GENETIC AND PHYSIOLOGICAL CHARACTERIZATION OF A RHIZOBIUM-ETLI MUTANTSTRAIN UNABLE TO SYNTHESIZE POLY-BETA-HYDROXYBUTYRATE

Rhizobium etli accumulates poly-beta-hydroxybutyrate (PHB) in symbiosi s and in free life. PHB is a reserve material that serves as a carbon and/or electron sink when optimal growth conditions are not met. It ha s been suggested that in symbiosis PHB can prolong nitrogen fixation u ntil the last stages of seed development, but experiments to test this proposition have not been done until now. To address these questions in a direct way, we constructed an R. etli PHB-negative mutant by the insertion of an Omega-Km interposon within the PHB synthase structural gene (phaC). The identification and sequence of the R. etli phaC gene are also reported here. Physiological studies showed that the PHB-neg ative mutant strain was unable to synthesize PHB and excreted more lac tate, acetate, pyruvate, beta-hydroxybutyrate, fumarate, and malate th an the wild-type strain. The NAD(+)/NADH ratio in the mutant strain wa s lower than that in the parent strain. The oxidative capacity of the PHB-negative mutant was reduced. Accordingly, the ability to grow in m inimal medium supplemented with glucose or pyruvate was severely dimin ished in the mutant strain. We propose that in free life PHB synthesis sequesters reductive power, allowing the tricarboxylic acid cycle to proceed under conditions in which oxygen is a limiting factor. In symb iosis with Phaseolus vulgaris, the PHB-negative mutant induced nodules that prolonged the capacity to fix nitrogen.