Joseph Pogliano - US grants

DNA segregation is a fundamental process that is poorly understood in bacteria. Recent work shows that E. coli plasmids are targeted to specific regions within the cell, rather than being randomly distributed in the cytoplasm as long thought. Multicopy plasmids form clusters that are targeted to the cell midpoint where they are duplicated. Following duplication, plasmids migrate with rapid kinetics from midcell to the future midpoints of the nascent daughter cells. Little is known about the mechanisms underlying plasmid localization or movement in bacteria. While a set of par genes important for DNA partitioning have been identified, how they contribute to plasmid localization is not understood. The goal of this study is to identify genes encoded by plasmids and their hosts that are directly responsible for mediating plasmid targeting, clustering and movement. Since bacterial DNA segregation is a fundamental process about which so little is currently known, the impact of this study will be extraordinarily broad, potentially affecting any area of research where bacteria are involved.

Some areas that will be impacted include microbial ecology and evolution, pathogenesis, genetics, genomics, and bioremediation. For example, understanding the details of DNA segregation could contribute directly to the development of new antibiotics designed to inhibit the process. Since one of the plasmids used here is a broad host range antibiotic resistance determinant, these studies will also impact our understanding on the evolution and spread of antibiotic resistance. This work will also have a significant impact on undergraduate education by contributing to the training of students in the biological sciences.

DESCRIPTION (provided by applicant): The goal of this proposal is to identify the mechanisms of DNA segregation for large, low copy number plasmids in Bacillus species. From studying plasmid pBtoxis in Bacillus thuringiensis, we identified a novel segregation protein belonging to the tubulin superfamily. TubZ is a divergent tubulin like protein that assembles dynamic polymers essential for the stable inheritance of plasmid pBtoxis. Tubulin-like proteins are commonly found on Bacillus virulence plasmids, but how they contribute to plasmid stability is unknown. We have also identified several families of divergent actin-like proteins involved in plasmid DNA segregation in Bacillus subtilis. AlfA of plasmid pLS32 and Alp7A both assemble polymers, but they appear to segregate plasmids by different mechanisms. The goal of this proposal is to understand the biochemical properties, in vivo assembly dynamics, and the functions of Bacillus tubulins and actins involved in plasmid DNA segregation. Since a wide variety of virulence factors are encoded on plasmids in pathogenic strains of Bacillus, such as pXO1 of B. anthracis, understanding how plasmids are transmitted from one generation to the next is important for understanding the evolution and spread of these key virulence determinants. PUBLIC HEALTH RELEVANCE: This study focuses on understanding how bacterial plasmids are inherited. Plasmids play key roles in bacterial pathogenesis, as they are often transmissible and encode antibiotic resistance and virulence traits, such as toxins and capsules. These studies could therefore help identify targets for new narrow spectrum antibiotics, since blocking plasmid stability will in certain infections decrease pathogenicity.

ParA systems play an important role in chromosome segregation and are responsible for the segregation of many bacterial plasmids. This proposal focuses on the ParA partitioning system of E. coli plasmid F, which consists of the SopA (ParA) ATPase, the SopB (ParB) DNA binding protein, and a DNA sequence, sopC, which contains the recognition sites for SopB binding. Our cell biological studies have demonstrated that SopA-GFP assembles into dynamic polymers that appear in the fluorescence microscope as a bright cloud surrounding the plasmid. This dynamic assembly of SopA is regulated by the SopB/sopC nucleoprotein complex in vivo and is essential for the ability of the Sop system to perform DNA segregation. The mechanism by which a ParA system performs DNA segregation remains unknown. We do not yet understand the mechanism of SopA polymerization or how polymerization contributes to each of the steps in plasmid segregation. We therefore propose to investigate the mechanism of ParA- mediated plasmid segregation in order to gain a better understanding of how this widespread family of proteins contributes to DNA segregation in bacteria. Specifically, we will characterize SopA polymerization in vivo using fluorescence recovery after photopbleaching (FRAP), total internal reflection fluorescence (TIRF) microscopy and speckle microscopy. Using time-lapse fluorescence microscopy and electron cryotomography, we will characterize the dynamic behavior of SopA during oscillation and plasmid separation. These studies will determine some of the basic features of SopA in vivo polymerization behavior and determine how polymerization is coupled to DNA segregation. The findings from this analysis of a representative ParAB plasmid partitioning system should have wide implications for our understanding of plasmid and chromosome segregation.

DESCRIPTION (provided by applicant): The timing of cell division is coordinated with other cyclic events, of other periodicities, in the lives of cells. From bacteria, to algae, to diverse cll types in mammals, the circadian biological clock controls the time of day during which cell division can occur. The mechanism and function of this time restriction, or gating, of cell divisio is poorly understood in any system, and such regulatory checkpoints in mammals are important factors in developmental programs and the progression of cancer. The overall goal of this project is to understand how and why circadian rhythms and cell division are interlocked. The circadian control of cell division in the cyanobacterium Synechococcus elongatus provides an elegant system in which to address questions that probe the interactions of the clock, the cytokinesis machinery, and the segregating chromosomes. This project seeks to answer: What is the biological role of the circadian checkpoint of cytokinesis? What are the components of the machinery that connect the circadian clock to cell division? Where are the clock oscillator components during the cell and circadian cycles and how are they inherited? And, is the partitioning of chromosomes in S. elongatus, whose ploidy levels oscillate with circadian rhythmicity, related to the gating of cell division? The specific aims will: (1) Define the components, through which the circadian clock regulates cell division, and determine the consequences of bypassing the cell division gate; (2) Discern the intracellular localization and dynamics of clock proteins during the circadian and cell division cycles; and (3) Elucidate the relationships among the clock, cytokinesis, and chromosome segregation. Existing mutants that bypass the circadian cell division checkpoint will be used to test hypotheses that address the role of the gate in protecting circadian precision, chromosome integrity, and/or cell-to-cell variations in gene expression. Mass spectrometry will identify factors that associate with the cell division machinery during the gating checkpoint. Time-lapse imaging of cells trapped in microfluidics chambers will enable simultaneous monitoring of cell division and circadian rhythms to assess the consequences of gating in individual wild-type cells and in mutants that bypass the circadian gate. Super-resolution Structured Illumination imaging will provide sufficient sensitivity and resolution to track the localization and dynamics of circadian oscillato proteins and tagged chromosomes in wild-type and mutant cells throughout the circadian and cell division cycles. Sorting and imaging flow cytometry methods will be used to assess ploidy changes in wild-type and mutant strains. This project will reveal how and why a circadian clock controls cell division, a coupling of timing circuits that occurs in mammalian cells as well as in cyanobacteria, and will provide novel insight into how cells inherit a sense of time.

DESCRIPTION (provided by applicant): Cytoskeletal proteins are of ancient origin, predating the divergence of prokaryotes and eukaryotes. Although these proteins play key roles in a variety of cellular processes, the proteins that make up the prokaryotic cytoskeleton are still poorly defined. In bacteria, only a few distinct families of tubulin have been characterized: FtsZ, a widely distributed protein critical for cell division, TubZ, involved in plasmid segregation and BtubA/BtubB, whose functions are still unknown. We recently discovered a divergent tubulin-like cytoskeletal protein, PhuZ, encoded by the very large (317 kb) Pseudomonas chlororaphis bacteriophage, 201?2-1. By expressing a GFP-tagged PhuZ at low levels in Pseudomonas, we could observe filament formation during lytic phage infection. We solved the structure of PhuZ to 1.67A resolution, and found a conserved tubulin fold with a novel, extended C-terminus that we showed to be critical for polymerization both in vitro and in vivo. Surprisingly, we found that PhuZ assembles a dynamic spindle that positions a single large complex of phage DNA at the center of the cell during lytic growth. Moreover, using PhuZ mutants designed from our structure, we could show that the dynamic nature of PhuZ filaments is required for phage centering. Bacterial viral particles appear to assemble around the periphery of this central DNA mass, creating a corona-like structure similar to the replication factories of herpes viruses, whic are distantly related to dsDNA bacteriophage. This is the first example of a prokaryotic spindle that performs a genome centering function analogous to the role of microtubule-based spindles of eukaryotes. Here, we propose to elucidate the biochemical, structural, and genetic basis of the ability of PhuZ to center DNA and the underlying mechanisms by which the polymer participates in viral lytic growth. Plausible roles for the polymer and centering include: defininga site to coordinate replication and packaging, facilitating phage head and or tail assembly, and facilitating cell lysis. Not only will we seek to answer these questions, but our work will also provide new insights into how tubulin family polymers can participate in such divergent functions as cell division, separation of plasmid DNA and organizing DNA into replication factories. Specifically, we propose the following research aims: 1. Examine the role of PhuZ in viral lytic growth. 2. Examine the possible connections between PhuZ assembly and DNA replication and movement, phage assembly and cell lysis in vivo. 3. Structurally and functionally characterize the mechanism and properties of PhuZ filaments assembled in vitro. 4. Identify phage and host proteins that interact with PhuZ and determine if they affect PhuZ polymerization, localization or other aspects of function. 5. Perform electron tomography and cryoTomography at various stages of infection to gain high resolution insights into the structural organization of PhuZ and viral capsids assembled in vivo during lytic growth.

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.

We recently described the discovery of a nucleus like structure assembled by phage 201?2-1 after infection of Pseudomonas chlororaphis. The phage nucleus is composed of a protein shell (GP105) that segregates phage and host bacterial proteins according to function, with metabolic enzymes and ribosomes in the cytoplasm and proteins related to DNA and RNA processing inside the phage nucleus. This compartment is centered by a bipolar spindle composed of the phage-encoded tubulin PhuZ. Capsids assemble on the bacterial membrane and then migrate to and dock on the surface of the phage nucleus where phage DNA is packaged. Ultimately, capsids assemble with tails to create mature particles and the cell lyses. The GP105 shell appears in our preliminary cryoEM as an irregularly shaped 5 nm wide border that encloses phage DNA. Remarkably, this shell allows selective entry or retention of specific proteins. This work raises a number of questions such as: Is the GP105 shell essential for phage replication? Are other proteins required for shell assembly? What is the structural organization of GP105 within the shell and how does it assemble and incorporate new subunits as it grows? Does it contain pores that allow diffusion of mRNA, proteins and small molecules in and out of the structure? How is the PhuZ spindle organized over time as it pushes the growing GP105 to midcell? Does the spindle participate in other aspects of phage development such as capsid movement? Here, we propose to use a combination of genetics, biochemistry, structural biology, and cell biology to study the nucleus like structure assembled by GP105 and the spindle assembled by PhuZ and determine how these two structures participate in viral lytic growth.