RS1 Element of Vibrio cholerae Can Propagate Horizontally as a Filamentous Phage Exploiting the Morphogenesis Genes of CTXΦ

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Abstract
In toxigenic Vibrio cholerae , cholera toxin is encoded by the CTX prophage, which consists of a core region carrying ctxAB genes and genes required for CTXΦ morphogenesis, and an RS2 region encoding regulation, replication, and integration functions. Integrated CTXΦ is often flanked by another genetic element known as RS1 which carries all open reading frames (ORFs) found in RS2 and an additional ORF designated rstC . We identified a single-stranded circularized form of the RS1 element, in addition to the CTXΦ genome, in nucleic acids extracted from phage preparations of 32 out of 83 (38.5%) RS1-positive toxigenic V. cholerae strains analyzed. Subsequently, the corresponding double-stranded replicative form (RF) of the RS1 element was isolated from a representative strain and marked with a kanamycin resistance (Km r ) marker in an intergenic site to construct pRS1-Km. Restriction and PCR analysis of pRS1-Km and sequencing of a 300-bp region confirmed that this RF DNA was the excised RS1 element which formed a novel junction between ig1 and rstC . Introduction of pRS1-Km into a V. cholerae O1 classical biotype strain, O395, led to the production of extracellular Km r transducing particles, which carried a single-stranded form of pRS1-Km, thus resembling the genome of a filamentous phage (RS1-KmΦ). Analysis of V. cholerae strains for susceptibility to RS1-KmΦ showed that classical biotype strains were more susceptible to the phage compared to El Tor and O139 strains. Nontoxigenic (CTX − ) O1 and O139 strains which carried genes encoding the CTXΦ receptor toxin-coregulated pilus (TCP) were also more susceptible (>1,000-fold) to the phage compared to toxigenic El Tor or O139 strains. Like CTXΦ, the RS1Φ genome also integrated into the host chromosomes by using the att RS sequence. However, only transductants of RS1-KmΦ which also harbored the CTXΦ genome produced a detectable level of extracellular RS1-KmΦ. This suggested that the core genes of CTXΦ are also required for the morphogenesis of RS1Φ. The results of this study showed for the first time that RS1 element, which encodes a site-specific recombination system in V. cholerae , can propagate horizontally as a filamentous phage, exploiting the morphogenesis genes of CTXΦ.