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Author (up) Bhattacharya, R.; Beck, D.J. file  url
openurl 
  Title Survival and SOS induction in cisplatin-treated Escherichia coli deficient in Pol II, RecBCD and RecFOR functions Type Journal Article
  Year 2002 Publication DNA Repair Abbreviated Journal DNA Repair (Amst)  
  Volume 1 Issue 11 Pages 955-966  
  Keywords Antineoplastic Agents/*pharmacology; Bacterial Proteins/physiology; Cell Division/drug effects/genetics/radiation effects; Cisplatin/*pharmacology; DNA Damage/drug effects/radiation effects; DNA Polymerase II/*physiology; DNA Polymerase III/physiology; DNA Repair/drug effects/radiation effects; DNA-Binding Proteins/physiology; Dose-Response Relationship, Drug; Drug Resistance, Bacterial/physiology; Escherichia coli/*drug effects/enzymology; Escherichia coli Proteins/pharmacology/*physiology; Exodeoxyribonuclease V; Exodeoxyribonucleases/*physiology; Lac Operon; SOS Response (Genetics)/*physiology; beta-Galactosidase/metabolism  
  Abstract Cisplatin is a potent anticancer agent forming intrastrand-crosslinks in DNA. The efficacy of cisplatin in chemotherapy can be limited by the development of tumor resistances such as elevated DNA repair or damage tolerance. In Escherichia coli, cisplatin treatment causes induction of the SOS regulon resulting in elevated levels of DNA Pol II, DNA Pol IV, DNA Pol V, the cell division inhibitor SfiA (SulA), homologous recombination (HR) and DNA repair. In this work, the roles of Pol II and HR in facilitating resistance of E. coli to cisplatin are studied. SOS induction levels were measured by beta-galactosidase assays in cisplatin-treated and untreated E. coli PQ30 that has the lacZ gene fused to the sfiA promoter. Comparative studies were carried out with derivatives of PQ30 constructed by P1 transduction that have transposon insertions in the polB gene, the recB gene blocking the RecBCD pathway of HR and genes of the RecFOR pathway of HR. Resistance of E. coli strains to cisplatin as determined by plating experiments decreased in the following order: parent PQ30 strain, polB > recO, recR, recF > recB. Both the RecBCD and RecFOR pathways of HR are important for survival when E. coli is exposed to cisplatin, because treatment of double mutants deficient in both pathways reduced colony forming ability to 37% in 6-9min in comparison to 39-120min for single mutants. Pol II and RecF appear to function in two distinct pathways to initiate replication blocked due to damage caused by cisplatin because function of Pol II was required for survival in mutants deficient in the RecFOR pathway after 2h of cisplatin treatment. In contrast, Pol II was not required for survival in recB mutants. SOS induction was delayed in RecFOR deficient mutants but occurred at high levels in the recB mutant soon after cisplatin treatment in a RecFOR-dependent way. An SfiA independent, DNA damage dependent pathway is apparently responsible for the filamentous cells observed after cisplatin or MMC treatments of these SfiA defective strains.  
  Call Number Serial 407  
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Author (up) Garrison-Schilling, K.L.; Grau, B.L.; McCarter, K.S.; Olivier, B.J.; Comeaux, N.E.; Pettis, G.S. file  url
doi  openurl
  Title Calcium promotes exopolysaccharide phase variation and biofilm formation of the resulting phase variants in the human pathogen Vibrio vulnificus Type Journal Article
  Year 2011 Publication Environmental Microbiology Abbreviated Journal Environ Microbiol  
  Volume 13 Issue 3 Pages 643-654  
  Keywords *Biofilms; Calcium/*pharmacology; Humans; Operon; Phenotype; Polysaccharides, Bacterial/*metabolism; Vibrio vulnificus/genetics/pathogenicity/*physiology  
  Abstract Vibrio vulnificus is a Gram-negative bacterium found in estuaries and coastal waters and is associated with human disease caused by ingestion of raw shellfish. Pathogenesis is directly related to the presence of capsular polysaccharide (CPS). Encapsulated virulent strains exhibit an opaque colony phenotype, while unencapsulated attenuated strains appear translucent. A third colony type, rugose, is caused by expression of rugose extracellular polysaccharide (rEPS) and forms robust biofilms. Vibrio vulnificus undergoes phase variation associated with altered levels of CPS and rEPS, and we show here that calcium (Ca(2)(+) ) significantly increases the rate of CPS and rEPS phase variation in this species. Interestingly, multiple phenotypic responses to increased [Ca(2)(+) ] were observed among strains, which suggests the existence of underlying cognate genetic or epigenetic differences. Certain translucent isolates contained deletions at the group I CPS operon, inferring increased [Ca(2)(+) ] upregulates existing phase variation mechanisms. Expanding on a previous observation (Kierek and Watnick, Proc. Natl. Acad. Sci. USA 100: 14357-14362, 2003), increased [Ca(2)(+) ] also enhanced biofilm formation for all phase variants. Our results show that Ca(2)(+) promotes both polysaccharide phase variation and biofilm formation of the resulting phase variants, thereby likely serving a dual role in persistence of V. vulnificus in the environment.  
  Call Number Serial 424  
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Author (up) Hellman, L.M.; Fried, M.G. file  url
openurl 
  Title Electrophoretic mobility shift assay (EMSA) for detecting protein-nucleic acid interactions Type Journal Article
  Year 2007 Publication Nature Protocols Abbreviated Journal Nat Protoc  
  Volume 2 Issue 8 Pages 1849-1861  
  Keywords Bacterial Proteins/metabolism; Cyclic AMP Receptor Protein/metabolism; DNA-Binding Proteins/*analysis/metabolism; Electrophoretic Mobility Shift Assay/*methods; Escherichia coli/genetics/metabolism; Escherichia coli Proteins/metabolism; Humans; Lac Operon/genetics; Lac Repressors; O(6)-Methylguanine-DNA Methyltransferase/metabolism; Promoter Regions, Genetic; RNA-Binding Proteins/*analysis; Repressor Proteins/metabolism; Transcription Factors/metabolism  
  Abstract The gel electrophoresis mobility shift assay (EMSA) is used to detect protein complexes with nucleic acids. It is the core technology underlying a wide range of qualitative and quantitative analyses for the characterization of interacting systems. In the classical assay, solutions of protein and nucleic acid are combined and the resulting mixtures are subjected to electrophoresis under native conditions through polyacrylamide or agarose gel. After electrophoresis, the distribution of species containing nucleic acid is determined, usually by autoradiography of 32P-labeled nucleic acid. In general, protein-nucleic acid complexes migrate more slowly than the corresponding free nucleic acid. In this protocol, we identify the most important factors that determine the stabilities and electrophoretic mobilities of complexes under assay conditions. A representative protocol is provided and commonly used variants are discussed. Expected outcomes are briefly described. References to extensions of the method and a troubleshooting guide are provided.  
  Call Number Serial 1994  
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Author (up) Lambert, G.; Kussell, E. file  url
doi  openurl
  Title Memory and fitness optimization of bacteria under fluctuating environments Type Journal Article
  Year 2014 Publication PLoS Genetics Abbreviated Journal PLoS Genet  
  Volume 10 Issue 9 Pages e1004556  
  Keywords *Adaptation, Biological; Algorithms; *Bacterial Physiological Phenomena; *Environment; Gene Expression Regulation, Bacterial; Lac Operon; Models, Biological; Phenotype; Stress, Physiological  
  Abstract Bacteria prudently regulate their metabolic phenotypes by sensing the availability of specific nutrients, expressing the required genes for their metabolism, and repressing them after specific metabolites are depleted. It is unclear, however, how genetic networks maintain and transmit phenotypic states between generations under rapidly fluctuating environments. By subjecting bacteria to fluctuating carbon sources (glucose and lactose) using microfluidics, we discover two types of non-genetic memory in Escherichia coli and analyze their benefits. First, phenotypic memory conferred by transmission of stable intracellular lac proteins dramatically reduces lag phases under cyclical fluctuations with intermediate timescales (1-10 generations). Second, response memory, a hysteretic behavior in which gene expression persists after removal of its external inducer, enhances adaptation when environments fluctuate over short timescales (< 1 generation). Using a mathematical model we analyze the benefits of memory across environmental fluctuation timescales. We show that memory mechanisms provide an important class of survival strategies in biology that improve long-term fitness under fluctuating environments. These results can be used to understand how organisms adapt to fluctuating levels of nutrients, antibiotics, and other environmental stresses.  
  Call Number Serial 1140  
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Author (up) Shanks, R.M.Q.; Lahr, R.M.; Stella, N.A.; Arena, K.E.; Brothers, K.M.; Kwak, D.H.; Liu, X.; Kalivoda, E.J. file  url
openurl 
  Title A Serratia marcescens PigP homolog controls prodigiosin biosynthesis, swarming motility and hemolysis and is regulated by cAMP-CRP and HexS Type Journal Article
  Year 2013 Publication PloS one Abbreviated Journal PLoS One  
  Volume 8 Issue 3 Pages e57634  
  Keywords Bacterial Proteins/*genetics/metabolism; Depsipeptides/*biosynthesis/genetics/pharmacology; Erythrocytes/drug effects; *Gene Expression Regulation, Bacterial; Genetic Complementation Test; Hemolysis/drug effects; Hexosyltransferases/genetics/metabolism; Humans; Membrane Proteins/genetics/metabolism; Movement/drug effects; Mutation; Operon; Prodigiosin/*biosynthesis; Sequence Homology, Amino Acid; Serratia marcescens/*genetics/metabolism; Signal Transduction; Transcription Factors/*genetics/metabolism  
  Abstract Swarming motility and hemolysis are virulence-associated determinants for a wide array of pathogenic bacteria. The broad host-range opportunistic pathogen Serratia marcescens produces serratamolide, a small cyclic amino-lipid, that promotes swarming motility and hemolysis. Serratamolide is negatively regulated by the transcription factors HexS and CRP. Positive regulators of serratamolide production are unknown. Similar to serratamolide, the antibiotic pigment, prodigiosin, is regulated by temperature, growth phase, HexS, and CRP. Because of this co-regulation, we tested the hypothesis that a homolog of the PigP transcription factor of the atypical Serratia species ATCC 39006, which positively regulates prodigiosin biosynthesis, is also a positive regulator of serratamolide production in S. marcescens. Mutation of pigP in clinical, environmental, and laboratory strains of S. marcescens conferred pleiotropic phenotypes including the loss of swarming motility, hemolysis, and severely reduced prodigiosin and serratamolide synthesis. Transcriptional analysis and electrophoretic mobility shift assays place PigP in a regulatory pathway with upstream regulators CRP and HexS. The data from this study identifies a positive regulator of serratamolide production, describes novel roles for the PigP transcription factor, shows for the first time that PigP directly regulates the pigment biosynthetic operon, and identifies upstream regulators of pigP. This study suggests that PigP is important for the ability of S. marcescens to compete in the environment.  
  Call Number Serial 1612  
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