The significance of adaptive pH tolerance responses in root nodule bacteria

The significance of adaptive pH tolerance responses in root nodule bacteria was investigated by examining whether root nodule bacteria show adaptive acid and alkaline tolerance responses and determining if acid- or alkaline-adapted root nodule bacteria are more tolerant of other stresses, namely ultra violet irradiation, high temperature, salinity and desiccation. For these experiments, Rhizobium meliloti strain WSM 419 and Bradyrhizobium lupini WU strain 425 were grown in buffered tryptone yeast (TY) broth at acidic, neutral and alkaline pH and then exposed to pH shock (pH 3.5 or pH 11.0), ultra violet light, heat shock (50°C), saline shock (1.2 M NaCl) and desiccation. The rate of cell growth in broth cultures was measured as the mean generation time (MGT) and the rate of cell death on exposure to stress was calculated as the decimal reduction time (D value). Strain WSM 419 and strain WU 425 both induced an acid tolerance response when grown at acidic pH before exposure to an acid shock at pH 3.5 in liquid media. Acid-grown WSM 419 (MGT 6.3 h) and WU 425 (MGT 23.4 h) died more slowly (D values 32 min and 86 min, respectively) than neutral-grown cells (D values 21 min and 40 min, respectively). An alkaline tolerance response was also induced in both WSM 419 and WU 425 when cultured at alkaline pH (MGT 4.8 h and 28.1 h, respectively) prior to exposure to extreme alkaline pH (11.0), with D values of 94 min and 10 min, respectively. The D values of neutral-grown WSM 419 and WU 425 when exposed to an alkaline shock were 46 min and 6.5 min, respectively. Unlike WU 425, WSM 419 grew better in alkaline conditions than in acidic conditions. Growth pH affects the ability of root-nodule bacteria to survive some environmental stresses other than pH. Acid-grown cells of WSM 419 (D value 57 min) and WU 425 (D value 9 min) were more tolerant of heat shock than neutral- and alkaline-grown cells. Furthermore, alkaline-grown cells of WSM 419, but not WU 425, were better able to withstand saline shock (D value 10 h) than acid- (D value 5.7 h) and neutral-grown cells (D value 5.6 h). Growth pH did not affect the tolerance of WSM 419 and WU 425 to UV light and desiccation. The effect of calcium on the expression of acid tolerance response of R. meliloti strain WSM 419 and its acid-sensitive mutant, TG 5-46, was assessed using cultures grown in the defined medium, JMM, at pH 7.0 and pH 6.0 containing 1.0 or 10 mM CaT+. WSM 419 generated an acid tolerance response in JMM and the concentration of calcium did not affect the expression of the tolerance response. The mutant, TG 5-46, did not show an adaptive acid tolerance response in JMM. The effect of growth of root nodule bacteria at an alkaline pH on their subsequent survival in alkaline soils was determined to examine the significance of the alkaline tolerance response in these bacteria on their ability to colonize soils. Cells of WSM 419, grown in TY broth at pH 7.0 and pH 9.0, were inoculated into Coogee soil at pH 7.0 (1:5 in H20), over burden top soil at pH 8.0 and over burden top soil limed with dolomite (15% w/w CaMgC03) at pH 9.1. Survival of the inoculum in the soils was estimated using the most probable number method based on the plant-infection test. Growth pH did not affect the survival of the bacteria in soil at pH 7.0 and pH 8.0. In soil at pH 9.1, there was no difference in survival of the bacteria up to 21 days after inoculation. Subsequently, alkaline-grown cells tended to increase in number but neutral-grown cells continued to decline. In conclusion, the rate of death of root nodule bacteria on exposure to stresses depends on the strain and the pH of culture medium. R. meliloti strain WSM 419 and B. lupini strain WU 425 showed adaptive acid and alkaline tolerance responses in laboratory media. Moreover, acid-adapted cells of both strains were more tolerant of heat shock than alkaline- and neutral-adapted cells. While alkaline-adapted WSM 419, but not WU 425, were more resistant to saline shock than acid- or neutral-adapted cells. The concentration of calcium in the growth medium did not effect the induction of acid tolerance response in WSM 419. The acid sensitive mutant, TG5-46, did not have an acid tolerance response. The ability of WSM 419 to induce an alkaline tolerance response does not appear to increase its capacity to survive and colonize alkaline soils