A pervasive large conjugative plasmid mediates multispecies biofilm formation in the intestinal microbiota increasing resilience to perturbations.

Although horizontal gene transfer is pervasive in the intestinal microbiota, we understand only superficially the roles of most exchanged genes and how the mobile repertoire affects community dynamics. Similarly, little is known about the mechanisms underlying the ability of a community to recover after a perturbation. Here, we identified and functionally characterized a large conjugative plasmid that is one of the most frequently transferred elements among Bacteroidales species and is ubiquitous in diverse human populations. This plasmid encodes both an extracellular polysaccharide and fimbriae, which promote the formation of multispecies biofilms in the mammalian gut. We use a hybridization-based approach to visualize biofilms in clarified whole colon tissue with unprecedented 3D spatial resolution. These biofilms increase bacterial survival to common stressors encountered in the gut, increasing strain resiliency, and providing a rationale for the plasmid's recent spread and high worldwide prevalence.

A ubiquitous mobile genetic element disarms a bacterial antagonist of the gut microbiota.

DNA transfer is ubiquitous in the gut microbiota, especially among species of Bacteroidales. In silico analyses have revealed hundreds of mobile genetic elements shared between these species, yet little is known about the phenotypes they encode, their effects on fitness, or pleiotropic consequences for the recipient's genome. Here, we show that acquisition of a ubiquitous integrative and conjugative element encoding an antagonistic system shuts down the native contact-dependent antagonistic system of Bacteroides fragilis . Despite inactivating the native antagonism system, mobile element acquisition increases fitness of the B. fragilis transconjugant over its progenitor by arming it with a new weapon. This DNA transfer causes the strain to change allegiances so that it no longer targets ecosystem members containing the same element yet is armed for communal defense.

Insights and strategies for improving equity in graduate school admissions.

Applying to graduate school can be particularly challenging for students from historically minoritized backgrounds due to a hidden curriculum in the graduate admissions process. To address this issue, a team of volunteer STEM trainees established the Científico Latino Graduate Student Mentorship Initiative (CL-GSMI) in 2019 to support applicants from historically minoritized backgrounds. CL-GSMI is designed to improve access to critical resources, including information, mentorship, and financial support, and has assisted 443 students in applying and matriculating to graduate school. Using program evaluation data from 2020 to 2021, we highlight areas in graduate school admissions that can be improved to promote equity and inclusion.

Analysis of Effector and Immunity Proteins of the GA2 Type VI Secretion Systems of Gut Bacteroidales.

Three distinct genetic architectures (GAs) of Type VI secretion systems (T6SSs) have been described in gut Bacteroidales species, each with unique genes and characteristics. Unlike the GA3 T6SSs, potent antagonism has not yet been demonstrated for the GA1 or GA2 T6SSs. We previously showed that the GA2 T6SS loci are contained on integrative and conjugative elements and that there are five subtypes. Collectively, GA2 are the most prevalent Bacteroidales T6SSs in the human populations analyzed. In this study, we provide a comprehensive bioinformatic analysis of the three variable regions of GA2 T6SS loci, which encode toxic effector and immunity proteins. In total, we identified 63 distinct effectors encoded within 31 nonredundant GA2 loci, 18 of which do not have described motifs or predicted functions. We provide experimental evidence for toxin activity for four different GA2 effectors, showing that each functions only when present in the periplasm, and experimentally confirm their cognate immunity proteins. Our data demonstrate that each GA2 locus encodes at least three distinct effectors with targets in both the cytoplasm and the periplasm. The data also suggest that the effectors of a given locus are loaded onto the tube by different mechanisms, which may allow all three effectors encoded within a single GA2 locus with distinct antibacterial activity to be loaded onto a single T6 tube, increasing the antagonistic effect. IMPORTANCE Humans are colonized with many gut Bacteroidales species at high density, allowing for extensive opportunities for contact-dependent antagonism. To begin to understand the antagonistic potential of the GA2 T6SSs of the gut Bacteroidales, we performed bioinformatic and experimental analyses of the three divergent regions containing the toxin effector and immunity genes. We show that each GA2 T6SS locus encodes at least three distinct toxic effectors including toxins linked to Rhs and Hcp with cytoplasmic targets, and unlinked effectors with targets in the periplasm. The diversity and modality of effectors exceeds that of the GA1 or GA3 T6SS loci (M. J. Coyne, K. G. Roelofs, and L. E. Comstock, BMC Genomics 17:58, 2016, https://doi.org/10.1186/s12864-016-2377-z) and suggests that these T6SSs have the potential to be potent antibacterial weapons in the human gut.

Bacteroidetocins Target the Essential Outer Membrane Protein BamA of Bacteroidales Symbionts and Pathogens.

Bacteroidetocins are a family of antibacterial peptide toxins that are produced by and target members of the phylum Bacteroidetes. To date, 19 bacteroidetocins have been identified, and four have been tested and shown to kill diverse Bacteroidales species (M. J. Coyne, N. Béchon, L. M. Matano, V. L. McEneany, et al., Nat Commun 10:3460, 2019, https://doi.org/10.1038/s41467-019-11494-1). Here, we identify the target and likely mechanism of action of the bacteroidetocins. We selected seven spontaneous mutants of four different genera, all resistant to bacteroidetocin A (Bd-A) and found that all contained mutations in a single gene, bamA. Construction of three of these bamA mutants in the wild-type (WT) strains confirmed they confer resistance to Bd-A as well as to other bacteroidetocins. We identified an aspartate residue of BamA at the beginning of exterior loop 3 (eL3) that, when altered, renders strains resistant to Bd-A. Analysis of a panel of diverse Bacteroidales strains showed a correlation between the presence of this aspartate residue and Bd-A sensitivity. Fluorescence microscopy and transmission electron microscopy (TEM) analysis of Bd-A-treated cells showed cellular morphological changes consistent with a BamA defect. Transcriptomic analysis of Bd-A-treated cells revealed gene expression changes indicative of cell envelope stress. Studies in mice revealed that bacteroidetocin-resistant mutants are outcompeted by their WT strain in vivo. Analyses of longitudinal human gut isolates showed that bamA mutations leading to bacteroidetocin resistance do not become fixed in the human gut, even in bacteroidetocin-producing strains and nonproducing coresident strains. Together, these data lend further support to the applicability of the bacteroidetocins as therapeutic peptides in the treatment of maladies involving Bacteroidales species. IMPORTANCE The bacteroidetocins are a newly discovered class of bacteriocins specific to Bacteroidetes with a spectrum of targets extending from symbiotic gut Bacteroides, Parabacteroides, and Prevotella species to pathogenic oral and vaginal Prevotella species. We previously showed that one such bacteroidetocin, Bd-A, is active at nanomolar concentrations, is water soluble, and is bactericidal, all desirable features in a therapeutic antibacterial peptide. Here, we identify the target of several of the bacteroidetocins as the essential outer membrane protein BamA. Although mutations in bamA can be selected in bacteria grown in vitro, we show both in a mouse model and in human gut ecosystems that bamA mutants leading to Bd-A resistance are fitness attenuated and are not selected. These features further support the potential usefulness of the bacteroidetocins as therapeutics for maladies associated with pathogenic Prevotella species, such as recurrent bacterial vaginosis, for which there are few effective treatments.

Mobile Type VI secretion system loci of the gut Bacteroidales display extensive intra-ecosystem transfer, multi-species spread and geographical clustering.

The human gut microbiota is a dense microbial ecosystem with extensive opportunities for bacterial contact-dependent processes such as conjugation and Type VI secretion system (T6SS)-dependent antagonism. In the gut Bacteroidales, two distinct genetic architectures of T6SS loci, GA1 and GA2, are contained on Integrative and Conjugative Elements (ICE). Despite intense interest in the T6SSs of the gut Bacteroidales, there is only a superficial understanding of their evolutionary patterns, and of their dissemination among Bacteroidales species in human gut communities. Here, we combine extensive genomic and metagenomic analyses to better understand their ecological and evolutionary dynamics. We identify new genetic subtypes, document extensive intrapersonal transfer of these ICE to Bacteroidales species within human gut microbiomes, and most importantly, reveal frequent population fixation of these newly armed strains in multiple species within a person. We further show the distribution of each of the distinct T6SSs in human populations and show there is geographical clustering. We reveal that the GA1 T6SS ICE integrates at a minimal recombination site leading to their integration throughout genomes and their frequent interruption of genes, whereas the GA2 T6SS ICE integrate at one of three different tRNA genes. The exclusion of concurrent GA1 and GA2 T6SSs in individual strains is associated with intact T6SS loci and with an ICE-encoded gene. By performing a comprehensive analysis of mobile genetic elements (MGE) in co-resident Bacteroidales species in numerous human gut communities, we identify 74 MGE that transferred to multiple Bacteroidales species within individual gut microbiomes. We further show that only three other MGE demonstrate multi-species spread in human gut microbiomes to the degree demonstrated by the GA1 and GA2 ICE. These data underscore the ubiquity and dissemination of mobile T6SS loci within Bacteroidales communities and across human populations.

Nanaerobic growth enables direct visualization of dynamic cellular processes in human gut symbionts.

Mechanistic studies of anaerobic gut bacteria have been hindered by the lack of a fluorescent protein system to track and visualize proteins and dynamic cellular processes in actively growing bacteria. Although underappreciated, many gut "anaerobes" are able to respire using oxygen as the terminal electron acceptor. The oxygen continually released from gut epithelial cells creates an oxygen gradient from the mucus layer to the anaerobic lumen [L. Albenberg et al., Gastroenterology 147, 1055-1063.e8 (2014)], with oxygen available to bacteria growing at the mucus layer. Here, we show that Bacteroides species are metabolically and energetically robust and do not mount stress responses in the presence of 0.10 to 0.14% oxygen, defined as nanaerobic conditions [A. D. Baughn, M. H. Malamy, Nature 427, 441-444 (2004)]. Taking advantage of this metabolic capability, we show that nanaerobic growth provides sufficient oxygen for the maturation of oxygen-requiring fluorescent proteins in Bacteroides species. Type strains of four different Bacteroides species show bright GFP fluorescence when grown nanaerobically versus anaerobically. We compared four different red fluorescent proteins and found that mKate2 yields the highest red fluorescence intensity in our assay. We show that GFP-tagged proteins can be localized in nanaerobically growing bacteria. In addition, we used time-lapse fluorescence microscopy to image dynamic type VI secretion system processes in metabolically active Bacteroides fragilis The ability to visualize fluorescently labeled Bacteroides and fluorescently linked proteins in actively growing nanaerobic gut symbionts ushers in an age of imaging analyses not previously possible in these bacteria.

Utilizing Ribose Compounds: How Bacteroides PUL It Off.

Bacteroides genomes encode a large repertoire of proteins dedicated to the utilization of diverse plant polysaccharides and host glycans. In this issue of Cell Host & Microbe, Glowacki et al. (2020) show that B. thetaiotaomicron can also extract the monosaccharide ribose from nucleosides and characterize proteins necessary for its utilization.

Streamlined Genetic Manipulation of Diverse Bacteroides and Parabacteroides Isolates from the Human Gut Microbiota.

Studies of the gut microbiota have dramatically increased in recent years as the importance of this microbial ecosystem to human health and disease is better appreciated. The Bacteroidales are the most abundant order of bacteria in the healthy human gut and induce both health-promoting and disease-promoting effects. There are more than 55 species of gut Bacteroidales with extensive intraspecies genetic diversity, especially in regions involved in the synthesis of molecules that interact with other bacteria, the host, and the diet. This property necessitates the study of diverse species and strains. In recent years, the genetic toolkit to study these bacteria has greatly expanded, but we still lack a facile system for creating deletion mutants and allelic replacements in diverse strains, especially with the rapid increase in resistance to the two antibiotics used for genetic manipulation. Here, we present a new versatile and highly efficient vector suite that allows the creation of allelic deletions and replacements in multiresistant strains of Bacteroides and Parabacteroides using a gain-of-function system based on polysaccharide utilization. These vectors also allow for easy counterselection independent of creating a mutant background strain, using a toxin from a type VI secretion system of Bacteroides fragilis Toxin production during counterselection is induced with one of two different molecules, providing flexibility based on strain phenotypes. This family of vectors greatly facilitates functional genetic analyses and extends the range of gut Bacteroidales strains that can be genetically modified to include multiresistant strains that are currently genetically intractable with existing genetic tools.IMPORTANCE We have entered an era when studies of the gut microbiota are transitioning from basic questions of composition and host effects to understanding the microbial molecules that underlie compositional shifts and mediate health and disease processes. The importance of the gut Bacteroidales to human health and disease and their potential as a source of engineered live biotherapeutics make these bacteria of particular interest for in-depth mechanistic study. However, there are still barriers to the genetic analysis of diverse Bacteroidales strains, limiting our ability to study important host and community phenotypes identified in these strains. Here, we have overcome many of these obstacles by constructing a series of vectors that allow easy genetic manipulation in diverse gut Bacteroides and Parabacteroides strains. These constructs fill a critical need and allow streamlined allelic replacement in diverse gut Bacteroidales, including the growing number of multiantibiotic-resistant strains present in the modern-day human intestine.

Mechanisms of Resistance to the Contact-Dependent Bacteriocin CdzC/D in Caulobacter crescentus.

The Cdz bacteriocin system allows the aquatic oligotrophic bacterium Caulobacter crescentus to kill closely related species in a contact-dependent manner. The toxin, which aggregates on the surfaces of producer cells, is composed of two small hydrophobic proteins, CdzC and CdzD, each bearing an extended glycine-zipper motif, that together induce inner membrane depolarization and kill target cells. To further characterize the mechanism of Cdz delivery and toxicity, we screened for mutations that render a target strain resistant to Cdz-mediated killing. These mutations mapped to four loci, including a TonB-dependent receptor, a three-gene operon (named zerRAB for zipper envelope resistance), and perA (for pentapeptide envelope resistance). Mutations in the zerRAB locus led to its overproduction and to potential changes in cell envelope composition, which may diminish the susceptibility of cells to Cdz toxins. The perA gene is also required to maintain a normal cell envelope, but our screen identified mutations that confer resistance to Cdz toxins without substantially affecting the cell envelope functions of PerA. We demonstrate that PerA, which encodes a pentapeptide repeat protein predicted to form a quadrilateral ß-helix, localizes primarily to the outer membrane of cells, where it may serve as a receptor for the Cdz toxins. Collectively, these results provide new insights into the function and mechanisms of an atypical, contact-dependent bacteriocin system.IMPORTANCE Bacteriocins are commonly used by bacteria to kill neighboring cells that compete for resources. Although most bacteriocins are secreted, the aquatic, oligotrophic bacterium Caulobacter crescentus produces a two-peptide bacteriocin, CdzC/D, that remains attached to the outer membranes of cells, enabling contact-dependent killing of cells lacking the immunity protein CdzI. The receptor for CdzC/D has not previously been reported. Here, we describe a genetic screen for mutations that confer resistance to CdzC/D. One locus identified, perA, encodes a pentapeptide repeat protein that resides in the outer membrane of target cells, where it may act as the direct receptor for CdzC/D. Collectively, our results provide new insight into bacteriocin function and diversity.

Bacterial antagonism in host-associated microbial communities.

Antagonistic interactions are abundant in microbial communities and contribute not only to the composition and relative proportions of their members but also to the longer-term stability of a community. This Review will largely focus on bacterial antagonism mediated by ribosomally synthesized peptides and proteins produced by members of host-associated microbial communities. We discuss recent findings on their diversity, functions, and ecological impacts. These systems play key roles in ecosystem defense, pathogen invasion, spatial segregation, and diversity but also confer indirect gains to the aggressor from products released by killed cells. Investigations into antagonistic bacterial interactions are important for our understanding of how the microbiota establish within hosts, influence health and disease, and offer insights into potential translational applications.

Contact-dependent killing by Caulobacter crescentus via cell surface-associated, glycine zipper proteins.

Most bacteria are in fierce competition with other species for limited nutrients. Some bacteria can kill nearby cells by secreting bacteriocins, a diverse group of proteinaceous antimicrobials. However, bacteriocins are typically freely diffusible, and so of little value to planktonic cells in aqueous environments. Here, we identify an atypical two-protein bacteriocin in the α-proteobacterium Caulobacter crescentus that is retained on the surface of producer cells where it mediates cell contact-dependent killing. The bacteriocin-like proteins CdzC and CdzD harbor glycine-zipper motifs, often found in amyloids, and CdzC forms large, insoluble aggregates on the surface of producer cells. These aggregates can drive contact-dependent killing of other organisms, or Caulobacter cells not producing the CdzI immunity protein. The Cdz system uses a type I secretion system and is unrelated to previously described contact-dependent inhibition systems. However, Cdz-like systems are found in many bacteria, suggesting that this form of contact-dependent inhibition is common.

De novo pyrimidine biosynthesis in the oomycete plant pathogen Phytophthora infestans.

The oomycete Phytophthora infestans, causal agent of the tomato and potato late blight, generates important economic and environmental losses worldwide. As current control strategies are becoming less effective, there is a need for studies on oomycete metabolism to help identify promising and more effective targets for chemical control. The pyrimidine pathways are attractive metabolic targets to combat tumors, virus and parasitic diseases but have not yet been studied in Phytophthora. Pyrimidines are involved in several critical cellular processes and play structural, metabolic and regulatory functions. Here, we used genomic and transcriptomic information to survey the pyrimidine metabolism during the P. infestans life cycle. After assessing the putative gene machinery for pyrimidine salvage and de novo synthesis, we inferred genealogies for each enzymatic domain in the latter pathway, which displayed a mosaic origin. The last two enzymes of the pathway, orotate phosphoribosyltransferase and orotidine-5-monophosphate decarboxylase, are fused in a multi-domain enzyme and are duplicated in some P. infestans strains. Two splice variants of the third gene (dihydroorotase) were identified, one of them encoding a premature stop codon generating a non-functional truncated protein. Relative expression profiles of pyrimidine biosynthesis genes were evaluated by qRT-PCR during infection in Solanum phureja. The third and fifth genes involved in this pathway showed high up-regulation during biotrophic stages and down-regulation during necrotrophy, whereas the uracil phosphoribosyl transferase gene involved in pyrimidine salvage showed the inverse behavior. These findings suggest the importance of de novo pyrimidine biosynthesis during the fast replicative early infection stages and highlight the dynamics of the metabolism associated with the hemibiotrophic life style of pathogen.

Aislamiento y caracterización de dos cepas de Fusarium oxysporum causantes de la pudrición seca en Solanum tuberosum en Colombia / Isolation and characterization of two strains of Fusarium oxysporum causing potato dry rot in Solanum tuberosum in Colombia

resumen(AU)

Background. Fusarium oxysporum has worldwide distribution and causes severe vascular wilt or root rot in many plants. Strains are classified into formae speciales based on their high degree of host specificity, of which multilocus sequence typing provides a fairly good estimate. Aims. The main aim of this study was to identify the causal agent of an infected potato tuber in Colombia. Methods. Two F. oxysporum isolates were recovered from a potato tuber showing symptoms of dry rot. Both macroscopic and microscopic morphology differences were observed between the two isolates. Koch's postulates were verified and in quantitative tuber pathogenecity trials, both isolates induced moderate dry rot. Ribosomal internal transcribed spacer (ITS) and partial intergenic spacer region (IGS) sequences were PCR-amplified, sequenced and shown to be identical for the two isolates. A maximum parsimony phylogeny was created using F. oxysporum IGS sequences available in the Genebank database, which does not include sequences from the formae speciales tuberosi. Results. Our two isolates were most closely related to a red clover (Trifolium pratense) pathogenic isolate and two non-pathogenic F. oxysporum isolates from birdsfoot trefoil (Lotus corniculatus) and Lycopersicon sp. rhyzosphere (99% identity). Conclusions. These experiments showed that our isolates are not restricted to potato and that a molecular marker is needed to differentiate the formae speciales since the IGS and EF-1alpha do not have the power to do it(AU)

Isolation and characterization of two strains of Fusarium oxysporum causing potato dry rot in Solanum tuberosum in Colombia.

BACKGROUND: Fusarium oxysporum has worldwide distribution and causes severe vascular wilt or root rot in many plants. Strains are classified into formae speciales based on their high degree of host specificity, of which multilocus sequence typing provides a fairly good estimate. AIMS: The main aim of this study was to identify the causal agent of an infected potato tuber in Colombia. METHODS: Two F. oxysporum isolates were recovered from a potato tuber showing symptoms of dry rot. Both macroscopic and microscopic morphology differences were observed between the two isolates. Koch's postulates were verified and in quantitative tuber pathogenecity trials, both isolates induced moderate dry rot. Ribosomal internal transcribed spacer (ITS) and partial intergenic spacer region (IGS) sequences were PCR-amplified, sequenced and shown to be identical for the two isolates. A maximum parsimony phylogeny was created using F. oxysporum IGS sequences available in the Genebank database, which does not include sequences from the formae speciales tuberosi. RESULTS: Our two isolates were most closely related to a red clover (Trifolium pratense) pathogenic isolate and two non-pathogenic F. oxysporum isolates from birdsfoot trefoil (Lotus corniculatus) and Lycopersicon sp. rhyzosphere (99% identity). CONCLUSIONS: These experiments showed that our isolates are not restricted to potato and that a molecular marker is needed to differentiate the formae speciales since the IGS and EF-1α do not have the power to do it.