Pseudomonas putida Fis binds to the lapF promoter in vitro and represses the expression of LapF.

The biofilm matrix of the rhizospheric bacterium Pseudomonas putida consists mainly of a proteinaceous component. The two largest P. putida proteins, adhesins LapA and LapF, are involved in biofilm development but prevail in different developmental stages of the biofilm matrix. LapA is abundant in the initial stage of biofilm formation whereas LapF is found in the mature biofilm. Although the transcriptional regulation of the adhesins is not exhaustively studied, some factors that can be involved in their regulation have been described. For example, RpoS, the major stress response sigma factor, activates, and Fis represses LapF expression. This study focused on the LapF expression control by Fis. Indeed, using DNase I footprint analysis a Fis binding site Fis-F2 was located 150 bp upstream of the lapF gene coding sequence. The mapped 5' end of the lapF mRNA localized the promoter to the same region, overlapping with the Fis binding site Fis-F2. Monitoring the lapF promoter activity by a ß-galactosidase assay revealed that Fis overexpression causes a 4-fold decrease in the transcriptional activity. Furthermore, mutations that diminished Fis binding to the Fis-F2 site abolished the repression of the lapF promoter. Thus, these data suggest that Fis is involved in the biofilm regulation via repression of LapF expression.

Fis overexpression enhances Pseudomonas putida biofilm formation by regulating the ratio of LapA and LapF.

Bacteria form biofilm as a response to a number of environmental signals that are mediated by global transcription regulators and alarmones. Here we report the involvement of the global transcription regulator Fis in Pseudomonas putida biofilm formation through regulation of lapA and lapF genes. The major component of P. putida biofilm is proteinaceous and two large adhesive proteins, LapA and LapF, are known to play a key role in its formation. We have previously shown that Fis overexpression enhances P. putida biofilm formation. In this study, we used mini-Tn5 transposon mutagenesis to select potential Fis-regulated genes involved in biofilm formation. A total of 90 % of the studied transposon mutants carried insertions in the lap genes. Since our experiments showed that Fis-enhanced biofilm is mostly proteinaceous, the amounts of LapA and LapF from P. putida cells lysates were quantified using SDS-PAGE. Fis overexpression increases the quantity of LapA 1.6 times and decreases the amount of LapF at least 4 times compared to the wild-type cells. The increased LapA expression caused by Fis overexpression was confirmed by FACS analysis measuring the amount of LapA-GFP fusion protein. Our results suggest that the profusion of LapA in the Fis-overexpressed cells causes enhanced biofilm formation in mature stages of P. putida biofilm and LapF has a minor role in P. putida biofilm formation.

Fis regulates the competitiveness of Pseudomonas putida on barley roots by inducing biofilm formation.

An important link between the environment and the physiological state of bacteria is the regulation of the transcription of a large number of genes by global transcription factors. One of the global regulators, Fis (factor for inversion stimulation), is well studied in Escherichia coli, but the role of this protein in pseudomonads has only been examined briefly. According to studies in Enterobacteriaceae, Fis regulates positively the flagellar movement of bacteria. In pseudomonads, flagellar movement is an important trait for the colonization of plant roots. Therefore we were interested in the role of the Fis protein in Pseudomonas putida, especially the possible regulation of the colonization of plant roots. We observed that Fis reduced the migration of P. putida onto the apices of barley roots and thereby the competitiveness of bacteria on the roots. Moreover, we observed that overexpression of Fis drastically reduced swimming motility and facilitated P. putida biofilm formation, which could be the reason for the decreased migration of bacteria onto the root apices. It is possible that the elevated expression of Fis is important in the adaptation of P. putida during colonization of plant roots by promoting biofilm formation when the migration of bacteria is no longer favoured.