## 2 Results
### LapA Domain vWFa Is Involved in the Peptide-Dependent Biofilm of P. putida PaW85  
We were interested in the behaviour of _P. putida_ PaW85-based strains. These strains do not contain mini-Tn7 insertion after the _glmS_ gene, and _lapA_ transcription is directly regulated by IPTG without indirect effects on the _fis_-overexpression in the cell (Table 1). It is essential to consider two facts: (i) LapA exposure on the outer membrane is complexly regulated and depends on proteolysis and the presence of effectors, (ii) the expression of LapA transporters (_lapBC_ and _lapE_) is not regulated in these strains. The biofilm was evaluated by every medium separately. In the M9-0.2 CAA medium, the biofilm of all PaW85-based strains was similar (Figure 4B). Although P-PANB forms more biofilm in the presence of IPTG than in its absence (_p_ = 0.025), P-PANB biofilm does not differ from P-PANB-Avwf\({}^{-}\) biofilm (_p_ > 0.108); there is no significant biological difference among the biofilms of these strains. The importance of LapA became evident in the M9-0.2 CAA+tryptone medium. Although the positive effect of _lapA_ expression by IPTG on the P-PANB biofilm was modest (1.2-fold, \(p\) = 0.0004), the biofilm of bacteria without the LapA vWFa domain (P-PANB-Avwf\({}^{-}\)) was significantly decreased (Figure 4C). Strain P-PANB-Avwf\({}^{-}\) had 1.4 times less biofilm in medium without IPTG than P-PANB (_p_ < 0.0001) and 1.6 times less biofilm in medium with IPTG (_p_ < 0.0001). Whereas the P-PANB biofilm was IPTG-dependent, the vWFa deletion biofilm was insensitive to _lapA_ expression by IPTG (P-PANB-Avwf; Figure 4C). In the LB medium, the P-PANB biofilm was increased 1.3-fold in the presence of IPTG (_p_ < 0.0001), whereas deletion of the vWFa domain resulted in the independence of IPTG (Figure 4D). The biofilm of the P-PANB-Avwf\({}^{-}\) strain was similar regardless of the presence or absence of IPTG (Figure 4D). The biofilm of the original strain PaW85 did not depend on the vWFa domain (Figure 4D). Figure 4: LapA and LapF amount in _P. putida_ Pav85-based strains and biofilm. (**A**) SDS-PAA gel electrophoresis of crude cell lysates, 40 \(\upmu\)g of total protein is loaded to each lane. The media in which cells were grown for preparing crude cell lysates are shown on the left of the gel image. Cells were grown with and without 1 mM IPTG. The strains used in the analysis are shown above and below the figure. PSmAlapA and PSmAlapF strains were used as negative controls for LapA and LapF. Arrows indicate LapA and LapF on the right side of the figure. Inclined arrows indicate an unknown protein complex. (**B**) Biofilm of PaW85-based strains in M9-0.2 CAA, (**C**) M9-0.2 CAA+tryptone, (**D**) LB, (**E**) M9-0.2 CAA+poly-L-lysine, (**F**) M9-0.2 CAA+cellulose and (**G**) M9-0.2 CAA+PGA in the presence and absence of 1 mM IPTG. Arithmetical averages of at least three biological parallels with 95% confidence intervals are given. Homogeneity groups are shown above the columns by lower-case letters. Identical letters denote nonsignificant differences (\(p\geq 0.05\)) between averages of biofilm. Multifactorial ANOVA was used for statistical analysis, analysed separately by media. Figure 5: SDS-PAA gels and biofilm of _P. putida_ PSm and PSm-E based strains and biofilm. (**A**) SDS-PAA gel electrophoresis of crude cell lysates, 40 ug of total protein is loaded to each lane. The media in which cells were grown for preparing crude cell lysates are shown on the left of the gel image. Cells were grown with and without 1 mM IPTG. The strains used in the analysis are shown above and below the figure. PSmAlapA and PSmAlapF strains were used as negative controls for LapA and LapF. Arrows indicate LapA and LapF on the right side of the figure. Inclined arrows indicate an unknown protein complex. (**B**) Biofilm of PSm-based strains in M9-0.2 CAA, (**C**) M9-0.2 CAA+tryptone and (**D**) LB in the presence and absence of 1 mM IPTG. An arithmetical average of at least three biological parallels with 95% confidence intervals are given.