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.
Highlights
Having reduced levelsof 8-aminolevulinic acid synthe- aminolevulinic acid, labeledheme is produced; the plant fractase,the first unique enzyme inthispathway,was tion is unable to convert this compound into heme (5, 7 ) .In obtained inRhizobium meliloti 102F34 by N-methyl-N’- addition,theactivity of thehemebiosynthetic enzyme 6
Enzyme activ- aminolevulinic acid synthetase, which catalyzes the condenity ranged from 3-13% of the wild type
A cloned segment of Rhizobium genomic DNA capable of complementing this lesion was identified in a previously described genebank fromR. meliloti preacid, dramatically increases in Rhizobium japonicumbacteroids during nodule development and this increase parallels that of total heme in the nodule [6]
Summary
Isolation of Heme Mutants-We observed that theE . coli hemA- mutant S905 grew poorly, yielding tiny colonies in M9 minimal medium with succinate as a carbon source and supplemented with low concentrations of 8-aminolevulinic acid. Coli hemA- mutant S905 grew poorly, yielding tiny colonies in M9 minimal medium with succinate as a carbon source and supplemented with low concentrations of 8-aminolevulinic acid. E first mutation, A34, was generated at 200 p g / m l mutagen, Activities shown are: Rm102F34, 0; Rm102F34 (pRK290), D, while the second, A36, was obtained at 50pg/ml Both mutants Rm102F34 (pRK290.790),A;a, both A34, 0, and A34 (pRK290), were detected on plates containing 0.05 pg/ml S-aminolevu- and A34 (pRK290.790),A. linic acid and 0.5 mg/ml casamino acids and were subsequently found to require 8-aminolevulinic acid in a varietyof media including RMin medium supplementedwith either succinate or mannitol as a carbon source and the succinatecontaining minimal medium described by Kiss et al [28].
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