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標題: Xanthomonas campestris 之 dif 序列以及其緊鄰的 trbP 基因與線狀噬菌體 phiLf 感染的關係
Possible involvement of dif site and the adjacent trbP gene of host Xanthomonas campestris in infection of filamentous phage phiLf
作者: 陳姿蓉
Chen, Zih-Rong
關鍵字: filamentous phage;線狀噬菌體;dif site;attP;trbP;複製終點;基因間區
出版社: 分子生物學研究所
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phiLf 為專一感染十字花科黑腐病菌之線狀噬菌體,其基因體為 6,009 個核苷酸組成的環狀單股DNA。 感染宿主後,以 RF DNA 為複製中間產物,不會造成宿主細胞溶裂或死亡。其 viral strand 上具有九個基因,基因排列順序為 gII-gX-gV-gVII-gIX-gVIII-gIII-gVI-gI。 gII、gX 和 gV 與 DNA 複製有關,gVII、gIX、gVIII、gIII 和 gVI 主導噬菌體之外套蛋白,而 gI 產物則負責噬菌體之裝配與釋放。 互補股上具有三個 open reading frames,分別命名為 orf137、orf155 和 orf102。
緊鄰 Xc17 染色體複製終點 dif (deletion induced filamentation) site 位置,有一段 4,445 bp 之 phiLf-homologous region (FHR)。 此區域之 DNA 序列可協助 (1) 宿主染色體上的 dif site (attB) 與 phiLf 上含有 dif 同源序列的 attP site 進行 site-specific integration,並且 (2) 藉由 dif site 外圍之FHR 區域進行 homologous recombination。
本研究利用 NCBI database 分析顯示:(1) P20H 染色體上不具有 FHR,(2) 任一線狀噬菌體及其 Xanthomonas 宿主至少具有一個 dif 同源性序列,(3) Xanthomonas 染色體緊鄰 dif site具有類似 E. coli 之 pilus acetylation 功能的 trbP 基因,而在 Xanthomonas 噬菌體 phiXo2 (感染 X. oryzae pv. oryzae) 和 Cf1c (感染 X. axonopodis pv. citri) 也發現類似 trbP 基因,但 phiLf、phiXo1 (感染 X. oryzae pv. oryzae) 以及 phiXv 和 phiXv2 (感染 X. axonopodis pv. vesicatoria) 並不具 trbP。 分子遺傳研究指出 (1) 破壞 P20H 染色體上的 trbP 基因並不會影響 phiLf 的感染與複製,(2) 刪除 P20H dif site 導致菌體呈現線形,但並不影響細菌生長,此與 E.coli 不同,其 dif 突變株之生長速率變慢,(3) 同時刪除 dif 與 trbP 的雙重突變株之生長速率與 P20H 相同,(4) phiLf 感染 dif 突變株比感染 P20H 後,釋出之噬菌體增加 5 倍。
此外,經 RT-PCR 結果顯示 phiLf 互補股上之 orf137 和 orf155 均可轉錄表現 RNA。 但是卻無法利用 Western blotting 技術以抗體偵測到兩者之蛋白質表現。 推測此二基因無表現蛋白質,或蛋白質之表現量極低,無法以 Western blotting 偵測獲得訊號。

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.
其他識別: U0005-0402200921484100
Appears in Collections:分子生物學研究所

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