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Journal of Bacteriology, August 2005, p. 5075-5083, Vol. 187, No. 15
0021-9193/05/$08.00+0     doi:10.1128/JB.187.15.5075-5083.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

The Stringent Response Is Required for Amino Acid and Nitrate Utilization, Nod Factor Regulation, Nodulation, and Nitrogen Fixation in Rhizobium etli

Arturo Calderón-Flores,¹ Gisela Du Pont,¹ Alejandro Huerta-Saquero,¹ Horacio Merchant-Larios,² Luis Servín-González,¹ and Socorro Durán^1*

Departamento de Biología Molecular y Biotecnología,¹ Departamento de Biología Celular y Fisiología, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, México D. F. 04510, Mexico²

Received 2 February 2005/ Accepted 11 May 2005

A Rhizobium etli Tn5 insertion mutant, LM01, was selected for its inability to use glutamine as the sole carbon and nitrogen source. The Tn5 insertion in LM01 was localized to the rsh gene, which encodes a member of the RelA/SpoT family of proteins. The LM01 mutant was affected in the ability to use amino acids and nitrate as nitrogen sources and was unable to accumulate (p)ppGpp when grown under carbon and nitrogen starvation, as opposed to the wild-type strain, which accumulated (p)ppGpp under these conditions. The R. etli rsh gene was found to restore (p)ppGpp accumulation to a relA spoT mutant of Escherichia coli. The R. etli Rsh protein consists of 744 amino acids, and the Tn5 insertion in LM01 results in the synthesis of a truncated protein of 329 amino acids; complementation experiments indicate that this truncated protein is still capable of (p)ppGpp hydrolysis. A second rsh mutant of R. etli, strain AC1, was constructed by inserting an element at the beginning of the rsh gene, resulting in a null allele. Both AC1 and LM01 were affected in Nod factor production, which was constitutive in both strains, and in nodulation; nodules produced by the rsh mutants in Phaseolus vulgaris were smaller than those produced by the wild-type strain and did not fix nitrogen. In addition, electron microscopy revealed that the mutant bacteroids lacked poly-ß-hydroxybutyrate granules. These results indicate a central role for the stringent response in symbiosis.

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* Corresponding author. Mailing address: Departamento de Biología Molecular y Biotecnología, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, Apdo. Postal 70228, México D.F. C.P. 04510, México. Phone: 52 (55) 56-22-38-80. Fax: 52 (55) 56-22-38-55. E-mail: sduranv{at}servidor.unam.mx.

This work is dedicated to the memory of Jorge Calderón.

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Journal of Bacteriology, August 2005, p. 5075-5083, Vol. 187, No. 15
0021-9193/05/$08.00+0     doi:10.1128/JB.187.15.5075-5083.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

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