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Amino acids are crucial in nitrogen cycling and to shape the metabolism of microorganisms. Among them, arginine is a versatile molecule able to sustain nitrogen, carbon, and even ATP supply and to regulate multicellular behaviors such as biofilm formation. Arginine modulates the intracellular levels of 3′–5′cyclic diguanylic acid (c-di-GMP), a second messenger that controls biofilm formation, maintenance and dispersion. In Pseudomonas putida , KT2440, a versatile microorganism with wide biotechnological applications, modulation of c-di-GMP levels by arginine requires the transcriptional regulator ArgR, but the connections between arginine metabolism and c-di-GMP are not fully characterized. It has been recently demonstrated that arginine can be perceived by the opportunistic human pathogen Pseudomonas aeruginosa through the transducer RmcA protein (Redox regulator of c-di-GMP), which can directly decrease c-di-GMP levels and possibly affect biofilm architecture. A RmcA homolog is present in P. putida , but its function and involvement in arginine perceiving or biofilm life cycle had not been studied. Here, we present a preliminary characterization of the RmcA-dependent response to arginine in P. putida in modulating biofilm formation, c-di-GMP levels, and energy metabolism. This work contributes to further understanding the molecular mechanisms linking biofilm homeostasis and environmental adaptation. [ABSTRACT FROM AUTHOR]

Bacterial biofilm represents a multicellular community embedded within an extracellular matrix attached to a surface. This lifestyle confers to bacterial cells protection against hostile environments, such as antibiotic treatment and host immune response in case of infections. The Pseudomonas genus is characterised by species producing strong biofilms difficult to be eradicated and by an extraordinary metabolic versatility which may support energy and carbon/nitrogen assimilation under multiple environmental conditions. Nutrient availability can be perceived by a Pseudomonas biofilm which, in turn, readapts its metabolism to finally tune its own formation and dispersion. A growing number of papers is now focusing on the mechanism of nutrient perception as a possible strategy to weaken the biofilm barrier by environmental cues. One of the most important nutrients is amino acid L-arginine, a crucial metabolite sustaining bacterial growth both as a carbon and a nitrogen source. Under low-oxygen conditions, L-arginine may also serve for ATP production, thus allowing bacteria to survive in anaerobic environments. L-arginine has been associated with biofilms, virulence, and antibiotic resistance. L-arginine is also a key precursor of regulatory molecules such as polyamines, whose involvement in biofilm homeostasis is reported. Given the biomedical and biotechnological relevance of biofilm control, the state of the art on the effects mediated by the L-arginine nutrient on biofilm modulation is presented, with a special focus on the Pseudomonas biofilm. Possible biotechnological and biomedical applications are also discussed. [ABSTRACT FROM AUTHOR]

Bis‐(3′‑5′)‐cyclic diguanylic acid (c‑di‑GMP) belongs to the class of cyclic dinucleotides, key carriers of cellular information in prokaryotic and eukaryotic signal transduction pathways. In bacteria, the intracellular levels of c‐di‐GMP and their complex physiological outputs are dynamically regulated by environmental and internal stimuli, which control the antagonistic activities of diguanylate cyclases (DGCs) and c‐di‐GMP specific phosphodiesterases (PDEs). Allostery is one of the major modulators of the c‐di‐GMP‐dependent response. Both the c‐di‐GMP molecule and the proteins interacting with this second messenger are characterized by an extraordinary structural plasticity, which has to be taken into account when defining and possibly predicting c‐di‐GMP‐related processes. Here, we report a structure‐function relationship study on the catalytic portion of the PA0575 protein from Pseudomonas aeruginosa, bearing both putative DGC and PDE domains. The kinetic and structural studies indicate that the GGDEF‐EAL portion is a GTP‐dependent PDE. Moreover, the crystal structure confirms the high degree of conformational flexibility of this module. We combined structural analysis and protein engineering studies to propose the possible molecular mechanism guiding the nucleotide‐dependent allosteric control of catalysis; we propose that the role exerted by GTP via the GGDEF domain is to allow the two EAL domains to form a dimer, the species competent to enter PDE catalysis. The catalytic portion (GGDEF‐EAL domains in tandem) of the Pseudomonas aeruginosa PA0575 protein (RmcA) is a phosphodiesterase (PDE) allosterically controlled by GTP. The crystal structure confirms the large plasticity of this motif; we propose that this feature allows the two EAL domains to form a dimer, the catalytically competent species, upon GTP binding to the GGDEF module. [ABSTRACT FROM AUTHOR]

• HD-GYPs belong to HD-hydrolases and control cyclic dinucleotides levels. • HD-GYPs are related to biofilm, virulence and control of motility. • HD-GYPs are heterogenous in the metal binding site in terms of number and nature. • Additional efforts are need to depict the molecular mechanism of HD-GYP function. The control of the intracellular level of cyclic dinucleotides is a major strategy to transduce external signals into a cellular response, particularly in bacteria. The HD-GYP metalloproteins, a subgroup of the larger family of histidine-aspartate (HD) hydrolases, can catalyze the cleavage of the phosphodiester bond(s) of cyclic dinucleotides. The HD signature is involved in metal binding in the active site, whereas the GYP motif is likely involved in recognition and interaction with other partners. The most representative substrate of HD-GYPs is the second messenger cyclic-di-GMP (c-di-GMP), a global regulator of bacterial biofilm, motility, and virulence. Hydrolysis of c-di-GMP into the linear form pGpG or into the monomeric guanosine derivative (GMP) reprograms the cellular phenotype, usually promoting biofilm dispersion and virulence. Recent data indicate that members of HD-GYP group can also hydrolyze the bacterial cyclic diGMP-AMP (cGAMP) dinucleotide or act as possible sensors of pGpG. The HD-GYP diversity is not limited to substrate recognition: the most striking trait is the extraordinary heterogeneity of the active sites characterized so far, showing different metals ions assisting catalysis, both in terms of their nature and number. In this review, we provide a critical overview of the structural, functional, and kinetic properties of the bacterial HD-GYP metalloproteins. [ABSTRACT FROM AUTHOR]

GGDEF-containing proteins respond to different environmental cues to finely modulate cyclic diguanylate (c-di-GMP) levels in time and space, making the allosteric control a distinctive trait of the corresponding proteins. The diguanylate cyclase mechanism is emblematic of this control: two GGDEF domains, each binding one GTP molecule, must dimerize to enter catalysis and yield c-di-GMP. The need for dimerization makes the GGDEF domain an ideal conformational switch in multidomain proteins. A re-evaluation of the kinetic profile of previously characterized GGDEF domains indicated that they are also able to convert GTP to GMP: this unexpected reactivity occurs when conformational issues hamper the cyclase activity. These results create new questions regarding the characterization and engineering of these proteins for in solution or structural studies. [ABSTRACT FROM AUTHOR]

The ability of many bacteria to form biofilms contributes to their resilience and makes infections more difficult to treat. Biofilm growth leads to the formation of internal oxygen gradients, creating hypoxic subzones where cellular reducing power accumulates, and metabolic activities can be limited. The pathogen Pseudomonas aeruginosa counteracts the redox imbalance in the hypoxic biofilm subzones by producing redox-active electron shuttles (phenazines) and by secreting extracellular matrix, leading to an increased surface area-to-volume ratio, which favors gas exchange. Matrix production is regulated by the second messenger bis-(3′,5′)-cyclic-dimeric-guanosine monophosphate (c-di-GMP) in response to different environmental cues. RmcA (Redox modulator of c-di-GMP) from P. aeruginosa is a multidomain phosphodiesterase (PDE) that modulates c-di-GMP levels in response to phenazine availability. RmcA can also sense the fermentable carbon source arginine via a periplasmic domain, which is linked via a transmembrane domain to four cytoplasmic Per-Arnt-Sim (PAS) domains followed by a diguanylate cyclase (DGC) and a PDE domain. The biochemical characterization of the cytoplasmic portion of RmcA reported in this work shows that the PAS domain adjacent to the catalytic domain tunes RmcA PDE activity in a redox-dependent manner, by differentially controlling protein conformation in response to FAD or FADH2. This redox-dependent mechanism likely links the redox state of phenazines (via FAD/FADH2 ratio) to matrix production as indicated by a hyperwrinkling phenotype in a macrocolony biofilm assay. This study provides insights into the role of RmcA in transducing cellular redox information into a structural response of the biofilm at the population level. Conditions of resource (i.e. oxygen and nutrient) limitation arise during chronic infection, affecting the cellular redox state and promoting antibiotic tolerance. An understanding of the molecular linkages between condition sensing and biofilm structure is therefore of crucial importance from both biological and engineering standpoints. [ABSTRACT FROM AUTHOR]