# Nutrient Sensing and Biofilm Modulation: The Example of L-arginine in _Pseudomonas_
## 5 Beyond Nutrient: L-arginine, Electrons and Oxygen  
Oxygen restriction and microaerophilic environments are often found in the _P. aeruginosa_ biofilm and, therefore, in chronic infection sites, such as the CF lung environment [83]. In particular, CF lungs promote the microaerophilic apparatus, including oxidase Cbb3-2, azurin and cytochrome c\({}_{551}\) and the _Arc_ operon encoding for the ADI pathway [83]. More generally, proteomic analysis carried out specifically in the anoxic portion of the biofilm displayed higher abundances of proteins from L-arginine and polyamine metabolism [84]. Chronic _P. aeruginosa_ infections in mammals are regulated by L-arginine and the derived NO metabolism. Microaerophilic environments, such as in chronic infection sites, favour L-arginine fermentation, which in turn contributes to the NO deficiency typical of a reduced host defense response [85].  
The metabolic reprogramming required to switch into the anaerobic metabolism contributes to the tolerance to antibiotics and resistance to specific molecules [76; 83]. Indeed, the activation of ANR [86; 87] and of the derived regulon, including L-arginine fermentation, represents a hallmark of the stress response leading to antibiotic tolerance and, more generally, is associated with chronic infections and persistence.  
Parallel to L-arginine fermentation, ANR also controls the long-term anaerobic survival, which involves pyruvate fermentation and the electron supply by phenazine redox cycling to obtain ATP [88; 89]. Phenazines take advantage of the extracellular DNA to shuttle electrons within the biofilm structure to support anaerobic energy metabolism [90; 91]. Briefly, a working phenazine oxidative cycle implies that in the deeper layers of the biofilm community, electrons accumulated for ATP production may be accepted by (oxidised) phenazines and delivered to oxidants (such as oxygen), which can be easily found in the outward part of the biofilm structure. Considering this, phenazines contribute to antibiotic tolerance together with the anaerobic stress response [92].  
L-arginine was found to sustain both swarming inhibition and phenazine biosynthesis, although the molecular determinants linking phenazines and L-arginine are far from being elucidated [93]. When the phenazines' electron-shuttling is poor due to mutations in the phenazines' metabolism pathway or to robust biofilm formation, a wrinkling phenotype develops to increase the surface/volume ratio and, therefore, increase the gas exchange (i.e., oxygen availability). Therefore, the wrinkling phenotype controlled by RmcA (see above) in PA14 is related to energy production and redox balance (i.e., NADH/NAD\({}^{+}\) ratio). Basically, it has been found that RmcA can be activated by oxidised phenazines to degrade c-di-GMP and inhibit colony wrinkling [67]. The authors hypothesised that electrons are perceived at the level of the PAS domain(s) located in the cytoplasmic portion of the protein. Given that other data showed that the periplasmic portion of PAO1 RmcA recognises and responds to L-arginine [64], it is not ruled out that _P. aeruginosa_ checks the sustainability of its anaerobic ATP supply using this one-component system to finally reshape the colony morphology by tuning the c-di-GMP levels (Figure 3).