Bacteriology Commons^™

Homocysteine Thiolactone From Translation Proofreading Is An Endogenous Ligand For Cell-Cell Signaling By The Receptor Sdia, Lorenzo Eugenio Leiva, Roberto A. Zúñiga, Gregory J. Phillips, Michael Ibba

Biology, Chemistry, and Environmental Sciences Faculty Articles and Research

Several widespread mechanisms enable cellular communication and coordinate the behavior of bacterial populations. The modules responsible for cellular communication are often found in matching pairs: an enzyme that synthesizes a signal molecule and a specific receptor that decodes it. A notable exception is Escherichia coli SdiA, a homolog of known N-acyl-homoserine lactone (AHL) receptors, for which no corresponding AHL synthase exists. SdiA is an orphan receptor that enables cross-talk by sensing AHLs from other bacterial species. In this work, we investigated whether homocysteine thiolactone (HTL), an AHL-like molecule arising from a proofreading reaction of methionyl tRNA synthetase (MetRS) to correct …

Regulation And Localization Of Urease In Sporosarcina Pasteurii: Implications For Coastal Protection, Amar Kosovac

UNF Graduate Theses and Dissertations

Coastal erosion is a persistent problem exacerbated by global warming. As the climate changes, the southeastern coast is likely to experience an increased frequency and intensity of storms, which can significantly damage the coastline. The coast is crucial as it serves as a habitat for numerous animals, supports tourism, and underpins infrastructure. Microbially induced calcite precipitation (MICP) is a novel method to enhance erosion resistance in sandy soils using the non-pathogenic bacterium Sporosarcina pasteurii. S. pasteurii produces urease to catalyze urea into bicarbonate, which drives the precipitation of calcium carbonate. Maximal expression of urease is a limiting factor for …

Transcription Factor Binding And Discovery Of Novel Gene In The Opportunistic Human Pathogen, Pseudomonas Aeruginosa, Alaina Westee

Symposium of Student Scholars

Pseudomonas aeruginosa (PA) is a gram-negative, ubiquitously-found bacterium that primarily causes nosocomial, or hospital-borne, infections within immunocompromised patients. Therefore, it has been deemed a critical priority pathogen by the World Health Organization (WHO) and the Center for Disease Control (CDC), generating a need for research on its fundamental biology. The Van Dyke laboratory focuses on proteins called transcription factors, which promote or repress gene expression to regulate an organism’s functioning. We are specifically studying the cadmium-responsive transcription factor, CadR, due to the importance of metals in virulence and the lack of information on PA’s response to metal fluctuations. We intend …

The Physiological Significance Of Nitric Oxide Synthase In Staphylococci, Ryan M. Singh

Theses & Dissertations

Staphylococci, particularly Staphylococcus epidermidis, are common causes of implant-associated infections that may result in notable morbidity and mortality. One of the ways the innate immune system attempts to combat these invading pathogens is via the respiratory burst, in which free radical species such as nitric oxide (NO^•), toxic to bacterial respiration, are released in large quantities. Despite the toxicity of NO^•, some bacteria, including staphylococci, encode an endogenous bacterial nitric oxide synthase (bNOS), whose physiological role has been unclear until now. In this dissertation, we demonstrate that S. epidermidis is able to overcome the toxicity …

Characterizing The Role Of Pa5189 Of Pseudomonas Aeruginosa In Deletion And Overexpression Mutants, Seh Na Mellick

Theses/Capstones/Creative Projects

In the context of rising multidrug resistance in biofilm-forming pathogens like Pseudomonas aeruginosa, this study investigates the role of the understudied transcription factor PA5189 in antibiotic resistance and biofilm formation. PA5189 deletion and overexpression mutants were created in a parent P. aeruginosa strain using pEX18Tc-based recombinant suicide vectors, with genotypic verification of putative triparental conjugants achieved through restriction digestion and PCR. The study revealed that PA5189 overexpression significantly increases resistance to commonly used broad spectrum antibiotics such as ciprofloxacin and imipenem. Additionally, differential expression of PA5189 was found to notably affect biofilm formation, with variations contingent on the nutrient …

Ribosome Hibernation Factor Induces Antibiotic Persisters Of Mycobacterium Tuberculosis In Neutrophil-Rich Lung Lesions, Jamie Corro

Electronic Theses & Dissertations (2024 - present)

Mycobacterium tuberculosis (Mtb), the etiological agent of tuberculosis, is a prescient global health threat and the leading cause of death by an infectious agent. Treatment of Mtb infection involves administering 2-4 antibiotics for a minimum of 6 months. This extended drug regimen is required to target a subpopulation of persistent bacilli. As their name implies, these bacteria can “persist” by acquiring nonhereditary and transient antibiotic tolerance. Although the precise mechanism is unknown, it’s often attributed to slowing cellular growth and metabolic processes. During zinc deprivation, bacteria can remodel their ribosomes by replacing their zinc-binding paralogues containing the CXXC motif (C+) …

Spatial Structure Formation By The Post-Transcriptional Gene Regulator Rsme In Pseudomonas Fluorescens Pf0-1, Anton Evans

Electronic Theses and Dissertations

Microorganisms are often found in microbial communities we call biofilms. Organisms living in these crowded environments have significant evolutionary pressure to retain access to the resources necessary to sustain life. My research uses the bacterium Pseudomonas fluorescens Pf0-1 to study how organisms evolve strategies to solve this crowding problem as aging colonies repeatedly generate mutant patches. These mutants expand the reach of the colony resulting in decreased local density as they push themselves up to the resource rich surface. These spatial structures result from social interactions between the mutant and the parental cells mediated through extracellular secretions, resulting in the …

Diagnosis Of Urinary Tract Infections And Rapid Molecular Characterization Of Antibiotic Resistance, Mohammed Harris

All Dissertations

Urinary tract infections (UTIs) are one of the most common infectious clinical entities in both community and hospital settings. They have a broad range of clinical severity yet inflict large epidemiological burden of morbidity and mortality on patients and the healthcare system with billions of dollars in cost of treatment. Understanding what methods are optimal for diagnosing UTIs are critical to mitigate the marked impact and cost of these infections.

Chapter 1 and 2 in this work surveys the broad array of diagnostic modalities for UTIs and highlights their advantages and limitations in the context of the current standard of …

Protocol To Identify The Core Gene Supported By An Essential Gene In E. Coli Bacteria Using A Genome-Wide Suppressor Screen, Isao Masuda, Ya-Ming Hou

Department of Biochemistry and Molecular Biology Faculty Papers

We describe here a genome-wide screening approach to identify the most critical core reaction among a network of many that are supported by an essential gene to establish cell viability. We describe steps for maintenance plasmid construction, knockout cell construction, and phenotype validation. We then detail isolation of suppressors, whole-genome sequencing analysis, and reconstruction of CRISPR mutants. We focus on E. coli trmD, which encodes an essential methyl transferase that synthesizes m1G37 on the 3'-side of the tRNA anticodon. For complete details on the use and execution of this protocol, please refer to Masuda et al. (2022).

Resolving The Repression Pathway Of Virulence Gene Hila In Salmonella, Alexandra King, Lon Chubiz Phd, Brenda Pratte, Lauren Daugherty

Undergraduate Research Symposium

Salmonella is a relatively abundant, virulent species of bacteria that is most known for spreading gastrointestinal diseases through food. These illnesses result in approximately 1.35 million infections, including over 25,000 hospitalizations each year, in the U.S. alone (CDC.gov). As antibiotic resistance becomes an increasingly urgent public health problem, the importance of developing alternative treatment methods is only becoming more crucial. One of the genes responsible for this virulence is known as hilA. HilA is the main transcriptional regulator of Salmonella Pathogenicity Island-1 gene (UniProt). SPI-1 plays an important role in the invasion of Salmonella into epithelial cells. The proteins encoded …

Characterization Of A Medium-Dependent Streptomyces Developmental Mutant, Dylan Gray, Jennifer A. Bennett

Undergraduate Distinction Papers

Streptomyces coelicolor is a soil-dwelling, Gram-positive Actinobacteria. Streptomyces species produce thousands of secondary metabolites which include many antibiotics used in medicine today. In Streptomyces development, as colonies grow, secondary metabolites are produced, and ultimately mature spores are dispersed. The production of each of these secondary metabolites in wild-type Streptomyces is determined by a distinct biosynthetic pathway. In order to identify new genes for antibiotic production and other aspects of development, an engineered Tn5-derived transposon was used to mutagenize wild-type cells in vivo and produce single, stable insertions.

Colonies that were visually distinct from the wild type were selected for further …

Sars-Cov-2 Reveals That Chimeric Agents Are The Bioweapons Of The Future, Rachel Craig

Senior Honors Theses

Bioweapons programs have existed since their development during the Cold War. These biowarfare programs initially utilized naturally occurring pathogens capable of infecting crops, livestock populations, and human populations. Anthrax is a widely exploited bioagent responsible for attacks ranging from the Germans’ deployment in World War I to the mailing of anthrax through the postal service in attempts on U.S. senators’ lives. With the development of genetic manipulations, the Soviet Union began modifying anthrax to resist detection and treatment. With the continued advancement of science and technology, a new bioagent has entered the scene – the man-made chimeric virus. Chimeric viruses …

Escherichia Coli Alanyl-Trna Synthetase Maintains Proofreading Activity And Translational Accuracy Under Oxidative Stress, Arundhati Kavoor, Paul Kelly, Michael Ibba

Biology, Chemistry, and Environmental Sciences Faculty Articles and Research

Aminoacyl-tRNA synthetases (aaRSs) are enzymes that synthesize aminoacyl-tRNAs to facilitate translation of the genetic code. Quality control by aaRS proofreading and other mechanisms maintains translational accuracy, which promotes cellular viability. Systematic disruption of proofreading, as recently demonstrated for alanyl-tRNA synthetase (AlaRS), leads to dysregulation of the proteome and reduced viability. Recent studies showed that environmental challenges such as exposure to reactive oxygen species can also alter aaRS synthetic and proofreading functions, prompting us to investigate if oxidation might positively or negatively affect AlaRS activity. We found that while oxidation leads to modification of several residues in Escherichia coli AlaRS, unlike …

Assesment Of Antibiotic Resistant Gene Expression In Clinical Isolates Of Pseudomonas Aeruginosa, Dustin Esmond

Biology Theses

Increasing prevalence of nosocomial infections by antimicrobial resistant pathogens resulting in higher mortality rates and financial burden is of great concern. Pseudomonas aeruginosa represents one of six highly virulent “ESKAPE” pathogens that exhibit considerable intrinsic drug resistance as well as mechanisms for acquiring further resistance. As many of these mechanisms are regulated through gene expression, we sought to identify regulatory strategies and patterns at play in 23 clinical isolates collected from Baku, Azerbaijan and Tyler, Texas, USA. Real-time quantitative polymerase chain reaction was performed on six gene targets implicated in resistance and contrasted with antibiotic phenotypes. We found AmpC cephalosporinase …

Examining Effects Of The Dna Regulator Lrp On Quorum Sensing Gene Expression In Pseudomonas Aeruginosa, Bradley Lumsden

Symposium of Student Scholars

Pseudomonas aeruginosa is an opportunistic human pathogen that has the capacity to express multiple virulence factors that are regulated through an extensive quorum sensing network. Three major quorum sensing systems have been identified in Pseudomonas species: the acyl homoserine lactones of las and rhl, and the Pseudomonas Quinolone Signal (PQS). We seek to investigate the involvement of a global regulator, Lrp with the expression of these three networks. Specifically, we will compare expression levels of las, rhl, and pqs in wild type P. aeruginosa (MPAO1) with an lrp transposon insertion mutant using quantitative PCR. Through this comparative …

Role Of A Cryptic Trna Gene Operon In Survival Under Translational Stress, Javier Santamaría-Gómez, Miguel Ángel Rubio, Rocío López-Igual, Ana B. Romero-Losada, Fernando M. Delgado-Chaves, Roque Bru-Martínez, Francisco J. Romero-Campero, Antonio Herrero, Michael Ibba, Jesús A. G. Ochoa De Alda, Ignacio Luque

Biology, Chemistry, and Environmental Sciences Faculty Articles and Research

As compared to eukaryotes, bacteria have a reduced tRNA gene set encoding between 30 and 220 tRNAs. Although in most bacterial phyla tRNA genes are dispersed in the genome, many species from distinct phyla also show genes forming arrays. Here, we show that two types of arrays with distinct evolutionary origins exist. This work focuses on long tRNA gene arrays (L-arrays) that encompass up to 43 genes, which disseminate by horizontal gene transfer and contribute supernumerary tRNA genes to the host. Although in the few cases previously studied these arrays were reported to be poorly transcribed, here we show that …

Identification Of Antibiotic Producing Soil Bacteria Against Bacillus Subtilis, Morgan Brockhouse, Dr. Lori Scott

Identifying and Characterizing Novel Antibiotic Producing Microbes From the Soil

This project is a collaboration with the Tiny Earth Project Initiative (TEPI), which is a global network of educators and students focused on student-sourcing antibiotic discovery from soil. Individual strains of soil bacteria were isolated and produced antibiotic against Bacillus subtilis. Two of these samples were sequenced using the 16S rRNA gene to reveal they are very closely related to the genus Pseudomonas.

The Role Of Manganese In Streptococcus Sanguinis, Tanya M. Puccio

Theses and Dissertations

Streptococcus sanguinis is primarily associated with oral health as a commensal bacterium. As an opportunistic pathogen, S. sanguinis is capable of colonizing heart valve vegetations, leading to the disease infective endocarditis. Previous studies from our lab have identified the high-affinity manganese transporter SsaACB as important for endocarditis virulence. The impact that manganese depletion has on S. sanguinis had never been evaluated and a secondary manganese transporter has not been identified. Thus, we employed the use of a fermentor to control large-scale growth over time and depleted manganese in an ΔssaACB mutant using a metal chelator, EDTA. The changes in …

Genetic And Biochemical Analysis Of A Conserved, Multi-Gene System Regulation Spore-Associated Proteins In Streptomyces Coelicolor, Joseph Sallmen

Electronic Theses and Dissertations

Streptomyces coelicolor is a Gram positive, filamentous soil dwelling bacterium that exhibits a complex life cycle including the transition from a vegetative mycelium to a sporulating, aerial mycelium. Early genetic studies identified two classes of genes that resulted in developmental blocks, bald and white. The bald (bld) phenotype occurs when colonies cannot produce aerial hyphae. White (whi) colonies exhibit incomplete sporulation and/or loss of production of the concurrently produced grey pigment. While some of the original mutants have been explored, the developmental functions of many of the identified genes are not well understood. Of particular interest …

Co-Occurrence Of Manganese Oxidase Genes Indicates Lateral Transfer Between Classes Of Proteobacteria, Jacob Olichney

Student Scholar Showcase

Pseudomonas putida GB-1 is a model organism for the study of manganese oxidation in bacteria, however, the frequency of co-localization of multiple known and suspected manganese oxidizing proteins, as well as their occurrence between species, is unknown. Eight different genes isolated from known manganese oxidizing bacteria (MnxG, MopA, McoA, PputGB1_2552, PputGB1_2553, MoxA, MofA, and Bacillus MnxG) were tested individually using BioPython and BLAST (Basic Local Alignment Search Tool) on multiple genomic databases. BLAST searches had an expect value cutoff of 1e-50, limiting gene homologs to those with high sequence similarity. The abundance of homologous genes across classes of proteobacteria point …

Impact Of Seasonal And Host-Related Factors On The Intestinal Microbiome And Cestode Community Of Sorex Cinereus And Sorex Monticola, Katelyn D. Cranmer

College of Graduate Studies: Theses & Dissertations

The intestinal microbiome of mammals plays a significant role in host health and response to environmental stimuli and can include both beneficial native bacteria as well as parasitic worms. In this study, I examined the intestinal cestode and bacterial communities of two closely related species of shrew, Sorex monticola and Sorex cinereus, over a six month period in 2016. Specimens were collected approximately every three weeks from May to October from the Sangre de Cristo Mountains in Cowles, New Mexico. A total of 79 shrews were prepared with the gastrointestinal tracts removed and flash-frozen in liquid nitrogen. An additional …

Identification And Heterologous Reconstitution Of A 5-Alk(En)Ylresorcinol Synthase From Endophytic Fungus Shiraia Sp. Slf14, Huiwen Yan, Lei Sun, Jinge Huang, Yixing Qiu, Fuchao Xu, Riming Yan, Du Zhu, Wei Wang, Jixun Zhan

Biological Engineering Faculty Publications

A new type III polyketide synthase gene (Ssars) was discovered from the genome of Shiraia sp. Slf14, an endophytic fungal strain from Huperzia serrata. The intron-free gene was cloned from the cDNA and ligated to two expression vectors pET28a and YEpADH2p-URA3 for expression in Escherichia coli BL21(DE3) and Saccharomyces cerevisiae BJ5464, respectively. SsARS was efficiently expressed in E. coli BL21(DE3), leading to the synthesis of a series of polyketide products. Six major products were isolated from the engineered E. coli and characterized as 1,3-dihydroxyphenyl-5-undecane, 1,3-dihydroxyphenyl-5-cis-6'-tridecene,1,3-dihydroxyphenyl-5-tridecane, 1,3-dihydroxyphenyl-5-cis-8'-pentadecene, 1,3-dihydroxyphenyl-5-pentadecane and 1,3-dihydroxyphenyl-5-cis-10'-heptadecene, respectively, …

Arthropod Transcriptional Activator Protein-1 (Ap-1) Aids Tick-Rickettsial Pathogen Survival In The Cold, Supreet Khanal, Vikas Taank, John F. Anderson, Hameeda Sultana, Girish Neelakanta

Biological Sciences Faculty Publications

Ixodes scapularis ticks transmit several pathogens to humans including rickettsial bacterium, Anaplasma phagocytophilum. Here, we report that A. phagocytophilum uses tick transcriptional activator protein-1 (AP-1) as a molecular switch in the regulation of arthropod antifreeze gene, iafgp. RNAi-mediated silencing of ap-1 expression significantly affected iafgp gene expression and A. phagocytophilum burden in ticks upon acquisition from the murine host. Gel shift assays provide evidence that both the bacterium and AP-1 influences iafgp promoter and expression. The luciferase assays revealed that a region of approximately 700 bp upstream of the antifreeze gene is sufficient for AP-1 binding to promote …

Functional Studies Of The E. Coli Proc And A Putative Ortholog Mrub_1345, Maureen Azar, Dr. Lori Scott

Meiothermus ruber Genome Analysis Project

This project is part of the Meiothermus ruber genome analysis project, which uses the bioinformatics tools associated with the Guiding Education through Novel Investigation –Annotation Collaboration Toolkit (GENI-ACT) to predict gene function. We investigated the biological function of Escherichia coli and Meiothermus ruber proC genes using the complementation assay. In this research project, mutants of varying severity to the functional state of the protein were developed. The results showed that two or more amino acid deletions reduced or eliminated ProC function. Amino acid substitutions, on the other hand, were not severe enough to impact ProC function. Double and triple mutants …

Regulation Of The Tubulin Homolog Ftsz In Escherichia Coli, Monika S. Buczek

Dissertations, Theses, and Capstone Projects

Escherichia coli is a well-known pathogen, and importantly, a widely used model organism in all fields of biological sciences for cloning, protein purification, and as a model for Gram-negative bacterial species. And yet, researchers do not fully understand how this bacterium replicates and divides. Every year additional division proteins are discovered, which adds complexity to how we understand E. coli undergoes cell division. Due to their specific roles in cytokinesis, some of these proteins may be potential targets for development of antibacterials or bacteriostatics, which are much needed for fighting the current global antibacterial deficit. My thesis work focuses on …

Mrub_2120, Mrub_2121, Mrub_2122, Mrub_2123 And Mrub_2124 Are Orthologs Of E. Coli Genes B3458, B3457, B3456, B3455 And B3454, Respectively, And Make Up An Operon That Codes For The Branched-Chain Amino Acid Abc Transporter In Meiothermus Ruber Dsm 1279, Aaron Jones, Madelyn Huber, Dr. Lori Scott

Meiothermus ruber Genome Analysis Project

In this project we investigated the biological function of the genes Mrub_2120, Mrub_2121, Mrub_2122, Mrub_2123 and Mrub_2124 (KEGG map number 02010). We predict these genes encode components of a branched-chain amino acid ATP Binding Cassette (ABC) transporter: 1) Mrub_2120 (DNA coordinates 2169247-2170416 on the reverse strand) encodes the branched-chain amino acid binding protein that is localized to the periplasm; 2) Mrub_2121 (DNA coordinates 2170433..2171353 on the reverse strand) encodes the first TMD; 3) Mrub_2122 (DNA coordinates 2171365..2172279 on the reverse strand) encodes the second TMD; 4) Mrub_2123 (DNA coordinates 2172276..2173028 on the reverse strand) encodes the first NBD; 5) Mrub_2124 …

The Role Of The Dosage Compensation Complex As A Pathway For Spiroplasma To Induce Male Lethality In Drosophila Melanogaster, Becky Cheng

CMC Senior Theses

Drosophila melanogaster and many other insects harbor intracellular bacterial symbionts that are transmitted vertically from infected host mothers to their offspring. Many of these bacteria alter host reproductive developmental processes in order to increase their transmission success. For example, Spiroplasma, a spirochete that naturally infects D. melanogaster, selectively kills males during mid-embryogenesis while sparing females. Previous studies suggested that Spiroplasma interacts genetically with the male-specific dosage compensation pathway, which causes ~2-fold up-regulation of most genes located on the male’s single X chromosome so that their expression matches the levels found in females who have two Xs. To further …

Antifungal Genome Mining And Genetics In Filamentous Actinomycete Bacteria Isolated From Local Soils, Jacob Alex, Kevin Lou, Bilal Makhdoom

Undergraduate Research Symposium Posters

Actinomycetes are gram positive, filamentous bacteria that produce useful antibiotics, antitumor agents, and agricultural products. A series of enrichments were undertaken to isolate actinomycetes from local soils, varying enrichment media, antibacterials, and soil treatments (including heat and CaCO3). Isolates were characterized by 16S rDNA sequencing, phenotypic and morphological observations, and antibiotic production. The genetic tractability of select isolates was analyzed using a panel of integrating vectors derived from ɸC31, ɸBT1, and OzzyJ phage using intergeneric conjugation. Further, a semi-degenerate multiplex PCR assay to detect ɸBT1 genomic integrants was designed and tested for the first time. Finally, PCR screens were used …

The Effect Of Transformed Escherichia Coli On The Mouse Intestine Microbiome: The Microbial Metabolic Enhancement Hypothesis, Bryar P. Kader

Senior Honors Theses

Metabolic disorders affect around thirty-four percent of the population in the United States. Among these disorders is lactose intolerance, which results from diminished production of the human lactase enzyme. This disorder and others like it are genetically determined and cannot be cured. However, the use of transformed bacteria implanted in the colon may provide a means by which the faulty pathway can be bypassed. To test whether transformed bacteria have the capability to aid in the digestion of normally indigestible compounds, a transformed strain of Escherichia coli overexpressing the beta-galactosidase enzyme encoded by the lacZ gene was colonized in the …

The Prevalence And Effect Of Wolbachia Infection On The Brown Widow Spider (Latrodectus Geometricus), Brittany Dane' Arrington

College of Graduate Studies: Theses & Dissertations

Wolbachia was recently identified in the Brown Widow Spider (Latrodectus geometricus). This symbiont has not previously been found in this genus, therefore the consequences on the host’s population dynamics are unknown. The frequency of endosymbiont infection can provide insight into the population dynamics of the host as this bacteria is known to alter host life history. In my 1^st chapter I determine the prevalence of Wolbachia pipentis infection in in the Brown Widow Spider (Latrodectus geometricus) from five locations in the Southeastern United States. Infection frequency differed significantly among sampling locations, ranging from 20% to …