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Natural products are complex organic molecules made by a variety of organisms including bacteria, fungi, and plants which often possess bioactivities that make them useful as drugs, drug leads, or probes for deciphering biological networks. Using bioinformatics tools developed in the Melançon lab and elsewhere, we have identified and partially annotated 6 natural product gene clusters of interest in Actinosynnema mirum, including 2 encoding analogs of ansamycin class anticancer compounds. To determine if these 6 gene clusters are transcriptionally active, we have designed and begun to optimize real-time PCR assays targeting 1 to 2 genes in each cluster by creating four cDNA libraries from mRNA samples collected at time points representing several growth phases of A. mirum. From this, we have determined that 2 of the 6 gene clusters of interest, including 1 of the clusters encoding an ansamycin analog, are transcriptionally active. Additionally, we have also cultured A. mirum in large scale and have obtained crude natural product extracts from it using standard isolation techniques, including organic extraction and adsorption onto Amberlite XAD-7 and XAD-16 resins. Extracts will be analyzed for the predicted compound using analytical HPLCESI mass spectrometry. Once compounds of interest are identified, they will be purified using preparative HPLC.
The effect of the ansamitocin analog will be assessed in an in vitro assay. If active, its structure will be determined by NMR. Other purified compounds will also be assessed for antibacterial and anticancer activities by our group and by collaborators.
CULTIVATION OF HETEROTROPHIC MARINE BACTERIA ALONG THE PACIFIC COAST OF NORTH AMERICAMaria Ortiz, Erin Schaadt, Lizzette Rojas, and Jesse Dillon.
California State University, Long Beach, Long Beach, CA.
Environmental factors vary geographically along coasts (i.e., temperature) and may affect the abundance and diversity of marine heterotrophic bacteria along the Pacific coast. This project involved isolation of cultures from seawater samples collected from sandy beaches from Baja California to northern Oregon in January and August of 2010. Monthly samples were collected at Huntington Beach. It was hypothesized that heterotrophic marine bacteria would display significant differences in diversity and abundance geographically (north and south of Point Conception) as well as seasonally (summer and winter). Bacteria were counted via serial dilution (MPNs) in marine broth to determine abundance. Cultures were isolated via streak plate serial dilution and were characterized by Gram staining and light microscopy. Nucleic acid extractions, PCR amplification, and sequencing of 16S rRNA genes were performed to construct a neighbor joining tree using ARB software. In parallel, salinity tolerance experiments were done on marine broth isolates (N = 77) using treatments of 3.5%, 15%, and 23% salinity. There was no significant difference in bacterial abundance between seasons after comparison of winter and summer MPNs (unpaired t-test, p = 0.057). Within the seasons, there were no strong relationships between temperature and bacterial abundance (winter R2 = 0.005, summer R2 = 0.127). The salinity experiment showed growth only in the 3.5% concentration.
Sequencing analysis revealed diverse assemblages of Gamaproteobacteria, Flavobacteria, Actinobacteria, Alphaproteobacteria, and Bacillus spp. along coastal North America. Current phylogenetic analyses indicate biogeographical influence south of Point Conception along with seasonality of Actinobacteria and species of Vibrio and Psychrobacter.
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Biological Sciences SAT-573
COMPARISON OF MATRIX COMPONENT AND DRUG SUSCEPTIBILITY BETWEEN CANDIDA ALBICANS AND
NON-ALBICANS SPECIESKeely Redhage, Hiram Sanchez, David Andea.
University of Wisconsin, Madison, Madison, WI.
Fungal infections are becoming an increasing problem in hospital settings leading to growing mortality rates and skyrocketing health-care costs. The most prominent fungal infection is due to Candida albicans and non-albicans species Candida glabrata, Candida parapsilosis, and Candida tropicalis. These pathogens, when attached to a surface such as a catheter, produce a biofilm which is highly resistant to antifungal drugs. Most of the drug resistance is due to the extracellular material (matrix) secreted by the biofilm. This study focuses on matrix components of nonalbicans species in comparison to C. albicans. Our hypothesis is that the matrix components of biofilms are key to antifungal resistance. Carbohydrates such, β-1,3/1,6 glucan, mannans, and proteins are present in matrix. In order to understand how these components contribute to drug resistance, matrix was produced by growing the organisms in roller bottles forming a biofilm, and the matrix was harvested after 48 hours. Samples were dialyzed and lyophilized.
The matrix components were analyzed with phenol sulfuric assay (for total carbohydrate content), BCA assay (for total protein content), and ELISAs for mannans and β-1,3/1,6 glucan (for specific carbohydrate content). Although all components of the matrix play a role in antifungal resistance, preliminary data show that carbohydrates are the most important components in the drug resistance mechanism. This was shown by XTT assays with a drug combination of tunicamycin and fluconazole where a correlation was found between the amount of matrix carbohydrates and drug susceptibility between strains.
ASPERGILLUS MOLD – BACTERIA COCULTURE: IMPROVED POLYCYCLIC AROMATIC HYDROCARBON
DEGRADATION THROUGH SYNERGISTIC METABOLISMMarissa Hartleb1, Mi-kyung Lee2.
Cornell University, Ithaca, NY, 2University of Wisconsin-Madison, Madison, WI.
1 Tens of thousands of Superfund sites, abandoned or inactive hazardous waste sites recognized by the Environmental Protection Agency (EPA), are found throughout the United States and hold dangerous contaminants such as polycyclic aromatic hydrocarbons (PAHs). PAHs are carcinogenic, mutagenic, and highly toxic and also have high persistence in soil. Some organisms have been found in Superfund sites that can metabolize these contaminants.
Bacterial organisms have been found which degrade PAHs with 2 to 3 benzene rings, while fungal organisms have been found which degrade PAHs with 4 or more rings. The hypothesis for this experiment is that by having the 2 types of organisms live and work together, the range and quality of PAH degradation will increase. This idea will be tested using a Transwell-24 system. This system will allow small metabolites to be exchanged between the two organisms but will disallow all physical interaction. The fungus Aspergillus nidulans is a common soil fungus known to degrade benzo[a]pyrene, a 5-ring PAH. The bacterium Burkholderia sp. Ch1-1, is found in Superfund sites and is known to degrade phenanthrene, a 3-ring PAH. It is suspected that the fungus will mineralize pyrene, a 4-ring PAH, which will then allow the bacterium to metabolize pyrene. If the hypothesis is correct, improved bioremediation strategies will become possible, as well as more accurate PAH biosensors which can be used to measure initial concern or subsequent success.
ANALYSIS OF GROWTH PATTERNS OF PSEUDOMONAS STUTZERI AND STREPTOCOCCUS EPIDERMIDIS: A
MULTISPECIES BIOFILMNnenna Kpando, Lea Means, Kimberly Yawn, Caroline Barbosa, Antonio Mendoza, Poonam Gulati, Youn-Sha Chan.
University of Houston-Downtown, Houston, TX.
The objective of this project is to analyze the growth and development patterns of bacteria living in communities known as biofilms. A biofilm is a communal of single-species or multiple-species microorganisms that adhere to a moist surface and to one another using extra polymeric substances that are synthesized by them. Bacteria can also have an independent growth pattern known as planktonic or free-swimming. The growth pattern of planktonic cells is well studied and shows a logistic growth pattern. Studies in our lab on single species biofilms of Streptococcus epidermidis, a Gram-positive, coccus-shaped bacterium, and Pseudomonas stutzeri, a Gram-negative, rod-shaped bacterium, have demonstrated a cyclic pattern of growth. We have developed a mathematical model for the cyclic growth pattern. This model, which is a Bessel function, predicts the cyclic nature of the growth and a dampening
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of the cycles with time. In nature, multispecies biofilms are commonly found. As such, in this project, the multiplespecies biofilm growth patterns of S. epidermidis and P. stutzeri are being studied as a mixed species biofilm. Based on our studies of the two bacteria, we predict that as resources deplete, P. stutzeri will overshadow and outlast S.
epidermidis. We plan to develop a predator-prey model between the two organisms that establishes growth peaks for one organism and troughs for the other over a period of time. The model could then be used to explain some of the dynamics of natural biofilms.
IDENTIFICATION OF BACTERIA OF THE RIO GRANDE WATERS BY DGGE ANALYSISMelissa Saucedo1, Rebecca Castro1, Brittan Wilson2, and Monica Mendez1.
Texas A&M International University, Laredo, TX, 2University of Baltimore, Baltimore, MD.
1 Previous studies examining the microbial community of the Rio Grande border have focused on culturable pathogenic bacteria, mainly E.coli. This study will focus on the identification of noncultured bacteria obtained from a previous study using denaturing gradient gel electrophoresis (DGGE) of the 16S rRNA gene amplified from the DNA extracts of three water sampling sites within the Laredo, Texas, and Nuevo Laredo, Mexico, border of the Rio Grande. The three sampling sites were Zacate Creek area (Site 1), International Bridge I (Site 2), and the Jefferson Water Treatment Plant (Site 3). Sites were chosen based on effluent discharge points that were greatest at Site 1. Phylotype bands were excised from a DGGE gel after DGGE-PCR using primers 1070F and 1406-GC. Eluted DNA from each band will be reamplified using DGGE-PCR followed by cloning using the TOPO-TA cloning kit. Plasmids will then be purified for sequencing using T3 and T7 primers. Sequences will then be used for bacterial identification using the NCBI-BLAST database. Although all sites were similar (80%, Dice coefficient) based on DGGE profiles, Site 1 contained the most unique types of bacteria and was significantly greater in heterotrophic bacterial counts (9.4 x 103 CFU/ml, p 0.01) than all other sites. We expect that Site 1 will have an even greater number of unique phylotypes identified assuming that the DGGE bands will contain a greater number of mixed templates. This study will allow us to identify potential microbial biomarkers for understanding fluxes of nutrients and contaminants in the Rio Grande waters.
DISCOVERING THE STRUCTURE OF AN IMPORTANT HUMAN HEALTH ENZYME IN ENTEROCOCCUSFAECIUM Amber Anderson, Gilbert John.
Oklahoma State University, Stillwater, OK.
Enterococcus faecium is an anaerobic bacterium that flourishes within the human intestine. This organism contains an important enzyme called azoreductase (AzoM), which is involved in cleaving the azo bond within azo dyes, producing a toxic, aromatic amine. Carcinogenic compounds can result from the reduction of dyes, which are commonly used to color food, clothing, drugs, and cosmetics. In terms of structure and function of the enzyme, very few intestinal bacteria containing azoreductase have been studied. This study used sophisticated crystallization methods to obtain the structure of AzoM. The pure AzoM protein was obtained from an Escherichia coli expression system, extracted using BugBuster Protein Extraction Kit, and protein purity was verified by SDS-PAGE gel electrophoresis. The protein sample was collected in two different buffer conditions, Tris and HEPES, and concentrated to over 10 mg/ ml. Initial protein crystallization was obtained by the sitting drop method using the 96-well Hamilton and Index Crystal screens. Positive samples were then optimized using additive screens as well as 24-well, custom-prepared conditions based on varying concentrations of salts, dyes, buffers, and pHs. Based on microscopic examination, crystals with sufficient size were subjected to X-ray crystallography using the Brookhaven National Lab, National Synchrotron Light Source. Computer software was used to create the 3D structure based on data from X-ray diffraction. Analysis of crystallization will provide an understanding of the structure and function of AzoM, ultimately impacting the environmental, medical, and biotechnology fields.
ISOLATION AND PURIFICATION OF AN ACTIVE COMPOUND PRODUCED BY STREPTOMYCES BACILLARIS
AGAINST STAPHYLOCOCCUS AUREUSMelisa Fuentes, Luis Mota-Bravo.
University of California, Irvine, Irvine, CA.
The emergence of multidrug resistance in Staphylococcus aureus has triggered an urgent need for new antibiotics and novel antibiotic combinations against both Gram-positive and Gram-negative bacteria. Actinobacteria, in
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Biological Sciences particular, the genus Streptomyces, produce almost two-thirds of today’s clinically useful antibiotics. The objective of this study is to isolate actinobacteria from local soil samples and investigate their potential as producers of antibiotics against Staphylococcus aureus by using direct overlay assays with S. aureus ATCC 25923. A neomycin producer, Streptomyces fradiae ATCC 10745, was used as a positive control. Inhibition zones surrounding actinobacteria colonies were measured after overnight incubation at 35 oC. Active compounds were extracted using ethyl acetate.
The extracts were separated by thin-layer chromatography (TLC) using different ratios of methanol-dichloromethane.