Microbiology Commons^™

Fecal Indicator Bacteria In Knitting Mill Creek With A Comparison Of Methods Used To Enumerate E. Coli, Manju Nagarajan

OES Theses and Dissertations

This research seeks to evaluate the impact of an urban, multi-use neighborhood on the bacteriological quality of water in Knitting Mill Creek, a blind arm of the Lafayette River, a sub-estuary of the lower Chesapeake Bay. A principal objective was to determine the effects of rainfall, a surrogate for run-off, on water-column concentrations of fecal-indicator bacteria (FIB). I collected water samples weekly (September 2009-December 2010) and biweekly (January 2010-December 2011) at a storm-sewer outfall and a nearby marina and quantified their E. coli and enterococci concentrations using Colilert-18 and Enterolert, most-probable-number assays. In addition, I took monthly samples for these …

An Investigation Of Bacterial Ribonucleases As An Antibiotic Target, Ashley Denise Frazier

Electronic Theses and Dissertations

Antibiotics have been commonly used in medical practice for over 40 years. However, the misuse and overuse of current antibiotics is thought to be the primary cause for the increase in antibiotic resistance.

Many current antibiotics target the bacterial ribosome. Antibiotics such as aminoglycosides and macrolides specifically target the 30S or 50S subunits to inhibit bacterial growth. During the assembly of the bacterial ribosome, ribosomal RNA of the 30S and 50S ribosomal subunits is processed by bacterial ribonucleases (RNases). RNases are also involved in the degradation and turnover of this RNA during times of stress, such as the presence of …

Toxin-Antitoxic Loci Vapbc-1 And Vapxd Contribute To Survival And Virulence In Nontypeable Haemophilus Influenzae, Dabin Ren, Anna N. Walker, Dayle A. Daines

Biological Sciences Faculty Publications

Background: Nontypeable Haemophilus influenzae (NTHi) is a significant human pathogen responsible for respiratory tract infections and the most common cause of recurrent otitis media. Type II toxin-antitoxin (TA) systems are genetic elements that code for a stable protein toxin and a labile antitoxin that are thought to be involved in metabolic regulation of bacteria by enabling a switch to a dormant state under stress conditions. The contribution to infection persistence of the NTHi TA loci vapBC-1 and vapXD was examined in this study.

Results: Deletions in vapBC-1, vapXD and vapBC-1 vapXD significantly decreased the survival of NTHi co-cultured with …

Modulation Of Alpha-Subunit Visit-Dg Sequence Residues Ser-347, Gly-351 And Thr-349 In The Catalytic Sites Of Escherichia Coli Atp Synthase., Laura Elaine Brudecki

Electronic Theses and Dissertations

Binding of inorganic phosphate (P_i) in ATP synthase catalytic sites is a crucial step for the synthesis of adenosine-5'-triphosphate (ATP). ATP is the fundamental means of cellular energy in almost every organism, and in order to gain insight into the regulation of ATP catalysis, critical amino acid residues responsible for binding P_i must be identified. Here, we investigate the role of highly conserved α-subunit VISIT-DG sequence residues αSer-347, αGly-351, and αThr-349 in P_i binding. Mutations αS347A/Q, αG351Q, αT349A/D/R, βR182A, and αT349R/βR182A were generated via site directed mutagenesis. Results from biochemical assays showed that αSer-347 is required …

Interaction Between Bacteriophage Dms3 And Host Crispr Region Inhibits Group Behaviors Of Pseudomonas Aeruginosa, Michael E. Zegans, Jeffrey C. Wagner, Kyle C. Cady, Daniel M. Murphy, John H. Hammond, George A. O'Toole

Dartmouth Scholarship

Bacteriophage infection has profound effects on bacterial biology. Clustered regular interspaced short palindromic repeats (CRISPRs) and cas (CRISPR-associated) genes are found in most archaea and many bacteria and have been reported to play a role in resistance to bacteriophage infection. We observed that lysogenic infection of Pseudomonas aeruginosa PA14 with bacteriophage DMS3 inhibits biofilm formation and swarming motility, both important bacterial group behaviors. This inhibition requires the CRISPR region in the host. Mutation or deletion of five of the six cas genes and one of the two CRISPRs in this region restored biofilm formation and swarming …

The Structural And Folding Characteristics Of The Plasmid-Encoded Toxin From Enteroaggregative Escherichia Coli, Patricia Scaglione

Electronic Theses and Dissertations

Plasmid-encoded toxin (Pet) from enteroaggregative Escherichia coli is a member of the autotransporter subfamily termed SPATE (serine protease autotransporters of Enterobacteriaceae). Autotransporters, which are the most common Gram-negative secreted virulence factors, contain three functional domains: an amino terminal leader sequence, a mature protein or passenger domain, and a carboxy-terminal β domain. The leader sequence targets the protein to the periplasmic space and the β domain then forms a β-barrel pore in the outer membrane of the bacterium which allows the passenger domain to enter the external milieu. In some cases the passenger domain is cleaved from the β-barrel at the …

A Serratia Marcescens Oxyr Homolog Mediates Surface Attachment And Biofilm Formation, Robert M. Q. Shanks, Nicholas A. Stella, Eric J. Kalivoda, Megan R. Doe

Dartmouth Scholarship

OxyR is a conserved bacterial transcription factor with a regulatory role in oxidative stress response. From a genetic screen for genes that modulate biofilm formation in the opportunistic pathogen Serratia marcescens, mutations in an oxyR homolog and predicted fimbria structural genes were identified. S. marcescens oxyR mutants were severely impaired in biofilm formation, in contrast to the hyperbiofilm phenotype exhibited by oxyR mutants of Escherichia coli and Burkholderia pseudomallei. Further analysis revealed that OxyR plays a role in the primary attachment of cells to a surface. Similar to what is observed in other bacterial species, S. marcescens OxyR …

Membrane Association And Multimerization Of Tcpt, The Cognate Atpase Ortholog Of The Vibrio Cholerae Toxin-Coregulated-Pilus Biogenesis Apparatus, Shital A. Tripathi, Ronald K. Taylor

Dartmouth Scholarship

The toxin-coregulated pilus (TCP) is one of the major virulence factors of Vibrio cholerae. Biogenesis of this type 4 pilus (Tfp) requires a number of structural components encoded by the tcp operon. TcpT, the cognate putative ATPase, is required for TCP biogenesis and all TCP-mediated functions. We studied the stability and localization of TcpT in cells containing in-frame deletions in each of the tcp genes. TcpT was detectable in each of the biogenesis mutants except the ΔtcpT strain. TcpT was localized to the inner membrane (IM) in a TcpR-dependent manner. TcpR is a predicted bitopic inner membrane protein …

Susceptibility Of Biofilms To Bdellovibrio Bacteriovorus Attack, Daniel Kadouri, George A. O'Toole

Dartmouth Scholarship

Biofilms are communities of microorganisms attached to a surface, and the growth of these surface attached communities is thought to provide microorganisms with protection against a range of biotic and abiotic agents. The capability of the gram-negative predatory bacterium Bdellovibrio bacteriovorus to control and reduce an existing Escherichia coli biofilm was evaluated in a static assay. A reduction in biofilm biomass was observed as early as 3 h after exposure to the predator, and an 87% reduction in crystal violet staining corresponding to a 4-log reduction in biofilm cell viability was seen after a 24-h exposure period. We observed that …

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 …

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.

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.

Comparison Of Methods For Dna Isolation From Food Samples For Detection Of Shiga Toxin-Producing Escherichia Coli By Real-Time Pcr, Loree C. Heller, Carisa R. Davis, K. Kealy Peak, David Wingfield, Andrew C. Cannons, Philip T. Amuso, Jacqueline Cattani

Bioelectrics Publications

In this study, food samples were intentionally contaminated with Escherichia coli O157:H7, and then DNA was isolated by using four commercial kits. The isolated DNA samples were compared by using real-time PCR detection of the Shiga toxin genes. The four kits tested worked similarly.

Mortality Of Escherichia Coli O157:H7 In Two Soils With Different Physical And Chemical Properties, D. N. Mubiru, Mark S. Coyne, John H. Grove

Plant and Soil Sciences Faculty Publications

Wild and domesticated animals can harbor a pathogenic Escherichia coli strain designated as O157:H7. Potential health problems could occur if strain O157:H7 is a more robust survivor in defecated waste than commonly used indicator bacteria. A laboratory study was conducted to assess E. coli O157:H7 survival relative to a nonpathogenie E. coli strain in two soils with different physical and chemical characteristics. Bacteria in the inoculated soils were enumerated on a weekly basis for 8 wk using a most probable number (MPN) technique. First-order decay models were used to describe bacteria mortality in the soils. Decay series were described slightly …

Differential Activation Of The Tcpph Promoter By Aphb Determines Biotype Specificity Of Virulence Gene Expression In Vibrio Cholerae, Gabriela Kovacikova, Karen Skorupski

Dartmouth Scholarship

Vibrio cholerae strains of the classical biotype express the genes encoding cholera toxin (CT) and toxin- coregulated pilus (TCP) under a variety of environmental conditions in vitro, whereas El Tor biotype strains express these genes only under specialized culture conditions. We show here that a single base-pair difference at positions 2 65 and 2 66 of the classical and El Tor tcpPH promoters, respectively, is responsible for the differential regulation of virulence gene expression in these two disease-causing biotypes. Analysis of tcpP-lacZ fusions in both V. cholerae and Escherichia coli indicated that transcriptional activation of the El Tor tcpPH promoter …

Construction Of An Escherichia Coli Lac Repressor-Based System To Study Human Cytomegalovirus Gene Expression, Laura Fernanda Cageao-Luchetti

Theses and Dissertations in Biomedical Sciences

An Escherichia coli lac repressor-based system was developed to study the roles of human cytomegalovirus (HCMV) genes during viral replication. To this end, a recombinant HCMV expressing the lac repressor was generated (RVlac), and an HCMV-specific promoter was targeted for conditional expression by inserting the lac operator sequence. The promoter of a nonessential gene was chosen in order to be able to assess parameters of repression and derepression of the operator-containing promoter in the endogenous locus, without having virus growth dependent on the specific inducer isopropylthiogalactoside (IPTG). The feasibility of this approach to conditionally express an HCMV promoter …

Estimation Of Diversity And Community Structure Through Restriction-Fragment-Length-Polymorphism Distribution Analysis Of Bacterial 16s Ribosomal-Rna Genes From A Microbial Mat At An Active, Hydrothermal Vent System, Loihi Seamount, Hawaii, Craig L. Moyer, Fred C. Dobbs, David M. Karl

OES Faculty Publications

PCR was used to amplify (eu)bacterial small-subunit (16S) rRNA genes from total-community genomic DNA. The source of total-community genomic DNA used for this culture-independent analysis was the microbial mats from a deep-sea, hydrothermal vent system, Pele's Vents, located at Loihi Seamount, Hawaii. Oligonucleotides complementary to conserved regions in the 16S rRNA-encoding DNA (rDNA) of bacteria were used to direct the synthesis of PCR products, which were then subcloned by blunt-end ligation into phagemid vector pBluescript II. Restriction fragment length polymorphism patterns, created by using tandem tetrameric restriction endonucleases, revealed the presence of 12 groups of 16S rRNA genes representing discrete …

Frequency Of Mug Negative Escherichia Coli In Kentucky Groundwater Samples, Mark S. Coyne, J. C. Shuler

Plant and Soil Sciences Faculty Publications

MUG negative Escherichia coli are a small fraction (2.5%) of the total E. coli in Kentucky groundwater samples. It is unlikely that they alone will cause a significant potential to underestimate fecal contamination using MUG as the primary criterion for that assessment. An unresolved question is how effectively MUG-based, defined-substrate tests address false negative water samples containing MUG positive E. coli.

Cole1 Copy Number Mutants., Londa Schmidt, Joseph Inselburg

Dartmouth Scholarship

A deletion mutant of the colicin E1-derived plasmid, pDMS6642, exhibited an approximately fourfold increase in copy number. We subsequently isolated hydroxylamine-induced mutants of that plasmid that had a further increase in copy number. Analysis of them suggests that the increased copy number of pDMS6642 is associated with transcriptional readthrough from a Tn3 transposon into the region of ColE1 containing information that influences plasmid replication. The hydroxylamine mutation in one copy number mutant appeared to increase the plasmid copy number by stimulating readthrough transcription from the Tn3 transposon into the ColE1 replication control region, whereas the other hydroxylamine mutation acts by …

Selection And Characterization Of Cole1 Plasmid Mutants That Exhibit Altered Stability And Replication., Joseph Inselburg

Dartmouth Scholarship

This report describes a method for isolating mutants of plasmid ColE1 that exhibit unstable maintenance and altered replication characteristics. It also describes the initial characterization of four mutants isolated by that method. A chimeric plasmid, pHSG124, containing a ColE1 derivative and a temperature-sensitive replication derivative of pSC101 was mutagenized in vitro, using hydroxylamine. By adjusting the growth conditions of transformants containing the mutagenized chimeric deoxyribonucleic acid, it was possible to rapidly screen colonies and identify those that had a high probability of carrying ColE1 mutants that exhibit unstable maintenance. Of those mutants, some exhibited altered copy number or accumulated catenated …

A Genetic And Physiological Study Of An Arsenite Resistant, Uncoupled Mutant Of Escherichia Coli, Daniel Gordon Smiley

Dissertations and Theses

Chromosomally determined arsenate resistance in Escherichia coli is well characterized. Little, however, is known about chromosomally determined arsenite resistance in E. coli. Accordingly, spontaneous arsenite resistant mutants were selected in a plasmid free strain of E. coli. One mutant strain was analyzed in detail, genetically and physiologically. The mutation confering arsenite resistance was shown to be a single gene mutation. Genetic mapping studies using conjugation and transduction showed that the mutation was closely linked to the ilv region of the E. coli map.

Physiological studies of this mutant showed that it shared characteristics common to those of mutants …

Plasmid-Mediated Resistance To Arsenite And Arsenate In Escherichia Coli, Thomas Patrick Trezona

Dissertations and Theses

Bacterial strains resistant to arsenite, arsenate (or both) are readily isolated from nature. The resistance, in many cases, can be shown to be determined by conjugal plasmids. The mechanisms by which these plasmids confer resistance to arsenic compounds are entirely unknown. This study was undertaken to shed light on some of the physiological and genetic aspects of this resistance using plasmid-bearing strains of Escherichia coli which are highly resistant to both arsenite and arsenate. Most studies were carried out using E. coli J53 (R773) or the plasmid-free strain J53.

Functional Relationship Between Parts Of The Replication Region Of Plasmid Cole1., Joseph Inselburg

Dartmouth Scholarship

The inhibition of plasmid ColE1 replication caused by a deletion of the ColE1 plasmid replication origin has been previously reported (T. Hashimoto-Gotoh and J. Inselburg, J. Bacteriol. 139:597-619). Evidence is presented showing that restoration of the deleted nucleotide sequence in the precise relationship it normally has to the rest of the replication region is essential for restoration of ColE1 replication capability to the deletion mutant.

Repression Of Β-Galactosidase Synthesis In Escherichia Coli By Salicylates, Joan Carlyn Olson

Dissertations and Theses

Salicylic acid, and to a lesser extent aspirin, have been shown to repress β-galactosidase synthesis in Escherichia coli. The repression is not due to decreased inducer uptake, nor does it result from competition with inducer for repressor. Dinitrophenol does not exert similar effects on β-galactosidase synthesis. Cyclic adenosine 31, 51-monophosphate partially relieves the repression. The extent or the relief by cyclic adenosine monophosphate seems to depend on the concentration of salicylic acid. This indicates there may be direct interaction between the actions of cyclic adenosine monophosphate and salicylic acid in E. coli cells.

Circadian Organization And Microbiology: Variance Spectra And A Periodogram On Behavior Of Escherichia Coli Growing In Fluid Culture, Franz Halberg, Robert L. Conner

Journal of the Minnesota Academy of Science

Over three decades ago Rogers and Greenbank (1) published observations on intermittent growth in a bacterial culture. These interesting data are analyzed further in this report by computational procedures ( 2-8) designed for detecting and evaluating the significance of nearly periodic phenomena. By such methods, circadian (about 24-hour) periodicity in a culture of E. coli is unmasked as a significant component of the recorded changes. These results extend the already broad scope of circadian temporal organization in microbiology.