Microbiology Commons^™

Characterization Of The Extracellular Proteome Of A Natural Microbial Community With An Integrated Mass Spectrometric / Bioinformatic Approach, Brian Keith Erickson

Doctoral Dissertations

Proteomics comprises the identification and characterization of the complete suite of expressed proteins in a given cell, organism or community. The coupling of high performance liquid chromatography (LC) with high throughput mass spectrometry (MS) has provided the foundation for current proteomic progression. The transition from proteomic analysis of a single cultivated microbe to that of natural microbial assemblages has required significant advancement in technology and has provided greater biological understanding of microbial community diversity and function.

To enhance the capabilities of a mass spectrometric based proteomic analysis, an integrated approach combining bioinformatics with analytical preparations and experimental data collection was …

Mechanisms Of Antimicrobial Peptide Resistance In Campylobacter, Ky Van Hoang

Doctoral Dissertations

Campylobacter is the major bacterial cause of human gastroenteritis in the United States and other developed countries. Poultry are considered a main source of human Campylobacter infections. Thus, reduction of Campylobacter load in poultry is significant in food safety and public health. However, no effective measure is commercially available to prevent Campylobacter colonization in poultry to date. Antimicrobial peptides (AMPs) are short and bactericidal peptides widely present in intestine to limit bacterial infections. Recently, AMPs have been increasingly recognized as a novel class of antibiotics (peptide antibiotics) to control foodborne pathogens. Notably, several potent anti-Campylobacter bacteriocins, a group of AMPs …

Growth Of Lactic Acid Bacteria: Infuence Of Protocooperation, Bacteriophage Infection, And Prebiotic Carbohydrates, Caitlin D. Goin

School of Biological Sciences: Dissertations, Theses, and Student Research

Lactic acid bacteria are a vital part of the fermented food industry and are the subject of much interest and research. Industry is especially interested in using modern molecular approaches to maintain and improve selected strains; several industrial uses could be improved by closer investigation, namely the protocooperation of S. thermophilus and L. bulgaricus, the role of CRISPRs in phage resistance, and the utilization of prebiotic carbohydrates. Questions such as what genes and pathways are shared during milk fermentation between S. thermophilus and L. bulgaricus, when and what genes are active during bacteriophage infection, and how and where …

Phylometrics: A Pipeline For Inferring Phylogenetic Trees From A Sequence Relationship Network Perspective, Samuel A. Smits, Cleber C. Ouverney

Faculty Publications, Biological Sciences

Background
Comparative sequence analysis of the 16S rRNA gene is frequently used to characterize the microbial diversity of environmental samples. However, sequence similarities do not always imply functional or evolutionary relatedness due to many factors, including unequal rates of change and convergence. Thus, relying on top BLASTN hits for phylogenetic studies may misrepresent the diversity of these constituents. Furthermore, attempts to circumvent this issue by including a large number of BLASTN hits per sequence in one tree to explore their relatedness presents other problems. For instance, the multiple sequence alignment will be poor and computationally costly if not relying on …

Genome Sequence Of The Model Mushroom Schizophyllum Commune, Robin A. Ohm, Jan F. De Jong, Luis G. Lugones, Andrea Aerts, Erika Kothe, Jason E. Stajich, Ronald P. De Vries, Eric Record, Anthony Levasseur, Scott E. Baker, Kirk A. Bartholomew, Pedro M. Coutinho, Susann Erdmann, Thomas J. Fowler, Allen C. Gathmen, Vincent Lombard, Bernard Henrissat, Nicole Knabe, Ursula Kues, Walt W. Lily

Biology Faculty Publications

Much remains to be learned about the biology of mushroom-forming fungi, which are an important source of food, secondary metabolites and industrial enzymes. The wood-degrading fungus Schizophyllum commune is both a genetically tractable model for studying mushroom development and a likely source of enzymes capable of efficient degradation of lignocellulosic biomass. Comparative analyses of its 38.5-megabase genome, which encodes 13,210 predicted genes, reveal the species's unique wood-degrading machinery. One-third of the 471 genes predicted to encode transcription factors are differentially expressed during sexual development of S. commune. Whereas inactivation of one of these, fst4, prevented mushroom formation, inactivation of another, …

Jsphylosvg: A Javascript Library For Visualizing Interactive And Vector-Based Phylogenetic Trees On The Web, Samuel A. Smits, Cleber C. Ouverney

Faculty Publications, Biological Sciences

Background
Many software packages have been developed to address the need for generating phylogenetic trees intended for print. With an increased use of the web to disseminate scientific literature, there is a need for phylogenetic trees to be viewable across many types of devices and feature some of the interactive elements that are integral to the browsing experience. We propose a novel approach for publishing interactive phylogenetic trees.

Methods/Principal Findings
We present a javascript library, jsPhyloSVG, which facilitates constructing interactive phylogenetic trees from raw Newick or phyloXML formats directly within the browser in Scalable Vector Graphics (SVG) format. It is …

Development Of Pyrf-Based Genetic System For Targeted Gene Deletion In Clostridium Thermocellum And Creation Of A Pta Mutant, Shital A. Tripathi, Daniel G. Olson, D. Aaron Argyros, Bethany B. Miller, Trisha F. Barrett, Daniel M. Murphy, Jesse D. Mccool, Anne K. Warner, Vineet B. Rajgarhia, Lee R. Lynd, David A. Hogsett, Nicky C. Caiazza

Dartmouth Scholarship

We report development of a genetic system for making targeted gene knockouts in Clostridium thermocellum, a thermophilic anaerobic bacterium that rapidly solubilizes cellulose. A toxic uracil analog, 5-fluoroorotic acid (5-FOA), was used to select for deletion of the pyrF gene. The ΔpyrF strain is a uracil auxotroph that could be restored to a prototroph via ectopic expression of pyrF from a plasmid, providing a positive genetic selection. Furthermore, 5-FOA was used to select against plasmid-expressed pyrF, creating a negative selection for plasmid loss. This technology was used to delete a gene involved in organic acid production, namely pta, which encodes …

An Analysis Of Global Gene Expression Resulting From Exposure To Energetic Materials, Vernon L. Mcintosh Jr.

Doctoral Dissertations

AN ANALYSIS OF GLOBAL GENE EXPRESSION RESULTING FROM EXPOSURE TO ENERGETIC MATERIALS

A Dissertation

Presented for the Doctor of Philosophy Degree

University of Tennessee, Knoxville

VERNON LASHAWN MCINTOSH JR.

August 2010

Dedication

This dissertation is dedicated to my family. My mother and father Debra and Vernon McIntosh instilled in me the respect for academic excellence and the drive maximize my potential. Early on, my younger brother Kyle started showing signs of a shared interest in biology thus my desire to be a positive role model for him kept me motivated. Last but certainly not least, my loving wife and best …

Alteration Of Microbial Communities Colonizing Leaf Litter In A Temperate Woodland Stream By Growth Of Trees Under Conditions Of Elevated Atmospheric Co2, John J. Kelly, Amit Bansal, Jonathan Winkelman, Lori R. Janus, Shannon Hell, Marie Wencel, Patricia Belt, Kevin A. Kuehn, Steven T. Rier, Nancy C. Tuchman

Faculty Publications

Elevated atmospheric CO₂ can cause increased carbon fixation and altered foliar chemical composition in a variety of plants, which has the potential to impact forested headwater streams because they are detritus-based ecosystems that rely on leaf litter as their primary source of organic carbon. Fungi and bacteria play key roles in the entry of terrestrial carbon into aquatic food webs, as they decompose leaf litter and serve as a source of nutrition for invertebrate consumers. This study tested the hypothesis that changes in leaf chemistry caused by elevated atmospheric CO₂ would result in changes in the size and …

Cattle Access Affects Periphyton Community Structure In Tennessee Farm Ponds., Robert Gerald Middleton

Masters Theses

Cattle farming is vital to the economy of the United States. Frequently, cattle are given access to ponds and streams for water. The relative impacts of cattle access in natural water sources on the periphyton community have been rarely investigated. Periphyton is the basis of the aquatic food web, and community composition can serve as a bioindicator of pollution. Thus, my objectives were to quantify the effects of cattle access in aquatic lentic systems on periphyton community structure and biovolume, identify taxa that were associated with cattle access, and identify abiotic mechanisms that might be driving assemblage changes. I conducted …

Use Of Proteomics Tools To Investigate Protein Expression In Azospirillum Brasilense, Gurusahai K. Khalsa-Moyers

Doctoral Dissertations

Mass spectrometry based proteomics has emerged as a powerful methodology for investigating protein expression. “Bottom up” techniques in which proteins are first digested, and resulting peptides separated via multi-dimensional chromatography then analyzed via mass spectrometry provide a wide depth of coverage of expressed proteomes. This technique has been successfully and extensively used to survey protein expression (expression proteomics) and also to investigate proteins and their associated interacting partners in order to ascertain function of unknown proteins (functional proteomics). Azospirillum brasilense is a free-living diazotrophic soil bacteria, with world-wide significance as a plant-growth promoting bacteria. Living within the rhizosphere of cereal …

Agricultural Soil Bacteria; A Study Of Collection, Cultivation, And Lysogeny, Katherine Elizabeth Sides

Masters Theses

The aim of this research project was to test new collection and cultivation techniques that may increase the range of cultivable diversity of soil bacteria. Fortified BioSep beads were employed in situ to capture soil bacteria, and the success of the beads was analyzed using Phylochip microarray analysis. In the cultivation phase, three different media substrates and increased incubation period were evaluated for the ability to select novel or rare bacteria. Over 700 agricultural soil bacterial isolates were classified, including a rare Gemmatimonadetes sp., a rare Verrucomicrobia sp., several Acidobacteria sp., and many novel isolates. Land management, media, and incubation …

Candida Albicans Cellwall Components And Farnesol Stimulate The Expression Of Both Inflammatory And Regulatory Cytokines In The Murine Raw264.7 Macrophage Cell Line, Suman Ghosh, Nina Howe, Katie Volk, Swetha Tati, Kenneth W. Nickerson, Thomas M. Petro

Kenneth Nickerson Papers

Candida albicans causes candidiasis, secretes farnesol, and switches from yeast to hyphae to escape from macrophages after phagocytosis. However, before escape, macrophages may respond to C. albicans’ pathogen-associated molecular patterns (PAMPs) through toll-like receptor 2 (TLR2) and dectin-1 receptors by expressing cytokines involved in adaptive immunity, inflammation, and immune regulation. Therefore, macrophages and the RAW264.7 macrophage line were challenged with C. albicans preparations of live wild-type cells, heat-killed cells, a live mutant defective in hyphae formation, a live mutant producing less farnesol, or an isolate producing farnesoic acid instead of farnesol. Interleukin-6 (IL-6), and IL-1b, IL- 10, and tumor …

Soil Organic Matter Fractions And Aggregate Distribution In Response To Tall Fescue Stands, I. P. Handayani, Mark S. Coyne, R. S. Tokosh

Plant and Soil Sciences Faculty Publications

The study was conducted to evaluate the influences of tall fescue management on soil organic matter fractions and macro- and microaggregate distribution. Soil samples were collected from four paired adjacent fields consisting of five years of tall fescue mono and poly stands in Western Kentucky. Soil samples from 0 to 15 cm and 15 to 30 cm soil depths were analyzed for soil organic C and N, particulate organic matter C (POM-C) and N (POM-N), macro- and micro aggregate distribution and C-associated with macro- and micro- aggregates. Significant effects were observed between stands for all the properties, except total C, …

The Construction And Analysis Of Marker Gene Libraries, S.M. Short, F. Chen, Steven Wilhelm

Microbiology Publications and Other Works

Marker genes for viruses are typically amplified from aquatic samples to determine whether specific viruses are present in the sample, or to examine the diversity of a group of related viruses. In this chapter, we will provide an overview of common methods used to amplify, clone, sequence, and analyze virus marker genes, and will focus our discussion on viruses infecting algae, bacteria, and heterotrophic flagellates. Within this chapter, we endeavor to highlight critical aspects and components of these methods. To this end, instead of providing a detailed experimental protocol for each of the steps involved in examining virus marker gene …

Determining Rates Of Virus Production In Aquatic Systems By The Virus Reduction Approach,, M.G. Weinbauer, J.M. Rowe, Steven Wilhelm

Microbiology Publications and Other Works

The reduction approach to assess virus production and the prokaryotic mortality by viral lysis stops new infection by reducing total virus abundance (and thus virus–host contacts). This allows for easy enumeration of viruses that originate from lysis of already infected cells due to the decreased abundance of free virus particles. This reoccurrence can be quantified and used to assess production and cell lysis rates. Several modifications of the method are presented and compared. The approaches have great potential for elucidating trends in virus production rates as well as for making generalized estimates of the quantitative effects of viruses on marine …

Spatial Structure And Activity Of Sedimentary Microbial Communities Underlying A Beggiatoa Spp. Mat In A Gulf Of Mexico Hydrocarbon Seep, Karen Lloyd, Daniel B. Albert, Jennifer F. Biddle, Jeffrey P. Chanton, Oscar Pizarro, Andreas Teske

Microbiology Publications and Other Works

Background

Subsurface fluids from deep-sea hydrocarbon seeps undergo methane- and sulfur-cycling microbial transformations near the sediment surface. Hydrocarbon seep habitats are naturally patchy, with a mosaic of active seep sediments and non-seep sediments. Microbial community shifts and changing activity patterns on small spatial scales from seep to non-seep sediment remain to be examined in a comprehensive habitat study.

Methodology/Principal Findings

We conducted a transect of biogeochemical measurements and gene expression related to methane- and sulfur-cycling at different sediment depths across a broad Beggiatoa spp. mat at Mississippi Canyon 118 (MC118) in the Gulf of Mexico. High process rates within the …

Diversity Of Oligotrichia And Choreotrichia Ciliates In Coastal Marine Sediments And In Overlying Plankton, Jan A.C. Vriezen Dr., Mary Doherty Dr., Maiko Tamura Dr., George B. Mcmanus Dr.

Jan A.C. Vriezen Dr.

Elucidating the relationship between ciliate communities in the benthos and the plankton is critical to understanding ciliate diversity in marine systems. Although data for many lineages are sparse, at least some members of the dominant marine ciliate clades Oligotrichia and Choreotrichia can be found in both plankton and benthos, in the latter either as cysts or active forms. In this study, we developed a molecular approach to address the relationship between the diversity of ciliates in the plankton and those of the underlying benthos in the same locations. Samples from plankton and sediments were compared across three sites along the …

Effects Of Tillage On Microbial Populations Associated To Soil Aggregation In Dryland Spring Wheat System, Andrew W. Lenssen, Thecan Caesar-Tonthat, Anthony J. Caesar, Upendra M. Sainju, John F. Gaskin

Andrew W. Lenssen

Tillage may influence the microbial populations involved in soil aggregation.We evaluated the effects of no till (NT) and conventional tillage (CT, tillage depth about 7 cm) continuous spring wheat system on culturable heterotrophic bacterial communities predominant in microaggregates (0.25e0.05 mm) and on soil-aggregating basidiomycete fungi in aggregate-size classes (4.75e2.00, 2.00e0.25, and 0.25e0.05 mm) at 0e20 cm depth of a Williams loam (fine-loamy, mixed, Typic Argiustolls) in dryland Montana, USA. Enzyme-linked immunosorbent assay used to quantify antigenic response to basidiomycete cellwalls,was higher in NT than in CT in 4.75e2.00 mm size class in 2007 and higher in all classes and years …

Quantitativepcrmethods Forrna Anddnainmarine Sediments: Maximizing Yieldwhile Overcoming Inhibition, Karen Lloyd, Barbara J. Macgregor, Andreas Teske

Karen Lloyd

For accurate quantification of DNA and RNA from environmental samples, yield loss during nucleic acid purification has to be minimized. Quantitative PCR (qPCR) and reverse transcription (RT)-qPCR require a trade-off between maximizing yield and removing inhibitors. We compared DNA and RNA yield and suitability for quantitative SYBR Green PCR and RT-PCR using the UltraClean and PowerSoil extraction kits and a bead-beating protocol with phenol/chloroform extraction steps. Purification methods included silica-column-based procedures from the MoBio kits, RNeasy MinElute, WizardPlus miniprep columns, and an acrylamide gel extraction. DNA and RNA purification with WizardPlus and RNeasy, respectively, led to significant losses of nucleic …

Spatial Structure And Activity Of Sedimentary Microbial Communities Underlying A Beggiatoa Spp. Mat In A Gulf Of Mexico Hydrocarbon Seep, Karen Lloyd, Daniel B. Albert, Jennifer F. Biddle, Jeffrey P. Chanton, Oscar Pizarro, Andreas Teske

Karen Lloyd

Background Subsurface fluids from deep-sea hydrocarbon seeps undergo methane- and sulfur-cycling microbial transformations near the sediment surface. Hydrocarbon seep habitats are naturally patchy, with a mosaic of active seep sediments and non-seep sediments. Microbial community shifts and changing activity patterns on small spatial scales from seep to non-seep sediment remain to be examined in a comprehensive habitat study. Methodology/Principal Findings We conducted a transect of biogeochemical measurements and gene expression related to methane- and sulfur-cycling at different sediment depths across a broad Beggiatoa spp. mat at Mississippi Canyon 118 (MC118) in the Gulf of Mexico. High process rates within the …

Determining Rates Of Virus Production In Aquatic Systems By The Virus Reduction Approach,, M.G. Weinbauer, J.M. Rowe, Steven Wilhelm

Steven Wilhelm

The reduction approach to assess virus production and the prokaryotic mortality by viral lysis stops new infection by reducing total virus abundance (and thus virus–host contacts). This allows for easy enumeration of viruses that originate from lysis of already infected cells due to the decreased abundance of free virus particles. This reoccurrence can be quantified and used to assess production and cell lysis rates. Several modifications of the method are presented and compared. The approaches have great potential for elucidating trends in virus production rates as well as for making generalized estimates of the quantitative effects of viruses on marine …

The Construction And Analysis Of Marker Gene Libraries, S.M. Short, F. Chen, Steven Wilhelm

Steven Wilhelm

Marker genes for viruses are typically amplified from aquatic samples to determine whether specific viruses are present in the sample, or to examine the diversity of a group of related viruses. In this chapter, we will provide an overview of common methods used to amplify, clone, sequence, and analyze virus marker genes, and will focus our discussion on viruses infecting algae, bacteria, and heterotrophic flagellates. Within this chapter, we endeavor to highlight critical aspects and components of these methods. To this end, instead of providing a detailed experimental protocol for each of the steps involved in examining virus marker gene …

Visible And Hyperspectral Imaging Systems For The Detection And Discrimination Of Mechanical And Microbiological Damage Of Mushrooms, Edurne Gaston

Doctoral

Horticultural products such as mushrooms are exposed to environmental conditions during their postharvest life, which may affect product quality. Loss of whiteness during storage is particularly important in the mushroom industry. Rough handling and distribution, fruiting body senescence and bacterial infections are among the main causes of mushroom discolouration. The aim of this work was to study the use of visible and hyperspectral imaging (HSI) systems for the detection and discrimination of mechanical and microbiological damage of mushrooms. This piece of research involved a) monitoring the browning of mushroom with visible computer imaging systems, b) investigating the effect of mechanical …

Evolutionary Aspects Of Urea Utilization By Fungi, Dhammika H. M. L. P Navarathna, Steven D. Harris, David D. Roberts, Kenneth W. Nickerson

Kenneth Nickerson Papers

The higher fungi exhibit a dichotomy with regard to urea utilization. The hemiascomycetes use urea amidolyase (DUR1,2), whereas all other higher fungi use the nickel-containing urease. Urea amidolyase is an energy-dependent biotincontaining enzyme. It likely arose before the Euascomycete/Hemiascomycete divergence c. 350 million years ago by insertion of an unknown gene into one copy of a duplicated methylcrotonyl CoA carboxylase (MccA). The dichotomy between urease and urea amidolyase coincides precisely with that for the Ni/Co transporter (Nic1p), which is present in the higher fungi that use urease and is absent in those that do not. We suggest that …

Oceanic Heterotrophic Bacterial Nutrition By Semilabile Dom As Revealed By Data Assimilative Modeling, Yw Luo, M. A.M. Friedrichs, Sc Doney, Mj Church, Hw Ducklow

VIMS Articles

Previous studies have focused on the role of labile dissolved organic matter (DOM) (defined as turnover time of similar to 1 d) in supporting heterotrophic bacterial production, but have mostly neglected semilabile DOM (defined as turnover time of similar to 100 to 1000 d) as a potential substrate for heterotrophic bacterial growth. To test the hypothesis that semilabile DOM supports substantial amounts of heterotrophic bacterial production in the open ocean, we constructed a 1-dimensional epipelagic ecosystem model and applied it to 3 open ocean sites: the Arabian Sea, Equatorial Pacific and Station ALOHA in the North Pacific Subtropical Gyre. The …

Thermocrinis Minervae Sp. Nov., A Hydrogen And Sulfur-Oxidizing, Thermophilic Member Of The Aquificales From A Costa Rican Terrestrial Hot Spring, Sara L. Caldwell, Yitai Liu, Isabel Ferrera, Terry Beveridge, Anna-Louise Reysenbach

Biology Faculty Publications and Presentations

A thermophilic bacterium, designated strain CR11T , was isolated from a filamentous sample collected from a terrestrial hot spring on the south-western foothills of the Rincón volcano in Costa Rica. The Gram-negative cells are approximately 2.4–3.9 mm long and 0.5–0.6 mm wide and are motile rods with polar flagella. Strain CR11T grows between 65 and 85 6C (optimum 75 6C, doubling time 4.5 h) and between pH 4.8 and 7.8 (optimum pH 5.9–6.5). The isolate grows chemolithotrophically with S0 , S2O2{ 3 or H2 as the electron donor and with O2 (up to 16 %, v/v) as the sole electron …

Early Cranial Patterning In The Direct-Developing Frog Eleutherodactylus Coqui Revealed Through Gene Expression, Ryan Kerney, Joshua Gross, James Hanken

Ryan Kerney

Genetic and developmental alterations associated with the evolution of amphibian direct development remain largely unexplored. Specifically, little is known of the underlying expression of skeletal regulatory genes, which may reveal early modifications to cranial ontogeny in direct-developing species. We describe expression patterns of three key skeletal regulators (runx2, sox9, and bmp4) along with the cartilage-dominant collagen 2a1 gene (col2a1) during cranial development in the direct- developing anuran, Eleutherodactylus coqui. Expression patterns of these regulators reveal transient skeletogenic anlagen that correspond to larval cartilages, but which never fully form in E. coqui. Suprarostral anlagen in the frontonasal processes are detected through …

Synthetic Genome: Now That We’Re Creators, What Should We Create?, Frederick M. Cohan

Frederick M. Cohan

No abstract provided.

The Ecology Of Speciation In Bacillus, Nora Connor, Johannes Sikorski, Alejandro P. Rooney, Sarah Kopac, Alexander F. Koeppel, Andrew Burger, Scott G. Cole, Elizabeth B. Perry, Danny Krizanc, Nicholas C. Field, Michele Slaton, Frederick M. Cohan

Frederick M. Cohan

No abstract provided.