Further Investigation of the Roles of Poly-3-Hydroxybutyrate (PHB) and Glycogen in Sinorhizobium mellioti-Medicago SP. Symbiosis

Abstract

Poly-3-hydroxybutyrate (PHB) and glycogen are major carbon storage compounds in Sinorhizobium meliloti. The roles of PHB and glycogen in rhizobia-legume symbiosis are not fully understood (1, 2, 3). Bacteroids with determinate nodules often accumulate high levels of PHB (up to 70% dry weight) (1). In contrast, bacteroids within indeterminate nodules do not accumulate PHB. In the symbiosis between S. meliloti and alfalfa, it has been reported that phbC mutants form bacteroids capable of fixing nitrogen as efficiently as the wild type (4, 5, 6), but are less competitive than wild type strain (5). There are two glycogen synthase-encoding genes in S. meliloti, glgA1 in a cluster of other glycogen synthesis pathway genes on the chromosome, and glgA2 on megaplasmid pSymb. Recently, it has been shown in Rhizobium tropici with Phaseolus vulgaris that there may be a link between glycogen synthase deficiency, decreased exopolysaccharide, and increased symbiotic performance (2). However, the reason for the increased symbiotic efficiency of the glgA mutant is uncertain (3). To determine the roles these compounds may play in the symbiotic process and in the overall physiology of the organism in the free-living and bacteroid states, mutants unable to synthesize PHB and/or glycogen were constructed. A glgA1 mutation was constructed by in-frame deletion, preserving the expression of the downstream pgm gene, while a glgA2 mutation was constructed by disruption of the gene with the Sp omega cassette (7). A pre-existing Tn5-generated mutation of the PHB synthase encoding gene phbC (8) was combined with glgA1 and glgA2 mutations to make all combinations of double mutants, and the triple mutant (Table 1). PHB was not detectable in free-living cells of any mutant containing the phbC mutation; glycogen was not detectable in any of the mutants containing the glgA1 mutation. The production of PHB decreased significantly in the glgA1 mutant (Rm11479), and the glgA double mutant (Rm11482). The production of glycogen increased significantly in the phbC mutant (Rm11105) in high carbon ratio media. Exopolysaccharide (EPS) was not detected in any of the mutants containing the phbC mutation, while the glgA double mutant (Rm11482) produced much more EPS in MOPS medium compared to the wild type (Rm1021), glgA1