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.

Solanimycin: Biosynthesis and Distribution of a New Antifungal Antibiotic Regulated by Two Quorum-Sensing Systems.

The increasing emergence of drug-resistant fungal infections has necessitated a search for new compounds capable of combating fungal pathogens of plants, animals, and humans. Microorganisms represent the main source of antibiotics with applicability in agriculture and in the clinic, but many aspects of their metabolic potential remain to be explored. This report describes the discovery and characterization of a new antifungal compound, solanimycin, produced by a hybrid polyketide/nonribosomal peptide (PKS/NRPS) system in Dickeya solani, the enterobacterial pathogen of potato. Solanimycin was active against a broad range of plant-pathogenic fungi of global economic concern and the human pathogen Candida albicans. The genomic cluster responsible for solanimycin production was defined and analyzed to identify the corresponding biosynthetic proteins, which include four multimodular PKS/NRPS proteins and several tailoring enzymes. Antifungal production in D. solani was enhanced in response to experimental conditions found in infected potato tubers and high-density fungal cultures. Solanimycin biosynthesis was cell density dependent in D. solani and was controlled by both the ExpIR acyl-homoserine lactone and Vfm quorum-sensing systems of the bacterial phytopathogen. The expression of the solanimycin cluster was also regulated at the post-transcriptional level, with the regulator RsmA playing a major role. The solanimycin biosynthetic cluster was conserved across phylogenetically distant bacterial genera, and multiple pieces of evidence support that the corresponding gene clusters were acquired by horizontal gene transfer. Given its potent broad-range antifungal properties, this study suggests that solanimycin and related molecules may have potential utility for agricultural and clinical exploitation. IMPORTANCE Fungal infections represent a major clinical, agricultural, and food security threat worldwide, which is accentuated due to the difficult treatment of these infections. Microorganisms represent a prolific source of antibiotics, and current data support that this enormous biosynthetic potential has been scarcely explored. To improve the performance in the discovery of novel antimicrobials, there is a need to diversify the isolation niches for new antibiotic-producing microorganisms as well as to scrutinize novel phylogenetic positions. With the identification of the antifungal antibiotic solanimycin in a broad diversity of phytopathogenic Dickeya spp., we provide further support for the potential of plant-associated bacteria for the biosynthesis of novel antimicrobials. The complex regulatory networks involved in solanimycin production reflect the high metabolic cost of bacterial secondary metabolism. This metabolic regulatory control makes many antibiotics cryptic under standard laboratory conditions, and mimicking environmental conditions, as shown here, is a strategy to activate cryptic antibiotic clusters.

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.