Research Interests

We refer to our lab as Computational Microbiology Laboratory and our research centers on comparative analyses of microbial (mainly prokaryotic) genomes. However, unlike standard comparative genomic studies, which compare gene contents among different genomes, our analyses focus mainly on genome properties other than protein- and RNA-coding genes. For example, different genomes often have distinct nucleotide and oligonucleotide compositions, repeat structures, and/or sequence patterns that affect structures of DNA or RNA molecules and physical organization of the chromosomes. Our goal is to understand how these genome features evolve and what physiological roles they play in the organisms. Our research relies to a large extent on statistics and computer science, and includes development of new methods, algorithms and software for sequence analysis. Our web site features a number of programs developed in our lab that we not only use in our research but also offer for use by other researchers.

     Our most recent project involved analyses of simple sequence repeats (SSRs), that is, uninterrupted tandem repeats of a single nucleotide or a short oligonucleotide like AAAAAAAA… or ATCCGATCCGATCCG… Such repeats are hypervariable due to slipped-strand mutations and a common source of phase variation, including antigenic variation in pathogens. By comparative analysis among hundreds of genomes, we found that repeats of mono-, di-, tri-, and tetranucleotides occur in different types of genomes (mostly host-adapted pathogens) than repeats of pentanucleotides and longer oligonucleotides (environmental organisms and opportunistic pathogens), and probably have different functions. Further investigation revealed that the function of SSRs is not limited to phase variation, and extends to genome reduction in host adapted pathogens, influence on DNA structure, and possibly expression of adjacent genes.

The sequestration and storage of metals, and the maturation and roles of metal-containing enzymes by bacterial pathogens are of keen interest. The bacterial pathogens under study include the persistent gastric pathogen Helicobacter pylori, other Helicobacters that colonize liver and colon environments, and some enteric diarrheal pathogens (e.g. Salmonella/Shigella). For example, all these bacteria sequester the metal nickel for use in nickel-containing enzymes, yet the mechanism of nickel storage and subsequent nickel allocation and insertion into the nickel enzyme sinks (hydrogenases and ureases) is not understood. Mutant analysis combined with pure protein studies and animal infection models are all used to study metal containing proteins in these pathogens. A key growth substrate used by all these pathogens in animals is molecular hydrogen produced by the host flora; the pathogens ability to use this small but highly energetic substrate is due to a nickel-containing H2-splitting enzyme. The nickel-dependent expression and maturation of this enzyme is studied, as well as unique ways (i.e. nickel chelation) to inhibit its activity. Another related area of research involves stress-combating proteins used by Helicobacter pylori to colonize the gastric mucosa of humans. Such colonization leads to a variety of inflammatory gastric diseases. The persistence of the pathogen in withstanding host defense mechanisms over a period of years or decades results in the most severe gastric diseases, including even gastric carcinomas. Growth of the bacterium in animals is partially inhibited by a battery of host-produced reactive oxygen species. Our goal is to identify and then characterize the expression of oxidative stress resistance proteins that enable the gastric pathogen to persistently survive the harsh host environment.

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Research Interests: The goal of the research in my laboratory is to characterize the regulation of bacterial gene expression at multiple levels, from DNA rearrangements that alter the DNA template to RNA repair, thereby contributing to a complete picture of gene regulation in bacteria.

Current areas of focus:

1) Gene regulation by the DEDD-motif family of specialized DNA recombinases. We previously identified a unique family of DNA recombinases, the DEDD-motif family, that mediate DNA rearrangements and frequently control expression of genes involved in interactions of bacteria with their environment. We utilize genetic and biochemical approaches to define the mechanisms for recombination mediated by the DEDD-motif recombinases and to characterize the genes regulated by the recombinase systems. The recombinase systems that we study include Irg mediated integration of the Meningococcal Disease Associated (MDA) phage in Neisseria meningitidis, and MooV-mediated site-specific integration and excision of IS492 in the marine bacterium Pseudomonas atlantica to control extracellularpolysaccharide production and biofilm formation.

2) Gene regulation through the activity of the alternative sigma factor, RpoN. We are utilizing genomics, genetics, and molecular biology approaches to define the RpoN regulon of Salmonella enterica subspecies enterica serovar Typhimurium. Transcription of many operons that encode proteins and structural RNAs involved in diverse cellular processes are dependent on RpoN-RNA polymerase holoenzyme. Our research has also revealed numerous intragenic, RpoN-dependent promoters and RpoN binding sites that appear to regulate expression of genes within different sigma factor regulatory networks through transcriptional interference, expression of regulatory small RNAs, or repression. The novel regulatory roles for RpoN in the global regulatory networks of S. Typhimurium are being investigated.

3) Potential gene regulation by the RNA repair system of S. Typhimurium. In a RpoN-dependent operon, S. Typhimurium encodes homologs of human and archaeal RNA ligase (RtcB) and RNA 3' phosphate cyclase (RtcA); these enzymes are involved in tRNA splicing and RNA repair pathways in humans and archaea, but their physiological functions in bacteria are unknown. We are using transcriptomics, proteomics, genetics, and molecular biology approaches to characterize the RNA repair system and its function in S. Typhimurium.

Research Interests:

Helicobacter pylori is a bacterial pathogen that colonizes the human gastric mucosa where it can cause a variety of stomach diseases, including peptic ulcer disease and gastric cancer. H. pylori uses a cluster of polar flagella for motility which is essential for colonization of the gastric musoca. All three of the sigma factors in H. pylori (RpoD, RpoN and FliA) are used to coordinate the expression of over 60 genes encoding structural components, enzymes and regulatory proteins needed for assembly and function of the flagellum. Current work in the lab is directed at characterizing the mechanisms by which expression of the various sets of flagellar genes are regulated.

A second project in the lab focuses on RpoN (σ⁵⁴) regulon of Salmonella enterica serovar Typhimurium (S. Typhimurium), which is a major food-borne pathogen. Transcription by σ⁵⁴-RNA polymerase holoenzyme (σ⁵⁴-holoenzyme) requires an activator protein.  S. Typhimurium possesses 13 RpoN-dependent activators, each targeting a specific set of genes.  Functions for many of the genes of the S. Typhimurium RpoN regulon are unknown, and one of the goals of the project is to determine the physiological roles of these genes.  In addition, we are characterizing RpoN-dependent promoters and other intragenic σ⁵⁴-holoenzyme binding sites in the S. Typhimurium genome to identify core and contextual sequences that are important for promoter activity, as well as determining the functions of these sites.

Our laboratory is interested in bacterial metabolism and physiology. Much of the work we do is performed in Salmonella enterica because we can do sophisticated genetic analyses of strains. We are currently focused on three areas of research. First, we study metabolic pathway integration. We identified the cobB gene of Salmonella enterica as a new member of the SIR2 family regulatory proteins in eukaryotes whose activities are needed for gene silencing and cell aging. Our report was an important contribution to this field of research and led to the identification of two enzymatic activities associated with these proteins.

Molecular approaches to studying microbial structures, cell biology, gene regulation, genomic stability, development, etc.

Research Interests:

Upon invasion of a host cell, intracellular pathogens must actively ensure their survival in an immediately hostile environment. One such survival tactic of some pathogenic bacteria is through the subversion of host membrane fusion machinery, thereby inhibiting phagolysosomal fusion and subsequent delivery of the bacterium to the host degradative lysosome. The foodborne pathogen, Salmonella enterica, and the causative agent of Legionnaire’s disease, Legionella pneumophila, are examples of such bacterial pathogens that utilize this particular survival tactic. While evading host cell defenses in this manner is key to the organism’s ability to cause infection and disease, the mechanisms underlying these evasion pathways remain poorly understood. Many studies have tentatively identified bacterial factors thought to be important for the disruption of normal host membrane dynamics, but the biochemical analysis of these factors remains lacking. By employing a powerful in vivo and in vitro model system of eukaryotic membrane fusion, my laboratory will investigate the biochemistry of eukaryotic membrane fusion, identify and biochemically characterize bacterial effectors capable of modulating membrane fusion, and finally analyze these activities within the context of pathogenesis.

Vacuoles of the budding yeast Saccharomyces cerevisiae serve the equivalent physiological function of the mammalian lysosome, and undergo constant rounds of fission and homotypic (self) fusion in response to cellular growth conditions. Isolation of these fusogenic organelles from yeast is now a straightforward task, and robust colorimetric assays have been developed to assay the multi-stage process of their fusion in vitro. As an excellent model of general eukaryotic SNARE-, Rab GTPase-, and SM protein-dependent intracellular membrane fusion, the yeast homotypic vacuole fusion system will comprise the backbone of our genetic, molecular, and biochemical approaches. Initial studies in the lab will characterize factors that allow an organism to drive a given membrane fusion event with a specific set of fusion machinery. The recent discovery that a yeast protein complex (the so-called HOPS complex) provides a proofreading activity to ensure proper homotypic vacuole fusion will be further studied. In addition, we will conduct genetic and biochemical screens of the intracellular pathogens Salmonella and Legionella to identify bacterially-produced inhibitors of vacuole fusion in vivo and in vitro. Mechanistic information gleaned from these studies will open new avenues towards the detailed study of basic bacterial pathogenesis.