Trehalose Biosynthesis in Rhizobium leguminosarum bv. trifolii and Its Role in Desiccation Tolerance

Abstract

Rhizobium leguminosarum bv. trifolii forms nitrogen-fixing root nodules on the pasture legume Trifolium repens, and T. repens seed is often coated with a compatible R. leguminosarum bv. trifolii strain prior to sowing. However, significant losses in bacterial viability occur during the seed-coating process and during storage of the coated seeds, most likely due to desiccation stress. The disaccharide trehalose is known to function as an osmoprotectant, and trehalose accumulation due to de novo biosynthesis is a common response to desiccation stress in bacteria. In this study we investigated the role of endogenous trehalose synthesis in desiccation tolerance in R. leguminosarum bv. trifolii strain NZP561. Strain NZP561 accumulated trehalose as it entered the stationary phase due to the combined actions of the TreYZ and OtsAB pathways. Mutants deficient in either pathway showed near-wild-type levels of trehalose accumulation, but double otsA treY mutants failed to accumulate any trehalose. The double mutants were more sensitive to the effects of drying, and their survival was impaired compared to that of the wild type when glass beads were coated with the organisms and stored at relative humidities of 5 and 32%. The otsA treY mutants were also less competitive for nodule occupancy. Gene expression studies showed that the otsA and treY genes were expressed constitutively and that expression was not influenced by the growth phase, suggesting that trehalose accumulation is controlled at the posttranscriptional level or by control of trehalose breakdown rates. Our results indicate that accumulated trehalose plays an important role in protecting R. leguminosarum bv. trifolii cells against desiccation stress and against stress encountered during nodulation.Rhizobium leguminosarum bv. trifolii forms nitrogen-fixing root nodules on the pasture legume Trifolium repens (white clover). Inoculation of legumes is an efficient and convenient way of introducing rhizobia into soil and subsequently into the rhizosphere of legumes. The seeds of many clover species (including white clover) are coated with compatible R. leguminosarum bv. trifolii strains prior to sowing. One of the main reasons for the ineffectiveness of legume inoculants in the field is the rapid death of rhizobia due to desiccation (12, 55). Significant losses in inoculant viability occur during the seedcoating process, which involves a drying stage, during storage of the coated seeds and after introduction into the field environment. These losses in bacterial viability are likely to be due mainly to desiccation stress.Bacteria accumulate osmoprotective compounds, referred to as compatible solutes or osmolytes, in response to osmotic or desiccation stress. Osmolytes can be obtained by uptake from the environment (exogenous) or through de novo biosynthesis (endogenous). De novo biosynthesis of trehalose, a nonreducing (␣,␣-1,1)-glucose disaccharide, is a common response to desiccation and osmotic stress in many bacteria (11,35,57). Trehalose has been shown to protec...

The Physiological Mechanisms of Desiccation Tolerance in Rhizobia

“…At the same time, no intracellular trehalose was detected in the otsA, treY mutant and cell recovery was 63%. Twenty four hours after drying, recovery of viable cells was 39% for NZP561 and < 0.001% for the double mutant, clearly indicating the role of trehalose in improved desiccation tolerance (McIntyre et al, 2007).…”

“…Trehalose is a non-reducing disaccharide which is believed to act as a compatible solute by maintaining membrane structure through the formation of biological glasses which protect proteins and other macromolecules from denaturation during desiccation (Crowe et al, 1984). The intracellular accumulation of trehalose in rhizobia has been observed to significantly improve desiccation tolerance (Streeter, 2003;McIntyre et al, 2007;Streeter, 2007).…”

“…Mutants of either otsA or treY genes were found to still be able to synthesise trehalose, however, double mutants (otsA,treY) failed to synthesise trehalose altogether (McIntyre et al, 2007). The genes coding for the three possible trehalose synthesis pathways (OtsA, TreYZ, and TreS) were present in the genome of S. meliloti 1021.…”

“…In Rhizobium leguminosarum bv. trifolii NZP561, peak trehalose concentrations are reached in the early stationary phase of growth and decline during stationary phase due to catabolism as carbon in the growth medium becomes depleted (McIntyre et al, 2007).…”

“…trifolii NZP561 involves the TreYZ and OtsAB pathways and mutants deficient in either pathway accumulate trehalose in amounts similar to the wild-type. However, double mutants fail to accumulate any trehalose and are also more sensitive to desiccation stress (McIntyre et al, 2007).…”