Genomic Characterization of Ralstonia solanacearum Phage φRSA1 and Its Related Prophage (φRSX) in Strain GMI1000

Abstract

φRSA1 is a wide-host-range bacteriophage isolated from Ralstonia solanacearum . In this study, the complete nucleotide sequence of the φRSA1 genomic DNA was determined. The genome was 38,760 bp of double-stranded DNA (65.3% G+C) with 19-bp 5′-extruding cohesive ends ( cos ) and contained 51 open reading frames (ORFs). Two-thirds of the φRSA1 genomic region encodes the phage structural modules, and they are very similar to those reported for coliphage P2 and P2-like phages. A φRSA1 minireplicon with an 8.2-kbp early-expressing region was constructed. A late-expression promoter sequence motif was predicted for these φRSA1 genes as 5′ TGTTGT-(X) ₁₃ -ACAACA. The genomic sequence similarity between φRSA1 and related phages φ52237 and φCTX was interrupted by three AT islands, one of which contained an insertion sequence element, suggesting that they were recombinational hot spots. φRSA1 was found to be integrated into at least three different strains of R. solanacearum , and the chromosomal integration site ( attB ) was identified as the 3′ portion of the arginine tRNA(CCG) gene. In the light of the φRSA1 gene arrangement, one possible prophage sequence previously detected on the chromosome of R. solanacearum strain GMI1000 was characterized as a φRSA1-related prophage (designated φRSX). φRSX was found to be integrated at the serine tRNA (GGA) gene as an att site, and its size was determined to be 40,713 bp. φRSX ORFs shared very high amino acid identity with their φRSA1 counterparts. The relationships and evolution of these P2-like phages are discussed.