# Genomic and Metabolic Characteristics of the Pathogenicity in Pseudomonas aeruginosa
## 4 Metabolic Pathways Associated with Pathogenicity
### Biofilm  
Bacterial biofilms are structured colonial aggregates of cells, most often embedded in a matrix of extracellular polymeric substances (EPS) [149]. These biofilms are resistant to antibiotics, phagocytosis, and surfactants, making them difficult to remove once established [150]. Biofilm formation in _P. aeruginosa_ is triggered upon association with a biotic or abiotic surface, leading to the specific production and secretion of extracellular matrix, that can include exopolysaccharides, DNA and/or protein [151]. The development of _P. aeruginosa_ PAO1 biofilms is divided in five stages. The first stage is the reversible attachment, where planktonic cells attach to the substratum by means of their flagella. In the second stage, there is a continuous influx of motile cells to the site of biofilm initiation. Subsequently, the bacteria form microcolonies. At this stage, type IV pili maintain twitching motility. After definitive fixation, _P. aeruginosa_ starts the expression of the _algC_, _algD_, and _algU_ genes, responsible for the synthesis of the alginate extracellular polymeric substances. At the third stage, cell proliferation and loss of motility organelles mark the beginning of the biofilm maturation process, during which exponential upregulation of certain genes is observed. In the fourth stage, biofilm maturation and thickening are now complete. The initial monolayer cell mat transforms into a three-dimensional structure and structurally adopts a mushroom-shaped multicellular assembly. The fifth and last state is the detachment of single cells or aggregates from the biofilm, leading to the dispersion of the planktonic cells back into the environment, which can re-form a new biofilm elsewhere. For dispersion and separation of bacterial cells from the biofilm, the swimming capacity of _P. aeruginosa_ and enzymatic degradation of the EPS matrix are important factors [151; 152]. In _P. aeruginosa_, the _Psl_ system, encoded by the _psl_ operon, which contains many _psl_ genes, contributes to the formation of biofilm, allowing to increase cell surface and intercellular adhesion [153]. The _psl_ locus encodes the synthesis of polysaccharides rich in galactose and mannose, which play an essential role in the cell surface, cell interactions, and biofilm formation. Pellicle (Pel) polysaccharide, a glucose-rich matrix material, is another biofilm formation system. It is encoded by the _pelABCDEF_ operon, containing six genes. When this operon is active, a glucose-rich, cellulase-sensitive matrix is created [154; 155]. The Pel and Psl systems are involved in the early stages of biofilm formation [152]. The _pel_ operon is necessary for the formation of biofilm on certain surfaces. It was demonstrated when comparing _P. aeruginosa_ strain PA14 and _pelA_ mutant of PA14_pelA_::Tn that the increase in biomass was significantly greater in wild type PA14 than in PA14_pelA_ at all time points after inoculation [105]. Regulation of biofilm formation involves multiple bacterial machineries, such as the QS systems (_las_ and _rhl_), two-component regulatory systems, and the nucleotide signaling molecule _c-di-GMP (Figure 5) [129; 147; 127]. The _las_ system intervenes in biofilm formation and maturation. The QS regulator LasR appears to bind to the promoter region of the _psl_ operon, suggesting that this QS system regulates _psl_ expression [156]. On the other hand, the _rhl_ system intervenes in the formation of biofilm by increasing biosynthesis of the Pel polysaccharide. For instance, the transcription of the _pelABCDEF_ operon has been reported to be drastically reduced in the _Rlll_ mutant, however, the _rhl_R mutant did not [157; 105]. In two-component regulatory systems (GacS/GacA), the RsmA represses the expression of the _pel_ and _psl_ genes. The interaction between GacS and RetS makes phosphorylation of GacS impossible and, consequently, there is no inhibition of RsmA, making RetS a negative regulator for biofilm formation [130].