The function of a lysogenic rhizobiophage in the symbiosis between Rhizobium and leguminous plants.

温带噬菌体PHIU通过在噬菌体(AttP)和细菌(AttB)位点之间的定点特异性重组来裂解豆科根瘤菌bv.trifolii 4S。在噬菌体DNA的6.0kb的EcoRI片段上鉴定了attP位点，并将其克隆到自杀载体pSUP202上的EcoRI位点上。重组质粒pCI6能以4.40×10~(-3)~(-3)的频率整合到菌株4S的染色体上。PCI6也能整合到对噬菌体PHIU感染不敏感的豆类根瘤菌K5的染色体上。用attP片段进行Southern杂交，发现4S和K5菌株染色体上pCI6的整合位置完全相同。以pSUP203为基础构建的融合表达载体pCINod1，可用于根瘤菌的基因转移。将含有pCINod1基因的大肠杆菌S17-1与菌株4S的共生缺陷突变体H1接合，接种白三叶草(Trifolium regens cv.Ladino)的根瘤。用透射电子显微镜观察结节的形态。根瘤菌通过侵染丝线侵入寄主植物细胞，形成类杆菌。从根瘤中分离到接合子，其中一株被命名为菌株CNH1。用attP进行Southern杂交发现，pCINod1和pCI6一样整合到了CNH1的染色体上。这些结果表明，整合到染色体上的nod基因在根瘤菌中发挥了有效的作用。

The temperate phage PHIU lysogenizes Rhizobium leguminosarum bv.trifolii 4S by site specific recombination between phage (attP) and bacterial (attB) sites. The attP site was identified on a 6.0 kb EcoRI fragment of the phage DNA and cloned into the EcoRI site on the suicide plasmid pSUP202. Plasmid pCI6, the resulting recombinant plasmid, could integrate into the chromosome of strain 4S at high frequency of 4.40 X 10^<-3>. The pCI6 could also integrate into the chromosome of R.leguminosarum bv.viciae K5 which was not sensitive to phage PHIU infection. Southern hybridization probed with attP fragment revealed that the integration site of pCI6 on the chromosome was completely identical between strains 4S and K5. It is suggested that pCI6 may be a useful chromosome integrative vector for gene transfer on Rhizobium.The fusion plasmid carrying the attP of phage PHIU and the nod-genes derived from R.leguminosarum bv.trifolii was constructed based on pSUP203 and designated as pCINodl. E.coli S17-1 harboring pCINod1 was conjugated with strain H1, a symbiotic defective mutant of strain 4S.The inoculation of the mating mixture resulted in nodule formation on the roots of white clover (Trifolium repens cv.Ladino). The nodules were observed by transmission electron microscopy. Rhizobium invaded into the host plant cells through infection threads and changed their form to bacteroids. Transconjugants were isolated from the nodules and one of them was designated as strain CNH1. Southern hybridization probed with attP revealed that pCINod1 integrated into the chromosome of strain CNH1 as same as pCI6. These results indicate that nod-genes integrated into the chromosome functioned efficiently in Rhizobium.