Microbes display broad diversity in cobamide preferences.

Cobamides, the vitamin B (cobalamin) family of cofactors, are used by most organisms but produced by only a fraction of prokaryotes, and are thus considered key shared nutrients among microbes. Cobamides are structurally diverse, with multiple different cobamides found in most microbial communities. The ability to use different cobamides has been tested for several bacteria and microalgae, and nearly all show preferences for certain cobamides.

Metagenomic analysis during capecitabine therapy reveals microbial chemoprotective mechanisms and predicts drug toxicity in colorectal cancer patients.

Purpose: Unpredictable chemotherapy side effects are a major barrier to successful treatment. Cell culture and mouse experiments indicate that the gut microbiota is influenced by and influences anti-cancer drugs. However, metagenomic data from patients paired to careful side effect monitoring remains limited.

High-throughput protein characterization by complementation using DNA barcoded fragment libraries.

Our ability to predict, control, or design biological function is fundamentally limited by poorly annotated gene function. This can be particularly challenging in non-model systems. Accordingly, there is motivation for new high-throughput methods for accurate functional annotation.

Fitness factors impacting survival of a subsurface bacterium in contaminated groundwater.

Many factors contribute to the ability of a microbial species to persist when encountering complexly contaminated environments, including time of exposure, the nature and concentration of contaminants, availability of nutritional resources, and possession of a combination of appropriate molecular mechanisms needed for survival. Herein we sought to identify genes that are most important for survival of Gram-negative Enterobacteriaceae in contaminated groundwater environments containing high concentrations of nitrate and metals using the metal-tolerant Oak Ridge Reservation isolate, Pantoea sp. MT58 (MT58).

High-throughput genetics enables identification of nutrient utilization and accessory energy metabolism genes in a model methanogen.

Archaea are widespread in the environment and play fundamental roles in diverse ecosystems; however, characterization of their unique biology requires advanced tools. This is particularly challenging when characterizing gene function. Here, we generate randomly barcoded transposon libraries in the model methanogenic archaeon and use high-throughput growth methods to conduct fitness assays (RB-TnSeq) across over 100 unique growth conditions.

Molecular mechanisms and environmental adaptations of flagellar loss and biofilm growth of Rhodanobacter under environmental stress.

Biofilms aid bacterial adhesion to surfaces via direct and indirect mechanisms, and formation of biofilms is considered as an important strategy for adaptation and survival in suboptimal environmental conditions. However, the molecular underpinnings of biofilm formation in subsurface sediment/groundwater ecosystems where microorganisms often experience fluctuations in nutrient input, pH, and nitrate or metal concentrations are underexplored. We examined biofilm formation under different nutrient, pH, metal, and nitrate regimens of 16 Rhodanobacter strains isolated from subsurface groundwater wells spanning diverse levels of pH (3.

Barcoded overexpression screens in gut Bacteroidales identify genes with roles in carbon utilization and stress resistance.

A mechanistic understanding of host-microbe interactions in the gut microbiome is hindered by poorly annotated bacterial genomes. While functional genomics can generate large gene-to-phenotype datasets to accelerate functional discovery, their applications to study gut anaerobes have been limited. For instance, most gain-of-function screens of gut-derived genes have been performed in Escherichia coli and assayed in a small number of conditions.

A phage tail-like bacteriocin suppresses competitors in metapopulations of pathogenic bacteria.

Bacteria can repurpose their own bacteriophage viruses (phage) to kill competing bacteria. Phage-derived elements are frequently strain specific in their killing activity, although there is limited evidence that this specificity drives bacterial population dynamics. Here, we identified intact phage and their derived elements in a metapopulation of wild plant-associated genomes.

Genomic and environmental controls on Castellaniella biogeography in an anthropogenically disturbed subsurface.

Castellaniella species have been isolated from a variety of mixed-waste environments including the nitrate and multiple metal-contaminated subsurface at the Oak Ridge Reservation (ORR). Previous studies examining microbial community composition and nitrate removal at ORR during biostimulation efforts reported increased abundances of members of the Castellaniella genus concurrent with increased denitrification rates. Thus, we asked how genomic and abiotic factors control the Castellaniella biogeography at the site to understand how these factors may influence nitrate transformation in an anthropogenically impacted setting.

An expanded transcriptome atlas for Bacteroides thetaiotaomicron reveals a small RNA that modulates tetracycline sensitivity.

Plasticity in gene expression allows bacteria to adapt to diverse environments. This is particularly relevant in the dynamic niche of the human intestinal tract; however, transcriptional networks remain largely unknown for gut-resident bacteria. Here we apply differential RNA sequencing (RNA-seq) and conventional RNA-seq to the model gut bacterium Bacteroides thetaiotaomicron to map transcriptional units and profile their expression levels across 15 in vivo-relevant growth conditions.

A weaponized phage suppresses competitors in historical and modern metapopulations of pathogenic bacteria.

Unlabelled: Bacteriophages, the viruses of bacteria, are proposed to drive bacterial population dynamics, yet direct evidence of their impact on natural populations is limited. Here we identified viral sequences in a metapopulation of wild plant-associated spp. genomes.

Randomly barcoded transposon mutant libraries for gut commensals II: Applying libraries for functional genetics.

The critical role of the intestinal microbiota in human health and disease is well recognized. Nevertheless, there are still large gaps in our understanding of the functions and mechanisms encoded in the genomes of most members of the gut microbiota. Genome-scale libraries of transposon mutants are a powerful tool to help us address this gap.

Randomly barcoded transposon mutant libraries for gut commensals I: Strategies for efficient library construction.

Randomly barcoded transposon mutant libraries are powerful tools for studying gene function and organization, assessing gene essentiality and pathways, discovering potential therapeutic targets, and understanding the physiology of gut bacteria and their interactions with the host. However, construction of high-quality libraries with uniform representation can be challenging. In this review, we survey various strategies for barcoded library construction, including transposition systems, methods of transposon delivery, optimal library size, and transconjugant selection schemes.

A chemically-defined growth medium to support Lactobacillus-Acetobacter sp. community analysis.

Lactobacilli and Acetobacter sp. are commercially important bacteria that often form communities in natural fermentations, including food preparations, spoilage, and in the digestive tract of the fruit fly Drosophila melanogaster. Communities of these bacteria are widespread and prolific, despite numerous strain-specific auxotrophies, suggesting they have evolved nutrient interdependencies that regulate their growth.

Simvastatin induces human gut bacterial cell surface genes.

Drugs intended to target mammalian cells can have broad off-target effects on the human gut microbiota with potential downstream consequences for drug efficacy and side effect profiles. Yet, despite a rich literature on antibiotic resistance, we still know very little about the mechanisms through which commensal bacteria evade non-antibiotic drugs. Here, we focus on statins, one of the most prescribed drug types in the world and an essential tool in the prevention and treatment of high circulating cholesterol levels.

A mutant fitness compendium in Bifidobacteria reveals molecular determinants of colonization and host-microbe interactions.

Bifidobacteria commonly represent a dominant constituent of human gut microbiomes during infancy, influencing nutrition, immune development, and resistance to infection. Despite interest as a probiotic therapy, predicting the nutritional requirements and health-promoting effects of Bifidobacteria is challenging due to major knowledge gaps. To overcome these deficiencies, we used large-scale genetics to create a compendium of mutant fitness in ().

Genome-wide fitness profiling reveals molecular mechanisms that bacteria use to interact with Trichoderma atroviride exometabolites.

Trichoderma spp. are ubiquitous rhizosphere fungi capable of producing several classes of secondary metabolites that can modify the dynamics of the plant-associated microbiome. However, the bacterial-fungal mechanisms that mediate these interactions have not been fully characterized.

Eliminating genes for a two-component system increases PHB productivity in Cupriavidus basilensis 4G11 under PHB suppressing, nonstress conditions.

Species of bacteria from the genus Cupriavidus are known, in part, for their ability to produce high amounts of poly-hydroxybutyrate (PHB) making them attractive candidates for bioplastic production. The native synthesis of PHB occurs during periods of metabolic stress, and the process regulating the initiation of PHB accumulation in these organisms is not fully understood. Screening an RB-TnSeq transposon library of Cupriavidus basilensis 4G11 allowed us to identify two genes of an apparent, uncharacterized two-component system, which when omitted from the genome enable increased PHB productivity in balanced, nonstress growth conditions.

Dynamic genetic adaptation of Bacteroides thetaiotaomicron during murine gut colonization.

To understand how a bacterium ultimately succeeds or fails in adapting to a new host, it is essential to assess the temporal dynamics of its fitness over the course of colonization. Here, we introduce a human-derived commensal organism, Bacteroides thetaiotaomicron (Bt), into the guts of germ-free mice to determine whether and how the genetic requirements for colonization shift over time. Combining a high-throughput functional genetics assay and transcriptomics, we find that gene usage changes drastically during the first days of colonization, shifting from high expression of amino acid biosynthesis genes to broad upregulation of diverse polysaccharide utilization loci.

Geochemical constraints on bacteriophage infectivity in terrestrial environments.

Lytic phages can be potent and selective inhibitors of microbial growth and can have profound impacts on microbiome composition and function. However, there is uncertainty about the biogeochemical conditions under which phage predation modulates microbial ecosystem function, particularly in terrestrial systems. Ionic strength is critical for infection of bacteria by many phages, but quantitative data is limited on the ion thresholds for phage infection that can be compared with environmental ion concentrations.

Large-scale genetic characterization of the model sulfate-reducing bacterium, Hildenborough.

Sulfate-reducing bacteria (SRB) are obligate anaerobes that can couple their growth to the reduction of sulfate. Despite the importance of SRB to global nutrient cycles and their damage to the petroleum industry, our molecular understanding of their physiology remains limited. To systematically provide new insights into SRB biology, we generated a randomly barcoded transposon mutant library in the model SRB Hildenborough (DvH) and used this genome-wide resource to assay the importance of its genes under a range of metabolic and stress conditions.

4'-Deoxypyridoxine disrupts vitamin B homeostasis in K12 through combined inhibition of cumulative B uptake and PLP-dependent enzyme activity.

Pyridoxal 5'-phosphate (PLP) is the active form of vitamin B and a cofactor for many essential metabolic processes such as amino acid biosynthesis and one carbon metabolism. 4'-deoxypyridoxine (4dPN) is a long known B antimetabolite but its mechanism of action was not totally clear. By exploring different conditions in which PLP metabolism is affected in the model organism K12, we showed that 4dPN cannot be used as a source of vitamin B as previously claimed and that it is toxic in several conditions where vitamin B homeostasis is affected, such as in a B auxotroph or in a mutant lacking the recently discovered PLP homeostasis gene, .

An integrated transcriptomics-functional genomics approach reveals a small RNA that modulates sensitivity to tetracyclines.

Gene expression plasticity allows bacteria to adapt to diverse environments, tie their metabolism to available nutrients, and cope with stress. This is particularly relevant in a niche as dynamic and hostile as the human intestinal tract, yet transcriptional networks remain largely unknown in gut spp. Here, we map transcriptional units and profile their expression levels in over a suite of 15 defined experimental conditions that are relevant , such as variation of temperature, pH, and oxygen tension, exposure to antibiotic stress, and growth on simple carbohydrates or on host mucin-derived glycans.

Multicopy suppressor screens reveal convergent evolution of single-gene lysis proteins.

Single-strand RNA (ssRNA) Fiersviridae phages cause host lysis with a product of single gene (sgl for single-gene lysis; product Sgl) that induces autolysis. Many different Sgls have been discovered, but the molecular targets of only a few have been identified. In this study, we used a high-throughput genetic screen to uncover genome-wide host suppressors of diverse Sgls.

Construction and characterization of a genome-scale ordered mutant collection of Bacteroides thetaiotaomicron.

Background: Ordered transposon-insertion collections, in which specific transposon-insertion mutants are stored as monocultures in a genome-scale collection, represent a promising tool for genetic dissection of human gut microbiota members. However, publicly available collections are scarce and the construction methodology remains in early stages of development.

Results: Here, we describe the assembly of a genome-scale ordered collection of transposon-insertion mutants in the model gut anaerobe Bacteroides thetaiotaomicron VPI-5482 that we created as a resource for the research community.