Extracellular proteins of Vibrio cholerae: molecular cloning, nucleotide sequence and characterization of the deoxyribonuclease (DNase) together with its periplasmic localization in Escherichia coli K-12.
The gene encoding the extracellular DNase of Vibrio cholerae was cloned into Escherichia coli K-12. A maximal coding region of 1.2 kb and a minimal region of 0.6 kb were determined by transposon mutagenesis and deletion analysis. The nucleotide sequence of this region contained a single open reading frame of 690 bp corresponding to a protein of Mr 26,389 with a typical N-terminal signal sequence of 18 aa which, when removed, would give a mature protein of Mr 24,163. This is in good agreement with the size of 24 kDa, calculated directly by Coomassie blue staining following sodium dodecyl sulphate-polyacrylamide gel electrophoresis and indirectly via a DNA-hydrolysis assay. The protein is located in the periplasmic space of E. coli K-12 unlike in V. cholerae where it is excreted into the extracellular medium. The introduction of the DNase gene into a periplasmic (tolA) leaky mutant of E. coli K-12 facilitates the release of the protein, further confirming the periplasmic location.
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004251:DNA Transposable Elements; D003851:Deoxyribonucleases / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D005110:Extracellular Space / Q000201:enzymology; D005822:Genetic Vectors; D009154:Mutation; D014170:Transformation, Genetic; D014734:Vibrio cholerae / Q000201:enzymology* / Q000235:genetics
D004251:DNA Transposable Elements; D003851:Deoxyribonucleases