title: Cloning and characterization of the Brucella ovis heat shock protein DnaK functionally expressed in Escherichia coli.
abstract: The Brucella ovis dnaK gene, homolog to the eukaryotic hsp70 genes, was cloned by using a Drosophila melanogaster probe. Comparison of B. ovis and Escherichia coli sequences revealed a similar organization for the dnaK and dnaJ genes and putative regulatory signals. In E. coli transfected with the cloned fragment, B. ovis hsp70 was expressed at 30 and 50 degrees C apparently under the control of its own promoter. The recombinant protein and a B. ovis native protein displaying the same molecular weight were both recognized by anti-E. coli DnaK serum. Native B. ovis protein was also recognized by sera of sheep either infected or vaccinated with an attenuated Brucella strain, suggesting that Brucella hsp70 could be up-regulated during host colonization. A thermosensitive E. coli dnaK mutant transfected with the cloned fragment recovered colony-forming ability at 42 degrees C, showing that the B. ovis DnaK protein could behave as a functional heat shock protein in E. coli.
authors.affiliation: Département Biologie-Santé, Institut National de la Santé et de la Recherche Médicale U-65, Université Montpellier II, France.
authors.forename: M F
authors.initials: MF
authors.lastname: Cellier
authors.forename: J
authors.initials: J
authors.lastname: Teyssier
authors.forename: M
authors.initials: M
authors.lastname: Nicolas
authors.forename: J P
authors.initials: JP
authors.lastname: Liautard
authors.forename: J
authors.initials: J
authors.lastname: Marti
authors.forename: J
authors.initials: J
authors.lastname: Sri Widada
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D015153:Blotting, Western; D002002:Brucella / Q000235:genetics*; D003001:Cloning, Molecular; D004269:DNA, Bacterial; D004926:Escherichia coli; D029968:Escherichia coli Proteins; D005816:Genetic Complementation Test; D018840:HSP70 Heat-Shock Proteins; D006360:Heat-Shock Proteins / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D015183:Restriction Mapping; D012689:Sequence Homology, Nucleic Acid; D013696:Temperature
chemical_list: D001426:Bacterial Proteins; D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D018840:HSP70 Heat-Shock Proteins; D006360:Heat-Shock Proteins; C063842:dnaK protein, E coli