Analysis of Ensifer aridi Mutants Affecting Regulation of Methionine, Trehalose, and Inositol Metabolisms Suggests a Role in Stress Adaptation and Symbiosis Development

Abstract

Isolated from desert, the nitrogen-fixing bacterium Ensifer aridi LMR001 is capable of survival under particularly harsh environmental conditions. To obtain insights in molecular mechanisms involved in stress adaptation, a recent study using RNAseq revealed that the RpoE2-mediated general stress response was activated under mild saline stress but appeared non-essential for the bacterium to thrive under stress and develop the symbiosis. Functions associated with the stress response included the metabolisms of trehalose, methionine, and inositol. To explore the roles of these metabolisms in stress adaptation and symbiosis development, and the possible regulatory mechanisms involved, mutants were generated notably in regulators and their transcriptions were studied in various mutant backgrounds. We found that mutations in regulatory genes nesR and sahR of the methionine cycle generating S-adenosylmethionine negatively impacted symbiosis, tolerance to salt, and motility in the presence of NaCl. When both regulators were mutated, an increased tolerance to detergent, oxidative, and acid stresses was found, suggesting a modification of the cell wall components which may explain these phenotypes and support a major role of the fine-tuning methylation for symbiosis and stress adaptation of the bacterium. In contrast, we also found that mutations in the predicted trehalose transport and utilization regulator ThuR and the trehalose phosphate phosphatase OtsB-encoding genes improved symbiosis and growth in liquid medium containing 0.4 M of NaCl of LMR001ΔotsB, suggesting that trehalose metabolism control and possibly trehalose-6 phosphate cellular status may be biotechnologically engineered for improved symbiosis under stress. Finally, transcriptional fusions of gfp to promoters of selected genes and expression studies in the various mutant backgrounds suggest complex regulatory interplay between inositol, methionine, and trehalose metabolic pathways.

Symbiosis under Abiotic Stress and Its Challenges

“…The salinity stress is closely related in soils from excessive natural accumulation or inorganic compound applications. The main salts are sodium-related compounds such as sodium chloride (NaCl), sodium nitrate (NaNO 3 ), sodium sulfate (Na 2 SO 4 ), sodium carbonates (NaHCO 3 , Na 2 CO 3 ), and sulfate-related compounds such as potassium sulfate (K 2 SO 4 ), calcium sulfate (CaSO 4 ), magnesium sulfate (MgSO 4 ) and other salts [3,38]; the symbionts like rhizobia helps to increase the tolerance through releasing metabolites or compounds such as plant-hormones (auxins, gibberellins, cytokinins), organic acids such as succinic acid and SA [38,[41][42][43]. In addition, EPS accumulation helps the symbiont survival and the communication between microbe-microbe and microbe-host.…”

“…For Ensifer, it has been reported similar to B. japonicum in the trehalose synthesis for increased salt tolerance. In addition, regulation of methionine and inositol metabolisms [43]. In the case of other rhizobia, it has been found that flavonoid-accumulation improves the interaction under salinity stress, and these flavonoids could be salt-depend or salt-independent induction; then, the host-interaction continuing under salt stress due to the nodulation is not flavonoid-depend in some nod genes like happen in R. tropici CIA899 [38].…”

“…These associations might have sufficient traits necessary to establish successful growth and N 2 fixation under the conditions prevailing in arid regions. Most of the reports indicated that those rhizobial strains isolated from alkaline conditions or alkali soils showed the best response under alkaline stress [45][46][47], and their mechanism to improve the tolerance during the symbiosis process is similar, or kind of related to salinity-stress reported up to date [41][42][43][45][46][47]. The existence of Rhizobium-legume symbioses, which can fix the appreciable amount of N 2 under arid conditions, is fascinating.…”

Bacterial Exopolysaccharides: From Production to Functional Features

“…In E. meliloti and E. aridi LMR001T, the MucR protein, which is controlled by the exopolysaccharide production regulatory system, has been demonstrated to be involved in the motility control network and flagellum biosynthesis [45,46] . It was also established that the gene regulator nesR, which encodes for the NesR and is an inductor of the ExpR family of type LuxR operon, regulates the genes involved in the production of the symbiotic exopolysaccharide EPS II, the regulation of motility, chemotaxis, and production of lowmolecular-weight succinoglycans.…”

“…It was also established that the gene regulator nesR, which encodes for the NesR and is an inductor of the ExpR family of type LuxR operon, regulates the genes involved in the production of the symbiotic exopolysaccharide EPS II, the regulation of motility, chemotaxis, and production of lowmolecular-weight succinoglycans. This latter represents another form of exopolysaccharide with significant symbiotic relevance [46][47][48][49][50][51] . It's noteworthy to mention that the control of motility occurs after the activity of ExpR regulator, which regulates two LuxR-like proteins called VisN and VisR [50,52,53] .…”