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Cloning of the adenylate cyclase genetic determinant of Bordetella pertussis and its expression in Escherichia coli and B. pertussis.
- Source :
-
Microbial pathogenesis [Microb Pathog] 1988 May; Vol. 4 (5), pp. 335-44. - Publication Year :
- 1988
-
Abstract
- A recombinant plasmid, pRMB1, identified from a gene library of B. pertussis, restored adenylate cyclase (AC) and haemolysin (HLY) activities to B. pertussis BP348 (a Tn5-insertion mutant deficient in both these activities). B. pertussis BP348 was considerably less virulent than wild type strains of B. pertussis when 3-week-old mice were challenged intranasally; possession of pRMB1 restored virulence. Neither AC nor HLY activities were expressed in E. coli harbouring pRMB1. However, expression of calmodulin-responsive AC was obtained in E. coli when restriction fragments of pRMB1 were subcloned into other vectors; expression depended on the lac and tac promoters of these vectors. The enzyme was not readily solubilized from urea extracts of E. coli and required sonication for efficient release. One plasmid conferred a specific AC enzymic activity to E. coli which was greater than that for wild type B. pertussis strains. Unlike extracts of B. pertussis, extracts from E. coli expressing enzymic AC activity, did not elevate cAMP levels in S49 lymphoma cells. A second plasmid, pRMB2, was identified from the gene library, which contained a trans-acting regulatory determinant required for expression of AC, HLY and other virulence-associated factors in B. pertussis.
- Subjects :
- Adenylyl Cyclases biosynthesis
Animals
Blotting, Southern
Bordetella pertussis enzymology
Bordetella pertussis pathogenicity
Cloning, Molecular
DNA, Bacterial genetics
Escherichia coli genetics
Genetic Complementation Test
Mice
Nucleic Acid Hybridization
Plasmids
Restriction Mapping
Virulence
Whooping Cough microbiology
Adenylyl Cyclases genetics
Bordetella pertussis genetics
Gene Expression Regulation
Subjects
Details
- Language :
- English
- ISSN :
- 0882-4010
- Volume :
- 4
- Issue :
- 5
- Database :
- MEDLINE
- Journal :
- Microbial pathogenesis
- Publication Type :
- Academic Journal
- Accession number :
- 2907365
- Full Text :
- https://doi.org/10.1016/0882-4010(88)90061-7