The effect of host‐encoded nucleoid proteins on transposition: H‐NS influences targeting of both IS903 and Tn10

Abstract

SummaryNucleoid proteins are small, abundant, DNA-binding proteins that profoundly affect the local and global structure of the chromosome, and play a major role in gene regulation. Although several of these proteins have been shown to enhance assembly of transpososomes before initiating transposition, no systematic survey has been carried out examining the in vivo role(s) of these proteins in transposition. We have examined the requirement of the six most abundant nucleoid proteins in transposition for three different transposons, IS 903 , Tn 10 and Tn 552 . Most notably, H-NS was required for efficient transposition of all three elements in a papillation assay, suggesting a general role for H-NS in bacterial transposition. Further studies indicated that H-NS was exerting its effect on target capture. Targeting preferences for IS 903 into the Escherichia coli chromosome were dramatically altered in the absence of H-NS. In addition, the alterations observed in the IS 903 target profile emphasized the important role that H-NS plays in chromosome organization. A defect in target capture was also inferred for Tn 10 , as an excised transposon fragment, a precursor to target capture, accumulated in in vivo induction assays. Furthermore, a transposase mutant that is known to increase target DNA bending and to relax target specificity eliminated this block to target capture. Together, these results imply a role for H-NS in target capture, either by providing regions of DNA more accessible to transposition or by stabilizing transpososome binding to captured targets immediately before strand transfer.

The global regulator H-NS acts directly on the transpososome to promote Tn10 transposition

“…One expectation of this hypothesis is that the intermolecular transposition frequency would be reduced in the hns12 strain because instead of the vast majority of H-NS-interacting transpososomes going on to form intermolecular STPs, some fraction of these would be partitioned to the intramolecular pathway to give knotted inversion circles. In support of this we have found that in a papillation assay (Swingle et al 2004), intermolecular transposition events were greatly reduced in the hns12 strain (data not shown).…”

“…In IS1 transposition, H-NS promotes transposition as it affects the amount of transposase (InsAB protein) produced, either by aiding in InsAB translation or by protecting InsAB from proteolysis (Rouquette et al 2004). H-NS also promotes IS903 and Tn552 transposition, but it is not yet known how it acts in these systems (Swingle et al 2004). It seems unlikely that H-NS would influence IS903 or Tn552 transposition via the same mechanism as that proposed here for Tn10 transposition because there is no evidence for IHF-mediated transpososome unfolding in these systems and they lack obvious IHF-binding sites close to the terminal inverted repeats (Derbyshire and Grindley 1992;Leschziner et al 1998).…”

“…In a strain expressing the wild-type form of H-NS, transposition events from a mini-Tn10-containing plasmid partition between intermolecular insertions and intratransposon insertions (Swingle et al 2004). The latter products are almost exclusively unknotted inversion circles (Benjamin and Kleckner 1989).…”

“…More recently, H-NS was shown to play a role in Tn10 transposition in vivo (Swingle et al 2004). In an hns deletion strain, the frequency of Tn10 transposition is significantly reduced.…”

Measurements of transposition frequency of insertion sequence IS1 by GFP hop-on assay

“…This allows investigation of a host mutation which disturbs the stability or mobility of the plasmids. It was reported that change of the local and global structure of the chromosome induced by binding of nucleoid proteins or chromosomal DNA replication affects target site selection of transposons (Peters and Craig, 2000;Swingle et al, 2004). The GFP hop-on assay system represents a powerful tool for a better understanding of the roles of chromosome architecture on transpositional recombination.…”