Abstract
Rhizobium tropici CIAT899 is a nodule-forming α-proteobacterium displaying intrinsic resistance to several abiotic stress conditions such as low pH and high temperatures, which are common in tropical environments. It is a good competitor for Phaseolus vulgaris (common bean) nodule occupancy at low pH values, however little is known about the genetic and physiological basis of the tolerance to acidic conditions. To identify genes in R. tropici involved in pH stress response we combined two different approaches: (1) A Tn5 mutant library of R. tropici CIAT899 was screened and 26 acid-sensitive mutants were identified. For 17 of these mutants, the transposon insertion sites could be identified. (2) We also studied the transcriptomes of cells grown under different pH conditions using RNA-Seq. RNA was extracted from cells grown for several generations in minimal medium at 6.8 or 4.5 (adapted cells). In addition, we acid-shocked cells pre-grown at pH 6.8 for 45 min at pH 4.5. Of the 6,289 protein-coding genes annotated in the genome of R. tropici CIAT 899, 383 were differentially expressed under acidic conditions (pH 4.5) vs. control condition (pH 6.8). Three hundred and fifty one genes were induced and 32 genes were repressed; only 11 genes were induced upon acid shock. The acid stress response of R. tropici CIAT899 is versatile: we found genes encoding response regulators and membrane transporters, enzymes involved in amino acid and carbohydrate metabolism and proton extrusion, in addition to several hypothetical genes. Our findings enhance our understanding of the core genes that are important during the acid stress response in R. tropici.
Highlights
The response to acidic stress conditions is understood best in enterobacteria, and Escherichia coli, S. enterica var
R. tropici CIAT899 was routinely grown in TY medium (Beringer, 1974) or the minimal medium described by Kingsley and Bohlool (Kingsley and Bohlool, 1992), adjusted to pH 6.8 [MM- buffered to pH 6.8 with 20 mM Hepes (N(2-Hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid))] or to pH 4.5 (MAM-buffered to pH 4.5 with 25 mM Homopipes (Homopiperazine-N,N′-bis-2-(ethanesulfonic acid))), Research Organics, Cleveland, OH, USA) at 30◦C
Mutants were grown for 5 days, selecting those that grew at neutral pH as well as the wildtype but did not grow at acidic pH or that showed a drastically reduced growth under the latter condition (Figure 1)
Summary
The response to acidic stress conditions is understood best in enterobacteria, and Escherichia coli, S. enterica var. Typhimurium, P. mirabilis and Y. enterocolitica (Castanie-Cornet et al, 1999; Kieboom and Abee, 2006; De Biase and Pennacchietti, 2012), all have effective systems to contend with acid stress. Well-known are the decarboxylation systems, which are composed of two components: a decarboxylase and an antiporter. Protons are consumed in the cytoplasm through the decarboxylation of specific amino acids and the corresponding antiporter exports the decarboxylation product and imports more of the required amino acid (Foster, 2004). The beststudied example is the glutamate decarboxylase (Gad) system depending upon the concerted action of glutamate decarboxylase (GadA/GadB) and of the glutamate/GABA antiporter, GadC (Foster, 1999; Audia et al, 2001; Lund et al, 2014).
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