## 3 Discussion  
The _P. putida_ biofilm is a dynamic phenotype depending on the growth phase of bacteria and the environment where the bacterium lives [1; 19]. Our previous research showed that peptides in growth media increase the _P. putida_ biofilm [19]. In addition to the fact that the peptides may act as an additional source of C and N, the peptides may induce a more substantial biofilm by being a structural component of the biofilm matrix [19]. To study biofilm, we used _P. putida_ strain F15, which carries the additional _fs_ gene under the control of P\({}_{tac}\) (IPTG). We used the _fs_-overexpression strain F15 because the use of strains lacking or down-regulating _fs_ was not possible due to the essentiality of the _fs_ gene or instability of the constructed strain. Surprisingly, this artificial strain served as an excellent tool for identifying the factors influencing _P. putida_ biofilm. Although it is an overexpression strain, it is possible to study the physiology of the bacterium. For example, Fis represses transcription of _lapF_ through a single binding site in front of _lapF_, thereby reducing cell surface hydrophobicity [2; 21]. Mutation of the Fis-binding site restored _lapF_ transcription, amount of LapF in cells, and ultimately, surface hydrophobicity [2; 21]. Hence, this direct effect of Fis on the bacterial phenotype through one binding site in front of another gene encouraged us to use F15 in LapA expression studies as well. The main aim of this study was to assess the involvement of surface adhesins, LapA and LapF, in the peptide-dependent _P. putida_ biofilm. We knew that the peptides increase the biofilm of the _P. putida_ wild-type strain PSm, and the so-called Fis-enhancing effect on the F15 biofilm only occurs in the presence of the peptides [19]. Moreover, we assessed the Fis-enhancing effect on biofilm in LB and suggested that the searchable factor could be LapA, as Fis activates _lapA_ transcription and increases the amount of LapA in F15 in LB [1; 20]. Surprisingly, Fis activates _lapA_ transcription in all studied growth media, but the increased quantity of LapA and Fis-enhanced biofilm appeared when growth media were supplemented with peptides (Figures 1-3). It indicates that in the absence of peptides in growth media, the post-transcriptional regulatory steps could affect the expression of _lapA_ more intensively. For example, in the absence of peptides in the growth medium, the translation of _lapA_ may be downregulated, LapA itself may be effectively degraded, or LapA may be released from the surface due to the activity of LapG. We can only speculate which factor might cause the peptide-dependent post-transcriptional regulation of _lapA_ expression. For example, the RsmA, a component of the GacS/A signalling pathway, binds RNAs and can regulate translation initiation, RNA stability, and/or transcript elongation [33] and the involvement in the regulation of the GacS/A signalling pathway _P. putida_ lap-genes expression has been previously shown [34; 35]. One of three RsmA homologs in _P. putida_, RsmE, down-regulates _lapA_ expression in LB medium [35]. On the other hand, LapA can be efficiently released from the outer membrane due to the increased amount of active LapG in the periplasm. The amount of active LapG depends not only on the expression of the _lapGD_ operon, the amount of protease in the periplasm, but also on the effectors such as c-di-GMP and ppGpp; and the availability of calcium [4; 36; 37]. However, we cannot exclude the possibility that other regulators are involved in the peptide-dependent expression of _lapA_. Thus, the post-transcriptional steps in _lapA_ expression, including translation and the amount of LapA on the cell surface, may depend on the components of the growth medium. The importance of LapG proteolytical activity appears in comparing P-PANB, PSm-E-PANB, and PSm-G-PANB biofilms in the growth medium without tryptone (M9-0.2 CAA).