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Author (up) Downing, K.J.; Thomson, J.A. file  url
  Title Introduction of the Serratia marcescens chiA gene into an endophytic Pseudomonas fluorescens for the biocontrol of phytopathogenic fungi Type Journal Article
  Year 2000 Publication Canadian Journal of Microbiology Abbreviated Journal Can J Microbiol  
  Volume 46 Issue 4 Pages 363-369  
  Keywords Chitinases/*genetics/metabolism; DNA-Binding Proteins/genetics/metabolism; Escherichia coli/genetics; Fabaceae/microbiology; *Pest Control, Biological; Plant Diseases/microbiology; Plants, Medicinal; Plasmids/genetics; Polymerase Chain Reaction/methods; Promoter Regions, Genetic; Pseudomonas fluorescens/*enzymology/*genetics/growth & development/isolation & purification; Repressor Proteins/genetics/metabolism; Rhizoctonia/*growth & development; *Saccharomyces cerevisiae Proteins; Serratia marcescens/enzymology/*genetics; *Telomere-Binding Proteins  
  Abstract An endophytic strain of Pseudomonas fluorescens was isolated from micropropagated apple plantlets and introduced into beans (Phaseolus vulgaris) via their root tips. It was shown to be present as an endophyte in the roots at a level of 1.2 x 10(5) CFU/g fresh weight. The gene coding for the major chitinase of Serratia marcescens, chiA, was cloned under the control of the tac promoter into the broad-host-range plasmid pKT240 and the integration vector pJFF350. Pseudomonas fluorescens carrying tacchiA either on the plasmid or integrated into the chromosome is an effective biocontrol agent of the phytopathogenic fungus Rhizoctonia solani on bean seedlings under plant growth chamber conditions.  
  Call Number Serial 1662  
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Author (up) Gatenby, A.A.; Boccara, M.; Baulcombe, D.C.; Rothstein, S.J. file  url
  Title Expression of a wheat alpha-amylase gene in Escherichia coli: recognition of the translational initiation site and the signal peptide Type Journal Article
  Year 1986 Publication Gene Abbreviated Journal Gene  
  Volume 45 Issue 1 Pages 11-18  
  Keywords DNA/genetics; Escherichia coli/genetics; Plant Proteins/*biosynthesis/genetics; Protein Biosynthesis; Protein Processing, Post-Translational; Protein Sorting Signals/metabolism; Recombinant Fusion Proteins/*biosynthesis/genetics/secretion; Recombinant Proteins/*biosynthesis; Species Specificity; Triticum/enzymology/genetics; alpha-Amylases/*biosynthesis/genetics/secretion; beta-Lactamases/genetics  
  Abstract Transcription of a full-length cDNA clone of wheat alpha-amylase using a lac promoter in Escherichia coli results in synthesis of a precursor alpha-amylase polypeptide of the correct size, indicating that translation initiates correctly. Recognition of the plant translational initiation site by E. coli ribosomes is 15-20% as efficient as the ribosome-binding site of the beta-lactamase gene when it is fused to alpha-amylase. The alpha-amylase signal peptide is recognised in E. coli resulting in secretion of the enzyme into the periplasmic space; deletion of the signal peptide prevents secretion. Replacement of the alpha-amylase signal peptide with a beta-lactamase signal peptide also enables the bacterial cell to secrete the enzyme. The presence of the beta-lactamase and the alpha-amylase signal peptides in tandem results in secretion of the enzyme and removal of both signal peptides.  
  Call Number Serial 498  
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Author (up) Gregg-Jolly, L.A.; Ornston, L.N. file  url
  Title Properties of Acinetobacter calcoaceticus recA and its contribution to intracellular gene conversion Type Journal Article
  Year 1994 Publication Molecular Microbiology Abbreviated Journal Mol Microbiol  
  Volume 12 Issue 6 Pages 985-992  
  Keywords Acinetobacter calcoaceticus/*genetics/metabolism; Amino Acid Sequence; *Bacterial Proteins; Base Sequence; Cloning, Molecular; DNA Transposable Elements; *DNA-Binding Proteins; Escherichia coli/genetics; Gene Conversion/*physiology; Genes, Bacterial/*genetics; Genetic Complementation Test; Genomic Library; Molecular Sequence Data; Mutation/physiology; Rec A Recombinases/chemistry/*genetics/metabolism; Restriction Mapping; Sequence Analysis, DNA; Sequence Homology, Amino Acid; Transformation, Bacterial  
  Abstract The Acinetobacter calcoaceticus pcaJ and catJ genes, nearly identical in DNA sequence, differ in transcriptional control and are separated by more than 20 kb of chromosomal DNA. The pcaJ3125 mutation is repaired frequently in organisms containing the wild-type catJ gene. This high-frequency repair is eliminated in strains lacking the catJ gene, which suggests that recombination between the homologous catJ and pcaJ genes contributes to the high-frequency repair of the pcaJ3125 mutation. We report here that the high-frequency repair also requires a functional recA gene. The A. calcoaceticus recA gene was cloned in Escherichia coli by complementation of a recA mutation in the host strain. The nucleotide sequence of a 1506 bp DNA fragment containing A. calcoaceticus recA was determined. The amino acid sequences of RecA from E. coli and A. calcoaceticus shared 71% identity. The DNA sequences differed in that a consensus binding site for binding of LexA repressor, represented upstream from recA in E. coli, is not evident in the corresponding region of the A. calcoaceticus DNA sequence. A Tn5 insertion was introduced into the A. calcoaceticus recA gene. Selection for Tn5-encoded kanamycin resistance allowed the inactivated recA gene to be recombined by natural transformation into the A. calcoaceticus chromosome. Strains that had acquired the mutant gene were sensitive to both MMS and u.v. light, were deficient in natural transformation, and failed to carry out catJ-dependent high-frequency repair of the pcaJ3125 mutation.  
  Call Number Serial 299  
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Author (up) Hellman, L.M.; Fried, M.G. file  url
  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) Huisman, O.; D'Ari, R. file  url
  Title An inducible DNA replication-cell division coupling mechanism in E. coli Type Journal Article
  Year 1981 Publication Nature Abbreviated Journal Nature  
  Volume 290 Issue 5809 Pages 797-799  
  Keywords Bacterial Proteins/physiology; *Cell Division; *DNA Replication; Escherichia coli/genetics/*physiology; Gene Expression Regulation/radiation effects; Genes, Regulator; Hot Temperature; Thymine/metabolism; Ultraviolet Rays  
  Abstract Cell division is a tightly regulated periodic process. In steady-state cultures of Enterobacteriaceae, division takes place at a well defined cell mass and is strictly coordinated with DNA replication. In wild-type Escherichia coli the formation of cells lacking DNA is very rare, and interruptions of DNA replication arrest cell division. The molecular bases of this replication-division coupling have been elusive but several models have been proposed. It has been suggested, for example, that the termination of a round of DNA replication may trigger a key event required for cell division. A quite different model postulates the existence of a division inhibitor which prevents untimely division and whose synthesis is induced to high levels when DNA replication is perturbed. The work reported here establishes the existence of the latter type of replication-division coupling in E. coli, and shows that the sfiA gene product is an inducible component of this division inhibition mechanism which is synthesized at high levels after perturbations of DNA replication.  
  Call Number Serial 410  
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Author (up) Richards, G.R.; Vanderpool, C.K. file  url
doi  openurl
  Title Molecular call and response: the physiology of bacterial small RNAs Type Journal Article
  Year 2011 Publication Biochimica et Biophysica Acta Abbreviated Journal Biochim Biophys Acta  
  Volume 1809 Issue 10 Pages 525-531  
  Keywords Carbon/chemistry; Cyclic AMP/metabolism; Escherichia coli/genetics; Gene Expression Regulation, Bacterial; Genes, Bacterial; Homeostasis; Iron/metabolism; Models, Biological; Models, Genetic; RNA, Bacterial/*genetics/*physiology; RNA, Untranslated/genetics  
  Abstract The vital role of bacterial small RNAs (sRNAs) in cellular regulation is now well-established. Although many diverse mechanisms by which sRNAs bring about changes in gene expression have been thoroughly described, comparatively less is known about their biological roles and effects on cell physiology. Nevertheless, for some sRNAs, insight has been gained into the intricate regulatory interplay that is required to sense external environmental and internal metabolic cues and turn them into physiological outcomes. Here, we review examples of regulation by selected sRNAs, emphasizing signals and regulators required for sRNA expression, sRNA regulatory targets, and the resulting consequences for the cell. We highlight sRNAs involved in regulation of the processes of iron homeostasis (RyhB, PrrF, and FsrA) and carbon metabolism (Spot 42, CyaR, and SgrS).  
  Call Number Serial 411  
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Author (up) Snapper, S.B.; Melton, R.E.; Mustafa, S.; Kieser, T.; Jacobs, W.R.J. file  url
  Title Isolation and characterization of efficient plasmid transformation mutants of Mycobacterium smegmatis Type Journal Article
  Year 1990 Publication Molecular Microbiology Abbreviated Journal Mol Microbiol  
  Volume 4 Issue 11 Pages 1911-1919  
  Keywords DNA, Bacterial/isolation & purification; Escherichia coli/genetics; *Mutagenesis; Mycobacterium/*genetics/growth & development/isolation & purification; Phenotype; *Plasmids; Restriction Mapping; Transformation, Bacterial  
  Abstract Recent development of vectors and methodologies to introduce recombinant DNA into members of the genus Mycobacterium has provided new approaches for investigating these important bacteria. While most pathogenic mycobacteria are slow-growing, Mycobacterium smegmatis is a fast-growing, non-pathogenic species that has been used for many years as a host for mycobacteriophage propagation and, recently, as a host for the introduction of recombinant DNA. Its use as a cloning host for the analysis of mycobacterial genes has been limited by its inability to be efficiently transformed with plasmid vectors. This work describes the isolation and characterization of mutants of M. smegmatis that can be transformed, using electroporation, at efficiencies 10(4) to 10(5) times greater than those of the parent strain, yielding more than 10(5) transformants per microgram of plasmid DNA. The mutations conferring this efficient plasmid transformation (Ept) phenotype do not affect phage transfection or the integration of DNA into the M. smegmatis chromosome, but seem to be specific for plasmid transformation. Such Ept mutants have been used to characterize plasmid DNA sequences essential for replication of the Mycobacterium fortuitum plasmid pAL5000 in mycobacteria by permitting the transformation of a library of hybrid plasmid constructs. Efficient plasmid transformation of M. smegmatis will facilitate the analysis of mycobacterial gene function, expression and replication and thus aid in the development of BCG as a multivalent recombinant vaccine vector and in the genetic analysis of the virulence determinants of pathogenic mycobacteria.  
  Call Number Serial 174  
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