Cyclic AMP competitively inhibits periplasmic phosphatases to coordinate nutritional growth with competence development of Haemophilus influenzae

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.

226 To test directly if AphA Hi is involved in KW20 competence, we constructed the aphA Hi deletion strain 227 ∆aphA Hi ::cat, where aphA Hi was replaced with a chloramphenicol resistance marker. ∆aphA Hi ::cat 228 grows similar as the WT KW20 in sBHI rich medium ( Figure S5A). We then tested the competence 229 phenotype under several conditions, i.e., during growth into stationary phase in sBHI medium, M-IV 230 starvation medium induced competence, and cAMP (1 mM) induced competence of Log phase sBHI 231 culture. Nucleotides (and nucleosides) inhibit the natural competence in H. influenzae (4). Given the 232 fact that AphA degrades nucleotides, we also tested the role of AphA Hi in AMP mediated inhibition 233 of KW20 competence. However, there was no obvious difference in competence efficiency between 234 wt KW20 and ∆aphA Hi ::cat strains under all the tested competence conditions (data not shown).

cAMP affects the catalytic activity of Hel Hi in a pH-dependent manner
236 The current model of nucleotide mediated inhibition of KW20 natural competence propose that 237 extracellular nucleotides are degraded to nucleosides, which enter the cytosol to inhibit the translation 238 of the master regulator of competence, Sxy Hi (3). Besides AphA Hi , KW20 encodes two additional 239 phosphatases, the periplasmic NadN (NadN Hi ) and the outer membrane anchored Hel (Hel Hi , e(P4)). 240 Hel Hi is a close structural homolog of AphA (30), and dephosphorylates NADP, NMN and 241 nucleotides (31). NadN Hi first degrades NAD to NMN and AMP, then both NMN and AMP are 242 further dephosphorylated by NadN Hi into nicotinamide riboside (NR) and adenosine, which traverse 243 the inner membrane (11). Therefore, NadN Hi and Hel Hi  246 To test if cAMP also competitively inhibits NadN Hi and Hel Hi , we purified His 6 -NadN Hi and His 6 -247 Hel Hi, and performed phosphatase assay with pNPP and cAMP. Surprisingly, cAMP inhibited the 248 phosphatase activity of His 6 -Hel Hi at pH 8.0, but stimulated it at pH 5.6 ( Figure 3E). To rule out 249 potential effect of the histidine tag, we purified a tag-less Hel Hi (Materials & Methods). However, the 274 Lineweaver-Burk plot of the doubling time (i.e., the reciprocal of growth-rate, in hour per cell 275 division) and substrate concentrations (i.e., in 1/[substrate concentration, µM]) was performed. Figure   276 4D showed characteristic straight lines converging to the Y-axis and indicates that cAMP reduces the 277 growth-rate in a competitive manner when NAD was studied. Conversely, when NAD was replaced 278 with NR or when the other essential growth factor hemin was assayed, the plotted curves were not 279 consistent with a competitive model ( Figure 4E, 4F). These data suggest that cAMP competitively 280 inhibits the function of NadN Hi Figure S5B). The three strains were grown in sBHI+NR broth to Log phase and shifted to M-IV 289 starvation medium to induce competence. Fifteen minutes after the shift, various concentrations of 290 AMP were added. As shown in Figure 5, AMP from as low as 10 µM reduced the M-IV induced 291 competence by two-orders of magnitude in wt KW20. The triple mutant was completely refractory to 292 the inhibitory effect of AMP (up to 4 mM tested), and the ∆hel Hi ∆nadN Hi double mutant showed an 293 intermediate effect ( Figure 5A). These data suggest that AphA Hi , NadN Hi and Hel Hi all contribute to 294 the inhibitory effect of AMP on KW20 competence. By contrast, M-IV induced competence of both 295 the double and triple mutants were completely inhibited by adenosine (from 10 µM, Figure 5B).
296 Since cAMP competitively inhibits the activities of AphA Hi and NadN Hi , we added cAMP (1-10 mM) 297 in the assay to see if cAMP counteracts AMP in KW20 competence development. We observed that 298 the AMP inhibited competence of wt KW20 was not restored by cAMP, consistent with previous 299 report (4) (data not shown; see discussion below). Instead, we tested if cAMP restores the competence 300 inhibited by GMP (1 mM), which has a weaker ability than AMP to outcompete the binding of 32 p-301 cAMP to AphA ( Figure 2C) and to inhibit competence (4). We found that cAMP restored the inhibited 302 competence in a dose dependent manner ( Figure 5C), consistent with previous report (4). Finally, we 303 argue that nucleosides should still inhibit KW20 competence despite the presence of cAMP. Indeed, 304 cAMP at the highest concentration (10 mM) only partially restored the KW20 competence inhibited 305 by 1 mM guanosine, and the restoration was worse with 5 mM guanosine ( Figure 5D). Taken together, 306 these data demonstrate the roles of AphA Hi , NadN Hi and Hel Hi in the utilization of nucleotides and 307 regulation of H. influenzae KW20 competence.
308 Discussion 309 Development of bacterial competence is often characterized by a simultaneously inhibited cell growth 310 (2), which results from an exhaustion of a key nutrient, typically a carbon source that consequently 311 stimulates the production of cAMP. In H. influenzae KW20, competence is also inhibited by 312 nucleotides, especially AMP and GMP, and their corresponding nucleosides, but not the nucleobases 313 (4). KW20 is a fastidious bacterium requiring several essential factors, including NAD, hemin, and 314 pyrimidines, to grow. How KW20 perceives and integrates these nutritional signals with competence 315 development remains incompletely understood. In this study, we found via in vitro and in vivo 316 methods that cAMP competitively inhibits the periplasmic non-specific phosphatases AphA Hi and 317 Hel HI in KW20. Importantly, we showed that cAMP competitively inhibits the KW20 growth-rate in 318 sBHI medium supplemented with NAD, but not NR, strongly suggesting that cAMP inhibits NadN Hi 319 as well. Since NadN Hi also degrades a variety of nucleotides, it is imaginable that cAMP binds to the 320 active site and competitively inhibits its activity. Moreover, only the triple deletion mutant 327 Under growth conditions with plenty of carbon sources, NAD(P), and nucleotides ( Figure 6A), Hel Hi 328 cleaves NADP to NAD (11, 14), and NadN Hi cleaves NAD to NMN and NR, providing the essential 329 factor V for KW20 growth (31). AphA Hi might contribute to generation of NR given its phosphatase 330 activity. However, neither Hel HI nor AphA Hi is essential for KW20 growth using NAD (data not 331 shown). Consistently, the double ∆hel Hi ∆nadN Hi and triple ∆hel Hi ∆nadN Hi ∆aphA Hi mutants showed 332 growth patterns similar to wt strain in the sBHI medium supplemented with NR ( Figure S5B). Besides 333 NAD, all three proteins degrade various nucleotides to nucleosides (26, 31), providing both the 334 essential pyrimidines, carbon and energy sources for cell growth.  (37). Given the chemical similarity of cAMP to nucleotides, we 398 propose that cAMP inhibits the phosphatase activities of these proteins, thus serving as a mechanism 399 that couples competence development with nucleotides scavenging. 400 Another unique aspect of V. cholerae is that deoxycytidine also inhibits the V. cholerae competence 401 (34), while deoxynucleosides do not inhibit KW20 competence (4). Therefore, it is possible that the 402 degraded DNA chain releases deoxynucleotides in the periplasm, and that these are further degraded 403 to deoxynucleosides that enter the cytosol and feedback inhibit the competence of V. cholerae. This 404 mechanism may determine how much DNA can be taken up by V. cholerae.
405 The differential use of purine and pyrimidine nucleotides in regulating bacterial competence in KW20 406 and V. cholerae elicits some interesting questions regarding how the natural competence systems 407 evolved. The competence systems seem to depend on both the native niches and the metabolic 408 features of the specific bacterium. KW20 cannot synthesize NAD or pyrimidines (3). Therefore, the 409 three proteins, NadN Hi , Hel Hi , and AphA Hi , likely play a house-keeping function to generate NR and 410 pyrimidine nucleosides from the native niche of KW20, the mucus, which is known to be rich in 411 nucleotides (38). The absolute requirement for pyrimidine and NAD for KW20 growth likely tuned 412 the three acid phosphatases to constantly scavenging and depleting NAD and pyrimidines before the 413 competence system is activated to take up external DNA as carbon, pyrimidine and energy sources.
414 Despite this, it is surprising that purine but not pyrimidine suppresses competence of KW20, although 415 KW20 has no CytR homolog but a PurR homolog. The scenario is even less clear for V. cholerae. 419 Among these, dTMP may be further degraded by periplasmic phosphatases to thymidine, which 420 enters the cytosol and is used for DNA synthesis. Since neither purine nor other pyrimidine 421 nucleosides were tested in V. cholerae, the picture remains incomplete.
422 Limitations of the study and perspectives 423 We purified a recombinant NadN Hi produced in E. coli cytosol; however, the protein showed no 424 phosphatase activity. This is potentially due to unique modifications in KW20, e.g. posttranslational