Genetic and Metabolic Engineering of Rhizobium Etli to Modify (Enhance) Nitrogen Fixation in the Symbiosis with Phaseolus Vulgaris

Abstract

Strains of Rhizobium etli, Rhizobium meliloti, and Rhizobium tropici decreased their capacity to grow after successive subcultures in minimal medium, with a pattern characteristic for each species. During the growth of R. etli CE3 in minimal medium (MM), a fermentation-like response was apparent: the O₂ content was reduced and, simultaneously, organic acids and amino acids were excreted and poly-ß-hydroxybutyrate (PHB) was accumulated. Some of the organic acids excreted into the medium were tricarboxylic acid (TCA) cycle intermediates, and, concomitantly, the activities of several TCA cycle and auxiliary enzymes decreased substantially or became undetectable. It is proposed that the fermentative state in Rhizobium species is triggered by a cell density signal that results in the regulation of some of the enzymes responsible for the flux of carbon through the TCA cycle and that this in turn determines how much carbon is available for the synthesis and accumulation of PHB. The fermentative state of free-living Rhizobium species may be closely related to the metabolism that these bacteria express during symbiosis (Encarnación et al., 1995).