Boron nutrition affects growth, adaptation to stressful environments, and exopolysaccharide synthesis of Ensifer meliloti

Abstract

Boron (B) is a micronutrient required for the development of symbiotic legume nodules but is apparently not essential for rhizobia, although it has been related to bacteria cell surface formation and adaptation to stress responses. In this paper, we explore whether B nutrition can influence the growth of several strains of Ensifer meliloti (formerly Sinorhizobium meliloti), including mutants defective in exopolysaccharide (EPS) production. All the strains were able to grow in B-deficient (-B) media, except for the non-nodulating Sm11605 mutant, which was unable to produce EPS. The addition of boric acid before the exponential phase of growth (in all strains, including Sm11605) showed that 50 μM H3BO3 (50 B) resulted in the highest growth rate. Moreover, the addition of B increased the tolerance of E. meliloti to salinity (up to 0.5 mM NaCl), to the cell surface stressor SDS (up to 100 μg mL−1 SDS), and to 0.5 mM H2O2. Furthermore, 50 B increased survival rate after 90 min of exposure to 1 mM H2O2, which can be crucial to overcoming transient oxidative burst due to ROS production by root legumes during the initial phases of infection. Given that rhizobia exopolysaccharides (EPSs) are involved in the responses to stress and in the adaptation to and endophytic environment, we hypothesized that B affects EPS production and it can be crucial for growth and survival under stressful environments. Indeed, the amount of EPS extracted from –B cultures was 4–5 times reduced. Gene expression analysis by using qRT-PCR revealed that the genes mucR (a positive regulator of EPS-I but negative regulator of EPS-II synthesis), and emmA (a negative regulator of EPS-I), were overexpressed in –B cells. Meanwhile, expG (a positive regulator of EPS-II), and the genes exoY and expA1 (involved in the synthesis of EPS-I and EPS-II, respectively) were downregulated. These results are consistent with a reduction of both EPS-I and EPS-II synthesis under B deficiency supporting our hypothesis.