The promoter region of lapA and its transcriptional regulation by Fis in Pseudomonas putida

Abstract

LapA is the biggest protein in Pseudomonas putida and a key factor for biofilm formation. Its importance and posttranslational regulation is rather thoroughly studied but less is known about the transcriptional regulation. Here we give evidence that transcription of lapA in LB-grown bacteria is initiated from six promoters, three of which display moderate RpoS-dependence. The global transcription regulator Fis binds to the lapA promoter area at six positions in vitro, and Fis activates the transcription of lapA while overexpressed in cells. Two of the six Fis binding sites, Fis-A7 and Fis-A5, are necessary for the positive effect of Fis on the transcription of lapA in vivo. Our results indicate that Fis binding to the Fis-A7 site increases the level of transcription from the most distal promoter of lapA, whereas Fis binding to the Fis-A5 site could be important for modifying the promoter area topology.

Colonization efficiency of Pseudomonas putida is influenced by Fis-controlled transcription of nuoA-N operon

“…Therefore the promoter area of nuoA or part of it was cloned in front of promoterless lacZ gene in p9TT B lacZ ([ 29 ], S1 Table ), which ensures low basal activity in P . putida strains [ 22 , 29 ]. The functionality of promoters was inspected by using p9TT B lacZ derivatives p9_P nuoA 1 and p9_P nuoA 2 that contained only one nuoA potential respective promoter P N-I and P N-II in front of the lacZ test gene ( S1 Table , Fig 3A ).…”

“…We have previously described that fis -overexpression increases both biofilm formation and the expression of lapA in P . putida [ 22 , 23 , 31 ]. LapA is a cell surface protein necessary for P .…”

“…It seems that the attachment of P . putida depends on two factors: detoxification of ROS and the presence of the main adhesin LapA as we have previously reported that fis -overexpression increases the expression of lapA [ 22 , 23 ]. Thus, the elevated amount of LapA on cells surface would enhance attachment to roots if the bacteria were capable to effectively detoxify ROS.…”

“…The promoter-probe vector p9TTBlacZ, which has low basal activity [ 22 , 29 ], was used for a β-galactosidase assay and the nuoA upstream regions were cloned in front of the promoterless lacZ gene. The constructs for the verification of potential promoters were cloned as follows.…”

“…Additionally, we have shown that the amount of LapA, a key adhesin for bacterial adherence and biofilm formation, is up-regulated in the fis -overexpression strain of P . putida [ 22 , 23 ]. Thus, the fis -overexpression should facilitate rather than reduce the adherence of P .…”