Plasmids carrying cloned fragments of RF DNA from the filamentous phage φLf can be integrated into the host chromosome via site-specific integration and homologous recombination

Abstract

Different regions of RF DNA from the filamentous bacteriophage phiLf were cloned in Escherichia coli vectors that can not be maintained in Xanthomonas. After introduction into X. campestris pv. campestris 17 (Xc17), most of these constructs were found to integrate into the host chromosome, either by recA-dependent homologous recombination or recA-independent site-specific integration. Mutations in himA, which codes for the alpha-subunit of the Integration Host Factor, does not affect the integration. Integration occurs into a chromosomal region which harbors a copy of a defective phage (4445 bp) that shares a high degree of identity with the phiLf genome. While various parts of the 4445-bp region are susceptible to homologous recombination, site-specific integration requires the attB sequence on the chromosome and the phage attP. The attB shows a high level of sequence identity (22 out of 28 bp) to the dif site required for E. coli Xer site-specific recombination, including the 6-bp central region, and 8/11 identity in both the left XerC-binding arm and the right XerD-binding arm, with the innermost 5 nt of the arms forming a dyad symmetry that is also present in dif. The attP has the same central region and shows 10/11 identity to the dif site in the left arm, but the sequence of the right arm is less conserved than that of attB. The smallest regions still capable of mediating integration are a cloned 72-bp phiLf attP-containing sequence and a 51-bp Xc17 attB-containing sequence, which was reinserted into the Xc17 chromosome after the 4445-bp region had been deleted, indicating that accessory sequences are not necessary and that the integrase required for site-specific integration is neither specified by the 4445-bp Xc17 chromosomal region nor encoded by the phiLf genome.

A horizontally acquired filamentous phage contributes to the pathogenicity of the plague bacillus

“…1). As observed for several other filamentous prophages (Lin et al ., 2001; Gonzalez et al ., 2002; Huber and Waldor, 2002), YpfΦ is inserted into the chromosomal dif site, a recombinational locus that functions in the resolution of chromosome dimers (Lesterlin et al ., 2004). This insertion reconstitutes an intact dif site and generates an imperfect 39 bp duplication of the sequence flanking the prophage (Fig.…”

Comprehensive prediction of chromosome dimer resolution sites in bacterial genomes

“…In this case, dimer resolution still depends on FtsK and dif is still located opposite the origin of replication between oriented polar sequences [27]. Several filamentous phages are known to hijack this site-specific recombination machinery of dif /XerCD for their integration into the host chromosome, containing pseudo- dif sequences within these phage genomes [28-34]. However, the dif sequence remains intact during such recombination process to ensure the integrity of chromosome dimer resolution machinery [35,36].…”

“…However, the dif sequence remains intact during such recombination process to ensure the integrity of chromosome dimer resolution machinery [35,36]. The dif -like sequences in phages often contain more variable central region that is longer than the canonical 6 bp [31,33,34], and the XerD binding arm is considerably degenerate [28]. …”

The dif/Xer Recombination Systems in Proteobacteria

“…For instance, the filamentous phages CTXΦ and VGJΦ in Vibrio cholerae , f237 in V. parahaemoliticus , CUS-1 in E. coli 018:K1:H7, YpfΦ in Yersinia pestis and Cf16-v1 and ΦLf in Xanthomonas campestris all integrate into the host chromosome at the dif site [24], [25], [26], [27], [28], [29], [30]. The mechanism of prophage genome integration has been described in detail in V. cholerae CTXΦ, the filamentous phage containing the cholera toxin-encoding gene [31], [32].…”