# Nutrient Sensing and Biofilm Modulation: The Example of L-arginine in _Pseudomonas_
## 4 L-arginine Sensing and Biofilm Regulation in _Pseudomonas_  
As mentioned previously, certain amino acids are emerging as key molecules in the regulation of attachment and biofilm development in different microorganisms. In _P. aeruginosa_, several amino acids promote robust biofilm formation and reduce swarming motility: arginine, ornithine, isoleucine, leucine, valine, phenylalanine and tyrosine [59]. In _P. putida_, arginine and its precursor aspartic acid have divergent effects on biofilm formation; while increasing concentrations of arginine stimulate surface attachment, aspartic acid causes the opposite effect. These phenotypes relate to changes in the c-di-GMP content [60]. Other amino acids also influence accumulation of the second messenger in this bacterium,with proline and valine reducing it and tryptophan causing an increase [44]. Interestingly, there is a synergistic effect between arginine and tryptophan [44]. All these data suggest the existence of complex signalling circuits that connect amino acid sensing, metabolic pathways and the cellular mechanisms controlling bacterial lifestyles. Some of those circuits are being uncovered. Once the diverse environmental cues are perceived, the intracellular levels of c-di-GMP change to reshape cellular metabolism; biosynthesis and degradation of this dinucleotide are due to the activity of diguanylate cyclases and phosphodiesterase, harboring the conserved GGDEF and EAL domains, respectively [14]. In _P. aeruginosa_, L-arginine supplementation leads to increased intracellular levels of c-di-GMP through the activity of SadC and RoeA diguanylate cyclases; the corresponding mutant strains showed a reduction in biofilm formation when grown with this amino acid [59]. Among the GGDEF-EAL-containing proteins, there are multidomain enzymes harboring putative nutrient-sensory domains, thus suggesting that selected nutrients may tune the c-di-GMP network by controlling enzyme activity [24]. Indeed, a sensory system of L-arginine able to modulate the c-di-GMP levels has been indirectly identified in _Salmonella typhimurium_[61] or hypothesised in other organisms [60,62]. It has also been proposed that cellular arginine pools, resulting both from biosynthesis and uptake of the amino acid from the environment, are sensed and transduced into changes in c-di-GMP contents [44]. This hypothesis is based on the fact that mutants defective in either arginine synthesis or in its transport show reduced second messenger levels. This would imply a connection between the metabolic state of cells and their \"decision\" to modify their lifestyle. Recently, transcriptional regulator ArgR has been shown to act as a key element in the arginine-dependent regulation of c-di-GMP turnover in _P. putida_. ArgR functions as a repressor of the arginine biosynthesis genes and as a positive regulator of arginine transport, thus altering the c-di-GMP levels. An _argR_ mutant loses the response to arginine in terms of c-di-GMP accumulation and shows reduced expression of some biofilm matrix elements [44]. In turn, expression of _argR_ is modulated by the second messenger, indicating the existence of a regulatory feedback loop (Figure 2). Figure 2: **Simplified model of the regulatory network connecting L-arginine metabolism, c-di-GMP signaling and biofilm formation in _P. putida_ KT2440. In the presence of arginine, ArgR positively controls arginine transport and negatively controls _de novo_ arginine biosynthesis; intracellular arginine indirectly influences the c-di-GMP levels (and, therefore, biofilm formation) and, in turn, c-di-GMP modulates the expression of argR, thus establishing a feedback loop [63]. **  
From a biochemical point of view, it has been shown that the GGDEF-EAL-containing RmcA protein from PAOI _P. aeruginosa_ is able to recognise L-arginine through the periplasmic PBP domain (periplasmic binding domain) harboring the Venus flytrap fold [64]. This protein also contains a GGDEF-EAL tandem moiety whose final activity is a GTP-dependent phosphodiesterase [65].