Biochemistry, Biophysics, and Structural Biology Commons^™

The Sos Response In Escherichia Coli K12: An Exploration Of Mutations In Lexa And Reca Using Fluorescence Microscopy, Steven Van Alstine

Doctoral Dissertations

Faithful replication of the genome is paramount for maintaining the fitness of an organism. Therefore, life has evolved inducible mechanisms to be able to repair damaged DNA and maintain evolutionary fitness. The SOS response is a highly conserved DNA damage inducible response that is tightly regulated. Multiple factors contribute to the ability of the cell to perform proper DNA repair and induction of the SOS response including the amount of RecA, mutations in RecA that affect competition for DNA, and other proteins that interact with the RecA filament. The complex relationship between RecA and LexA is the subject of this …

Mechanisms Of Substrate Selectivity And Transport By Escherichia Coli Atp- Binding Cassette (Abc) Transporter Metni, Yuchun Chen

Master's Theses

ABC transporters are central in many cellular functions including nutrient uptake, signal transduction membrane assembly, and cellular detoxification. Both structural and functional studies have revealed insights into the high-affinity uptake mechanism of the MetNI methionine ABC transporter. Using the energy from ATP binding and hydrolysis, the MetNI-Q system can import L-Met, D-Met, and other methionine derivatives against concentration gradients. Many mechanistic studies of ABC transporter propose a model in which cognate binding proteins sequester substrates in the periplasm and deliver them to the transporter. In contrast, recent in vivo and crystallographic studies of MetNI-Q suggest that some substrates may be …

Studying The Lysine Acetylation Of Aconitase Isozymes In E. Coli, Sara Ottinger

Chemistry & Biochemistry Undergraduate Honors Theses

The contents of this thesis have been modified from the publication “Araujo J, Ottinger S, Venkat S, Gan Q and Fan C (2022) Studying Acetylation of Aconitase Isozymes by Genetic Code Expansion. Front. Chem. 10:862483”. Though studies have found multiple lysine sites in which acetylation takes place in Escherichia Coli aconitase, acetylation’s effects on the enzyme’s activity have yet to be studied. Aconitase is the dehydratase-hydratase found in the citric acid and glyoxylate cycles responsible for the reversible isomerization of citrate to isocitrate via cis-aconitate intermediate. There are two isoforms of aconitase in E. coli: AcnA and AcnB. In …

Extracellular Secretion Of A Cutinase With Polyester-Degrading Potential By E. Coli Using A Novel Signal Peptide From Amycolatopsis Mediterranei, Yeqi Tan, Gemma K. Kinsella, Gary T. Henehan, Barry J. Ryan

Articles

Recent studies in this laboratory showed that an extracellular cutinase from A. mediterranei (AmCut) was able to degrade the plastics polycaprolactone and polybutylene succinate. Such plastics can be slow to degrade in soils due to a lack of efficient polyester degrading organisms. AmCut also showed potential for the biocatalytic synthesis of esters by reverse hydrolysis.

The gene for AmCut has an upstream leader sequence whose transcript is not present in the purified enzyme. In this study, we show using predictive modelling, that this sequence codes for an N-terminal signal peptide that directs transmembrane expression via the Sec secretion pathway.

E. …

Validation Of Anti-Oxidative Stress Genes From Genome-Wide Screening Of Escherichia Coli, Carson Ercanbrack

Chemistry & Biochemistry Undergraduate Honors Theses

The primary purpose of this project is to evaluate the genes that play a role in the oxidative stress response in Escherichia coli. In doing so, the entire genome of E. coli was subject to throughput in which individual genes were determined to have a role in the bacteria’s oxidative stress response. Moreover, this project focused on the validation of the genes that were able to pass the initial throughput stage. The genes were subject to two forms of validation. In the first validation technique, candidate genes were overexpressed and minimum inhibitory concentrations of hypochlorous acid were taken. Following, a …

Impact Of Metazooplankton Filter Feeding On Escherichia Coli Under Variable Environmental Conditions, Niveen S. Ismail, Brittney M. Blokker, Tyler R. Feeney, Ruby H. Kohn, Jingyi Liu, Vivian E. Nelson, Mariah C. Ollive, Sarah B. L. Price, Emma J. Underdaha

Engineering: Faculty Publications

The fecal indicator bacterial species Escherichia coli is an important measure of water quality and a leading cause of impaired surface waters. We investigated the impact of the filter-feeding metazooplankton Daphnia magna on the inactivation of E. coli. The E. coli clearance rates of these daphnids were calculated from a series of batch experiments conducted under variable environmental conditions. Batch system experiments of 24 to 48 h in duration were completed to test the impacts of bacterial concentration, organism density, temperature, and water type. The maximum clearance rate for adult D. magna organisms was 2 ml h1 organism1. Less than …

Ni Site Structure And Function In Biological Sensing And Enzyme Activity, Hsin-Ting Huang

Doctoral Dissertations

Ni(II) is one of the important cofactors involved in various enzyme functions. For organisms utilizing Ni(II), a regulation system is required to maintain Ni(II) homeostasis and prevent toxicity. The focus of this dissertation is on investigating the relationship between the Ni(II) site structure and the function of proteins, a Ni(II) sensor and a Ni(II) enzyme. RcnR, a Ni(II)/Co(II) sensor in E. coli, controls the expression of the Ni(II)/Co(II) exporter proteins, RcnAB. Due to the lack of structural information, the mechanism of metal induced allosteric regulation and metal selection is not fully elucidated. Results presented here show that binding of …

Maximizing And Modeling Malonyl-Coa Production In Escherichia Coli, Tatiana Thompson Silveira Mello

LSU Master's Theses

In E. coli, fatty acid synthesis is catalyzed by the enzyme acetyl-CoA carboxylase (ACC), which converts acetyl-CoA into malonyl-CoA. Malonyl-CoA is a major building block for numerous of bioproducts. Multiple parameters regulate the homeostatic cellular concentration of malonyl-CoA, keeping it at a very low level. Understanding how these parameters affect the bacterial production of malonyl-CoA is fundamental to maximizing it and its bioproducts. To this end, competing pathways consuming malonyl-CoA can be eliminated, and optimal nutritional and environmental conditions can be provided to the fermentation broth. Most previous studies utilized genetic modifications, expensive consumables, and high-cost quantification methods, making …

How A Cell Knows Where To Divide: Oscillation Of Mind In Vivo, Colby Ferreira

Senior Honors Projects

Over two-million people in the United States are infected by antibiotic resistant bacteria each year. Of this number 23,000 die from these infections and other complications. Due to this, novel antibiotic targets are constantly being investigated. One process in prokaryotes that holds promise is cellular division. Bacterial cells grow and reproduce using a series of proteins known as the cell division machinery. This machinery enables the division of the parental cell into two identical daughter cells. The cell division machinery is similar between bacterial taxa, making it an ideal target for new classes of antibiotics. Therefore, understanding the molecular mechanisms …

Post-Translational Modifications And Functional Studies Of Dksa In Escherichia Coli, Andrew Charles Isidoridy

Legacy Theses & Dissertations (2009 - 2024)

DksA is a bacterial gene regulator that functions synergistically with the stress alarmone ppGpp to mediate the stringent response. DksA also functions independently of ppGpp to regulate transcription of a number of genes. DksA function is dependent on its binding affinity to RNA polymerase and requires specific interactions between RNAP and catalytic amino acids located on the coiled coil tip, D74 and A76. While much of the previous work on DksA has focused on understanding the mechanisms of action and the numerous gene targets for transcriptional regulation, little is known about the mechanisms by which DksA expression and function may …

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 …

Synthesis And Incorporation Of 1,2-Alkanolamine-Functionalized Lysine As A Non-Canonical Amino Acid Into Gfp, Chesley M. Rowlett

Scholar Week 2016 - present

Synthesis of specific post translational modifications in proteins can be difficult but achievable via genetic code expansion techniques. An attempt has been made to synthesize and incorporate D-cThrK into green fluorescent protein (GFP) at an amber mutation site in Escherichia coli via the coordination of pyrrolysyl- tRNA synthetase and its cognate tRNApyl. The incorporation of this non-canonical amino acid and potential chemical transformations following it allow the synthesis of proteins with post translational lysine modifications, making a variety of basic and biotechnological applications available.

Examination Of Orthologous Genes (Mrub_2518 And B3728, Mrub_2519 And B3727, Mrub_2520 And B3726, Mrub_2521 And B3725) Responsible For Abc Phosphate Transporters In Two Species M. Ruber And E. Coli, Margaret Meyer, Dr. Lori Scott

Meiothermus ruber Genome Analysis Project

In this project we investigated the biological function of the genes b3725, b3726, b3727, b3728 and Mrub_2518, Mrub_2519, Mrub_2520 and Mrub_2521 (KEGG map number 02010). We predict that these genes encode the components of a Phosphate ABC transporter: Orthologous genes Mrub_2518 (DNA coordinates 2565359..2566438) and b3728 encodes the periplasmic phosphate binding component; Orthologous genes Mrub_2519 (DNA coordinates 2566499..2567485) and b3727, and Mrub_2520 (DNA coordinates 2567496..2568326) and b3726 encode for the two transmembrane proteins; Orthologous genes Mrub_2521 (DNA coordinates 2568338..2569159) and b3725 encode for the ATP binding protein within the cytoplasm. Within the two species, M. ruber and E. coli, …

Confirmation That Mrub_1751 Is Homologous To E. Coli Xylf, Mrub_1752 Is Homologous To E. Coli Xylh, And Mrub_1753 Is Homologous To E. Coli Xylg, Ben Price, Dr. Lori Scott

Meiothermus ruber Genome Analysis Project

In this project we investigated the biological function of the genes Mrub_1751, Mrub_1752 and Mrub_1753 (KEGG map number 02010). We predict these genes encode components of a D-xylose ATP Binding Cassette (ABC) transporter: 1) Mrub_1752 (DNA coordinates 1809004-1810224 on the forward strand) encodes the permease component (aka transmembrane domain), predicted to be an ortholog and 2) Mrub_1753 (DNA coordinates 1810227-1811000 on the forward strand) encodes the ATP-binding domain (aka nucleotide binding domain); and 3) Mrub_1751 (DNA coordinates 1807855-1808892 on the forward strand) encodes the solute binding protein. The ABC-transporter for M. ruber to transport D-xylose is homologous with the transporter …

Developing Algorithms For Quantifying The Super Resolution Microscopic Data: Applications To The Quantification Of Protein-Reorganization In Bacteria Responding To Treatment By Silver Ions, Sai Divya Challapalli

Graduate Theses and Dissertations

Histone-like nucleoid structuring proteins (HNS) play significant roles in shaping the chromosomal DNA, regulation of transcriptional networks in microbes, as well as bacterial responses to environmental changes such as temperature fluctuations. In this work, the intracellular organization of HNS proteins in E. coli bacteria was investigated utilizing super-resolution fluorescence microscopy, which surpasses conventional microscopy by 10–20 fold in spatial resolution. More importantly, the changes of the spatial distribution of HNS proteins in E. coli, by addition of silver ions into the growth medium were explored. To quantify the spatial distribution of HNS in bacteria and its changes, an automatic method …

Genetic Engineering Studies Of Escherichia Coli And Microalgae For Expression Of Hydrolytic Enzymes And Development Of High Throughput Screening Technique, Shreyas S. Yedahalli

Electronic Thesis and Dissertation Repository

The field of biochemical engineering has made substantial progress through major advances in genetic and metabolic engineering with applications in various sectors such as energy, food science, pharmaceuticals, etc. The hosts used for this work are constantly broadening. A host particularly important for energy applications are microalgae. The potential to enhance microalgae genetically for energy applications is not well explored and was therefore investigated in this thesis. Non-photosynthetic micro-organisms and photosynthetic microalgae offer a potential approach to enhance sustainable biochemical production. In this study expression vectors for Escherichia coli (E. coli) and Chlorella vulgaris (C. vulgaris) …

The Selective Survival Of Escherichia Coli In Freshwater Beach Sand, Natalie Ann Rumball

Theses and Dissertations

The quantification of Escherichia coli or E. coli is the most common method used to detect recent fecal pollution in recreational water, as this species is known for its high abundance in fecal matter and assumed host-associated nature. However, it has been determined that some strains are capable of long-term survival and potential propagation in non-host environments, such as the beach sand. These long-term environmental survivors are host-independent and are not associated with the same health risks as those E. coli from recent fecal pollution. However, they have been shown to impact how water quality is perceived as they are …

Production And Characterization Of Islet Amyloid Polypeptide Using Recombinant Technology, Emilio Duverna

Graduate Theses and Dissertations

Islet amyloid polypeptide (IAPP) also called amylin is an amyloid-forming protein; IAPP is a proteinaceous hormone that comprises 37 amino acid residues. It is secreted along with insulin from the pancreatic β-cells to help it regulate the uptake and removal of glucose in the bloodstream. IAPP has been observed in the amyloid deposits found in pancreatic β-cells of most patients suffering from type II diabetes mellitus. This research project aims at producing recombinant amylin peptide. To achieve this goal, we used the pBAD plasmid vector which we introduced into Escherichia coli to express the peptide. Although the vector was successfully …

The Effects Of Salinity, Ph, Temperature, And Dissolved Oxygen On Sensitivity Of Pcr Identification Of T4 Bacteriophage, Joesph F. Cannon, Nicholas A. Thurn, Paul E. Richardson

Journal of the South Carolina Academy of Science

Bacteriophages are used as indicators of pathogenic bacteria in drinking, and wastewaters. They also show potential in limiting aquatic bacterial populations through their lytic properties. The effect of different water characteristics (salinity, pH, dissolved oxygen, and temperature) on the sensitivity of the PCR identification of virus particles were analyzed to determine at what levels bacteriophage can be detected in environmental samples. Results from this preliminary study indicate that a PCR bacteriophage detection technique has potential as a relatively efficient and economical indicator of coliform contamination in multiple aquatic environments. While further evaluation is needed, the protocol appears to function in …

Detection Of Viable Microorganisms Using Propidium Monoazide, Erik J. Mcfarland, Adrian Ponce Dr.

STAR Program Research Presentations

Propidium monoazide (PMA) is a molecular tool used to assess viability of microorganisms. Currently, PMA is thought to discern viability through membrane permeability; PMA enters only membrane compromised cells, irreversibly crosslinks to theirDNAand precipitates theDNAout of solution, preventing it from being amplified during polymerase chain reaction (PCR). Using PMA on a sample of live and dead microorganisms results in only theDNAof living organisms being amplified and identified. Therefore, a comparison ofPCRresults with and without PMA allows one to determine the live fraction and total population, respectively.

Current literature provides conflicting evidence as to the effectiveness of the technique. Our research …

The Fate Of Iron Released From Heme By Hemeoxygenase-1, Jonathan Mark Gardner

Undergraduate Honors Capstone Projects

A strain of Escherichia coli was genetically modified to co-express human heme oxygenase-1 and ferritin. The E. coli were then grown with varying amounts of hemin to see if the iron released upon degradation of the hemin by heme oxygenase-1 is loaded into ferritin. Following incubation, the ferritin was purified and the amount of iron loaded into ferritin determined. It was found that ferritin purifed from E. coli expressing human heme oxygenase-1 contained more iron than E. coli that did not contain human heme oxygenase-1. It was concluded that some of the iron released upon degradation of hemin by heme …

Development Of 31p Nuclear Magnetic Resonance Methods For The Study Of Phosphate Metabolisms In E. Coli And B. Subtilis, Fabian M. Dayrit, Emilyn Q. Espiritu, Noreen Gonzalez, Nina Rosario L. Rojas, Jennifer T. Aguilan, Antonio M. Basilio, Edward T. Chainani, Enrico Cruz, Bernadette Matanguihan

Environmental Science Faculty Publications

31P NMR experiments were performed on Escherichia coli and Bacillus subtilis at various temperatures under aerobic and anaerobic conditions. The total soluble intracellular phosphate concentration was estimated to be 2 x 10^-17 mole/cell, while intracellular orthophosphate concentration was around 1 x 10^-17 mole/cell. Addition of glucose resulted in a general decrease in intracellular pH and was accompanied by the formation of sugar monophosphates. The concentrations of soluble intracellular phosphates, both inorganic and organic phosphates, were estimated by integration versus methylene diphosphonic acid (MDPA) standard. Although intracellular and extracellular orthophosphate could be observed, these appear to exchange rapidly on the NMR …