Involvement of H-NS in Transpositional Recombination Mediated by IS 1

Abstract

IS1, the smallest active transposable element in bacteria, encodes a transposase that promotes inter-and intramolecular transposition. Host-encoded factors, e.g., histone-like proteins HU and integration host factor (IHF), are involved in the transposition reactions of some bacterial transposable elements. Host factors involved in the IS1 transposition reaction, however, are not known. We show that a plasmid with an IS1 derivative that efficiently produces transposase did not generate miniplasmids, the products of intramolecular transposition, in mutants deficient in a nucleoid-associated DNA-binding protein, H-NS, but did generate them in mutants deficient in histone-like proteins HU, IHF, Fis, and StpA. Nor did IS1 transpose intermolecularly to the target plasmid in the H-NS-deficient mutant. The hns mutation did not affect transcription from the indigenous promoter of IS1 for the expression of the transposase gene. These findings show that transpositional recombination mediated by IS1 requires H-NS but does not require the HU, IHF, Fis, or StpA protein in vivo. Gel retardation assays of restriction fragments of IS1-carrying plasmid DNA showed that no sites were bound preferentially by H-NS within the IS1 sequence. The central domain of H-NS, which is involved in dimerization and/or oligomerization of the H-NS protein, was important for the intramolecular transposition of IS1, but the N-and C-terminal domains, which are involved in the repression of certain genes and DNA binding, respectively, were not. The SOS response induced by the IS1 transposase was absent in the H-NSdeficient mutant strain but was present in the wild-type strain. We discuss the possibility that H-NS promotes the formation of an active IS1 DNA-transposase complex in which the IS1 ends are cleaved to initiate transpositional recombination through interaction with IS1 transposase.IS1 is an insertion element present in chromosomes and plasmids of enteric bacteria (for a review, see reference 38). IS1 (768 bp long) carries imperfect terminal inverted repeats (IRL and IRR) that are about 30 bp long (17,40). IS1 mediates the formation of cointegrates between the IS1-carrying plasmid and a target plasmid in which two copies of IS1 are duplicated at the two junctions in direct orientation (10,39). This element encodes two open reading frames, insA and BЈ-insB (16,32,33). Transcription occurs from a promoter present in the left-terminal region (IRL) preceding insA (31). A translational frameshift occurs in the Ϫ1 direction at the AAAAAAC (A 6 C) sequence in the overlapping region between the two open reading frames, producing the InsA-BЈ-InsB transframe protein, IS1 transposase (7, 49). Unless frameshifting occurs, IS1 produces InsA protein from insA which can bind to the IRs (51, 74) and inhibits transposition (30,75). An IS1 mutant (IS1-31) with a single adenine insertion at the frameshifting site efficiently produces transposase and therefore can frequently transpose and mediate cointegration (49). The plasmid with this IS1 mutant generates min...

Presence of a Characteristic D-D-E Motif in IS1Transposase

“…The bacterial strains used were Escherichia coli K-12 derivatives JM109 (50), JE6638 (polA1) (National Institute of Genetics collection), and NM554 (33). pNS17 is the pUC-based ampicillin resistance (Ap r ) plasmid carrying mini-IS1 (46). Plasmid pSEK80 (40) is the R100-based spectinomycin resistance (Sp r ) plasmid used as the target for mini-IS1 transposition.…”

“…pNS17 is an ampicillin resistance (Ap r ) plasmid carrying mini-IS1, which has the chloramphenicol resistance (Cm r ) gene replacing the transposase-coding region in IS1 (46). Mini-IS1 in this plasmid can transpose to the spectinomycin resistance (Sp r ) plasmid pSEK80 harbored within the same cell of E. coli strain NM554 when transposase is supplied from the kanamycin resistance (Km r ) plasmid pKEN200G, which produces IS1 transposase without translational frameshifting due to the presence of the sequence GA 2 GA 3 C in place of the A 6 C sequence (46). The IS1 transposase gene in pKEN200G can be expressed from a promoter induced by arabinose.…”

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

“…Several members of the bacterial nucleoid-associated family of proteins, including integration host factor (IHF), HU, and histone-like nucleoid structuring protein (H-NS) have been shown to modulate selected transposition systems (Craigie et al 1985;Morisato and Kleckner 1987;Surette and Chaconas 1989;Signon and Kleckner 1995;Shiga et al 2001;Rouquette et al 2004). It is particularly intriguing that H-NS should act in this fashion, because one of its main roles is to facilitate adaptation to environmental stresses (Dorman 2004).…”

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

“…2C, middle panel). This was A nucleoid-associated DNA binding protein H-NS is required for transposition of IS1 (Shiga et al, 2001). We examined the effect of hns-defi ciency on IS1 transposition by the GFP hop-on assay.…”