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Abstract

To study the role of adenylate cyclase in competence development, a partial clone of the Haemophilus influenzae cya gene was isolated by complementation of a Acya Escherichia coli strain. Adenylate cyclase was believed to have a role in competence development because it catalyzes production of adenosine 3':5'-cyclic monophosphate (cAMP), a known regulator of competence. To prove that adenylate cyclase was essential for competence development, transposon mutagenesis was used to form the cya- H. influenzae strain RR668, with an insertion in the region of cya coding for the catalytic domain. Characterization of this mutant has shown that cya is an essential gene for spontaneous late log competence, and for competence induced by starvation conditions. The partial preliminary sequence of the cloned gene had significant amino acid homology to the cya genes from enteric bacteria and the more closely related bacterium Pasteurella multocida. Examination of the cya sequence also revealed a possible CRP binding site (with 55% homology to the consensus Escherichia coli site) located upstream of the putative start codon GTG. The presence of the presumptive CRP site indicated that H. influenzae, like other bacterial species, may regulate cAMP synthesis by CRP mediated feedback repression of transcription. If the start codon was correctly identified as GTG, then this is the first known bacterial cya gene to use GTG as a start codon instead of ATG or TTG.