Browsing by Subject "Bacteriology"
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Item A Novel sRNA Member of the Carbon Storage Regulatory System of Escherichia Coli(2002-12-01) Weilbacher, Thomas; Jerry SimeckaWeilbacher, Thomas S., A Novel sRNA Member of the Carbon Storage Regulatory System of Escherichi coli. Master of Science (Microbiology & Immunology), December, 2002, 57 pp., 2 tables, 12 illustrations, bibliography, 44 titles. Small untranslated RNAs (sRNAs) perform a variety of important functions in bacterial systems. The 245 nt sRNA of Escherichia coli K-12, CsrC, was uncovered using a genetic screen for genes that regulate glycogen biosynthesis. CsrC RNA binds multiple copies of CsrA, a protein that post-transcriptionally regulates central carbon flux, biofilm formation, and motility in E. coli. CsrC antagonizes the regulatory effects of CsrA, presumably by sequestering this protein. The discovery of CsrC is intriguing, in that a similar sRNA, CsrB, performs essentially the same function. Both of these sRNAs possess similar imperfect repeat sequences (18 in CsrB, 9 in CsrC), primarily localized in the loops of predicted hairpins, which may serve as CsrA binding elements. Transcription of csrC increases as the culture approaches the stationary phase of growth and is activated by CsrA and the response regulator UvrY. Complementation and in vitro transcription-translation experiments reveal that CsrA effects on csrC are mediated indirectly, through UvrY. Because CsrB and CsrC antagonize the activity of CsrA and are dependent on CsrA for their synthesis, a csrB null mutation causes a modest compensatory increase in CsrC levels and vice versa. An updated model for the signaling circuitry of the Csr system is discussed.Item Lethality of Staphylococcus in Murine Pneumonia is Due to Alpha-Toxin and Other Secreted Factors Regulated by AGR and SAR(2003-08-01) Overheim, Katie A.; Dan Dimitrijevich; Glenn Dillon; James CaffreyOverheim, Katie A., Lethality of Staphlococcus aureus in Murine Pneumonia is Due to Alpha-Toxin and Other Secreted Factors Regulated by agr and sar. Doctor of Philosophy (Biomedical Sciences), August, 2003, 91 pp, 6 Tables, 9 illustrations, bibliography, 106. The purpose of these studies was to determine if the S. aureus global regulators agr and sar play a role in staphylococcal pneumonia and if the virulence factors regulated by them contributed to the severity of staphylococcal pneumonia. To determine this, we established a pneumonia model in mice in order to identify if S. aureus global regulators agr and sar play a role in the pathogenesis of staphylococcal pneumonia. As well, we took steps to identify the extracellular factors responsible for the lethality in a murine model of staphylococcal pneumonia and determine if these factors involved in disease process could be used as targets for immune therapy. My work revealed that lethal pneumonia in a mouse model is dependent on the S. aureus global regulators agr and sar. This study also revealed that the lethality associated with our model is due to secreted factors, regulated by S. aureus global regulators agr and sar. Further investigation demonstrated the alpha-toxin is a major virulence factor involved in the lethality in our model. By generating an alpha-toxin deficient strain in S. aureus RN6390, we show a reduced virulence in our disease model. As well, antiserum to alpha-toxin, when administered with a lethal dose of S. aureus RN6390, we show a reduced virulence in our disease model. As well, antiserum to alpha-toxin, when administered a lethal dose of S. aureus RN6390, we show a reduced virulence in our disease model. As well, antiserum to alpha-toxin, when administered with a lethal dose of S. aureus RN6390 protected animals from death. By evaluating the role of alpha-toxin’s ability to contribute to lethality, we assessed numerous strains of S. aureus in our pneumonia model. We discovered that there was a correlation to alpha-toxin production levels and lethality in our pneumonia model. However, our study also demonstrated that alpha-toxin is not the only factor involved in the disease process.Item Positive Regulation of Acetate Metabolism and Motility by the RNA-Binding Protein CsrA in Escherichia coli(2000-08-01) Wei, Bangdong L.; Jerry Simecka; Ming-Chi Wu; Stephen R. GrantWei, Bangdong L., Positive Regulation of Acetate Metabolism and Motility by the RNA-binding Protein CsrA in Escherichia coli. Doctor of Philosophy (Biomedical Sciences), August, 2000, 118 pp., 5 tables, 19 illustrations, bibliography, 175 titles. The carbon storage regulatory (Csr) system consists of a small RNA-binding effector protein, CsrA, and non-coding RNA, CsrB. CsrA acts as a global regulator and modulates specific mRNA stability in Escherichia coli. It regulates central carbon metabolism, physiology, and cell surface properties on a broad scale. In this study, the regulatory roles of csrA in acetate metabolism and motility were examined. The csrA gene was demonstrated to positively regulate acetyl-CoA synthetase and isocitrate lyase, while it did not affect phosphotransacetylase, isocitrate dehydrogenase, or citrate synthase. As a result, growth of csrA rpoS mutant strains was very poor on acetate as a sole carbon source. Surprisingly, growth also was inhibited specifically by the addition of modest amounts of acetate to rich media. Cultures grown in the presence of ≥25 mM acetate consisted substantially of glycogen biosynthesis (glg) mutants, which were no longer inhibited by acetate. Several classes of glg mutations were mapped to known and novel loci. The TCA cycle intermediates or pyruvate, but not glucose, galactose or glycerol, restored growth and prevented the glg mutations in the presence of acetate. Furthermore, amino acid uptake was inhibited by acetate specifically in the csrA rpoS strain. Apparently, central carbon flux imbalance, inhibition of amino acid uptake, and a deficiency in acetate metabolism are combined to cause metabolic stress by depleting the TCA cycle. The csrA gene was essential for motility and flagellum biosynthesis. Further studies elucidated the molecular mechanism by which CsrA positively regulates flagellum synthesis. Purified recombinant CsrA protein, which was isolated as a ribonucleoprotein complex consisting of one single CsrB molecule and ~18 CsrA subunits, directly stimulated the coupled transcription-translation of flhDC::lacZ in S-30 extracts and bound specifically to the 5’ non-coding segment of flhDC mRNA in mobility shift assay. The steady state level of flhDC mRNA was higher and its half-life was ~3-fold greater in a csrA wild type versus a csrA::kanR mutant strain, as shown by RT-PCR. Thus, CsrA is able to stimulate flhDC gene expression by a post-transcriptional mechanism that resembles its function in repression.Item Quorum Sensing in Sinorhizobium meliloti(2008-12-01) Patankar, Arati V.; Juan E. Gonzales; Jerry W. Simecka; Stephen O. MathewPatankar, Arati V., Quorum Sensing in Sinorhizobium meliloti. Doctor of Philosophy (Microbiology and Immunology), December 2008, 170 pp., 14 tables, 23 illustrations, bibliography, 212 titles. The overall goal of this study was to elucidate the role of a series of transcriptional regulators and potential signal molecules in the coordination of gene regulation in Sinorhizobium meliloti. The agriculturally important gram-negative soil bacterium S. meliloti, forms a symbiotic association with its host legume, Medicago sativa (alfalfa); thereby serving as a good model for studying host-bacterial interactions. Often, bacteria associated with eukaryotic hosts utilize global gene regulatory systems to coordinate their behavior in order to establish pathogenic or symbiotic associations. Quorum sensing is one such form of bacterial gene regulation which is mediated by signaling molecules and regulatory proteins in a population density dependent manner. In S. meliloti, the process of quorum sensing has been shown to play an important role in the relationship with its host plant. Control of essential processes such as plant nodulation and exopolysaccharide production has been attributed to the Sin/ExpR quorum-sensing system of S. meliloti. Interestingly, S. meliloti contains four additional (SMc04032, SMc00658, Smc00878 and SMc00877) putative quorum-sensing response regulators whose regulatory network was not known. The predicted protein sequences of these genes contain features typical of the LuxR family of proteins i.e., an N-terminal signal binding domain and C-terminal helix-turn-helix DNA biding domain. In order to identify their regulatory role, mutants of the response regulators were constructed and their expression profile was determined by employing genome-wide microarray and real-time PCR expression analysis. Through these analyses, it was determined that the SMc004032 locus controls expression of genes involved in the active methyl cycle, while the SMc00658, SMc00878 and SMc00877 loci control expression of genes from the denitrification of pathway of S. meliloti. Further, through phenotypic studies it was established that SMc04032 impacts stress response adaptation, and effective competition for plant nodulation. This suggests that SMc04032 could play a role in bacterial survival in the soil as well as within the host. The ability to denitrify is highly variable in different strains of S. meliloti. Through growth and enzymatic assays, it was established that the wild-type strain of this study, S. meliloti Rm8530, is a partial dentrifier in which, the capacity to metabolize nitrate is impaired. It was further determined that SMc00658, SMc00878 and SMc0877 modulated nitrite reductase activity under aerobic conditions, implying that these genes are involved in aerobic denitrification and therefor probably play a role in detoxification in S. meliloti. Based on the sequenced-genome analysis, S. meliloti possess homologs of other mediators of quorum sensing, that might be responsible for the synthesis of novel signal molecules. Bioreporter strains and mass spectrometry analysis were employed to identify production of cyclic dipeptides in S. meliloti. These compounds have been previously reported as quorum-sensing signal molecules in several bacteria. The results presented in this study provide a better understanding of S. meliloti’s metabolic and physiological properties and will be fundamental in future studies of bacterial interaction with its host and survival within its ecological niche.Item Resistance of Bacillus Subtilis Spores Lacking Either Nucleotide Excision Repair or Spore Photoproduct Lyase to Ultraviolet (UV) Radiation from Artificial or Natural Sources(1996-06-01) Xue, Yaming; Tony Romeo; Ming-Chi Wu; Wayne L. NicholsonXue, Yaming, Resistance of Bacillus subtilis spores lacking either nucleotide excision repair or spore photoproduct lyase to ultraviolet (UV) radiation from artificial or natural sources. Master of Science (Biomedical Sciences), June 1996, pp., 4 tables, 12 illustrations, references, 38 titles. Exposure of bacterial spores to UV radiation causes the accumulation of a unique pyrimidine dimer in the DNA, “spore photoproduct” (SP). In Bacillus subtilis, two distinct DNA repair pathways are used for removal of SP: general nucleotide excision repair (the uvr parthway), or the SP-specific enzyme SP lyase (the spl pathway). Spores of four strains of Bacillus subtilis differing in their repair capabilities were irradiated under either artificial or solar UV. To determine the biologically-relevant cumulative UV dose under each irradiation condition, a sporocidal dosimeter was constructed. The results showed: (i) Both uvr and spl pathways contributed to the survival of spores under all tested conditions. The spl pathway was more efficient than uvr pathway in repairing the DNA damage caused by UV-C and solar UV-A, but no significant difference was noted in repairing DNA damage caused by UV-B or full-spectrum solar UV. (ii) Exposure of spores to solar UV can cause cellular lethal damage which is reparable by neither repair pathway.Item The Effects of Two Staphylococcal Global Regulators (agr and sar) on Acid Phosphatase production in Staphylococcus aureus(2003-05-01) Agouna-Deciat, Bahrka Olivier; Jerry Simecka; Michael SmithAgouna-Deciat, B. Olivier, The Effect of Two Staphylococcal Global Regulators (agr and sar) on Acid Phosphatase Production in Staphylococcus aureus. Master of Science (Molecular Biology and Immunology), May 2003, 75 pp., 3 tables, 14 illustrations, 16 titles. Staphylococcus aureus produces an extensive number of cell-surface associated proteins, extracellular proteins and enzymes that contribute to its virulence. The key to better preventative or curative approaches resides in identifying and targeting the very genes and their products that play major roles in the survival of the bacteria within the host and the establishment of diseases. Two well known regulatory loci, the accessory gene regulatory (agr) and the staphylococcal accessory regulator (sar), control the expression of most S. aureus genes that encode for its virulence factors. Other virulence gene regulators have recently been isolated. Over 40 proteins and enzymes produced by S. aureus have been identified and several of them have been linked to staphylococcal pathogenesis. In this study, we attempt to determine the role of agr and sar in the regulation of the production of a secreted staphylococcal acid phosphatase (Sap) suspected to contribute to virulence.Item Transmission and Establishment of Helicobacter pylori Infection in Childhood(2013-08-01) Cervantes, Diana; David SterlingTransmission of Helicobacter pylori infection is thought to occur predominately during childhood in both developed and developing countries. Various bacterial, host and environmental factors influence H. pylori transmission and establishment of infection. The most likely routes of H. pylori transmission include gastro-oral, fecal-oral and oral-oral. Therefore, conditions such as close, personal contact with a child’s infected mother and siblings may favor H. pylori transmission. The immunological response mounted against H. pylori infection by a child, possibly modulated by age and passive immunity, may alter the establishment of infection. The current research, utilizing data from The Pasitos Cohort Study, aimed to estimate the effect that H. pylori infected mothers, H. pylori infected siblings and breastfeeding may have in the acquisition and establishment of H. pylori infection in children. The Pasitos Cohort Study, a birth cohort established on the U.S.-Mexico border, recruited pregnant women from El Paso, Texas and Juarez, Mexico to identify risk factors for H. pylori infection during childhood. Assessment of active H. pylori infection for 615 children (472 index children and 143 younger siblings) was performed from April 1998 until December 2005 accounting for an average of 3.8 years of follow-up for the index child and an average of 2.5 years of follow-up for younger siblings of the index. The Pasitos Cohort Study provides advantages such as longitudinal assessment of H. pylori status for determination of directionality of H. pylori infection in familial transmission. In addition, differentiation between infection acquisition, short-term and long-term establishment of infection was attempted. The current study found that infected older siblings in the household were positively associated with both the acquisition and long-term infection establishment of H. pylori in younger siblings. Mothers and breastfeeding may also be positively associated with H. pylori outcomes but due to highly uncertain results obtained, further studies are need to assess their association with infection acquisition, short-term infection and long-term infection in children.