Oxidative Stress, Mitochondria and Fertility

Samantha Legere and Steven Gorsich, Central Michigan University, Mt. Pleasant, MI 48858

Human fertility problems in male sperm were explored with Saccharomyces cervisiae as the model organism. The DMN1 mutant has a deficiency in the protein involved with mitochondrial fission. As such, it acts similarly to cells that have undergone oxidative stress, such as being treated with H2O2. S. cerevisiae cells are known to be extremely sensitive to oxidative damage when Ynl241cp, which will be referred to as zwf1p, is deficient. This study focused upon mitochondria during sporulation. The similarities between S. cerevisiae and humans allows for study of spermatogenesis and infertility due to oxidative stress. Sperm vitality, and thus fertility in general, is dependent on mitochondria. These organelles are what power the tail of the sperm, allowing them to swim to and fertilize the egg. It was hypothesized that zwf1p may protect cells from oxidative stress damage. The strains used were FY S288C #114 and FY S288C #12F8. Standard methods were used to grow, transform, and manipulate yeast strains. GFP was used to visualize the mitochondrion under fluorescence microscopy. Both wild-type and DMN1 mutant cell types were observed with and without an overexpression of zwf1p. The cells were grown for 12–18 h at 25°C in sds-ura media and transferred to YPG at 0.5 OD600 U/ml. After 18–36 h at 30°C, cells were resuspended in 5 ml of sporulation medium and shaken (225 rpm, gyratory G2 tabletop shaker) at 25°C for 9 days. Aliquots of sporulating cells were examined daily with fluorescence and differential interference contrast (DIC) microscopy. The overexpression of zwf1 was found to decrease abnormal mitochondria morphology in wild-type and DMN1 mutant strains. In regards to mitochondria morphology: wild-type was mostly fragmented and ‘bead on
string,’ DMN1 mutant was clustered and mass, wild-type with zwf1p had increased fragmented and DMN1 mutant with zwf1p had an increase in tubular, fragmented and ‘bead on string.’ Thus, zwf1p increased mitochondrial fission and counteracted the mimic of oxidative stress.

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Effect of common fragile site flexibility peaks on mitotic recombination in S. cerevisiae

Valerie Sponyoe and Anne Casper, Eastern Michigan University

Common fragile sites (CFS) are areas of the chromosome that are susceptible to DNA breakage when placed under replication stress. CFS breaks near tumor suppressor genes can lead to cancer. Previously we have shown that in cells under replication stress, breaks at a yeast fragile site stimulate homologous recombination (HR) events that lead to loss of heterozygousity. We hypothesize that a sequence motif called a flexibility peak found in human fragile sites will also stimulate HR. To test this hypothesis, we have inserted this flexibility peak into the yeast Saccharomyces cerevisiae. This organism allows us to view the effects of this sequence on recombination events.

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Genetic instability at Fragile Site FS2 stimulates mitotic recombination in Saccharomyces cerevisiae

Shaylynn Miller and Anne Casper, Eastern Michigan University

Common fragile sites are pervasive in the DNA of many known species, including humans. The instability of fragile site DNA sequences can lead to mitotic recombination in the event of a strand break. If this occurs near cancer-suppressing genes, such as tumor suppressor genes, abnormal cell growth could occur. FS instability increases when the cell is experiencing replication stress. In Saccharymyces cerevisiae this can be induced through the use of a modified galactose-dependent promoter region of polymerase α. Homologous recombination (HR) is one mechanism for repairing DNA breaks.  Mitotic HR near FS2 was analyzed; more recombination was found at FS2 during periods of replication stress.

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Determining Accuracy of DNA Synthesis during Repair by Homologous Recombination

Mikael K Dunn and Shahana Ahmed, Eastern Michigan University

The yeast Saccharmyces cerevisiae, inhibition of DNA polymerase alpha causes breaks at specific sites – called fragile sites.  We will identify yeast cells that repaired the break by Homologous Recombination (HR) during mitosis, and then evaluate whether the DNA synthesis that happened as part of the repair process was accurate.  We hypothesize that DNA synthesis during HR is inaccurate.  To test this hypothesis, we are using a mutant lys2 reporter gene next to the fragile site. If DNA synthesis during repair is accurate, lys2 will still be mutant. Inaccurate DNA synthesis during repair can restore the gene to its wild-type version.

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Measuring Break Frequencies of Human Common Fragile Sites carried on Yeast Artificial Chromosomes

Catherine Kaminski, Alicia Layer, Katherine Dzuiba, Anne Casper, Eastern Michigan University, Ypsilanti, MI, 48197

Fragile sites (FS) are areas of the genome more susceptible to DNA breakage in cells under replication stress. These regions are associated with tumor suppressor genes and oncogenes, which can lead to tumorigenesis when mutated. Yeast Artificial Chromosomes (YACs) carrying inserts of human DNA from FS regions and control, non-FS regions were studied. We hypothesize that break frequencies in the YACs containing human FS DNA will be higher in cells under replication stress than in cells under normal conditions.  We also hypothesize that the YACs with human FS DNA will be more unstable than YACs with non-FS DNA.  Preliminary data on two FS-containing YACs supports our first hypothesis.

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Insertion of human common fragile site FRA16D flexibility peak in yeast Saccharomyces cerevisiae does not stimulate mitotic recombination

Quinn Ellison, Valerie Sponyoe, Ellen Younkin, Anne Casper, Eastern Michigan University.

Common fragile sites (CFS) are regions of mammalian chromosomes that form DNA breaks under conditions of replication stress. CFS are prevalent throughout the human genome and some lie near or within tumor suppressor genes or oncogenes. Breaks at CFS can lead to gene deletions and amplifications that can result in the genesis of cancer cells. One potential mechanism for the propensity of CFS to form DNA breaks under replication stress is the failure to resolve secondary structures, such as AT-rich hairpin loops, that form during stressed DNA replication. Flexibility peaks are AT-rich sequences that may be especially prone to forming secondary structures and contributing to double-strand DNA break formation. We have used the Saccharomyces cerevisiae yeast model system to investigate this potential stimulus of fragile site instability. Previous work has shown that under replication stress, the native yeast fragile site FS2 is a hotspot for the initiation of mitotic recombination events that can lead to loss of heterozygosity (LOH), including reciprocal crossovers (RCO) and break-induced replication (BIR). The flexibility peak sequence Flex1 from FRA16D was inserted into an experimental yeast strain in place of the native FS2 and compared to a control strain with the FS2 sequence removed. The Flex1 strain did not show an increased rate of mitotic recombination events leading to LOH over the control strain (p= 0.44).These preliminary results indicate that the presence of a flexibility peak sequence from FRA16D is not sufficient to stimulate an increase in mitotic recombination events that lead to LOH, and therefore this flexibility peak is unlikely to be a stimulus of fragile site instability.

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Assessing Maritime Vessel Ballast Waters for Indicator Microorganisms

Timothy J. Stieve, Rebecca L. Ives, Yiseul Kim, Tiong Gim Aw and Joan B. Rose, Michigan State University, East Lansing, MI, 48825

Maritime vessels of the past used metals, rocks, or sand to balance a ship on open seas. Today, a more efficient and economical approach is to take on ballast water to counterbalance the weight of ship and cargo. This results in 3 to 5 billion tons of water exchanged internationally every year. Uptake of water on such a large scale can transport marine animals, viruses, bacteria, and other biological organisms across the globe. If International Maritime Organization (IMO) regulations controlling ballast water exchange are not followed, these stowaways have the ability to negatively impact the aquatic ecosystems they are released into and also the health of the communities surrounding these ports. This study assessed the levels of fecal pollution indicator microorganisms in ballast and port water, in order to shed light on whether the water is consistent with their respective discharge standards. Water samples were processed from 5 ships and 3 harbors in Los Angeles and Long Beach, California. The methods used include: Colilert®, Enterolert®, male specific coliphage assay, and Escherichia coli (E. coli) detection by membrane filtration on mTEC agar. Results showed male specific coliphage, an indicator of fresh fecal contamination, was present in harbor waters but essentially absent from all ballast waters.  Enterococcus was found at levels above the discharge standard of <100 CFU/100ml in two of the five ships. The discharge standard for E. coli of <250 CFU/100 mL was met in four of the five ballast waters. For harbor water, a minimum protective standard of <104 MPN/100ml for Enterococcus and <10,000 MPN/100ml for total coliform is implemented by the California Department of Health Services for water adjacent to public beaches. All three harbors exceeded the standard for Enterococcus, while one of the three exceeded total coliform standard.

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Cyanobacterial Communities from Alvar Substrates on Drummond Island, Michigan

Travis Bean and Gregory M. Colores, Biology Department, Central Michigan University, Mount Pleasant MI, 48859

Despite its rarity, there is little research on the alvar habitat and in particular, associated microbial communities. Many unique species are adapted to the harsh alvar environment impacted by sporadic rainfall and large fluctuations in temperature. Alvars are areas of calcareous bedrock, typically calcite (CaCO3) or dolomite (CaMg(CO3)2), with little to no soil or organic matter. The development of soil upon the alvar facilitates the transition from bare substrate to an environment that can support vascular plant growth. Cyanobacteria are some of the earliest colonizers of bedrock and are structurally and functionally important in soil development. Large filamentous cyanobacteria form matrices capable of trapping soil particles, retaining moisture, and preventing erosion. Cyanobacteria are also important in areas with little to no anthropogenic nitrogen inputs as a producer of fixed nitrogen that is needed by successional species. Alvar rock samples and soil crust samples were collected from two sites, found on Drummond Island, Michigan. Endolithic and soil crust cyanobacteria were successfully cultivated from both alvar sites using a liquid cyanobacterial growth medium, BG-11. Lithic samples and soil crust enrichments were imaged using light and scanning electron microscopy. Geochemical analysis of the alvar samples revealed that the two sites differ in the degree of weathering, but are geologically similar.  The results of microscopy show substrate composition, diversity within the cyanobacterial communities, and the possible roles that cyanobacteria play in stabilizing the delicate soils of the alvar.

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Gene Conversion at Fragile Site Breaks in Saccharomyces Cerevisiae

Thomas F Coates, Kaelen Medeiros and Anne Casper, Eastern Michigan University

Fragile sites are regions within the genome that are highly susceptible to breaking during replication. Breaks at these fragile sites can be repaired using a number of different mechanisms, one of which is homologous recombination (HR), which can lead to loss of heterozygosity (LOH). LOH, in turn, can contribute to cancer development if it occurs in tumor suppressor alleles. We hypothesize that fragile site breaks are frequently repaired by a HR mechanism called gene conversion. We are in the process of testing this hypothesis by inducing breaks at a common fragile site in a Saccharomyces cerevisae model yeast strain and analysing the methods used to repair these breaks in instances where LOH has occurred.

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The association of fecal indicator molecular measurements from Lake St. Clair sediment cores with nutrient loading and climate

Yolanda M Brooks1, Melissa M Baustian2, Mark M Baskaran3, Joan B Rose1
1Michigan State University, East Lansing, MI, 2The water Institute of the Gulf, Baton Rouge, LA, 3Wayne State University, Detroit, MI

Advances in water quality monitoring have decreased waterborne disease incidence in the last century. Large-scale temporal studies have rarely determined how anthropogenic attributes and climate are associated to fecal pollution levels. Sediment core can investigate watershed health on a large scale. This study’s objectives were to measure genetic marker concentrations from fecal indicators, Escherichia coli uidA (EC-uidA) and enterococci 23S rRNA (ENT-23), in sediment cores from Anchor Bay in northwest Lake St. Clair (AB), and the mouth of the Clinton River (CR); to estimate chronostratigraphy in each core with Cs-137; and to assess the association of indicators to climate (average yearly temperature and cumulative yearly precipitation) and anthropogenic attributes (watershed population; and C, N, and P concentrations).
The estimated sedimentation rates were 0.42g/cm2-y (core represented ~117yrs) and 0.35g/cm2-y (core represented ~ 255yrs) for CR and AB, respectively. In CR, nutrients (C, N, P) gradually increased over time. In AB, nutrients spiked during ~1856 – 1919, and then C and N became steady until ~1990 where they increased, while P gradually increased. The AB ENT-23 concentrations were at steady state, ~5 * 10^4 copies/g-dry wt from 1757-1878, and increased to ~8 * 10^5 copies/g-dry wt from 1902-2010. ENT-23 concentrations in CR had three steady state concentrations, ~5 * 10^4 copies/g-dry wt from 1895-1966; ~3 * 10^5 copies/g-dry wt from 1966-1987; and 2 * 10^6 copies/g-dry wt from 1990 -2011. EC-uidA concentrations ranged in CR and AB from 1.42 * 10^6 – 1.69 * 10^7 copies/g-dry wt, and 1.81 * 10^6 – 8.46 * 10^6 copies/g-dry wt, respectively. Linear regression analysis determined that indicator concentrations were significantly affected by indicator species (ENT-23 concentrations were significantly smaller than EC-uidA), anthropogenic attributes, and location (p<0.05), and were not statistically significant associated to precipitation (p>0.05). These results indicate that population growth, and nutrient concentrations were indicative of increased pollution in the Lake St. Clair watershed in the last 100yrs.
*These findings will also be presented at Michigan State University Graduate Academic Conference.

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Inositol Transport and Catablism in Sinorhizobium meliloti

Ee Leng Choong, Mary Thwaites, and Silvia Rossbach
Biological Sciences, Western Michigan University, Kalamazoo, USA

Atmospheric nitrogen is in a form that is unavailable to plants. Nevertheless, there are nitrogen fixing bacteria that can be found either free-living or in association with plants. Sinorhizobium meliloti is a symbiotic nitrogen-fixing bacterium that infects legume plants such as alfalfa and induces specialized organs known as root nodules.  In the nodule, the bacteria will fix the atmospheric nitrogen for use by the host plant, and in return, the plants supply the bacteria with carbon sources. Specialized carbon sources such as inositols may play a role in determining bacterial composition of the rhizosphere. The focus of this study is to characterize essential genes that are involved in inositol transport and catabolism in S. meliloti. The S. meliloti ibpA gene was found to be the first gene in an operon that encodes a periplasmic binding protein of an ABC transporter responsible for the transport of inositol isomers. A mutant with an insertion in the ibpA gene was tested for the ability to use inositol as sole carbon source. The ibpA mutant shows a delayed growth, but it regained growth after three days. It was concluded that the ibpA-iatA-iatP operon encodes a major inositol transporter, but that there may be also a second, minor inositol transporter present in S. meliloti. A candidate gene was identified based on homology, and mutants with insertions in this gene were constructed and their phenotypes were analyzed.  The inositol (iol) genes are required for bacterial growth with inositol and S. meliloti mutant strains with Tn5 insertion in each of the individual iol genes were previously characterized with growth studies testing myo-inositol as the sole carbon source. Interestingly, the iolI gene involved in the catabolism of another isomer of inositol, D-chiro-inositol, still remains unidentified in S. meliloti. A candidate gene was discovered based on homology with the iolI gene of Bacillus subtilis and a mutant with an inserted antibiotic resistance cassette was constructed and its phenotype was analyzed.  Plant inoculation studies will show whether inositol transport genes and D-chiro-inositol catabolism genes of S. meliloti are involved in establishing and maintaining an efficient nitrogen fixing symbiosis between rhizobia and legume plants.

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Investigating Catabolism of the Methylated Sugar Alcohol Pinitol in the Soil Bacterium Sinorhizobium meliloti

Olivia Walser, Mary Thwaites, Silvia Rossbach, Western Michigan University, Kalamazoo, MI

In the mutualistic symbiosis between Sinorhizobium meliloti and the legume plant alfalfa, the bacteria fix atmospheric nitrogen for the plant and the plant provides bacteria with nutrients. Our recent work on this plant-microbe symbiosis model has been with the genes involved in the catabolism of pinitol, a methylated inositol derivative. We have identified a gene region of interest based on bioinformatics and conducted growth studies on mutant strains using pinitol as sole carbon source, the results of which allowed us to develop a model for pinitol degradation. We also did a survey of the recently sequenced genomes of other Sinorhizobium strains and found that there is one strain which does not have the gene region which we have characterized. We conducted a growth study using minimal media and found that this particular strain does not grow when provided pinitol as sole carbon source. This result not only supports the notion that the genes we identified are required for pinitol degradation, and shows that this strain may also be a convenient tool for further characterizing this gene region. We constructed a vector which constitutively expresses these genes and complemented this strain by homologous complementation using a triparental mating. We verified that our mating was successful via PCR and our next steps will be to conduct a growth study to observe the phenotype of this now complemented strain.

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Effects of Perfluorooctanoic Acid (PFOA) and Perfluorooctane Sulfonic Acid (PFOS) on Rhodobacter sphaeroides Enzyme Activity

Erin M. Mulroney and Ranson Olson, Britton D., Lake Superior State University, Sault Ste. Marie, MI 49783

Perfluorooctanoic acid (PFOA) and perfluorooctane sulfonic acid (PFOS) are man-made environmental contaminants that have been identified in numerous biological sampling projects, and that these perfluorinated compounds (PFCs) are still produced overseas, there is a need to investigate the potential effects that these chemicals have on a cellular level. Rhodobacter sphaeroides is a good model to demonstrate such effects because it has been shown to be sensitive to both PFOA and PFOS and it possesses similar metabolic processes to those found in eukaryotic mitochondria. The results of this study demonstrate a change in the enzyme activity of R. sphaeroides for both the carbon and phosphorus cycling based on increased peroxidase, phenol oxidase, and acid and alkaline phosphatase activity.  As oxidative stress is suspected to be induced by PFCs, peroxidase activity of R. sphaeroides was measured and compared to PFC-growth insensitive Escherichia coli. It was shown that the peroxidase activity in R. sphaeroides increased in the presence of the PFOS, while it did not change significantly with PFOA.  It is unknown why E. coli is insensitive to PFC, and so to investigate whether biotransformation might be a possible mechanism, the peroxidase activity was measured in a R. sphaeroides strain grown in the supernatant of E. coli cultures pre-incubated in PFC. It was shown that activity was lowered in cultures grown with the supernatant containing PFOS, suggesting a possible method for reducing the harmful effects of this PFC. Overall, this study supports oxidative stress as a potential cellular effect of PFOS, while PFOA mechanisms remain unknown.

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Occurrence of Coliphage and Salmonella Phage on Romaine Lettuce to Indicate Fecal Contamination

Olivia Stenzel, Wengert, Samantha, Ives, Rebecca, Rose, Joan, Michigan State University, East Lansing, MI 48825

It is estimated that there are 48 million cases of foodborne illness every year in the United States and approximately 46% of those illnesses can be traced back to produce. A common cause of foodborne illness is due to enteric pathogens and fecal contamination on fresh produce.  Bacteriophages, which are a type of virus that infects bacteria, are commonly used as indicators of fecal contamination. The purpose of this study was to determine the occurrence of recent fecal contamination using phage on romaine lettuce from the distributor to the grocery store and also to determine if the contamination varied between inner and outer lettuce leaves. Romaine lettuce was sampled from two locations after harvest including food chain distribution centers and then grocery stores.  Samples were tested for F-specific coliphage and Salmonella phage with the hosts Escherichia coli Famp and Salmonella typhimurium respectively.  Lettuce samples then underwent an initial elution step followed by testing using a double agar overlay in a modified version of EPA method 1602, and an enrichment using a modified version of EPA method 1601. One positive sample for phage was detected on the outer leaves from one head of lettuce from the distribution center resulting in 5 MPN/ g wet weight.  All other inner and outer leaves from the distribution center and grocery store came back negative for any cultivable phage. Results suggest that fecal contamination was minimal and that the phage were inactivated by the time the lettuce reached the grocery store. More samples and analyses are needed and a survival study of phage on lettuce to provide insight into inactivation of viruses on lettuce.

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Cloning of NOD1 and NOD2 in Danio rerio

Kendra Garcia, Emerald Butko, Natasha DelCid and L. Rob Peters
Aquinas College, Grand Rapids, MI

NOD1 and NOD2 encode cytosolic innate immune receptors that bind either, or both, intracellular gram-positive and gram-negative bacteria. Gene variants of human NOD2 are implicated in Crohn’s disease, a form of inflammatory bowel disease. This and other observations demonstrate the importance of NOD receptors in intestinal immunity. The zebrafish (Danio rerio) model system allows for unparalleled imaging in the developing embryo, as zebrafish embryos are transparent and develop ex vivo. The recent characterization of transgenic zebrafish harboring fluorescent immune cells poises the zebrafish model as extremely useful in understanding the relationship between commensal microbes, intestinal epithelial cells, and immune cells in vertebrates. As zebrafish NOD1 and NOD2 have a role in pathogen clearance, we aim to use the zebrafish model to assess the role of these receptors in the development of intestinal immunity. Dominant negative NOD1 and NOD2 gene variants have been identified and characterized in humans and mice, some of which are associated with Crohn’s disease. Here we show data on the cloning of zebrafish NOD1 and NOD2. We will use these cloned genes to generate and express putative dominant negative NOD1 and/or NOD2 proteins in zebrafish intestinal epithelial cells.  We will test the ability of these NOD mutations to block endogenous NOD1 or NOD2 function in zebrafish.

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EBV LMP1 Induces the Expression of SUMO-1 and SUMO-2/3 in LMP1-positive Lymphomas and Cells.

Emma K Fath1, Salahuddin, Sadia1,2, Shackelford, Julia1, Pagano, Joseph S1, Bentz, Gretchen L1,3, 1Chapel Hill, North Carolina 27514, 2Islamabad, Pakistan, 3Macon, Georgia 31201.

Epstein-Barr virus latent membrane protein (LMP1) interacts with the SUMO-conjugating enzyme Ubc9, which induces protein sumoylation and may contribute to LMP1-mediated oncogenesis. After analyzing human lymphoma tissues and EBV-positive cell lines, we now document that there is a strong correlation between LMP1 and sumo-1/2/3 levels, which also correlated with increased cell migration. Together, these results suggest a second mechanism, induction of sumo1/2/3 levels, by which LMP1 deregulates sumoylation processes and contributes to viral pathogenesis.

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Characterization of Microorganisms from Alfalfa Seed Inoculants

Benedicte Diatta, Kalianna Keenoy and Silvia Rossbach, Department of Biological Sciences, Western Michigan University, Kalamazoo, MI 49008-5410

Atmospheric nitrogen fixation can be accomplished by free living microorganisms or by symbiosis between legume plants and rhizobia. The symbiosis between the alfalfa plant and Sinorhizobium meliloti is one example of the symbiotic relationship between legume plants and rhizobia. During the symbiosis, the rhizobia fix nitrogen for the plant and the plant in return provides carbohydrates for the bacteria. In order to maximize the plant’s yield in agriculture, recent technologies used include seed inoculants. Seed inoculation methods rely on coating the seeds with plant-growth promoting bacteria. Besides nitrogen-fixing bacteria, the seeds may also be coated with microorganisms aiding in mineral solubilization, biological control or nutritional requirements.
The goal of this study was to identify the different bacteria present in coated alfalfa seeds and their impact on alfalfa plant yield. Bacteria were isolated from the coated seeds and purified. Several morphologically different bacteria were isolated from the coated seeds. Their 16S rRNA genes were amplified and sequenced. Several Bacillus spp. were identified. Surprisingly, no rhizobial strains were found among the isolated strains. Nevertheless, we performed a plant inoculation assay with the coated seeds and nodules were observed, suggesting the presence of rhizobia in the inoculants. We also screened for the presence of nitrogen fixation (nifH) genes in the bacterial isolates. The results indicated that two strains contained the nifH genes. Another plant inoculation assay with uncoated alfalfa seeds is under investigation in order to evaluate the impact of each isolated strain on alfalfa growth.

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Bioremediation of Crude Oil Spills Impacts Microbial Communities and the Magnetic Susceptibility of Sediments

Carol L. Beaver1,  Anja Williams1, Estella Atekwana2, Gamal Abdel Aal2,
Farag Mewafy2, Lee Slater3, and Silvia Rossbach1, 1Department of Biological Sciences, Western Michigan University, Kalamazoo, Michigan, USA , 2School of Geology, Oklahoma State University, USA, 3Earth and Environmental Sciences, Rutgers University-Newark, USA

A worldwide increase in the production, shipment, and storage of crude oil has led to an increase in oil spills, and efforts to physically remove this oil from the environment are often insufficient. As a result, hydrocarbon contamination that is not eliminated by suction, excavation, or by dispersants remains in the water and sediments to be degraded by microbes. Initially, the bioremediation of oil occurs aerobically, but as oxygen is depleted, microbes must switch to alternate electron acceptors. Gradients of terminal electron acceptors form over time, which in conjunction with the toxic properties of oil, have an effect on the microbial community composition at hydrocarbon-impacted sites. These gradients of terminal electron acceptors also change the geochemical and geophysical properties of these sediments. Eventually, iron-reducing and methanogenic communities predominate at the center of oil plumes, where iron-reducing bacteria may precipitate magnetite. Thus, we are investigating if magnetic susceptibility measurements (MS) of crude oil-contaminated sediments can be used to monitor the production of magnetite resulting from bioremediation. For our experiments, we obtained one core retrieved from the center of an oil plume and one from an uncontaminated area downstream of an oil plume at a crude oil spill site in Bemidji, MN. MS measurements were taken in the wells remaining from the cores. Total DNA was extracted from samples taken from the cores, and 16S rRNA gene high throughput sequencing was used to assess the microbial communities. Results showed that microbial communities shifted towards methanogenic and fermenting populations in the oil plume at the water table, unlike the uncontaminated core, which did not. Interestingly, the highest peaks in MS were measured in the contaminated core around the water table, corresponding to the oil plume and the methanogenic zone. The peaks in MS may have been due to magnetite formed from iron reduction that occurred prior to methanogenesis, or from iron reduction that is occurring concurrently with methanogenesis. In either case, MS was higher where hydrocarbons are being degraded. Thus, we conclude that MS may be used to monitor bioremediation processes at crude oil spill sites.

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The Study of Esherichia coli Survival in Deer Pellets

Daniel Ginzburg, Jessica Fry, Rebecca Ives, Joan B. Rose, Department of Fisheries and Wildlife, 3 Natural Resources, Michigan State University, East Lansing, MI

Pathogenic Escherichia coli (E. coli) can be transmitted through the environment from wildlife such as deer to cattle, and then to humans via contact or the food chain.  Information on the survival of E.coli shed in fecal material is needed as an input in transmission models.  E. coli is an important fecal indicator organism that can be used as a surrogate for pathogenic E.coli. In this study, survival rates of naturally occurring E.coli in whitetail deer fecal matter were examined over a period of 31 days at selected intervals using 3 different temperatures 4°C, 20°C, and 35°C. Samples were taken from a deer population that is maintained at Michigan State University.  Fecal pellets were homogenized with a 100ml of sterile phosphate buffered water in a stomacher, centrifuged and tested using mTEC membrane filtration (USEPA METHOD 1603). All three temperatures were found to have regrowth occur after the first day.  At 4°C, E. coli was inactivated at the fastest rate of 0.1925 logarith10 reduction per day. E. coli logarith10 reduction per day was 0.0616 and 0.1528 for 20C storage and 35°C storage, respectively. Moisture content ranged from 70% to about 40% for all temperatures, with the 35°C losing the most moisture over the study period. We conclude that E. coli survival varies over the temperature ranges of 4°C, 20°C, and 35°C with loss of the population at 4°C occurring at approximately day 16 but populations ranging from 105 – 108 CFU/g dry weight remaining by day 31 at 20°-35°C. Recommendations of future work include testing the spread of E. coli from single specimens in the environment and the variation of E. coli survival among different species, including sheep from which pellet samples are easier to collect.

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