Bacteriology Commons^™

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 …

Virulence Factors Of The Group A Streptococci And Genes That Regulate Their Expression, Wayne Hines

Biological Sciences Faculty Publications

Group A streptococci produce a wide variety of extracellular (cell-associated and released) virulence factors. The function of these factors varies and includes roles in adhesion, spreading, tissue destruction, immune system evasion, and cell toxicity. How these factors are regulated with regard to one another is important for this organism's ability to bring about the variety of diseases this microbe is capable of causing. Therefore, along with the multitude of virulence factors, there are a number of regulatory systems that regulate, either directly or indirectly, the production of these factors and therefore influence the pathogenesis of group A streptococcal infections.

A Survey Of Antibiotic Resistance Among Coliform Bacteria Isolated From The Missouri River, Sara E. Mcdonnell, Amy M. Treonis

Biology Faculty Publications

The prevalence of antibiotic resistant microorganisms in the environment is not well known but could represent a challenge to maintaining public health in the future. Fecal waste from livestock facilities, where antibiotics are routinely used, is recognized as a significant source of pollution to surface waters in the United States. We collected water from the Missouri River across a winter to spring seasonal change in order to survey the density of coliform bacteria. We tested bacterial isolates for resistance to penicillin, tetracycline, ampicillin, erythromycin, and ciprofloxacin. Coliform density in Missouri River water generally was high throughout the study (0-405 cfu …

Identification And Characterization Of A Nontypeable Haemophilus Influenzae Putative Toxin-Antitoxin Locus, Dayle A. Daines, Justin Jarisch, Arnold L. Smith

Biological Sciences Faculty Publications

Background: Certain strains of an obligate parasite of the human upper respiratory tract, nontypeable Haemophilus influenzae (NTHi), can cause invasive diseases such as septicemia and meningitis, as well as chronic mucosal infections such as otitis media. To do this, the organism must invade and survive within both epithelial and endothelial cells. We have identified a facilitator of NT(Hi) survival inside human cells, virulence-associated protein D (vapD_Hi, encoded by gene H10450). Both vapD_Hi and a flanking gene, H10451, exhibit the genetic and physical characteristics of a toxin/antitoxin ( TA) locus, with VapD_Hi serving as the …

Isolation And Characterization Of Mycobacteria From Striped Bass Morone Saxatilis From The Chesapeake Bay, Martha W. Rhodes, Howard Kator, Ilsa Kaattari, David Gauthier, Wolfgang Vogelbein, Christopher A. Ottinger

Biological Sciences Faculty Publications

Mycobacteriosis in striped bass Morone saxatilisof Chesapeake Bay, USA, was first diagnosed in 1997 based on the presence of granulomatous inflammation and acid-fast bacteria in skin and spleen. To confirm histopathology, bacteriological detection and identification of mycobacteria were begun using splenic tissue from fish with and without skin ulcerations. On the basis of initial studies using a variety of selective and nonselective media, decontamination, homogenization and incubation conditions, a simple and quantitative recovery method using aseptic necropsy of splenic tissue was developed. Optimal recovery was obtained by spread-plating homogenates on Middlebrook 7H10 agar with incubation for 3 mo at …