Mutagenesis of Zebrafish (Danio rerio) nod1 and nod2

Jamaal Tarpeh, 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. Ligand-binding by these receptors plays a critical role in activating downstream signaling proteins that upregulate the production of inflammatory molecules critical in protecting against disease. While it has been established that Nod receptors play an important role in innate immunity, this role has not been fully characterized. Characterizing the role of Nod receptors in the etiology of intestinal immune disorders is made challenging due to the intricate relationship between the commensal microbiota and the intestinal innate immune system. The zebrafish (Danio rerio) embryo is an amenable model system for studying the interaction of the immune system with the intestinal microbiota and the concurrent development of the immune system and colonization of the zebrafish embryo with the microbiota. The transparency and ex vivo development of the embryo is highly beneficial in studying real-time biological processes (Kanther et al. 2010). Since a role for Nod receptors has been established in intestinal immunity, we will use the zebrafish model system to further characterize the role of these receptors in the intestinal innate immune system. To do this, we plan to create dominant negative Nod receptors and express the receptors in zebrafish intestinal epithelial cells to block the function of the endogenous proteins.

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Symbiotic Human Gut Bacteria with Reciprocal Metabolic Priorities for Host Mucosal Glycans

Nicholas A. Pudlo1, Karthik Urs1, Supriya Suresh Kumar1, J. Bruce German2, David A. Mills2 and Eric C. Martens1, 1 University of Michigan Medical School, Ann Arbor, MI 48109, 2 University of California, Davis, Davis, CA 95616

Many symbiotic gut bacteria possess the ability to degrade multiple polysaccharides and glycans, thereby providing nutritional advantages to their hosts. Like microorganisms adapted to other complex nutrient environments, some gut symbionts give different metabolic priorities to substrates present in mixtures. We investigated the responses of B. thetaiotaomicron (Bt), a common human commensal that metabolizes over a dozen different groups of glycans, including the O-linked structures that are abundant in secreted mucin. Experiments in which mucin glycans were presented simultaneously with other carbohydrates, show that degradation of these endogenous host carbohydrates is consistently repressed in the presence of alternative substrates, even by Bt previously acclimated to growth in pure mucin. Experiments with media containing systematically varied carbohydrate cues and genetic mutants reveal that transcriptional repression of genes involved in mucin glycan metabolism is imposed by simple sugars released from polysaccharides and, in one case that was tested, is mediated through a non-coding region of the corresponding mRNA in a transcript-autonomous fashion. Repression of mucin glycan-responsive gene clusters in two other human gut bacteria, Bacteroides massiliensis and Bacteroides fragilis, exhibited variable and sometimes reciprocal responses relative to Bt, revealing that symbionts vary in their preference for mucin glycans and that these differences are manifest at the level of controlling individual gene clusters. Our results reveal that sensing and metabolic triaging of glycans is a complex process that varies among species, underscoring the ideas that such phenomena are likely to be a hidden driver of microbiota community dynamics and may dictate which microorganisms preferentially commit to various niches in a constantly changing nutritional environment.

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RDP: Databases and Tools for rRNA Analysis
 
Benli Chai, Qiong Wang1, Jordan A. Fish1, Yanni Sun2, C. Titus Brown2, James M. Tiedje1 and James R. Cole1, 1Center for Microbial Ecology, 2Department Computer Science and Engineering, Michigan State University, East Lansing, MI  48824-4320

The Ribosomal Database Project (RDP) offers aligned and annotated rRNA sequence data and analysis services to the research community through http://rdp.cme.msu.edu. RDP recently released new alignments of bacterial and archaeal 16S rRNA gene sequence alignments and a fungal 28S gene sequence alignment using the latest Infernal 1.1 aligner with specially-tuned covariance models (CMs). As part of RDP’s efforts to support the fungal research community following the release of the RDP Fungal 28S Classifier (Liu et al. 2011. link), most  RDP tools, including the RDP Hierarchy Browser, Sequence Match, Probe Match, and RDPipeline, have been updated to work with the new fungal 28S sequences.
 
The RDPipeline expands upon our existing high-throughput tool offerings and is designed to accommodate the latest benchtop high-throughput sequencing technologies. The RDPipeline integrates with researchers’ existing myRDP accounts for streamlined analysis job submission and monitoring.  The new RDPipeline includes both improved performance in optimizing back-end job load distribution and increased capacity for larger datasets. It also provides additional user-friendly features such as a “my jobs” page for each user to track the job status, download results, and retrieve process parameters for past analysis tasks submitted to RDPipeline. Other enhancements include optimized paired-end read assembly (Assembler). Tested on Illumina MiSeq paired-end data, this tool outperformed its peers in selectively filtering out error-containing sequence reads, and also better handles different types of paired-end overlaps. A new data validation mechanism implemented in RDPipeline provides feedback if incorrect data input is submitted before an analysis job starts running--a feature especially valuable for inexperienced users.
 
In addition to web-based services, RDP now distributes many of its process/analysis tools as stand-alone, open-source versions through https://github.com/rdpstaff. Tutorials are provided to guide researchers through the otherwise complex data processing steps in well-defined, task-oriented workflows with detailed instructions. RDP’s mission includes user support; email [email protected] or call +1(517) 432-4997.

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The Role of GST (Glutathione S-Transferases) Activity of Burkholderia cepacia and Serratia marcescens in Tetracycline Detoxification

Jennifer R Connors, Aparupa Sengupta, Rupali Datta, Susan Bagley, Michigan Technological University, Houghton, MI, 49931

Antibiotic accumulation in the water supply and soil from unregulated disposal has become a worldwide problem for human health and ecological disturbance that deserves serious attention. This accumulation is also contributing to the multi-antibiotic resistance of bacteria: a condition that is causing enhanced mortalities every passing year. Using the results from previous studies and literature reviews, the involvement of the antioxidant mechanism in tetracycline detoxification was established. Hence we hypothesized that the detoxification of tetracycline could be performed by B. cepacia and S. marcescens using a particular biochemical mechanism. The level of detoxification of tetracycline by these bacteria was measured through the interaction between the reactive oxygen species (ROS), glutathione (GSH), and glutathione S- transferases (GSTs) during the exponential phase of growth. GSTs catalyze the conjugation of reduced GSH to xenobiotic substrates. Protein was extracted from the bacteria, grown with tetracycline, and the level of GST was measured using a GST assay. The results produced were indicative of a connection between oxidative stress (yields ROS) and the isozymes (GSTs) responsible for the degradation of tetracycline. The tetracycline-tolerant bacteria B. cepacia and S. marcescens showed significantly higher activity of GSTs compared to the control. The results found suggest that the GSTs metabolic pathway is a possible method for tetracycline detoxification by vetiver plants and their associated TC- tolerant bacteria.

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Cyanobacterial Communities from Alvar Substrates on Drummond Island, Michigan

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

Despite its rarity, there is little research on the alvar habitat and in particular, associated microbial communities. Alvars are areas of calcerous 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. Alvar rock samples and soil crust samples were collected from two sites found on Drummond Island, Michigan. Samples were subject to microscopy to characterize the alvar material. Samples were enriched using liquid media with and without fixed nitrogen and cyanobacteria were identified using microscopy and 16S rRNA sequencing. Geochemical analysis of the alvar samples revealed that the two sites differ in the degree of weathering, but are geologically similar. Scanning electron microscopy with energy dispersive spectroscopy showed that both sites contain Mg making them both dolomite substrates. 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 revealing a diverse array of cyanobacterial species. Liquid enrichments primarily contained Leptolyngbya, Pleurocapsa, Microcoleus, Nostoc, Calothrix and Phormidium spp. Molecular analysis of unenriched samples found some of the same species along with Microsystis, Oscillatoria, and Spirulina spp. A BLASTn search of 16S rRNA sequences found that a number of the organisms detected had closest relatives from species found in Antarctic and Colorado Plateau environments. Overall, the results of microscopy and molecular detection revealed a diverse array of cyanobacteria likely play an important role in developing and stabilizing the delicate soils of the alvar.

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Metagenomic Analysis of Viral Communities Associated with Irrigation Water: A Potential Pre-Harvest Source of Fresh Produce Contamination

Samantha L. Wengert, Aw, Tiong G. Rose, Joan B. , Michigan State University, East Lansing, MI 48824

Foodborne outbreaks associated with fresh produce have become increasingly relevant in the United States. Despite the increased importance of fresh produce as a vehicle for human pathogens, there is currently limited knowledge about where in the supply chain contamination occurs. Potential pre-harvest sources of contamination include irrigation water, soil, manure and human handling.  Viruses are small biological nanoparticles and the host specificity of viruses suggests that they could be a promising library-independent tool to determine the major microbial sources in food systems. The objective of this study was to generate a comprehensive view of the virome in irrigation water and lettuce using an established method of virus recovery and metagenomics approach. Viruses were concentrated from 100L of irrigation water in Yuma AZ, using a low cost disposable hollow fiber ultrafiltration system.  In addition, Colilert and Enterolert tests were performed to detect the presence of indicator bacteria Escherichia coli (E.coli) and enterococci accordingly.  Virus recovery from romaine and iceberg lettuce included an elution step with Tris-glycine buffer, shaking for 20 min, followed by concentration of the virus particles by polyethylene glycol (PEG) and centrifugation.  Samples were then passed through 0.22-µm filters and treated with DNase prior to viral nucleic acid extraction. Sequencing was performed using Illumina HiSeq technology and results were analyzed using BLASTx against the NCBI Viral Reference Sequence (RefSeq) database and MEGAN software. The concentrations of E.coli and enterococci were low and ranged from 2.0 to 4.1 and 3.1 to 8.4 MPN/100 ml with a geometric mean of 3.3 and 4.9 MPN/100ml, respectively. However, a wide variety of viruses were recovered from the three irrigation water samples analyzed. Caudovirales and Microviradae bacteriophage families were the most commonly recovered viruses. In addition, the percentage of known contigs was far smaller (7%) in comparison to those without hits (90%), suggesting possible novel viruses. For future work, we will be analyzing the rest of the irrigation water samples as well as our recovery from pre and post-harvest lettuce.

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Developing Scientists as Teachers; Developing Students as Scientists: A Dual Approach to Transforming the Culture of Undergraduate Biology Education

John R Geiser1, Susan Stapleton1,2, Renee’ Schwartz3, 1Department of Biological Sciences, 2Department of Chemistry, Western Michigan University, Kalamazoo, MI, 49008, 3Department of Middle and Secondary Education, College of Education, Georgia State University

President Obama challenged the nation to increase the number of qualified STEM graduates by one million within 10  years. To do so, undergraduate STEM programs must change the way they approach education. In Vision and Change for Undergraduate Biology Education: A Call to Action, biology departments are encouraged to make the learning experience more authentic, more like what scientists do. We will address the challenge and transform the culture of biology education at Western Michigan University by developing scientists as teachers who develop students as scientists. Our dual approach is based on research recommended practices for fostering student engagement and promoting institutional change. The innovation potential for the institution is well-grounded in practices already begun at Western Michigan University. We build on the momentum of our prior work to merge research experiences for undergraduates, curriculum development, and professional development. Our primary goal is to increase persistence of undergraduates in STEM majors, with specific focus on biological sciences.

Our philosophy of transformation acknowledges that for students to develop as scientists, scientists must develop as teachers. This project develops scientists as teachers who embrace the essence of authentic science practices within science instruction. By engaging all students in relevant science research, we develop students as scientists who persist toward a STEM degree. A cultural change emerges as scientist/teacher/student identities are fostered, the outcome of which is a learning environment reflective of real and engaging science.

We propose the following program elements. (1) Professional Development. We will establish learning communities for faculty, teaching assistants, and undergraduate peer leaders. Additional professional development includes a course for teaching assistants to learn methods of active/inquiry strategies. This course is already developed as part of our current HHMI Undergraduate Science Education program. We will establish a science teaching journal club, a “scientists as teachers” speaker series, and summer workshops with follow up that includes on-site classroom-based support and feedback from peers and experts.  (2) Curriculum Development. We will incorporate multiple research experiences for all biology majors so that students see themselves as scientists from early in their undergraduate program through the end. We focus on two introductory biology courses and advanced capstone courses. The classroom is transformed into a scientific community where students are scientists: asking questions, conducting investigations, and generating and communicating evidence-based arguments to their peers. (3) Assessment Plan. Our rigorous assessment plan will identify impacts of the sustainable program.

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Phenotypic Selection of Pseudomonas aeruginosa in a Cystic Fibrosis Lung Modifies Sensitivity to Hydrogen Peroxide

Erin M. Mulroney, Lake Superior State University, Sault Ste. Marie, MI

Before eventual respiratory failure and death, Cystic Fibrosis (CF) patients are subjected to chronic lung infection and inflammation throughout their lives. The nature of the genetic mutation causing this disease results in a buildup of thick mucus within CF lungs and provides a favorable environment for the opportunistic bacteria, Pseudomonas aeruginosa, to successfully colonize. Nearly all CF patients acquire chronic infection of this bacterium, where a mucoid phenotype is selected for within their lungs. The mucoid phenotype is identified by an overproduction of alginate and associated with a poor patient prognosis. Due to the amount of oxidative stress bacteria is subjected to in this environment, it was hypothesized that mucoid P. aeruginosa would be more resistant to hydrogen peroxide treatment than its isogenic non-mucoid pair. Surprisingly, the opposite trend was observed. Although treatment with hydrogen peroxide can induce a mucoid phenotype, this research demonstrates that it also produces an increase in sensitivity. Furthermore, this sensitivity was shown to be independent of alginate production. More investigation will be needed to determine additional P. aeruginosa regulatory consequences when conversion to a mucoid phenotype occurs.

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