Asymmetry of Shufflon-specific Recombination Sites in Plasmid R64 Inhibits Recombination between Direct sfx Sequences

Atsuko Gyohda,Shujuan Zhu,Nobuhisa Furuya,Teruya Komano

doi:10.1074/jbc.m513654200

Atsuko Gyohda, Shujuan Zhu + Show 2 more

Open Access

https://doi.org/10.1074/jbc.m513654200

Copy DOI

Abstract

The shufflon of plasmid R64 consists of four DNA segments separated and flanked by seven sfx recombination sites. Rci-mediated recombination between any inverted sfx sequences causes inversion of the DNA segments independently or in groups. The R64 shufflon selects one of seven pilV genes encoding type IV pilus adhesins, in which the N-terminal region is constant, while the C-terminal regions are variable. The R64 sfx sequences are asymmetric. The sfx central region and right arm sequences are conserved, but left arm sequences are not. Here we constructed a symmetric sfx sequence, in which the sfx left arm sequence was changed to the inverted repeat of the right arm sequence and made artificial shufflon segments carrying symmetric sfx sequences in inverted or direct orientations. The symmetric sfx sequence exhibited the highest inversion frequency in a shufflon segment flanked by two inverted sfx sequences. Rci-dependent deletion of a shufflon segment flanked by two direct symmetric sfx sequences was observed, suggesting that asymmetry of R64 sfx sequences inhibits recombination between direct sfx sequences. In addition, intermolecular recombination between symmetric sfx sequences was also observed. The extra C-terminal domain of Rci was shown to be essential for inversion of the R64 shufflon using asymmetric sfx sequences but not essential for recombination using symmetric sfx sequences, suggesting that the Rci C-terminal segment helps the binding of Rci to asymmetric sfx sequences. Rci protein lacking the C-terminal domain bound to both arms of symmetric sfx sequence but only to the right arm of asymmetric sfx sequence.

Highlights

While most site-specific recombination systems consist of two recombination sites and the cognate recombinase gene, several multiple DNA inversion systems containing more than two recombination sites as well as the recombinase gene have been reported after the discovery of shufflon in IncI1 plasmid R64 [4, 5]
A symmetric sfx site, in which the sfx left arm sequence was changed to the inverted repeat of the right arm sequence, was constructed and artificial shufflon segments carrying symmetric sfx site in inverted or direct orientations were made
Construction and Activity of Symmetric sfx Sequence—In our previous study [11], we found that the R64 sfx sequences are asymmetric and that the sfx left arm sequences greatly affect inversion frequency

Summary

Introduction

When pSYM210 carrying one symmetric sfxaa201 sequence in pACYC184 was introduced into E. coli cells harboring pKK097, a series of multimer bands were Cated that small plasmids are produced by the recombination In vitro recombination assay was performed using superbetween sfxaa201 and sfxa112 of pDEL301, or between coiled pSYM201 DNA and purified Rci protein (Fig. 3C). For pSYM201 carrying from pAG105 by multiple inversions in the presence of the symmetric sfxaa201, a series of multimer bands were formed in wild-type Rci (Fig. 4A, lane 3).

Results

Conclusion