The Essential HupB and HupN Proteins of Pseudomonas putida Provide Redundant and Nonspecific DNA-bending Functions

Abstract

A protein mixture containing two major components able to catalyze a ␤-recombination reaction requiring nonspecific DNA bending was obtained by fractionation of a Pseudomonas putida extract. N-terminal sequence analysis and genomic data base searches identified the major component as an analogue of HupB of Pseudomonas aeruginosa and Escherichia coli, encoding one HU protein variant. The minor component of the fraction, termed HupN, was divergent enough from HupB to predict a separate DNA-bending competence. The determinants of the two proteins were cloned and hyperexpressed, and the gene products were purified. Their activities were examined in vitro in ␤-recombination assays and in vivo by complementation of the Hbsu function of Bacillus subtilis. HupB and HupN were equally efficient in all tests, suggesting that they are independent and functionally redundant DNA bending proteins. This was reflected in the maintenance of in vivo activity of the 54 Ps promoter of the toluene degradation plasmid, TOL, which requires facilitated DNA bending, in ⌬hupB or ⌬hupN strains. However, hupB/hupN double mutants were not viable. It is suggested that the requirement for protein-facilitated DNA bending is met in P. putida by two independent proteins that ensure an adequate supply of an essential cellular activity.

Regulation of Cyclic Lipopeptide Biosynthesis inPseudomonas fluorescensby the ClpP Protease

“…Several ORFs were identified (Fig. 2), including the chaperone and protein-folding trigger factor (tig); the ATPase chaperone, clpX; the lon protease; the DNA binding and -bending hupB; and a partial sequence of ppiD isomerase, a gene involved in protein folding (3,19,24,35). The organization of these genes in strain SS101 is identical to that found in various other fully sequenced Pseudomonas species and strains (Fig.…”

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

“…For example, it is known in E. coli that Fis is involved in the regulation of genes from 15 different categories, including metabolism of several carbon sources, and motility (Bradley et al, 2007). In some cases the inactivation of particular global regulator(s) can be complemented by a protein with a similar function [for example, integration host factor (IHF) can replace HU in the Sin recombinase synapse on the resH site (Rowland et al, 2006)], but in other organisms the inactivation of the same regulators can be lethal, as seen in an hupN and hupB double mutant of P. putida (Bartels et al, 2001) and an hbs mutant of B. subtilis (Micka & Marahiel, 1992). Similarly, although Fis-minus mutants of E. coli and S. typhimurium are viable (Bradley et al, 2007;Johnson et al, 1988;Kelly et al, 2004;Osuna et al, 1995), Fis is essential to P. putida.…”

LacZ-promoter fusions: the effect of growth

“…In contrast, an inverse relation between the level of expression and growth rate, as observed here, would be indicative of a growth-rate-independent rate of transcription (10). Such considerations may be helpful when discussing growth-rate-dependent expression (2,3,4,5,8) and, at least, show the importance of careful use of terminology. At any time, it should be kept in mind that the rate of transcription is a complex parameter containing many different contributions.…”

“…Two of these ORFs encode the two subunits of the IHF protein of P. putida (5). Two others match the HupB and HupN protein sequences (2). But, in addition, a fifth gene was found that clearly encoded an extra HU-like protein.…”

“…The failure of in vivo and in vitro screenings to detect either the function or the sequence of the hupP gene of P. putida (2) and P. aeruginosa (6) is intriguing. In the case of P. putida, such a screening was based on the enrichment of DNA-binding proteins from a cell lysate that stimulated a β-recombination reaction dependent on non-specific DNA bending.…”