## 3 Discussion
Namely, LapA quantity is elevated in all strains by IPTG supplementation (Figures 4A, 5A and 6A), while only PSm-G-PANB, the _lapG_ deletion strain, had a robust biofilm in the M9-0.2 CAA medium (Figures 4B, 5B and 6B). Thus, proteolytic cleavage of LapA from the outer membrane probably affects peptide-dependent biofilm formation more than the transporters. However, we cannot exclude the possibility that P-PANB biofilm is a result of a lack of LapA transporters, indicating peptide dependent regulation of _lapBC_ and/or _lapE_ genes. The P-PANB strain constructed from PaW85 had only a modest, albeit significant, effect on biofilm in peptide-containing media upon induction of _lapA_ transcription by IPTG (Figure 4). However, the Avwf\({}^{-}\)-strains of PaW85, without the vWFa domain in LapA, had a significantly decreased biofilm relative to the strains with the correct LapA (Figure 4). The importance of the vWFa-domain to the biofilm was particularly evident when P-PANB and P-PANB-Avwf\({}^{-}\) were compared. Both strains had _lapA_ transcription under the control of P_tac_ promoter (IPTG). Still, the absence of the vWFa-domain in LapA eliminated the effect of the elevated amount of LapA by IPTG in the media containing the peptides (Figure 4). We decided to use previously constructed PSmAlapE and PSmAlapG strains as a negative and positive control of inducible _lapA_ expression. In the first case, increasing the amount of LapA or the absence of the vWFa domain should not significantly affect the biofilm because in the absence of LapE, LapA does not reach the cell surface. Indeed, in general, the biofilm of _lapE_-minus strains did not depend on the induction of _lapA_ transcription by IPTG or the absence of vWFa (Figure 5). In addition, experiments on the _lapG_ deletion strain confirm the importance of the vWFa domain for _P. putida_ biofilm formation (Figure 6). The LapA strain without the vWFa domain resulted in less biofilm than the strains with correct LapA, although the absence of _lapG_ always produced a stronger biofilm than the wild-type strain (Figures 5 and 6). In sum, we can conclude that not only does the presence of LapA cause the peptide-dependent _P. putida_ biofilm, but LapA must also have the correct vWFa domain. Since the absence of the LapA vWFa domain negatively affected the biofilm in _P. putida_ wild-type strains, we investigated the effect of the lack of this LapA domain on the F15 biofilm (Figure 7). So far, we hypothesised that, among other factors, Fis could increase the F15 biofilm via _lapA_ because Fis activates _lapA_ transcription to some extent [20]. The complete loss of the Fis-enhancing effect for biofilm in LB and M9-0.2 CAA+tryptone medium was astonishing when we deleted the vWFa-domain sequence from the F15 _lapA_ gene, F15-Avwf\({}^{-}\) (Figure 7). Therefore, the enhancing effect of the Fis to _P. putida_ biofilm described so far must be entirely or predominantly dependent on LapA. Consequently, Fis enhances _lapA_ transcription, at least to some extent, and the LapA vWFa domain increases biofilm when peptides are present in the medium. However, the question arises why the lack of vWFa domain had a pronounced impact on the F15 biofilm, whereas wild-type PaW85 with elevated LapA had only a modest effect (Figures 4 and 7). It seems that Fis may more broadly control the _lapA_ expression and its persistence on the cell surface. For example, Fis may indirectly regulate LapA persistence on the cell surface through other genes, whereas the wild-type strain undergoes native strong down-regulation. As a result, the wild-type bacterium may lose most of the expressed _lapA_ to the environment. Indeed, according to our preliminary data, Fis represses transcription of the _lapGD_ operon (data not shown), and it may explain at least partly a substantial biofilm enhancement with elevated Fis in _P. putida_ F15. However, the transport and maintenance of LapA on the cell surface seems to be more critical for biofilm than transcriptional regulation.