Stress response regulators identified through genome-wide transcriptome analysis of the (p) ppGpp-dependent response in Rhizobium etli

Background: The alarmone (p) ppGpp mediates a global reprogramming of gene expression upon nutrient limitation and other stresses to cope with these unfavorable conditions. Synthesis of (p) ppGpp is, in most bacteria, controlled by RelA/SpoT (Rsh) proteins. The role of (p) ppGpp has been characterized primarily in Escherichia coli and several Gram-positive bacteria. Here, we report the first in-depth analysis of the (p) ppGpp-regulon in an alpha-proteobacterium using a high-resolution tiling array to better understand the pleiotropic stress phenotype of a relA/rsh mutant. Results: We compared gene expression of the Rhizobium etli wild type and rsh (previously rel) mutant during exponential and stationary phase, identifying numerous (p) ppGpp targets, including small non-coding RNAs. The majority of the 834 (p) ppGpp-dependent genes were detected during stationary phase. Unexpectedly, 223 genes were expressed (p) ppGpp-dependently during early exponential phase, indicating the hitherto unrecognized importance of (p) ppGpp during active growth. Furthermore, we identified two (p) ppGpp-dependent key regulators for survival during heat and oxidative stress and one regulator putatively involved in metabolic adaptation, namely extracytoplasmic function sigma factor EcfG2/PF00052, transcription factor CH00371, and serine protein kinase PrkA. Conclusions: The regulatory role of (p) ppGpp in R. etli stress adaptation is far-reaching in redirecting gene expression during all growth phases. Genome-wide transcriptome analysis of a strain deficient in a global regulator, and exhibiting a pleiotropic phenotype, enables the identification of more specific regulators that control genes associated with a subset of stress phenotypes. This work is an important step toward a full understanding of the regulatory network underlying stress responses in alpha-proteobacteria.