Biochemical and symbiotic properties of histidine‐requiring mutants of Rhizobium leguminosarum biovar trifolii

Abstract

A .S . Y A DA V, R .K . V A SH IS H AT , L . D. KU Y KE ND A LL AN D F. M. H AS HE M . 1998. A perturbation of the histidine biosynthetic pathway in legume microsymbionts can abolish their symbiotic competence. Twenty-one histidine-requiring (His − ) mutants were isolated from berseem clover-nodulating, symbiotically-competent (Nod + , Fix + ) Rhizobium leguminosarum bv. 'trifolii' strain RTH 48 Sm r by N-methyl-N?-nitro-Nnitrosoguanidine (MNNG) mutagenesis followed by enrichment. These mutants were analysed for their biochemical defect and the corresponding effect, if any, on their symbiotic abilities. Cross-feeding, supplementation and enzymatic studies identified three types of mutants. Group 1 mutants, His-2 and His-12, grew with histidine supplementation but not with the addition of either L-histidinol or L-histidinol phosphate to the medium ; they lacked histidinol dehydrogenase (EC 1.1.1.23) activity and consequently formed only ineffective, or 'non-fixing' nodules. Group 2 mutant, His-17, grew when supplemented with either L-histidinol or L-histidine, had low histidinol phosphate phosphatase (EC 3.1.3.15) activity (37% of wild-type), and consequently failed to nodulate berseem clover. Group 3, the remaining 18 mutants, grew when supplemented with L-histidinol phosphate, L-histidinol or histidine, and did not nodulate. Typically, reversion rates were between 10 −7 and 10 −8 . Defects in early steps of the pathway abolished nodulating ability, whereas lesions in the last step did not. The last step, however, was required for symbiotic nitrogen fixation. It is hypothesized that histidine may be supplied by the host in sufficient quantity for nodulation by histidinol dehydrogenase mutants to occur, whereas the amount provided in the nodule may be insufficient to support bacteroid development and nitrogen fixation.

Genome-scale metabolic modelling of lifestyle changes in Rhizobium leguminosarum

“…In addition, mutants of R. leguminosarum bv. trifolii lacking histidinol dehydrogenase activity formed ineffective nodules on clover (77). To investigate the requirement for histidine biosynthesis, we compared the amino acid composition of the Nif and Fix proteins, which are highly expressed in bacteroids, with the overall amino acid composition of the Rlv3841 proteome (Table S7).…”

Auxotrophy in rhizobia revisited

“…This step has the additional advantage of permitting the auxotrophs to exhaust their pools of required growth factors. For preparing the hypertonic MM, either 0.6 M sucrose 63 or 0.5 M sorbitol 64 or a combination of both, with reduced concentrations 65 have been used. By introducing these modifi cations, it has been possible to screen populations at densities up to 5x10 8 cells ml -1 i.e.…”

“…The method is based on the fact that the killing power of carbenicillin is amplifi ed by the addition of lysozyme. In rhizobia, a combination of ampicillin or penicillin with lysozyme 55,56,[63][64][65] has been useful for the enrichment. For the isolation of auxotrophs, the wild type strain has to be either penicillin or ampicillin sensitive [63][64][65] .…”

“…In rhizobia, a combination of ampicillin or penicillin with lysozyme 55,56,[63][64][65] has been useful for the enrichment. For the isolation of auxotrophs, the wild type strain has to be either penicillin or ampicillin sensitive [63][64][65] . The aim of choosing an ampicillin or penicillin-sensitive strain for the isolation of auxotrophs is to ensure killing of prototrophic survivors after mutagenesis and to curtail the duration of ampicillin/penicillin treatment during enrichment of auxotrophs.…”

“…These results indicated that both mutants are defective in the purine biosynthetic pathway and that metabolic fl ow through this pathway is essential for nodule morphogenesis. Yadav et al (1998) 65 analyzed his -mutants for their biochemical and symbiotic defects in R. leguminosarum bv. trifolii and it was suggested that a perturbation of the histidine biosynthetic pathway can abolish the symbiotic competence of Rhizobium.…”