## 2 Results  
### LapA Is Essential for Peptide-Dependent Biofilm  
Knowing the peptide-dependency of _P. putida_ biofilm [19], we tested whether it is based on LapA or LapF. We previously reported that LapA is a crucial factor in forming _P. putida_ biofilm in LB and that LapF is required for _P. putida_ cell surface hydrophobicity [1; 2; 20]. Thus, we assessed the biofilm formation of PSm and F15 deletant strains \(\Delta\)lapA, \(\Delta\)lapF, and double deletant \(\Delta\)lapAF in the following media: M9-0.2 CAA; M9-0.2 CAA+tryptone, and LB (Table S2 and Figure 1). The two terms are used hereafter: Fis-enhanced biofilm and peptide-dependent biofilm. Fis-enhanced biofilm appears in _P. putida_ F15 strain when the expression of _fis_ is increased by IPTG compared to the biofilm of F15 without IPTG supplementation (compare biofilms of _P. putida_ F15 in Figure 1B,C). Peptide-dependent biofilm appears in _P. putida_ strains when the peptides are present in growth media compared to the biofilms formed in growth media  
Figure 1: Biofilm of _P. putida_ strains PSm, F15 and deletion-mutants \(\Delta\)lapA, \(\Delta\)lapF, and \(\Delta\)lapAF constructed from them. “-” indicates original strain without deletions. Cells were grown in (**A**) M9-0.2 CAA, (**B**) M9-0.2 CAA+tryptone, and (**C**) LB medium. Cells were grown with or without 1 mM IPTG. Arithmetic means of at least three independent sets of measurements are shown. The 95% confidence intervals of the arithmetic means and the homogeneous groups are shown above the columns by lower-case letters, tested by following Bonferroni’s test for unequal n. Identical letters in homogenous groups denote nonsignificant differences (\(p\geq 0.05\)) between averages of biofilm. without peptides (compare biofilms in Figure 1A,C) [19]. If the arithmetic means of the samples are significantly different (\(p<0.05\)), it is considered a Fis-enhanced or peptide-dependent biofilm. Otherwise, the increased amount of Fis in the cells or the peptides in the growth medium does not affect biofilm. The F15 has been a helpful strain in evaluating the positive effects of peptides on _P. putida_ biofilm. The Fis-enhanced biofilm was formed in the presence of the peptides [19], the _lapA_ transcription was activated by _fis_-overexpression [20], and _lapF_ transcription was repressed [21]. We hypothesised that if LapA or LapF are involved in the peptide-dependence of biofilm, then the peptide-dependence of biofilm should be absent in strains where _lapA_ or _lapF_ genes are deleted, even in cells with an elevated amount of Fis. We observed that the deletion of _lapA_ decreased the biofilm of _P. putida_ wild-type strain PSm, regardless of the medium (Figure 1). The _lapA_ deletion strain PSm\(\Delta\)lapA had 1.3-1.9 times decreased biofilm than the wild-type strain PSm (\(p<0.032\)), except for the _lapA_, _lapF_ double deletion derivative PSm\(\Delta\)lapAF in M9-0.2 CAA+tryptone, whose biofilm was similar to PSm (Figure 1). The _lapF_-deletion strain PSm\(\Delta\)lapF formed biofilms similarly to the wild-type strain PSm in the presence of peptides in media (Figure 1B,C). In the case of _fis_-overexpression strain F15, the Fis-enhanced biofilm appeared in the tryptone-containing growth media with a 2.3-fold difference in M9-0.2 CAA+tryptone (\(p<0.0001\)) and a 2.0-fold difference in LB (\(p<0.0001\)), but not in M9-0.2 CAA (1.1-fold difference, \(p=1\); Figure 1). The strain with the deleted _lapA_ gene F15\(\Delta\)lapA did not produce a Fis-enhanced biofilm (Figure 1B,C). At the same time, the _lapF_ deletion strain F15\(\Delta\)lapF retained the positive effect of _fis_-overexpression on the F15 biofilm in M9-0.2 CAA+tryptone and LB (3.9 and 2.0-fold differences, respectively, \(p<0.0001\)), indicating that _lapA_ expression is needed for the enhanced biofilm by Fis in the presence of peptides in the medium (Figure 1B,C). We previously observed the positive effect of poly-L-lysine (pK) on _P. putida_ F15 biofilm; it restored the Fis-enhancing effect for _P.