Transcriptional Regulation of Capsule Synthesis in Systemic Streptococcal Pathogens

Brett R. Hanson and Melody N. Neely, Wayne State University School of Medicine, Scott Hall 540 E. Canfield, Detroit, MI 48201

Systemic streptococcal pathogens, such as S. agalactiae and S. pneumoniae are a significant cause of morbidity and mortality worldwide and present a major challenge to the health care system.  Production of a polysaccharide capsule enables streptococci to initiate systemic disease and is therefore an important target of study.  The highly conserved and uncharacterized putative transcriptional activator CpsA is thought to be responsible for transcriptional regulation of the capsule operon in streptococci.  Part of the LytR_CpsA_psR protein family, the 450 amino acid triple-pass integral membrane protein CpsA has a distinctive predicted membrane topology with less than 30 amino acids present in the cytoplasm.  Unique to CpsA, this topology indicates a novel mechanism of transcriptional regulation.  To confirm the predicted membrane topology of CpsA, various regions of the protein were fused to either beta-galactosidase or alkaline phosphatase.  Using colorimetric assays we confirmed that the predicted membrane topology is correct.  To characterize the ability of CpsA to bind to the capsule operon promoter, full length and various truncated forms of CpsA were purified and used in Electrophoretic Mobility Shift Assays (EMSA).  Using EMSA, we showed that the region of CpsA predicted to bind DNA was necessary and sufficient for specific binding to the capsule promoter. Modulation of transcription due to differential environmental conditions was determined by fusing the capsule operon promoter to the reporter gene alkaline phosphatase.  Changes in capsule level were correlated to promoter expression using buoyant density centrifugation.  Using these tools we demonstrated that transcription of the capsule operon, and subsequently capsule level, is altered during exposure to differential pH, temperature, and as a function of growth phase.    Characterization of this enigmatic protein will provide insight into the role of capsule synthesis and variability in virulence and may provide a new therapeutic target for the control of systemic streptococcal infection.

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ZWF1 and YDR049W Reduce the Oxidative Damage Caused by Furfural

Sandra A Allen and Steven Gorsich. Central Michigan University, Mount Pleasant, MI 48858

Currently our nation is facing many environmental and economical concerns, raising the need for an alternative and renewable fuel source. One such fuel source that has been proposed and tested is bio-ethanol. Unfortunately before an ethanol-based fuel industry can be achieved, several complications must be addressed. While the current process of producing bio-ethanol using Saccharomyces cerevisiae (baker’s yeast) grown with a corn starch substrate fermentation is quite efficient, our nation lacks the amount of corn needed to produce the desired amount of bio-ethanol for fuel. In order to overcome this problem, researchers have made developing new ways of fermenting ethanol from yeast their aim. However, the alternative substrates that would replace cornstarch substrates, such as biomass waste, produce multiple growth inhibitors during the fermentation process that prevents yeast from efficiently producing ethanol. The object of this research is to genetically engineer a more robust yeast strain by over-expressing genes found in screens for tolerance to growth inhibitors. When over-expressed ZWF1, a pentose phosphate pathway gene, was found to grow with a shorter lag time and have less cellular morphology damage than wild-type S. cerevisiae when grown in the presence of the growth inhibitor, furfural. Similarly to the finding of ZWF1, another gene of interest, YDR049W, an uncharacterized gene, was found in various screens for growth inhibitor tolerance. The over-expression of YDR049W has yielded a shorter lag period as compared with wild-type S. cerevisiae. Because YDR049W is a proposed transcription factor with an unknown target, it was hypothesized that the over-expression of both ZWF1 and YDR049W would have an additive affect. It was found that the lag time in yeast with both genes over-expressed was shortened as compared to strains of yeast with the genes over-expressed individually. Currently we are examining this strain of yeast over-expressed with both genes to determine if it too can recover from some of the cellular damages caused by the inhibitor, furfural.

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The Synergistic Effects of Probiotic Microorganisms on the Microbial Production of Butyrate In Vitro

Khadija A. Abbas, Elizabeth Dorman and Dan Clemens, Eastern Michigan University, Ypsilanti, MI  48197

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Formation of Clone Libraries as a High Resolution Finger Printing Technique to Determine Microbial Diversity

Alicia Ketcham and Dan Clemens, Eastern Michigan University, Ypsilanti, MI 48197

Microbial communities cultivated on unglazed ceramic tiles in artificial streams were analyzed to examine microbial diversity and dynamics in response to different levels of light and phosphorous. Results from culture independent molecular techniques indicated that the microbial communities consisted largely of bacteria from the Proteobacteria phylum. Another interesting but unexplainable result was the presence of chloroplast hits, and their higher prevalence in streams containing low light and phosphorous conditions. Further molecular analysis will help identify the specific organisms and how they respond to these nutrient and light conditions.

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Identification of HopM1 Targets using a Yeast Model System

Vanessa Revindran and John R. Geiser, Western Michigan University, Kalamazoo, MI

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Identifying Escherichia coli Factors Involved in Curli Biogenesis

Michael J O’Connor, Matthew P. Badtke and Matthew R Chapman, University of Michigan, Ann Arbor, MI 48109

Escherichia coli are capable of producing curli, long fibers of amyloid protein which bind the dye Congo red.  These fibers are essential for biofilm formation and assist in host pathogenesis.  Amyloid formation is traditionally associated with several neurodegenerative diseases including Alzheimer’s, Parkinson’s and Huntington’s.  However, it has now been shown that several organisms, including E. coli, use amyloids like curli to their benefit.  Researchers have identified six major genes that influence curli production.  The function of several of these genes has been examined, however the role of “curli specific gene” E (csgE) is not known.  The purpose of this project is to investigate the function of csgE in curli biogenesis. It was hypothesized that CsgE transports curli subunits to the outside of the cell, protecting them from degradation by a protease. Wild-type E. coli produce curli and exhibit a red phenotype when grown on Congo red plates, whereas E. coli without csgE cannot produce curli and are white.  A transposon was used to create random genomic mutations in E. coli cells without csgE.  Cells were screened for colonies that were restored in their Congo red phenotype and turned from white to red in color. This color change indicated colonies which had restored curli production.  The DNA of these strains was sequenced to identify the mutated genes.  This process has identified 17 unique insertions that appear to restore curli production.  The interactions of these proteins with CsgE are currently being explored.

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The Effects of Light and Seasonality on Microbe Populations in Three Ecological Areas Along the Huron River

Rachel E Hutchins, Alicia M Ketcham, Shirley M Demko, Stephanie Cholyk and Daniel Clemens, Eastern Michigan University, Ypsilanti, MI 48197

The Huron River watershed is comprised of three main land usages: agricultural, natural, and urban.  Theoretical speculation deduces that land usage effects the biochemical conditions within the watershed, therefore effecting water quality. Recent studies support that water quality can be assessed by examining the composition of the microbial communities found in biofilms.  We analyzed and compared samples collected from the three site locations over the course of three seasons; Fall (August), Winter (December), and Spring (April).  Microbial diversity was determined by isolating, amplifying and genotyping the 16S rRNA gene.  The results show that microbial community structure varies as seasons, material compositions, and site locations vary.

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Microbial and Molecular survey of Grand Traverse Bay Watershed

Marc P Verhougstraete and Joan B Rose, Michigan State University, East Lansing, MI 48824

The Grand Traverse Bay drains 2520 km2 of mixed use landmass and has recently experienced lower lake levels, non-desirable invasive species infestation, and increased pollution loading from stormwater runoff resulting in elevated bacterial levels at area beaches. Monitoring efforts have identified hotspots of water quality impairments throughout the watershed. Based on these findings, I collected samples from eight Mitchell Creek sites (n=111) and the beaches at Bryant Park (n=6), Traverse City State Park (n=16), and Milliken Park (n=16) during the summer of 2009. Each sample was assayed for Escherichia coli (E. coli), enterococci, Clostridium perfringens (C. perfringens), somatic coliphage, and the Enterococcus surface protein (esp). In the Mitchell Creek, E. coli, enterococci, C. perfringens, and coliphage geometric mean concentrations were 385.7 (±627.4), 873.9 (±3109.2), 24.9 (±59.8), and 69.5 (±499.6) organisms/100 ml, respectively. E. coli, enterococci, C. perfringens, and coliphage geometric mean concentrations at Bryant Park beach were 13.9 (±13.3), 15.5 (±25.1), 1.02 (±1.38), 11.1 (±2.98) organisms/100 ml, respectively. E. coli, enterococci, C. perfringens, and coliphage geometric mean concentrations at Traverse City State Park beach were 10.0 (±20.2), 17.3 (±122.2), 0.60 (±0.95), 10.7 (±2.80) organisms/100 ml, respectively. E. coli, enterococci, C. perfringens, and coliphage geometric mean concentrations at Milliken Park beach were 9.68 (±434.9), 21.6 (±316.3), 1.13 (±23.6), 20.1 (±1321.2) organisms/100 ml, respectively. Surface water samples (n=112) were assayed for the presence of the esp gene. PCR and gel electrophoresis identified 11 samples (10%) positive for the gene (7 Mitchell Creek samples, 1 Milliken Beach sample, 2 Bryant Park samples, and 1 sample from a storm drain discharging at Bryant Park). Water quality trends indicate that fecal inputs are entering Mitchell Creek near sampling site MC6. Bacterial levels recorded at MC6 are elevated in comparison to up stream samples and remain elevated through the mouth of the creek. Furthermore, esp was detected downstream of MC6 but not detected upstream.

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Organization and Regulation of the Inositol Catabolism Genes in Sinorhizobium meliloti

Petra R. A. Kohler and Silvia Rossbach, Western Michigan University, Kalamazoo, MI 49008

Nutritional mediation is part of the complex signaling processes required to establish and maintain the nitrogen fixing symbiosis Rhizobiaceae and legume plants. Known symbiosis specific nutritional mediators, the rhizopines, are derivatives of the sugar alcohol inositol. Inositol has nine possible stereoisomeres, of which the myo-, scyllo-, D- and L-chiro isomeric forms are naturally occurring inositols. Legumes exude inositol into the rhizosphere, where it serves a carbon source for several soil bacteria. The rhizobial inositol catabolism has only been studied in parts but its requirement for rhizopines utilization has been demonstrated. Our research focuses on the elucidation of the rhizobial inositol catabolism and its potential role during plant-bacteria interactions using the Sinorhizobium meliloti – alfalfa model. The putative S. meliloti inositol catabolism genes (iol) genes are scattered over the genome. The idhA gene, encoding the myo-inositol dehydrogenase, is located on one of the symbiotic plasmids (pSymB), whereas the iolR, iolC, iolD, iolE and iolB genes are organized in one cluster on the chromosome, all oriented in the same direction. The iolA gene is located 400 kb apart on the chromosome. S. meliloti mutant analysis confirmed the requirement of the individual iol genes for the growth with different inositol sterioisomers as sole carbon sources. Complementation of the mutant phenotypes, reporter gene assays and enzymatic studies were carried out to investigate the regulation of the inositol pathway and confirmed the organization of the iolCDEB genes as one operon. An in vitro plant assay showed that the inositol catabolism genes iolABCDE as well as the regulatory gene iolR are essential for competition during nodule occupancy. Taken together our data are supporting the notion that inositol serves nutritional and signaling purposes.

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The Diversity and Dynamics of Wetland Microbial Biofilm Communities in Response to Nitrogen and Phosphorous

Greg Sheremeta and Daniel Clemans, Eastern Michigan University, Department of Biology, Ypsilanti, MI

Microbial identity plays a crucial role in the diverse biochemical cycles within our wetlands and ecosystems. In this research study we examined the microbial diversity of biofilm communities in response to different nitrogen and phosphorous levels at the man-made Paint Creek Wetland. Using culture-independent molecular techniques based on 16S rDNA, we will compare the microbial dynamics and diversity of the biofilms grown at the Paint Creek wetlands with those formed at the natural wetland within Loesell Field laboratory.

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Study of Antibacterial Efficacy of AOT Against A Wide Range Of Micro-organisms

Keshav P Sah, John Reid and Dan Clemens, Eastern Michigan University, Ypsilanti, MI 48197, USA

AOT is an anionic surfactant and a common ingredient in consumer products, including stool softeners. This study was aimed at finding the role of surfactant AOT as an antibacterial against a wide range of micro-organisms from pathogenic, probiotics to yeast. It involved culture and identification of organisms, MIC (Minimum Inhibitory Concentration), followed by determination of MBC (Minimum Bactericidal Concentration) of AOT. In this study, AOT was found to be an effective antibacterial substance against a wide range of micro-organisms including pathogenic organisms. Its antibacterial effect is limited to the Gram-positive organisms tested, including several Staphylococci. It is interesting to note that one strain of Lactobacillus was found to be resistant to AOT.

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Identifying and Characterizing the ToxT and H-NS Binding Sites in the Cholera Toxin Promoter of Vibrio cholerae

Jennifer Stone and Jeffrey H. Withey, Department of Immunology and Microbiology, Wayne State University School of Medicine, Detroit, MI 48201

Vibrio cholerae is a gram-negative curved rod that causes the severe diarrheal disease cholera.  After ingestion by the host, the bacterium produces virulence factors including cholera toxin (CT), which is directly responsible for the large volumes of water loss in the intestine during acute cholera.  The regulatory network controlling virulence gene expression is complex and responds to various environmental signals and transcription factors.  Ultimately ToxT, a member of the AraC/XylS transcription regulator family, is responsible for activating transcription of the virulence genes.  V. cholerae virulence gene promoters all contain one or more copies of the toxbox, a 13 base pair DNA sequence that ToxT recognizes.  The 5' half of the toxbox sequence is well conserved and contains an invariant tract of four consecutive T nucleotides, whereas the 3' half of the toxbox sequence is not highly conserved other than being A/T rich.  The binding of ToxT to toxboxes is required to activate the transcription of virulence genes and these binding sites have been characterized in several virulence gene promoters.  However, the toxboxes required for activating transcription from the cholera toxin promoter have not been identified. The cholera toxin promoter contains a series of heptad repeats (GATTTTT) each of which matches the 5' half of the toxbox consensus sequence and is a potential binding site for ToxT.  Using site-directed mutagenesis and high resolution Copper-Phenanthroline footprinting, we have determined which of these heptad repeats are required for CT expression. The A/T rich regions of the cholera toxin promoter also provide binding sites for H-NS, a global transcriptional repressor in gram-negative bacteria.  The current model suggests that H-NS is de-repressed by ToxT to activate transcription of the CT genes, ctxAB.  Our goal is to understand the interplay between ToxT and H-NS and their interaction with the ctxAB promoter.

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The CpsY Regulon Protects Streptococcus iniae from PMN Phagocytic Killing

Jonathan P. Allen and Melody N. Neely, Wayne State University School of Medicine, Detroit, MI 48201

Systemic streptococcal pathogens are a major cause of severe invasive infections.  We have developed an infectious disease model for identification of streptococcal virulence genes using the systemic pathogen, Streptococcus iniae, and a natural host, the zebrafish (Danio rerio).  Using this model, we previously identified a transcriptional regulator, CpsY, that is required for systemic survival and dissemination of the pathogen to the brain.  The cpsY gene is located adjacent to the capsule biosynthesis operon, but has been shown to have little effect on capsule levels, suggesting an involvement in the regulation of other virulence genes.  An in-frame deletion mutant of cpsY (DcpsY) is highly attenuated in whole blood.  This attenuation is due to an increased susceptibility to phagocytic killing by neutrophils.  We have shown that both DcpsY and wild-type S. iniae are trafficked to the mature phagosome of neutrophils.  Quantitative real-time PCR data suggests that CpsY may be regulating genes whose repression results in a strengthening of the bacterial cell wall, which is important for bacterial survival in mature phagosomes.  Identification of the pathogenic mechanisms used by systemic streptococcal pathogens can the virulence factors involved is a key step in the development of new therapeutic strategies to combat invasive streptococcal infection.

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Chromosomally Encoded Serum-Resistance in Salmonella typhimurium

Kristie C. Mitchell and James L. VandenBosch, Eastern Michigan University, Ypsilanti, MI 48197

Salmonella typhimurium is a leading cause of infectious gastroenteritis and results in systemic disease when it escapes into the bloodstream.  One of the most important components of human serum in fighting bacterial infections is the complement system.  Outside of cells, S. typhimurium exhibits resistance to killing by complement (serum-resistance), a trait conferred by several known virulence genes primarily those encoding lipopolysaccharide length but also including ancillary contributions from rck, pagC, and traT. This study investigates a potential complement-resistance gene located on the chromosome of S. typhimurium.  S. typhimurium strain EM876 has a TnphoA insertion in the chromosome and also has reduced serum-resistance.  Inverse PCR and subsequent DNA sequence analysis reveal the insertion to be in the gene glpQ. glpQ codes for a periplasmic enzyme involved in the glycerol degradation pathway.  Complementation assays using a clone of glpQ on a multicopy plasmid are consistent with a role for glpQ in serum-resistance.  Significantly, glpQ expression is temperature-dependent, preferentially expressed at 37°C rather than 30°C.  This is similar to traT and suggests that multiple genes are regulated by temperature and, together, contribute to the overall serum-resistance of S. typhimurium.

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Mechanism of bile action on Vibrio cholerae virulence gene expression

Sarah Plecha and Jeffrey H. Withey, Department of Immunology and Microbiology, Wayne State University School of Medicine, Detroit, MI 48201

The severe diarrheal illness cholera is caused by the gram-negative curved bacillus Vibrio cholerae.  There are two biotypes of serogroup O1 V. cholerae that are capable of causing cholera: classical and El Tor.  Growing the bacteria under virulence-inducing conditions or during infection of a host initiates a complex regulatory cascade that results in the production of ToxT, a regulatory DNA binding protein that activates the transcription of genes encoding toxin-coregulated pilus (TCP), cholera toxin (CT) and other virulence genes.  Previous studies have shown that bile and the unsaturated fatty acid (UFA) components of bile reduce virulence gene expression in both classical and El Tor biotypes.  However, the mechanism for the bile-mediated reduction of TCP and CT expression has not been defined, and the specific effect of bile and its UFA components on ToxT has also not been determined.  Through the use of transposon mutagenesis and error-prone PCR, mutants will be made that either have knockouts of genes important in virulence regulation or have amino acid substitutions in ToxT both resulting in an insensitivity to bile.  Flow cytometry methods will be used to screen and sort these mutants using a virulence gene promoter fused to green fluorescent protein (GFP).  Given that bile and its UFA components are present in the upper small intestine where V. cholerae colonizes, bile is most likely an important chemical signal that V. cholerae recognizes in the host that causes a reduction in virulence gene expression in certain niches.  Determining the genes involved in the response to bile and the amino acid residues of ToxT affected by bile will give a clearer understanding of the regulatory networks controlling virulence gene expression.

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