Bacteriology Commons^™

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).

Damped Oscillating Phosphoryl Transfer Reaction In The Cyanobacterial Circadian Clock, Hye In Jang, Pyonghwa Kim, Yongick Kim

Chemistry Faculty Research

Most organisms have circadian clocks to ensure the metabolic cycle to resonate with the rhythmic environmental changes without “damping,” or losing robustness. Cyanobacteria is the oldest and simplest form of life that is known to harbor this biological intricacy. Its KaiABC-based central oscillator proteins can be reconstituted inside a test tube, and the post-translational modification cycle occurs with 24 h periodicity. KaiC’s two major phosphorylation sites, Ser-431 and Thr-432, become phosphorylated and dephosphorylated by interacting with KaiA and KaiB, respectively. Here, we mutate Thr-432 into Ser to find the oscillatory phosphoryl transfer reaction damps. Previously, this mutant KaiC was reported …

Enrichment Media Selection And Co-Culture Potential Among Exoelectrogen Bacteria Vary With Ecological Factors, Nur Syafira Khoirunnisa, Syaiful Anwar, Untung Sudadi, Dwi Andreas Santosa

Makara Journal of Science

Staphylococcus saprophyticus ICBB 9554 and Citrobacter freundii ICBB 9763 are exoelectrogen bacteria applied as microbial fuel cells (MFC). We selected enrichment media for both these exoelectrogen bacteria, characterized their synergistic traits, and evaluated the growth conditions under different ecological factors. In this study, different enrichment media, such as those containing sugar, molasses, and palm sugar (2, 4, and 6% w/v) were tested for S. saprophyticus ICBB 9554. Meanwhile, technical sodium acetate (NaAc), commercial vinegar 25%, and cider vinegar (5, 10, and 15 mM acetate concentration) were tested for C. freundii ICBB 9763. Both the exoelectrogen bacteria were tested for the …

Tools For Quantifying Bacterial Motility Using Digital Holographic Microscopy As Applied To Studying The Simulated Microgravity Environment, Jacqueline Marie Acres

Dissertations and Theses

Digital holographic microcopy (DHM) is a label-free technique that has gained attention in recent years as a tool for volumetric imaging. One application of DHM is for the study of microbial motility with the advantage being that organisms may freely move within their environment. Images created from DHM are in the form of holograms. Holograms are time recordings showing XY information with the Z information contained within. Z information can be retrieved from the holograms directly through a variety of numerical techniques or through reconstruction. Datasets generated from DHM are large and processing remains a challenging task. Here, we show …

Fish Farming Wastewater Treatment Using Algae-Cyanobacterial Inocula, Yolanys Aranda

Graduate Industrial Research Symposium

Fish is one of the most important nutrient sources of the human diet, and its low-cost aquaculture production is advantageous. During processing, large quantities of organic and inorganic nutrients are produced. Discharging fish farming wastewater to the environment without proper treatment can support harmful algal growth. Therefore, aquaculture effluent must be treated prior to discharge to the receiving waters. In this study, six different cyanobacteria and algae species, including Chlorococcumminutum, Porphyridiumcruentum, Chlorella vulgaris, Microcystis aeruginosa, Chlamydomonas reinhardtii, and Fischerellamuscicola were studiedto treatfish aquaculture wastewater, using mix-culture bacteria as the inoccolum. All samples were incubated for 21 days. Parameters such as …

Investigating The Diversity Of Wolbachia Across The Spiny Ants (Polyrhachis), Jenna L. Webb, Leland C. Graber, Manuela O. Ramalho, Corrie S. Moreau

Biology Faculty Publications

Among insects, Wolbachia is an exceedingly common bacterial endosymbiont with a range of consequences of infection. Despite the frequency of Wolbachia infection, very little is known about this bacteria's diversity and role within hosts, especially within ant hosts. In this study, we analyze the occurrence and diversity of Wolbachia across the spiny ants (Polyrhachis), a large and geographically diverse genus. Polyrhachis samples from throughout the host genus' phylogenetic and biogeographical range were first screened for single infections of Wolbachia using the wsp gene and Sanger sequencing. The multilocus sequence typing (MLST) scheme was then used on these singly infected samples …

Conformational Rearrangements In The Sensory Rcsf/Omp Complex Mediate Signal Transduction Across The Bacterial Cell Envelope, Sarah Rosemarie Lach Phd

Dissertations and Theses (Open Access)

Timely detection and repair of envelope damage are paramount for bacterial survival. The Regulator of Capsule Synthesis (Rcs) stress response is a complex signaling cascade that monitors gram-negative cell envelope integrity and can transduce the stress signals across the multilayered envelope to regulate gene expression in the cytoplasm. The outer membrane (OM) lipoprotein RcsF is the sensory component, but how RcsF functions remains elusive. RcsF interacts with the β-barrel assembly machinery (Bam) complex, which assembles RcsF in complex with OM proteins (OMPs), resulting in RcsF’s partial cell surface exposure. RcsF can also interact with the periplasmic domain of the negative …

The Immunomodulating Effects Of Delta-9 Tetrahydrocannabinol (Thc) And Cannabidiol (Cbd) In The Context Of Infection, Rose D. Goodman, Kate Rouse, Victor Jimenez

Annual Research Symposium

No abstract provided.

Evaluation Of Qpcr To Detect Shifts In Population Composition Of The Rhizobial Symbiont Mesorhizobium Japonicum During Serial In Planta Transfers, Kenjiro W. Quides, Yoobeen Lee, Teresa Hur, Hagop S. Atamian

Biology, Chemistry, and Environmental Sciences Faculty Articles and Research

Microbial symbionts range from mutualistic to commensal to antagonistic. While these roles are distinct in their outcome, they are also fluid in a changing environment. Here, we used the Lotus japonicus–Mesorhizobium japonicum symbiosis to investigate short-term and long-term shifts in population abundance using an effective, fast, and low-cost tracking methodology for M. japonicum. We use quantitative polymerase chain reaction (qPCR) to track previously generated signature-tagged M. japonicum mutants targeting the Tn5 transposon insertion and the flanking gene. We used a highly beneficial wild type and moderately beneficial and non-beneficial mutants of M. japonicum sp. nov. to demonstrate the specificity …

Genetic Determinants Involved In Acquisition Of Iron In The Opportunistic Pathogen Mycobacterium Abscessus, Manal F. Farhat

Dissertations, Theses, and Capstone Projects

The opportunistic pathogen Mycobacterium abscessus subsp. abscessus (Mab) has become an emerging public health threat due to the increasing number of Mab-associated chronic pulmonary disease cases. Infection cure rates are only 50% due to the complex resistance of Mab to most antimicrobial drugs and chemotherapy.

Deeper knowledge of Mab biology is needed to illuminate potential candidates for development of improved therapeutics against Mab infections. The ESX-3 type VII protein secretion system of Mab plays an important role in host inflammatory and pathological responses during infection.

In this study, we demonstrate a functional link between the ESX-3 secretion …

Untangling The Complex Interactions Between Turtle Ants And Their Microbial Partners, Manuela O. Ramalho, Corrie S. Moreau

Biology Faculty Publications

Background: To understand the patterns of biodiversity it is important to consider symbiotic interactions as they can shape animal evolution. In several ant genera symbiotic interactions with microbial communities have been shown to have profound impacts for the host. For example, we know that for Camponotini the gut community can upgrade the host's diet and is shaped by development and colony interactions. However, what is true for one ant group may not be true for another. For the microbial communities that have been examined across ants we see variation in the diversity, host factors that structure these communities, and the …

Clonal Diversity, Antibiotic Resistance, And Virulence Factor Prevalence Of Community Associated Staphylococcus Aureus In Southeastern Virginia, Katelyn D. Cranmer, Mohan D. Pant, Suzanne Quesnel, Julia A. Sharp

EVMS School of Health Professions Faculty Publications

Staphylococcus aureus is a significant human pathogen with a formidable propensity for antibiotic resistance. Worldwide, it is the leading cause of skin and soft tissue infections (SSTI), septic arthritis, osteomyelitis, and infective endocarditis originating from both community- and healthcare associated settings. Although often grouped by methicillin resistance, both methicillin-resistant (MRSA) and methicillin-sensitive (MSSA) strains are known to cause significant pathologies and injuries. Virulence factors and growing resistance to antibiotics play major roles in the pathogenicity of community-associated strains. In our study, we examined the genetic variability and acquired antibiograms of 122 S. aureus clinical isolates from SSTI, blood, and urinary …

Identification Of Novel Biosynthetic Gene Clusters Encoding For Polyketide/Nrps-Producing Chemotherapeutic Compounds From Marine-Derived Streptomyces Hygroscopicus From A Marine Sanctuary, Hannah Ruth Flaherty

Honors Theses and Capstones

Nearly one out of six deaths in 2020, around ten million people, were caused by cancer, making it a leading cause of death worldwide (WHO, 2022). This major public health issue, in addition to the rise of multidrug-resistant (MDR) pathogens, provides a high demand for the discovery of new pharmaceutical drugs to be used clinically to treat these conditions. The Streptomyces genus accounts to produce 39% of all microbial metabolites currently approved for human health, indicating its potential as an important species to study for antimicrobial and anticancer agents. The long linear genome of Streptomyces contains specialized sequences known as …

Trends In Antibiotic Susceptibility Of Staphylococcus Aureus Isolates In A Pediatric Hospital: An Analysis Of The Impact Of The Sars-Cov-2 Pandemic, Juan Miguel S. Gonzalez Rivero

Honors Undergraduate Theses

Infections caused by the organism Staphylococcus aureus are one of the most common causes for community-associated and healthcare-acquired infections (HAI). Isolates of this bacterium found within the healthcare setting often demonstrate a higher prevalence of antibiotic resistance making these infections difficult to treat. Historically, considerable focus has been placed on methicillin-resistant S. aureus (MRSA), which are strains resistant to β-lactam antibiotics like penicillin, oxacillin and cephalosporins; however, methicillin-sensitive (MSSA) strains may also possess resistance to several first-line antibiotics. Resistance to antibiotics can be acquired through horizontal gene transfer (HGT) by means of mobile genetic elements or by random DNA mutations …

Effects Of Altering Physiologically Relevant Cholesterol Levels And Media Types On Porin Gene Expression In Klebsiella Pneumoniae And The Resulting Impact On Antibiotic Resistance, Megan R. Camden

UNF Graduate Theses and Dissertations

Klebsiella pneumoniae is a Gram-negative, non-motile bacterium belonging to the Enterobacteriaceae family. K. pneumoniae is a rising threat in the clinical setting, as there has been a large increase in the presence of antibiotic resistant isolates. While much research is conducted on laboratory and clinical strains of bacteria, not much is known regarding the impact that human physiology can have on bacterial gene expression, and in response, to antibiotic susceptibility. The goal of this study is to determine if physiologically relevant cholesterol levels and media types impact porin gene expression and antibiotic resistance in K. pneumoniae. To accomplish this, …

Probing The Porphyromonas Gingivalis Nitrosative Stress Response Network, Alexandra Galina

Theses and Dissertations

Objective: To persist in the oral cavity, the perio-pathogen Porphyromonas gingivalis must have an efficient nitrosative stress response mechanism. To better understand how P. gingivalis responds to nitrite (NO₂^-) we must determine what genes are involved in the nitrosative stress response network.

Methods: Through genomic screening, a list of genes was generated that were hypothesized to be part of the nitrosative stress response network and believed to be involved in nitrate/nitrite metabolism or redox state of the cells. Protein coding regions were replaced with the ermF sequence by allelic exchange mutagenesis. 15 mutants were created …

Treponema Denticola Synthesizes C-Di-Amp And Encodes The Cdaa-Type Diadenylate Cyclase Cdaa, Claire R. O'Brien

Theses and Dissertations

Periodontitis is a form of oral disease characterized by dysbiosis of the oral microbiome, leading to inflammation, bone resorption, and in severe cases, entire tooth loss, affecting 42% of adults in the US. One of the bacteria most associated with periodontal disease progression is Treponema denticola (Td), an oral spirochete which inhabits the mouth in small quantities during health but which can dominate the biofilms that form during periodontal disease. The ability of Td to survive in a disease environment and contribute to the progression of disease requires the use of robust signaling networks. Analysis of Td cultures …

Functional Residues Of Cjpata, Robert Lamont

Theses and Dissertations (Comprehensive)

Antibiotic resistance in pathogenic bacteria continues to challenge clinicians and threaten the lives of infected individuals. For this reason, new classes of antibiotics or new targets for antibiotic therapy are needed to circumvent this global health crisis. In Gram-negative bacteria, the enzymes peptidoglycan O-acetyltransferase A and B (PatA and PatB), are responsible for adding acetyl groups to the C-6 hydroxyl group of N-acetyl muramic acid (MurNAc) in peptidoglycan (PG). These acetyl groups are responsible for inhibiting the activity of lysozyme from host organisms that hydrolyze the β1-4 glycosidic linkage between MurNAc and N-acetylglucosamine (GlcNAc). The acetyl groups …

The Type Iv Pilus Secretin Bfpb: Structural Analysis And Binding Interactions, Janay I. Little

Theses and Dissertations

Enteropathogenic Escherichia coli (EPEC) causes severe diarrhea in young children. The type IV pilus (T4P) of EPEC, known as the bundle-forming pilus (BFP), plays an important role in EPEC pathogenesis. T4Ps are a family of surface appendages that are important for adhesion, colonization, biofilm formation, virulence, twitching motility and many other functions. One essential component of the BFP system is the secretin, BfpB. Secretins are a large family of integral outer membrane proteins found in T4Ps as well as type II and type III secretion systems, and filamentous phages. Details of the secretin structure have been limited to the overall …

Distribution Patterns Of Allorhizobium Vitis In Missouri Vineyards And Non-Vineyard Soils, Jacquelyn M. Wray

Graduate Theses/Dissertations

Crown gall disease causes significant economic loss to the grape and wine industry. Preventive strategies are most effective for mitigating the loss of grapevines in vineyards, as there is no known cure for this disease. The bacterium Allorhizobium vitis carrying a tumor-inducing (Ti) plasmid is the causative agent. A. vitis bacteria live systemically in the grapevine before causing visible symptoms and can survive in residual plant tissues and soil for more than two years. Diagnostic methods have been developed to detect A. vitis bacteria in grapevines and soil. However, more reliable, specific, and high-throughput diagnostics are still needed for screening …