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871400.9929Tales from the tomb: the microbial ecology of exposed rock surfaces. Although a broad diversity of eukaryotic and bacterial taxa reside on rock surfaces where they can influence the weathering of rocks and minerals, these communities and their contributions to mineral weathering remain poorly resolved. To build a more comprehensive understanding of the diversity, ecology and potential functional attributes of microbial communities living on rock, we sampled 149 tombstones across three continents and analysed their bacterial and eukaryotic communities via marker gene and shotgun metagenomic sequencing. We found that geographic location and climate were important factors structuring the composition of these communities. Moreover, the tombstone-associated microbial communities varied as a function of rock type, with granite and limestone tombstones from the same cemeteries harbouring taxonomically distinct microbial communities. The granite and limestone-associated communities also had distinct functional attributes, with granite-associated bacteria having more genes linked to acid tolerance and chemotaxis, while bacteria on limestone were more likely to be lichen associated and have genes involved in photosynthesis and radiation resistance. Together these results indicate that rock-dwelling microbes exhibit adaptations to survive the stresses of the rock surface, differ based on location, climate and rock type, and seem pre-disposed to different ecological strategies (symbiotic versus free-living lifestyles) depending on the rock type.201829235707
866710.9916Glacier-Fed Stream Biofilms Harbor Diverse Resistomes and Biosynthetic Gene Clusters. Antimicrobial resistance (AMR) is a universal phenomenon the origins of which lay in natural ecological interactions such as competition within niches, within and between micro- to higher-order organisms. To study these phenomena, it is crucial to examine the origins of AMR in pristine environments, i.e., limited anthropogenic influences. In this context, epilithic biofilms residing in glacier-fed streams (GFSs) are an excellent model system to study diverse, intra- and inter-domain, ecological crosstalk. We assessed the resistomes of epilithic biofilms from GFSs across the Southern Alps (New Zealand) and the Caucasus (Russia) and observed that both bacteria and eukaryotes encoded twenty-nine distinct AMR categories. Of these, beta-lactam, aminoglycoside, and multidrug resistance were both abundant and taxonomically distributed in most of the bacterial and eukaryotic phyla. AMR-encoding phyla included Bacteroidota and Proteobacteria among the bacteria, alongside Ochrophyta (algae) among the eukaryotes. Additionally, biosynthetic gene clusters (BGCs) involved in the production of antibacterial compounds were identified across all phyla in the epilithic biofilms. Furthermore, we found that several bacterial genera (Flavobacterium, Polaromonas, Superphylum Patescibacteria) encode both atimicrobial resistance genes (ARGs) and BGCs within close proximity of each other, demonstrating their capacity to simultaneously influence and compete within the microbial community. Our findings help unravel how naturally occurring BGCs and AMR contribute to the epilithic biofilms mode of life in GFSs. Additionally, we report that eukaryotes may serve as AMR reservoirs owing to their potential for encoding ARGs. Importantly, these observations may be generalizable and potentially extended to other environments that may be more or less impacted by human activity. IMPORTANCE Antimicrobial resistance is an omnipresent phenomenon in the anthropogenically influenced ecosystems. However, its role in shaping microbial community dynamics in pristine environments is relatively unknown. Using metagenomics, we report the presence of antimicrobial resistance genes and their associated pathways in epilithic biofilms within glacier-fed streams. Importantly, we observe biosynthetic gene clusters associated with antimicrobial resistance in both pro- and eukaryotes in these biofilms. Understanding the role of resistance in the context of this pristine environment and complex biodiversity may shed light on previously uncharacterized mechanisms of cross-domain interactions.202336688698
936120.9916Evolutionary consequences of bacterial resistance to a flagellotropic phage. Bacteria often rapidly evolve resistance to bacteriophages (phages) by mutating or suppressing the phage-receptors, the factors that phages first target to initiate infection. Flagellotropic phages infect bacteria by initially binding to the flagellum. Since motility is an important fitness factor that allows bacteria to efficiently explore their environment, losing flagellar function to evade infection by flagellotropic phages represents a crucial trade-off. In this study, we investigated the evolutionary responses of Escherichia coli when exposed to the flagellotropic phage χ. Using an experimental evolution approach, E. coli cells were repeatedly subjected to environments rich in phage χ but selective for motility. Unlike traditional well-mixed cultures, we employed swim-plate assays to simulate spatial confinement and promote motility. Whole genome sequencing of evolved populations revealed early emergence of non-motile, χ-resistant mutants with mutations disrupting motility-related genes. Motile mutants emerged in later passages, possessing mutations in the flagellin gene fliC. Swim-plate assays showed a diverse range of motility among these mutants, with some displaying slower, and others faster, expansion speeds compared to the ancestral strain. Single-cell tracking experiments indicated an increased tumble bias in χ-resistant mutants, suggesting an adaptive response involving altered flagellar rotation. Our findings demonstrate that motility can undergo trade-offs and trade-ups with phage resistance, shedding light on the complex evolutionary dynamics between motile bacteria and flagellotropic phages.202540654869
825930.9915Secondary Metabolite Transcriptomic Pipeline (SeMa-Trap), an expression-based exploration tool for increased secondary metabolite production in bacteria. For decades, natural products have been used as a primary resource in drug discovery pipelines to find new antibiotics, which are mainly produced as secondary metabolites by bacteria. The biosynthesis of these compounds is encoded in co-localized genes termed biosynthetic gene clusters (BGCs). However, BGCs are often not expressed under laboratory conditions. Several genetic manipulation strategies have been developed in order to activate or overexpress silent BGCs. Significant increases in production levels of secondary metabolites were indeed achieved by modifying the expression of genes encoding regulators and transporters, as well as genes involved in resistance or precursor biosynthesis. However, the abundance of genes encoding such functions within bacterial genomes requires prioritization of the most promising ones for genetic manipulation strategies. Here, we introduce the 'Secondary Metabolite Transcriptomic Pipeline' (SeMa-Trap), a user-friendly web-server, available at https://sema-trap.ziemertlab.com. SeMa-Trap facilitates RNA-Seq based transcriptome analyses, finds co-expression patterns between certain genes and BGCs of interest, and helps optimize the design of comparative transcriptomic analyses. Finally, SeMa-Trap provides interactive result pages for each BGC, allowing the easy exploration and comparison of expression patterns. In summary, SeMa-Trap allows a straightforward prioritization of genes that could be targeted via genetic engineering approaches to (over)express BGCs of interest.202235580059
867140.9915Adapting to UV: Integrative Genomic and Structural Analysis in Bacteria from Chilean Extreme Environments. Extremophilic bacteria from extreme environments, such as the Atacama Desert, Salar de Huasco, and Antarctica, exhibit adaptations to intense UV radiation. In this study, we investigated the genomic and structural mechanisms underlying UV resistance in three bacterial isolates identified as Bacillus velezensis PQ169, Pseudoalteromonas sp. AMH3-8, and Rugamonas violacea T1-13. Through integrative genomic analyses, we identified key genes involved in DNA-repair systems, pigment production, and spore formation. Phylogenetic analyses of aminoacidic sequences of the nucleotide excision repair (NER) system revealed conserved evolutionary patterns, indicating their essential role across diverse bacterial taxa. Structural modeling of photolyases from Pseudoalteromonas sp. AMH3-8 and R. violacea T1-13 provided further insights into protein function and interactions critical for DNA repair and UV resistance. Additionally, the presence of a complete violacein operon in R. violacea T1-13 underscores pigment biosynthesis as a crucial protective mechanism. In B. velezensis PQ169, we identified the complete set of genes responsible for sporulation, suggesting that sporulation may represent a key protective strategy employed by this bacterium in response to environmental stress. Our comprehensive approach underscores the complexity and diversity of microbial adaptations to UV stress, offering potential biotechnological applications and advancing our understanding of microbial resilience in extreme conditions.202540565314
866650.9915Decoding the genetic drivers of marine bacterial blooms through comparative genomics. BACKGROUND: While oligotrophic bacteria are known to dominate most marine microbial habitats, under certain conditions, such as during phytoplankton blooms, copiotrophs can dramatically increase in abundance and reach towering proportions of the bacterial communities. We are uncertain whether the bacteria exhibiting this capacity, which we denote as "bloomers," have specific functional characteristics or if, instead, they are randomly selected from the broader pool of copiotrophs. To explore the genomic determinants of this ecological trait, we conducted a comparative genomic analysis of bacterial genomes from microcosm experiments where grazer and viral presence was reduced and nutrient availability was increased, conditions that triggered bacterial blooms. RESULTS: We tested which functional genes were overrepresented in the bacteria that responded to the treatments, examining a total of 305 genomes from isolates and metagenome-assembled genomes (MAGs) that were categorized as copiotrophs or oligotrophs according to their codon usage bias (CUB). The responsive bacteria were enriched in genes related to transcriptional regulation in response to stimuli (mostly via two-component systems), transport, secretion, cell protection, catabolism of sugars and amino acids, and membrane/cell wall biosynthesis. These genes confer on them capabilities for adhesion, biofilm formation, resistance to stress, quorum sensing, chemotaxis, nutrient uptake, and fast replication. They were overrepresented mainly in copiotrophic genomes from the families Alteromonadaceae, Vibrionaceae, Rhodobacteraceae, Sphingomonadaceae, and Flavobacteriaceae. Additionally, we found that these responsive bacteria, when abundant, could affect biogeochemical cycling, particularly the phosphorus cycle. CONCLUSIONS: In this study, we provide insights into the functional characteristics that enable certain bacteria to rapidly respond to changes in the environment and bloom. We also hint at the ecological meaning and implications of these phenomena that could affect biogeochemical cycles in the oceans. Video Abstract.202541029845
47460.9914Novel antibiotic resistance profiles in bacteria isolated from oil fly larvae Helaeomyia petrolei living in the La Brea Tar Pits. Larvae from the petroleum oil fly, Helaeomyia petrolei, live in the asphaltene and polyaromatic hydrocarbon rich asphalt seeps of Rancho La Brea, Los Angeles, California. These larvae pass high amounts of viscous asphalt through their digestive system, and their gut microbiota is exposed to these extreme conditions. Environmental stress response mechanisms can co-select for antibiotic resistance, and in the current study we used 16S rRNA and genomic sequencing along with the Comprehensive Antibiotic Resistance Database (CARD) tools to characterize antibiotic resistance profiles from six bacteria previously isolated from the oil fly larval intestinal tract, linking phenotypic and genotypic resistance profiles. The isolates contain a core set of antibiotic resistance determinants along with determinants that are rarely found in these species. Comparing these oil fly isolates to the phenotypic prevalence data generated by the CARD Resistance Gene Identifier revealed sixteen instances where the oil fly bacteria appeared to carry a resistance not seen in related taxa in the database, suggesting a novel suite of resistance families in the oil fly isolates compared to other members of the same taxa. Results highlight the functional duality of genes that simultaneously code for antibiotic resistance and survival under extreme conditions, and expand our understanding of the ecological and evolutionary role of antibiotic resistance genes in environmental habitats.202439718641
823970.9914Surviving bacterial sibling rivalry: inducible and reversible phenotypic switching in Paenibacillus dendritiformis. Natural habitats vary in available nutrients and room for bacteria to grow, but successful colonization can lead to overcrowding and stress. Here we show that competing sibling colonies of Paenibacillus dendritiformis bacteria survive overcrowding by switching between two distinct vegetative phenotypes, motile rods and immotile cocci. Growing colonies of the rod-shaped bacteria produce a toxic protein, Slf, which kills cells of encroaching sibling colonies. However, sublethal concentrations of Slf induce some of the rods to switch to Slf-resistant cocci, which have distinct metabolic and resistance profiles, including resistance to cell wall antibiotics. Unlike dormant spores of P. dendritiformis, the cocci replicate. If cocci encounter conditions that favor rods, they secrete a signaling molecule that induces a switch to rods. Thus, in contrast to persister cells, P. dendritiformis bacteria adapt to changing environmental conditions by inducible and reversible phenotypic switching. IMPORTANCE: In favorable environments, species may face space and nutrient limits due to overcrowding. Bacteria provide an excellent model for analyzing principles underlying overcrowding and regulation of density in nature, since their population dynamics can be easily and accurately assessed under controlled conditions. We describe a newly discovered mechanism for survival of a bacterial population during overcrowding. When competing with sibling colonies, Paenibacillus dendritiformis produces a lethal protein (Slf) that kills cells at the interface of encroaching colonies. Slf also induces a small proportion of the cells to switch from motile, rod-shaped cells to nonmotile, Slf-resistant, vegetative cocci. When crowding is reduced and nutrients are no longer limiting, the bacteria produce a signal that induces cocci to switch back to motile rods, allowing the population to spread. Genes encoding components of this phenotypic switching pathway are widespread among bacterial species, suggesting that this survival mechanism is not unique to P. dendritiformis.201121628502
865780.9914The Phytoplankton Taxon-Dependent Oil Response and Its Microbiome: Correlation but Not Causation. Phytoplankton strongly interact with their associated bacteria, both attached (PA), and free-living (FL), and bacterial community structures can be specific to phytoplankton species. Similarly, responses to environmental stressors can vary by taxon, as exemplified by observed shifts in phytoplankton community structure from diatoms to phytoflagellates after the Deepwater Horizon (DWH) oil spill. Here, we assess the extent to which associated bacteria influence the phytoplankton taxon-specific oil response by exposing xenic and axenic strains of three phytoplankton species to oil and/or dispersant. The dinoflagellates Amphidinium carterae and Peridinium sociale, and the diatom Skeletonema sp., all harbored significantly distinct bacterial communities that reflected their host oil response. Oil degrading bacteria were detected in both PA and FL communities of the oil resistant dinoflagellates, but their FL bacteria were more efficient in lipid hydrolysis, a proxy for oil degradation capability. Inversely, the growth rate and photosynthetic parameters of the diatom Skeletonema sp. was the most impacted by dispersed oil compared to the dinoflagellates, and oil-degrading bacteria were not significantly associated to its microbiome, even in the dispersed oil treatment. Moreover, the FL bacteria of Skeletonema did not show significant oil degradation. Yet, the lack of consistent significant differences in growth or photosynthetic parameters between the xenic and axenic cultures after oil exposure suggest that, physiologically, the associated bacteria do not modify the phytoplankton oil response. Instead, both oil resistance and phycosphere composition appear to be species-specific characteristics that are not causally linked. This study explores one aspect of what is undoubtedly a complex suite of interactions between phytoplankton and their associated bacteria; future analyses would benefit from studies of genes and metabolites that mediate algal-bacterial exchanges.201930915045
841690.9913Protective role of the vulture facial skin and gut microbiomes aid adaptation to scavenging. BACKGROUND: Vultures have adapted the remarkable ability to feed on carcasses that may contain microorganisms that would be pathogenic to most other animals. The holobiont concept suggests that the genetic basis of such adaptation may not only lie within their genomes, but additionally in their associated microbes. To explore this, we generated shotgun DNA sequencing datasets of the facial skin and large intestine microbiomes of the black vulture (Coragyps atratus) and the turkey vulture (Cathartes aura). We characterized the functional potential and taxonomic diversity of their microbiomes, the potential pathogenic challenges confronted by vultures, and the microbial taxa and genes that could play a protective role on the facial skin and in the gut. RESULTS: We found microbial taxa and genes involved in diseases, such as dermatitis and pneumonia (more abundant on the facial skin), and gas gangrene and food poisoning (more abundant in the gut). Interestingly, we found taxa and functions with potential for playing beneficial roles, such as antilisterial bacteria in the gut, and genes for the production of antiparasitics and insecticides on the facial skin. Based on the identified phages, we suggest that phages aid in the control and possibly elimination, as in phage therapy, of microbes reported as pathogenic to a variety of species. Interestingly, we identified Adineta vaga in the gut, an invertebrate that feeds on dead bacteria and protozoans, suggesting a defensive predatory mechanism. Finally, we suggest a colonization resistance role through biofilm formation played by Fusobacteria and Clostridia in the gut. CONCLUSIONS: Our results highlight the importance of complementing genomic analyses with metagenomics in order to obtain a clearer understanding of the host-microbial alliance and show the importance of microbiome-mediated health protection for adaptation to extreme diets, such as scavenging.201830309375
8669100.9913The ins and outs of metal homeostasis by the root nodule actinobacterium Frankia. BACKGROUND: Frankia are actinobacteria that form a symbiotic nitrogen-fixing association with actinorhizal plants, and play a significant role in actinorhizal plant colonization of metal contaminated areas. Many Frankia strains are known to be resistant to several toxic metals and metalloids including Pb(2+), Al(+3), SeO2, Cu(2+), AsO4, and Zn(2+). With the availability of eight Frankia genome databases, comparative genomics approaches employing phylogeny, amino acid composition analysis, and synteny were used to identify metal homeostasis mechanisms in eight Frankia strains. Characterized genes from the literature and a meta-analysis of 18 heavy metal gene microarray studies were used for comparison. RESULTS: Unlike most bacteria, Frankia utilize all of the essential trace elements (Ni, Co, Cu, Se, Mo, B, Zn, Fe, and Mn) and have a comparatively high percentage of metalloproteins, particularly in the more metal resistant strains. Cation diffusion facilitators, being one of the few known metal resistance mechanisms found in the Frankia genomes, were strong candidates for general divalent metal resistance in all of the Frankia strains. Gene duplication and amino acid substitutions that enhanced the metal affinity of CopA and CopCD proteins may be responsible for the copper resistance found in some Frankia strains. CopA and a new potential metal transporter, DUF347, may be involved in the particularly high lead tolerance in Frankia. Selenite resistance involved an alternate sulfur importer (CysPUWA) that prevents sulfur starvation, and reductases to produce elemental selenium. The pattern of arsenate, but not arsenite, resistance was achieved by Frankia using the novel arsenite exporter (AqpS) previously identified in the nitrogen-fixing plant symbiont Sinorhizobium meliloti. Based on the presence of multiple tellurite resistance factors, a new metal resistance (tellurite) was identified and confirmed in Frankia. CONCLUSIONS: Each strain had a unique combination of metal import, binding, modification, and export genes that explain differences in patterns of metal resistance between strains. Frankia has achieved similar levels of metal and metalloid resistance as bacteria from highly metal-contaminated sites. From a bioremediation standpoint, it is important to understand mechanisms that allow the endosymbiont to survive and infect actinorhizal plants in metal contaminated soils.201425495525
8647110.9913Eco-evolutionary strategies for relieving carbon limitation under salt stress differ across microbial clades. With the continuous expansion of saline soils under climate change, understanding the eco-evolutionary tradeoff between the microbial mitigation of carbon limitation and the maintenance of functional traits in saline soils represents a significant knowledge gap in predicting future soil health and ecological function. Through shotgun metagenomic sequencing of coastal soils along a salinity gradient, we show contrasting eco-evolutionary directions of soil bacteria and archaea that manifest in changes to genome size and the functional potential of the soil microbiome. In salt environments with high carbon requirements, bacteria exhibit reduced genome sizes associated with a depletion of metabolic genes, while archaea display larger genomes and enrichment of salt-resistance, metabolic, and carbon-acquisition genes. This suggests that bacteria conserve energy through genome streamlining when facing salt stress, while archaea invest in carbon-acquisition pathways to broaden their resource usage. These findings suggest divergent directions in eco-evolutionary adaptations to soil saline stress amongst microbial clades and serve as a foundation for understanding the response of soil microbiomes to escalating climate change.202439019914
8300120.9912The Copper Resistome of Group B Streptococcus Reveals Insight into the Genetic Basis of Cellular Survival during Metal Ion Stress. In bacteria, copper (Cu) can support metabolic processes as an enzymatic cofactor but can also cause cell damage if present in excess, leading to intoxication. In group B Streptococcus (GBS), a system for control of Cu efflux based on the prototypical cop operon supports survival during Cu stress. In some other bacteria, genetic systems additional to the cop operon are engaged during Cu stress and also contribute to the management of cellular Cu homeostasis. Here, we examined genetic systems beyond the cop operon in GBS for regions that contribute to survival of GBS in Cu stress using a forward genetic screen and probe of the entire bacterial genome. A high-density mutant library, generated using pGh9-ISS1, was used to expose GBS to Cu stress and compare it to nonexposed controls en masse. Eight genes were identified as essential for GBS survival in Cu stress, whereas five genes constrained GBS growth in Cu stress. The genes encode varied factors including enzymes for metabolism, cell wall synthesis, transporters, and cell signaling factors. Targeted mutation of the genes validated their roles in GBS resistance to Cu stress. Excepting copA, the genes identified are new to the area of bacterial metal ion intoxication. We conclude that a discrete and limited suite of genes beyond the cop operon in GBS contributes to a repertoire of mechanisms used to survive Cu stress in vitro and achieve cellular homeostasis. IMPORTANCE Genetic systems for copper (Cu) homeostasis in bacteria, including streptococci, are vital to survive metal ion stress. Genetic systems that underpin survival of GBS during Cu stress, beyond the archetypal cop operon for Cu management, are undefined. We show that Streptococcus resists Cu intoxication by utilizing a discrete and limited suite of genes beyond the cop operon, including several genes that are new to the area of bacterial cell metal ion homeostasis. The Cu resistome of GBS defined here enhances our understanding of metal ion homeostasis in GBS.202235404113
7691130.9912Antimicrobial Chemicals Associate with Microbial Function and Antibiotic Resistance Indoors. Humans purposefully and inadvertently introduce antimicrobial chemicals into buildings, resulting in widespread compounds, including triclosan, triclocarban, and parabens, in indoor dust. Meanwhile, drug-resistant infections continue to increase, raising concerns that buildings function as reservoirs of, or even select for, resistant microorganisms. Support for these hypotheses is limited largely since data describing relationships between antimicrobials and indoor microbial communities are scant. We combined liquid chromatography-isotope dilution tandem mass spectrometry with metagenomic shotgun sequencing of dust collected from athletic facilities to characterize relationships between indoor antimicrobial chemicals and microbial communities. Elevated levels of triclosan and triclocarban, but not parabens, were associated with distinct indoor microbiomes. Dust of high triclosan content contained increased Gram-positive species with diverse drug resistance capabilities, whose pangenomes were enriched for genes encoding osmotic stress responses, efflux pump regulation, lipid metabolism, and material transport across cell membranes; such triclosan-associated functional shifts have been documented in laboratory cultures but not yet from buildings. Antibiotic-resistant bacterial isolates were cultured from all but one facility, and resistance often increased in buildings with very high triclosan levels, suggesting links between human encounters with viable drug-resistant bacteria and local biocide conditions. This characterization uncovers complex relationships between antimicrobials and indoor microbiomes: some chemicals elicit effects, whereas others may not, and no single functional or resistance factor explained chemical-microbe associations. These results suggest that anthropogenic chemicals impact microbial systems in or around buildings and their occupants, highlighting an emergent need to identify the most important indoor, outdoor, and host-associated sources of antimicrobial chemical-resistome interactions. IMPORTANCE The ubiquitous use of antimicrobial chemicals may have undesired consequences, particularly on microbes in buildings. This study shows that the taxonomy and function of microbes in indoor dust are strongly associated with antimicrobial chemicals-more so than any other feature of the buildings. Moreover, we identified links between antimicrobial chemical concentrations in dust and culturable bacteria that are cross-resistant to three clinically relevant antibiotics. These findings suggest that humans may be influencing the microbial species and genes that are found indoors through the addition and removal of particular antimicrobial chemicals.201830574558
308140.9912Linearmycins Activate a Two-Component Signaling System Involved in Bacterial Competition and Biofilm Morphology. Bacteria use two-component signaling systems to adapt and respond to their competitors and changing environments. For instance, competitor bacteria may produce antibiotics and other bioactive metabolites and sequester nutrients. To survive, some species of bacteria escape competition through antibiotic production, biofilm formation, or motility. Specialized metabolite production and biofilm formation are relatively well understood for bacterial species in isolation. How bacteria control these functions when competitors are present is not well studied. To address fundamental questions relating to the competitive mechanisms of different species, we have developed a model system using two species of soil bacteria, Bacillus subtilis and Streptomyces sp. strain Mg1. Using this model, we previously found that linearmycins produced by Streptomyces sp. strain Mg1 cause lysis of B. subtilis cells and degradation of colony matrix. We identified strains of B. subtilis with mutations in the two-component signaling system yfiJK operon that confer dual phenotypes of specific linearmycin resistance and biofilm morphology. We determined that expression of the ATP-binding cassette (ABC) transporter yfiLMN operon, particularly yfiM and yfiN, is necessary for biofilm morphology. Using transposon mutagenesis, we identified genes that are required for YfiLMN-mediated biofilm morphology, including several chaperones. Using transcriptional fusions, we found that YfiJ signaling is activated by linearmycins and other polyene metabolites. Finally, using a truncated YfiJ, we show that YfiJ requires its transmembrane domain to activate downstream signaling. Taken together, these results suggest coordinated dual antibiotic resistance and biofilm morphology by a single multifunctional ABC transporter promotes competitive fitness of B. subtilisIMPORTANCE DNA sequencing approaches have revealed hitherto unexplored diversity of bacterial species in a wide variety of environments that includes the gastrointestinal tract of animals and the rhizosphere of plants. Interactions between different species in bacterial communities have impacts on our health and industry. However, many approaches currently used to study whole bacterial communities do not resolve mechanistic details of interspecies interactions, including how bacteria sense and respond to their competitors. Using a competition model, we have uncovered dual functions for a previously uncharacterized two-component signaling system involved in specific antibiotic resistance and biofilm morphology. Insights gleaned from signaling within interspecies interaction models build a more complete understanding of gene functions important for bacterial communities and will enhance community-level analytical approaches.201728461449
8672150.9912Pangenomic and functional investigations for dormancy and biodegradation features of an organic pollutant-degrading bacterium Rhodococcus biphenylivorans TG9. Environmental bacteria contain a wealth of untapped potential in the form of biodegradative genes. Leveraging this potential can often be confounded by a lack of understanding of fundamental survival strategies, like dormancy, for environmental stress. Investigating bacterial dormancy-to-degradation relationships enables improvement of bioremediation. Here, we couple genomic and functional assessment to provide context for key attributes of the organic pollutant-degrading strain Rhodococcus biphenylivorans TG9. Whole genome sequencing, pangenome analysis and functional characterization were performed to elucidate important genes and gene products, including antimicrobial resistance, dormancy, and degradation. Rhodococcus as a genus has strong potential for degradation and dormancy, which we demonstrate using R. biphenylivorans TG9 as a model. We identified four Resuscitation-promoting factor (Rpf) encoding genes in TG9 involved in dormancy and resuscitation. We demonstrate that R. biphenylivorans TG9 grows on fourteen typical organic pollutants, and exhibits a robust ability to degrade biphenyl and several congeners of polychlorinated biphenyls. We further induced TG9 into a dormant state and demonstrated pronounced differences in morphology and activity. Together, these results expand our understanding of the genus Rhodococcus and the relationship between dormancy and biodegradation in the presence of environmental stressors.202234688761
8291160.9912Pseudomonas Can Survive Tailocin Killing via Persistence-Like and Heterogenous Resistance Mechanisms. Phage tail-like bacteriocins (tailocins) are bacterially produced protein toxins that mediate competitive interactions between cocolonizing bacteria. Both theoretical and experimental research has shown there are intransitive interactions between bacteriocin-producing, bacteriocin-sensitive, and bacteriocin-resistant populations, whereby producers outcompete sensitive cells, sensitive cells outcompete resistant cells, and resistant cells outcompete producers. These so-called rock-paper-scissors dynamics explain how all three populations occupy the same environment, without one driving the others extinct. Using Pseudomonas syringae as a model, we demonstrate that otherwise sensitive cells survive bacteriocin exposure through a physiological mechanism. This mechanism allows cells to survive bacteriocin killing without acquiring resistance. We show that a significant fraction of the target cells that survive a lethal dose of tailocin did not exhibit any detectable increase in survival during a subsequent exposure. Tailocin persister cells were more prevalent in stationary- rather than log-phase cultures. Of the fraction of cells that gained detectable resistance, there was a range from complete (insensitive) to incomplete (partially sensitive) resistance. By using genomic sequencing and genetic engineering, we showed that a mutation in a hypothetical gene containing 8 to 10 transmembrane domains causes tailocin high persistence and that genes of various glycosyltransferases cause incomplete and complete tailocin resistance. Importantly, of the several classes of mutations, only those causing complete tailocin resistance compromised host fitness. This result indicates that bacteria likely utilize persistence to survive bacteriocin-mediated killing without suffering the costs associated with resistance. This research provides important insight into how bacteria can escape the trap of fitness trade-offs associated with gaining de novo tailocin resistance.IMPORTANCE Bacteriocins are bacterially produced protein toxins that are proposed as antibiotic alternatives. However, a deeper understanding of the responses of target bacteria to bacteriocin exposure is lacking. Here, we show that target cells of Pseudomonas syringae survive lethal bacteriocin exposure through both physiological persistence and genetic resistance mechanisms. Cells that are not growing rapidly rely primarily on persistence, whereas those growing rapidly are more likely to survive via resistance. We identified various mutations in lipopolysaccharide biogenesis-related regions involved in tailocin persistence and resistance. By assessing host fitness of various classes of mutants, we showed that persistence and subtle resistance are mechanisms P. syringae uses to survive competition and preserve host fitness. These results have important implications for developing bacteriocins as alternative therapeutic agents.202032312747
9079170.9912Review, Evaluation, and Directions for Gene-Targeted Assembly for Ecological Analyses of Metagenomes. Shotgun metagenomics has greatly advanced our understanding of microbial communities over the last decade. Metagenomic analyses often include assembly and genome binning, computationally daunting tasks especially for big data from complex environments such as soil and sediments. In many studies, however, only a subset of genes and pathways involved in specific functions are of interest; thus, it is not necessary to attempt global assembly. In addition, methods that target genes can be computationally more efficient and produce more accurate assembly by leveraging rich databases, especially for those genes that are of broad interest such as those involved in biogeochemical cycles, biodegradation, and antibiotic resistance or used as phylogenetic markers. Here, we review six gene-targeted assemblers with unique algorithms for extracting and/or assembling targeted genes: Xander, MegaGTA, SAT-Assembler, HMM-GRASPx, GenSeed-HMM, and MEGAN. We tested these tools using two datasets with known genomes, a synthetic community of artificial reads derived from the genomes of 17 bacteria, shotgun sequence data from a mock community with 48 bacteria and 16 archaea genomes, and a large soil shotgun metagenomic dataset. We compared assemblies of a universal single copy gene (rplB) and two N cycle genes (nifH and nirK). We measured their computational efficiency, sensitivity, specificity, and chimera rate and found Xander and MegaGTA, which both use a probabilistic graph structure to model the genes, have the best overall performance with all three datasets, although MEGAN, a reference matching assembler, had better sensitivity with synthetic and mock community members chosen from its reference collection. Also, Xander and MegaGTA are the only tools that include post-assembly scripts tuned for common molecular ecology and diversity analyses. Additionally, we provide a mathematical model for estimating the probability of assembling targeted genes in a metagenome for estimating required sequencing depth.201931749830
8299180.9912Regulatory cross-talk supports resistance to Zn intoxication in Streptococcus. Metals such as copper (Cu) and zinc (Zn) are important trace elements that can affect bacterial cell physiology but can also intoxicate bacteria at high concentrations. Discrete genetic systems for management of Cu and Zn efflux have been described in several bacterial pathogens, including streptococci. However, insight into molecular cross-talk between systems for Cu and Zn management in bacteria that drive metal detoxification, is limited. Here, we describe a biologically consequential cross-system effect of metal management in group B Streptococcus (GBS) governed by the Cu-responsive copY regulator in response to Zn. RNAseq analysis of wild-type (WT) and copY-deficient GBS subjected to metal stress revealed unique transcriptional links between the systems for Cu and Zn detoxification. We show that the Cu-sensing role of CopY extends beyond Cu and enables CopY to regulate Cu and Zn stress responses that effect changes in gene function for central cellular processes, including riboflavin synthesis. CopY also supported GBS intracellular survival in human macrophages and virulence during disseminated infection in mice. In addition, we show a novel role for CovR in modulating GBS resistance to Zn intoxication. Identification of the Zn resistome of GBS using TraDIS revealed a suite of genes essential for GBS growth in metal stress. Several of the genes identified are novel to systems that support bacterial survival in metal stress and represent a diverse set of mechanisms that underpin microbial metal homeostasis during cell stress. Overall, this study reveals a new and important mechanism of cross-system complexity driven by CopY in bacteria to regulate cellular management of metal stress and survival.202235862444
8293190.9911Identification of Bicarbonate as a Trigger and Genes Involved with Extracellular DNA Export in Mycobacterial Biofilms. Extracellular DNA (eDNA) is an integral biofilm matrix component of numerous pathogens, including nontuberculous mycobacteria (NTM). Cell lysis is the source of eDNA in certain bacteria, but the source of eDNA remains unidentified for NTM, as well as for other eDNA-containing bacterial species. In this study, conditions affecting eDNA export were examined, and genes involved with the eDNA export mechanism were identified. After a method for monitoring eDNA in real time in undisturbed biofilms was established, different conditions affecting eDNA were investigated. Bicarbonate positively influenced eDNA export in a pH-independent manner in Mycobacterium avium, M. abscessus, and M. chelonae The surface-exposed proteome of M. avium in eDNA-containing biofilms revealed abundant carbonic anhydrases. Chemical inhibition of carbonic anhydrases with ethoxzolamide significantly reduced eDNA export. An unbiased transposon mutant library screen for eDNA export in M. avium identified many severely eDNA-attenuated mutants, including one not expressing a unique FtsK/SpoIIIE-like DNA-transporting pore, two with inactivation of carbonic anhydrases, and nine with inactivation of genes belonging to a unique genomic region, as well as numerous mutants involved in metabolism and energy production. Complementation of nine mutants that included the FtsK/SpoIIIE and carbonic anhydrase significantly restored eDNA export. Interestingly, several attenuated eDNA mutants have mutations in genes encoding proteins that were found with the surface proteomics, and many more mutations are localized in operons potentially encoding surface proteins. Collectively, our data strengthen the evidence of eDNA export being an active mechanism that is activated by the bacterium responding to bicarbonate. IMPORTANCE: Many bacteria contain extracellular DNA (eDNA) in their biofilm matrix, as it has various biological and physical functions. We recently reported that nontuberculous mycobacteria (NTM) can contain significant quantities of eDNA in their biofilms. In some bacteria, eDNA is derived from dead cells, but that does not appear to be the case for all eDNA-containing organisms, including NTM. In this study, we found that eDNA export in NTM is conditionally dependent on the molecules to which the bacteria are exposed and that bicarbonate positively influences eDNA export. We also identified genes and proteins important for eDNA export, which begins to piece together a description of a mechanism for eDNA. Better understanding of eDNA export can give new targets for the development of antivirulence drugs, which are attractive because resistance to classical antibiotics is currently a significant problem.201627923918