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Applied and Environmental Microbiology, October 1998, p. 3954-3960, Vol. 64, No. 10
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

A New Genetic Locus in Sinorhizobium meliloti Is Involved in Stachydrine Utilization

Donald A. Phillips,1 Eve S. Sande,1,dagger J. A. C. Vriezen,2 Frans J. de Bruijn,2 Daniel Le Rudulier,3 and Cecillia M. Joseph1,*

Department of Agronomy and Range Science, University of California, Davis, California 956161; DOE-Plant Research Laboratory and Department of Microbiology, Michigan State University, East Lansing, Michigan 488242; and Laboratoire de Biologie Végétale et Microbiologie, URA CNRS 1114, Université de Nice-Sophia Antiopolis, 06108 Nice Cedex 2, France3

Received 24 February 1998/Accepted 12 July 1998

Stachydrine, a betaine released by germinating alfalfa seeds, functions as an inducer of nodulation genes, a catabolite, and an osmoprotectant in Sinorhizobium meliloti. Two stachydrine-inducible genes were found in S. meliloti 1021 by mutation with a Tn5-luxAB promoter probe. Both mutant strains (S10 and S11) formed effective alfalfa root nodules, but neither grew on stachydrine as the sole carbon and nitrogen source. When grown in the absence or presence of salt stress, S10 and S11 took up [14C]stachydrine as well as wild-type cells did, but neither used stachydrine effectively as an osmoprotectant. In the absence of salt stress, both S10 and S11 took up less [14C]proline than wild-type cells did. S10 and S11 appeared to colonize alfalfa roots normally in single-strain tests, but when mixed with the wild-type strain, their rhizosphere counts were reduced more than 50% (P <=  0.01) relative to the wild type. These results suggest that stachydrine catabolism contributes to root colonization. DNA sequence analysis identified the mutated locus in S11 as putA, and the luxAB fusion in that gene was induced by proline as well as stachydrine. DNA that restored the capacity of mutant S10 to catabolize stachydrine contained a new open reading frame, stcD. All data are consistent with the concept that stcD codes for an enzyme that produces proline by demethylation of N-methylproline, a degradation product of stachydrine.

* Corresponding author. Mailing address: Department of Agronomy and Range Science, University of California, Davis, CA 95616. Phone: (530) 752-1891. Fax: (530) 752-4361. E-mail: CMJoseph{at}UCDavis.edu.

dagger Present address: NifTAL Center, Paia, Maui, HI 98779-9744.

Applied and Environmental Microbiology, October 1998, p. 3954-3960, Vol. 64, No. 10
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.


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