Microbiology Commons^™

Role Of Spontaneous Current Oscillations During High-Efficiency Electrotransformation Of Thermophilic Anaerobes, Michael V. Tyurin, Charles R. Sullivan, Lee R. Lynd

Dartmouth Scholarship

Current oscillations at about 24 MHz were observed during electrotransformation (ET) of the thermophilic anaerobes Clostridium thermocellum ATCC 27405, C. thermocellum DSM 1313, and Thermoanaerobacterium saccharolyticum YS 485, using a pulse gated by a square signal generated by a custom generator. In experiments in which only the field strength was varied, all three of these strains resulted in a one-to-one correspondence between the appearance of current oscillations and successful ET. Oscillations accompanied ET of both C. thermocellum strains only at field strengths of >12 kV/cm, and ET was only observed above the same threshold. Similarly, for T. saccharolyticum, oscillations were …

Sara Positively Controls Bap-Dependent Biofilm Formation In Staphylococcus Aureus, María P. Trotonda, Adhar C. Manna, Ambrose L. Cheung, Iñigo Lasa, José R. Penadés

Dartmouth Scholarship

The biofilm-associated protein Bap is a staphylococcal surface protein involved in biofilm formation. We investigated the influence of the global regulatory locus sarA on bap expression and Bap-dependent biofilm formation in three unrelated Staphylococcus aureus strains. The results showed that Bap-dependent biofilm formation was diminished in the sarA mutants by an agr-independent mechanism. Complementation studies using a sarA clone confirmed that the defect in biofilm formation was due to the sarA mutation. As expected, the diminished capacity to form biofilms in the sarA mutants correlated with the decreased presence of Bap in the bacterial surface. Using transcriptional fusion and …

Requirements For Vibrio Cholerae Hapr Binding And Transcriptional Repression At The Hapr Promoter Are Distinct From Those At The Apha Promoter, Wei Lin, Gabriela Kovacikova, Karen Skorupski

Dartmouth Scholarship

Virulence gene expression in certain strains of Vibrio cholerae is regulated in response to cell density by a quorum-sensing cascade that influences the levels of the LuxR homolog HapR through small regulatory RNAs that control the stability of its message. At high cell density, HapR represses the expression of the gene encoding the virulence gene activator AphA by binding to a site between −85 and −58 in the aphA promoter. We show here that a second binding site for HapR lies within the hapR promoter from which it functions to repress its own transcription. This site, as determined by gel …

Sara Is An Essential Positive Regulator Of Staphylococcus Epidermidis Biofilm Development, Maria A. Tormo, Miguel Marti, Jaione Valle, Adhar C. Manna

Dartmouth Scholarship

Staphylococcus epidermidis biofilm formation is associated with the production of the polysaccharide intercellular adhesin (PIA)--poly-N-acetylglucosamine polysaccharide (PNAG) by the products of the icaADBC operon. Recent evidence indicates that SarA, a central regulatory element that controls the production of Staphylococcus aureus virulence factors, is essential for the synthesis of PIA/PNAG and the ensuing biofilm development in this species. Based on the presence of a sarA homolog, we hypothesized that SarA could also be involved in the regulation of the biofilm formation process in S. epidermidis. To investigate this, we constructed nonpolar sarA deletions in two genetically unrelated S. epidermidis clinical strains, …

A Three-Component Regulatory System Regulates Biofilm Maturation And Type Iii Secretion In Pseudomonas Aeruginosa, Sherry L. Kuchma, John P. Connolly, George A. O'Toole

Dartmouth Scholarship

Biofilms are structured communities found associated with a wide range of surfaces. Here we report the identification of a three-component regulatory system required for biofilm maturation by Pseudomonas aeruginosa strain PA14. A transposon mutation that altered biofilm formation in a 96-well dish assay originally defined this locus, which is comprised of genes for a putative sensor histidine kinase and two response regulators and has been designated sadARS. Nonpolar mutations in any of the sadARS genes result in biofilms with an altered mature structure but do not confer defects in growth or early biofilm formation, swimming, or twitching motility. After …

Efficacy Of Innovative Anti-Microbial Decontamination Of Minimally Processed Vegetables, Christina Busquets I Tortosa

Masters

Chlorine has widespread use commercially as a disinfectant wash for raw vegetables. However, it is an extremely corrosive gas and it may have severe health and environmental implications. The aim of this thesis was to find safe, alternative decontamination treatments for ready-to-eat Iceberg lettuce. The efficacy of 1% acetic acid, 2% citric acid; calcium lactate concentrations of 0.5%, 1.5%, 2.5% and 3% at wash temperature of 4,25 and 50°C, ozone alone (1 mg 1-1) and in combination with calcium lactate (2.5%) were compared with chlorine as decontaminating treatments. Microbiological analyses were performed following decontamination treatments using the pour plate method …

The Effects Of Dams In The Big Sandy Watershed Using A Novel Bacteria-Based Bioindicator Of Water Quality, Kathleen Riha Loughman

Theses, Dissertations and Capstones

During 2003, water samples from the Big Sandy watershed were collected in conjunction with the United States Army Corps of Engineers (USACE). Objectives were to determine the effects of dams on bacterial populations and to test a bioindicator of water quality based on antibiotic-resistant and fecal indicator bacteria. Thirty-five samples were taken each season within the Big Sandy Watershed, which includes six USACE dams. Total cultivable, ciprofloxacin-resistant, erythromycin-resistant, tetracycline- resistant, total coliform, and fecal coliform bacteria were enumerated. Data on water chemistry and physical parameters were collected by the USACE in the spring and summer seasons. Antibiotic-resistant bacteria and fecal …

High-Temperature Fluorescent In Situ Hybridization For Detecting Escherichia Coli In Seawater Samples, Using Rrna-Targeted Oligonucleotide Probes And Flow Cytometry, Ying Zhong Tang, Karina Yew Hoong Gin, Tok Hoon Lim

OES Faculty Publications

Fluorescence in situ hybridization (FISH) is a widely used method to detect environmental microorganisms. The standard protocol is typically conducted at a temperature of 46°C and a hybridization time of 2 or 3 h, using the fluorescence signal intensity as the sole parameter to evaluate the performance of FISH. This paper reports our results for optimizing the conditions of FISH using rRNA-targeted oligonucleotide probes and flow cytometry and the application of these protocols to the detection of Escherichia coli in seawater spiked with E. coli culture. We obtained two types of optimized protocols for FISH, which showed rapid results with …

Effect Of Humic Substance Photodegradation On Bacterial Growth And Respiration In Lake Water, Alexandre M. Anesio, William Granéli, George R. Aiken, David J. Kieber, Kenneth Mopper

Chemistry & Biochemistry Faculty Publications

This study addresses how humic substance (HS) chemical composition and photoreactivity affect bacterial growth, respiration, and growth efficiency (BGE) in lake water. Aqueous solutions of HSs from diverse aquatic environments representing different dissolved organic matter sources (autochthonous and allochthonous) were exposed to artificial solar UV radiation. These solutions were added to lake water passed through a 0.7-μm pore-size filter (containing grazer-free lake bacteria) followed by dark incubation for 5, 43, and 65 h. For the 5-h incubation, several irradiated HSs inhibited bacterial carbon production (BCP) and this inhibition was highly correlated with H₂O₂ photoproduction. The H_{2 …}

Periplasm Organization In Treponema Denticola As Studied By Cryo-Electron Tomography, Jacques Izard, C.-E. Hsieh, C. A. Mannella, Ronald J. Limberger, M. Marko

Department of Food Science and Technology: Faculty Publications

As a spirochete, the genus Treponema is one of the few major bacterial groups whose natural phylogenic relationships are evident at the level of gross phenotypic characteristics such as their morphology. Treponema spp. are highly invasive due to their unique motility in dense media, and their ability to penetrate cell layers. This feature is associated with the helical cell body and the presence of flagellar filaments in the periplasm. Treponema denticola is an oral pathogen involved in endodontic infections and periodontal diseases. The presence and quantity of T. denticola in the subgingival biofilm is correlated with the severity of periodontal …

Electrotransformation Of Clostridium Thermocellum, Michael V. Tyurin, Sunil G. Desai, Lee R. Lynd

Dartmouth Scholarship

Electrotransformation of several strains of Clostridium thermocellum was achieved using plasmid pIKm1 with selection based on resistance to erythromycin and lincomycin. A custom-built pulse generator was used to apply a square 10-ms pulse to an electrotransformation cuvette consisting of a modified centrifuge tube. Transformation was verified by recovery of the shuttle plasmid pIKm1 from presumptive transformants of C. thermocellum with subsequent PCR specific to the mls gene on the plasmid, as well as by retransformation of Escherichia coli. Optimization carried out with strain DSM 1313 increased transformation efficiencies from <1 to (2.2 􏰍 0.5) 􏰎 105 transformants per 􏰃g of plasmid DNA. Factors conducive to achieving high transformation efficiencies included optimized periods of incubation both before and after electric pulse application, chilling during cell collection and washing, subculture in the presence of isoniacin prior to electric pulse application, a custom-built cuvette embedded in an ice block during pulse application, use of a high (25-kV/cm) field strength, and induction of the mls gene before plating the cells on selective medium. The protocol and preferred conditions developed for strain DSM 1313 resulted in transformation efficiencies of (5.0 􏰍 1.8) 􏰎 104 transformants per 􏰃g of plasmid DNA for strain ATCC 27405 and 􏰁1 􏰎 103 transformants per 􏰃g of plasmid DNA for strains DSM 4150 and 7072. Cell viability under optimal conditions was 􏰁50% of that of controls not exposed to an electrical pulse. Dam methylation had a beneficial but modest (7-fold for strain ATCC 27405; 40-fold for strain DSM 1313) effect on transformation efficiency. The effect of isoniacin was also strain specific. The results reported here provide for the first time a gene transfer method functional in C. thermocellum that is suitable for molecular manipulations involving either the introduction of genes associated with foreign gene products or knockout of native genes.

Cloning And Functional Analysis Of The Putative Tetracycline Resistance Gene Hp1165 In Helicobacter Pylori, Yuhuan Li

All-Inclusive List of Electronic Theses and Dissertations

Tetracycline has been used in combination with other antibiotics in treatment of Helicobacter pylori infection. However, tetracycline resistance has developed in clinical isolates of H. pylori, rendering treatment failure. Mutations in the 16S rRNA gene have been reported to mediate tetracycline resistance in some patient isolates. Nevertheless, the diversity of tetracycline resistance cases suggests multiple genes are involved. HPl 165, a putative tetracycline resistance gene in the genome of H. pylori 26695, displays 49.8% identity to the tetracycline efflux gene tetA (P) from Clostridium perfringens. To determine the function of the HPl 165 gene and gain a better understanding of …

Identification Of Sarv (Sa2062), A New Transcriptional Regulator, Is Repressed By Sara And Mgra (Sa0641) And Involved In The Regulation Of Autolysis In Staphylococcus Aureus, Adhar C. Manna, Susham S. Ingavale, Marybeth Maloney, Willem Van Wamel, Ambrose L. Cheung

Dartmouth Scholarship

The expression of genes involved in the pathogenesis of Staphylococcus aureus is known to be controlled by global regulatory loci, including agr, sarA, sae, arlRS, lytSR, and sarA-like genes. Here we described a novel transcriptional regulator called sarV of the SarA protein family. The transcription of sarV is low or undetectable under in vitro conditions but is significantly augmented in sarA and mgrA (norR or rat) (SA0641) mutants. The sarA and mgrA genes act as repressors of sarV expression, as confirmed by transcriptional fusion and Northern analysis data. Purified SarA and MgrA proteins bound specifically to separate regions of the …

The Assessment Of Antibiotic Resistance And Detection Of Class 1 And Class 2 Integrons In Fecal Coliform Bacteria Isolated From The Triplett Creek Watershed, Rowan County, Kentucky, Tina Howard Ousley

Morehead State Theses and Dissertations

A thesis presented to the faculty of the College of Science and Technology at Morehead State University in partial fulfillment of the requirements for the Degree of Master of Science by Tina Howard Ousley on July 15, 2004.

Sadb Is Required For The Transition From Reversible To Irreversible Attachment During Biofilm Formation By Pseudomonas Aeruginosa Pa14, Nicky C. Caiazza, George A. O'Toole

Dartmouth Scholarship

Current models of biofilm formation by Pseudomonas aeruginosa propose that (i) planktonic cells become surface associated in a monolayer, (ii) surface-associated cells form microcolonies by clonal growth and/or aggregation, (iii) microcolonies transition to a mature biofilm comprised of exopolysaccharide-encased macrocolonies, and (iv) cells exit the mature biofilm and reenter the planktonic state. Here we report a new class of P. aeruginosa biofilm mutant that defines the transition from reversible to irreversible attachment and is thus required for monolayer formation. The transposon insertion carried by the sadB199 mutant was mapped to open reading frame PA5346 of P. aeruginosa PA14 and encodes …

Pcr And 16s Rdna Sequencing For Identifying And Comparing Gram-Negative Bacteria, Molly Truax

Honors Capstones

Capstone submitted as a graduation requirement for the BSU Honors Program.

Caenorhabditis Elegans: A Look At Koch's Postulates And Host-Pathogen Interactions, Nicole Helmrichs

Honors Capstones

Capstone submitted as a graduation requirement for the BSU Honors Program.

Methods For Detection Of Salmonella Spp., Parichaya Attaphongse

All Graduate Plan B and other Reports, Spring 1920 to Spring 2023

Worldwide concern about food safety and associated health costs is increasing, and Salmonella contamination of foods is one of the most important causes of foodborne disease outbreaks. Although conventional detection methods for Salmonella offer high sensitivity and low cost, they require many different steps and are very time consuming. Ideally, methods to detect Salmonella in foods should be rapid, provide high specificity and sensitivity, be cost-effective, and low labor-intensive. Many rapid tests have been developed to address these goals, including DNA-based tests such as nucleic acid hybridization and PCR, immunoassay-based tests such as ELISA, ELF A and immunomagnetic method, and …

Rapid Detection Of Salmonella Without Enrichment, Emily J. Harrington

All Graduate Theses and Dissertations, Spring 1920 to Summer 2023

Salmonella is one of the leading foodborne pathogens causing illness today. Because of this, Salmonella rapid detection methods are under immense study for use in food. The traditional method, using the Food and Drug Administration- approved Bacterial Analytical Manual procedure, takes 4-6 days for Salmonella detection in food. Other rapid methods still take at least 16 h for detection due to their enrichment steps.

The hypothesis of this study was that the use of immobilized antibodies coupled with polymerase chain reaction (PCR) can be used for the rapid capture and detection of Salmonella spp. in food without the need for …

Isolation And Characterization Of A Generalized Transducing Phage For Pseudomonas Aeruginosa Strains Pao1 And Pa14, Jonathan M. Budzik, William A. Rosche, Arne Rietsch, George A. O'Toole

Dartmouth Scholarship

A temperate, type IV pilus-dependent, double-stranded DNA bacteriophage named DMS3 was isolated from a clinical strain of Pseudomonas aeruginosa. A clear-plaque variant of this bacteriophage was isolated. DMS3 is capable of mediating generalized transduction within and between P. aeruginosa strains PA14 and PAO1, thus providing a useful tool for the genetic analysis of P. aeruginosa.

Development And Characterization Of Eukaryotic Biomimetic Liposomes, Bradley Jay Taylor

All Graduate Theses and Dissertations, Spring 1920 to Summer 2023

This study developed and characterized phospholipid vesicles, or liposomes, that mimic cell surfaces. Microemulsified liposomes contained biotinylated phosphatidylethanolamine, allowing them to be immobilized to avidin-coated glass. Laminin (LN), glycosphingolipids (GMl and GM3), and Escherichia coli's mechanosensitive channel of large conductance (EcoMscL) were embedded into liposome membranes. It was determined whether these embedded molecules exhibited their physiological roles of adhesion, cell recognition, and mechanosensation, respectively. Confocal laser scanning microscopy (CLSM) was employed to examine the interaction of fluorescently probed proteins, toxins, and bacteria with the immobilized microemulsified liposomes. Capture of individual and simultaneous multiple species of bacteria by GMl, GM3, …

Physiology Of The Thermophilic Acetogen Moorella Thermoacetica, Harold L. Drake, Steven L. Daniel

Faculty Research & Creative Activity

Moorella thermoacetica (originally isolated as Clostridium thermoaceticum) has served as the primary acetogenic bacterium for the resolution of the acetyl coenzyme A (acetyl-CoA) orWood–Ljungdahl pathway, a metabolic pathway that (i) autotrophically assimilates CO2 and (ii) is centrally important to the turnover of carbon in many habitats. The purpose of this article is to highlight the diverse physiological features of this model acetogen and to examine some of the consequences of its metabolic capabilities.

Physiology Of The Thermophilic Acetogen Moorella Thermoacetica, Harold Drake, Steven Daniel

Faculty Research & Creative Activity

Moorella thermoacetica (originally isolated as Clostridium thermoaceticum) has served as the primary acetogenic bacterium for the resolution of the acetyl coenzyme A (acetyl-CoA) orWood–Ljungdahl pathway, a metabolic pathway that (i) autotrophically assimilates CO2 and (ii) is centrally important to the turnover of carbon in many habitats. The purpose of this article is to highlight the diverse physiological features of this model acetogen and to examine some of the consequences of its metabolic capabilities.

A Dominant-Negative Fur Mutation In Bradyrhizobium Japonicum, Heather P. Benson, Kristin Levier, Mary Lou Guerinot

Dartmouth Scholarship

In many bacteria, the ferric uptake regulator (Fur) protein plays a central role in the regulation of iron uptake genes. Because iron figures prominently in the agriculturally important symbiosis between soybean and its nitrogen-fixing endosymbiont Bradyrhizobium japonicum, we wanted to assess the role of Fur in the interaction. We identified a fur mutant by selecting for manganese resistance. Manganese interacts with the Fur protein and represses iron uptake genes. In the presence of high levels of manganese, bacteria with a wild-type copy of the furgene repress iron uptake systems and starve for iron, whereas fur mutants fail to …

Detection Of Bacteria To Dental Tissue By Low-Light Imaging, G-Hong Robert Hsu

Loma Linda University Electronic Theses, Dissertations & Projects

Bacteria have been shown to play an important role in the development of pulp and periradicular disease. The source of the infective bacteria is most often the oral cavity. Previous studies have detected bacteria in teeth that are structurally intact that seemingly have no communication intraorally. Anachoresis has been proposed as a mechanism for localizing circulating bacteria to such teeth. In several previous bacteriologic anachoresis studies, bacteria have been found to localize in inflamed dental tissues. The criticism most commonly associated with these investigations has been the potential contribution of contaminant bacteria resulting in false positive findings.

Low-light image analysis …

Oxalate Metabolism By The Acetogenic Bacterium Moorella Thermoacetica, Steven L. Daniel, Christine Pilsl, Harold L. Drake

Faculty Research & Creative Activity

Whole-cell and cell-extract experiments were performed to study the mechanism of oxalate metabolism in the acetogenic bacterium Moorella thermoacetica. In short-term, whole-cell assays, oxalate consumption was low unless cell suspensions were supplemented with CO2, KNO3, or Na2S2O3. Cell extracts catalyzed the oxalate-dependent reduction of benzyl viologen. Oxalate consumption occurred concomitant to benzyl viologen reduction; when benzyl viologen was omitted, oxalate was not appreciably consumed. Based on benzyl viologen reduction, specific activities of extracts averaged 0.6 μmol oxalate oxidized min−1 mg protein−1. Extracts also catalyzed the formate-dependent reduction of NADP+; however, oxalate-dependent reduction of NADP+ was negligible. Oxalate- or formate-dependent reduction …

Oxalate Metabolism By The Acetogenic Bacterium Moorella Thermoacetica, Steven Daniel, Christine Pilsl, Harold Drake

Faculty Research & Creative Activity

Whole-cell and cell-extract experiments were performed to study the mechanism of oxalate metabolism in the acetogenic bacterium Moorella thermoacetica. In short-term, whole-cell assays, oxalate consumption was low unless cell suspensions were supplemented with CO2, KNO3, or Na2S2O3. Cell extracts catalyzed the oxalate-dependent reduction of benzyl viologen. Oxalate consumption occurred concomitant to benzyl viologen reduction; when benzyl viologen was omitted, oxalate was not appreciably consumed. Based on benzyl viologen reduction, specific activities of extracts averaged 0.6 μmol oxalate oxidized min−1 mg protein−1. Extracts also catalyzed the formate-dependent reduction of NADP+; however, oxalate-dependent reduction of NADP+ was negligible. Oxalate- or formate-dependent reduction …

Microbial Endemism And Biogeography, Brian P. Hedlund, James T. Staley

Life Sciences Faculty Research

The topic of microbial biogeography is almost 100 years old, however, when confronted with questions about the existence and extent of endemism in the microbial world, many microbiologists respond with opinions and theoretical arguments rather than examples of well-conducted studies. We begin this chapter with an overview of this debate as it applies to free-living prokayotes in part because there are relatively few good microbial biogeography studies. Furthermore, the arguments help to frame microbial biogeography in the larger context of biodiversity in that if endemism is common, then many more species exist.

Estimation Of Microbial Diversity In Poultry Litter Using Terminal Restriction Fragment Length Polymorphism And Isolation Of Phosphate Accumulating Bacteria From Poultry Litter, Yoganand Vadari

Masters Theses & Specialist Projects

The contamination of fresh water by phosphates in poultry litter results in substantial eutrophication of fresh water causing fish kills and other types of environmental damage. The poultry indus try in Kentucky is expanding rapidly. The number of broilers is increasing as more poultry farms are established in the state producing waste that needs disposal. Investigations were made to study the possibility of using microorganisms normally found in poultry litter to sequester phosphate, thereby delaying phosphate runoff after litter is applied to croplands. Little is known, however, about the microflora of poultry litter. Terminal restriction fragment length polymorphism of 16S …

Ultrastructure Of Mycobacterium Marinum Granuloma In Striped Bass Morone Saxatilis, D. T. Gauthier, W. K. Vogelbein, C. A. Ottinger

Biological Sciences Faculty Publications

An emerging epizootic of mycobacteriosis currently threatens striped bass Morone saxatilis populations in Chesapeake Bay, USA. Several species of mycobacteria, including Mycobacterium marinum, species resembling M. avium, M. gordonae, M. peregrinum, M. scrofulaceum and M. terrae, and the new species M. shottsii have been isolated from diseased and healthy bass. In this study, we describe the ultrastructure of developing M. marinum granulomas in experimentally infected bass over a period of 45 wk. The primary host response to injected mycobacteria was formation of large macrophage aggregations containing phagocytosed bacilli. M. marinum were always contained within phagosomes. Close …