Kit Pogliano - US grants

DESCRIPTION: The goal of this proposal is to achieve a detailed understanding of a key step in the sporulation of Bacillus subtilis, engulfment. During engulfment the edges of the sporulation septum migrate around and fully enclose the forespore; ultimately the forespore is fully enclosed within the mother cell cytoplasm and is surrounded by two membranes. Engulfment is essential for both spore morphogenesis and for full activation of late sporulation-specific transcription factors in both cells of the developing sporangium. Although several sporulation-specific proteins have been implicated in engulfment, its mechanism remains obscure. Bacterial cells lack the cytoskeletal proteins associated with similar eukaryotic events, and hence must use a noval mechanism to drive the engulfment of one cell by another. We will use the following specific aims to further our understanding of engulfment: Characterization of the known engulfment proteins to determine their subcellular distribution, to identify proteins with which they interact, to determine their biochemical activities, and to more precisely determine the stage at which they are required for engulfment. Identification of additional proteins required for engulfment, using both genetic and biochemical approaches. The development of a system to observe engulfment in living bacteria, using video microscopy and protein fusions to the green fluorescent protein (GFP) of Aquoria victoria. It is hoped that by identifying a large number of proteins required for engulfment, and by studying their biochemical activities, interactions and subcellular distributions, that we can begin to understand the mechanism by which one bacterial cell can engulf another. Engulfment provides a unique model system for the study of regulated membrane movements in bacteria.

The localization of membrane proteins to specific regions of the cell is a conserved and essential feature of both prokaryotic and eukaryotic cells. In bacteria, protein localization is essential for cell division, DNA replication, as well as for chemotaxis, pathogenesis and the development of specialized cell types. However, in contrast to eukaryotic cells, little is known about the mechanism by which proteins reach their correct subcellular location in bacterial cells. Many cases of protein localization in bacteria involve the localization of membrane proteins to specific regions of the cytoplasmic membrane, often at the cell pole or septum. During B. subtilis sporulation, protein localization involves the specific localization of proteins to one of two separate membranes within the cell, similar to eukaryotic protein localization.
The long term goals of the research described here are to understand the mechanism by which bacterial membrane proteins assemble at their correct locations within the cell. This project uses genetic and cell biological methods to investigate the mechanism by which proteins localize to specific membrane regions during sporulation, and to investigate the possibility that B. subtilis contains two integral membrane protein insertion complexes that serve to direct membrane proteins to different locations within the cell. It will also develop new methods to investigate if proteins made before division are restricted to one or the other cell after division, or if they are differently localized in the two cells, as these processes may provide a simple mechanism to generate daughter cells of differing fate. They also may control the activity of proteins that act in a vectoral manner across a newly-synthesized septum.
These studies will provide further insight into the mechanism by which integral membrane proteins reach their correct subcellular address in bacterial cells. Thus far, the mechanisms of integral membrane protein insertion and assembly have been almost exclusively studied in Gram negative bacteria, primarily in E. coli. These studies will be extended to the Gram positive bacterium B. subtilis, which represents a separate bacterial kingdom that includes many human pathogens such as the Mycobacteria, Streptococcus sp. and Staphylococcus sp., and many bacteria of industrial importance, such as the Actinomycetes.
The project will contribute to training graduate and undergraduate students in microbial cell biology and genetics.

The phagocytosis-like process of engulfment is the hallmark of endospore formation in bacteria from the genera Bacillus and Clostridium, which produce unusually durable endospores that are the infectious agent of Anthrax and Botulism. Engulfment mediates a dramatic change in cellular architecture, rearranging the sporangium from two cells that lie side by side, to an endospore in which one cell (the forespore) lies within the cytoplasm of another (the mother cell). The studies supported by this grant have provided new insights into the mechanisms by which coordinated peptidoglycan synthesis and degradation mediate this dramatic example of the architectural plasticity of bacterial cells, and they are providing insight into the role essential proteins play in this process. We here propose to visualize the dramatic morphological rearrangements of sporulation and the protein complexes that mediate them, by using a new implementation of cryo-electron tomography that uses a focused ion beam to produce thin lamella of bacterial cells, producing images that reveal structures within cells at nanometer resolution. We also propose to pursue preliminary data that has revealed that sporulation entails a dramatic and previously unrecognized metabolic differentiation of the two cells, after which the future spore is completely dependent on the mother cell for the precursors for biosynthesis. This process, which appears to be mediated by a massive forespore-specific proteolysis event, effectively converts the two cells into synthrophic partners, providing an accessible model for studying coupled metabolism, which is prevalent in microbial communities and biofilms. We here propose to study these processes, using genetics, metabolomics, fluorescence and cryo- electron microscopy, integrating these experimental efforts with computational analysis and modeling. These studies will reveal the cellular and metabolic landscape of sporulation in unprecedented detail, capitalizing on the dispensability and streamlined machinery of sporulation to provide insight into conserved processes that are often essential for growth.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The phagocytosis-like process of engulfment is a key step in the sporulation pathway of the endospore forming bacteria, such as Bacillus subtilis and B. anthracis. During engulfment, the membrane of one bacterial cell (the mother cell) migrates around another, until the second cell is fully enclosed within the mother cell cytoplasm. We propose using the NCMIR to provide a higher resolution view of key steps in engulfment, including the steps of membrane migration, membrane fission and the assembly of protein complexes that play a synapse-like role coordinating signal transduction in the two cells.

DESCRIPTION (provided by applicant): Bacillus subtilis produces a wide array of extracellular metabolites that can inhibit the growth of bacteria and fungi or modify their behavior to attenuate the production of antibacterial products by potentially dangerous neighbors. We here propose to use the new technique of imaging mass spectrometry and classical analytical chemistry to systematically identify the extracellular metabolome of B. subtilis, with a focus on characterizing the interactive metabolome that is induced by other bacterial species. We will investigate the role these compounds play in two distinct outcomes of the interaction of B. subtilis with other species. The first is an impasse, in which B. subtilis forms closely abutting colonies with other species that produce a variety of antibacterial compounds (such as P. aeruginosa). The second, more frequent behavior is contact-dependent predation, in which B. subtilis moves towards, invades and destroys neighboring colonies, leading to death of the prey species and expanding the territory of the B. subtilis colony. These reproducible behaviors are conserved in different undomesticated B. subtilis strains. We will determine if these behaviors depend on the interactive metabolome and investigate the effects individual compounds have on target cell viability and behavior. We will further investigate the genetic requirements for interspecies interactions to identify stress responses, developmental and biosynthetic pathways that contribute to these distinct outcomes and we will use fluorescence microscopy to visualize the cellular consequences of interspecies interactions. These studies will illuminate the mechanistic basis for interspecies interactions and identify secondary metabolites that affect viability or behavior of other species that represent potential new antibacterial drugs. PUBLIC HEALTH RELEVANCE: Bacteria produce many extracellular metabolites that mediate their interaction with other species, many of which have antibacterial and antifungal activities. We will here elucidate the chemical, genetic and cellular mechanisms by which these molecules allow Bacillus subtilis to interact with other bacterial species, producing outcomes ranging from coexistence to the invasion and destruction of neighboring colonies. Interspecies interactions are critical in medicine and the metabolites that facilitate destruction of other species represent promising new pharmaceutical leads.

DESCRIPTION (provided by applicant): We developed a new approach to facilitate identification of natural products with antibacterial activities that rapidly discriminates between different mechanisms of action (MOA). This approach, bacterial cytological profiling (BCP), uses quantitative fluorescence microscopy to measure the effects of antibiotic treatment on individual cells. Antibiotics that target different cellular pathways and different steps within a pathway generate unique cytological profiles, allowing identification of the likely MOA of new compounds within a few hours. We have now developed a complimentary approach that will allow us to identify molecules that inhibit proteins that are not currently targeted by antibacterial drugs. Ths approach, which we call rapid inhibition profiling (R.I.P.), entails the rapid, inducible depletionof a target protein, followed by cytological profiling. Our preliminary data demonstrate that depletion of a drug target by R.I.P. produces cytological effects identical to those produced by the corresponding drug. Furthermore, depletion of essential proteins that are not targeted by current antibacterial drugs produces novel cytological profiles that can be subsequently used to identify molecules that inhibit these new targets. We here propose to more fully develop the R.I.P. technology by employing it on a genome wide basis in E. coli and B. subtilis. This will create a comprehensive reference set of profiles associated with the inhibition of essential cellular pathways that are not the targets of current antibacterial drugs. The genome-wide R.I.P. analysis will also provide insight into the function of conserved genes and our preliminary data suggests that it will provide insight into proteins that coordinate two or more biosynthetic pathways, providing interesting starting points for future basic research. We will then use this more complete reference data set with BCP to screen a unique and diverse collection of natural product extracts to identify those that inhibit these new drug targets and kill multidrug resistant bacteria. Together with our collaborators at Fundaci¿n Medina, we will then purify and characterize our highest priority lead molecules (those that kill multidrug resistant bacteria) wit a goal of advancing them into toxicity trials.

? DESCRIPTION (provided by applicant): The increasing prevalence of bacterial pathogens that are resistant to most of the clinically approved antibiotics is an alarming situation that has spurred renewed interest in antibiotic discovery programs. Since most antibiotics are derived from natural products produced by microorganisms, there is now intense interest in using new methods to screen genetically and chemically diverse collections of bacteria. However, identifying new molecules from bacterial extracts is confounded by the overwhelming presence of previously identified molecules as well as the fact that most of the biosynthetic potential of a organism is typically not expressed under laboratory growth conditions. We have characterized a unique collection of microbes obtained from deep within four different caves of New Mexico. Since these bacteria were isolated from remote, underexplored locations that are only just beginning to be mined for antibiotics, there is an increased probability of identifying molecules with unique chemical structures and new modes of action. The goal of this project is to use two new powerful platforms to identify and purify molecules active against multidrug resistant (MDR) bacteria from this diverse collection of cave bacteria. First, we will use our recently developed bacterial cytological profiling (BCP) approach to identify natural products with antibacterial activities in crude organic extracts or directly on plates. BCP uses quantitative fluorescence microscopy to measure the effects of antibiotic treatment on individual cells. Antibiotics that target different cellular pathways and different steps within a pathway generate unique cytological profiles, allowing identification of the likely cellular target of newly isolated compounds in a few hours. BCP works in complex crude extracts and subsequent fractions, allowing it to be used to guide natural product purification. We will sequence strains producing antibiotics and then use target directed genome mining (TDGM) and heterologous biosynthetic gene cluster (BGC) overproduction to identify novel antibacterial producing BGCs. Heterologous overexpression of normally silent BGCs will allow us to identify molecules missed by traditional screening.