Genetic analysis of the export of an extracellular DNase of Vibrio cholerae using DNase-beta-lactamase fusions.
A series of C-terminal deletions of the dns-encoded extracellular deoxyribonuclease (DNS) of Vibrio cholerae, fused to the mature form TEM beta-lactamase (Bla) has been used to analyse the export of the DNase in both V. cholerae and Escherichia coli. All hybrid proteins were localized to the periplasmic space in E. coli and V. cholerae, with specific cleavage of the DNS-Bla fusion occurring in V. cholerae. Periplasmic accumulation of wt DNS was also seen in V. cholerae when present on a multicopy plasmid. DNS fusions retaining all six Cys residues of DNS displayed both DNase and Bla enzymatic activity. While hybrid proteins were unable to be secreted across the outer membrane in V. cholerae, the cleaved (active) DNS portion of these proteins was exported. Taken together, these data suggest that the periplasmic form seen in E. coli is a normal intermediate also seen in V. cholerae, and that the lack of secretion machinery in E. coli prevents further export across the outer membrane. Although the DNS portion of the protein fusions must be able to interact with secretion genes, the whole fusion proteins are not exported.
Focareta
T
T
Department of Microbiology and Immunology, University of Adelaide, Australia.
Manning
P A
PA
D000595:Amino Acid Sequence; D000668:Ampicillin Resistance / Q000235:genetics; D001483:Base Sequence; D001692:Biological Transport / Q000235:genetics; D015153:Blotting, Western; D003851:Deoxyribonucleases / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D014734:Vibrio cholerae / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics; D001618:beta-Lactamases / Q000235:genetics*
D011993:Recombinant Fusion Proteins; D003851:Deoxyribonucleases; D001618:beta-Lactamases