two kinds of players. The change in oxytocin might be traceable during the match but not before a match and thus perhaps depends on a more subtle context of competition, but not on the assumption of competition.

evolutionary biology↗

bioRxiv2020

Sedimentation rate and organic matter dynamics shape microbiomes across a continental margin

Marine sedimentation rate and bottom-water O2 concentration control the remineralization/sequestration of organic carbon across continental margins; but whether/how they shape microbiome architecture (the ultimate effector of all biogeochemical phenomena), across shelf/slope sediments, is unknown. Here we reveal distinct microbiome structures and functions, amidst comparable pore-fluid chemistries, along ~3 m sediment-horizons underlying the seasonal (shallow coastal) and perennial (deep sea) oxygen minimum zones (OMZs) of the Arabian Sea, situated across the western-Indian margin (water-depths: 31 m and, 530 and 580 m, respectively). Along the perennial- and seasonal-OMZ sediment-cores microbial communities were predominated by Gammaproteobacteria/Alphaproteobacteria and Euryarchaeota/Firmicutes respectively. As a perennial-OMZ signature, a cryptic methane production-consumption cycle was found to operate near the sediment-surface; overall diversity, as well as the relative abundances of simple-fatty-acids-requiring anaerobes (methanogens, anaerobic methane-oxidizers, sulfate-reducers and acetogens), peaked in the topmost sediment-layer and then declined via synchronized fluctuations until the sulfate-methane transition zone was reached. The entire microbiome profile was reverse in the seasonal-OMZ sediment-horizon. We discerned that in the perennial-OMZ sediments organic carbon deposited was higher in concentration, and marine components-rich, so it potentially degraded readily to simple fatty acids; lower sedimentation rate afforded higher O2 exposure time for organic matter degradation despite perennial hypoxia in the bottom-water; thus, the resultant abundance of reduced metabolites sustained multiple inter-competing microbial processes in the upper sediment-layers. Remarkably, the whole geomicrobial scenario was opposite in the sediments of the seasonal/shallow-water OMZ. Our findings create a microbiological baseline for understanding carbon-sulfur cycling across distinct marine depositional settings and water-colum n oxygenation regimes.

microbiology↗

bioRxiv2020

Ecogenomics of groundwater viruses suggests niche differentiation linked to specific environmental tolerance

Viruses are ubiquitous microbiome components, shaping ecosystems via strain-specific predation, horizontal gene transfer and redistribution of nutrients through host lysis. Viral impacts are important in groundwater ecosystems, where microbes drive many nutrient fluxes and metabolic processes, however little is known about the diversity of viruses in these environments. We analyzed four groundwater plasmidomes and identified 200 viral sequences, which clustered into 41 ~ genus-level viral clusters (equivalent to viral genera) including 9 known and 32 putative new genera. We use publicly available bacterial whole genome sequences (WGS) and WGS from 261 bacterial isolates from this groundwater environment to identify potential viral hosts. We linked 76 of the 200 viral sequences to a range of bacterial phyla, the majority associated with Proteobacteria, followed by Firmicutes, Bacteroidetes and Actinobacteria. The publicly available microbial genome sequences enabled mapping bacterial hosts to a breadth of viral sequences. The WGS of groundwater isolates increased depth of host prediction by allowing identification of hosts at the strain level. The latter included 4 viruses that were almost entirely (>99% query coverage, >99% identity) identified as integrated in the genomes of specific Pseudomonas, Acidovorax and Castellaniella strains, resulting in very high-confidence host assignments. Lastly, 21 of these viruses encoded putative auxiliary metabolite genes for metal and antibiotic resistance, which might drive their infection cycles and/or provide selective advantage to infected hosts. Exploring the groundwater virome provides a necessary foundation for integration of viruses into ecosystem models where they act as key players in microbial adaption to environmental stress. ImportanceTo our knowledge, this is the first study to identify the bacteriophage distribution in a groundwater ecosystem shedding light on their prevalence and distribution across metal-contaminated and background sites. Our study is uniquely based on selective sequencing of solely the extrachromosomal elements of a microbiome followed by analysis for viral signatures, thus establishing a more focused approach for phage identifications. Using this method, we detect several novel phage genera along with those previously established. Our approach of using the whole genome sequences of hundreds of bacterial isolates from the same site enabled us to make host assignments with high confidence, several at strain levels. Certain phage-encoded genes suggest they provide an environment-specific selective advantage to their bacterial hosts. Our study lays the foundation for future research on directed phage isolations using specific bacterial host strains to further characterize groundwater phages, their lifecycles, and its effects on groundwater microbiome and biogeochemistry.

microbiology↗

bioRxiv2020

Systematic Discovery of Pseudomonad Genetic Factors Involved in Sensitivity to Tailocins

Tailocins are bactericidal protein complexes produced by a wide variety of bacteria to compete against closely related strains. Like tailed bacteriophages, with whom they share an evolutionary and morphological relationship, tailocins bind and kill a narrow spectrum of target cells. Thanks to their high specificity, tailocins have garnered recent attention for their potential as precision antibacterial agents. Nevertheless, the field currently lacks a systematic investigation of genetic determinants of tailocin sensitivity. Here, we employed barcoded transposon-insertion mutant libraries and comparative genomics to assess genetic contributions to tailocin sensitivity in pseudomonads. Our mutant screens identified O-specific antigen (OSA) composition and display as most important in defining sensitivity to our tailocins. Additionally, the screens suggest lipopolysaccharide (LPS) thinning as a mechanism by which resistant strains can become more sensitive to tailocins. Our comparative genomics analyses show a loose relationship between OSA biosynthetic genes and tailocin sensitivity, as well as sensitivity nuances that require further investigation. Overall, our data reinforces the model that LPS molecules can act as either a receptor for, or shield against, tailocin binding and killing. This work offers insight into the specificity of tailocins and tailocin-mediated competition, informing the potential use of tailocins in microbiome manipulation and antibacterial therapy.

microbiology↗

bioRxiv2020

GroEL/ES Buffers Entropic Traps In Folding Pathway During Evolution Of A Model Substrate

The folding landscape of proteins can change during evolution with the accumulation of mutations that may introduce entropic or enthalpic barriers in the protein folding pathway, making it a possible substrate of molecular chaperones in vivo. Can the nature of such physical barriers of folding dictate the feasibility of chaperone-assistance? To address this, we have simulated the evolutionary step to chaperone-dependence keeping GroEL/ES as the target chaperone and GFP as a model protein in an unbiased screen. We find that the mutation conferring GroEL/ES dependence in vivo and in vitro encode an entropic trap in the folding pathway rescued by the chaperonin. Additionally, GroEL/ES can edit the formation of non-native contacts similar to DnaK/J/E machinery. However, this capability is not utilized by the substrates in vivo. As a consequence, GroEL/ES caters to buffer mutations that predominantly cause entropic traps, despite possessing the capacity to edit both enthalpic and entropic traps in the folding pathway of the substrate protein.

molecular biology↗