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

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

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biology, 2017.This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections.Cataloged from student-submitted PDF version of thesis.Includes bibliographical references.In the battle for resources within microbial communities, antagonistic interactions between bacterial species are often mediated by diffusible inhibitory compounds, which can be diffusible or delivered in a contact-dependent manner. Bacteriocins are one ubiquitous type of such antimicrobials, and collectively constitute a very diverse group of ribosomally-synthesized diffusible proteinaceous toxins. The majority of well-characterized bacteriocin systems belong to a limited group of bacterial clades and environments. In contrast, interbacterial interactions are poorly characterized in the nutrient-poor aquatic environments where the a-proteobacterium Caulobacter crescentus thrives. Here, I describe the discovery and characterization of a new type of bacteriocin in C. crescentus. The Cdz toxin is composed of two small hydrophobic proteins, each harboring an extended glycine-zipper motif often found in amyloids. These proteins are retained on the surface of producer cells where they form large, insoluble aggregates. I show that Cdz mediates cell contact-dependent killing of closely related species. The Cdz bacteriocin uses a type I secretion system and is unrelated to previously described contact-dependent inhibition systems. Delivering the bacteriocin directly to the recipient, rather than secreting it into the extracellular milieu, likely enables C. crescentus to avoid the dilemma of producing an expensive common good that would rapidly diffuse away in its aqueous environment. Furthermore, Cdz-like systems are found in many clades of bacteria, including pathogens such as Klebsiella pneumoniae and Pseudomonas aeruginosa, suggesting that this form of contact-dependent inhibition is widespread. Using a cationic membrane stain, I showed that the Cdz bacteriocin causes inner membrane depolarization in the target cells. To further characterize the mechanism of delivery and toxicity, I conducted a suppressor screen to identify mutations that confer Cdz resistance to a target strain. I identified the putative surface receptor for the Cdz toxins, PerA, a protein harboring several pentapeptide-repeat motifs thought to adopt a quadrilateral [beta]-helical fold. PerA plays an important role in envelope homeostasis. Additionally, I identified two envelope-remodeling genes whose upregulation confers resistance to Cdz-mediated killing. Taken together, my work has expanded the repertoire of bacteriocins, demonstrating that these antimicrobials can be contact-dependent and, consequently, advantageous in a wider range of environments than previously anticipated.by Leonor García-Bayona.Ph. D

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

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.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

Mechanisms of Resistance to the Contact-Dependent Bacteriocin CdzC/D inCaulobacter 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 β-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.National Institutes of Health (Grant R01GM082899

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

Distant relatives of a eukaryotic cell-specific toxin family evolved a complement-like mechanism to kill bacteria

Abstract Cholesterol-dependent cytolysins (CDCs) comprise a large family of pore-forming toxins produced by Gram-positive bacteria, which are used to attack eukaryotic cells. Here, we functionally characterize a family of 2-component CDC-like (CDCL) toxins produced by the Gram-negative Bacteroidota that form pores by a mechanism only described for the mammalian complement membrane attack complex (MAC). We further show that the Bacteroides CDCLs are not eukaryotic cell toxins like the CDCs, but instead bind to and are proteolytically activated on the surface of closely related species, resulting in pore formation and cell death. The CDCL-producing Bacteroides is protected from the effects of its own CDCL by the presence of a surface lipoprotein that blocks CDCL pore formation. These studies suggest a prevalent mode of bacterial antagonism by a family of two-component CDCLs that function like mammalian MAC and that are wide-spread in the gut microbiota of diverse human populations