Leong S A, Ditta G S, Helinski D R
J Biol Chem. 1982 Aug 10;257(15):8724-30.
A symbiotically important gene system in rhizobial species is the heme biosynthetic pathway. A mutant having reduced levels of delta-aminolevulinic acid synthetase, the first unique enzyme in this pathway, was obtained in Rhizobium meliloti 102F34 by N-methyl-N'-nitro-N-nitrosoguanidine mutagenesis. Enzyme activity ranged from 3-13% of the wild type. Alfalfa plants inoculated with the Rhizobium synthetase mutant grew no better than uninoculated controls and formed only small white nodules which had no acetylene-reducing capacity. A cloned segment of Rhizobium genomic DNA capable of complementing this lesion was identified in a previously described gene bank from R. meliloti prepared with the broad host range plasmid cloning vector pRK290 (Ditta. G., Stanfield, S., Corbin, D., and Helinski, D. R. (1980) Proc. Natl. Acad. Sci. U. S. A. 77, 7347-7351). Symbiotic effectiveness could be restored in the mutant by supplementing plants with exogenous delta-aminolevulinic acid or by introducing into the mutant the wild type delta-aminolevulinic acid synthetase gene cloned into the pRK290 plasmid. The recombinant plasmid carrying the synthetase gene was also able to weakly complement an Escherichia coli hemA mutant. Transposon mutagenesis of this plasmid with Tn5 further localized the delta-aminolevulinic acid synthetase gene to a 1.4-kilobase region contained within a 4-6-kilobase Bam HI fragment. Full complementation of hemA was observed when this fragment was subcloned under E. coli trp and Tet promoter control on a pBR322 replicon. A temperature-sensitive mutant of this latter plasmid, which was unable to complement hemA at high temperature, produced enzyme having temperature-sensitive synthetase activity in vitro. This result confirmed that the cloned complementing DNA contained the structural gene for delta-aminolevulinic acid synthetase and not a biosynthetic regulatory gene.
根瘤菌属物种中一个具有共生重要性的基因系统是血红素生物合成途径。通过N-甲基-N'-硝基-N-亚硝基胍诱变，在苜蓿中华根瘤菌102F34中获得了一种突变体，该突变体中δ-氨基乙酰丙酸合成酶（此途径中的首个独特酶）的水平降低。酶活性为野生型的3%-13%。用根瘤菌合成酶突变体接种的苜蓿植株生长情况并不比未接种的对照更好，且仅形成了没有乙炔还原能力的小白结节。在先前用广宿主范围质粒克隆载体pRK290构建的苜蓿中华根瘤菌基因文库中，鉴定出了一个能够互补此缺陷的根瘤菌基因组DNA克隆片段（迪塔，G.，斯坦菲尔德，S.，科尔宾，D.，和赫林斯基，D.R.（1980年）美国国家科学院院刊77，7347-7351）。通过给植株补充外源δ-氨基乙酰丙酸，或通过将克隆到pRK290质粒中的野生型δ-氨基乙酰丙酸合成酶基因导入突变体，可使突变体恢复共生有效性。携带合成酶基因的重组质粒也能够微弱地互补大肠杆菌hemA突变体。用Tn5对该质粒进行转座子诱变，进一步将δ-氨基乙酰丙酸合成酶基因定位到一个4-6千碱基的Bam HI片段内包含的1.4千碱基区域。当该片段在pBR322复制子上的大肠杆菌trp和Tet启动子控制下进行亚克隆时，观察到了hemA的完全互补。后一种质粒的一个温度敏感突变体，在高温下无法互补hemA，在体外产生了具有温度敏感合成酶活性的酶。这一结果证实，克隆的互补DNA包含δ-氨基乙酰丙酸合成酶的结构基因，而非生物合成调节基因。
