The seed is a habitat with limited resources and space. Although it is widely accepted that microbial competition is a key driver of the assembly of seed-associated microbial communities, the underlying mechanisms of this competition are not well understood. The initial objective of this work was to assess the importance of contact-independent microbial competition between the phytopathogenic bacterium Xanthomonas campestris pv. campestris 8004 (Xcc8004) and 30 strains representative of the bacterial populations most commonly associated with radish (Raphanus sativus) seeds. We identified Pseudomonas lactucae CFBP 13502 as a potent inhibitor of Xcc8004, mediated by exometabolites, specifically induced by certain seed-borne strains. Transcriptomic analysis linked this inducible activity to the upregulation of a gene cluster encoding a lipopeptide siderophore. Targeted gene deletion in P. lactucae CFBP 13502 confirmed that this cluster is essential for antagonism against Xcc8004. Furthermore, iron supplementation abolished this inhibitory effect, strongly supporting iron chelation as the underlying mechanism. Through comparative metabolomics, we elucidated the structure of a family of lipopeptide siderophores, produced by P. lactucae CFBP 13502, which we named lactuchelins. Our findings provide molecular evidence of competitive exclusion mechanisms at the seed microbiome interface, highlighting lactuchelins as a promising avenue for the development of seed-based biocontrol strategies against seed-borne phytopathogens.

For years, the airway microbiota have been theorized to be gatekeepers of respiratory health, as pathogens entering the airway make contact with resident microbes prior to or coincident with their interaction with host cells. Thus, modification of the native airway community may serve as a means of altering the local environment in favor of health. In this work, we hypothesize that synthetic bacterial communities introduced into the airway can serve as prophylactic countermeasures against infection by Burkholderia thailandensis in mice. We demonstrate that understanding of antagonistic interactions between a pathogen and airway microbiota in vitro can guide identification of probiotics with protective capabilities in vivo. Specifically, we show that niche overlap between the probiotic and pathogen is indicative of probiotic performance in vivo. This work serves as a foundation for the rational design of probiotic communities for protection against and treatment of respiratory infections.

Bacteriophages (phages) are emerging as programmable biological therapeutics in oncology, extending beyond their traditional antimicrobial applications. This review proposes a phage-microbiome-immune-oncology axis that links microbial dynamics, immune modulation, and engineered phages to guide precision cancer prevention and therapy. Phages can eliminate cancer-associated bacteria, remodel the tumor microenvironment, enhance antitumor immunity, and deliver targeted therapeutic payloads. However, several critical challenges must be addressed to realize this therapeutic potential, particularly host immune responses that limit repeat dosing, inefficient tumor penetration, and the need for rigorous clinical validation. By examining phage-host-tumor interactions through robust model systems and highlighting translational opportunities, this review establishes phage therapy as a promising frontier in precision oncology that warrants accelerated clinical development.

Anthropogenic perturbations to the nitrogen cycle, primarily through use of synthetic fertilizers, is driving an unprecedented increase in the emission of nitrous oxide (N2O), a potent greenhouse gas and an ozone depleting substance, causing urgency in identifying the sources and sinks of N2O. Microbial denitrification is a primary contributor to biotic production of N2O in anoxic regions of soil, marine systems, and wastewater treatment facilities. Here, through comprehensive genome analysis, we show that pathway partitioning is a ubiquitous mechanism of complete denitrification within microbial communities. We have investigated mechanisms and consequences of process partitioning of denitrification through detailed physiological characterization and kinetic modeling of a synthetic community of Rhodanobacter thiooxydans FW510-R12 and Acidovorax sp. GW101-3H11. We have discovered that these two bacterial isolates, from a heavily nitrate (NO3-) contaminated superfund site, complete denitrification through the exchange of nitrite (NO2-) and nitric oxide (NO). The process partitioning of denitrification and other processes, including amino acid metabolism, contribute to increased cooperativity within this denitrifying community. We demonstrate that certain contexts, such as high NO3-, cause unbalanced growth of community members, due to differences in their substrate utilization kinetics. The altered growth characteristics of community members drives accumulation of toxic NO2-, which disrupts denitrification causing N2O off gassing.

Objectives/Goals: The goal of this work is to understand the physiological profile of phage susceptibility and identify candidate phage defense mechanisms. Additionally, it aims to determine the host receptors targeted by bacteriophages to infect E. coli O157:H7 through random bar code transposon-site sequencing (RB-TnSeq). Methods/Study Population: A collection of 109 E. coli O157:H7 strains from environmental, food, and animal sources were analyzed, representing phylogenetic lineages corresponding to clades 2, 3, 5, 6, 7, and 8. Phage susceptibility profiles were determined using 23 bacteriophages, assessing plaque morphology. Using the O157:H7 genomes, a genomic analysis was conducted with the Prokaryotic Antiviral Defense Locator (PADLOC), which identified putative phage defense systems through sequence homology. Additionally, 5 RB-TnSeq libraries were generated in representative strains to study loss-of-function mutations. These libraries will be screened against a subset of diverse phage to identify the receptors involved in phage adsorption. Results/Anticipated Results: The phage resistance patterns showed susceptibility varied across clades, suggesting distinct mechanisms. Several defense systems were identified using PADLOC, including restriction-modification, Cas, Lamassu, and Druantia. Phage defense candidate (PDC) systems were identified, showing homology to known systems, though their specific function remains unknown. Clade 7.2 exhibited higher phage resistance and a greater presence of PDCs compared to the other clades. Five saturated RB-TnSeq libraries were constructed in O157:H7, achieving 84.5–89% gene coverage. These libraries will facilitate the identification of receptors involved in phage adsorption and resistance. Discussion/Significance of Impact: This study deepens our understanding of phage resistance in E. coli O157:H7 by identifying key defense systems and receptors. The discovery of novel antiviral mechanisms offers promising targets for phage-based interventions, potentially enhancing strategies for controlling this dangerous pathogen.

Microbiology plays an important role in most sectors. Future progress in critical areas requires diverse workforce development. We outline a pathway program that aims to provide equitable exposure to high-impact research experiences and course-based instruction to provide crucial training in growing areas of microbiology (phage discovery, synthetic biology and data science/AI).

We report 26 subsurface sediment and 9 groundwater metagenomes from the Oak Ridge Reservation at Oak Ridge, TN, USA. Samples were collected from various depths and phases (attached vs planktonic) to study subsurface microbial metabolism, the effect of contamination on microbial communities, and differences across groundwater and sediment microbial communities.

Plasmids isolated or characterized from environmental samples serve as a resource that can be used to develop genetic tools for characterizing recently isolated or less-studied microbes. In this report, we leveraged sequences from a previously characterized groundwater plasmidome and developed a screen to identify novel plasmid origins. Putative origin sequences were used to construct a barcoded plasmid library, which contained both known and newly predicted origins. This library was tested against a panel of representative bacterial strains and led to the identification of 3 novel origins that putatively replicate in gram-negative bacteria not previously associated with these origin sequences. We empirically validated one of the newly identified origins, 6911, to be functional in both the model bacterial strain, Escherichia coli BW25113, as well as in Pantoea sp. MT58, a fast growing and metal tolerant, environmentally important bacterium from the widespread Pantoea genus. We confirmed that a plasmid bearing origin 6911 as the sole origin could replicate and had a copy number of 9 (± 2) in Pantoea sp. MT58. We successfully used a plasmid based on the new origin to express the reporter protein GFP, and two non-native metabolite pathways for the natural product, indigoidine and the terpenoid compound, isoprenol. By pairing functional novel origins of replication to non-model organisms this pipeline can expand the tool kit for genetic manipulations of both model and less-studied bacteria. ![Abstract figure.][1] Abstract figure. We developed a host-origin pair identification pipeline by constructing a plasmid library containing both literature-sourced and computationally predicted origins of replication. By conjugating this library into non-model microbes, we identified functional origins in diverse bacteria, and could leverage these findings to develop genetically tractable hosts for microbial engineering. ### Competing Interest Statement The authors have declared no competing interest. Office of Science, https://ror.org/00mmn6b08, DE-AC02-05CH11231 [1]: pending:yes

Microbial taxonomic diversity declines with increased environmental stress. Yet, few studies have explored whether phylogenetic and functional diversities track taxonomic diversity along the stress gradient. Here, we investigated microbial communities within an aquifer in Oak Ridge, Tennessee, USA, which is characterized by a broad spectrum of stressors, including extremely high levels of nitrate, heavy metals like cadmium and chromium, radionuclides such as uranium, and extremely low pH (< 3). Both taxonomic and phylogenetic α-diversities were reduced in the most impacted wells, while the decline in functional α-diversity was modest and statistically insignificant, indicating a more robust buffering capacity to environmental stress. Differences in functional gene composition (i.e., functional β-diversity) were pronounced in highly contaminated wells, while convergent functional gene composition was observed in uncontaminated wells. The relative abundances of most carbon degradation genes were decreased in contaminated wells, but genes associated with denitrification, adenylylsulfate reduction, and sulfite reduction were increased. Compared to taxonomic and phylogenetic compositions, environmental variables played a more significant role in shaping functional gene composition, suggesting that niche selection could be more closely related to microbial functionality than taxonomy. Overall, we demonstrated that despite a reduced taxonomic α-diversity, microbial communities under stress maintained functionality underpinned by environmental selection.

This protocol is a modification to the Qiagen PowerMax Soil Kit for extracting DNA from sediment and water filters. It reduces bead-beating to obtain higher quality DNA and less fragmented DNA. Running the DNA out on an agarose gel with a high molecular weight DNA ladder or on a femtopulse is recommended to check DNA quality. This method was published in Wu et al. 2023 Distinct Depth-discrete Profiles of Microbial Communities and Geochemical Insights in the Subsurface Critical Zone AEM (https://doi.org/10.1128/aem.00500-23) and is similar to protocols found in Hug et al. 2015 ISMEJ (https://doi.org/10.1038/ismej.2015.2) and Kantor et al. 2013 mSphere (https://doi.org/10.1128/mbio.00708-13). This was originally developed using the kit by MoBio, but MoBio was bought out by Qiagen and you can purchase the kit from Qiagen now.

Seeds harbor diverse microbial communities, including beneficial microbes that play a vital role in protecting plants from seed-borne pathogens. Despite their critical importance, the molecular mechanisms driving intermicrobial competition within the seed microbiome remain poorly understood, limiting the potential to optimize seed inoculation strategies. In this study, we evaluated the inhibitory potential of 30 seed-borne bacterial strains against the phytopathogen Xanthomonas campestris pv. campestris 8004 (Xcc8004). We identified Pseudomonas lactucae CFBP13502 as a potent inhibitor of Xcc8004, mediated by exometabolites specifically induced in the presence of Lysobacterales (formerly Xanthomonadales). Transcriptomic analysis of CFBP13502 revealed upregulation of a gene cluster involved in the biosynthesis of a lipopeptide siderophore biosynthesis. Gene deletion confirmed that this cluster is essential for the growth inhibition of Xcc8004. Furthermore, iron supplementation abolished this inhibitory effect, providing strong evidence for the role of iron chelation. Through comparative metabolomics, we elucidated the structure of a novel family of lipopeptide siderophores, which we named lactuchelins, produced by CFBP13502. Our findings provide the first molecular evidence of competitive exclusion mechanisms at the seed microbiome interface, highlighting lactuchelins as a promising avenue for the development of seed-based biocontrol strategies against seed-borne phytopathogens. ### Competing Interest Statement The authors have declared no competing interest.

Gene redundancy complicates systematic characterization of gene function as single-gene deletions may not produce discernible phenotypes. We report dual transposon sequencing (dual Tn-seq), a platform for assaying the fitness of a comprehensive double mutant pool in parallel. Dual Tn-seq couples random barcode transposon site sequencing with the Cre-lox system, enabling deep sampling of 73% of the 1.3 million possible double gene deletions in Streptococcus pneumoniae. The genetic interactions identified span a wide range of biochemical processes, revealing new factors in presumably well-studied pathways, exemplified by a cytidine triphosphate synthase PyrJ. Moreover, this approach should permit further investigation of growth condition-specific genetic interactions. Because dual Tn-seq does not require the construction of a large array of single mutants, it should be readily adaptable to various microorganisms.

Spirulina is the common name for the edible, nonheterocystous, filamentous cyanobacterium Arthrospira platensis that is grown industrially as a food supplement, animal feedstock, and pigment source. Although there are many applications for engineering this organism, until recently no genetic tools or reproducible transformation methods have been published. While recent work showed the production of a diversity of proteins in A. platensis, including single-domain antibodies for oral delivery, there remains a need for a modular, characterized genetic toolkit. Here, we independently establish a reproducible method for the transformation of A. platensis and engineer this bacterium to produce acetaminophen as proof-of-concept for small molecule production in an edible host. This work opens A. platensis to the wider scientific community for future engineering as a functional food for nutritional enhancement, modification of organoleptic traits, and production of pharmaceuticals for oral delivery.

ABSTRACT 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 Methanococcus maripaludis and use high-throughput growth methods to conduct fitness assays (RB-TnSeq) across over 100 unique growth conditions. Using our approach, we identified new genes involved in nutrient utilization and response to oxidative stress. We identified novel genes for the usage of diverse nitrogen sources in M. maripaludis including a putative regulator of alanine deamination and molybdate transporters important for nitrogen fixation. Furthermore, leveraging the fitness data, we inferred that M. maripaludi s can utilize additional nitrogen sources including ʟ-glutamine, ᴅ-glucuronamide, and adenosine. Under autotrophic growth conditions, we identified a gene encoding a domain of unknown function (DUF166) that is important for fitness and hypothesize that it has an accessory role in carbon dioxide assimilation. Finally, comparing fitness costs of oxygen versus sulfite stress, we identified a previously uncharacterized class of dissimilatory sulfite reductase-like proteins (Dsr-LP; group IIId) that is important during growth in the presence of sulfite. When overexpressed, Dsr-LP conferred sulfite resistance and enabled use of sulfite as the sole sulfur source. The high-throughput approach employed here allowed for generation of a large-scale data set that can be used as a resource to further understand gene function and metabolism in the archaeal domain. IMPORTANCE Archaea are widespread in the environment, yet basic aspects of their biology remain underexplored. To address this, we apply randomly barcoded transposon libraries (RB-TnSeq) to the model archaeon Methanococcus maripaludis . RB-TnSeq coupled with high-throughput growth assays across over 100 unique conditions identified roles for previously uncharacterized genes, including several encoding proteins with domains of unknown function (DUFs). We also expand on our understanding of carbon and nitrogen metabolism and characterize a group IIId dissimilatory sulfite reductase-like protein as a functional sulfite reductase. This data set encompasses a wide range of additional conditions including stress, nitrogen fixation, amino acid supplementation, and autotrophy, thus providing an extensive data set for the archaeal community to mine for characterizing additional genes of unknown function.

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. To address these challenges, we develop Barcoded Overexpression BActerial shotgun library sequencing (Boba-seq). We demonstrate the power of this approach by assaying genes from diverse gut Bacteroidales overexpressed in Bacteroides thetaiotaomicron. From hundreds of experiments, we identify new functions and phenotypes for 29 genes important for carbohydrate metabolism or tolerance to antibiotics or bile salts. Highlights include the discovery of a d-glucosamine kinase, a raffinose transporter, and several routes that increase tolerance to ceftriaxone and bile salts through lipid biosynthesis. This approach can be readily applied to develop screens in other strains and additional phenotypic assays. Here, the authors developed a novel high-throughput, DNA barcoded overexpression platform, BOBA-seq, to uncover new gene functions among gut anaerobes involved in carbon utilization and stress tolerance.

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 sub-optimal environmental conditions. However, the molecular underpinnings of biofilm formation in subsurface sediment/groundwater ecosystems where microorganisms often experience fluctuations in nutrient input, pH, nitrate or metal concentrations is underexplored. We examined biofilm formation under different nutrient, pH, metal, and nitrate regimes of 16 Rhodanobacter strains isolated from subsurface groundwater wells spanning diverse pH (3.5 to 5) and nitrate levels (13.7 to 146 mM). Eight Rhodanobacter strains demonstrated significant biofilm growth under low pH, suggesting adaptation to survive and grow at low pH. Biofilms intensified under aluminum stress, particularly in strains possessing fewer genetic traits associated with biofilm formation warranting further investigation. Through RB-TnSeq, proteomics, use of specific mutants and transmission electron microscopy analysis, we discovered flagellar loss under aluminum stress, indicating a potential relationship between motility, metal tolerance, and biofilm growth. Comparative genomic analyses revealed absence of flagella and chemotaxis genes, and presence of putative Type VI secretion system in the high biofilm-forming strain FW021-MT20. This study identifies genetic determinants associated with biofilm growth in a predominant environmental genus, Rhodanobacter, under metal stress and identifies traits aiding survival and adaptation to contaminated subsurface environments.

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). Survival fitness experiments in which a randomly barcoded transposon insertion (RB-TnSeq) library of MT58 was exposed directly to contaminated Oak Ridge Reservation groundwater samples from across a nitrate and mixed metal contamination plume were used to identify genes important for survival with increasing exposure times and concentrations of contaminants, and availability of a carbon source. Genes involved in controlling and using carbon, encoding transcriptional regulators, and related to Gram-negative outer membrane processes were among those found to be important for survival in contaminated Oak Ridge Reservation groundwater. A comparative genomics analysis of 75 Pantoea genus strains allowed us to further separate the survival determinants into core and non-core genes in the Pantoea pangenome, revealing insights into the survival of subsurface microorganisms during contaminant plume intrusion.

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. We report the isolation and characterization of several Castellaniella strains from the ORR subsurface. Five of these isolates match at 100% identity (at the 16S rRNA gene V4 region) to two Castellaniella amplicon sequence variants (ASVs), ASV1 and ASV2, that have persisted in the ORR subsurface for at least 2 decades. However, ASV2 has consistently higher relative abundance in samples taken from the site and was also the dominant blooming denitrifier population during a prior biostimulation effort. We found that the ASV2 representative strain has greater resistance to mixed metal stress than the ASV1 representative strains. We attribute this resistance, in part, to the large number of unique heavy metal resistance genes identified on a genomic island in the ASV2 representative genome. Additionally, we suggest that the relatively lower fitness of ASV1 may be connected to the loss of the nitrous oxide reductase (nos) operon (and associated nitrous oxide reductase activity) due to the insertion at this genomic locus of a mobile genetic element carrying copper resistance genes. This study demonstrates the value of integrating genomic, environmental, and phenotypic data to characterize the biogeography of key microorganisms in contaminated sites.

As nuclear technology evolves in response to increased demand for diversification and decarbonization of the energy sector, new and innovative approaches are needed to effectively identify and deter the proliferation of nuclear arms, while ensuring safe development of global nuclear energy resources. Preventing the use of nuclear material and technology for unsanctioned development of nuclear weapons has been a long-standing challenge for the International Atomic Energy Agency and signatories of the Treaty on the Non-Proliferation of Nuclear Weapons. Environmental swipe sampling has proven to be an effective technique for characterizing clandestine proliferation activities within and around known locations of nuclear facilities and sites. However, limited tools and techniques exist for detecting nuclear proliferation in unknown locations beyond the boundaries of declared nuclear fuel cycle facilities, representing a critical gap in non-proliferation safeguards. Microbiomes, defined as “characteristic communities of microorganisms” found in specific habitats with distinct physical and chemical properties, can provide valuable information about the conditions and activities occurring in the surrounding environment. Microorganisms are known to inhabit radionuclide-contaminated sites, spent nuclear fuel storage pools, and cooling systems of water-cooled nuclear reactors, where they can cause radionuclide migration and corrosion of critical structures. Microbial transformation of radionuclides is a well-established process that has been documented in numerous field and laboratory studies. These studies helped to identify key bacterial taxa and microbially-mediated processes that directly and indirectly control the transformation, mobility, and fate of radionuclides in the environment. Expanding on this work, other studies have used microbial genomics integrated with machine learning models to successfully monitor and predict the occurrence of heavy metals, radionuclides, and other process wastes in the environment, indicating the potential role of nuclear activities in shaping microbial community structure and function. Results of this previous body of work suggest fundamental geochemical-microbial interactions occurring at nuclear fuel cycle facilities could give rise to microbiomes that are characteristic of nuclear activities. These microbiomes could provide valuable information for monitoring nuclear fuel cycle facilities, planning environmental sampling campaigns, and developing biosensor technology for the detection of undisclosed fuel cycle activities and proliferation concerns.