The Haemophilus influenzae adenylate cyclase gene: cloning, sequence, and essential role in competence

Abstract

Competence for transformation in Haemophilus influenzae is stimulated by cyclic AMP (cAMP) and requires the cAMP-dependent catabolite regulatory protein CRP. Thus, understanding the control of competence will require understanding how cAMP levels are regulated. As a first step, we have cloned the H. influenzae adenylate cyclase gene (cya) by complementing the Lac -phenotype of Acya Escherichia coli. Its sequence specifies an 843-amino-acid protein which has significant identity to other known bacterial adenylate cyclases (41 to 43% and 61% identical to the cya genes of enteric bacteria and of Pasteurella multocida, respectively). As seen in other bacterial cya genes, there is evidence for regulation similar to that demonstrated for E. coli: the presence of a strong consensus CRP binding site within the promoter of the gene may provide feedback control of cAMP levels by repressing cya transcription, and translation may be limited by the weak ribosome binding site and by initiation of protein synthesis with GUG rather than AUG or the UUG used in other bacterial cya genes. We confirmed the essential role of cAMP in competence by constructing and characterizing H. influenzae cya mutants. This strain failed to develop competence either spontaneously or after transfer to a competenceinducing medium. However, it became as competent as its wild-type parent in the presence of exogenous cAMP. This result suggests that the failure of exogenously added cAMP to induce optimum competence in wild-type cells is not due to a limitation to the entry of cAMP into the cells. Rather, it strongly favors models in which competence induction requires both an increase in intracellular cAMP and a second as yet unidentified regulatory event. H. influenzae strains mutant in cya or crp were unable to ferment xylose or ribose. This confirms that H. influenzae, like E. coli, uses cAMP and CRP to regulate nutrient uptake and utilization and lends increasing support to the hypothesis that DNA uptake is a mechanism of nutrient acquisition.Although it is almost 50 years since bacterial transformation revealed that DNA carries genetic information (3), we still know very little about how or why some bacteria are able to take up exogenous DNA. Specifically, we do not know how transformable bacteria decide to become competent, we do not know how DNA is translocated across the cell envelope, and we do not know why these transformation systems ever evolved in the first place. In order to answer the last of these questions, we are investigating the first, guided by the principle that the regulatory mechanisms controlling competence development evolved to maximize its benefits and minimize its costs.DNA uptake could have evolved to provide cells with nutrients, with templates for DNA repair, or with novel genetic

How Phosphotransferase System-Related Protein Phosphorylation Regulates Carbohydrate Metabolism in Bacteria

“…In H. influenzae, only fructose, but not glucose, is taken up via a PTS, and a ptsI mutant can therefore ferment glucose (499). Interestingly, the development of competence, which in H. influenzae requires cAMP and Crp (105,196), was lowered about 70-fold in a crr mutant (305) and 250-to 500-fold in a ptsI mutant (305,499). The addition of extracellular cAMP restored the development of competence.…”

Isolation and characterization of multiple adenylate cyclase genes from the cyanobacterium Anabaena sp. strain PCC 7120

“…It has never been reported that a prokaryote possesses so many different kinds of cya. Most bacterial adenylate cyclases have been isolated from members of the enterobacterial family, such as E. coli, which has a single cya gene (1,13,14,33,49). As exceptions, it has been reported that R. meliloti possesses at least three cya genes (3) and that Stigmatella aurantiaca possesses two cya genes (13).…”

tfoX (sxy)-dependent transformation of Aggregatibacter (Actinobacillus) actinomycetemcomitans

“…The environmental signal for the development of competence in H. influenzae is not known either, but an increase in adenosine 3′,5′-cyclic monophosphate (cAMP) level triggers the expression of the early competence gene tfoX (Wise et al, 1973;Dorocicz et al, 1993;Redfield et al, 2005). The TfoX protein then activates other competence genes (the competence regulon) through CRP at CRP-S sites (Redfield et al, 2005;Cameron and Redfield, 2006).…”

“…Several genes are known to affect transformation in H. influenzae (Caster et al, 1970;Caster and Smith, 1972;Notani et al, 1972;Concino and Goodgal, 1982;Kooistra et al, 1983;Smith, 1985, 1986;Tomb et al, 1989Tomb et al, , 1991Larson and Goodgal, 1991;Chandler, 1992;Dorocicz et al, 1993;Clifton et al, 1994;Gwinn et al, 1996Gwinn et al, , 1997Gwinn et al, , 1998Chandler and Smith, 1996;Karudapuram and Barcak, 1997;Dougherty and Smith, 1999;Dubnau, 1999;Ma and Redfield, 2000;Chen and Dubnau, 2004;VanWagoner et al, 2004;Redfield et al, 2005Redfield et al, , 2006Cameron and Redfield, 2006;Smeets et al, 2006). The cAMP-CRP (cyclic-AMP receptor protein) complex helps to promote their expression and induce competence (the ability to take up and incorporate DNA) (Chandler, 1992;Redfield et al, 2005;Cameron and Redfield, 2006).…”