The integron integrase efficiently prevents the melting effect of Escherichia coli single-stranded DNA-binding protein on folded attC sites

Abstract

Integrons play a major role in the dissemination of antibiotic resistance genes among bacteria. Rearrangement of gene cassettes occurs by recombination between attI and attC sites, catalyzed by the integron integrase. Integron recombination uses an unconventional mechanism involving a folded single-stranded attC site. This site could be a target for several host factors and more precisely for proteins able to bind single-stranded DNA. One of these, Escherichia coli single-stranded DNA-binding protein (SSB), regulates many DNA processes. We studied the influence of this protein on integron recombination. Our results show the ability of SSB to strongly bind folded attC sites and to destabilize them. This effect was observed only in the absence of the integrase. Indeed, we provided evidence that the integrase is able to counterbalance the observed effect of SSB on attC site folding. We showed that IntI1 possesses an intrinsic property to capture attC sites at the moment of their extrusion, stabilizing them and recombining them efficiently. The stability of DNA secondary structures in the chromosome must be restrained to avoid genetic instability (mutations or deletions) and/or toxicity (replication arrest). SSB, which hampers attC site folding in the absence of the integrase, likely plays an important role in maintaining the integrity and thus the recombinogenic functionality of the integron attC sites. We also tested the RecA host factor and excluded any role of this protein in integron recombination.

FIG 1

attC recombination sites and a model for attC folding. (A) Schematic representation of a double-stranded (ds) attC site. Inverted repeats R″, L″, L′, and R′ are indicated by gray boxes. The dotted line represents the variable central part. Conserved nucleotides are indicated. The asterisk shows the conserved G nucleotide, which corresponds to the extrahelical base (EHB) in the folded attC site bottom strand. The black arrow shows the cleavage point. (B) Secondary structures of VCR_2/1 and attC_aadA7 site bottom strands (bs). Structures were determined by the UNAFOLD online interface at the Pasteur Institute. The four structural features of attC sites, namely, the UCS (unpaired central segment), the EHB, the stem, and the VTS (variable terminal structure), are indicated. The black arrow shows the cleavage points, and the asterisk shows the extrahelical G bases. Primary sequences of the attC sites are shown (except for the VTS of the VCR_2/1 site). (C) Representation of both ssDNA and dsDNA pathways for attC site folding. In the ssDNA pathway (1) (red), horizontal gene transfer (conjugation, transformation, and phage infection) and replication (template of lagging strand) lead to the production of ssDNA favoring attC hairpin formation. In the dsDNA pathway (2) (green), an increase of supercoiling ensures cruciform extrusion. Origins of replication are shown as blue ovals, and replication complexes are shown as yellow ovals.

FIG 2

Comparison of SSB binding to several VCR_2/1 site derivatives. (A) Nucleotide sequences and secondary structures of the bottom strands (bs) of VCR_2/1 site derivatives. Structures were determined by the UNAFOLD online interface at the Pasteur Institute. VCR_dbs is a VCR derivative used in EMSA in which the variable terminal structure (VTS) was replaced by GAA. The black arrow shows the cleavage point in the R box, and the asterisk shows the extrahelical G nucleotide. We introduced mutations (boldface type) into this VCR_dbs site in order to generate, after folding, paired (pd) and unpaired (unpd) VCR_dbs site derivatives. The ΔG (free energy) values of VCR_dbs, paired VCR_dbs, and unpaired VCR_dbs folding are −21.7, −37.2, and −5.3 kcal mol⁻¹, respectively. (B) EMSA of the binding of SSB to VCR_2/1 site derivatives. Equal quantities of radiolabeled VCR site derivative DNA fragments (0.6 pmol) were incubated with increasing amounts (0, 13, 26, 52, and 104 nM) of SSB. Complexes I and II probably represent several degrees of SSB multimerization. (C) Densitometry analysis of EMSA. Binding of SSB was measured by monitoring the fraction (×100) of the labeled DNA substrate bound by SSB in complexes I and II to labeled (bound and free) DNA, detected by an automatic peak search of Image Gauge.

FIG 3

In vivo effects of SSB and IntI1 on attC site folding. (A) Experimental setup of the replication slippage assay. The model proposed is that folding of the attC site between the pair of flanking direct repeats (EcoRI restriction sites) (black rectangles) permits a replication slippage event. This results in precise or nearly precise attC deletion, thus reconstituting a functional cat resistance gene (Cm^r reversion frequencies). The precise attC deletion was measured by sequencing (attC deletion frequencies). (B) Effects of SSB and IntI1 on Cm^r reversion and attC deletion frequencies (see Materials and Methods). The replication slippage assay was used to study the effects of IntI1Y312F and SSB on attC_aadA7 site folding. The Cm^r reversion frequencies and attC deletion frequencies are represented in the absence (−) and in the presence (+) of IntI1 for the GJ1885 wild-type strain and the GJ1885 ssb-200 strain (GJ1890). The results represent means of four independent experiments (see Materials and Methods). Error bars show the standard deviations.

FIG 4

Specificity of the in vivo effects of SSB and IntI1 on attC site folding. (A) Secondary structures of the attC_aadA7 site bottom strand (bs) and the unpaired attC_aadA7 bottom-strand (bs) site derivative. Structures were determined by the UNAFOLD online interface at the Pasteur Institute. The black arrow shows the cleavage point. Primary sequences of the attC sites are shown. The ΔG (free energy) values of attC_aadA7 bs and unpaired attC_aadA7 bs folding are −19.1 and −5.1 kcal mol⁻¹, respectively. (B) The replication slippage assay was used to study the effect of SSB on folding of the attC_aadA7 site (black bar) and the unpaired attC_aadA7 site derivative (gray bar). The relative deletion rates obtained for the ssb-200 strain versus GJ1885 (ssb-WT) are indicated. The results represent means of four independent experiments. (C) The replication slippage assay was used to study the effect of IntI1Y312F on folding of the attC_aadA7 site (black bar) and the unpaired attC_aadA7 site derivative (gray bar). The relative deletion rates obtained in the presence of integrase versus in its absence (intI1+/intI1−) are indicated. The results represent means of four independent experiments.

FIG 5

In vitro effect of IntI1 on attC_aadA7 (A), VCR_2/1 (B), attI1 (C), and attC_aadA7m (D) site folding. The OD₂₆₀ of each fragment (50 μg/ml) was measured at 37°C either after prechilling (prechilled attC_aadA7, VCR_2/1, attI1, and attC_aadA7m) or after preheating for 3 min at 95°C (preheated attC_aadA7, VCR_2/1, attI1, and attC_aadA7m) in the absence of IntI1 as well as after the addition of IntI1 (2 pmol) 30 min after the beginning of the measurement with prechilling (prechilled attC_aadA7 + IntI1, VCR_2/1 + IntI1, attI1 + IntI1, and attC_aadA7m + IntI1) or after preheating at 95°C (preheated attC_aadA7 + IntI1, VCR_2/1 + IntI1, attI1 + IntI1, and attC_aadA7m + IntI1). The OD₂₆₀ measurement of the proteins alone was previously performed and subtracted from the measurements obtained with the mixture in order to determine accurate values for the fragment solution. Results represent means of three independent experiments. Error bars show the standard deviations.

FIG 6

In vitro effect of SSB on attC_aadA7 (A) and VCR_2/1 (B) site folding. The OD₂₆₀ of each fragment (50 μg/ml) was measured at 37°C either after prechilling (prechilled attC_aadA7 and VCR_2/1) or after preheating for 3 min at 95°C (preheated attC_aadA7 and VCR_2/1) in the absence of SSB as well as after addition of SSB (2 pmol) 30 min after the beginning of the measurement with prechilling (prechilled attC_aadA7 + SSB and VCR_2/1 + SSB) or after preheating at 95°C (preheated attC_aadA7 + SSB and VCR_2/1 + SSB). The OD₂₆₀ measurement of the proteins alone was previously performed and subtracted from the measurements obtained with the mixture in order to determine accurate values for the fragment solution. Results represent means of three independent experiments. Error bars show the standard deviations.

FIG 7

In vivo effect of SSB on cassette excision. (A) Experimental setup of the cassette excision assay. The dapA gene is interrupted by a synthetic cassette (black line) encountered by the attI1 (black triangle) and attC_aadA7 (white triangle) sites. Recombination leads to the excision of the integron cassette and restores a functional dapA gene. (B) Excision frequencies of the attI1 × attI1, attC_aadA7 × attC_ereA2, and attI1 × attC_aadA7 cassettes in the WT or ssb-200 strain (bottom) and in the presence of the pCL1920 control vector (vector) or the pHYD620 plasmid (ssb⁺), which overexpresses the SSB protein (bottom). Black bars represent the results obtained in the presence of IntI1, and gray bars represent those obtained in the absence of IntI1. “nd” indicates “nondetected” recombination events. Results represent means of three independent experiments. Error bars show the standard deviations.

FIG 8

In vivo effect of RecA on cassette excision. Shown are excision frequencies of the attI1 × attI1, attC_aadA7 × attC_ereA2, and attI1 × attC_aadA7 cassettes in the WT or recA-deleted strain (bottom) and in the presence of the pGB2 control vector (vector) or the pGB2-recA plasmid (recA⁺), which overexpresses the RecA protein (top). Black bars represent the results obtained in the presence of IntI1, and gray bars represent the results obtained in the absence of IntI1. “nd” indicates “nondetected” recombination events. Results represent means of three independent experiments. Error bars show the standard deviations.