Microbiology Commons^™

Lysine Biosynthesis In Bacteria: A Metallodesuccinylase As A Potential Antimicrobial Target, Danuta M. Gillner, Daniel P. Becker Ph.D., Richard C. Holz

Chemistry: Faculty Publications and Other Works

In this review, we summarize the recent literature on dapE-encoded N-succinyl-l,l-diaminopimelic acid desuccinylase (DapE) enzymes, with an emphasis on structure–function studies that provide insight into the catalytic mechanism. Crystallographic data have also provided insight into residues that might be involved in substrate and hence inhibitor recognition and binding. These data have led to the design and synthesis of several new DapE inhibitors, which are described along with what is known about how inhibitors interact with the active site of DapE enzymes, including the efficacy of a moderately strong DapE inhibitor.

Understanding The Posttranslational Regulation Of The Response Regulator Rcsb And Acetyl Phosphate As An Acetyl Group Donor In E. Coli, Linda Hu

Dissertations

The observation that Nepsilon-lysine acetylation occurs on a hundreds of proteins in bacteria is a recent discovery. To study the mechanisms that regulate acetylation and to determine if acetylation affects physiology, I studied the Escherichia coli response regulator and transcription factor RcsB, which was reported to be acetylated in vitro. To monitor RcsB activity, I measured transcription from the rprA promoter, which requires RcsB. I confirmed that RcsB is activated by phosphorylation through the Rcs phosphorelay and acetyl phosphate and showed that acetyl phosphate could phosphorylate RcsB. However, a mutant that accumulates acetyl phosphate (ackA) exhibited reduced rprA transcription instead …

The Role Of Linker 2 (L2) Region In Rhtrim5Α Assembly And Hiv-1 Restriction, Jayalaxmi Sastri

Dissertations

The cellular restriction factor TRIM5alpha inhibits infection by numerous retroviruses in a species specific manner. TRIM5alpha protein from rhesus macaques (rhTRIM5alpha) and a related protein TRIM-Cyp from Owl monkeys restrict infection by HIV-1 while human TRIM5α (huTRIM5alpha) restricts infection by N-tropic murine leukemia virus (N-MLV) but not HIV-1. Several models have been proposed for retroviral restriction by TRIM5 proteins (TRIM5alpha and TRIMCyp). These models collectively suggest that TRIM5 proteins mediate restriction by recognizing specific determinants in the viral capsid and directly binding the capsid. Following binding, the TRIM5 proteins self-associate into large assemblies around the viral capsid, which leads to …

Characterization Of The Novel Response Regulator Sype: A Dual Regulator Of Biofilm Formation And Colonization In Vibrio Fischeri, Andrew Robert Morris

Dissertations

Biofilms, or surface-associated microbial communities encased in a matrix, represent a common mode of growth for many bacterial species. Importantly, biofilms often play significant roles in the association of bacteria with their animal hosts, including promoting attachment to and colonization of host tissues. The symbiosis between the bacterium Vibrio fischeri and its animal host Euprymna scolopes provides a natural model system for investigating the impact of biofilms on host infection. In particular, V. fischeri produces a specific biofilm to successfully initiate host colonization.

Biofilm formation by V. fischeri is a tightly regulated process involving a complex network of regulatory genes. …

Evading Innate And Adaptive Immunity During Adenovirus Cell Entry, Shauna Marvin

Dissertations

Adenovirus (Ad), a non-enveloped, dsDNA virus, enters cells via clathrin-mediated endocytosis. For viral genome delivery to the nucleus, Ad must penetrate endosomal membranes to create defects sufficient for the passage of the 90 nm diameter capsid across cell membranes. Recent observations suggest that adenovirus type 5 (Ad5) capsid uncoating occurs at the cell surface upon binding to both the coxsackievirus and adenovirus receptor and α_v integrins. This uncoating event leads to the exposure of the capsid membrane lytic protein VI. Using the cytosolic protein galectin-3 (gal3) as a marker of membrane rupture, we demonstrate that Ad5 membrane rupture occurs …

Going Beyond Phosphorylation: Acetylphosphate-Mediated Ran Polymerase Acetylation And Caper Phosphorylation Co-Regulate Transcription Of Escherichia Coli Stress-Responsive Gene Cpxp, Bruno P. Lima

Dissertations

The ability of bacteria to sense and adapt to environmental changes has al-lowed these organisms to thrive in all parts of the globe and to establish many complex interactions with the environments they live as well as other members of these environments. Crucial to monitoring extracellular conditions is a group of signaling pathways known as two-component signal transduction system (2CST). These systems relay information from the extracellular environment to the interior of the cell via a transfer of phosphoryl groups from the sensory protein know as sensor kinase (SK) to the output protein known as response regulator (RR).

In addition …

Identifying And Characterizing The Degradative Pathway Of The Retroviral Restriction Factor Rhtrim5Α, Rachel Nelson

Master's Theses

Human immunodeficiency virus 1 (HIV-1) is a lentivirus that progresses to acquired immunodeficiency syndrome (AIDS). The protein TRIM5alpha from rhesus macaques (rhTRIM5alpha) restricts HIV-1 by blocking infection after entry of the virion into cells. Treatment of rhTRIM5alpha expressing cells with inhibitors to a cellular degradation pathway, the proteasome, partially relieves restriction but does not inhibit rhTRIM5alpha protein turnover. The role of a second degradation pathway, the autophagy-lysosomal pathway, in TRIM5alpha mediated restriction has not been explored.

In the present study, we demonstrate that rhTRIM5alpha is degraded by chaperone mediated autophagy (CMA). Inhibition of CMA alters rhTRIM5alpha localization and turnover, while …

Exosomes: Antiviral Agents In The Human Lung, Jennifer Alexandra Novak

Master's Theses

Hundreds of interferon effectors comprise the immediate host response to virus infection. One family of interferon stimulated genes is the IFITM (Interferon-Induced Transmembrane) proteins. IFITM3, in particular, has been shown to block virus cell entry of Influenza A Virus, SARS Coronavirus, and West Nile Virus, among others. IFITM3 has also been identified as an essential factor for host protection against Influenza A Virus in mice and humans. However, the mechanism by which IFITM3 functions remains unclear.

IFITM3 may function both intracellularly and extracellularly to block virus infection in the human lung. The extracellular milieu contains microscopic vesicles termed exosomes which …

Assessing The Effects Of Titanium Dioxide Nanoparticles On Microbial Communities In Stream Sediment Using Artificial Streams And High Throughput Screening, Alexandra Ozaki

Master's Theses

Nano-titanium dioxide (nano-TiO2) is an engineered nanomaterial used in a wide array of commercial products. The production and use of large amounts of nano-TiO2 is resulting in the unintended release to the environment. Nano-TiO2 is known to be cytotoxic due primarily to its ability to generate reactive oxygen species, and negative impacts on a variety of organisms have been demonstrated, but the effects of nano-TiO2 on complex microbial communities under ecologically relevant conditions have rarely been tested. We conducted a controlled manipulative experiment using recirculating model streams dosed with a one-time amendment of 1mg L-1 nano-TiO2 (specifically P25). Within one …

Antibodies To Inhibit Clostridium Difficile Adhesion To Human Gut Epithelial Cell Line, Wonbeom Paik

Master's Theses

Clostridium difficile infection (CDI) is a major hospital-acquired diarrhea that can cause life-threatening complications such as pseudomembranous colitis. CDI is caused by colonization of host with C. difficile, a Gram positive, anaerobic bacterium known to produce toxins that cause disease. Normally, the gut microbiota protects the host from CDI, but disruption of the microbial composition through antibiotic treatment can leave one vulnerable for CDI. To date, no vaccines for preventing CDI are available.

In this study, the potential of antibodies directed against specific surface molecules of C. difficile to block bacterial adherence to host gut epithelial cells was studied, in …

The Effect Of Glucose On Transcription At Cpxr- And Ompr-Regulated Promoters, Andrew Charles Cosgrove

Master's Theses

While reversible acetylation of proteins has been well studied in eukaryotes and is now recognized in bacteria, global protein acetylation in bacteria is a recently appreciated phenomenon. Protein acetylation is known to affect almost every aspect of cellular physiology in eukaryotes and there is proteomic evidence that this may also hold true in bacteria. In eukaryotes, lysines are acetylated by acetyltransferases that use acetyl-CoA as the acetyl group source, and de-acetylated by deacetylases. In bacteria, this reversible process uses enzymes homologous to those used by eukaryotes.

Our lab has recently found that acetylation of RNA polymerase (RNAP) can activate transcription …

Characterization Of The Clostridium Difficile Biofilm, Michelle Laning

Master's Theses

Clostridium difficile is a Gram-positive, spore forming bacterium and an opportunistic gastrointestinal pathogen. Clostridium difficile infection (CDI) is a common nosocomial infection. Upon antibiotic treatment, patients can be colonized with C. difficile from ingestion of spores. After colonization, cells produce toxins creating symptoms from diarrhea to pseudomembranous colitis, and many patients develop recurrent infections. Interactions between host epithelium and the bacteria is a critical step in infection, yet the interaction is not well understood. It is not known how C. difficile evades host immune responses. These are important mechanisms to answer in advancing CDI treatment. A biofilm could contribute to …

Understanding The Regulation Of Metabolic Enzyme Acetylation In E. Coli, Arti Walker-Peddakotla

Master's Theses

Global protein acetylation is a newly discovered phenomenon in bacteria. Of the more than 250 acetylations reported in E. coli, many are of metabolic enzymes. Thus, acetylation could represent a novel posttranslational mechanism of metabolic control. Yet, almost nothing is known about the regulation of these acetylations or of their metabolic outcomes. Here, we report that the cAMP receptor protein (CRP) regulates protein acetylation in E. coli and provide evidence that protein acetylation modulates the flux of carbon through central metabolism. When we grew cells in mixed amino acids supplemented with glucose and cAMP, global protein acetylation increased in a …

Mechanisms Of Adenovirus Membrane Permeabilization, Oana Maier

Dissertations

For a successful infection to occur, a virus must first penetrate host cell membranes to access intracellular sites of viral replication. Currently the mechanism through which adenovirus, a non-enveloped, dsDNA virus, disrupts the endosomal membrane during cell entry is not well characterized.

Recent studies suggest that adenovirus protein VI, which is released from the interior of the capsid during cell entry, has all of the in vitro membrane lytic activity of the virion. We found that protein VI binds membranes via an amino-terminal 80 residue α-helical domain. Critical to this interaction are conserved hydrophobic and basic lysine residues within this …

Cofactors In Coronavirus Entry, Ana Shulla

Dissertations

Viruses have evolved complex ways to penetrate host barriers and cause disease. One of the most important barriers the virus has to cross is the cellular membrane. Enveloped viruses accomplish this task by viral glycoprotein-mediated binding to host cells and fusion of virus and host cell membranes. For the coronaviruses, viral spike (S) proteins execute these cell entry functions. In my dissertation research I focused on understanding the coronavirus spike proteins as well as other cofactors required for S-mediated entry into cells.

The S proteins are set apart from other viral and cellular membrane fusion proteins by their extensively palmitoylated …

Detection Of The Sire1 Env-Like Protein, A Potential Retroviral Protein In Soybean, Garen Gaston

Master's Theses

Long terminal repeat (LTR) retroelements are mobile DNAs that are ubiquitous in eukaryotic genomes and include retroviruses, endogenous retroviruses and retrotransposons. Though retrotransposons, which are noninfectious, can make up as much as 80% of plant genomes, no traces of retroviral elements have been confirmed in plants. However, the 2000-copy soybean retrotransposon SIRE1 has an extra open reading frame, that has been described as env-like, because its conceptual translation product is predicted to have features of retroviral envelope proteins, which sponsor infectivity. Demonstrating that this region encodes an envelope protein would extend the range of endogenous retroviruses to plants. Demonstrating also …

Role Of Type Ii Transmembrane Serine Proteases In Coronavirus Production, Gitanjali Cuckemane Subramanya

Master's Theses

Proteolytic cleavage of Coronavirus spike proteins at the appropriate time and location results in efficient virus entry activation. Type II transmembrane serine proteases (TTSPs), specifically, transmembrane protease serine 2 (TMPRSS2) when expressed on target cells, enhance SARS coronavirus entry by activating spike cleavage. This study investigated the effect of TTSPs expressed in Coronavirus producer cells. Murine Hepatitis Virus strain A59 (MHV A59) viruses produced in the absence of TMPRSS2 required target cell protease activity - either cell surface serine proteases or endosomal acidophilic proteases - for entry. MHV A59 viruses produced in the presence of TMPRSS2 were less infectious overall, …

Hypervirulent Clostridium Difficile Strains: Adherence, Toxin Production And Sporulation, Michelle Marie Merrigan

Dissertations

Clostridium difficile is a leading cause of nosocomial infections, and recently emerged "hypervirulent" C. difficile strains have caused epidemics worldwide. We hypothesized that multiple factors were responsible for this phenotype, particularly the interaction of C. difficile with the epithelial cell, as well as toxin production and sporulation.

To test if host interaction varied in HV strains, we developed a quantitative host-cell adherence assay, and found that while C. difficile strains varied in adherence to human intestinal epithelial cells, hypervirulent strains were not significantly more adherent than other strains. The bacterial surface protein SlpA varied in both size and amount between …

Coronavirus Replicase Proteins: Multifunctional Mediators Of Replication And Innate Immunity Evasion, Mark Anthony Clementz

Dissertations

Coronaviruses are positive-sense, single-stranded RNA viruses. The majority of the RNA encodes non-structural proteins (nsps) that are translated as a large polyprotein, which is cleaved by the papain-like (PLP) and picornavirus 3C-like (3CLpro) proteases. The nsps modify host membranes to produce double membrane vesicles (DMVs) upon which the replicase-transcriptase assembles and synthesizes viral RNA. nsp3, nsp4, and nsp6 are integral membrane proteins believed to be involved in DMV formation. Work presented here demonstrates that nsp4 is subjected to N-linked glycosylation and mutation of N258 to threonine in nsp4 confers a temperature sensitive phenotype to MHV-A59 infectious clone virus. This virus …

Characterization Of Tram, A Required Bacteroides Fragilis Conjugal Apparatus Protein That Interacts With The B. Fragilis Putative Coupling Protein Bcta And The B. Fragilis Relaxase Protein Bmph, Mai Thi Thanh Nguyen

Dissertations

Bacteroides spp. organisms, the most prominent bacteria of the human colon, are reservoirs of antibiotic resistance genes which promiscuously transfer within and from the genus via conjugation. The B. fragilis clinical isolate LV23 harbors a 37kb conjugative transposon, BTF-37. Two important conjugal apparatus proteins encoded by BTF-37 are BctA, a predicted coupling protein (CP) that powers DNA translocation, and ORF7 (TraM). In this study, we demonstrated that TraM exhibits characteristics of a required conjugal apparatus protein including 1) upregulated expression under conjugation conditions; 2) localization to the bacterial inner membrane; 3) interaction with the putative coupling protein BctA and the …

Morphogenesis Of The Bacillus Anthracis Spore, Michael Mallozzi

Dissertations

With the ability to shut down metabolism, survive exposure to high degrees of heat, ultra-violet and ionizing radiation, digestive enzymes, and antimicrobials, the bacterial spore is one of the most-resistant forms of life on earth. Although most spores are harmless, several species' spores cause serious diseases such as tetanus, anthrax, gas gangrene, botulism, and diarrhea. Spore-forming bacteria differentiate into spores in response to stresses (especially starvation) in a multi-stage developmental pathway involving the coordinated expression of hundreds of genes and the formation of specialized protective organelles which surround and protect the spore during dormancy. The Bacillus anthracis spore (the causative …

Deconstructing Ihf-Mediated Inhibition Of The Complex Acs Promoter, David S. Thach

Dissertations

acs encodes a high affinity enzyme that permits survival during carbon starvation. As befits a survival gene, its transcription is subject to complex regulation. Previously, the Wolfe lab reported that CRP activates acs transcription by binding tandem DNA sites located upstream of the major acsP2 promoter and that the nucleoid protein IHF binds three specific sites located just upstream. The most proximal site (IHF III) exhibits reduced transcription compared to the full-length promoter or to a construct lacking all three IHF sites. The goal of my research was to understand how IHF III inhibits CRP-dependent acs transcription. First, I helped …

Vh Gene Usage In Vha2 Allotype-Producing B Cells From Mutant Alicia Rabbits, Anusorn Boonthum

Dissertations

No abstract provided.

Interleukin-2 Activated Lymphocytes Use Cd11b/Cd18 For Adhesion To Candida Albicans, Christopher B. Forsyth

Dissertations

No abstract provided.

Physiological Role Of The Short Form Of The Histidine Protein Kinase, Cheas, In Bacterial Chemotaxis, Barry Patrick Mcnamara

Dissertations

No abstract provided.

Structure-Function Analysis Of The Catalytic Domain Of The Histidine Kinase Chea, Dolph David Ellefson

Dissertations

No abstract provided.

V, D And J Gene Rearrangements On The Unexpressed Igh Allele In Rabbit B Cells, Chainarong Tunyaplin

Dissertations

No abstract provided.

Nonsense Codon-Mediated Degradation Of Immunoglobulin Heavy-Chain [Mu] Messenger Rna In B Cells, Tianhong Li

Dissertations

No abstract provided.

Effects Of Rearing Environment On The Somatic Diversification Of Rabbit Immunoglobulin Vdj Genes, David P. Brashinger

Master's Theses

No abstract provided.

Vh And D Gene Usage In B-Lineage Cells From Young Rabbits, Martha L. Friedman

Master's Theses

No abstract provided.