OSU Microbiology
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Faculty Bios

Pathogenicity of Gram-negative Pathogens.

Neil R. Baker

Neil R. Baker

baker.2@osu.edu

Associate Professor,
Ph.D., University of Maryland, 1975.

Pathogenicity of gram-negative pathogens.

My research is primarily concerned with secondary pathogens of the respiratory tract. Pseudomonas aeruginosa, has been of particular concern because of the high mortality rates associated with hospital-acquired infections and the high incidence in cystic fibrosis patients. More recently we have expanded our interests to other Gram-negative bacteria.

Recent investigations have concerned identification of the receptors on eukaryotic cells for bacteria. The primary interest has been identification of receptors for Pseudomonas aeruginosa. Two classes of receptors have been identified. We are currently trying to identify the bacterial adhesins responsible for the binding. At least two adhesins have been characterized: pili and flagella. The role of pili has been documented by several investigators. How flagella may function as an adhesin is less clear, but our results indicate that it is required for adherence. We have hypothesized that after the organisms have attached to cells by contractile pili, they are brought to the cell surface where secondary adhesins create a stronger attachment. Flagellin appears to be a major secondary adhesin.

An exciting finding that may be related to the targeting of the flagellin is posttranslational modification of flagellin. A segment of DNA located downstream of the flagellin gene appears to be involved in the modification of flagellin. This process is distinct from the phosphorylation of flagellin that has been noted by others, and may involve glycosylation–a rare event in bacteria. The regulation and significance of the modificationare a major areas of focus.

An new area of investigation is genetic immunization to prevent the occurence of secondary infection following traumatic injury. Preliminary data using the P. aeruginosa flagellin gene in a eucaryotic expression system indicates that we can induce a strong immune response to bacterial antigens following injection of the plasmid DNA. Current studies are concentrating on defining the optimum conditions for immunization and determining the efficacy of these vaccines.

Recent Publications

 

Baker, N. and Verran, J. (2004). The future of Microbiology Laboratory Classes: Wet, dry or in combination. Nature Rev. Microbiol. 2 :338-342.
Denis-Mize KS, Price BM, Baker N.R., Galloway DR. 2000. Analysis of immunization with DNA encoding Pseudomonas aeruginosa exotoxin A. FEMS Immunol Med Microbiol. 27:147-54.
Wahl, S. and Baker, N.R. Identification of a locus involved in the posttranslational modification of flagellin from Pseudomonas aeruginosa. Molecular Microbiology – In preparation

Wahl, S. and Baker, N.R. Analysis of type a flagellin from Pseudomonas aeruginosa strain PAO1. In preparation.

Baker, N.R., Gehring, K. and Wahl, S. Evidence for a direct role of flagellin in the adherence of Pseudomonas aeruginosa to glycosphingolipids. In preparation.

Baker, N. R. 1992. Mucosal adherence of Pseudomonas aeruginosa. In R.B.Fick (ed.), Pseudomonas aeruginosa the opportunist: pathogenesis and disease. CRC Press, Inc.

Baker, N.R., V. Minor, C. Deal, M.S. Shahrabadi, D.A. Simpson, and D.E.Woods. 1991. Pseudomonas aeruginosaexoenzyme-S is an adhesin. Infect.Immun. 59:2859-2863.

Baker, N. R. 1991. The role of exoenzyme-S in the adherence of Pseudomonas aeruginosa. Pediat. Pulmon. S6: 136-137.

Baker, N.R., G. Hansson, H. Leffler, G. Riise, and C. Svanborg-Eden. 1990.Glycosphingolipid receptors for Pseudomonas aeruginosa. Infect. Immun.58:2361-2366

Faculty Bios

Microbial Genomics, Emphasizing the Molecular Biology of Polysaccharide Degradation and Bacterial Adhesion to Plant Surfaces.

Mark Morrison

morrison.234@osu.edu

Associate Professor
Ph.D., University of Illionois, 1991

Microbial genomics, emphasizing the molecular biology of polysaccharide degradation and bacterial adhesion to plant surfaces.

An obligatory step in cellulose degradation by anaerobic bacteria is the adhesion of the bacterium to the polysaccharide. In many anaerobic bacteria the adhesion protein, and the enzymes required for polysaccharide hydrolysis, are organized into a complex and interesting structure called the cellulosome. The genetics and biochemical characteristics of cellulosomes are quite diverse, but very little is known about the events underpinning the synthesis and assembly of these interesting complexes. My laboratory is currently examining the production and characteristics of these complexes in a variety of anaerobic cellulolytic and non-cellulolytic bacteria. My lab is also interested in the adhesion of bacteria to plant surfaces, and we have identified a novel form of cellulose-binding protein, produced by the gram-positive, cellulose-degrading anaerobe Ruminococcus albus. This protein is a member of the Pil-protein family, and is very similar to the type 4 fimbrial proteins of Gram-negative, pathogenic bacteria. These studies have provided new insights into the adhesion of bacteria to plant surfaces, and call attention to the likely existence of genetically analogous adhesion determinants in both pathogenic and non-pathogenic bacteria. A number of cellulose-degrading microorganisms are now the subject of genome sequencing projects, which will facilitate a more global analysis of the genetics and molecular biology controlling this key process in the cellulose degradation.

My laboratory is also studying the microbial interactions that support optimal rates and extent of cellulose degradation. One example is the requirement by Ruminococcus albus for micromolar amounts of phenylacetic and phenylpropionic acids, which stimulate the formation of cellulosome-like complexes and maximal rates of cellulose degradation. Other members of the microbial community produce these phenyl-substituted fatty acids, and my laboratory is currently investigating how R. albus responds to these compounds, by using techniques such as differential display RT-PCR and proteomics. A second example is the synergistic association between cellulose-degrading eubacteria and methanogenic archaea, via the process known as interspecies hydrogen transfer. Many cellulose-degrading bacteria will produce hydrogen during fermentation, which permits the bacterium to produce additional ATP by substrate-level phosphorylation, and affords faster rates of growth and cellulose degradation. However, the cellulose-degrading bacteria are dependent on the consumption of hydrogen by autotrophic methanogens to maintain this energetically favorable pathway of fermentation. Now that the genomes of several methanogens and cellulose-degrading bacteria have been completely sequenced, it will be possible to investigate this synergistic association more completely. An ultimate goal for our research is to use genomics and related methods to understand the molecular details of these microbial interactions and improve the bioconversion of cellulosic wastes into useful products such as ethanol.

Recent Publications

Morrison, M. and Miron, J. 2000. MiniReview: Adhesion to cellulose by Ruminococcus albus: a combination of cellulosomes and Pil-proteins? FEMS Microbiol. Letts. 185: 109-115

Heng, N.C.K., Bateup, J.M., Loach, D.M., Wu, X., Jenkinson, H.F., Morrison, M. and Tannock, G.W. 1999. The influence of different functional elements of plasmid pGT232 on the maintenance of recombinant plasmids in Lactobacillus reuteri populations in vitro and in vivo. Appl. Environ. Microbiol. 65: 5378-5385.

Larson, M.A. and Morrison, M. 1999. Application of the differential display RT-PCR technique to examine conditional gene expression in Ruminococcus albus. In: Bell, C.R. et al. (Eds.) Microbial Biosystems: New Frontiers. Proc. 8th Int. Symp. Microb. Ecol

Pegden, R. S., Larson, M.A., Grant, R.J., and Morrison, M. 1998. Adherence of the gram-positive bacterium Ruminococcus albus to cellulose, and identification of a novel form of cellulose-binding protein which belongs to the Pil-family of proteins. J. Bacteriol. 180: 5921-5927.

White, B.A., Cann, I.K.O., Mackie, R.I., and Morrison, M. 1997. Cellulase and xylanase genes from ruminal bacteria. Domain analysis suggests a non-cellulosome model for organization of the cellulase complex. pp. 69-80. In: Onodera, R. et al. (Eds.) “Rumen Microbes and Digestive Physiology of Ruminants ” Japan Scientific Societies Press, Tokyo, Japan.

Faculty Bios

Associate Professor

Juan D. Alfonzo

alfonzo.1@osu.edu

B.S., Indiana University- Bloomington, IN
Ph.D., Indiana University- Bloomington, IN
Post-doctoral Research, University of California, Los Angeles

The Alfonzo Lab

Editing & Modification of tRNA: Roles in Mitochondrial Biogenesis and Disease.

We are interested in RNA processing events that are unique to trypanosomes and could be exploited as targets for the design of therapies against protozoan diseases. To date, the Protozoa are responsible for the infection and death of millions of people worldwide. I am especially interested in two facets of RNA processing in trypanosomes: tRNA editing and tRNA modification.

The Mechanism of tRNA Editing

RNA editing involves any process by which the information content of a given transcript (mRNA, tRNA, etc.) is changed so that it will differ from that encoded in the genome. RNA editing occurs by a variety of chemically distinct post-transcriptional mechanisms including nucleotide insertion, deletions, base deaminations, and nucleotide exchange (to name a few) and plays an important role in the regulation of gene expression. In trypanosomes, I discovered that the nucleus-encoded tRNATrp undergoes an essential cytosine to uridine editing of the wobble position of the anticodon upon importation into the mitochondrion. Most notable, the deamination of cytosine to uridine at the anticodon allows the decoding of UGA codons as tryptophan. We are interested in the mechanism of tRNA editing and plan to use a genetic and biochemical approach to achieve a complete structure/function analysis of this process in trypanosomes

The Modifications of tRNA and Contribution to Mitochondrial Function

The functional importance of RNA modifications is evident from the large number of phylogenetically conserved modifications that occur in various cellular RNAs. Little is known about the role and extent of RNA modifications in early divergent eukaryotes. In addition, there are increasing numbers of reports linking tRNA modifications with mitochondrial diseases. I plan to use trypanosomes as a model system to study the role that specific tRNA modifications play in mitochondrial function. These studies will be the basis for examining the role site-specific tRNA modifications plays in mitochondrial function. The long-term goal is to utilize the exquisite specificity of these editing and modification enzymes for the long-term applications in gene therapy in humans.

In general, the emphasis of my laboratory will focus on the translation of RNA structure into RNA function in a biological system. Specifically, I hope to dissect the pathways involved in tRNA maturation, including editing and modification, to address issues involving molecular recognition. I firmly believe this research will not only uncover useful knowledge of basic cellular processes, but also provides the added incentive of great overall implications towards applications in medicine.

Faculty Bios

Biochemistry, Molecular Biology, Molecular Ecology, Archaea.

  F. Robert Tabita

Professor; Ohio Eminent Scholar of Microbiology
Professor, Natural Resources
Professor, Plant Biology
Director, Plant-Microbe Genomics Facility
Director, Plant Molecular Biology/Biotechnology Program
Ph.D., Syracuse University, 1971.

 

Biochemistry, molecular biology, molecular ecology, archaea.

My laboratory is concerned with the molecular regulation, biochemistry,and enzymology of carbon dioxide assimilation.  All organisms require CO2 and many enzyme-catalyzed reactions employ CO2 as a reactant for processes as important and varied as carbohydrate metabolism, lipid biosynthesis,and the production of important metabolic intermediates for the cell. With the realization that many microorganisms require CO2 in order to elicit pathogenesis, it is not surprising that CO2metabolism and its control has great health relevance.  Carbon dioxide may also be employed as the sole source of carbon by a large and diverse group of organisms on this planet.  For this reason, CO2 fixation is a process that is associated with global issues of agricultural productivity, carbon cycling, and industrial productivity.  CO2 is also recognized as the chief green house gas and has been implicated in the general warming of the earth’s biosphere.  For all these reasons,research on various aspects of CO2 fixation control, biochemistry,and ecology have attracted wide interest.  The following pages summarize our ongoing and future efforts to probe various aspects of this issue.

Molecular Regulation

We are specifically interested in how CO2 fixation structural genes are regulated in bacteria and various eukaryotic organisms. The bulk of our work over the years has concentrated on two or three model systems, both of which enable organisms to use CO2 as the sole source of carbon for growth.  One system, the Calvin-Bassham-Benson (CBB) reductive pentose phosphate pathway, is undoubtedly the means by which most organic matter is produced on earth.  For these studies, we have concentrated on the metabolically versatile nonsulfur purple bacteria, especially Rhodobacter sphaeroides , Rhodobacter capsulatus , and Rhodopseudomonas palustris . In these organisms, we have found that the structural ( cbb ) genes are clustered in distinct operons, some of which are localized in different genetic elements, as in R. sphaeroides .  In this organism, the entire cbb regulon is under the control of a specific transcriptional regulator gene, cbbR , whose product, CbbR, positively controls the transcription of the two major operons required for CO2 fixation. CbbR must be activated in the cell, probably after binding a specific effector molecule, in order to turn on transcription.  We are currently studying aspects of the biochemistry of CbbR so that we can understand how the cell is able to convert this constitutively synthesized protein to the activated state.  Unlike R. sphaeroides, there are two cbbR genes in R. capsulatus , each of which specifically regulates its cognate cbb operon.   Current studies indicate that several factors, in addition to CbbR, impinge on control in these organisms.  Of particular note is a global two-component signal transduction system that integrates the control of CO2 assimilation with the nitrogen fixation ( nif ) system and other processes important for energy generation.  Indeed, molecular signals are apparently received at the surface of the cell, undoubtedly reflecting the redox state of some signal molecule (? in Fig. 1), thus stimulating a membrane-bound sensor kinase (RegB/PrrB) to become autophosphorylated. RegB~P then transfers its phosphate to a response regulator protein, RegA/PrrA; the phosphorylated form of this molecule (RegB~P) then binds to DNA, affecting transcription.  This has become a fairly complicated regulatory system, as good evidence for the presence of other regulator molecules has also been obtained.  An interesting aspect of our studies on cbb control was the finding that the nitrogen fixation ( nif ) system, and its control, is intimately involved, with the Reg (Prr) system important for this interaction.  Indeed, knocking out the cbb system, under conditions where CO2 is normally used as an electron acceptor and not a carbon source, causes both organisms to derepress nitrogenase synthesis and the nifHDK genes, so that reducing equivalents may now be dissipated as a result of the H+-reducing hydrogenase activity of nitrogenase.  Thus, the organism exquisitely controls how it handles environmental signals related to carbon and nitrogen metabolism.  Our current model for this complex regulatory process is summarized below for R. sphaeroides (Fig. 1); basically this conceptual model holds for R. capsulatus andR. palustris as well. Note, manipulating the regulation of this system allows the organisms to produce copious quantities of hydrogen gas, a biofuel of enormous significance.

 

Fig. 1. Conceptual model showing the interplay of various factors involved in signal transduction and regulation of cbb gene expression in R. sphaeroides . The link between the CO2 ( cbb ) and nitrogen regulatory system, including the nitrogen fixation ( nif ) genes is shown. Primary signals are received at the cytoplasmic membrane. This is thought to affect the redox potential of some key component (?) influencing RegB/PrrB autophosphorylation and the subsequent formation of RegA~P (PrrA~P). RegA~P (PrrA~P) interacts directly with the cbb and nif operator-promoter regions (Dubbs and Tabita, submitted for publication). Positive regulation is thus conferred both by the CbbR protein and RegA~P(PrrA~P), the phosphorylated response regulator of the Reg (Prr) two-component regulatory system. CbbR is converted to CbbR (the transcriptionally active form of this molecule), presumably by virtue of binding a coinducer molecule produced under CO2 fixation conditions or other growth conditions that favor cbb transcription. The expression of glnB is affected by the cbb system (Qian and Tabita 1998) with glnB influencing nif derepression through the Ntr system and NifA. Blockage of the CBB pathway results in hydrogen evolution by virtue of the hydrogenase activity of the derepressed nitrogenase complex (Joshi and Tabita 1996).The nitrogenase complex and its inherent hydrogenase activity thus serves to remove excess reducing equivalents not dissipated in strains unable to use CO2 as an electron acceptor. p, refers to promoter-operator regions that are activated in a positive manner (+).  From Tabita (1999).

 

The Rhodopseudomomas palustris Microbial Cell Project/Genomes to Life Program

OSU recently initiated a new program to study mechanisms by which a single organism controls the ability to regulate many aspects of metabolism simultaneously ( http://www.osu.edu/researchnews/archive/micromet.htm ). For this purpose, the Department of Energy awarded a large grant to a consortium of investigators from seven institutions to begin the Rhodopseudomonas palustris Microbial Cell Project. Ohio State, with F. R. Tabita, the Principal Investigator, is the lead institute of the consortium.This project involves the genetically tractable and metabolically versatile organism, Rhodopseudomonas palustris , whose entire genomic sequence was recently completed. Postgenomic approaches will be used to determine how the organism regulates the ability to produce useful forms of energy while also removing greenhouse gases (such as carbon dioxide) and degrading toxic compounds. Rps. palustris is metabolically similar, but more versatile than other nonsulfur purple bacteria ( Rhodobacter ) under study in the laboratory (see above).

Enzymology

A. RubisCO

We are deeply involved in efforts to determine how the structure influences the function of key enzymes and proteins important for CO2 fixation.  How the activity of these proteins is regulated in the cell is also of prime importance to our laboratory.  Over the years, we have primarily focused on RubisCO, which is the key enzyme of the CBB pathway.  This enzyme is a very poor catalyst, yet it is the protein that actually fixes the bulk of CO2 on this planet.  This is probably why RubisCO is the most abundant protein found on earth, making aspects of its biochemistry and molecular control (see above) so topical.  The major issue that we study is the basis by which RubisCO discriminates between CO2 and O2 , two gaseous substrates that compete for the active same active site on the protein. This is a very important issue as O2normally prevents efficient CO2 fixation. Despite a wealth of structural and mechanistic information, it is still not clear how closely related RubisCO molecules possess different specificities for CO2 and O2 .  Taking a combined molecular biological and chemical approach we are attacking this problem by constructing novel mutant enzymes, and have developed prokaryotic genetic selection procedures to facilitate these efforts (Smith and Tabita 2003).  An added bonus has been the finding, from genomic sequencing studies, that anoxic hyperthermophilic archaea contain RubisCO genes.  We have recently found that at least some of these putative RubisCO sequences encode for bona fide RubisCO activity.  As these enzymes are derived from organisms that never encounter molecular oxygen, these proteins are proving to be very interesting; i.e., they serve as model systems to understand how the active site of RubisCO may have evolved. The structure of the Methanococcus jannaschii and Archaeoglobus fulgidus RubisCO enzymes have been modeled (Fig. 2), providing a ready system for current and future structure-function studies of the archaeal enzyme. More recently, we have shown that these enzymes actually produce physiologically significant RubisCO proteins (Finn and Tabita 2003) and we have elucidated the probable role of this enzyme in archaea.

 

Fig. 2. Tertiary structure prediction of archaeal RubisCO molecules. The predicted tertiary structure of the M. jannaschii sequence (A) and the A. fulgidus 2 sequence (B) is compared to the known structure of (C), the Synechococcus large subunit.  The Synechococcus small subunit is also shown to the lower left of the structure (in amber).  Label sizes and shading reflect the distance from the viewer, smaller and darker being further from the viewer. The main features are highlighted as follows: yellow = active site residues within 3.3 � of the bound transition state analogue CABP in the Synechococcus enzyme, and the equivalent residues in the M. jannaschii and A. fulgidus sequences; red = loop-6; cyan = highly divergent ?-helix-6 residues; purple = residues that appear to be absent in the M. jannaschii and A. fulgidussequences (eight residues at the N-terminus of the Synechococcus enzyme were not resolved in the structure determination and therefore are not shown here).  Mg 2 + is represented as a green sphere and CABP as a ball and stick model in C.  From Watson et al. (1999)

 

B. Other CBB Enzymes

Other enzymes of the CBB pathway are also under study.  RubisCO activase is an enzyme that catlyzes the removal of inhibitory phosphorylated compounds from the active site of RubisCO. Another key enzyme of the pathway that is studied is phosphoribulokinase, the enzyme that catalyses the formation of the substrate for RubisCO.  Transketolase and pentose 5-phosphate-3-epimerase are two enzymes whose genes, cbbT and cbbE , respectively, are found in the cbb regulon (Fig.1).  These two enzymes catalyze important 5-carbon sugar phosphate transformations and are also studied. Transketolase has particularly been the focus of recent studies, in which the involvement of a specific cysteine residue in cofactor binding was elucidated (Bobst and Tabita submitted)

C. RTCA Cycle Enzymes

We also study another model CO2 fixation system, the reductive tricarboxylic acid (RTCA) pathway, in which several interesting and unique CO2 fixation catalysts are focal points. This is a pathway found in many bacteria and eukaryotic CO2 fixing organisms, and many of the key reactions are important in archaea as well. Virtually nothing is known of the molecular regulation of the RTCA cycle and we have recently developed an interesting model system, the green sulfur bacterium Chlorobium tepidum , for these studies. This organism, unlike other organisms that use this pathway, has a fairly well defined genetic system and the organism grows rapidly. We have recently isolated all the relevant and important enzymes of this pathway, including pyruvate synthase, alpha-ketoglutarate synthase, ATP-citrate lyase, and PEP carboxylase, along with several important electron carriers including rubredoxin, two ferredoxins, and two cytochromes. The structural genes for these proteins have all been isolated and we have begun to study aspects of the molecular regulation of this interesting CO2 fixation process. The key CO2 fixation catalysts are redox sensitive proteins which show interesting and novel interactions with electron carriers such as rubredoxin (Fig. 3). This work has recently been published in the Journal of Biological Chemistry (Yoon et al. 1999; 2001) and other manuscripts are in preparation.

Fig. 3. EPR spectrum of C. tepidum rubredoxin before (A) and after (B) reduction with pyruvate ferredoxin oxidoreductase/pyruvate synthase.

An interesting aspect of our work with C. tepidum was the finding of an unusual RubisCO-like protein (RLP) which is involved with sulfur metabolism and the stress response (Hanson and Tabita 2001, 2003).

Molecular Ecology

We have collaborated with marine scientists at the University of South Florida to understand how the regulation of key CO2 fixation genes, like the RubisCO genes, are controlled in the oceans. This work, a combination of ship-board and laboratory investigations, is devoted to a primary problem, namely the sequestration of CO2 in the environment. Procedures for the direct examination of RubisCO transcripts in the open ocean were developed and applied to the global CO2 fixation problem. As these studies unfolded, it became possible to identify organisms which contribute to active CO2 fixation and sequestration by amplifying and sequencing specific RubisCO transcripts via RT-PCR technology. These studies have been performed in concert with physiological and biochemical studies with marine cyanobacteria and algae, such that a coherent picture of how carbon dioxide assimilation may be controlled in these organisms, both in the environment and in the laboratory.

We also have a collaboratine project to ascertain the role and study the significance of a RubisCO in hydrothermal vent archaeal extremophiles. These studies promise to provide much information as to how CO2 fixation pathways may evolve in potential extraterrestial systems .

 

Applied Studies

Knowledge of the biochemistry and molecular control of CO2 fixation has stimulated us to use this knowledge to consider the possibility that useful compounds of economic and industrial importance might be synthesized using this cheap and ubiquitous gas as the starting material. In one study, we have considered the possibilty that CO2 might be converted to ethanol using a genetically engineered strain with the requisite genes. Other offshoots of this technology are also being developed for the production of value-added products.

 

Future Studies

In addition to continuing the molecular, biochemical, and ecological efforts described above, we are embarking on new approaches to learn how CO2 fixation control relates to the overall regulation of metabolism in representative organisms. Taking advantage of the many genomes that have been sequenced from CO2 -fixing prokaryotes, including bacteria and archaea which are studied in our laboratory, we have initiated studies employing microarray and proteomics technology to investigate the global expression of CO2 -responsive genes in these organisms. This technology promises to allow us the capability of identifying a large complement of genes important for CO2 fixation that we would not normally identify with standard procedures. Moreover, since we have mutants at our disposal that are known to influence carbon assimilation, it should be feasible to greatly improve our efforts to examine overlapping metabolic schemes. Finally, it is our intention to use this technology to examine genes that are turned on during CO2 -dependent pathogenesis in selected organisms of health significance.

Seminars & Schedules

Study of Bacterial Gene

Study of bacterial gene expression heavily relies on E. coli-based in vivo and in vitro systems with RNA polymerase at their core. Although methods utilizing RNA polymerase assembled from individually expressed subunits or single-subunit expression vectors have met with considerable success, our experience indicates that co-overexpression vectors, featuring most or all RNA polymerase subunits have substantial advantages, particularly in efficiency of assembly of some mutants, deemed “defective” based on in vitro assembly attempts (see for example Artsimovitch et al, Co-overexpression of Escherichia coli RNA polymerase subunits allows isolation and analysis of mutant enzymes lacking lineage-specific sequence insertions, JBC, 2003, 278(14):12344-55). Here we present the latest co-overexpression vector for E. coli RNA polymerase, pVS10, and a sample protocol for purification of the enzyme produced using this vector (although it should be compatible with any classical RNA polymerase purification protocols as well).

pVS10 vector contains ORFs for 4 subunits, comprising E. coli core RNA polymerase rpoA (a), rpoB (b), rpoC (b’), and rpoZ (w). This plasmid has been successfully used as a platform for genetic manipulations of rpo genes and production of the wild-type as well as a number of mutant enzymes. It has been tested for expression in BL21(DE3), XJb(DE3), and DH5a(DE3) cell lines using both IPTG– and autoinduction (e.g. Overnight Express, Novagen) protocols. Yield varies with the toxicity of the construction, the identity of the strain and the induction conditions, usually within 1-10 mg/L of culture.

Induction and growth conditions:

The autoinduction using the Overnight Express (Novagen) reagents was carried out according to the manufacturer’s protocol with modifications to preculture preparation. BL21(DE3) cells transformed with pVS10 plasmid were inoculated from the frozen stock into 30 ml of LB medium (Miller) supplemented with ampicillin at 100 mg/L in 125 ml flask and grown with agitation at 37C till OD600 =0.6, when 0.6 ml of this culture were used to inoculate 600 ml of LB medium (Miller) supplemented with carbenicillin at 100 mg/L in 2 L flask, supplemented with Overnight Express reagents per Novagen protocol. The culture was incubated at 32C with agitation for 18 hours, and cells were harvested by centrifugation (4C, 5000 g, 10 min).

Lysis and Ni-affinity chromatography:

Cell pellet was frozen (-80C) and thawed (on ice), cells were resuspended in 50 ml of lysis buffer (500 mM NaCl, 50 mM Tris-HCl (pH 6.9), 5% glycerol, 0.2 mM b-ME) supplemented with 1X Complete EDTA-free protease inhibitors cocktail (Roche), and 1 mg/ml lysozyme. Suspension was incubated on ice for 60 min with occasional swirling, supplemented with Tween 20 to 0.2% and sonicated briefly to disrupt the cells. Extract was cleared from cell debris by centrifugation (27 000 g, 15 min, 4C). Cleared extract was combined with 4 ml of 50 % Ni-NTA slurry in lysis buffer and incubated with agitation for 30 min at 4C. The slurry was poured into a disposable gravity flow column (Bio-Rad) and drained. The column was washed with 20 volumes (40 ml) of lysis buffer, and 10 volumes of heparin column loading buffer (50 mM Tris-HCl, pH 6.9, 5 % glycerol, 0.5 mM EDTA, 1 mM b-ME) supplemented with 10 mM imidazole. Elution was carried out by adding 1 ml aliquotes of heparin column loading buffer, supplemented with 250 mM imidazole. Fractions with more than 0.2 mg/ml of protein (by Bradford) were analyzed using SDS-PAGE, the ones containing RNAP were pooled and if needed concentrated to total volume of 4-5 ml using Amicon Ultra 100K concentrators (Millipore).

Heparin-affinity chromatography:

RNAP-containing pool of fractions was loaded onto a 5 ml Heparin HiTrap column (Amersham) at 0.5 ml/min using low-pressure LC system (AKTAPrime, GE) (can use Heparin 16/10 columns for larger scale preps, load at 1 ml/min).

Column loaded with polymerase preparation was washed with 5 column volumes (25 ml) of loading buffer, elution was carried out using linear gradient of NaCl in loading buffer : 0-1.5 M NaCl over 120 ml at 1 ml/min. Peak fractions were collected, pooled, and dialyzed against loading buffer (alternatively the ionic strength can be reduced by several cycles of concentration-dilution with loading buffer using Amicon Ultra 100K concentrators).

Ion-exchange chromatography:

Polymerase fractions collected in previous step were loaded onto 6ml ResourceQ column (GE) using the same system at 0.5 ml/min, washing and elution were carried out as described for Heparin Hi-Trap column. Peak fractions were analyzed by SDS-PAGE, pooled, and dialyzed against the DNA-agarose loading buffer (10 mM Tris-HCl (pH 7.9), 0.1 mM Na-EDTA, 0.2 mM b-ME, 5% glycerol).

DNA-affinity chromatography:

Polymerase fractions collected in previous step were loaded onto 5 ml DNA-Agarose HiTrap column (GE) using AKTAPrime LC system at 0.2 ml/min. Loaded column was washed with 4 volumes (20 ml) of DNA-agarose loading buffer, and purified core RNAP was eluted using linear gradient of NaCl in loading buffer 0-1.5 M over 100 ml at 0.2 ml/min. Core RNAP containing fractions were analyzed by SDS-PAGE, pooled and dialyzed against RNAP storage buffer.

Faculty Bios

Jessica Dyszel

Jessica Dyszel

I received my Bachelors degree in microbiology from Southern Illinois University. As an undergraduate I worked in an independent food-testing laboratory assisting in research projects and performing routine testing. Working as a laboratory technician furthered my interest in microbiology and prompted me to attend graduate school. When searching for a university to attend, the balanced program at Ohio State stood out. The diversity of the professors, their backgrounds, and research made the program very attractive when compared to other schools. During a campus visit I met many of the professors and graduate students and was surprised by the overall friendliness and enthusiasm of the department. After I joined the department, lab rotations allowed me to explore various fields and interests as well as meet more of the students and professors.

In 2002 I joined the lab of Brian Ahmer. Currently I am working on the identification of genes regulated by SdiA, a LuxR homolog, in Salmonella typhimurium. One of the greatest advantages to being at a large university is access to equipment and facilities, that an individual lab could not support. The classes that I have taken are constantly under revision to include the most recent information and involve the students in the process. I am sure that the education that I receive from the department will serve me well in the future.

Faculty Bios

Environmental and Industrial Microbiology

Olli H. Tuovinen

Olli H. Tuovinen

tuovinen.1@osu.edu

Professor
Ph.D., University of London, 1973

Environmental and Industrial Microbiology.

Several projects are underway in my laboratory that combine microbial metabolism and ecology with environmental disciplines. Examples of current studies include (1) the biodegradation of pesticides and polynuclear aromatic hydrocarbons, (2) microbial ecology and biogeochemistry of acid mine drainage, (3) microbial solubilization and weathering of minerals, and (4) microbial processes in landfills.
(1) Biodegradation is by far the single most important factor in the attenuation of pesticides in soils. Biodegradation rates vary with bioavailability and presence of organisms with degradative capabilities in soils and subsurface environments. We have previously investigated the degradation of phenoxyalkanoic acid and chloroacetamide herbicides and our current focus is on the pre-emergent weed killer atrazine. This compound has a symmetric, N-heterocyclic aromatic structure and has no known natural analog in the nature.
Annual applications of atrazine are known to enhance biodegradation rates in agricultural soils, and this acclimation effect has also been reported for many other herbicides. Biodegradation rate constants and the corresponding half-lives may vary by two orders of magnitude between soils with different histories of atrazine application, including reference soils with no prior exposure. Such kinetic differences reflect phenotypic and genotypic variation in soil microbial communities.
Complete biodegradation of atrazine involves dehalogenation, N-dealkylation, deamination, and ring-cleavage steps. Several genes of the atrazine degradative pathways have been described in the literature. The genes provide the basis for the characterization of soil microbial communities by molecular techniques such as DNA probes and PCR amplification, cloning, and sequencing. In addition to agricultural soils, we have also used these approaches for describing atrazine-degrading microbial communities in natural and constructed wetlands.
Biodegradation is also most important in the natural attenuation of polynuclear aromatic hydrocarbons; these compounds are highly nonpolar and sorb on soil and sediment particles. The scope in our studies is to assess the biodegradation potential of polynuclear aromatic hydrocarbons in compost materials and in sediments impacted by hydrocarbon pollution and stormwater runoff. The biodegradation potential yields useful information for assessing the fate and kinetics of the biodegradation of these compounds in environments impacted by anthropogenic sources. Such information may be useful in designing bioremediation strategies for solid waste materials.
(2) Mine drainage constitutes a serious water pollution problem in many metal and coal mine areas. Treatment methods of acid mine water include constructed wetland systems to remove acidity and metal ions. Our approach to elucidating microbial ecology in constructed acid mine wetlands has been based on using 16S rDNA-based techniques. With emphasis on non-culturable approaches, we have use PCR amplification with universal and genus/species specific phylogenetic primers, cloning and sequencing, RFLP, DGGE, and FISH to characterize microbial communities in a constructed wetland system treating acid coal mine water. Geochemical and mineralogical changes with time have been described by analysis of pore water and iron precipitates in aerobic and anaerobic zones of the constructed wetland.
(3) The dominant bacterial groups in constructed mine drainage wetland systems are aerobic, iron- and sulfur-oxidizing bacteria. These acidophilic bacteria are also involved in the oxidative dissolution of sulfide minerals in ore deposits and coal refuse piles. A long-term study in my laboratory has been to characterize dissolution processes using research-grade minerals as well as ores and concentrates. These bacteria have application for processing of copper-, uranium-, and precious metal-containing ore materials in the mining industry. We have also shown that anaerobic sulfate reducing-bacteria can also be involved in biogeochemical transformations by using solid-phase electron acceptors.
(4) Microbial processes in conventional landfills are usually limited by low moisture content. This dry tomb technology of landfill management is contrasted by landfill bioreactor approaches, which aim to accelerate waste decomposition and landfill gas production through leachate circulation. Our studies in landfill bioreactor technology are aimed at optimizing environmental conditions conducive to anaerobic microbial activities and assessing nutrient deficiency conditions for landfill materials.
Projects in my laboratory have prospective in remediation and other applications with interdisciplinary approaches, but the foundations are firmly based on microbial biochemistry, ecology, and physiology. Many of the research problems under study in my laboratory involve biogeochemical redox reactions, interfacial reactions, and molecular ecological approaches that help design microbiological strategies competitive in environmental investigations.

 

Recent Publications

Hao, Y., W.A. Dick, and O.H. Tuovinen. 2002. PCR amplification of 16S rDNA sequences in Fe-rich sediment of coal refuse drainage. Biotechnology Letters 24:1049-1053.

 

Nicomrat, D., W.A. Dick, and O.H. Tuovinen. 2002. Molecular bacterial ecology of Fe(III)-precipitates in a constructed wetland treating acid coal mine water. Paper No. 614. In: Transactions of the 17th World Congress of Soil Science, p. 614-1 to 614-9. Bangkok, Thailand.

 

Ostrofsky, E.B., J.B. Robinson, S.J. Traina, and O.H. Tuovinen. 2002. Analysis of atrazine-degrading microbial communities in soils using most-probable-number enumeration, DNA hybridization, and inhibitors. Soil Biology and Biochemistry 34:1449-1459.

 

Anderson , K.L., K.A. Wheeler, J.B. Robinson, and O.H. Tuovinen. 2002. Atrazine mineralization potential in two wetlands. Water Research 36:4785-4794.

 

Bevilaqua, D., A.L.L.L. Leite, O. Garcia Jr., and O.H. Tuovinen. 2002. Oxidation of chalcopyrite by Acidithiobacillus ferrooxidans and Acidithiobacillus thiooxidans in shake flasks. Process Biochemistry 38:589-594.

 

Stamper, D.M., M. Radosevich, K.B. Hallberg, S.J. Traina, and O.H. Tuovinen. 2002. Ralstonia basilensis M91-3, a denitrifying soil bacterium capable of using s -triazines as nitrogen sources. Canadian Journal of Microbiology 48:1089-1098.

 

Karnachuk, O.V., S.Y. Kurochkina, D. Nicomrat, Y.A. Frank, D.A. Ivasenko, E.A. Phyllipenko, and O.H. Tuovinen. 2003. Copper resistance in Desulfovibrio strain R2. Antonie van Leeuwenhoek 83:99-106.

 

Carlstrom, C.J., and O.H. Tuovinen. 2003. Mineralization of phenanthrene and fluoranthene in yardwaste compost. Environmental Pollution 124:81-91.

 

Li, Y., W.A. Dick, and O.H. Tuovinen. 2003. Evaluation of fluorochromes for imaging bacteria in soil. Soil Biology and Biochemistry 36:737-744.

 

Liu, Q., K.M. Mancl, and O.H. Tuovinen. 2003. Biomass accumulation and carbon utilization in layered sand filter biofilm systems receiving milkfat/detergent mixtures. Bioresource Technology 89:275-279.

 

Kang, Y.W., K. M. Mancl and O.H. Tuovinen. 2003. Biological treatment of turkey processing wastewater with coarse/fine sand filtration. In: Animal, Agricultural and Food Processing Wastes (Proceedings of the Ninth International Symposium), p. 44-49. American Society of Agricultural Engineers, St. Joseph, MI.

 

Xi, J., K.M. Mancl, and O.H. Tuovinen. 2003. Transformations and accumulation of carbon in gavel/sand filters treating cheese processing wastewater. In: Animal, Agricultural and Food Processing Wastes (Proceedings of the Ninth International Symposium), p. 341-349. American Society of Agricultural Engineers, St. Joseph, MI.

 

Radosevich, M., and O.H. Tuovinen. 2004. Microbial degradation of atrazine in soils, sediments and surface waters. In: Pesticide decontamination and detoxification (ACS Symposium Series No. 863) (J.J. Gan, P.C. Zhu, S.D. Aust, and A.T. Lemley, Eds.), p. 129-139. American Chemical Society, Washington, D.C.

 

Li, Y., W.A. Dick, and O.H. Tuovinen. 2004. Fluorescence microscopy for visualization of soil microorganisms � a review. Biology and Fertility of Soils 39:301-311.

 

�tyriakov�, I., T.M. Bhatti, J.M. Bigham, I. �tyriak, A. Vuorinen, and O.H. Tuovinen. 2004. Weathering of phlogopite by Bacillus cereus and Acidithiobacillus ferrooxidans. Canadian Journal of Microbiology 50:213-219.

 

Rowan, M.A., K. M. Mancl, and O.H. Tuovinen. 2004. Clogging incidence of drip irrigation emitters distributing effluents of differing levels of treatment. In: On-site Wastewater Treatment X(Proceedings of the Tenth National Symposium on Individual and Small Community Sewage Systems), p. 84-91. American Society of Agricultural Engineers, St. Joseph, MI.

 

Xi, J., K.M. Mancl, and O.H. Tuovinen. 2005. Carbon transformation during sand filtration of cheese processing wastewater. Applied Engineering in Agriculture 21:271-274.

 

Karnachuk, O.V., N.V. Pimenov, S.K. Yusupov, Y.A. Frank, A.H. Kaksonen, J.A. Puhakka, M.V. Ivanov, E.B. Lindstr�m, and O.H. Tuovinen. 2005. Sulfate reduction potential in sediments in the Norilsk mining area, northern Siberia. Geomicrobiology Journal 22:11-25.

 

Kang, Y.K., K.M. Mancl, and O. H. Tuovinen. 2005. Feasibility of renovating turkey processing wastewater using fixed film bioreactors. In: Proceedings of the 14th Annual Technical Education Conference, 9 p. National Onsite Wastewater Recycling Association, Cleveland, OH.

 

Gaur, R.S., L. Cai, K.M. Mancl, and O.H. Tuovinen. 2005. Pretreatment of animal fat through coarse sand filters. In: Proceedings of the 14th Annual Technical Education Conference, 10 p. National Onsite Wastewater Recycling Association, Cleveland, OH.

 

Rowan, M., K.M. Mancl, and O.H. Tuovinen. 2005. Performance of drip irrigation emitters distributing primary and secondary wastewater effluent. In: Proceedings of the 14th Annual hnical Education Conference, 9 p. National Onsite Wastewater Recycling Association, Cleveland, OH.

 

Rzhepishevska, O.I., E.B. Lindstr�m, O.H. Tuovinen, and M. Dopson. 2005. Bioleaching of sulfidic tailing samples with a novel, vacuum-positive pressure driven bioreactor. Biotechnology and Bioengineering 92:559-567.

 

Stamper, D.M., J.A. Krzycki, D. Nicomrat, S.J. Traina, and O.H. Tuovinen. 2005. Ring-cleaving cyanuric acid amidohydrolase activity in the atrazine-mineralizing Ralstonia basilensis M91-3. Biocatalysis and Biotransformation 23:387-396.

 

Nicomrat, D., W.A. Dick, and O.H. Tuovinen. 2006. Assessment of the microbial community in a constructed wetland that receives acid coal mine drainage. Microbial Ecology, in press.

 

Wang, H., J.M. Bigham, and O.H. Tuovinen. 2006. Formation of schwertmannite and its transformation to jarosite in the presence of acidophilic iron-oxidizing microorganisms. Materials Science and Engineering C, in press.

Faculty Bios

Understanding the Relationship Between Ribosome Structure and Function

  Kurt Fredrick

fredrick.5@osu.edu

Assistant Professor

B.A. Biology, Gustavus Adolphus College, 1992
Ph.D. Microbiology, Cornell University, 1997
Postdoctoral research, University of California Santa Cruz, 1997-2003

 

We are interested in how the ribosome works. The ribosome is a large (~2.5 MDa), two-subunit, RNA-based machine that translates the genetic code in all organisms.

In recent years, numerous X-ray crystal structures of the ribosome and its isolated subunits and many cryo-EM reconstitutions of functional ribosomal complexes have been reported. Today, a primary challenge for the field is to elucidate the functional roles of specific structural elements of the ribosome. Since the ribosome is the most common target of natural antibiotics, gaining a better understanding of how the ribosome functions should contribute substantially to the development of new antibiotics. In our group, we use mutagenesis and antibiotics to address questions of ribosome structure and function. One of our primary interests is translocation, the coupled movement of tRNA and mRNA within the ribosome. In recent work, we have shown that destabilization of the codon-anticodon helix accompanies movement of tRNA from the P/P to P/E state, an important transition during translocation.

McGarry, K. G., Walker, S. E., Wang, H. and Fredrick, K. 2005. Destabilization of the P site codon-anticodon helix results from movement of tRNA into the P/E hybrid state within the ribosome. Mol. Cell 20: 613-622.

Abdi, N. M. and Fredrick, K. 2005. Contribution of 16S rRNA nucleotides forming the 30S subunit A and P sites to translation in E. coli . RNA 11: 1624-1632.

Yassin, A., Fredrick, K. and A. S. Mankin. 2005. Deleterious mutations in small subunit ribosomal RNA identify functional sites and potential targets for antibiotics. PNAS 102: 16620-16625.

Noller, H. F., Hoang, L. and Fredrick, K. 2005. The 30S ribosomal P site: A function of 16S rRNA. FEBS Letters 579: 855-858.

Hoang, L., Fredrick, K. and H. F. Noller. 2004. Creating ribosomes with an all-RNA 30S subunit P site. PNAS 101: 12439-12443.

Fredrick, K. and H. F. Noller. 2003. Catalysis of ribosomal translocation by sparsomycin. Science 300: 1159-1162.

Fredrick, K. and H. F. Noller. 2002. Accurate translocation of mRNA by the ribosome requires a peptidyl group or its analog on the tRNA moving into the 30S P site. Mol. Cell 9: 1125-1131.

Noller, H. F., Yusupov, M. M., Yusupova, G. Z., Baucom, A., Lieberman, K., Lancaster, L., Dallas, A., Fredrick, K., Earnest, T. N. and J. H. D. Cate. 2001. Structure of the ribosome at 5.5 � resolution and its interaction with functional ligands . Cold Spring Harbor Symp. Quant. Biol. 66: 57-66.

Fredrick, K., Dunny, G. M. and H. F. Noller. 2000. Tagging ribosomal protein S7 allows rapid identification of mutants defective in assembly and function of 30S subunits. J. Mol. Biol. 298: 379-394.

Faculty Bios

Associate Professor Of Microbiology

Brian Ahmer

Ahmer Dot 1 At Osu Dot Edu

B.S., Colorado State University,
Ph.D., Washington State University
Post-doc with Fred Heffron at Oregon Health Sciences University

Associate Professor Of Microbiology

Detection of other microbial species and the host environment by Salmonella.

We are studying metabolic and environmental inputs to virulence gene regulation in Gram-negative pathogens, primarily Salmonella. The two regulators studied most intensely, sdiA and sirA, are located adjacent to each other on the chromosome.

SdiA. SdiA is a transcription factor of the LuxR family that detects bacterial pheromones (N-acyl homoserine lactones, or AHLs). SdiA detects only the pheromones made by other species of bacteria. In the figure below wild-type Salmonella is using SdiA to detect pheromones synthesized by Yersinia enterocolitica. This was done by fusing one of the genes that SdiA regulates to the genes for luciferase. The Salmonella and Yersinia are streaked perpendicular to each other on an LB agar plate and you can see that the Salmonella lights up near the Yersinia. The second picture used a fusion to lacZ instead of luciferase. LacZ activity is indicated by the blue color. This experiment used motility agar so that the Salmonella and Yersinia could swim to each other in the plate. In both pictures note that only the wild-type Salmonella can detect the Yersinia. The sdiA mutant is “blind”. Also, Salmonella can only detect the wild-type Yersinia. The yenI mutant of Yersinia cannot make pheromones. Using a genetic screen we have determined that in Salmonella, SdiA activates a horizontal acquisition named srgE for which the function is unknown, and a small cluster of genes known as the rck operon. This operon is located on the Salmonella virulence plasmid and confers adhesion to epithelial cells and resistance to killing by mammalian complement systems. We are currently using a technique called RIVET to determine when and where Salmonella detects other microbial species in host animals. Our most recent discovery is that Salmonella can detect Yersinia enterocolitica in mice and pigs, and Aeromonas hydrophila in turtles (see papers below).

Papers on SdiA:

Jessica L. Dyszel, Jenee N. Smith, Darren E. Lucas, Jitesh A. Soares, Matthew C. Swearingen, Mathew A. Vross, Glenn M. Yong, and Brian M. M. Ahmer. 2010. Salmonella enterica serovar Typhimurium can detect acyl homoserine lactone production by Yersinia enterocolitica in mice. Journal of Bacteriology 192: 29-37.

Jessica L. Dyszel, Jitesh A. Soares, Matthew C. Swearingen, Amber Lindsay, Jenee N. Smith, Brian M. M. Ahmer. 2010. E. coli K-12 and EHEC Genes Regulated by SdiA. PLoS ONE 5(1): e8946.

J. T. Noel, J. Joy, Jenee N. Smith, M. Fatica, K. R. Schneider, Brian M. M. Ahmer, and Max Teplitski. 2010. Salmonella SdiA recognizes N-acyl homoserine lactone signals from Pectobacterium carotovorum in vitro but not in a bacterial soft rot. Molecular Plant-Microbe Interactions 23(3): 273-282.

Jenee N. Smith, Jessica L. Dyszel, Jitesh A. Soares, Craig D. Ellermeier, Craig Altier, Sara D. Lawhon, L. Garry Adams, Vjollca Konjufca, Roy Curtiss III, James M. Slauch, and Brian M. M. Ahmer. 2008. SdiA, an N-Acylhomoserine Lactone Receptor, Becomes Active during the Transit of Salmonella enterica through the Gastrointestinal Tract of Turtles. PLoS ONE 3(7): e2826.

Brian M. M. Ahmer, Jenee N. Smith, Jessica L. Dyszel, and Amber Lindsay. 2007. Methods in cell-to-cell signaling in Salmonella. In Schatten, Heide; Eisenstark, Abe (Eds.), Salmonella: Methods and Protocols (pp. 307-322). Humana Press, New Jersey.

Amber Lindsay and Brian M. M. Ahmer.  2005.  The effect of sdiA on biosensors of N-acylhomoserine lactones.  Journal of Bacteriology 187: 5054-5058.

Brian M. M. Ahmer. 2004. Cell to cell signaling in Escherichia coli and Salmonella enterica. Molecular Microbiology, 52: 933-945.

Jenee N. Smith and Brian M. M. Ahmer. 2003. Detection of other microbial species by Salmonella:  Expression of the SdiA regulon. Journal of Bacteriology 185: 1357-1366.

Bindhu Michael, Jenee N. Smith, Simon Swift, Fred Heffron, and Brian M. M. Ahmer. 2001. SdiA of Salmonella enterica is a LuxR homolog that detects mixed microbial communities.  Journal of Bacteriology 183: 5733-5742.

Brian M. M. Ahmer, Jeroen van Reeuwijk, Cynthia D. Timmers, Peter J. Valentine, and Fred Heffron. 1998. Salmonella typhimurium encodes an SdiA homolog, a putative quorum sensor of the LuxR family, that regulates genes on the virulence plasmid.  Journal of Bacteriology 180: 1185-1193.

SirA. The gene downstream of sdiA is present throughout the gamma-proteobacteria and regulates virulence genes in all of these organisms. However, unlike SdiA, we do not know the signal for SirA. The signal that is activating virulence gene expression in so many pathogens is a very significant topic. So far we know that SirA is phosphorylated by a sensor kinase named BarA and we know many of the genes that are activated by SirA. Interestingly, two of the genes activated by SirA are regulatory RNAs named csrB and csrC. They in turn inhibit the activity of an RNA binding protein named CsrA. When CsrA is not inhibited, it regulates carbon storage genes and many of the same virulence genes that SirA regulates. The hilA and hilC genes in the diagram below are regulators that control one of the Type III secretion systems that is a primary virulence factor of Salmonella. Thus, SirA is activating transcription of virulence genes and controlling the translation of those same genes via CsrA. This regulatory network is an active area of investigation in our lab.

Papers on SirA:

Yakhya Dieye, Jessica L. Dyszel, Rebin Kader, and Brian M. M. Ahmer.  2007.  Systematic analysis of the regulation of type three secreted effectors in Salmonella enterica serovar Typhimurium.  BMC Microbiology, 7: 3.

Max Teplitski, Ali Al-Agely, and Brian M. M. Ahmer.  2006.  Contribution of the SirA regulon to biofilm formation in Salmonella enterica serovar Typhimurium.  Microbiology, 152: 3411-3423.

Max Teplitski, Robert I. Goodier, and Brian M. M. Ahmer.  2006.  Catabolite repression of the SirA regulatory cascade in Salmonella.  International Journal of Medical Microbiology, 296: 449-466.

Max Teplitski and Brian M. M. Ahmer.  2005.  The control of secondary metabolism, motility, and virulence by the two-component regulatory system BarA/SirA of Salmonella and other g-proteobacteria, pp 107-132, In B. M. Pruess (ed.), “Complex regulatory networks in enteric bacteria,” Research Signpost, Trivandrum, India.

Max Teplitski, Robert I. Goodier, and Brian M. M. Ahmer.  2003.  Pathways leading from BarA/SirA to motility and virulence gene expression in SalmonellaJournal of Bacteriology, 185: 7257-7265.

Robert I. Goodier and Brian M. M. Ahmer. 2001. SirA orthologs affect both motility and virulence.  Journal of Bacteriology 183: 2249-2258.

Brian M. M. Ahmer, Mimi Tran, and Fred Heffron. 1999. The virulence plasmid of Salmonella typhimurium is self-transmissable. Journal of Bacteriology 181: 1364-1368.

Brian M. M. Ahmer, Jeroen van Reeuwijk, Patricia R. Watson, Tim S. Wallis, and Fred Heffron. 1999. Salmonella SirA is a global regulator of genes mediating enteropathogenesis.  Molecular Microbiology 31: 971-982.

Current Lab Members (Winter 2010):

Jay Soares, Ph.D., Fabien Habyarimana, Ph.D., Darren Lucas, Matt Swearingen, and Mohamed Ali.

Ahmer Lab alumni:

Rob Goodier (postdoc 1999-2001), currently at Q-One Biotech, Scotland
Yakhya Dieye (postdoc 2002-2005), currently at Ondek, Perth Australia
Max Teplitski (postdoc 2002-2005), currently an Assistant Professor at University of Florida
Amber Lindsay (M.S. 2006), currently a Senior Microbiology Technician at Battelle, Columbus OH
Jenee Smith (Ph.D. 2007), currently a researcher at Battelle, Columbus OH
Jessica Dyszel (Ph.D. 2009), currently Director of Research at Richter International, Columbus OH

Undergraduate Research Opportunities:

Dr. Ahmer’s lab has opportunities for undergraduate research. If you have received an A in Micro 520 or 581 and have a cumulative GPA of at least 3.0, send an email.

Faculty Bios

Immune mechanisms that determine outcome of “New world” cutaneous and visceral leishmaniasis.

Abhay Satoskar

satoskar.2@osu.edu

Lab page

Associate Professor
M.B.B.S., University of Bombay (India), 1989
M.D., University of Bombay (India), 1992
PhD., University of Strathclyde, Glasgow (U.K.), 1996

Immune mechanisms that determine outcome of “New world” cutaneous and visceral leishmaniasis.

The leishmaniases comprise several diseases caused by intracellular protozoan parasites belonging to Leishmaniaspecies leading to a wide spectrum of clinical manifestations and a global health problem. Among the parasitic infections, this disease is responsible for the highest number of DALYs (Disability adjusted life years; a measure of health burden) after malaria. “Old world” cutaneous leishmaniasis usually manifests as a localized self-healing skin lesion with long-term protective immunity in humans. In contrast, some forms of “New world” cutaneous leishmaniasis manifests as a chronic infection that is associated with mutilation of ear and disfiguring scars or as a severe mucocutaneous disease involving nasal and oropharyngeal mucosa with extensive tissue destruction. Visceral leishmaniasis is the most severe clinical form, characterized by hepatosplenomegaly, fever, abdominal pain and weight loss.

Our laboratory is interested in understanding the immune mechanisms that determine outcome of “New world” cutaneous and visceral leishmaniasis caused by L. mexicana and L. donovani respectively. We are particularly interested in studying the role of cytokines in regulation of immune responses during these two species of Leishmaniaand the use of cytokine and cytokine receptor gene deficient mice has been a very powerful tool in these studies. As cytokines can modulate functions of several cells of the immune system in vivo, we are now using cell-specific gene deficient mice lacking specific cytokine receptors on specific immune cells such as macrophages and T cells. These mice are generated using cre/lox technology that enables us to delete a gene in cell-specific manner. We believe that these studies will enable us to determine how cytokines regulate immune responses in vivo during leishmaniasis. With regards to L. donovani, our studies have focused on understanding the regulation of effector cell responses in murine visceral leishmaniasis caused by L. donovani. Of particular interest to our group is the determining the immune mechanisms that mediate protection and/or induce immunopathology during VL. More recently, in collaboration with the McGill University, we have initiated studies that focus on the development of amastigote-specific single candidate vaccine against visceral and “New world” cutaneous leishmaniasis that cause considerable morbidity and mortality in humans. Another area of research in our laboratory is understanding the immunological basis of gender-related differences in susceptibility to Leishmania. In these studies, we are interested in determining the roles of sex-hormones in modulation of immune response and determining the outcome of Leishmania infection. Our long-term goal is to identify the basic mechanisms by which cytokines regulate T cell responses and host immunity to cutaneous leishmaniasis caused by L. mexicana and visceral leishmaniasis caused by L. donovani and utilize this knowledge to develop a vaccine against these diseases.

Recent Publications

Xu D, McSorley SJ, Tetley L, Chatfield S, Dougan G, Chan WL, Satoskar, AR, David JR, Liew FY. Protective effect on Leishmania major infection of MIF, TNF-alpha and IFN-gamma administered orally via attenuated Salmonella typhimurium. J. Immunol. 1998; 160: 1285-1289.

Satoskar AR, Khamis Al-Q, Alexander J. Sex-determined resistance against Leishmania mexicana is associated with the preferential induction of a Th1-like response and IFN-gamma production by female but not male DBA/2 mice. Immun Cell Biol. 1998; 76: 159-166.

Satoskar, AR, Okano M, Connaughton S, David JR, Labow M. Enhanced Th2-like responses in IL-1 type 1 receptor-deficient mice. Europ J Immunol.1998; 28: 2066-2074.

Stamm L, Raisanen-Solokowski A, Okano M, Russell M, David JR, Satoskar AR. Mice with STAT6-targeted disruption develop a Th1 response and control cutaneous leishmaniasis. J. Immunol. 1998; 161: 6180-6188.

Bozza M, Soares M, Bozza P, Satoskar AR, Brombacher F, Titus R, Shoemaker C, David JR. The selective PACAP-type I receptor agonist Maxadilan from Sand fly saliva protects mice against lethal endotoxemia. Europ J Immunol. 1998; 28: 3120-3127.

Bozza M, Satoskar AR, Lin G, Lu B, Humbles AA, Gerard C, David JR. Targeted disruption of migration inhibitory factor gene reveals its critical role in sepsis. J. Exp. Med. 1999; 189: 341-346.

Satoskar AR, Stamm LM, Zhang, XM, Satoskar, AA, Okano, M, David JR, Terhorst C, Wang B. Mice lacking natural killer (NK) cells develop an efficient Th1 response and control cutaneous L. major infection. J. Immunol. 1999; 162:6747-6754.

Satoskar, AR, Stamm, LM, Zhang, X., Okano, M, David, JR, Terhorst, C., and Wang, B. NK cell deficient mice develop Th1-like response but fail to mount an efficient antigen-specific IgG2a antibody response. J. Immunol. 1999; 163: 5298-5302.

Stamm, LM, Satoskar, AA, Ghosh, S, David, JR and Satoskar, AR. STAT4-mediated IL-12 signaling pathway is critical for the development of protective immunity in cutaneous leishmaniasis. Europ. J. Immunol. 1999; 29:2524-2529.

Alexander, J, Satoskar AR and Russell, DG. Leishmania species: models of intracellular parasitism. J. Cell Sci.1999; 112:2993-3002. (Review).

Satoskar, AR, Rodig, S, Telford, SR, Satoskar, AA, Ghosh, S., von Lichtenberg, F., and David, JR. Interleukin 12 gene deficient mice are susceptible to L. donovani infection but have diminished hepatic immunopathology. Europ. J. Immunol. 2000; 30:834-839..

Monteforte, G., Takeda, K., Akira, S, David, JR and Satoskar, AR. Interleukin-18 is not critical for the development of Th1 response and control of cutaneous L. major infection. J. Immunol. 2000; 164: 5890-5893.

Alexander, J., Carter, KC., Al-Fasi, N., Satoskar, AR, and Brombacher, F. Effective drug therapy against visceral leishmaniasis is dependent on endogenous IL-4. Europ. J. Immunol. 2000; 30:2935-2943.

Hattori, H, Okano, M, Yoshino, T, Akagi, T, Nakayama, E, Saito, C, Satoskar, AR, Ogawa, T, Azuma, M and Nishizaki, K. Expression of co-stimulatory CD80/CD86-CD28/CD152 molecules in the nasal mucosa of patients with perennial allergic rhinitis. Clin. Exp. Allergy. 2001; 31:1242.

Okano, M, Azuma, M, Yoshino, T, Hattori, H, Nakada, M, Satoskar, AR, Harn Jr, DA, Nakayama, E, Akagi, T, and Nishizaki, K. Differential role of CD80 and CD86 molecules in the induction and the effector phases of allergic rhinitis in mice. Am. J. Res. Crtic. Care Med. 2001; 164: 1501.

Szabo, SJ, Sullivan, BM, Stemmann, C., Satoskar, AR, Sleckman, BP, and Glimcher, LH. Distinct effects of T-bet in Th1 lineage commitment and IFN-γ production in CD4 and CD8 T cells. Science. 2002; 295:338.

Greenwald, RJ, McAdam, AJ, Van der Woude, D, Satoskar, AR, and Sharpe, AH. Inducible co-stimulator protein regulates both Th1 and Th2 responses to cutaneous leishmaniasis. J. Immunol. 2002; 168: 991.

Rodriguez-Sosa, M, David, JR, Bojalil, R., Satoskar, AR, and Terrazas, LI. Susceptibility to the larval stage of the helminth parasite Taenia crassiceps is mediated by the Th2 response induced via STAT6 signaling. J. Immunol. (Cutting Edge) 2002; 168:3135.

Brown, JA, Greenwald, RJ, Scott, S, Schweitzer, A.N, Satoskar, AR, Chung, C, Schopf, LR, van der Woude, D, Sypek, JP, and Sharpe, AH. T helper differentiation in resistant and susceptible B7-deficient mice infected with Leishmania major. Eur. J. Immunol. 2002; 32:1764.

Hattori H, Okano M, Yamamoto T, Yoshino T, Yamashita Y, Watanabe T, Satoskar, AR, Harn DA, and Nishizaki K. Intranasal application of purified protein derivative suppresses the initiation but not the exacerbation of allergic rhinitis in mice. Clin. Exp. Allergy. 2002; 32:951.

Costa, CH, Stewart, JM, Gomes, RBB, Garcez, LM, Ramos, PK, Bozza, M, Satoskar, AR, Dissennayake, S, Santos, RS, Silva, MRB, Shaw, JJ, David, JR, and Maguire, JH. Asymptomatic human carriers of Leishmania chagasi. Am. J. Trop. Med. Hyg. 2002; 66:334-337.

Rodriguez-Sosa, M, Satoskar, AR, Calderon, R., Gomez-Garcia, L, Saaverda, R., Bojalil, R., and Terrazas, LI. Chronic helminth infection induces alternatively activated macrophages expressing high levels of CCR5 with low interleukin-12 production and Th2-biasing ability. Infect. Immun. 2002; 70:3656.

Parish, CL, Finkelstein, DI, Tripanichkul, W, Satoskar, AR, Drago, J, and Horn, MK. The role of Interleukin-1, Interleukin-6 and glia in inducing growth of neuronal terminal arbors in mice. J. Neurosci. 2002; 22:8034.

Wurster, AL, Rodgers, VL, Satoskar, AR., Whitters, MJ, Young, DA, Collins, M, and Grusby, MJ. Interleukin-21 is a T helper (Th) cell 2 cytokine that specifically inhibits the differentiation of naïve Th cells into interferon gamma-producing Th1 cells. J. Exp. Med. 2002; 196: 969.

Pien, GC, Nguyen, KB, Malmgaard, L, Satoskar, AR, and Biron, CA. A unique mechanism for innate cytokine promotion of T cell responses to viral infections. J. Immunol. 2002; 169:5827.

Rodriguez-Sosa, M, Rosas, LE, David, JR, Bojalil, R, Satoskar, AR*, and Terrazas, LI*. Macrophage migration inhibitory factor plays a critical role in mediating protection against the helminth parasite Taenia crassiceps. (*Joint senior co-authors) Infect. Immun. 2003; 71:1247.

Rodriguez-Sosa, M, Rosas, LE, Terrazas, LI, Lu, B, Gerard, C, and Satoskar, AR*. CC chemokine receptor 1 enhances susceptibility to Leishmania major during early phase of infection. Immunol. Cell Biol. 2003; 80:114.

Rodriguez-Sosa, M, Satoskar, AR, David, JR, and Terrazas, LI. Altered T helper responses in CD40 and interleukin-12 deficient mice reveal a critical role for Th1 responses in eliminating the helminth parasite Taenia crassiceps. Int. J. Parasitol. 2003; 33: 701.

Rosas, L, Keiser, T, Pyles, R, Durbin, J, and Satoskar, AR*. Development of protective immunity against cutaneous leishmaniasis is dependent on STAT1-mediated IFN signaling pathway. Eur. J. Immunol. 2003; 33: 1799.

Pan, J.H., Sukhova, G.K., Satoskar, A.R., David, J.R., Yang, J.T., Fu, H., Metz, C., Baugh, J.A., Bucala, R., Fang, K., Libby, P. and Shi, G.P. Regulation of cysteine protease expression by macrophage migration inhibitory factor. Circulation. 2004; 109:3149-3153.

Rodriguez-Sosa, M., Rosas, L.E., Saavedra, R., Satoskar, A.R. and Terrazas, L.I. STAT4-dependent IL-12 signaling pathway is required for resistance to the helminth parasite Taenia crassiceps. Infect. Immun. 2004; 71:1247-1254.

Wang, N., Satoskar, A.R., Faubion, W., Howie, D., Okamoto. S., Feske, S., Gullo, C., Clarke, K., Rodriguez Sosa, M., Sharpe, A.H. and Terhorst, C. SLAM controls T cell and macrophage functions. J. Exp. Med. 2004; 199:1255-1264.

Bhardwaj, N., Rosas, L.E., Lafuse, W.P., and Satoskar, A.R. Leishmania inhibits STAT1-mediated IFN-γ signaling in macrophages: Increased tyrosine phosphorylation of dominant negative STAT1β by Leishmania mexicana. Int. J. Parasitol. 2005; 35:7582.

Howie, D., Laroux, F.S., Morra, M., Satoskar, A.R., Rosas, L.E., Faubion, W.A., Julien, A., Rietdijk, S., Coyle, A.J., Fraser, C., and Terhorst, C. The SLAM family receptor Ly108 controls T cell and neutrophil functions. J. Immunol. 2005; 174:5931-5935.

Morra, M., Barrington, R.A., Abadia-Molina, A., Okamoto, S., Julien, A., Gullo, C., Kalsy, A., Edwards, M.J., Chen, G., Spolski, R., Leonard, W.J., Huber, B.T., Borrow, P., Biron, C.A., Satoskar, A.R., Carroll, M.C., and Terhorst, C. Defective B cell responses in the absence of SH2D1A. Proc. Natl Acad. Sci. USA. 2005; 102:4819-4823.

Okano, M., Hattori, H., Yoshino, T., Sugata, Y., Yamamoto, M., Fujiwara, T., Satoskar, A.A., Satoskar, A.R., and Nishizaki, K.Nasal exposure to Staphylococcal enterotoxin enhances the development of allergic rhinitis in mice. Clin. Exp. Allergy. 2005; 35:506-514.

Powell, N.D., Papenfuss, T.L., McClain, M.A., Gienapp, I.E., Shawler, T.M., Satoskar, A.R., and Whitacre, C.C. Macrophage migration inhibitory factor is necessary for progression of experimental autoimmune encephalomyelitis. J. Immunol. 2005; 175:5611-5614.

Rosas, L.E., Barbi, J., Lu, B., Fujiwara, N., Gerard, C., Sanders, V.M., and Satoskar A.R. CXCR3-/- mice mount an efficient Th1 response but fail to control L. major infection. Eur. J. Immunol. 2005; 35:515-523.

Rosas, L.E., Keiser, T., Barbi, J., Satoskar, A.A., Septer, A., Kcazmarek, J., Lezama-Davila, C.M., and Satoskar, A.R. Genetic background influences immune responses and disease outcome of cutaneous L. mexicana infection in mice. Int. Immunol. 2005; 17:1347-1357.

Hattori, H., Okano, M., Kariya, S., Nishizaki, K. and Satoskar, A. R. CD40-CD40L interaction is involved in pathogenesis of SEA induced allergic rhinitis. Amer. J. Rhinol. 2006; 20:165-169.

Liang, S.C., Greenwald, R.J., Latchman, Y.E., Rosas, L., Satoskar, A. R, Freeman, G.J. and Sharpe, A.H. PD-L1 and PD-L2 have distinct roles in regulating host immunity to cutaneous leishmaniasis. Eur. J. Immunol. 2006; 36:58-64.

Reyes, J., Terrazas, L.I., Espinoza, B., Gomez-Garcia, L., Cruz-Robles, D., Rivera-Montoya, I., Snider, H., Satoskar, A. R. and Rodriguez-Sosa, M. Macrophage migration inhibitory factor (MIF) plays a critical role in host defense against acute Trypanosoma cruzi infection. Infect. Immun. 2006; 74:3170-3179.

Rosas, L.E., Barbi, J., Snider, H., Satoskar, A.A., Lugo-Villarino, G., Keiser,T., Papenfuss, T, Durbin, J, Radzioch, D, Glimcher, LH and Satoskar, A. R. Cutting edge: STAT1 and T-bet play distinct roles in determining outcome of visceral leishmaniasis caused by Leishmania donovani J. Immunol. 2006; 177:22-25.

Rosas, L.E., Satoskar, A.A., Roth, K., Keiser, T., Barbi, J., Hunter, C.A, de Sauvage, F. and Satoskar, A. R. IL-27R (WSX-1/TCCR) gene deficient mice display enhanced resistance to Leishmania donovani infection but develop severe liver immunopathology. Am. J. Pathol. 2006; 168:158-169.