Possible involvement of dif site and the adjacent trbP gene of host Xanthomonas campestris in infection of filamentous phage phiLf

• Main Authors: Zih-Rong Chen, 陳姿蓉
• Other Authors: 翁淑芬
• Format: Others
• Language: zh-TW
• Online Access: http://ndltd.ncl.edu.tw/handle/77707772896136050907

碩士 === 國立中興大學 === 分子生物學研究所 === 97 === Filamentous phage phiLf specifically infects Xanthomonas campestris pv. campestris (Xcc). It has a single-stranded circular DNA genome of 6,009 nucleotides, uses replicative form (RF) DNA as the replication intermediate, and performs a non-lytic life cycle. The phiLf viral strand encodes nine genes organized into the order gII-gX-gV-gVII-gIX-gVIII-gIII-gVI-gI, with gII, gX and gV shown to be required for replication, gVII, gIX, gVIII, gIII, and gVI encoding coat proteins, and gI responsible for assembly and morphogenesis. On the complementary strand, three open reading frames, orf137, orf155, and orf102, have been assigned. A 4,445-bp phiLf-homologous region (FHR) is present on the chromosome of Xcc strain Xc17, next to the dif (deletion induced filamentation) site that is the end of chromosome replication. This region has been shown to facilitate 1) site specific integration between dif site (attB) and the dif-homologous attP site in phiLf, and 2) homologous recombination via the FHR region outside dif. In this study, analysis of the sequences available in the database revealed that there is 1) no FHR on P20H chromosome, 2) at least one dif homologous sequence in each of the filamentous phages as well as their Xanthomonas hosts, and 3) a gene homologous to trbP, involved in pilus acetylation in E. coli, next to dif in each of the Xanthomonas strains as well as filamentous phages phiXo2 (infecting X. oryzae pv. oryzae) and Cf1c (infecting X. axonopodis pv. citri), but not the genome of phiLf, phiXo1(infecting X. oryzae pv. oryzae), phiXv and phiXv2 (both infecting X. axonopodis pv. vesicatoria). Molecular genetic study indicated that 1) mutation in the trbP gene of P20H causes no effects on phiLf infection and propagation, 2) deletion of dif causes filamentation of P20H without affecting cell growth, dislike the dif mutant of E. coli which grew slower than the wild-type cells, 3) dif and trbP double mutant grew at the same rate as P20H, and 4) dif mutant is capable of producing 5 times higher titer of phiLf than P20H. Expression of orf137 and orf155 of phiLf was also studied, and the results indicated that while RNA transcripts were detectable by RT-PCR, no proteins were detected in Western blotting experiments, suggesting that either no proteins were expressed or the levels expressed were too low to be detected.