Ensifer aridi LMR001T Symbiosis and Tolerance to Stress Do Not Require the Alternative Sigma Factor RpoE2

Abstract

The recently proposed species Ensifer aridi represents an interesting model to study adaptive mechanisms explaining its maintenance under stressful pedo-climatic conditions. To get insights into functions associated with hyperosmotic stress adaptation in E. aridi, we first performed RNAseq profiling of cells grown under sub-lethal stresses applied by permeating (NaCl) and non-permeating (PEG8000) solutes that were compared to a transcriptome from unstressed bacteria. Then an a priori approach, consisting of targeted mutagenesis of the gene encoding alternative sigma factor (rpoE2), involved in the General Stress Response combined with phenotyping and promoter gfp fusion-based reporter assays of selected genes was carried out to examine the involvement of rpoE2 in symbiosis and stress response. The majority of motility and chemotaxis genes were repressed by both stresses. Results also suggest accumulation of compatible solute trehalose under stress and other metabolisms such as inositol catabolism or the methionine cycling-generating S-adenosyl methionine appears strongly induced notably under salt stress. Interestingly, many functions regulated by salt were shown to favor competitiveness for nodulation in other rhizobia, supporting a role of stress genes for proper symbiosis’ development and functioning. However, despite activation of the general stress response and identification of several genes possibly under its control, our data suggest that rpoE2 was not essential for stress tolerance and symbiosis’ development, indicating that E. aridi possesses alternative regulatory mechanisms to adapt and respond to stressful environments.

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

“…However, the mutation of the rpoE2 gene suggested that the response was complex and that alternative regulatory mechanisms may be recruited by the bacterium to cope with various stresses. Among genes that were found differentially regulated, a strong repression of motility genes could be identified and validated phenotypically by swimming assays [15]. We also found that salt stress altered regulation for a number of genes involved in the methionine cycle generating S-adenosylmethionine (SAM), the universal methyl donor, in inositol transport and catabolism or in trehalose de novo biosynthesis and transport [15].…”

“…Seeds of Vachellia tortilis subsp. raddiana and endemic Moroccan Vachellia gummifera were surface sterilized, germinated, and cultivated, as described previously [15]. Briefly, after germination (5 days in the dark), young seedlings were transferred in pots containing sterilized sand and Ensifer aridi strains were inoculated using 1 mL of washed cells at an optical density (600 nm) ≈ 0.7 corresponding to exponential growth phase.…”

“…Microorganisms 2022, 10, 298 2 of 19 Among the most rapid methods to identify genes and functions associated to a particular stress, transcriptomics represents a method of choice. Genome-wide transcriptional responses resulting from abiotic stresses have been studied using microarray technology or NGS-based RNAseq in several rhizobial genera including, for example, Bradyrhizobium [7], Rhizobium [8,9], Mesorhizobium [10][11][12][13][14], or Sinorhizobium (Ensifer) [15][16][17][18][19][20]. The bacterial response to an osmotic upshift is complex and varies according to the strain and species.…”

“…According to a transcriptome analysis performed in E. aridi to explore the response of this bacterium to hyperosmotic stress, a number of genes were found differentially regulated, including the alternative extra-cytoplasmic function sigma factor RpoE2 which suggested activation of the general stress response (GSR) [15]. However, the mutation of the rpoE2 gene suggested that the response was complex and that alternative regulatory mechanisms may be recruited by the bacterium to cope with various stresses.…”

“…Among genes that were found differentially regulated, a strong repression of motility genes could be identified and validated phenotypically by swimming assays [15]. We also found that salt stress altered regulation for a number of genes involved in the methionine cycle generating S-adenosylmethionine (SAM), the universal methyl donor, in inositol transport and catabolism or in trehalose de novo biosynthesis and transport [15]. To further explore the possible roles that these metabolisms have in stress adaptation and symbiosis of E. aridi, deletion mutants of regulatory genes involved in the methionine cycle (nesR, sahR), the trehalose transport and utilization (thuR), the inositol catabolism (iolR), or in the trehalose denovo biosynthesis (otsB) were generated and phenotyped.…”