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Abstracts - Fall 2016
 
 
 
 
Oral Presentations
 
 
 
 
*Best Oral or Poster Presentation, Graduate
#Best Oral or Poster Presentation, Undergraduate
 

Structural Phenotypes of Carboxysomes in the Cyanobacteria Fremyella diplosiphon and Synechococcus elongatus PCC 7942
Authors:
Rohnke Brandon A.; Montgomery, Beronda L.
Michigan State University
East Lansing, MI 48824
Abstract Text: 
            Cyanobacteria utilize a carbon concentrating mechanism (CCM) to enhance carbon fixation. One step of CCM involves carboxysomes, which are proteinaceous bacterial microcompartments that house the enzyme Rubisco and serve as a barrier to diffusion of carbon dioxide substrate out of the structure. Some cyanobacteria tune photosynthetic pigment composition to external light cues to increase photosynthetic efficiency. Given the functions of carboxysomes and external light cues in maintaining efficient carbon fixation levels, we investigated whether carboxysome structure and function are regulated by external light cues. Using Transmission Electron Microscopy (TEM), we identified structural phenotypes of carboxysomes in wild-type (WT) Fremyella diplosiphon cells.  A photoreceptor mutant that lacks a response to red light vs. green light cues (i.e., an ?rcaE mutant strain) exhibits smaller, and perhaps more numerous, carboxysomes than WT. Although the ?rcaE mutant exhibits high intracellular levels of reactive oxygen species (ROS), the carboxysome phenotype is not reversed when cells are treated with an antioxidant to reduce ROS levels, suggesting the carboxysome defect is not caused by cellular redox conditions. In addition to carboxysomes, another type of inclusion body, i.e. phosphate bodies, were reduced in size in the ?rcaE mutant strain. Phosphate bodies were identified as dark spots in unstained cells visualized in whole-cell TEM analysis and confirmed using Energy Dispersive X-Ray (EDX) analysis. Carboxysomes and phosphate bodies have been suggested to physically interact. In Synechococcus elongatus PCC 7942, carboxysome number has also been shown to increase in response to increasing light intensity. Utilizing negative-stain TEM, we found that carboxysomes seem to cluster around phosphate bodies under high light, but that the number of phosphate bodies remains the same independent of light intensity. We utilized both traditional TEM and a novel application of negative staining techniques to survey carboxysome size, number, cellular location, and correlation with phosphate bodies under various light conditions.

 

 

 

*MS2 Bacteriophage VLPs Displaying Multiple HPV L2 Epitopes Show Protective Antibody Responses against three HPV Types
Lukai Zhai and Ebenezer Tumban
Department of Biological Sciences, Michigan Technological University, Houghton, MI
Human papillomaviruses (HPVs) are the causative agents of human neoplasias such as warts and cancers. Although three prophylactic vaccines have been approved to protect against HPV infections, these vaccines protect mostly against the HPV types included in the vaccines. The new Gardasil-9 protects against HPV types that cause 90% and 86% of HPV-associated cervical and penile cancers, respectively; however, the vaccine has not yet been recommended to recipients of first-generation HPV vaccines and as such, recipients of first generation HPV vaccines are protected only against HPV types that cause 70% and 53-79% of cervical and penile cancers, respectively. With this in consideration, there is still a need to develop a broadly protective HPV vaccine. Our group had previously shown that immunization with MS2 bacteriophage VLPs displaying a single conserved L2 epitope offers protection from vaginal infection with a broad range of diverse HPV types associated with cancer.  However, there was suboptimal protection against some HPV types. To enhance protection against these HPV types, we have developed bacteriophage virus-like particles (VLPs) displaying three epitopes from HPVs. Mice immunized with these VLPs elicit immune response against these HPV epitopes and show broad protective antibody response against three HPV pseudoviruses. Taken together, these results suggest that immunization with multiple L2 epitope MS2 VLPs is an excellent approach to broaden antibody responses against multiple HPV types.

 

Microbiological Quality and Antibiotic Resistance of Shrimp Retailed in Rochester, MI
Mamo, Andrina V, Walia, Dr. Satish
Department of Biological Sciences, Oakland University, Rochester, MI 48309
In 2009, the United States imported 589,670 metric tons of farmed shrimp worth more than $6 billion from Asia. Antibiotics are used in the shrimp ponds to stimulate growth and to retard the incidence of diseases caused by overcrowded, factory farm conditions. The indiscriminate use of these antibiotics may select bacteria resistant to multiple antibiotics, and such bacteria may transfer their antibiotic resistance determinants to pathogenic bacteria. The aim of the study was to investigate the prevalence and the antimicrobial resistance patterns of gram-negative bacteria isolated from shrimp retailed in Rochester, Michigan. In this preliminary study, a total of 16 shrimp samples were collected from retail grocery stores located in Rochester, MI. Out of 16 samples, 63% showed positive growth for Vibrio spp. as indicated by growth on TCBS agar. In this study eighty-three gram-negative bacterial isolates were tested for antibiotic susceptibility against six antibiotics (gentamicin [GEM], tetracycline [TET], ampicillin [AMP], ciprofloxacin [CIP], chloramphenicol [CHL], and ceftazidime [CEF]) by Kirby-Bauer method of disk diffusion. Bacteria showed high rates of resistance against ampicillin (58%), while a moderate rate was observed for gentamicin (35%) and intermediate resistance towards tetracycline (20%) and ciprofloxacin (20%). A low resistance rate was recorded against chloramphenicol (11%). Ceftazidime clavulanic acid resistance was found in 25% of the samples. Seven percent of the isolates were resistant to 4 antibiotics, displaying 3 different patterns. 22 out of 83 samples (27%) were resistant to a combination of 3 or more antibiotics. Only one isolate, AM 100, was resistant to 5 antibiotics. (GEM, TET, AMP, CHL, CEF). The heterotrophic bacterial count on Tryptic Soy Agar plates were ranging from 7.8 x 102 CFU/g to 4.18 x 103 CFU/g, meeting the microbiological quality standards for human consumption. The results of this study revealed the ubiquitous nature of Vibrio spp. in shrimp at retail. To reduce the potential risk of Vibrio infections due to handling or consumption of undercooked seafood, good manufacturing practice as well as safe handling and processing should be encouraged.

 

 

Assessing the Immunogenicity of Potential Zika virus B cell Epitopes on Phage Virus-like Particles
Rupsa Basu, Alice Contreras, Lukai Zhai, and Ebenezer Tumban
Department of Biological Sciences, Michigan Technological University, Houghton, Michigan 49931
Zika virus (ZIKV) is an arthropod-borne flavivirus that is transmitted predominantly through mosquitoes.  Within the last decade, ZIKV infection has spread around the world especially into Latina America. ZIKV infection is associated with guillain-barré syndrome, microcephaly, and miscarriage. Currently, there are no drugs or vaccines to protect against ZIKV infections. Moreover, there is no information on epitopes that could be targeted for peptide vaccine design.  Previous bioinformatics studies had identified B-cell epitopes on ZIKV envelope proteins. However, the immunogenicity of these epitopes has never been assessed. In order to assess the immunogenicity of these epitopes and their ability to neutralize ZIKV infection, we displayed the epitopes on highly immunogenic virus-like particles (VLPs) platforms derived from MS2 and Q? phages. We assessed, in mice, the immunogenicity of the ZIKV peptides displayed on VLPs as well as the immunogenicity of a ZIKV envelope protein. Immunizations with epitopes representing amino acid 294-315, 421-437 and 377-388 displayed on phage VLPs or immunization with ZIKV envelope protein elicited anti-ZIKV antibody responses. Our preliminary data show that these anti-ZIKV antibodies can neutralize, albeit low titers, ZIKV strain MR-766. This suggests that these epitopes are immunogenic ZIKV epitopes.

 

 
Poster Presentations
 

# Exopolysaccharide (EPS)-producing bacteria for Heavy Metal Removal in Aqueous Solution
Adam Oest, Mitchell Fenner, Alysia Sanchez, Dustin Overfield, Ali Alsaffar and Sonia M. Tiquia-Arashiro
Department of Natural Sciences, University of Michigan-Dearborn, Dearborn, MI 48128

Abstract
Microorganisms can remove metals from the environment by passive adsorption of metals on the charged macromolecules of the cell envelope, and can be considered promising as chelating agents for the removal of positively-charged heavy metal ions from water solutions.  In this study, four EPS-producing strains from the St. Clair River were tested for their ability to remove 8 different metals (As, Pb, Cu, Mn, Zn, Cd, Cr, Ni) in single- and multiple-metal solutions.  Dialysis assays were carried out with the strains in aqueous solution containing 10 mg/L solution of individual metals and the pH of the metal solutions was maintained at 4.5-5. Samples were withdrawn at different time intervals and metal removal was determined using an atomic absorption spectrometer. In the single-metal solutions, the strains showed metal uptake (0.47-319 mg/g dry mass after 5 hours), with the exception of chromium and arsenic. The strains were poorer at removing metals from the multiple-metal solutions. With nearly every metal showing less removal (0.11-297 mg/g dry mass) than the single-metal tests.  In multi-metal systems, the binding process was either noninteractive, synergistic or competitive between metal ions for different strains. Strains R3 and R19 were better at removing metals (0.47-319 mg/g dry mass) compared to L2 and L30 (0.83 to107.29 mg/g dry mass) with highest metal removal found on R19 which is likely due to the R isolates producing more EPS.  This research shows the potential in using native sediment microbes for building processes aimed to recover valuable metals from metal-contaminated wastewaters.

 

 

 

Determination of Antibiotic Minimum Inhibitory Concentrations (MIC) of Bacterial Strains Extracted From Saint Clair River Sediments
Aleksa Fortuna and Sonia M. Tiquia-Arashiro
Department of Natural Sciences, University of Michigan-Dearborn, Dearborn MI 48128

Abstract
Aquatic environments have been highlighted an important factor in the dissemination of antibiotic resistance genes (ARGs).  ARGs are ubiquitous in nature and elevated numbers are found in aquatic environments.  The source of this increase is due to routine discharge of antibiotics and resistance genes via wastewater or runoff from livestock facilities and agriculture. In this study, the susceptibility of 17 R2A and 3 LB isolates from Saint Clair River was examined against six antibiotics using MIC Etest strips. The strains were inoculated on Mueller-Hinton agar with the antibiotic strip fixed on the agar, and incubated at room temperature for 24 hours. The R2A isolates were generally more sensitive to the antibiotics than the LB isolates, displaying MIC readings as low as 1-2 µg/ml for chloramphenicol, erythromycin, kanamycin, streptomycin and tetracycline.  This is possibly due to the fact that R2A isolates are slow-growing strains and may be less efficient in activating resistant mechanisms than LB isolates. All isolates were resistant to benzylpenicillin (MIC >256 µg/ml), due to widespread discharge of ARGs coding for ?-lactamase in the environment. Of the six antibiotics, kanamycin and streptomycin were the most inhibitory to both isolates with MICs ranging from 0.25-32 µg/ml.  Overall, the tolerance displayed by the isolates against the antibiotics showed the importance the environment plays in the dissemination of antibiotic resistance. Further studies are needed to elucidate the levels of antibiotic contamination in the sediments to enable better risk analyses and facilitate measures for preventing dissemination and development of antibiotic resistance.

 

 

 

# 16S rRNA Sequence Analysis of Pb-Resistant Isolates and Their Potential as Bioflocculants
Alysia Sanchez, Wentao Xu, Drashti Patel and Sonia Tiquia-Arashiro
Department of Natural Sciences, University of Michigan-Dearborn, Dearborn, MI 48128
Abstract
Lead (Pb) is a major pollutant and highly toxic to humans, animals, plants, and microbes.  Biological approaches based on metal-resistant microorganisms have received a great deal of attention as an alternative remediation process. This study aims to identify 92 Pb-resistant bacterial strains previously isolated from Saint Clair River sediments using 16S rRNA gene sequencing and to examine their bioflocculation potential. Forty-five strains were enriched in LB medium while the remaining Forty-seven were enriched in R2A medium. Of the 92 strains, 80 were successfully sequenced and identified. The LB isolates closely identified with the generaEnterococcus, Alcaligenes, Providencia, and Serratia, while the R2A isolates closely identified with Raoultella, Klebsiella, Burkholderiaand Pantoea; all of which had at least 95% similarity to 16S rRNA sequences of known strains in GenBank.  The phylogenetic tree showed that some LB isolates (LB24 and LB45) were closely related to the R2A isolates (93-95%) while the majority was more distantly related (60-90%). Nucleotide similarity between sequences varied from 64%-95%. To test the strains’ potential to scavenge metals in aqueous solution, a bioflocculation assay was performed. The R2A isolates were better able to sequester heavy metals compared to LB isolates. Only 3 LB isolates showed flocculation ability >40% whereas 14 R2A isolates displayed >40% flocculation. Of the identified isolates, Raoultella orinthinolytica showed the highest flocculating activity (58.2%). Flocculation of Raoultella spp. ranged from 6.8%-58.2% and seemed to vary between species. Due it’s the high experimental flocculation activity, Raoultella orinthinolytica could be considered for use in remediation of metal-contaminated wastewater.

 

 

 

Anaerobic Degradation Potential of Toxic Hydrocarbons by Bacterial Strains Isolated from Saint Clair River Sediments
Alysia Sanchez, Drashti Patel and Sonia Tiquia-Arashiro
Abstract
Contamination of industrially-derived organic chemicals in Saint Clair River is well documented. Conventional decontamination methods involve physio-chemical processes that do not result in complete destruction of contaminants. Microbes have been known to metabolize toxic organic compounds and can be exploited for treatment of contaminated sediments and water.  This study aims to discover the ability of 20 bacterial strains previously isolated from Saint Clair sediments to metabolize 34 toxic organic compounds under anaerobic conditions. Isolates were inoculated in triplicates on MT2 Biolog plates containing the hydrocarbons as the sole carbon source, and subsequently incubated at room temperature for 72h under anaerobic conditions. The extent of degradation varied, and was inferred by growth as measured by difference in absorbance (595 nm) at time 0 and after 72h of incubation. All isolates were able to grow, utilizing between 19 and 34 of the hydrocarbons.  Growth rates ranged up to 0.03 h-1. L2 was the most successful at utilizing the hydrocarbons; it degraded all 34 hydrocarbons with growth rates in the highest range observed (>0.02 h-1). R6 was the least successful degrader, only managing to degrade 19 compounds to a lesser extent (14 of 19 isolates had a growth rate <0.01h-1). In general, LB isolates performed better than R2A isolates; they degraded a greater number of toxic compounds to a higher extent. In conjunction with metal removal studies, experiments on these isolates may prove to be candidates not only for remediation of metals but also for remediation of toxic hydrocarbons in water and sediments.

 

 

 

Spatial and temporal variation in enzymatic activities of St. Claire river and lake sediments via API ZYM method
Drashti Patel, Renee Gismondi, Mitchell Fenner and Sonia M. Tiquia-Arashiro
Department of Natural Sciences, University of Michigan-Dearborn
4901 Evergreen Rd, Dearborn MI 48128
Abstract
Spatial and temporal variations in enzymatic activities of lake and river sediments were determined using API ZYM assay. API ZYM is a semi-quantitative micromethod designed to determine activities of 19 different enzymes.  Three sites were selected on St Claire River (SC1, SC2, and SC3) and three sites on Lake St. Clair (LC1, LC2, LC3). A 1:10 sediment : water extract was prepared; and 30 ml of the extract was dispensed in each of the 20 cupules.  Each of the 19 cupules contained substrate for the 19 enzymes and and one cupule served as a control. Once incubated one drop of ZYM A and ZYM B were added to the cupules and results were collected after 10 mins. Results were analyzed with a variety of tests. T-test was used to compare activities between river and lake. There was no significant distinction between the river and the lake except for lipase and leucine aminopeptidase, of which St. Claire river sediments had more enzymatic activities. ANOVA test was used to compare lake and river site sediments for enzymatic acitvities. Leucine aminopeptidase and valine aminopeptidase activities were significantly among the three sites. SC3 had highest activities of leucine, valine aminopeptidase and ?-glucuronidase. Leucine aminopeptidase activity was significantly different in each of the three lake sites and with the highest activity in LC3. The research results give an important insight on different contaminants lake and river water are being exposed to and how these contaminants influence the diversity of the microbial community.

 

 

 

Association of Metal Tolerance with Multiple Antibiotic Resistance of Bacteria Isolated from Saint Clair River Sediments
Dustin Overfield, Mitchell Fenner, Adam Oest, Alysia Sanchez and Sonia Tiquia-Arashiro
Department of Natural Sciences, University of Michigan-Dearborn, 4901 Evergreen Rd, Dearborn, MI 48128
Abstract
Industrialization has increased heavy metal exposure in ecosystems.  Widespread use of antibiotics is also a concern due to increased development of antibiotic resistance among bacteria.  Saint Clair River is one ecosystem known to have widespread metal contamination.  In the present study, 20 bacterial strains (17 R2A and 3 LB strains) isolated from Saint Clair sediments were examined to assess the association of metal tolerance with multiple antibiotic resistance.  Minimum inhibitory concentrations (MIC) for eight metals and zone of inhibition assays for 8 antibiotics were determined.  All isolates showed resistance to penicillin and novobiocin.  Most were resistant to erythromycin (95%) and neomycin (75%), and susceptible to chloramphenicol (55%) and streptomycin (80%).  The MIC of the metals varied between 11 and 2831 mg/L, with highest values found for Mn, Pb, and Zn.  LB isolates were more resistant to metals (106-2831 mg/L) than the R2A isolates (11-1442 mg/L).  R2A isolates were more resistant to antibiotics than the metals, while the LB isolates were highly resistant to both.  Resistances were more prevalent in these fastidious strains because they require more growth factors making them able to more readily activate resistance mechanisms.  Tolerance to As2+, Cd2+, Cu2+, Pb2+, Mn2+, Ni2+ and Zn 2+ and positive correlations with multiple antibiotic resistance were expressed by most isolates.  Further studies will focus on examining interactions of selection mechanisms involving both chromosomal and plasmid genetic elements to account for the frequent incidence of metal tolerance and multiple antibiotic resistance among the bacterial isolates from Saint Clair River sediments.

 

 

 

Aerobic Biodegradation of Toxic Hydrocarbons by Bacterial Isolates using Biolog MT2 Microplate Assay
Mitchell Fenner, Dustin Overfield, Adam Oest, and Sonia M. Tiquia-Arashiro
Department of Natural Sciences, University of Michigan-Dearborn, Dearborn, MI 48128
Abstract
Microorganisms have been known to metabolize many organic contaminants as sources of carbon and energy, converting them into products such as CO2 and water. These biotransformations can be exploited for treatment of contaminated soils and water. In this study, the ability of 20 bacterial strains to utilize 34 toxic organic compounds as their sole carbon sources was assessed under aerobic conditions using the Biolog MT2 assay. The strains were isolated from St. Clair River and have known resistance to various metals. The absorbance (595 nm) over time was determined using a plate reader. This absorbance data for the strains in each hydrocarbon was compared against the absorbance of strains in enrichment media to determine the relative growth efficiency.  Increase in absorbance between time 0 and 72 hours was considered positive growth. All 20 strains grew in dimethyl sulfoxide, and chloroform. Most strains grew in carbon tetrachloride, tetrachloroethylene, methanol, 2,4-D and aminonaphthalene.  Interestingly, the insecticide DDT showed an initial decrease in absorbance followed by a steady increase in absorbance from 24 hours to 72 hours.  Almost no strains grew in quinoline, naphthalene or 1-4 dichlorobenzene.  Isolates R29 and R30 were the most resilient and degraded 21 and 19 compounds, respectively. The number of species determined to have grown in each hydrocarbon may be an overestimate, given how variable the absorbance measurements were from 0-72 hours. Even so, these results are important as they show the resiliency of these strains as well as their potential use in bioremediation of toxic organic compounds.

 

 

# The Antimicrobial Effect of Garlic and Garlic Supplements and the Characterization of Contaminants in the Supplements
Avery Wagner and Jennifer Hess, Aquinas College
Allicin (S-Prop-2-en-1-yl prop-2-ene-1-sulfinothioate) is released by crushed garlic, and inhibits the growth of bacteria. Garlic was tested as an antimicrobial against a variety of bacteria, alongside cranberry juice, honey, and garlic and honey combined. Garlic was found to be the most effective antimicrobial with a 1:64 minimum inhibitory concentration.  Garlic nutrient supplements were also tested to examine both their antimicrobial abilities and to see if they contained contaminants.  Our group had previously found one brand to be contaminated four years ago, so both a capsule from that pack and a capsule from a newly purchased pack of the same brand were tested. Two of four brands had contaminants, so these contaminants were tested with various selective and differential media tests before molecular biology methods were employed. PCR reactions were performed and sequencing information was obtained, and the Brand #1 old capsule’s contaminant was B. subtilis, Brand #1 new was B. thuringiensis, and Brand #2 was also B. subtilis.

 

 

 

Anti-Staphylococcal Activity of 3,4 Dihydroquinazolines
Martha A. Hutchens, Christina L. Magyar, Tyler J. Wall, R. Adam Mosey
Affiliation of all authors: Lake Superior State University, Sault Ste. Marie, MI, 49783
Abstract
Methicillin resistant Staphylococcus aureus (MRSA) is a widespread opportunistic pathogen that is responsible for many serious infections in healthcare settings and in the wider community.  There are a limited number of drugs that are effective against MRSA, and the threat that the bacterium will develop resistance to these drugs fuels the constant search for new ones.  We have developed a new method for synthesizing a class of organic compounds, 3,4 dihydroquinazolines, that previously were very difficult to produce.  Nineteen new 3,4 dihydroquinazolines were screened for activity against MRSA.  Two of these compounds showed MIC90 values approaching the range of clinical utility.  This is a promising beginning to the discovery of new anti-staphylococcal drugs; with future structure-activity studies we will be able to design and synthesize compounds that have even greater efficacy.

 

 

 

Tropism of Pythium insidiosum Zoospores Towards
Hyaluronic Acid and Collagen
Keith Barker
10/3/2016
Department of Biology
Lake Superior State University
Sault Ste. Marie, MI
Pythium insidiosum is a fungus-like water mold that can cause an infection known as pythiosis in humans. Pythiosis occurs when an open wound is exposed to water contaminated with P. insidiosum’s motile zoospores. This project explores if the water borne zoospore is attracted to hyaluronic acid or collagen. To do this, a sample of P. insidiosum was placed in a petri dish with either a slide coated in hyaluronic acid or collagen on one end and either a blank slide or a slide with grass on it  at the other end. The grass is used because it is known to attract zoospores. In this assay, the zoospores were allowed to swim freely for 24 hours before the slides were removed and observed for the number of germ tubes that formed from the zoospores. It was found that more germ tubes formed on the hyaluronic acid slides than the blank slides and that many more germ tubes formed on the grass slides than all the other slides. A possible explanation for hyaluronic acid having more germ tubes is that hyaluronic acid acts as a chemical signaler for the movement of P. insidiosum zoospores.

 

 

 

Isolation of Biocide Resistant Organisms from Hydraulic Fracturing Flow-back Water
Authors:
Baldwin, Andrew L. /Dr.  Techtmann, Stephen.
Michigan Technological University.

Abstract:
Hydraulic fracturing (HF) is the process of injecting pressurized water and chemicals into a subterraneous rock. This is used to create fractures that release natural gas or oil. The fluids used in HF contain biocides to control the growth of subsurface microbes. Flow-back water is the pressurized fluids that have been injected into the well and returned to the surface. Understanding how the HF fluids interact with the subsurface community is important to understanding the effectiveness of the chemicals and understanding how microbes may become resistant to the biocides.
The goal of this project is to investigate the diversity of biocide resistant strains present in flow-back water to both gain a better understanding of the subsurface community and to evaluate the effectiveness of biocides. 
Samples of flow-back water taken from a handful of wells in North West Texas. Flow-back water was exposed to 100ppm concentration of Glutaraldehyde, DBNPA and a combination of both, and plated into Marine and Hyper-Saline (HS) broth at 30C. Then High Temperature Marine (HTM) and High Temperature Hyper-Saline (HTHS) broth at 70C.
A total of 39 isolates were obtained and 16S sequenced. Eighteen unique species were identified from the 39 isolates. 6 species were found to be resistant to Glutaraldehyde alone, 3 species to DBNPA alone, and another 3 to a combination of both. Another 6 species were found without biocides treatments. The major taxa include soil bacteria like Bacillus and Halomonas. The isolates also include marine taxa like Marinobacter and Idiomarina. The extremophile taxa include Chromohalobacter and Geobacillus.
The 16S results show a variety of viable bacteria present in the flow-back water. These species have all survived exposure to biocides in-situ and/or ex-situ, indicating some level of resistance to these biocides. Including resistance to both common biocides, even when used together. The taxa closely related to marine species, which due to the geographic isolation, is striking. Further research with whole genome sequences is needed for comparison of our isolates to others in the same species and taxa.

 

 
   

Deciphering the Language of Symbiotic Communication
1,2Price, Paul A., 2Dillon, Brett A., 2Tanner, Houston R., 2and Griffitts, Joel S.
1*Eastern Michigan University, Ypsilanti, MI 48197
2Brigham Young University, Provo, UT 84602


Abstract:  Many of the symbiotic partnerships that have shaped our world have been continuously evolving for millions of years. In the legume-rhizobium symbiosis, plants have evolved specialized organs known as root nodules to accommodate their nitrogen-fixing bacterial symbionts. Nodule development is orchestrated as the plant and microbe exchange a series of chemical signals, a sort of symbiotic dialogue, that drive the expansion of root cortical tissues and the differentiation of internalized rhizobia into nitrogen-fixing cells called bacteroids. Bacteroid development appears to be driven by an array of plant-derived nodule-specific cysteine rich (NCR) antimicrobial peptides that result in an arrest in bacterial cell division, genomic endoreduplication, a 5- to 10-fold increase in bacteroid size, and a gentle membrane permeabilization. The interplay between plant-produced NCR peptides and bacterial response mechanisms dictates the productivity of a symbiosis. In the Sinorhizobium meliloti and Medicago truncatula symbiosis, for example, we often observe legume–rhizobium pairs that produce symbiotic root nodules but fail to fix nitrogen, providing a unique system for examining the mechanisms involved in the formation of productive nodules. In one such failed pairing, we have identified a rhizobia-encoded metallopeptidase gene, designated hrrP, that is directly responsible for blocking nitrogen fixation through the degradation of a range of plant-encoded NCR peptides. Interestingly, the ability of hrrP to suppress nitrogen fixation is conditioned upon the genotypes of both the host plant and the hrrP-expressing rhizobial strain. Our work is helping define some of the first “words” in a complex symbiotic communication system involving a varied ensemble of host-derived signaling peptides and bacterial modifier enzymes capable of adjusting the signaling potency of these peptides. hrrP homologs encoded on a variety of Sinorhizobium accessory plasmids provide further support for this model, as these homologs display an array of phenotypic outcomes that are dependent on their cellular localization and proteolytic capabilities.

 
 

# Microbial Community Dynamics during Lake Ice Formation

Timothy M Butler, Anna-Catherina Wilhelm, Andrew Baldwin, Amber Dwyer, Stephen Techtmann

Fresh water is such as the great Lakes of the Midwest supply water to millions of people. Many of these freshwater environments experience dramatic environmental changes on a seasonal basis, with some aquatic environments remaining ice covered for months at a time.  Little is known about changes in microbial community structure and abundance during times of persistent ice cover.  The goal of this study is to characterize temporal dynamics of microbial communities during ice formation.  To accomplish this goal, samples were collected in triplicate five days per week, every week from surface water in the Keweenaw Waterway between November and April.  Temperature, pH, Dissolved Oxygen, and Salinity were measured at the time of sampling.  Samples were collected for analysis of microbial abundance, and microbial community structure.  Abundance of bacteria, and archaea were determined using qPCR.  Microbial community structure was determined using high-throughput sequencing of the 16S rRNA gene.  The microbial community at initial time points was dominated by members of the Actinobacteria (24% of recovered reads) and Acidomicrobiales (12%). We expect the microbial community to be quite stable from day-to-day, but undergo dramatic shifts in microbial abundance and diversity during the transitions into and out of ice cover.  The microbial community during the peak ice cover was dominated by Actinobacteria and Acidomicrobiales. The diversity of the microbial community was expected to decrease, however, what was found was no increase or decrease in diversity. From this data and further testing a more detailed picture of the biogeochemical cycling specifically nitrogen cycling and methane cycling will become apparent. The results of this on-going study will help to elucidate how microbial abundance and diversity change over drastic seasonal transition and how ice cover affects microbial abundance and diversity.  In the long term we hope to link temporal changes in environmental parameters with functional differences to better understand how seasonal variations affects biogeochemical cycling.

 

Prediction Driven Functional Annotation of Hypothetical Proteins in the Major Facilitator Superfamily of S. aureus NCTC 8325
Marklevitz, Jessica*, 1, UG
Harris, Laura K.1, 2
Presenter; 1 Davenport University, Lansing, MI, 48933; UG Undergraduate Researcher; 2 Faculty
            Antibiotic resistance Staphylococcus aureus strains cause several life threatening infections.  New drug treatment options are needed, but are slow to develop because 50% of the S. aureus genome is hypothetical.  The goal of this is to aid in the annotation of the S. aureus NCTC 8325 genome by identifying hypothetical proteins related to the Major Facilitator Superfamily (MFS).  The MFS is a broad protein group with members involved in drug efflux mechanisms causing resistance.  To do this, sequences for three MFS proteins with x-ray crystal structures in E. coli were PSI-BLASTed against the S. aureus NCTC 8325 genome to identify homologs.  Eleven identified hypothetical protein homologs underwent BLASTP against the non-redundant NCBI database to fit homologs specific to each hypothetical protein.  ExPASy characterized the physiochemical features, CDD-BLAST and Pfam identified domains, and the SOSUI server defined transmembrane helices of each hypothetical protein.  Based on size (300 – 700 amino acids), number of transmembrane helices (>7), CD06174 and MFS domains in CDD-BLAST and Pfam, respectively, and close relation to well-defined homologs, SAOUHSC_00058, SAOUHSC_00078, SAOUHSC_00952, SAOUHSC_02435, SAOUHSC_02752, and ABD31642.1 are members of the MFS.  Further multiple-alignment and phylogeny analyses show SAOUHSC_00058 to be a quinolone resistance protein (NorB), SAOUHSC_00058 a siderophore biosynthesis protein (SbnD), SAOUHSC_00952 a glycolipid permease (LtaA), SAOUHSC_02435 a macrolide MFS transporter, SAOUHSC_02752 a chloramphenicol resistance (DHA1), and ABD31642.1 is a Bcr/CflA family drug resistance efflux transporter.  These findings provide better annotation for the existing genome, and identify proteins related to antibiotic resistance in S. aureus NCTC 8325.

 
 

Using Codon Usage to Distinguish Among Lactobacilli Species
School, Kuana*, 1, UG
Harris, Laura K.1, 2
Presenter; 1 Davenport University, Lansing, MI, 48933; UG Undergraduate Researcher; 2 Faculty

When the genome for strain 30SC was released, it was classified as a Lactobacillus acidophilus.  Recently, the National Center for Biotechnology Information (NCBI) changed the classification of 30SC to a L. amylovorous.  Proper categorization of 30SC is important due to its known probiotic properties and strong survival ability in the digestive tract.  Here, CodonW calculated codon usage properties for 18 DNA (cluster of orthologous groups of proteins category L) and 34 membrane (category S) proteins from 30SC that had homologs identified by the protein-protein Basic Local Alignment Search Tool (BLASTP) with a 70% or greater identity in both reference strains L. amylovorous GRL1118 and L. acidophilus NCFM.  This study showed statistically significant decreases in the Frequency of Optimal Codon Usage (FOP), Codon Bias Index (CBI), and %(G+C)3, all which measure aspects of an organism’s codon usage preference, in NCFM compared to 30SC and GRL1118.  However, no change was observed between 30SC and GRL1118.  There were no differences in Effective Number of Codons (Nc) or Codon Adaptive Index (CAI) for any organism.  The differences in FOP, CBI, and %(G+C)3 might be due to changes in amino acids preferences, as seen by statistically significant changes in aromaticity for both proteins groups and general average hydropathicity (GRAVY) for membrane proteins for NCFM but not 30SC or GRL1118.  These results support NCBI’s re-classification of 30SC to a L. amylovorous from L. acidophilus. Further work includes applying this approach to other categories of proteins and expanding the analysis to include relative synonymous codon usage (RSCU).

 
 

Characterization of Hypothetical Proteins with High mRNA Expression in Methicillin Resistant S. aureus
Justice, Sean*, 1, UG
Harris, Laura K.1, 2
Presenter; 1 Davenport University, Lansing, MI, 48933; UG Undergraduate Researcher; 2 Faculty

             
Community acquired Methicillin resistant Staphylococcus aureus (MRSA) is a growing clinical concern.  Despite this concern, close to half of MRSA’s proteins are annotated as hypothetical.  To examine hypothetical proteins that may contribute to antibiotic resistance, mRNA expression data was used to identify 16 hypothetical proteins expressed more in MRSA over an antibiotic sensitive strain (p<0.05).  If the hypothetical protein was >100 amino acids, it underwent physiochemical characterization to predict the protein’s stability.  The remaining proteins were: SACOL0468, SACOL0350, SACOL1531, SACOL1859, SACOL1178, SACOL0323, and SACOL2451.  These proteins underwent BLASTP, Pfam, and STRING to identify homologs, domains, and ligands, respectively. Protein models were generated by the (PS)2 algorithm. From these results, SACOL0468 and SACOL1178 may increase pathogenicity.  SACOL0350 and SACOL0323 may be associated with phage proteins.  More data is needed on protein families in general for further in silico characterization of the remaining three proteins as the data SACOL1531 generated is inconsistent among programs and most algorithms returned no findings for SACOL1859 and SACOL2481.  These three proteins are good candidates for experimental structure determination using nuclear magnetic resonance to increase what is known for the characterization of other hypothetical proteins. Further, future work will use these methods to characterize hypothetical proteins expressed more in sensitive strains than in MRSA.
   
 

Biofilm Inhibition and Destruction Using Newly Synthesized Compounds
Kory R. Korcal, Christina L. Magyar, Tyler J. Wall, R. Adam Mosey, Martha A. Hutchens
Affiliation of all authors: Lake Superior State University, Sault Ste. Marie, MI, 49783
Abstract
Methicillin resistant Stapylococcus aureus (MRSA) is a dangerous, infectious pathogen that is capable of organizing into biofilms. A biofilm is a biological system; a polysaccharide matrix with embedded bacteria. Having a drug that impairs the ability of S. aureus to form or maintain biofilms could be a powerful weapon against MRSA infections. We have developed a new, easier method for synthesizing a class of organic compounds, 3,4 dihydroquinazolines. Twenty-six of these new compounds were screened against the biofilm that MRSA produces to evaluate their ability to (1) prevent S. aureus from forming biofilms in the first place (biofilm inhibition) and (2) destroy S. aureus in a pre-existing biofilm (biofilm destruction). Out of the 26 compounds screened, six compounds showed significant biofilm destruction and/or inhibition properties. Further studies on two compounds showed minimum biofilm inhibition concentration values approaching the range of clinical utility. This is a promising beginning in the search for new drugs to treat MRSA and other bacterial infections maintained by biofilms.

 
 
 

Thermostable Mannan-Binding Proteins Identified by Functional Genomics
Gunther, Lindsay A; Masood, Qirat; and Sara Blumer-Schuette
Oakland University, Rochester, MI 48307
Caldicellulosiruptor bescii is a cellulolytic thermophile, effective at hydrolyzing and fermenting plant biomass (Yang et al., 2009). Polysaccharides associate with the non-catalytic subunits, known as substrate-binding proteins (SBP), of an ATP-binding cassette transporter and are translocated into the cell for fermentation (Davidson, 2006). SBPs have been annotated in the closely related genus member Caldicellulosiruptor saccharolyticus;initiating functional genomic studies into the specificity of polysaccharide binding affinity (Vanfossen et. al, 2009). BLAST revealed the putative glucomannan-binding SBPs Csac_0297 and Csac_2493 showed homology to Athe_2554 and Athe_2310 of C. bescii (Blumer-Schuette, unpublished).
Both full-length and truncated Athe_2310 and Athe_2554 were ligated into pET28b+. Full-length transcripts of both genes showed minimal soluble protein production, indicating that the hydrophobic transmembrane domains may create inclusion bodies.
Our initial aim is to determine the binding affinities of the truncated transcripts, Athe_2310TR and Athe_2554TR. Differential Scanning Fluorimetry (DSF) and Ultraviolet Difference Spectroscopy (UV-Diff) were used to determine protein-carbohydrate interactions. Melting temperature (Tm) values were calculated from DSF data. UV-Diff evaluates the absorbance of proteins incubated with increasing concentrations of sugar ligands. The peaks and troughs visible in the difference assays correlate to aromatic ring involvement.
An increase in Tm was seen when various carbohydrates were present for both proteins; this suggests a possible wide substrate affinity. Structural differences in the binding pocket could allow for interaction between longer polysaccharides with various glycosidic bonds. We hypothesize that glucomannan-binding SBPs in C. bescii can be altered to confer novel affinities for eukaryotic N-linked glycans. The purpose of creating SBPs that bind novel carbohydrate ligands is to investigate the directed evolution of C. bescii and its orthologs by uncovering the differences in substrate choice as a means of selection.

References:

Abbott DW, Boraston AB. Quantitative approaches to the analysis of carbohydrate-binding module function. Methods Enzymol. 2012 Dec;510:211-31.
Davidson AL, Chen J. ATP-binding cassette transporters in bacteria. Annu Rev Biochem. 2004;73:241-68.

 
   
 
     
     
     
     
     
     
 
 
     
     
 
 
 
 
     
 

 

 



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