Unusual structure of the attB site of the site-specific recombination system of Lactobacillus delbrueckii bacteriophage mv4

Abstract

The temperate phage mv4 integrates its genome into the chromosome of Lactobacillus delbrueckii subsp. bulgaricus by site-specific recombination within the 3' end of a tRNA(Ser) gene. Recombination is catalyzed by the phage-encoded integrase and occurs between the phage attP site and the bacterial attB site. In this study, we show that the mv4 integrase functions in vivo in Escherichia coli and we characterize the bacterial attB site with a site-specific recombination test involving compatible plasmids carrying the recombination sites. The importance of particular nucleotides within the attB sequence was determined by site-directed mutagenesis. The structure of the attB site was found to be simple but rather unusual. A 16-bp DNA fragment was sufficient for function. Unlike most genetic elements that integrate their DNA into tRNA genes, none of the dyad symmetry elements of the tRNA(Ser) gene were present within the minimal attB site. No inverted repeats were detected within this site either, in contrast to the lambda site-specific recombination model.

FIG. 1

attP × attB site-specific recombination test in E. coli. (A) Schematic representation of cointegrate formation between pMC1 and pAMattB. Restriction maps of the plasmids used in the test are shown. Pa and Bb, PCR primers for attR amplification; Ba and Pb, PCR primers for attL amplification. The arrows beside the attachment sites indicate the orientation of the homologous core sequences. (B) Electrophoresis in a 0.5% agarose gel (ethidium bromide stained) of XbaI-digested plasmid DNA isolated from E. coli TG2 transformed with pMC1 (int⁺ attP) (lane 1), pMC2 (int⁻ attP) (lane 2), pAM239 (lane 3), pAMattB (lane 4), pMC1 and pAMattB (lane 5), pMC1 and pAM239 (lane 6), and pMC2 and pAMattB (lane 7). L, 1-kb DNA ladder (Bethesda Research Laboratories). The photograph is a negative image. Arrows indicate the 1.6- and 8.1-kb XbaI fragments of the cointegrate.

FIG. 1

attP × attB site-specific recombination test in E. coli. (A) Schematic representation of cointegrate formation between pMC1 and pAMattB. Restriction maps of the plasmids used in the test are shown. Pa and Bb, PCR primers for attR amplification; Ba and Pb, PCR primers for attL amplification. The arrows beside the attachment sites indicate the orientation of the homologous core sequences. (B) Electrophoresis in a 0.5% agarose gel (ethidium bromide stained) of XbaI-digested plasmid DNA isolated from E. coli TG2 transformed with pMC1 (int⁺ attP) (lane 1), pMC2 (int⁻ attP) (lane 2), pAM239 (lane 3), pAMattB (lane 4), pMC1 and pAMattB (lane 5), pMC1 and pAM239 (lane 6), and pMC2 and pAMattB (lane 7). L, 1-kb DNA ladder (Bethesda Research Laboratories). The photograph is a negative image. Arrows indicate the 1.6- and 8.1-kb XbaI fragments of the cointegrate.

FIG. 2

Site-specific recombination in E. coli between the attP site and truncated or mutated attB sites. The attB sequences are presented in Table 2. Electrophoresis in a 0.5% agarose gel (ethidium bromide stained) of EcoRV-digested plasmid DNA isolated from E. coli TG2 transformed with pMC1 (int⁺ attP) (lane 1), pAM239 (lane 2), pMC1 and pAMattB (lane 3), pMC1 and pAMattB1 (lane 4), pMC1 and pAMattB2 (lane 5), pMC1 and pAMattB3 (lane 6), and pMC1 and pAMattB4 (lane 7) is shown. Plasmids pAM239, pAMattB, and pAMattB1 to -4 have no EcoRV site. Plasmid pMC1 and the cointegrate each contain one EcoRV site. L, 1-kb DNA ladder (Bethesda Research Laboratories). The photograph is a negative image. Arrows indicate the linear cointegrate (a), linear pMC1 (b), open circular form (c), and supercoiled circular form of pAMattB (d).

FIG. 3

DNA sequences of tRNA genes used as insertion sites by genetic elements. (A) Insertion sites for which the core sequence overlaps the anticodon loop sequence. The names of the genetic elements and tRNA genes into which they integrate are indicated on the left. The tRNA sequences and their 3′ flanking regions are shown in uppercase and lowercase letters, respectively. The core sequences are boxed and the boundaries of the minimal sequences required for attB function are indicated by brackets. The overlap regions, when determined experimentally, are shown in boldface type. (B) Insertion sites for which the core sequence is located downstream from the anticodon loop sequence.