Improved Soybean Root Association of N-Starved Bradyrhizobium japonicum

Abstract

In this study, we addressed the effects of N limitation in Bradyrhizobium japonicum for its association with soybean roots. The wild-type strain LP 3001 grew for six generations with a growth rate of 1.2 day ؊1 in a minimal medium with 28 mM mannitol as the carbon source and with the N source [(NH 4 ) 2 SO 4 ] limited to only 20 M. Under these conditions, the glutamine synthetase (GS) activity was five to six times higher than in similar cultures grown with 1 or 0.1 mM (NH 4 ) 2 SO 4 . The NtrBC-inducible GSII form of this enzyme accounted for 60% of the specific activity in N-starved rhizobia, being negligible in the other two cultures. The exopolysaccharide (EPS) and capsular polysaccharide (CPS) contents relative to cell protein were significantly higher in the N-starved cultures, but on the other hand, the poly-3-hydroxybutyrate level did not rise in comparison with N-sufficient cultures. In agreement with the accumulation of CPS in N-starved cultures, soybean lectin (SBL) binding as well as stimulation of rhizobial adsorption to soybean roots by SBL pretreatment were higher. The last effect was evident only in cultures that had not entered stationary phase. We also studied nodC gene induction in relation to N starvation. In the chromosomal nodC::lacZ fusion Bj110-573, nodC gene expression was induced by genistein 2.7-fold more in N-starved young cultures than in nonstarved ones. In stationary-phase cultures, nodC gene expression was similarly induced in N-limited cultures, but induction was negligible in cultures limited by another nutrient. Nodulation profiles obtained with strain LP 3001 grown under N starvation indicated that these cultures nodulated faster. In addition, as culture age increased, the nodulation efficiency decreased for two reasons: fewer nodules were formed, and nodulation was delayed. However, their relative importance was different according to the nutrient condition: in older cultures the overall decrease in the number of nodules was the main effect in N-starved cultures, whereas a delay in nodulation was more responsible for a loss in efficiency of N-sufficient cultures. Competition for nodulation was studied with young cultures of two wild-type strains differing only in their antibiotic resistance, the N-starved cultures being the most competitive.

Regulation of Polyhydroxybutyrate Synthesis in the Soil Bacterium Bradyrhizobium diazoefficiens

“…1H). These results, in agreement with previous investigations (3,27,40), indicated that the microoxia and the stationary phase of oxic cultures were permissive conditions for PHB synthesis while the exponential phase of the oxic cultures was nonpermissive. In addition, the oxygen-limiting bacteroid state inside root nodules is also a permissive condition (3).…”

“…These results indicate that PhaR exerts its activity under conditions of oxygen limitation, in which accumulation of the substrates for PHB synthesis, similarly to reducing power and acetyl-CoA levels, may take place. In addition, the lack of PHB granules in our phaR mutant was accompanied by a higher production of EPS, a correlation previously observed in E. meliloti (16), R. etli (15), and N-limited B. diazoefficiens wild-type cultures (27,40). However, a plausible explanation of how phaR also controls EPS synthesis is lacking.…”

“…Because late-stationary-phase cultures are poor competitors (40) and we wanted to compare permissive and nonpermissive conditions, we prepared the inocula from oxic cultures either in the exponential phase (nonpermissive) or in the middle stationary phase (permissive). As a result, we observed in two independent experiments that under nonpermissive conditions the levels of competitiveness of the two strains were similar, while under permissive conditions the phaR mutant was more competitive than the wild type, occupying 74% of the nodules (Fig.…”

Exploiting inter-organismal chemical communication for improved inoculants

“…When B. japonicum cells are cultured in N-starved media, the production of exopolysaccharides, capsular polysaccharides and the expression of nodC:lacZ is reduced, which results in the production of reduced nodule number by older cultures, or in delayed nodule formation in younger culture (Lopez-Garcia et al 2001 greenhouse experiments demonstrated that N-starvation during culture of an effective rhizobia strain did not enhance their competitiveness against an established and less competitive strain (Lopez-Garcia et al 2002), suggesting that number and position are more important than genetic/physiological aspects of competitiveness for inoculated strains. Using nod gene expression to indicate Nod factor production, Loh and Lopez-Garcia's experiments (Loh et al 2001;Lopez-Garcia et al 2001) demonstrated that although Nod factor production by a culture increased as cell density increased, production per cell decreased.…”

Growth and Nitrogen Fixation of DifferentMedicago Sativa-Sinorhizobium MelilotiAssociations Under Conditions of Mineral Elements Shortage

“…It is well known that legumes possess a systemic regulatory control able to detect the presence of combined nitrogen in the rhizosphere and block nodulation in response. Some publications (7,15,22), indicated that rhizobia nitrogen starvation has a positive influence on the symbiosis, leading to high N/C ratio and stimulating synthesis of surface polysaccharide, nod gene induction (19), all of which resulted in increased nodulation efficiency and competitiveness. Under carbon limitation, there is induction of a nonspecific attachment mechanism involving neither host plant lectins nor nod genes (31).…”