Cyclic AMP competitively inhibits periplasmic phosphatases to coordinate nutritional growth with competence development of Haemophilus influenzae
Authors: Kronborg Kristina , Zhang Yong Everett 
Published: August 2022

Natural competence is an important means of horizontal gene transfer that bacteria use to gain new physiological traits such as multiple drug resistance. Most naturally competent bacteria tightly regulate the window of competence state to maximize their ecological fitness under specific conditions. Here we study the inhibitory effect of purine nucleotides on the natural competence in Haemophilus influenzae Rd KW20. We first identified a periplasmic acid phosphatase AphAEc of E. coli as a new cyclic AMP (cAMP)-binding protein. cAMP competitively inhibits AphAEc. Subsequently, we found that cAMP also competitively inhibits AphAHi and two additional periplasmic phosphatases NadNHi and HelHi of KW20. HelHi cleaves NADP to NAD, and NadNHi cleaves NAD to NMN and NR (nicotinamide ribose), providing the essential growth factor V for KW20. Consistently, we found that cAMP inhibits growth of KW20 in sBHI medium supplemented with NAD, but not NR. Moreover, the combined deletion of aphAHi, nadNHi, and helHi, but not the single or double deletion mutants, made KW20 immune to the inhibition of nucleotides on competence development. However, nucleosides still inhibited the competence of the triple mutant. Finally, cAMP in a dose-dependent manner restored the competence inhibited by the nucleotide GMP, but not by the nucleoside guanosine. Altogether, we revealed an antagonistic mechanism of cAMP and nucleotides in regulating cell growth and competence of H. influenzae. Similar mechanisms are discussed in other H. influenzae related organisms and Vibrio cholerae.

Author summary

Haemophilus influenzae is an important human pathogen, causing respiratory tract infection including pneumonia. Extensive drug resistance is observed in Haemophilus influenzae species, which is attributed to their well-described natural competence system. Natural competence is a physiological state that some bacteria, under certain conditions, become active to take up external DNA and integrate it into the chromosome. External DNA may contain an antibiotic resistance gene and thereby confer antibiotic resistance on Haemophilus influenzae. Therefore, it is important to understand how natural competence of Haemophilus influenzae is regulated by external cues. Previously, it was found that the secondary messenger cyclic AMP (cAMP) activates while nucleotides inhibit the competence development of Haemophilus influenzae Rd KW20. However, the interplay between cAMP and nucleotides is unclear. Here we show that cAMP competitively inhibits three periplasmic phosphatases of Haemophilus influenzae Rd KW20 and thereby inhibits the utilization of nutritional nucleotides and the essential growth factor NAD. Via this mechanism, cAMP activates the competence development of Haemophilus influenzae Rd KW20 only after external nucleotides are sufficiently depleted, coupling growth arrest with competence development. Similar mechanisms are anticipated to function in bacteria closely related to Haemophilus influenzaee, and also Vibrio cholerae, another important human pathogen.