Integration specificities of two lambdoid phages (21 and e14) that insert at the same attB site

Abstract

It was shown previously that phage 21 and the defective element e14 integrate at the same site within the icd gene of Escherichia coli K-12 but that 21 integrase and excisionase excise e14 in vivo very infrequently compared to excision of 21. We show here that the reverse is also true: e14 excises itself much better than it excises an adjacent 21 prophage. In vitro integrase assays with various attP substrates delimit the minimal attP site as somewhere between 366 and 418 bp, where the outer limits would include the outermost repeated dodecamers suggested as arm recognition sites by S. J. Schneider (Ph.D. dissertation, Stanford University, Stanford, Calif., 1992). We speculate that the reason 21 attP is larger than attP (240 bp) is because it must include a 209-bp sequence homologous to the 3 end of the icd transcript in order to allow icd expression in lysogens. Alteration of portions of 21 attP to their e14 counterparts shows that 21 requires both the arm site and core site sequences of 21 but that replacements by e14 sequences function in some positions. Consistent with Schneider's in vivo results, and like all other known integrases from lambdoid phages, 21 requires integration host factor for activity.The lambdoid phages are a group of natural temperate phages whose best-known member is coliphage . They have a common genetic map and are related to one another by frequent natural recombination but exhibit extensive variation in function and nucleotide sequence (6). One example of such variation is seen in their integrase genes, which mediate insertion into the chromosome by site-specific recombination. Each lambdoid phage has a specific preferred site of insertion on the chromosome. Whereas some phages, like and 434, insert at the same chromosomal site and closely resemble each other in their integrase and excisionase genes and phage attachment sites, many distinct insertion specificities are found within the group. Between phages that insert at different sites, like and 21, the protein components are not interchangeable. Thus, each integrase protein has its own specificity of site recognition. The integrase genes of all lambdoid phages have sufficient sequence similarity to imply origin from a common ancestor, from which the whole spectrum of integration specificities must have evolved.The biochemical pathway of integrase-mediated site-specific recombination is well known (Fig. 1). For , the required extent of specific sequence is about 21 bp for the bacterial partner (attB) and 240 bp for the phage partner (attP). The actual crossover event takes place between attB and a similar sequence (core sequence) within attP and entails an exchange between the top two strands, forming a Holliday junction intermediate, followed by a homology-dependent process equivalent to branch migration through a 7-bp overlap (O) segment (identical in attB and the attP core) and resolution by exchange in the lower two strands at a position displaced 7 bp from the initiating exchange. The overlap segment is flanked by oppositely or...

Distribution, Functional Expression, and Genetic Organization of Cif, a Phage-Encoded Type III-Secreted Effector from Enteropathogenic and EnterohemorrhagicEscherichia coli

“…In strain E22, the Cif-encoding lambdoid prophage was found integrated between the gal and bio operons, precisely between the ybhC and ybhB genes. This integration site is the same as that for phage lambda, and the att core sequences were also identical to those of phage lambda (15,34,40). A lambdoid prophage, Sp3, is also integrated at the same chromosome locus in EHEC O157:H7 strain Sakai, although it does not encode the cif gene (13).…”

The Adaptation of Temperate Bacteriophages to Their Host Genomes

“…Integration into protein coding sequences has been described within icd (Wang et al 1997) and ompW in E. coli (Creuzburg et al 2011) and lepA in S. enterica (Hermans et al 2006). Indeed, we find these three loci among the most occupied hotspots (fig.…”

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“…The element has at one end 216 bp of homology with the C-terminal end of the host icd gene, and the actual crossover for integration (the attachment site) occurs between the first 11 bp at one end of the homology in e14 and an 11 bp sequence inside the host icd gene [12]. The integration event fused the e14 " icd replacement region" to the N-terminal portion of the host icd gene, causing only two amino acid changes in the isocitrate dehydrogenase protein [14]. The element is capable of excision if the host SOS response is triggered.…”

“…e14 shares its integration site with phage 21 and has a similar integration machinery to that of phage 21; both have slightly overlapping int and xis genes. These two genes are transcribed leftward and lie about 3 kb from the e14 att site [12,14]. However, e14 and phage 21 must have different specificities of site recognition since phage 21 Int and Xis cannot cure cells of the e14 element as demonstrated by Wang et al [14].…”