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Here, we present the complete genome sequence of Sporosarcina pasteurii type strain DSM33, a model organism for microbially induced calcium carbonate precipitation. This genome consists of a single 3.3-Mb chromosome and is an improvement upon draft S. pasteurii genome sequences currently available in public databases.

Microbiota that originate in the seed can have consequences for the education of the plant immune system, competitive exclusion of pathogens from the host tissue, and host access to critical nutrients. Our research objective was to investigate the consequences of the environmental conditions of the parent plant for bacterial seed microbiome assembly and transmission across plant generations. Using a fully factorial, three-generational experimental design, we investigated endophytic seed bacterial communities of common bean lines ( Phaseolus vulgaris L.) grown in the growth chamber and exposed to either control conditions, drought, or excess nutrients at each generation. We applied 16S rRNA microbiome profiling to the seed endophytes and measured plant health outcomes. We discovered stable transmission of 22 bacterial members, regardless of the parental plant condition. This study shows the maintenance of bacterial members of the plant microbiome across generations, even under environmental stress. Overall, this work provides insights into the ability of plants to safeguard microbiome members, which has implications for crop microbiome management in the face of climate change.IMPORTANCESeed microbiomes initiate plant microbiome assembly and thus have critical implications for the healthy development and performance of crops. However, the consequences of environmental conditions of the parent plant for seed microbiome assembly and transmission are unknown, but this is critical information, given the intensifying stressors that crops face as the climate crisis accelerates. This study provides insights into the maintenance of plant microbiomes across generations, with implications for durable plant microbiome maintenance in agriculture on the changing planet., Competing Interests: The authors declare no conflict of interest.

As white-footed mice, Peromyscus leucopus , are considered the primary animal reservoir of Borreliella burgdorferi sensu stricto ( Bb ), the main agent of Lyme disease (LD) in the United States, these animals represent the most relevant model to study borrelial spirochetes in the context of their natural life cycle. Previous studies have consistently demonstrated that although white-footed mice respond immunologically to the invasion of the Lyme pathogen, P. leucopus adults do not develop a clinically detectable disease. This tolerance, which is common for mammalian reservoirs of different pathogens, contrasts with detrimental anti-borrelial responses of C3H mice, a widely used animal model of LD, which always result in a clinical manifestation (e.g., arthritis). The current investigation is a follow-up of our recent study that already showed a relative quiescence of the spleen transcriptome for Bb -infected white-footed mice compared to the infected C3H mice. In an effort to identify the mechanism behind this tolerance, in this study, we have evaluated an extensive list of hematological and biochemical parameters measured in white-footed mice after their 70-day-long borrelial infection. Despite missing reference intervals for Peromyscus mice, our sex- and age-matched uninfected controls allowed us to assess the blood and serum parameters. In addition, for our assessment, we also utilized behavioral, immunological, and histological analyses. Collectively, by using the metrics reported herein, the present results have demonstrated clinical unresponsiveness of P. leucopus mice to the borrelial infection, presenting no restriction to a long-term host-pathogen co-existence.

Myxococcus xanthus uses short-range C-signaling to coordinate multicellular mound formation with sporulation during fruiting body development. A csgA mutant deficient in C-signaling can cheat on wild type (WT) in mixtures and form spores disproportionately, but our understanding of cheating behavior is incomplete. We subjected mixtures of WT and csgA cells at different ratios to co-development and used confocal microscopy and image analysis to quantify the arrangement and morphology of cells. At a ratio of one WT to four csgA cells (1:4), mounds failed to form. At 1:2, only a few mounds and spores formed. At 1:1, mounds formed with a similar number and arrangement of WT and csgA rods early in development, but later the number of csgA spores near the bottom of these nascent fruiting bodies (NFBs) exceeded that of WT. This cheating after mound formation involved csgA forming spores at a greater rate, while WT disappeared at a greater rate, either lysing or exiting NFBs. At 2:1 and 4:1, csgA rods were more abundant than expected throughout the biofilm both before and during mound formation, and cheating continued after mound formation. We conclude that C-signaling restricts cheating behavior by requiring sufficient WT cells in mixtures. Excess cheaters may interfere with positive feedback loops that depend on the cellular arrangement to enhance C-signaling during mound building. Since long-range signaling could not likewise communicate the cellular arrangement, we propose that C-signaling was favored evolutionarily and that other short-range signaling mechanisms provided selective advantages in bacterial biofilm and multicellular animal development., Importance: Bacteria communicate using both long- and short-range signals. Signaling affects community composition, structure, and function. Adherent communities called biofilms impact medicine, agriculture, industry, and the environment. To facilitate the manipulation of biofilms for societal benefits, a better understanding of short-range signaling is necessary. We investigated the susceptibility of short-range C-signaling to cheating during Myxococcus xanthus biofilm development. A mutant deficient in C-signaling fails to form mounds containing spores (i.e., fruiting bodies) but cheats on C-signaling by wild type in starved cell mixtures and forms spores disproportionately. We found that cheating requires sufficient wild-type cells in the initial mix and can occur both before mound formation and later during the sporulation stage of development. By restricting cheating behavior, short-range C-signaling may have been favored evolutionarily rather than long-range diffusible signaling. Cheating restrictions imposed by short-range signaling may have likewise driven the evolution of multicellularity broadly., Competing Interests: The authors declare no conflict of interest.

This report describes the genome sequences of seven Mannheimia haemolytica isolates recovered from feedlot cattle. These genome sequences will enhance our understanding of Mannheimia haemolytica evolution and antimicrobial resistance (AMR)-associated genes they carried over a two-decade span., Competing Interests: The authors declare no conflict of interest.

Medically important pathogenic fungi invade vertebrate tissue and are considered primary when part of their nature life cycle is associated with an animal host and are usually able to infect immunocompetent hosts. Opportunistic fungal pathogens complete their life cycle in environmental habitats or occur as commensals within or on the vertebrate body, but under certain conditions can thrive upon infecting humans. The extent of host damage in opportunistic infections largely depends on the portal and modality of entry as well as on the host's immune and metabolic status. Diseases caused by primary pathogens and common opportunists, causing the top approximately 80% of fungal diseases [D. W. Denning, Lancet Infect Dis, 24:e428-e438, 2024, https://doi.org/10.1016/S1473-3099(23)00692-8], tend to follow a predictive pattern, while those by occasional opportunists are more variable. For this reason, it is recommended that diseases caused by primary pathogens and the common opportunists are named after the etiologic agent, for example, histoplasmosis and aspergillosis, while this should not be done for occasional opportunists that should be named as [causative fungus] [clinical syndrome], for example, Alternaria alternata cutaneous infection. The addition of a descriptor that identifies the location or clinical type of infection is required, as the general name alone may cover widely different clinical syndromes, for example, "rhinocerebral mucormycosis." A list of major recommended human and animal disease entities (nomenclature) is provided in alignment with their causative agents. Fungal disease names may encompass several genera of etiologic agents, consequently being less susceptible to taxonomic changes of the causative species, for example, mucormycosis covers numerous mucormycetous molds.

Spirobacillus cienkowskii ( Spirobacillus, hereafter) is a widely distributed bacterial pathogen that has significant impacts on the population dynamics of zooplankton ( Daphnia spp .) , particularly in months when Daphnia are asexually reproducing. However, little is known about Spirobacillus' virulence, transmission mode, and dynamics. As a result, we cannot explain the dynamics of Spirobacillus epidemics in nature or use Spirobacillus as a model pathogen, despite Daphnia's tractability as a model host. Here, we work to fill these knowledge gaps experimentally. We found that Spirobacillus is among the most virulent of Daphnia pathogens, killing its host within a week and reducing host fecundity. We further found that Spirobacillus did not transmit horizontally among hosts unless the host died or was destroyed (i.e., it is an "obligate killer"). In experiments aimed at quantifying the dynamics of horizontal transmission among asexually reproducing Daphnia , we demonstrated that Spirobacillus transmits poorly in the laboratory. In mesocosms, Spirobacillus failed to generate epidemics; in experiments wherein individual Daphnia were exposed, Spirobacillus' transmission success was low. In the (limited) set of conditions we considered, Spirobacillus' transmission success did not change with host density or pathogen dose and declined following environmental incubation. Finally, we conducted a field survey of Spirobacillus' prevalence within egg cases (ephippia) made by sexually reproducing Daphnia . We found Spirobacillus DNA in ~40% of ephippia, suggesting that, in addition to transmitting horizontally among asexually reproducing Daphnia , Spirobacillus may transmit vertically from sexually reproducing Daphnia . Our work fills critical gaps in the biology of Spirobacillus and illuminates new hypotheses vis-à-vis its life history., Importance: Spirobacillus cienkowskii is a bacterial pathogen of zooplankton, first described in the 19 th century and recently placed in a new family of bacteria, the Silvanigrellaceae . Spirobacillus causes large epidemics in lake zooplankton populations and increases the probability that zooplankton will be eaten by predators. However, little is known about how Spirobacillus transmits among hosts, to what extent it reduces host survival and reproduction (i.e., how virulent it is), and what role virulence plays in Spirobacillus ' life cycle. Here, we experimentally quantified Spirobacillus' virulence and showed that Spirobacillus must kill its host to transmit horizontally. We also found evidence that Spirobacillus may transmit vertically via Daphnia' s seed-like egg sacks. Our work will help scientists to (i) understand Spirobacillus epidemics, (ii) use Spirobacillus as a model pathogen for the study of host-parasite interactions, and (iii) better understand the unusual group of bacteria to which Spirobacillus belongs., Competing Interests: The authors declare no conflict of interest.

Tupanviruses, members of the family Mimiviridae , infect phagocytic cells. Particle uncoating begins inside the phagosome, with capsid opening via the stargate. The mechanism through which this opening takes place is unknown. Once phagocytized, metal ion flux control and ROS are induced to inactivate foreign particles, including viruses. Here, we studied the effect of iron ions, copper ions, and H 2 O 2 on Tupanvirus particles. Such treatments induced stargate opening in vitro , as observed by different microscopy techniques. Metal-treated viruses were found to be non-infectious, leading to the hypothesis that stargate opening likely resulted in the release of the viral seed, which is required for infection initiation. To the best of our knowledge, this is the first description of a giant virus capsid morphological change induced by transition metals and H 2 O 2 , which may be important to describe new virulence factors and capsid uncoating mechanisms., Competing Interests: The authors declare no conflict of interest.

Mucoromycota fungi and their Mollicutes-related endobacteria (MRE) are an ideal system for studying bacterial-fungal interactions and evolution due to the long-term and intimate nature of their interactions. However, methods for detecting MRE face specific challenges due to the poor representation of MRE in sequencing databases coupled with the high sequence divergence of their genomes, making traditional similarity searches unreliable. This has precluded estimations on the diversity of MRE associated with Mucoromycota. To determine the prevalence of previously undetected MRE in fungal genome sequences, we scanned 389 Mucoromycota genome assemblies available from the National Center for Biotechnology Information for the presence of MRE sequences using publicly available tools to map contigs from fungal assemblies to publicly available MRE genomes. We demonstrate a higher diversity of MRE genomes than previously described in Mucoromycota and a lack of cophylogeny between MRE and the majority of their fungal hosts. This supports the late invasion hypothesis regarding MRE acquisition across most of the examined fungal families. In contrast with other Mucoromycota lineages, MRE from the Gigasporaceae displayed some degree of cophylogeny with their hosts, which may indicate that horizontal transmission is restricted between members of this family or that transmission is strictly vertical. These results underscore the need for a refined process to capture sequencing data from potential fungal endosymbionts to discern their evolution and transmission. Screens of fungal genomes for MRE can help improve the quality of fungal genome assemblies while identifying new MRE lineages to further test hypotheses on their origin and evolution.IMPORTANCEMollicutes-related endobacteria (MRE) are obligate intracellular bacteria found within Mucoromycota fungi. Despite their frequent detection, MRE roles in host functioning are still unknown. Comparative genomic investigations can improve our understanding of the impact of MRE on their fungal hosts by identifying similarities and differences in MRE genome evolution. However, MRE genomes have only been assembled from a small fraction of Mucoromycota hosts. Here, we demonstrate that MRE can be present yet undetected in publicly available Mucoromycota genome assemblies. We use these newfound sequences to assess the broader diversity of MRE and their phylogenetic relationships with respect to their hosts. We demonstrate that publicly available tools can be used to extract novel MRE sequences from assembled fungal genomes leading to insights on MRE evolution. This work contributes to a greater understanding of the fungal microbiome, which is crucial to improving knowledge on the dynamics and impacts of fungi in microbial ecosystems., Competing Interests: The authors declare no conflict of interest.

A major challenge faced by Vibrio cholerae is constant predation by bacteriophage (phage) in aquatic reservoirs and during infection of human hosts. To overcome phage predation, V. cholerae has acquired and/or evolved a myriad of phage defense systems. Although several novel defense systems have been discovered, we hypothesized that more were encoded in V. cholerae given the low diversity of phages that have been isolated, which infect this species. Using a V. cholerae genomic library, we identified a Type IV restriction system consisting of two genes within a 16-kB region of the Vibrio pathogenicity island-2, which we name TgvA and TgvB ( T ype I-embedded g mrSD -like system of V PI-2). We show that both TgvA and TgvB are required for defense against T2, T4, and T6 by targeting glucosylated 5-hydroxymethylcytosine (5hmC). T2 or T4 phages that lose the glucose modifications are resistant to TgvAB defense but exhibit a significant evolutionary tradeoff, becoming susceptible to other Type IV restriction systems that target unglucosylated 5hmC. We also show that the Type I restriction-modification system that embeds the tgvAB genes protects against phage T3, secΦ18, secΦ27, and λ, suggesting that this region is a phage defense island. Our study uncovers a novel Type IV restriction system in V. cholerae , increasing our understanding of the evolution and ecology of V. cholerae, while highlighting the evolutionary interplay between restriction systems and phage genome modification.IMPORTANCEBacteria are constantly being predated by bacteriophage (phage). To counteract this predation, bacteria have evolved a myriad of defense systems. Some of these systems specifically digest infecting phage by recognizing unique base modifications present on the phage DNA. In this study, we discover a Type IV restriction system encoded in V. cholerae, which we name TgvAB, and demonstrate it recognizes and restricts phage that have 5-hydroxymethylcytosine glucosylated DNA. Moreover, the evolution of resistance to TgvAB render phage susceptible to other Type IV restriction systems, demonstrating a significant evolutionary tradeoff. These results enhance our understanding of the evolution of V. cholerae and more broadly how bacteria evade phage predation., Competing Interests: The authors declare no conflict of interest.

The human pathogen Pseudomonas aeruginosa , a leading cause of hospital-acquired infections, inhabits and forms sessile antibiotic-resistant communities called biofilms in a wide range of biotic and abiotic environments. In this study, we examined how two global sensory signaling pathways-the RhlR quorum-sensing system and the CbrA/CbrB nutritional adaptation system-intersect to control biofilm development. Previous work has shown that individually these two systems repress biofilm formation. Here, we used biofilm analyses, RNA-seq, and reporter assays to explore the combined effect of information flow through RhlR and CbrA on biofilm development. We find that the Δ rhlR Δ cbrA double mutant exhibits a biofilm morphology and an associated transcriptional response distinct from wildtype and the parent Δ rhlR and Δ cbrA mutants indicating codominance of each signaling pathway. The Δ rhlR Δ cbrA mutant gains suppressor mutations that allow biofilm expansion; these mutations map to the crc gene resulting in loss of function of the carbon catabolite repression protein Crc. Furthermore, the combined absence of RhlR and CbrA leads to a drastic reduction in the abundance of the Crc antagonist small RNA CrcZ. Thus, CrcZ acts as the molecular convergence point for quorum- and nutrient-sensing cues. We find that in the absence of antagonism by CrcZ, Crc promotes the expression of biofilm matrix components-Pel exopolysaccharide, and CupB and CupC fimbriae. Therefore, this study uncovers a regulatory link between nutritional adaption and quorum sensing with potential implications for anti-biofilm targeting strategies.IMPORTANCEBacteria often form multicellular communities encased in an extracytoplasmic matrix called biofilms. Biofilm development is controlled by various environmental stimuli that are decoded and converted into appropriate cellular responses. To understand how information from two distinct stimuli is integrated, we used biofilm formation in the human pathogen Pseudomonas aeruginosa as a model and studied the intersection of two global sensory signaling pathways-quorum sensing and nutritional adaptation. Global transcriptomics on biofilm cells and reporter assays suggest parallel regulation of biofilms by each pathway that converges on the abundance of a small RNA antagonist of the carbon catabolite repression protein, Crc. We find a new role of Crc as it modulates the expression of biofilm matrix components in response to the environment. These results expand our understanding of the genetic regulatory strategies that allow P. aeruginosa to successfully develop biofilm communities., Competing Interests: The authors declare no conflict of interest.

The ubiquity and ease with which microbial cells disperse over space is a key concept in microbiology, especially in microbial ecology. The phenomenon prompted Baas Becking's famous "everything is everywhere" statement that now acts as the null hypothesis in studies that test the dispersal limitation of microbial taxa. Despite covering the content in lectures, exam performance indicated that the concepts of dispersal and biogeography challenged undergraduate students in an upper-level Microbial Ecology course. Therefore, we iteratively designed a hands-on classroom activity to supplement the lecture content and reinforce fundamental microbial dispersal and biogeography concepts while also building quantitative reasoning and teamwork skills. In a class period soon after the lecture, the students formed three-to-five-person teams to engage in the activity, which included a hands-on dispersal simulation and worksheet to guide discussion. The simulation involved stepwise neutral immigration or emigration and then environmental selection on a random community of microbial taxa represented by craft poms. The students recorded the results at each step as microbial community data. A field guide was provided to identify the taxonomy based on the pom phenotype and a reference to each taxon's preferred environmental niches. The worksheet guided a reflection of student observations during the simulation. It also sharpened quantitative thinking by prompting the students to summarize and visualize their and other teams' microbial community data and then to compare the observed community distributions to the idealized expectation given only selection without dispersal. We found that the activity improved student performance on exam questions and general student satisfaction and comfort with the biogeography concepts. Activity instructions and a list of needed materials are included for instructors to reproduce for their classrooms., Competing Interests: The authors declare no conflict of interest.

Quantitative microbial risk assessment (QMRA) is a growing interdisciplinary field addressing exposures to microbial pathogens and infectious disease processes. Risk science is inherently interdisciplinary, but few of the contributing disciplinary programs offer courses and training specifically in QMRA. To develop multidisciplinary training in QMRA, an annual 10-day long intensive workshop was conducted from 2015 to 2019-the Quantitative Microbial Risk Assessment Interdisciplinary Instructional Institute (QMRA III). National leaders in the fields of public health, engineering, microbiology, epidemiology, communications, public policy, and QMRA served as instructors and mentors over the course of the program. To provide cross-training, multidisciplinary teams of 5-6 trainees were created from the approximately 30 trainees each year. A formal assessment of the program was performed based on observations and surveys containing Likert-type scales and open-ended prompts. In addition, a longitudinal alumni survey was also disseminated to facilitate the future redevelopment of QMRA institutes and determine the impact of the program. Across all years, trainees experienced statistically significant increases ( P < 0.05) in their perceptions of their QMRA abilities (e.g., use of specific computer programs) and knowledge of QMRA constructs (e.g., risk management). In addition, 12 publications, three conference presentations, and two research grants were derived from the QMRA III institute projects or tangential research. The success of QMRA III indicates that a short course format can effectively address many multidisciplinary training needs. Key features of QMRA III, including the inter-disciplinary training approach, hands-on exercises, real-world institute projects, and interaction through a mentoring process, were vital for training multidisciplinary teams housing multiple forms of expertise. Future QMRA institutes are being redeveloped to leverage hybrid learning formats that can further the multidisciplinary training and mentoring objectives., Competing Interests: The authors declare no conflict of interest.

Vibrio cholerae is a Gram-negative gastrointestinal pathogen responsible for the diarrheal disease cholera. Expression of key virulence factors, cholera toxin and toxin-coregulated pilus, is regulated directly by ToxT and indirectly by two transmembrane transcription regulators (TTRs), ToxR and TcpP, that promote the expression of toxT . TcpP abundance and activity are controlled by TcpH, a single-pass transmembrane protein, which protects TcpP from a two-step proteolytic process known as regulated intramembrane proteolysis (RIP). The mechanism of TcpH-mediated protection of TcpP represents a major gap in our understanding of V. cholerae pathogenesis. The absence of tcpH leads to unimpeded degradation of TcpP in vitro and a colonization defect in a neonate mouse model of V. cholerae colonization. Here, we show that TcpH protects TcpP from RIP via direct interaction. We also demonstrate that α-linolenic acid, a dietary fatty acid, promotes TcpH-dependent inhibition of RIP via co-association of TcpP and TcpH molecules within detergent-resistant membranes (DRMs) in a mechanism requiring the TcpH transmembrane domain. Taken together, our data support a model where V. cholerae cells use exogenous α-linolenic acid to remodel the phospholipid bilayer in vivo , leading to co-association of TcpP and TcpH within DRMs where RIP of TcpP is inhibited by TcpH, thereby promoting V. cholerae pathogenicity., Importance: Vibrio cholerae continues to pose a significant global burden on health and an alternative therapeutic approach is needed, due to evolving multidrug resistance strains. Transcription of toxT , stimulated by TcpP and ToxR, is essential for V. cholerae pathogenesis. Our results show that TcpP, one of the major regulators of toxT gene expression, is protected from proteolysis by TcpH, via direct interaction. Furthermore, we identified a gut metabolite, α-linolenic acid, that stimulates the co-association of TcpP and TcpH within detergent-resistant membranes (also known as lipid-ordered membrane domains), thereby supporting TcpH-dependent antagonism of TcpP proteolysis. Data presented here extend our knowledge of RIP, virulence gene regulation in V. cholerae , and, to the best of our knowledge, provides the first evidence that lipid-ordered membranes exist within V. cholerae . The model presented here also suggests that TTRs, common among bacteria and archaea, and co-component signal transduction systems present in Enterobacteria , could also be influenced similarly., Competing Interests: The authors declare no conflict of interest.

Nasopharyngeal carriage of staphylococci spreads potentially pathogenic strains into (peri)oral regions and increases the chance of cross-infections. Some laboratory strains can also move rapidly on hydrated agar surfaces, but the biological relevance of these observations is not clear. Using soft-agar [0.3% (wt/vol)] plate assays, we demonstrate the rapid surface dispersal of (peri)oral isolates of Staphylococcus aureus and Staphylococcus epidermidis and closely related laboratory strains in the presence of mucin glycoproteins. Mucin-induced dispersal was a stepwise process initiated by the passive spreading of the growing colonies followed by their rapid branching (dendrites) from the colony edge. Although most spreading strains used mucin as a growth substrate, dispersal was primarily dependent on the lubricating and hydrating properties of the mucins. Using S. aureus JE2 as a genetically tractable representative, we demonstrate that mucin-induced dendritic dispersal, but not colony spreading, is facilitated by the secretion of surfactant-active phenol-soluble modulins (PSMs) in a process regulated by the agr quorum-sensing system. Furthermore, the dendritic dispersal of S. aureus JE2 colonies was further stimulated in the presence of surfactant-active supernatants recovered from the most robust (peri)oral spreaders of S. aureus and S. epidermidis . These findings suggest complementary roles for lubricating mucins and staphylococcal PSMs in the active dispersal of potentially pathogenic strains from perioral to respiratory mucosae, where gel-forming, hydrating mucins abound. They also highlight the impact that interspecies interactions have on the co-dispersal of S. aureus with other perioral bacteria, heightening the risk of polymicrobial infections and the severity of the clinical outcomes., Importance: Despite lacking classical motility machinery, nasopharyngeal staphylococci spread rapidly in (peri)oral and respiratory mucosa and cause cross-infections. We describe laboratory conditions for the reproducible study of staphylococcal dispersal on mucosa-like surfaces and the identification of two dispersal stages (colony spreading and dendritic expansion) stimulated by mucin glycoproteins. The mucin type mattered as dispersal required the surfactant activity and hydration provided by some mucin glycoproteins. While colony spreading was a passive mode of dispersal lubricated by the mucins, the more rapid and invasive form of dendritic expansion of Staphylococcus aureus and Staphylococcus epidermidis required additional lubrication by surfactant-active peptides (phenol-soluble modulins) secreted at high cell densities through quorum sensing. These results highlight a hitherto unknown role for gel-forming mucins in the dispersal of staphylococcal strains associated with cross-infections and point at perioral regions as overlooked sources of carriage and infection by staphylococci., Competing Interests: The authors declare no conflict of interest.

Bacteria have developed diverse strategies for defending their cell envelopes from external threats. In Firmicutes, one widespread strategy is to use Bce modules-membrane protein complexes that unite a peptide-detoxifying ABC transporter with a stress response coordinating two-component system. These modules provide specific, front-line defense for a wide variety of antimicrobial peptides and small molecule antibiotics as well as coordinate responses for heat, acid, and oxidative stress. Because of these abilities, Bce modules play important roles in virulence and the development of antibiotic resistance in a variety of pathogens, including Staphylococcus , Streptococcus , and Enterococcus species. Despite their importance, Bce modules are still poorly understood, with scattered functional data in only a small number of species. In this review, we will discuss Bce module structure in light of recent cryo-electron microscopy structures of the B. subtilis BceABRS module and explore the common threads and variations-on-a-theme in Bce module mechanisms across species. We also highlight the many remaining questions about Bce module function. Understanding these multifunctional membrane complexes will enhance our understanding of bacterial stress sensing and may point toward new therapeutic targets for highly resistant pathogens., Competing Interests: The authors declare no conflict of interest.

We provide a collection of 78 bacterial isolates from the rhizosphere of switchgrass ( Panicum virgatum L .) at the Lux Arbor Reserve in Delton, MI, a site of the Great Lakes Bioenergy Research Center (GLBRC), Michigan State University, MI, USA. We include information on isolation conditions and full-length 16S rRNA sequences., Competing Interests: The authors declare no conflict of interest.

Rapid climate change in the Arctic is altering microbial structure and function, with important consequences for the global ecosystem. Emerging evidence suggests organisms in higher trophic levels may also influence microbial communities, but whether warming alters these effects is unclear. Wolf spiders are dominant Arctic predators whose densities are expected to increase with warming. These predators have temperature-dependent effects on decomposition via their consumption of fungal-feeding detritivores, suggesting they may indirectly affect the microbial structure as well. To address this, we used a fully factorial mesocosm experiment to test the effects of wolf spider density and warming on litter microbial structure in Arctic tundra. We deployed replicate litter bags at the surface and belowground in the organic soil profile and analyzed the litter for bacterial and fungal community structure, mass loss, and nutrient characteristics after 2 and 14 months. We found there were significant interactive effects of wolf spider density and warming on fungal but not bacterial communities. Specifically, higher wolf spider densities caused greater fungal diversity under ambient temperature but lower fungal diversity under warming at the soil surface. We also observed interactive treatment effects on fungal composition belowground. Wolf spider density influenced surface bacterial composition, but the effects did not change with warming. These findings suggest a widespread predator can have indirect, cascading effects on litter microbes and that effects on fungi specifically shift under future expected levels of warming. Overall, our study highlights that trophic interactions may play important, albeit overlooked, roles in driving microbial responses to warming in Arctic terrestrial ecosystems., Importance: The Arctic contains nearly half of the global pool of soil organic carbon and is one of the fastest warming regions on the planet. Accelerated decomposition of soil organic carbon due to warming could cause positive feedbacks to climate change through increased greenhouse gas emissions; thus, changes in ecological dynamics in this region are of global relevance. Microbial structure is an important driver of decomposition and is affected by both abiotic and biotic conditions. Yet how activities of soil-dwelling organisms in higher trophic levels influence microbial structure and function is unclear. In this study, we demonstrate that predicted changes in abundances of a dominant predator and warming interactively affect the structure of litter-dwelling fungal communities in the Arctic. These findings suggest predators may have widespread, indirect cascading effects on microbial communities, which could influence ecosystem responses to future climate change., Competing Interests: The authors declare no conflict of interest.

Bacterial phage shock protein (PSP) systems stabilize the bacterial cell membrane and protect against envelope stress. These systems have been associated with virulence, but despite their critical roles, PSP components are not well characterized outside proteobacteria. Using comparative genomics and protein sequence-structure-function analyses, we systematically identified and analyzed PSP homologs, phyletic patterns, domain architectures, and gene neighborhoods. This approach underscored the evolutionary significance of the system, revealing that its core protein PspA (Snf7 in ESCRT outside bacteria) was present in the last universal common ancestor and that this ancestral functionality has since diversified into multiple novel, distinct PSP systems across life. Several novel partners of the PSP system were identified: (i) the Toastrack domain, likely facilitating assembly of sub-membrane stress-sensing and signaling complexes, (ii) the newly defined HTH-associated α-helical signaling domain-PadR-like transcriptional regulator pair system, and (iii) multiple independent associations with ATPase, CesT/Tir-like chaperone, and Band-7 domains in proteins thought to mediate sub-membrane dynamics. Our work also uncovered links between the PSP components and other domains, such as novel variants of SHOCT-like domains, suggesting roles in assembling membrane-associated complexes of proteins with disparate biochemical functions. Results are available at our interactive web app, https://jravilab.org/psp.IMPORTANCEPhage shock proteins (PSP) are virulence-associated, cell membrane stress-protective systems. They have mostly been characterized in Proteobacteria and Firmicutes. We now show that a minimal PSP system was present in the last universal common ancestor that evolved and diversified into newly identified functional contexts. Recognizing the conservation and evolution of PSP systems across bacterial phyla contributes to our understanding of stress response mechanisms in prokaryotes. Moreover, the newly discovered PSP modularity will likely prompt new studies of lineage-specific cell envelope structures, lifestyles, and adaptation mechanisms. Finally, our results validate the use of domain architecture and genetic context for discovery in comparative genomics., Competing Interests: The authors declare no conflict of interest.

Most of Earth's trees rely on critical soil nutrients that ectomycorrhizal fungi (EcMF) liberate and provide, and all of Earth's land plants associate with bacteria that help them survive in nature. Yet, our understanding of how the presence of EcMF modifies soil bacterial communities, soil food webs, and root chemistry requires direct experimental evidence to comprehend the effects that EcMF may generate in the belowground plant microbiome. To this end, we grew Pinus muricata plants in soils that were either inoculated with EcMF and native forest bacterial communities or only native bacterial communities. We then profiled the soil bacterial communities, applied metabolomics and lipidomics, and linked omics data sets to understand how the presence of EcMF modifies belowground biogeochemistry, bacterial community structure, and their functional potential. We found that the presence of EcMF (i) enriches soil bacteria linked to enhanced plant growth in nature, (ii) alters the quantity and composition of lipid and non-lipid soil metabolites, and (iii) modifies plant root chemistry toward pathogen suppression, enzymatic conservation, and reactive oxygen species scavenging. Using this multi-omic approach, we therefore show that this widespread fungal symbiosis may be a common factor for structuring soil food webs.IMPORTANCEUnderstanding how soil microbes interact with one another and their host plant will help us combat the negative effects that climate change has on terrestrial ecosystems. Unfortunately, we lack a clear understanding of how the presence of ectomycorrhizal fungi (EcMF)-one of the most dominant soil microbial groups on Earth-shapes belowground organic resources and the composition of bacterial communities. To address this knowledge gap, we profiled lipid and non-lipid metabolites in soils and plant roots, characterized soil bacterial communities, and compared soils amended either with or without EcMF. Our results show that the presence of EcMF changes soil organic resource availability, impacts the proliferation of different bacterial communities (in terms of both type and potential function), and primes plant root chemistry for pathogen suppression and energy conservation. Our findings therefore provide much-needed insight into how two of the most dominant soil microbial groups interact with one another and with their host plant., Competing Interests: The authors declare no conflict of interest.

The impacts of microsporidia on host individuals are frequently subtle and can be context dependent. A key example of the latter comes from a recently discovered microsporidian symbiont of Daphnia , the net impact of which was found to shift from negative to positive based on environmental context. Given this, we hypothesized low baseline virulence of the microsporidian; here, we investigated the impact of infection on hosts in controlled conditions and the absence of other stressors. We also investigated its phylogenetic position, ecology, and host range. The genetic data indicate that the symbiont is Ordospora pajunii , a newly described microsporidian parasite of Daphnia . We show that O. pajunii infection damages the gut, causing infected epithelial cells to lose microvilli and then rupture. The prevalence of this microsporidian could be high (up to 100% in the lab and 77% of adults in the field). Its overall virulence was low in most cases, but some genotypes suffered reduced survival and/or reproduction. Susceptibility and virulence were strongly host-genotype dependent. We found that North American O. pajunii were able to infect multiple Daphnia species, including the European species Daphnia longispina , as well as Ceriodaphnia spp. Given the low, often undetectable virulence of this microsporidian and potentially far-reaching consequences of infections for the host when interacting with other pathogens or food, this Daphnia-O. pajunii symbiosis emerges as a valuable system for studying the mechanisms of context-dependent shifts between mutualism and parasitism, as well as for understanding how symbionts might alter host interactions with resources., Importance: The net outcome of symbiosis depends on the costs and benefits to each partner. Those can be context dependent, driving the potential for an interaction to change between parasitism and mutualism. Understanding the baseline fitness impact in an interaction can help us understand those shifts; for an organism that is generally parasitic, it should be easier for it to become a mutualist if its baseline virulence is relatively low. Recently, a microsporidian was found to become beneficial to its Daphnia hosts in certain ecological contexts, but little was known about the symbiont (including its species identity). Here, we identify it as the microsporidium Ordospora pajunii . Despite the parasitic nature of microsporidia, we found O. pajunii to be, at most, mildly virulent; this helps explain why it can shift toward mutualism in certain ecological contexts and helps establish O. pajunii is a valuable model for investigating shifts along the mutualism-parasitism continuum., Competing Interests: The authors declare no conflict of interest.

We examined the dynamics of soil microbiomes under heat press disturbance from an underground coal mine fire in Centralia, PA. Here, we present metagenomic sequencing and assembly data from soil microbiomes across seven consecutive years at repeatedly sampled fire-affected sites along with unaffected reference sites., Competing Interests: The authors declare no conflict of interest.

It has been nearly a century since the isolation and use of penicillin, heralding the discovery of a wide range of different antibiotics. In addition to clinical applications, such antibiotics have been essential laboratory tools, allowing for selection and maintenance of laboratory plasmids that encode cognate resistance genes. However, antibiotic resistance mechanisms can additionally function as public goods. For example, extracellular beta-lactamases produced by resistant cells that subsequently degrade penicillin and related antibiotics allow neighboring plasmid-free susceptible bacteria to survive antibiotic treatment. How such cooperative mechanisms impact selection of plasmids during experiments in laboratory conditions is poorly understood. Here, we show in multiple bacterial species that the use of plasmid-encoded beta-lactamases leads to significant curing of plasmids in surface-grown bacteria. Furthermore, such curing was also evident for aminoglycoside phosphotransferase and tetracycline antiporter resistance mechanisms. Alternatively, antibiotic selection in liquid growth led to more robust plasmid maintenance, although plasmid loss was still observed. The net outcome of such plasmid loss is the generation of a heterogenous population of plasmid-containing and plasmid-free cells, leading to experimental confounds that are not widely appreciated.IMPORTANCEPlasmids are routinely used in microbiology as readouts of cell biology or tools to manipulate cell function. Central to these studies is the assumption that all cells in an experiment contain the plasmid. Plasmid maintenance in a host cell typically depends on a plasmid-encoded antibiotic resistance marker, which provides a selective advantage when the plasmid-containing cell is grown in the presence of antibiotic. Here, we find that growth of plasmid-containing bacteria on a surface and to a lesser extent in liquid culture in the presence of three distinct antibiotic families leads to the evolution of a significant number of plasmid-free cells, which rely on the resistance mechanisms of the plasmid-containing cells. This process generates a heterogenous population of plasmid-free and plasmid-containing bacteria, an outcome which could confound further experimentation., Competing Interests: The authors declare no conflict of interest.

During prolonged resource limitation, bacterial cells can persist in metabolically active states of non-growth. These maintenance periods, such as those experienced in stationary phase, can include upregulation of secondary metabolism and release of exometabolites into the local environment. As resource limitation is common in many environmental microbial habitats, we hypothesized that neighboring bacterial populations employ exometabolites to compete or cooperate during maintenance and that these exometabolite-facilitated interactions can drive community outcomes. Here, we evaluated the consequences of exometabolite interactions over the stationary phase among three environmental strains: Burkholderia thailandensis E264, Chromobacterium subtsugae ATCC 31532 , and Pseudomonas syringae pv. tomato DC3000. We assembled them into synthetic communities that only permitted chemical interactions. We compared the responses (transcripts) and outputs (exometabolites) of each member with and without neighbors. We found that transcriptional dynamics were changed with different neighbors and that some of these changes were coordinated between members. The dominant competitor B. thailandensis consistently upregulated biosynthetic gene clusters to produce bioactive exometabolites for both exploitative and interference competition. These results demonstrate that competition strategies during maintenance can contribute to community-level outcomes. It also suggests that the traditional concept of defining competitiveness by growth outcomes may be narrow and that maintenance competition could be an additional or alternative measure., Importance: Free-living microbial populations often persist and engage in environments that offer few or inconsistently available resources. Thus, it is important to investigate microbial interactions in this common and ecologically relevant condition of non-growth. This work investigates the consequences of resource limitation for community metabolic output and for population interactions in simple synthetic bacterial communities. Despite non-growth, we observed active, exometabolite-mediated competition among the bacterial populations. Many of these interactions and produced exometabolites were dependent on the community composition but we also observed that one dominant competitor consistently produced interfering exometabolites regardless. These results are important for predicting and understanding microbial interactions in resource-limited environments., Competing Interests: The authors declare no conflict of interest.

The RDP Classifier is one of the most popular machine learning approaches for taxonomic classification due to its robustness and relatively high accuracy. Both the RDP taxonomy and RDP Classifier have been updated to incorporate newly described taxa and recent changes to prokaryotic nomenclature., Competing Interests: The authors declare no conflict of interest.

The human papillomavirus (HPV) oncoprotein E7 is a relatively short-lived protein required for HPV-driven cancer development and maintenance. E7 is degraded through ubiquitination mediated by cullin 1 (CUL1) and the ubiquitin-conjugating enzyme E2 L3 (UBE2L3). However, E7 proteins are maintained at high levels in most HPV-positive cancer cells. A previous proteomics study has shown that UBE2L3 and CUL1 protein levels are increased by the knockdown of the E3 ubiquitin ligase membrane-associated ring-CH-type finger 8 (MARCHF8). We have recently demonstrated that HPV16 upregulates MARCHF8 expression in HPV-positive keratinocytes and head and neck cancer (HPV+ HNC) cells. Here, we report that MARCHF8 stabilizes the HPV16 E7 protein by degrading the components of the S-phase kinase-associated protein 1-CUL1-F-box ubiquitin ligase complex in HPV+ HNC cells. We found that MARCHF8 knockdown in HPV+ HNC cells drastically decreases the HPV16 E7 protein level while increasing the CUL1 and UBE2L3 protein levels. We further revealed that the MARCHF8 protein binds to and ubiquitinates CUL1 and UBE2L3 proteins and that MARCHF8 knockdown enhances the ubiquitination of the HPV16 E7 protein. Conversely, the overexpression of CUL1 and UBE2L3 in HPV+ HNC cells decreases HPV16 E7 protein levels and suppresses tumor growth in vivo . Our findings suggest that HPV-induced MARCHF8 prevents the degradation of the HPV16 E7 protein in HPV+ HNC cells by ubiquitinating and degrading CUL1 and UBE2L3 proteins.IMPORTANCESince human papillomavirus (HPV) oncoprotein E7 is essential for virus replication; HPV has to maintain high levels of E7 expression in HPV-infected cells. However, HPV E7 can be efficiently ubiquitinated by a ubiquitin ligase and degraded by proteasomes in the host cell. Mechanistically, the E3 ubiquitin ligase complex cullin 1 (CUL1) and ubiquitin-conjugating enzyme E2 L3 (UBE2L3) components play an essential role in E7 ubiquitination and degradation. Here, we show that the membrane ubiquitin ligase membrane-associated ring-CH-type finger 8 (MARCHF8) induced by HPV16 E6 stabilizes the E7 protein by degrading CUL1 and UBE2L3 and blocking E7 degradation through proteasomes. MARCHF8 knockout restores CUL1 and UBE2L3 expression, decreasing E7 protein levels and inhibiting the proliferation of HPV-positive cancer cells. Additionally, overexpression of CUL1 or UBE2L3 decreases E7 protein levels and suppresses in vivo tumor growth. Our results suggest that HPV16 maintains high E7 protein levels in the host cell by inducing MARCHF8, which may be critical for cell proliferation and tumorigenesis., Competing Interests: The authors declare no conflict of interest.

Bile acids (BAs) are cholesterol-derived molecules that aid in digestion and nutrient absorption, regulate host metabolic processes, and influence physiology of the gut microbiota. Both the host and its microbiome contribute to enzymatic modifications that shape the chemical diversity of BAs in the gut. Several bacterial species have been reported to conjugate standard amino acids to BAs, but it was not known if bacteria conjugate BAs to other amine classes. Here, we show that Bacteroides fragilis strain P207, isolated from a bacterial bloom in the J-pouch of a patient with ulcerative colitis pouchitis, conjugates standard amino acids and the neuroactive amines γ-aminobutyric acid (GABA) and tyramine to deoxycholic acid. We extended this analysis to other human gut isolates and identified species that are competent to conjugate GABA and tyramine to primary and secondary BAs, and further identified diverse BA-GABA and BA-tyramine amides in human stool. A longitudinal metabolomic analysis of J-pouch contents of the patient from whom B. fragilis P207 was isolated revealed highly reduced levels of secondary bile acids and a shifting BA amide profile before, during, and after onset of pouchitis, including temporal changes in several BA-GABA amides. Treatment of pouchitis with ciprofloxacin was associated with a marked reduction of nearly all BA amides in the J-pouch. Our study expands the known repertoire of conjugated bile acids produced by bacteria to include BA conjugates to GABA and tyramine and demonstrates that these molecules are present in the human gut. IMPORTANCE BAs are modified in multiple ways by host enzymes and the microbiota to produce a chemically diverse set of molecules that assist in the digestive process and impact many physiological functions. This study reports the discovery of bacterial species that conjugate the neuroactive amines, GABA and tyramine, to primary and secondary BAs. We further present evidence that BA-GABA and BA-tyramine conjugates are present in the human gut, and document a shifting BA-GABA profile in a human pouchitis patient before, during, and after inflammation and antibiotic treatment. GABA and tyramine are common metabolic products of the gut microbiota and potent neuroactive molecules. GABA- and tyramine-conjugated BAs may influence receptor-mediated regulatory mechanisms of humans and their gut microbes, and absorption of these molecules and their entry into enterohepatic circulation may impact host physiology at distal tissue sites. This study defines new conjugated bile acids in the human gut., Competing Interests: The authors declare no conflict of interest.

Wastewater-based epidemiology (WBE) expanded rapidly in response to the COVID-19 pandemic. As the public health emergency has ended, researchers and practitioners are looking to shift the focus of existing wastewater surveillance programs to other targets, including bacteria. Bacterial targets may pose some unique challenges for WBE applications. To explore the current state of the field, the National Science Foundation-funded Research Coordination Network (RCN) on Wastewater Based Epidemiology for SARS-CoV-2 and Emerging Public Health Threats held a workshop in April 2023 to discuss the challenges and needs for wastewater bacterial surveillance. The targets and methods used in existing programs were diverse, with twelve different targets and nine different methods listed. Discussions during the workshop highlighted the challenges in adapting existing programs and identified research gaps in four key areas: choosing new targets, relating bacterial wastewater data to human disease incidence and prevalence, developing methods, and normalizing results. To help with these challenges and research gaps, the authors identified steps the larger community can take to improve bacteria wastewater surveillance. This includes developing data reporting standards and method optimization and validation for bacterial programs. Additionally, more work is needed to understand shedding patterns for potential bacterial targets to better relate wastewater data to human infections. Wastewater surveillance for bacteria can help provide insight into the underlying prevalence in communities, but much work is needed to establish these methods.IMPORTANCEWastewater surveillance was a useful tool to elucidate the burden and spread of SARS-CoV-2 during the pandemic. Public health officials and researchers are interested in expanding these surveillance programs to include bacterial targets, but many questions remain. The NSF-funded Research Coordination Network for Wastewater Surveillance of SARS-CoV-2 and Emerging Public Health Threats held a workshop to identify barriers and research gaps to implementing bacterial wastewater surveillance programs., Competing Interests: RUH is managing member of AquaVitas, LLC and founder of the ASU non-profit project OneWaterOneHealth operating in the same intellectual space. LPS is employed by Ceres Nanosciences.

Extracellular electron transfer is a process by which bacterial cells can exchange electrons with a redox-active material located outside of the cell. In Shewanella oneidensis , this process is natively used to facilitate respiration using extracellular electron acceptors such as Fe(III) or an anode. Previously, it was demonstrated that this process can be used to drive the microbial electrosynthesis (MES) of 2,3-butanediol (2,3-BDO) in S. oneidensis exogenously expressing butanediol dehydrogenase (BDH). Electrons taken into the cell from a cathode are used to generate NADH, which in turn is used to reduce acetoin to 2,3-BDO via BDH. However, generating NADH via electron uptake from a cathode is energetically unfavorable, so NADH dehydrogenases couple the reaction to proton motive force. We therefore need to maintain the proton gradient across the membrane to sustain NADH production. This work explores accomplishing this task by bidirectional electron transfer, where electrons provided by the cathode go to both NADH formation and oxygen (O 2 ) reduction by oxidases. We show that oxidases use trace dissolved oxygen in a microaerobic bioelectrical chemical system (BES), and the translocation of protons across the membrane during O 2 reduction supports 2,3-BDO generation. Interestingly, this process is inhibited by high levels of dissolved oxygen in this system. In an aerated BES, O 2 molecules react with the strong reductant (cathode) to form reactive oxygen species, resulting in cell death.IMPORTANCEMicrobial electrosynthesis (MES) is increasingly employed for the generation of specialty chemicals, such as biofuels, bioplastics, and cancer therapeutics. For these systems to be viable for industrial scale-up, it is important to understand the energetic requirements of the bacteria to mitigate unnecessary costs. This work demonstrates sustained production of an industrially relevant chemical driven by a cathode. Additionally, it optimizes a previously published system by removing any requirement for phototrophic energy, thereby removing the additional cost of providing a light source. We also demonstrate the severe impact of oxygen intrusion into bioelectrochemical systems, offering insight to future researchers aiming to work in an anaerobic environment. These studies provide insight into both the thermodynamics of electrosynthesis and the importance of the bioelectrochemical systems' design., Competing Interests: The authors declare no conflict of interest.

Importance: The Gram-negative bacterium Bacteroides fragilis is a common member of the human gut microbiota that colonizes multiple host niches and can influence human physiology through a variety of mechanisms. Identification of genes that enable B. fragilis to grow across a range of host environments has been impeded in part by the relatively limited genetic tractability of this species. We have developed a high-throughput genetic resource for a B. fragilis strain isolated from a UC pouchitis patient. Bile acids limit microbial growth and are altered in abundance in UC pouches, where B. fragilis often blooms. Using this resource, we uncovered pathways and processes that impact B. fragilis fitness in bile and that may contribute to population expansions during bouts of gut inflammation., Competing Interests: The authors declare no conflict of interest.

Importance: A key to producing next-generation biofuels is to engineer microbes that efficiently convert non-food materials into drop-in fuels, and to engineer microbes effectively, we must understand their metabolism thoroughly. Zymomonas mobilis is a bacterium that is a promising candidate biofuel producer, but its metabolism remains poorly understood, especially its metabolism when exposed to oxygen. Although Z. mobilis respires with oxygen, its aerobic growth is poor, and disruption of genes related to respiration counterintuitively improves aerobic growth. This unusual result has sparked decades of research and debate regarding the function of respiration in Z. mobilis . Here, we used a new set of mutants to determine that respiration is essential for aerobic growth and likely protects the cells from damage caused by oxygen. We conclude that the respiratory pathway of Z. mobilis should not be deleted from chassis strains for industrial production because this would yield a strain that is intolerant of oxygen, which is more difficult to manage in industrial settings., Competing Interests: The authors declare no conflict of interest.

Importance: As intracellular pathogens, Brucella must contend with a variety of host-derived stressors when infecting a host cell. The inner membrane, cell wall, and outer membrane, i.e. the cell envelope, of Brucella provide a critical barrier to host assault. A conserved regulatory mechanism known as two-component signaling (TCS) commonly controls transcription of genes that determine the structure and biochemical composition of the cell envelope during stress. We report the identification of previously uncharacterized TCS genes that determine Brucella ovis fitness in the presence of cell envelope disruptors and within infected mammalian host cells. Our study reveals a new molecular mechanism of TCS-dependent gene regulation, and thereby advances fundamental understanding of transcriptional regulatory processes in bacteria., Competing Interests: The authors declare no conflict of interest.

Importance: The inherent complexity of biological systems is a major barrier to our understanding of cellular physiology. Bacteria with markedly fewer genes than their close relatives, or reduced genome bacteria, are promising biological models with less complexity. Reduced genome bacteria can also have superior properties for industrial use, provided the reduction does not overly restrict strain robustness. Naturally reduced genome bacteria, such as the alphaproteobacterium Zymomonas mobilis , have fewer genes but remain environmentally robust. In this study, we show that Z. mobilis is a simplified genetic model for Alphaproteobacteria, a class with important impacts on the environment, human health, and industry. We also identify genes that are only required in the absence of atmospheric oxygen, uncovering players that maintain and utilize the cellular energy state. Our findings have broad implications for the genetics of Alphaproteobacteria and industrial use of Z. mobilis to create biofuels and bioproducts., Competing Interests: Jason M. Peters and Amy B. Banta have filed for patents related to Mobile-CRISPRi technology and bacterial promoters.

The draft genome of Flavobacterium tructae strain S12, isolated from hatchery-reared Chinook salmon ( Oncorhynchus tshawytscha ) fingerlings, consisted of 5,695,942 bp, a G + C content of 35.6%, 4,775 predicted open reading frames, a putative type IX secretion system, collagenase, and hemolysin. F. tructae strains can be used as models for emerging Flavobacterium pathogens., Competing Interests: The authors declare no conflict of interest.

A collection of 44 isolates isolated from the epicuticular wax of stems of energy sorghum is available at the Great Lakes Bioenergy Researcher Center, Michigan State University, MI, USA. We enriched bacteria with putative plant-beneficial phenotypes and include information on their phenotypic diversity, taxonomy, and whole-genome sequences., Competing Interests: The authors declare no conflict of interest.

A collection of 47 bacteria isolated from the mucilage of aerial roots of energy sorghum is available at the Great Lakes Bioenergy Research Center, Michigan State University, Michigan, USA. We enriched bacteria with putative plant-beneficial phenotypes and included information on phenotypic diversity, taxonomy, and whole genome sequences., Competing Interests: The authors declare no conflict of interest.

Importance: To counteract infection with phage, bacteria have evolved a myriad of molecular defense systems. Some of these systems initiate a process called abortive infection, in which the infected cell kills itself to prevent phage propagation. However, such systems must be inhibited in the absence of phage infection to prevent spurious death of the host. Here, we show that the cyclic oligonucleotide based anti-phage signaling system (CBASS) accomplishes this by sensing intracellular folate molecules and only expressing this system in a group. These results enhance our understanding of the evolution of the seventh Vibrio cholerae pandemic and more broadly how bacteria defend themselves against phage infection., Competing Interests: The authors declare no conflict of interest.

Importance: Many free-swimming bacteria propel themselves through liquid using rotary flagella, and mounting evidence suggests that the inhibition of flagellar rotation initiates biofilm formation, a sessile lifestyle that is a nearly universal surface colonization paradigm in bacteria. In general, motility and biofilm formation are inversely regulated by the intracellular second messenger bis-(3´-5´)-cyclic dimeric guanosine monophosphate (c-di-GMP). Here, we identify a protein, PlzD, bearing a conserved c-di-GMP binding PilZ domain that localizes to the flagellar pole in a c-di-GMP-dependent manner and alters the foraging behavior, biofilm, and virulence characteristics of the opportunistic human pathogen, Vibrio vulnificus . Our data suggest that PlzD interacts with components of the flagellar stator to decrease bacterial swimming speed and changes in swimming direction, and these activities are enhanced when cellular c-di-GMP levels are elevated. These results reveal a physical link between a second messenger (c-di-GMP) and an effector (PlzD) that promotes transition from a motile to a sessile state in V. vulnificus ., Competing Interests: The authors declare no conflict of interest.

A suite of molecular sensory systems enables Caulobacter to control growth, development, and reproduction in response to levels of essential elements. The bacterial enhancer-binding protein (bEBP) NtrC and its cognate sensor histidine kinase, NtrB, are key regulators of nitrogen assimilation in many bacteria, but their roles in Caulobacter metabolism and development are not well defined. Notably, Caulobacter NtrC is an unconventional bEBP that lacks the σ 54 -interacting loop commonly known as the GAFTGA motif. Here we show that deletion of Caulobacter crescentus ntrC slows cell growth in complex medium and that ntrB and ntrC are essential when ammonium is the sole nitrogen source due to their requirement for glutamine synthetase expression. Random transposition of a conserved IS3-family mobile genetic element frequently rescued the growth defect of ntrC mutant strains by restoring transcription of the glnBA operon, revealing a possible role for IS3 transposition in shaping the evolution of Caulobacter populations during nutrient limitation. We further identified dozens of direct NtrC-binding sites on the C. crescentus chromosome, with a large fraction located near genes involved in polysaccharide biosynthesis. The majority of binding sites align with those of the essential nucleoid-associated protein, GapR, or the cell cycle regulator, MucR1. NtrC is therefore predicted to directly impact the regulation of cell cycle and cell development. Indeed, loss of NtrC function led to elongated polar stalks and elevated synthesis of cell envelope polysaccharides. This study establishes regulatory connections between NtrC, nitrogen metabolism, polar morphogenesis, and envelope polysaccharide synthesis in Caulobacter . IMPORTANCE Bacteria balance cellular processes with the availability of nutrients in their environment. The NtrB-NtrC two-component signaling system is responsible for controlling nitrogen assimilation in many bacteria. We have characterized the effect of ntrB and ntrC deletion on Caulobacter growth and development and uncovered a role for spontaneous IS element transposition in the rescue of transcriptional and nutritional deficiencies caused by ntrC mutation. We further defined the regulon of Caulobacter NtrC, a bacterial enhancer-binding protein, and demonstrate that it shares specific binding sites with essential proteins involved in cell cycle regulation and chromosome organization. Our work provides a comprehensive view of transcriptional regulation mediated by a distinctive NtrC protein, establishing its connection to nitrogen assimilation and developmental processes in Caulobacter ., Competing Interests: The authors declare no conflict of interest.

Mycobacteria can colonize environments where the availability of metal ions is limited. Biological or inorganic chelators play an important role in limiting metal availability, and we developed a model to examine Mycobacterium smegmatis survival in the presence of the chelator sodium citrate. We observed that instead of restricting M. smegmatis growth, concentrated sodium citrate killed M. smegmatis . RNAseq analysis during sodium citrate treatment revealed transcriptional signatures of metal starvation and hyperosmotic stress. Notably, metal starvation and hyperosmotic stress, individually, do not kill M. smegmatis under these conditions. A forward genetic transposon selection was conducted to examine why sodium citrate was lethal, and several sodium-citrate-tolerant mutants were isolated. Based on the identity of three tolerant mutants, mgtE , treZ , and fadD6, we propose a dual stress model of killing by sodium citrate, where sodium citrate chelate metals from the cell envelope and then osmotic stress in combination with a weakened cell envelope causes cell lysis. This sodium citrate tolerance screen identified mutants in several other genes with no known function, with most conserved in the pathogen M. tuberculosis . Therefore, this model will serve as a basis to define their functions, potentially in maintaining cell wall integrity, cation homeostasis, or osmotolerance. IMPORTANCE Bacteria require mechanisms to adapt to environments with differing metal availability. When Mycobacterium smegmatis is treated with high concentrations of the metal chelator sodium citrate, the bacteria are killed. To define the mechanisms underlying killing by sodium citrate, we conducted a genetic selection and observed tolerance to killing in mutants of the mgtE magnesium transporter. Further characterization studies support a model where killing by sodium citrate is driven by a weakened cell wall and osmotic stress, that in combination cause cell lysis., Competing Interests: R.B.A. is the founder and owner of Tarn Biosciences, Inc., a company that is working to develop new antimycobacterial drugs.

Nitro-containing compounds have emerged as important agents in the control of tuberculosis (TB). From a whole-cell high-throughput screen for Mycobacterium tuberculosis (Mtb) growth inhibitors, 10 nitro-containing compounds were prioritized for characterization and mechanism of action studies. HC2209, HC2210, and HC2211 are nitrofuran-based prodrugs that need the cofactor F 420 machinery for activation. Unlike pretomanid which depends only on deazaflavin-dependent nitroreductase (Ddn), these nitrofurans depend on Ddn and possibly another F 420 -dependent reductase for activation. These nitrofurans also differ from pretomanid in their potent activity against Mycobacterium abscessus . Four dinitrobenzamides (HC2217, HC2226, HC2238, and HC2239) and a nitrofuran (HC2250) are proposed to be inhibitors of decaprenyl-phosphoryl-ribose 2'-epimerase 1 (DprE1), based on isolation of resistant mutations in dprE1 . Unlike other DprE1 inhibitors, HC2250 was found to be potent against non-replicating persistent bacteria, suggesting additional targets. Two of the compounds, HC2233 and HC2234, were found to have potent, sterilizing activity against replicating and non-replicating Mtb in vitro , but a proposed mechanism of action could not be defined. In a pilot in vivo efficacy study, HC2210 was orally bioavailable and efficacious in reducing bacterial load by ~1 log in a chronic murine TB infection model., Competing Interests: R.B.A. is the founder and owner of Tarn Biosciences, Inc., a company that is working to develop new antimycobacterial drugs.

In its natural aquatic environment, the bacterial pathogen Vibrio cholerae , the causative agent of the enteric disease cholera, is in constant competition with bacterial viruses known as phages. Following ICP3 infection, V. cholerae cultures that exhibited phage killing always recovered overnight, and clones isolated from these regrowth populations exhibited complete resistance to subsequent infections. Whole-genome sequencing of these resistant mutants revealed seven distinct mutations in genes encoding for enzymes involved in O1 antigen biosynthesis, demonstrating that the O1 antigen is a previously uncharacterized putative receptor of ICP3. To further elucidate the specificity of the resistance conferred by these mutations, they were challenged with the V. cholerae -specific phages ICP1 and ICP2. All seven O1 antigen mutants demonstrated pan-resistance to ICP1 but not ICP2, which utilizes the OmpU outer membrane protein as a receptor. We show that resistant mutations to ICP1 and ICP3 evolve at a significantly higher frequency than ICP2, but these mutations have a significant fitness tradeoff to V. cholerae and are unable to evolve in the presence of an antimicrobial that mimics host cell defensins., Competing Interests: The authors declare no conflict of interest.

We have discovered a new cluster of genes that is found exclusively in the Actinobacteria phylum. This locus includes genes for the 2-aminophenol meta -cleavage pathway and the shell proteins of a bacterial microcompartment (BMC) and has been named aromatics (ARO) for its putative role in the breakdown of aromatic compounds. In this study, we provide details about the distribution and composition of the ARO BMC locus and conduct phylogenetic, structural, and functional analyses of the first two enzymes in the catabolic pathway: a unique 2-aminophenol dioxygenase, which is exclusively found alongside BMC shell genes in Actinobacteria, and a semialdehyde dehydrogenase, which works downstream of the dioxygenase. Genomic analysis reveals variations in the complexity of the ARO loci across different orders. Some loci are simple, containing shell proteins and enzymes for the initial steps of the catabolic pathway, while others are extensive, encompassing all the necessary genes for the complete breakdown of 2-aminophenol into pyruvate and acetyl-CoA. Furthermore, our analysis uncovers two subtypes of ARO BMC that likely degrade either 2-aminophenol or catechol, depending on the presence of a pathway-specific gene within the ARO locus. The precise precursor of 2-aminophenol, which serves as the initial substrate and/or inducer for the ARO pathway, remains unknown, as our model organism Micromonospora rosaria cannot utilize 2-aminophenol as its sole energy source. However, using enzymatic assays, we demonstrate the dioxygenase's ability to cleave both 2-aminophenol and catechol in vitro , in collaboration with the aldehyde dehydrogenase, to facilitate the rapid conversion of these unstable and toxic intermediates. IMPORTANCE Bacterial microcompartments (BMCs) are proteinaceous organelles that are widespread among bacteria and provide a competitive advantage in specific environmental niches. Studies have shown that the genetic information necessary to form functional BMCs is encoded in loci that contain genes encoding shell proteins and the enzymatic core. This allows the bioinformatic discovery of BMCs with novel functions and expands our understanding of the metabolic diversity of BMCs. ARO loci, found only in Actinobacteria, contain genes encoding for phylogenetically remote shell proteins and homologs of the meta -cleavage degradation pathway enzymes that were shown to convert central aromatic intermediates into pyruvate and acetyl-CoA in gamma Proteobacteria. By analyzing the gene composition of ARO BMC loci and characterizing two core enzymes phylogenetically, structurally, and functionally, we provide an initial functional characterization of the ARO BMC, the most unusual BMC identified to date, distinctive among the repertoire of studied BMCs., Competing Interests: The authors declare no conflict of interest.

Immune networks that control antimicrobial and inflammatory mechanisms have overlapping regulation and functions to ensure effective host responses. Genetic interaction studies of immune pathways that compare host responses in single and combined knockout backgrounds are a useful tool to identify new mechanisms of immune control during infection. For disease caused by pulmonary Mycobacterium tuberculosis (Mtb) infections, which currently lacks an effective vaccine, understanding the genetic interactions between protective immune pathways may identify new therapeutic targets or disease-associated genes. Previous studies have suggested a direct link between the activation of NLRP3-Caspase1 inflammasome and the NADPH-dependent phagocyte oxidase complex during Mtb infection. Loss of the phagocyte oxidase complex alone resulted in increased activation of Caspase1 and IL-1β production during Mtb infection, resulting in failed disease tolerance during the chronic stages of disease. To better understand this interaction, we generated mice lacking both Cybb , a key subunit of the phagocyte oxidase, and Caspase1/11 . We found that ex vivo Mtb infection of Cybb -/- Caspase1/11 -/- macrophages resulted in the expected loss of IL-1β secretion but an unexpected change in other inflammatory cytokines and bacterial control. Mtb infected Cybb -/- Caspase1/11 -/- mice rapidly progressed to severe TB, succumbing within 4 weeks to disease characterized by high bacterial burden, increased inflammatory cytokines, and the recruitment of granulocytes that associated with Mtb in the lungs. These results uncover a key genetic interaction between the phagocyte oxidase complex and Caspase1/11 that controls protection against TB and highlight the need for a better understanding of the regulation of fundamental immune networks during Mtb infection., Competing Interests: The authors declare no conflict of interest.