(p)ppGpp Regulates a Bacterial Nucleosidase by an Allosteric Two-Domain Switch.

The stringent response alarmones pppGpp and ppGpp are essential for rapid adaption of bacterial physiology to changes in the environment. In Escherichia coli, the nucleosidase PpnN (YgdH) regulates purine homeostasis by cleaving nucleoside monophosphates and specifically binds (p)ppGpp. Here, we show that (p)ppGpp stimulates the catalytic activity of PpnN both in vitro and in vivo causing accumulation of several types of nucleobases during stress. The structure of PpnN reveals a tetramer with allosteric (p)ppGpp binding sites located between subunits. pppGpp binding triggers a large conformational change that shifts the two terminal domains to expose the active site, providing a structural rationale for the stimulatory effect. We find that PpnN increases fitness and adjusts cellular tolerance to antibiotics and propose a model in which nucleotide levels can rapidly be adjusted during stress by simultaneous inhibition of biosynthesis and stimulation of degradation, thus achieving a balanced physiological response to constantly changing environments.

cAMP competitively inhibits periplasmic phosphatases to coordinate nutritional growth with competence of Haemophilus influenzae.

Most naturally competent bacteria tightly regulate the window of the competent state to maximize their ecological fitness under specific conditions. Development of competence by Haemophilus influenzae strain Rd KW20 is stimulated by cAMP and inhibited by purine nucleotides, respectively. In contrast, cAMP inhibits cell growth, but nucleotides are important for KW20 growth. However, the mechanisms underlying the abovementioned reciprocal effects are unclear. Here, we first identified a periplasmic acid phosphatase AphAEc of Escherichia coli as a new cAMP-binding protein. We show cAMP competitively inhibits the phosphatase activities of AphAEc and its homolog protein AphAHi in the KW20 strain. Furthermore, we found cAMP inhibits two other periplasmic nonspecific phosphatases, NadNHi (which provides the essential growth factor V, NAD) and HelHi (eP4, which converts NADP to NAD) in KW20. We demonstrate cAMP inhibits cell growth rate, especially via NadNHi. On the other hand, the inhibitory effect of purine nucleotide AMP on competence was abolished in the triple deletion mutant ΔhelHiΔnadNHiΔaphAHi, but not in the single, double deletion or complemented strains. Adenosine, however, still inhibited the competence of the triple deletion mutant, demonstrating the crucial role of the three phosphatases in converting nucleotides to nucleosides and thus inhibiting KW20 competence. Finally, cAMP restored the competence inhibited by GMP in a dose-dependent manner, but not competence inhibited by guanosine. Altogether, we uncovered these three periplasmic phosphatases as the key players underlying the antagonistic effects of cAMP and purine nucleotides on both cell growth and competence development of H. influenzae.

Unresolved mystery of cyclic nucleotide second messengers, periplasmic acid phosphatases and bacterial natural competence.

We recently characterized the competitive inhibition of cyclic AMP (cAMP) on three periplasmic acid phosphatases, AphAHi, NadNHi, and eP4 (HelHi), in Haemophilus influenzae Rd KW20. This inhibitory effect is vital for orchestrating the nutritional growth and competence development in KW20. Initially discovered in Escherichia coli, the function of AphA remains however obscure. This study investigates the regulation of E. coli aphA expression under nutrient starvation conditions. Using transcriptional reporters with truncated aphA promoter sequences, we found that starvations of carbon and phosphate, but not amino acid, stimulated aphA expression through distinct promoter regions. Deletions of crp or cyaA abolished aphA expression, confirming their crucial roles. Conversely, CytR deletion increased aphA expression, suggesting CytR's role as a repressor of aphA expression. Additionally, we extended the study of three other second messengers, i.e., cyclic GMP, cyclic UMP, and cyclic CMP, each sharing structural similarities with cAMP. Notably, cGMP competitively inhibits AphAHi's acid phosphatase activity akin to cAMP. In contrast, both cUMP and cCMP stimulate AphAHi's phosphatase activity in a concentration dependent manner. Collectively, these data imply a complicated connection between nucleotide metabolism, AphA, cyclic purine and pyrimidine nucleotides in bacterial nutrient uptake and natural competence.

Direct detection of stringent alarmones (pp)pGpp using malachite green.

The alarmone (p)ppGpp serves as the signalling molecule for the bacterial universal stringent response and plays a crucial role in bacterial virulence, persistence, and stress adaptation. Consequently, there is a significant focus on developing new drugs that target and modulate the levels of (p)ppGpp as a potential strategy for controlling bacterial infections. However, despite the availability of various methods for detecting (p)ppGpp, a simple and straightforward detection method is needed. In this study, we demonstrated that malachite green, a well-established compound used for phosphate detection, can directly detect (p)ppGpp and its analogues esp., pGpp. By utilizing malachite green, we identified three new inhibitors of the hydrolase activity of SpoT, one of the two RelA-SpoT homolog (RSH) proteins responsible for making and hydrolyzing (p)ppGpp in Escherichia coli. These findings highlight the convenience and practicality of malachite green, which can be widely employed in high-throughput studies to investigate (pp)pGpp in vitro and discover novel regulators of RSH proteins.

Basal level of ppGpp coordinates Escherichia coli cell heterogeneity and ampicillin resistance and persistence.

The universal stringent response alarmone ppGpp (guanosine penta and tetra phosphates) plays a crucial role in various aspects of fundamental cell physiology (e.g., cell growth rate, cell size) and thus bacterial tolerance to and survival of external stresses, including antibiotics. Besides transient antibiotic tolerance (persistence), ppGpp was recently found to contribute to E. coli resistance to ampicillin. How ppGpp regulates both the persistence and resistance to antibiotics remains incompletely understood. In this study, we first clarified that the absence of ppGpp in E. coli (ppGpp0 strain) resulted in a decreased minimal inhibition concentration (MIC) value of ampicillin but, surprisingly, a higher persistence level to ampicillin during exponential growth in MOPS rich medium. High basal ppGpp levels, thus lower growth rate, did not produce high ampicillin persistence. Importantly, we found that the high ampicillin persistence of the ppGpp0 strain is not due to dormant overnight carry-over cells. Instead, the absence of ppGpp produced higher cell heterogeneity, propagating during the regrowth and the killing phases, leading to higher ampicillin persistence. Consistently, we isolated a suppressor mutation of the ppGpp0 strain that restored the standard MIC value of ampicillin and reduced its cell heterogeneity and the ampicillin persistence level concomitantly. Altogether, we discussed the fundamental role of basal level of ppGpp in regulating cell homogeneity and ampicillin persistence.

Ribose 5-phosphate: the key metabolite bridging the metabolisms of nucleotides and amino acids during stringent response in Escherichia coli?

The bacterial stringent response and its effector alarmone guanosine penta- or tetra - phosphates (p)ppGpp are vital for bacterial tolerance and survival of various stresses in environments (including antibiotics) and host cells (virulence). (p)ppGpp does so by binding to its numerous target proteins and reprograming bacterial transcriptome to tune down the synthesis of nucleotides and rRNA/tRNA, and up-regulate amino acid biosynthesis genes. Recent identification of more novel (p)ppGpp direct binding proteins in Escherichia coli and their deep studies have unveiled unprecedented details of how (p)ppGpp coordinates the nucleotide and amino acid metabolic pathways upon stringent response; however, the mechanistic link between nucleotide and amino acid metabolisms remains still incompletely understood. Here we propose the metabolite ribose 5'-phosphate as the key link between nucleotide and amino acid metabolisms and a working model integrating both the transcriptional and metabolic effects of (p)ppGpp on E. coli physiological adaptation during the stringent response.

Transcription of the Alginate Operon in Pseudomonas aeruginosa Is Regulated by c-di-GMP.

Overproduction of the exopolysaccharide alginate contributes to the pathogenicity and antibiotic tolerance of Pseudomonas aeruginosa in chronic infections. The second messenger, c-di-GMP, is a positive regulator of the production of various biofilm matrix components and is known to regulate alginate synthesis at the posttranslational level in P. aeruginosa. We provide evidence that c-di-GMP also regulates transcription of the alginate operon in P. aeruginosa. Previous work has shown that transcription of the alginate operon is regulated by nine different proteins, AmrZ, AlgP, IHFα, IHFß, CysB, Vfr, AlgR, AlgB, and AlgQ, and we investigated if some of these proteins function as a c-di-GMP effector. We found that deletion of algP, algQ, IHFα, and IHFß had only a marginal effect on the transcription of the alginate operon. Deletion of vfr and cysB led to decreased transcription of the alginate operon, and the dependence of the c-di-GMP level was less pronounced, indicating that Vfr and CysB could be partially required for c-di-GMP-mediated regulation of alginate operon transcription. Our experiments indicated that the AmrZ, AlgR, and AlgB proteins are absolutely required for transcription of the alginate operon. However, differential radial capillary action of ligand assay (DRaCALA) and site-directed mutagenesis indicated that c-di-GMP does not bind to any of the AmrZ, AlgR, and AlgB proteins. IMPORTANCE The proliferation of alginate-overproducing P. aeruginosa variants in the lungs of cystic fibrosis patients often leads to chronic infection. The alginate functions as a biofilm matrix that protects the bacteria against host immune defenses and antibiotic treatment. Knowledge about the regulation of alginate synthesis is important in order to identify drug targets for the development of medicine against chronic P. aeruginosa infections. We provide evidence that c-di-GMP positively regulates transcription of the alginate operon in P. aeruginosa. Moreover, we revisited the role of the known alginate regulators, AmrZ, AlgP, IHFα, IHFß, CysB, Vfr, AlgR, AlgB, and AlgQ, and found that their effect on transcription of the alginate operon is highly varied. Deletion of algP, algQ, IHFα, or IHFß only had a marginal effect on transcription of the alginate operon, whereas deletion of vfr or cysB led to decreased transcription and deletion of amrZ, algR, or algB abrogated transcription.

Identifying the Binding Proteins of Small Ligands with the Differential Radial Capillary Action of Ligand Assay (DRaCALA).

The past decade has seen tremendous progress in the understanding of small signaling molecules in bacterial physiology. In particular, the target proteins of several nucleotide-derived secondary messengers (NSMs) have been systematically identified and studied in model organisms. These achievements are mainly due to the development of several new techniques including the capture compound technique and the differential radial capillary action of ligand assay (DRaCALA), which were used to systematically identify target proteins of these small molecules. This paper describes the use of the NSMs, guanosine penta- and tetraphosphates (p)ppGpp, as an example and video demonstration of the DRaCALA technique. Using DRaCALA, 9 out of 20 known and 12 new target proteins of (p)ppGpp were identified in the model organism, Escherichia coli K-12, demonstrating the power of this assay. In principle, DRaCALA could be used for studying small ligands that can be labeled by radioactive isotopes or fluorescent dyes. The critical steps, pros, and cons of DRaCALA are discussed here for further application of this technique.

Evolution of the bacterial nucleosidase PpnN and its relation to the stringent response.

In our recent publication (Zhang et al., 2019), we demonstrate an interesting mode of regulation of purine metabolism unique to Proteobacteria. In this microreview, we would like to reflect on the ideas put forward, with special focus on protein domain architecture of the enzyme involved, its orthologues in plants, and the implications of the differential effects observed between binding of the two alarmone molecules, ppGpp (guanosine 3',5'-bisdiphosphate) and pppGpp (guanosine-5'-triphosphate-3'-diphosphate). In our previous work, we showed that the Escherichia coli nucleotide 5'-monophosphate nucleosidase, PpnN, which is conserved in Proteobacteria, cleaves its preferred substrate, guanosine monophosphate (GMP), at a much higher rate in the presence of both pppGpp and ppGpp (Figure 1A). Structural analysis reveals that binding of pppGpp leads to a conformational change in the protein that exposes its active site, suggesting this is the reason for the observed increase in activity. Finally, point mutation of the alarmone-interacting residues show a defect in binding, resulting in (i) increased basal catalytic activity of PpnN and higher competitive fitness of E. coli in an environment with fluctuating nutrient levels, and (ii) increased bacterial sensitivity towards antibiotics. In contrast, complete loss of the ppnN gene has the inverse effect, i.e. reduced competitive growth and improved antibiotic tolerance. We used these observations to propose a model in which E. coli uses PpnN to balance the need of fitness (fast growth) against tolerance towards antibiotics to improve survival.