Fis negatively affects binding of Tn4652 transposase by out-competing IHF from the left end of Tn4652

Abstract

Transposition activity in bacteria is generally maintained at a low level. The activity of mobile DNA elements can be controlled by bacterially encoded global regulators. Regulation of transposition of Tn4652 in Pseudomonas putida is one such example. Activation of transposition of Tn4652 in starving bacteria requires the stationary-phase sigma factor RpoS and integration host factor (IHF). IHF plays a dual role in Tn4652 translocation by activating transcription of the transposase gene tnpA of the transposon and facilitating TnpA binding to the inverted repeats of the transposon. Our previous results have indicated that besides IHF some other P. putida-encoded global regulator(s) might bind to the ends of Tn4652 and regulate transposition activity. In this study, employing a DNase I footprint assay we have identified a binding site of P. putida Fis (factor for inversion stimulation) centred 135 bp inside the left end of Tn4652. Our results of gel mobility shift and DNase I footprint studies revealed that Fis out-competes IHF from the left end of Tn4652, thereby abolishing the binding of TnpA. Thus, the results obtained in this study indicate that the transposition of Tn4652 is regulated by the cellular amount of P. putida global regulators Fis and IHF.

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

“…Bacteria were electrotransformed as described by Sharma & Schimke (1996). E. coli strains DH5a (Invitrogen) and CC118lpir (Herrero et al, 1990) were used for DNA cloning procedures; BL21(DE3) (Studier & Moffatt, 1986) was used for the overexpression of P. putida Fis by a previously published protocol (Teras et al, 2009).…”

“…They obtained the insertion of MAR2xT7 in 4468 genes which make up 75 % of all the genes in this organism, and suggest that fis of P. aeruginosa is a candidate essential gene, since only one mutant that carried an insertion in the fis gene was detected, and this insertion was located in a stop codon of the fis gene (Liberati et al, 2006). Indeed, we have not succeeded in creating a fis knockout mutant of P. putida in our laboratory (Teras et al, 2009).…”

“…For the production of polyclonal mouse anti-Fis antibody, P. putida Fis(His) was overexpressed and purified with Ni-NTA agarose matrix (Qiagen), as described previously (Teras et al, 2009). The polyclonal antibodies against Fis (PP4821) were produced and purified by LabAs.…”

“…Fis is quite well conserved in these families; for example, in E. coli and P. putida, Fis proteins share 64.8 % identity and 81.7 % similarity at the deduced amino acid sequence level as determined via The Comprehensive Microbial Resource (CMR) website (http://cmr.jcvi.org/ tigr-scripts/CMR/CmrHomePage.cgi). Although Fis is well studied in E. coli, the role of this protein in pseudomonads has been examined only in a few recently published papers (Kugelberg et al, 2005;Teras et al, 2009;Yeung et al, 2009;Yuste et al, 2006). Interestingly, although the fis knockout mutants of E. coli and S. typhimurium are viable and grow well (Bradley et al, 2007;Johnson et al, 1988;Kelly et al, 2004;Osuna et al, 1995), it seems that pseudomonads do not tolerate the lack of functional Fis protein (Liberati et al, 2006;Teras et al, 2009;Yeung et al, 2009).…”

“…Although Fis is well studied in E. coli, the role of this protein in pseudomonads has been examined only in a few recently published papers (Kugelberg et al, 2005;Teras et al, 2009;Yeung et al, 2009;Yuste et al, 2006). Interestingly, although the fis knockout mutants of E. coli and S. typhimurium are viable and grow well (Bradley et al, 2007;Johnson et al, 1988;Kelly et al, 2004;Osuna et al, 1995), it seems that pseudomonads do not tolerate the lack of functional Fis protein (Liberati et al, 2006;Teras et al, 2009;Yeung et al, 2009). Liberati et al (2006) generated a library of random transposon insertion mutations in Pseudomonas aeruginosa PA14.…”