Heme biosynthesis in Rhizobium. Identification of a cloned gene coding for delta-aminolevulinic acid synthetase from Rhizobium meliloti

Abstract

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.