Enzymatic and Molecular Analyses of Bacterioplankton from the Rouge River

Peter Arno, Lyndsey Hage, and Sonia Tiquia, Department of Natural Sciences, University of Michigan-Dearborn, Dearborn, MI 48128

Analysis of the enzymatic activity and bacterial populations within an ecosystem provides insight into the general health and condition of that ecosystem. In this experiment, surface and ground water samples from the Rouge River were collected and analyzed for enzymatic activity and cloning and sequencing of 16S rRNA genes. Concentrations of different enzymes were measured by using the API ZYM kit. In the ground water samples the following enzymes with their average concentrations were present: Alkaline phosphatase, acid phosphatase, phosphohydrolase, lipase, esterase-lipase, esterase, leucine amino-peptidase, valine amino-peptidase, chymotrypsin, trypsin, galactosidase, a-Glucosidase, b-Glucosidase and b-Glucuronidase. Phosphohydrolase was the enzyme present in the highest concentration. Enzymes that were tested for in the ground water samples, but were not present were: Cystine Amino-Peptidase, N-Acetyl-b-Glucosamminidase, a-Mannosidase, and a-Gucosidase. In the surface water samples, the following enzymes were present: Alkaline phosphatase, acid phosphatase, phosphohydrolase, lipase, esterase-lipase, esterase, chymotrypsin, a-Galactosidase, b-Glucosidase, a-Glucosidase, and b-Glucuronidase. Lipase was the enzyme present in the highest concentration. Enzymes that were tested in the surface water samples, but were not present were: Leucine Amino-Peptidase, Valine Amino-Peptidase, Cystine Amino-Peptidase, b-Glucosidase, N-Acetyl-b-Glucosamminidase, a-Glucosidase, b-Glucosidase, a-Mannosidase, and a-Gucosidase. A total of 15 enzymes were found in ground water samples and a total of 10 enzymes were found in surface water samples. MOBIO Powerwater kits were used to extract environmental DNA from the water samples, which underwent polymerase chain reactions to amplify the 16S rRNA genes, and the gene inserted into competent cells using TOPO cloning kits. Fifteen transformants were recovered, and their 16S rRNA genes sequenced for comparison with 16S genes in the GeneBank database. Of the 15 transformants, three generated no match within the database, 11 matched uncultured bacterial clones, and one matched a Xanthomonas subspecies.

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Microbial Assessment and Physico-Chemical Characteristics of Surface and Groudwater from the Rouge River

Gennifer Cooper, Alia Sabbagh and Sonia Tiquia, Department of Natural Sciences, University of Michigan-Dearborn, Dearborn, MI 48128

Bacteria, which contain an abundance of diverse microbes, are a necessary component in maintaining a healthy ecosystem. Five sampling sites around the Rouge River were chosen to collect both surface and ground water for the purpose of classifying and comparing a number of parameters between the two different water samples. These parameters included the microbial, chemical, and field data. Surface water had significantly different results from ground water. Nitrate, which is formed when nitrogen and oxygen combine, was higher in surface water with a value of 50 mg/L compared to the ground water with a value of 10 mg/L. This correlation also appears with the amounts of dissolved oxygen present in surface and ground water, with values of 6.88 mg/L and 4.18 mg/L respectively. The high nitrate level found in surface water decreases the amount of fungal growth compared to ground water. Most plants such as fungi are often not able to readily utilize nitrate, which corresponds to the fungal count of 24 CFU/mL for surface water compared to 700 CFU/mL obtained for ground water. Likewise, algae does not consume nitrate, which leads to higher algae counts found in ground water than in and surface water. Actinomycetes were found in higher concentrations in ground water, with a value of approximately 37,000 CFU/mL, which is due to their abundance in soil; whereas a value of approximately 700 CFU/mL was found in surface water. Bacteria was found in higher concentrations in ground water with a value of approximately 3,700 CFU/mL, compared to the value of 1,000 CFU/mL found in the surface water. Bacteria, as mentioned earlier, are necessary components that support the growth of algae, fungi, and actinomycetes, all of which are a part of sustaining a healthy environment in both surface and ground water.

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Community Structure of Ammonia Oxidizing Bacteria and Archaea in Sediments from the Gulf of Mexico Continental Margin

Dylan Frabutt, Matthew Flood, Dalton Floyd and Sonia M. Tiquia, Department of Natural Sciences, University of Michigan-Dearborn, Dearborn, MI 48128

DNA was extracted from sediment samples taken from three different sampling sites along the Mexican continental margin (100, 200 and 1300 m deep respectively) in order to test for the presence and diversity of the amoA genes among both bacterial and archeal populations within the first 0.5 cm section of the sediment cores. The amoA genes, being examined, encode for the active site of the ammonia monooxygenase; an enzyme that is used to produce hydroxylamine from ammonium during the first step of nitrification. Since the amoA gene is essential for nitrification, its diversity can reflect the relative diversity of all nitrifiers in the sediment and may help correlate their presence with the selection pressures applied by environmental parameters such as redox potential and substrate availability. The environmental DNA obtained from the sediment samples was then amplified using PCR and primers for the amoA gene, cloned into a vector, inserted into chemically competent cells. The transformed cells were then cultured and then amplified using PCR and primers for the amoA gene, purified and then sequenced. The sequences obtained were used to construct clone libraries, from which diversity matrices (Shannon-Weiner [H’] index, Simpson’s index [1/D], and Chao1 index) rarefaction curves, and sample coverage were generated for each using DOTUR (Distance-based Operational Taxonomic Unit and Richness Determination) program. Our data suggests that the depth of the sampling site correlated to diversity.

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Geochemistry and Diversity of Bacterial Populations in Uranium-Contaminated Groundwater

Matthew Flood, Dylan Frabutt, Dalton Floyd and Sonia M. Tiquia, Department of Natural Sciences, University of Michigan-Dearborn, Dearborn, MI 48128

To understand the link between bacterial diversity and geochemistry in uranium contaminated groundwater, microbial communities were assessed based on clone libraries of 16S rDNA genes from the USDOE Oak Ridge Field Research Center (FRC) site. Four groundwater wells(GW835, GW836, FW113-47, and FW215-49), with a wide range of pH (3 to 7), nitrate (44 to 23,400 mg l-1) uranium (0.73 to 60.36 mg l-1) and other metal contamination, were investigated. Results indicated that bacterial diversity correlated with the geochemistry of groundwater. Microbial diversity decreased in relation to the contamination levels of the wells. The highly contaminated well (FW113-47) had lower gene diversity than less contaminated wells (FW215-49, GW835 and GW836). The high concentrations of contaminants present in well FW113-47 stimulated the growth of uranium (Shewanella and Pseudomonas), nitrate (Pseudomonas, Rhodanobacter, and Xanthomonas) and iron (Stenotrophomonas) reducing organisms unique to this well FW113-47. The clone libraries consisted primarily of sequences closely related to the phylum Proteobacteria, with FW-113-47 almost exclusively containing this phylum. Metal reducing bacteria were present in all four wells, which may suggest that there is potential for successful bioremediation of the groundwater at the Oak Ridge FRC. The microbial community information gained from this study and previous studies at the site can be used to develop predictive multivariate and Geographical Information System (GIS) based models for microbial populations at the Oak Ridge FRC. This will allow for better understanding of what organisms are likely to occur where and when based on geochemistry, and how these relate to bioremediation processes at the site.

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Multi-Drug-Resistant Coagulase-negative Staphylococci (CoNS) from Food Animals

Kanika Bhargava and Yifan Zhang, Department of Nutrition and Food Science, Wayne State University, Detroit, MI 48202

Food animals are important reservoir of antimicrobial-resistant coagulase-negative staphylococci (CoNS) that can pose potential threat to humans;however, there are limited studies on the antimicrobial susceptibilities of CoNS of animal origin. In the present study, 87 coagulase-negative staphylococci (CoNS) recovered from food animals were characterized by antimicrobial susceptibility testing, resistance gene identification by PCR, and conjugation tests. High percentages of CoNS were resistant to tetracycline (67.8%), cefoxitin (54%) and erythromycin (36.7%). Importantly, 54% of CoNS demonstrated resistance to at least 3 antimicrobial classes, including 6 isolates resistant to 6 classes of antimicrobial. The common genes for the above mentioned resistance phenotypes were mec(A), tet(M), and erm(A), which were identified from 68.9%,  62.7%, and 56.25% of the isolates, respectively.  tet(M) was conjugatively transferable from 11 tetracycline-resistant CoNS to a Enterococcus strain, underlining the potential of antimicrobial resistance transfer from Staphylococcus to the commensal bacteria in human. This study suggests that multi-drug-resistance are common in animals and adds to our knowledge of the antimicrobial resistance patterns in CoNS in the food production environment.

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Evaluation of a Household Drinking Water Disinfection Device with MS2 and Vibrio cholerae

Emaly S. Leak, Tiong G. Aw  and  Joan B. Rose, Department of Fisheries & Wildlife, Michigan State University, East Lansing, MI 44824

Obtaining safe drinking water is problematic in many developing nations.  Point of use devices (POU) are becoming increasingly popular for providing clean drinking water to people around the world.  The HaloPure disinfection canister designed by HaloSource was created to provide household water treatment for middle-class families in India.  The device disinfects using N-halamine bromine media.  Previous studies with an older model of the device (AquaSure) have found that it can provide approximately 5 log10 reduction in MS2 bacteriophage and 88.5% reduction in microcystin toxin.  The older model with N-halamine chlorine media achieved approximately 4 log10 reduction in S. typhiumurium and 6 log10 reduction in V. cholerae.  The new model device was tested by adding MS2 and V. cholerae stock cultures to 10L of well water.  MS2 is commonly used as a model for human enteric viruses.  V. cholerae is responsible for hundreds of thousands of deaths each year in developing countries, so it is an important disinfection target of POU devices.  Ten percent raw sewage was added as another treatment, to simulate organic compounds that are commonly found in untreated water.  The water mixture is allowed to flow through the device, and samples are collected before (influent) and after treatment (at 0, 15, and 45 minutes).  Samples are analyzed with the agar overlay method for quantification of MS2, and with membrane filtration for V. cholerae.  Initial results show that the new device, XTP 21, can provide approximately 3.75 log10 reduction of MS2, and 6.69 log10 reduction of V. cholerae (well water matrix, no sewage added).  Future plans include challenging the device with Salmonella typhiumurium, human adenovirus type-2, Hepatitis A virus, and murine norovirus.

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Evaluation of bacteria, virus, and parasite removal in biofilter system and comparison to biosand filter

Christopher D Wendt, Rebecca L. Ives, Stephanie E. Longstaff and Joan Rose, Michigan State University, East Lansing, MI, 48824

Background
The original biosand filter reduced microbial contamination in water using gravel and sand layers that could be hard to use and difficult to clean.  In the new biofilter system the sand stage is replaced by a carbon filter and foam sleeve/disk (depending on the configuration).  The process uses flocculation, foam disk or sleeve with biofilm, passage through a carbon filter, treatment with chlorine and into a holding reservoir for consumption.  Configurations were challenged to assess overall removal and comparison to the conventional biosand filter.

Methods 
Objective 1 compared removal difference of unit 1 (foam sleeve fitted around the carbon filter) and unit 2 (foam disk sitting above filter cartridge).  Water was passed through the systems until a biofilm developed.  Systems were then challenged with C. parvum , R. terrigena , P22 virus, and MS2 virus to determine average log reduction for each organism.  Objective 2 compared Unit 3 (biosand filter) to Units 1 & 2 without the development of a biofilm.

Results 
Objective 1 (O1):  Units 1 & 2 removed R. terrigena by at least 8 log, and C. parvum, P22 and MS2 virus by at least 6 log.  At sites 2 ? 4, Unit 2 removed bacteria and parasites 0.09 ? 0.9 Log Reduction Value (LRV) more than Unit 1.  R. terrigena saw 5.8 ? 6.72 LRV, C. parvum by 5.5 ? 5.6 LRV, P22 by 4.38-4.50 LRV and MS2 by 0.54 ? 2.92 LRV at sites 2 - 4. 
Objective 2 (O2): Removal for Unit 3 was 1-2 log for P22, 3-4 log for MS2, 9 log for R. terrigena, 5-6 log for C. parvum.  Site 3 (between foam disc and filter) showed biofilm contributed 1 -2 LRV for P22 and C. parvum.

Conclusions 
O1:  Units 1 & 2 performed similarly overall, the majority of removal occurred after flocculation and passage through the cartridge filter.  Unit 2 performed better at the major point of difference between the two units.
O2:  Comparing the foam filter units to the biosand unit produced mixed results.  Unit 3 was better at removing C. parvum, whereas Unit 1 removed more virus than Unit 2.  This comparison was conducted before the development of biofilm in any unit.  Pre and post biofilm development results suggest that biofilm is a large contributor to reduction (data for MS2 and R. terrigena is less conclusive).

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The Role of Binding Proteins SusE and SusF in Starch Utilization by Bacteroides thetaiotaomicron

Elizabeth A Cameron, Nicole M Koropatkin, Eric C Martens, University of Michigan Medical School, Ann Arbor, MI 48109

An essential function of the human gut microbiota is to break down indigestible carbohydrates into short chain fatty acids, providing the host with a significant source of nutrients and increasing overall colonic health.  A dominant bacterial phylum in the human gut microbiota, the Bacteroidetes, degrades a wide variety of host and dietary-derived carbohydrates via the expression of unique membrane-associated extracellular protein complexes which bind, degrade and import glycans. The starch utilization system (Sus) of B. thetaiotaomicron (B. theta) was the first such complex described and is essential for growth on starch, a significant dietary glycan. Two proteins within this complex, SusE and SusF belong to a highly abundant family of proteins within the human gut microbiota, yet their function within the Sus and homologous systems remains unknown. We have combined structural biology and microbial genetics to study the role of these two proteins in starch utilization by B. theta.  In vitro assays have confirmed that these proteins bind starch with high affinity and provide an advantage in competitive growth assays.  We have demonstrated that deleting susF confers a growth defect on starches of higher α1,6-branch density, where no growth defect was observed for a susE deletion strain.  This suggests that SusE and SusF play different roles in starch utilization, and may be accommodating structural diversity in starch molecules.  The crystal structures of SusE and SusF revealed that the proteins share a high level of structural similarity, although a striking difference is the presence of an additional starch binding domain in SusF, absent from SusE. We hypothesize that this extra domain is a major factor contributing to the phenotypic differences observed between strains lacking susE or susF.  Current efforts are directed toward examining the amount of SusE and SusF expressed on the surface of starch grown B. theta cells. These results have implications for understanding the role of SusE and SusF within the Sus complex, as well as this larger class of proteins in the human gut Bacteroidetes.

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Resistant Starch Utilization by Eubacterium rectale and Faecalibacterium prausnitzii, Two Abundant Butyrate Producers in the Human Distal Gut

Mallory A Maynard and Nicole M Koropatkin, Department of Microbiology and Immunology,University of Michigan, Ann Arbor, MI 48109

The microbiota of the human distal gut is known to be a critical aspect of structural polysaccharide breakdown and digestion.  Certain strains of bacteria in the human distal gut are comprised of enzymes that we as humans lack and these specific enzymes in turn breakdown these plant polysaccharides.  Two strains of interest are strains E. rectale and F. prausnitzii which are part of the phylum Firmicutes.  These strains become more abundant in the microbiota when the host consumes a diet rich in resistant starch resulting in increased butyrate production. Increased butyrate production is desirable due to its capabilities as an anti-inflammatory which may help protect the human intestinal tract from several chronic diseases.  In order to determine whether these butyrate-producing bacteria are capable of directly degrading resistant starch, we are studying the growth of E. rectale and F. prausnitzii with resistant starches Hylon 7 and Hi-Maize 260 (National Starch).  We then produced growth curves by measuring bacterial growth on plate dilutions every 12 hours.  Additionally, we will be taking a proteomics approach to identify the secreted and outermembrane enzymes that confer this starch-degrading ability.  If we can experimentally determine the mechanism of resistant starch degradation that aids in increased butyrate production, then we can possibly help as a first step towards greater protection against chronic diseases such as IBS, colon cancer, and Crohns Disease.

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UreF Interactions with other Proteins for Activating Urease, a Bacterial Virulence Factor

Jodi L. Boer and Robert P. Hausinger, Michigan State University, East Lansing, MI 48824

Urease is a virulence factor in bacteria such as Helicobacter pylori that hydrolyze urea to raise the local pH by forming ammonia and carbonic acid.  The model urease system from Klebsiella aerogenes has been studied in great detail by expressing recombinant ureA, ureB, and ureC (enzyme subunit genes) and ureD, ureE, ureF, and ureG (urease-associated genes) in Escherichia coli.  The current work is focused on the role of K. aerogenes UreF.  Using a multiple sequence alignment and the H. pylori UreF crystal structure, the codons of 15 K. aerogenes UreF residues were mutated to alanine in the full-length gene cluster and in a translational fusion of ureE and ureF (producing a soluble and functional dimer of UreEF).  Nine of these mutants activated urease to < 10% of its wild-type activity in vivo and the rest generated < 70% activity.  In vivo pull-down assays utilizing the UreEF construct in the context of the entire set of urease genes recovered a complex of all urease-related proteins, whereas similar samples for most mutants were deficient in UreG.  In vitro pull-down assays using isolated UreEF to bind purified UreABC:UreD demonstrated hindered interactions for all mutant proteins, with E94A and H214A variants of UreEF exhibiting very low binding.  Pull-down assays using a purified Strep-tagged version of UreG (UreGStr) and isolated UreEF and its variants confirmed several of the in vivo experiments.  Pull-down experiments using maltose binding protein (MBP)-UreD, a soluble form of UreD, resulted in the formation of MBP-UreD:UreEF binding in a 1:2 ratio, with the mutant proteins unexpectedly binding at even higher ratios, indicating that UreEF remains a dimer when bound to MBP-UreD. Overall, these results point to several key residues in UreF playing important roles in binding UreD and UreG.

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Plastics such as polystyrene are resistant to degradation and pose a threat to the environment.  A new approach to managing polystyrene waste is needed.  The goal of this research is to identify and characterize polystyrene degrading bacteria.  Environmental bacterial isolates from a common yard soil sample and from soil of the former Ford Rouge Steel plant in Dearborn were screened for polystyrene degradation.  Soil bacteria were incubated with sterile polystyrene (packing foam peanuts) in solutions of Bushnell Haas (BH), 9:1 or 1:1 of either BH: Yeast Extract Peptide Glucose (YEPG) or BH: Nutrient Broth media for five weeks.  Bacteria were transferred to identical media for an additional 8-week period.  From the second enrichment culture, a few foam peanuts exhibited a small loss of mass.  The sensitive Water Soluble Tetrazolium (WST-1) growth test was also performed on individual bacterial isolates or bacterial mixtures.  Cells were placed in sterile 96 well polystyrene plates with only PMM (no-carbon) medium and incubated for 4 weeks.  Cells unable to catabolize polystyrene did not proliferate and exhibited a negative WST-1 result (colorless).  Positive results in the WST-1 test were a dark red medium coloration.  At least 5 individual isolates were WST-1 positive, presumably able to use polystyrene as source of energy for proliferation.  Selected co-incubated bacteria inhibited the 5 polystyrene degraders recruitment of polystyrene.  One polystyrene degrader, Pseudomonas putida G7, caused polystyrene darkening and bacterial proliferation on the surface of sterile foam peanuts embedded in PMM-agar plates.  Initial rDNA sequence identification of the plastic degraders suggests they are not derived from a single genus.  By characterizing and optimizing culture conditions for polystyrene bacterial degradation, a new means of plastic removal may be realized.

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Live/Dead Verification Technology for Ballast Water Organisms

Jason P. Gizicki and Jeffrey L. Ram, Department of Physiology, Wayne State University

Background and objectives:  Non-native species, such as zebra mussels, have caused great ecological and economic harm to the Great Lakes. About half of such organisms arrived as "stowaways" in foreign ballast water discharged from ocean-going ships.  To prevent future ballast water introductions of non-native organisms and pathogens (e.g., Vibrio cholera and viruses), regulations have been developed by international, US, and state agencies for ballast water to be sterilized prior to discharge.  The objective of this project is to develop live/dead verification systems that will enable assessment of the effectiveness of shipboard ballast water treatment systems and estimate risk associated with any survivors.

Methods: Initial studies focused on live/dead methods for microorganisms and tested effects of propidium monoazide (PMA) treatment of live v. dead cells on PCR-amplifiable DNA extracted from the cells.  PMA is a membrane-impermeable DNA-cross-linking agent to which cells become permeable upon death.  Cultures of Escherichia coli, Vibrio cholera, Enterococcus sp., and Clostridium perfringens were killed with heat (15 min, 95 oC) or NaOH (24 hr, pH 12); treated (or not, for control) with PMA; DNA extracted; and amplified with universal primers and real-time qPCR.

Results: For all 4 species of bacteria, PMA treatment of live cultures prior to DNA extraction typically caused a shift towards higher threshold detection cycle number (CT) compared to non-PMA treated cultures.  DNA from dead cultures (killed by either heat or NaOH) that were not treated with PMA amplified nearly as well as DNA from comparable live cultures.  However, PCR of DNA from dead cultures was greatly inibited by PMA, with typical shifts of CT of >10 cycles, indicating <0.1% of PCR-amplifiable DNA compared to non-PMA treated dead cultures, a significantly greater shift (p<0.05) than PMA effects on live cultures.

Conclusions:  The PMA-PCR procedure has the capability of distinguishing between live and dead bacterial cultures. Generality of the method with environmental bacteria, phytoplankton (algae), and microinvertebrates will be tested.  For risk analysis, sequencing of PCR products of “surviving” amplifiable DNA may identify the organisms most resistant to ballast water treatments.

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Detection of Escherichia coli Beta-glucuronidase Gene From Enviornmental Samples Using Combinations of Conventional and Quantitative PCR Approaches

Jessica L. Koehler and Ola Olapade, Albion College, Albion, MI, 49224

The rapid detection of coliform bacteria is very essential for several environmental and public health reasons.  Currently, among known indicator organisms, Escherichia coli is the most reliable indicator for fecal contamination in aquatic systems.  E. coli is a reliable indicator because its gene i.e., uidA and product beta-glucuronidase, are specific only to the bacteria.  Comparatively, current culture-based techniques for determining water quality are too time consuming.  Therefore, this study was designed to examine the potential of polymerase chain reaction [PCR] application for detecting E. coli contamination in aquatic systems.  Triplicate water samples were collected periodically from three locations along the Kalamazoo River and examined with conventional and quantitative PCR [qPCR] assays as well as viable bacterial counts.  The DNA from one sample at each site was diluted to 10-2 and 10-4 to determine detection limits based on the three methods.  Dilution standards were established between 100 and 108/mL of E. coli cells, and used to establish a standard curve to normalize the qPCR and compare to the viable counts and conventional PCR results.  In general, the conventional PCR appeared more sensitive than the viable count method, although there were some discrepancies that may point to presence of impurities in the DNA samples.  The results from several of the qPCR assays were inconclusive, with only a few indicating acceptable r2 values. The future plan is to continue using all three methods for analyses until all assays are perfected and a detection limit established for the examination of various types of environmental samples.

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Analysis of a Cyclic Peptide Library to Identify Proteins That Effect hilA Expression and Invasion in Salmonella enterica serovar Typhimurium

Philip Kaseska, Brad Geal, Andrew Lux and Aaron Baxter, Grand Valley State University, Allendale, MI 49401

Salmonella enterica serovar Typhimurium is a gram-negative bacterium that produces a self-limiting, localized gastroenteritidis in humans upon ingestion of contaminated food or water. Pathogenesis demands Salmonella recognition of multiple environmental conditions which activate a 40 kb region of DNA known as Salmonella Pathogenicity Island 1 (SPI-1). The expressed SPI-1 proteins manipulate normal host cell function and direct the uptake of the bacteria into targeted cells. The expression of the SPI-1 genes is controlled via the sensing of various environmental conditions through multiple pathways. When conditions are optimal for invasion, these various inducing signals are directed toward the SPI-1 transcriptional activator hilA. In collaboration with Brad Jones at the University of Iowa, we have acquired a plasmid library that produces small random cyclic peptides. Currently we have identified some plasmids which reduce expression of a hilA::lacZY reporter in E. coli and in Salmonella Typhimurium. The effects of these plasmids are being further quantified through in vivo invasion assays.

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Identification and Characterization of HopM1 in Yeast

Vanessa Revindran and J.R. Geiser, Western Michigan University, Kalamazoo, MI, 49008.

We have created a model expression system to study the action of plant pathogen effectors in the yeast Saccharomyces cerevisiae. The expression system utilizes the inducible GAL1 promoter, has a V5 epitope for microscopy and a 6xHIS tag for purification. The expression constructs were created via PCR amplification of HopM1 followed by cloning into a Gateway Systemä expression vector.
HopM1 when expressed in yeast is lethal on solid media at 21°C, but not at 30°C and 37°C.  In liquid media, colony forming units have decreased to 73% of the initial inoculation by 24 hours at 21°C.  By 48 hours, colony forming units have decreased to 38% of the amount present at time zero. At 30°C, we do not see a decrease in the number of colony forming units over 48 hours.  We have examined the protein levels of HopM1 to determine the effect temperature had on expression. As demonstrated by Western blot, HopM1 protein is present at 21°C, 30°C and 37°C.  At 21°C (temperature that causes death), a full length protein of 78kDA is observed.  At 30°C and 37C, the majority of HopM1 protein exists as degraded fragments. This is not surprising because, in tomato plants, the effects of Pseudomonas syringae pathovar tomato DC3000 are seen at temperatures lower than 23° C, and at 31°C no effects are observed. We have isolated 16 suppressor strains that are capable of surviving the HopM1 imposed lethality at 21°C. All the strains have been examined for HopM1 protein expression.  Of the 16 strains, 11 show full-length HopM1 at 21°C and the remaining 5 show a degradation pattern.
We are currently trying to determine structure specific localization of HopM1. Results will be presented to show that HopM1 appears as punctate like structures in the yeast cytoplasm and colocalizes to the mitochondria as well. Further characterization of the suppressor strains is continuing to determine specific localization of HopM1 and to determine if the suppressors have any effect on the localization of HopM1. Work is also continuing to clone and identify a genetic target of HopM1.

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