Pleiotropic effects of a rel mutation on stress survival of Rhizobium etli CNPAF512

Abstract

The rel gene of Rhizobium etli (relRet), the nodulating endosymbiont of the common bean plant, determines the cellular level of the alarmone (p)ppGpp and was previously shown to affect free-living growth and symbiosis. Here, we demonstrate its role in cellular adaptation and survival in response to various stresses. Results: Growth of the R. etli relRet mutant was strongly reduced or abolished in the presence of elevated NaCl levels or at 37°C, compared to the wild type. In addition, depending on the cell density, decreased survival of exponentially growing or stationary phase relRet mutant cells was obtained after H2O2, heat or NaCl shock compared to the wild-type strain. Survival of unstressed stationary phase cultures was differentially affected depending on the growth medium used. Colony forming units (CFU) of relRet mutant cultures continuously decreased in minimal medium supplemented with succinate, whereas wild-type cultures stabilised at higher CFU-levels. Microscopic examination of stationary phase cells indicated that the relRet mutant was unable to reach the typical coccoid morphology of the wild type in stationary phase cultures. Assessment of stress resistance of reisolated bacteroids showed increased sensitivity of the relRet mutant to H2O2 and a slightly increased resistance to elevated temperature(45°C) or NaCl shock, compared to wild-type bacteroids. Conclusion: The relRet gene is an important factor in regulating rhizobial physiology, during free-living growth as well as in symbiotic conditions. Additionally, differential responses to several stresses applied to bacteroids and free-living exponential or stationary phase cells point to essential physiological differences between the different states.