Exploring the Metabolic Response of Pseudomonas putida to L-arginine.

Beyond their role as protein-building units, amino acids are modulators of multiple behaviours in different microorganisms. In the root-colonizing beneficial bacterium Pseudomonas putida (recently proposed to be reclassified as alloputida) KT2440, current evidence suggests that arginine functions both as a metabolic indicator and as an environmental signal molecule, modulating processes such as chemotactic responses, siderophore-mediated iron uptake or the levels of the intracellular second messenger cyclic diguanylate (c-di-GMP). Using microcalorimetry and extracellular flux analysis, in this work we have studied the metabolic adaptation of P. putida KT2440 to the presence of L-arginine in the growth medium, and the influence of mutations related to arginine metabolism. Arginine causes rapid changes in the respiratory activity of P. putida, particularly magnified in a mutant lacking the transcriptional regulator ArgR. The metabolic activity of mutants affected in arginine transport and metabolism is also altered during biofilm formation in the presence of the amino acid. The results obtained here further support the role of arginine as a metabolic signal in P. putida and the relevance of ArgR in the adaptation to the amino acid. They also serve as proof of concept on the use of calorimetric and extracellular flux techniques to analyse metabolic responses in bacteria and the impact of different mutant backgrounds on such responses.

Molecular insights into RmcA-mediated c-di-GMP consumption: Linking redox potential to biofilm morphogenesis in Pseudomonas aeruginosa.

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.

The phosphodiesterase RmcA contributes to the adaptation of Pseudomonas putida to l-arginine.

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.

Exploring Innovative Approaches to Isolate a One-Component c-di-GMP Transducer: A Pilot Study.

Environmental nutrients control bacterial biofilm homeostasis, by regulating the intracellular levels of c-di-GMP. One component transducers can sense different classes of small molecules through a periplasmic domain; the nutrient recognition triggers the subsequent regulation of the downstream cytosolic diguanylate cyclase (GGDEF) or phosphodiesterase (EAL) domains, via transmembrane helix(ces), to finally change c-di-GMP levels.Protein studies on such transducers have been mainly carried out on isolated domains due to the presence of the transmembrane portion. Nevertheless, the cleavage of GGDEF and EAL-containing proteins could be detrimental since both tertiary and quaternary structures could be allosterically controlled; to by-pass this limitation, studies on the corresponding full-length proteins are highly desired.We have in silico selected a GGDEF-EAL transducer from Dyella thiooxydans (ann. A0A160N0B7), whose periplasmic binding domain was predicted to bind to arginine, a nutrient often associated with chronic infections and biofilm. This protein has been used as an in vitro tool for the identification of the best approach for its isolation, including (i) protein engineering to produce a water-soluble version via QTY (Glutamine, Threonine, and Tyrosine) code or (ii) nanodiscs assembly. The results on this "prototype" may represent the proof-of-concept for future isolation of other transmembrane proteins sharing the same architecture, including more complex nutrient-based transducers controlling c-di-GMP levels.

Nutrient Sensing and Biofilm Modulation: The Example of L-arginine in Pseudomonas.

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.

Studying GGDEF Domain in the Act: Minimize Conformational Frustration to Prevent Artefacts.

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.