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Applied and Environmental Microbiology, June 2007, p. 3984-3992, Vol. 73, No. 12
0099-2240/07/$08.00+0     doi:10.1128/AEM.00412-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

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

Helen J. McIntyre,1 Holiday Davies,2 Timothy A. Hore,1,{dagger} Simon H. Miller,1,{ddagger} Jean-Pierre Dufour,2,§ and Clive W. Ronson1*

Department of Microbiology and Immunology,1 Department of Food Science, University of Otago, P.O. Box 56, Dunedin, New Zealand2

Received 21 February 2007/ Accepted 11 April 2007

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.

* Corresponding author. Mailing address: Department of Microbiology and Immunology, University of Otago, 720 Cumberland St., Dunedin, New Zealand. Phone: (64) 3 4797701. Fax: (64) 3 4798540. E-mail: clive.ronson{at}otago.ac.nz

{triangledown} Published ahead of print on 20 April 2007.

{dagger} Present address: Comparative Genomics Group, Research School of Biological Sciences, Australian National University, Canberra, Australia.

{ddagger} Present address: BIOMERIT Research Centre, Department of Microbiology, National University of Ireland, Cork, Ireland.

§ Deceased 26 February 2007.

Applied and Environmental Microbiology, June 2007, p. 3984-3992, Vol. 73, No. 12
0099-2240/07/$08.00+0     doi:10.1128/AEM.00412-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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