Secretion of the Bordetella pertussis adenylate cyclase from Escherichia coli containing the hemolysin operon

• Published in: Biochemistry (Easton) 1990-01, Vol.29 (1), p.140-145
• Main Authors: Masure, H. Robert, Au, Douglas C, Gross, Mary K, Donovan, Maura G, Storm, Daniel R
• Format: Article
• Language: English
• Subjects: adenylate cyclase; Adenylyl Cyclases - genetics; Adenylyl Cyclases - metabolism; Biological and medical sciences; Bordetella pertussis - enzymology; Bordetella pertussis - genetics; Escherichia coli; Escherichia coli - enzymology; Escherichia coli - genetics; Fundamental and applied biological sciences. Psychology; Gene expression; Gene Expression Regulation, Bacterial; Gene Expression Regulation, Enzymologic; Hemolysin Factors - genetics; Hemolysin Proteins - metabolism; hemolysins; Lac Operon - physiology; Molecular and cellular biology; Molecular genetics; Operon - physiology; operons; Plasmids - genetics; Promoter Regions, Genetic
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi00453a017

The extracellular calmodulin-sensitive adenylate cyclase produced by Bordetella pertussis is synthesized as a 215-kDa precursor. This polypeptide is transported to the outer membrane of the bacteria where it is proteolytically processed to a 45-kDa catalytic subunit which is released into the culture supernatant [Masure, H.R., & Storm, D.R. (1989) biochemistry 28, 438-442]. The gene encoding this enzyme, cyaA, is part of the cya operon that also includes the genes cyaB, cyaD, and cyaE. A comparison of the predicted amino acid sequences encoded by cyaA, cyaB, and cyaD with the amino acid sequences encoded by hlyA, hlyB, and hlyD genes from the hemolysin (hly) operon from Escherichia coli shows a large degree of sequence similarity [Glaser, P., Sakamoto, H., Bellalou, J., Ullmann, A., & Danchin, A. (1988) EMBO J. 7, 3997-4004]. Complementation studies have shown that HlyB and HlyD are responsible for the secretion of HlyA (hemolysin) from E. coli. The signal sequence responsible for secretion of hemolysin has been shown to reside in its C-terminal 27 amino acids. Similarly, CyaB, CyaD, and CyaE are required for the secretion of CyaA from Bordetella pertussis. We placed the cyaA gene and a truncated cyaA gene that lacks the nucleotides that code for a putative C-terminal secretory signal sequence under the control of the lac promoter in the plasmid pUC-19. These plasmids were transformed into strains of E. coli which contained the hly operon. The truncated cyaA gene product, lacking the putative signal sequence, was not secreted but accumulated inside the cell.