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Applied and Environmental Microbiology, October 2005, p. 5858-5864, Vol. 71, No. 10
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.10.5858-5864.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Development of a Functional Genomics Platform for Sinorhizobium meliloti: Construction of an ORFeome

Brenda K. Schroeder,1 Brent L. House,1,2 Michael W. Mortimer,1 Svetlana N. Yurgel,1 Scott C. Maloney,1 Kristel L. Ward,1 and Michael L. Kahn1,2*

Institute of Biological Chemistry,1 School of Molecular Biosciences, Washington State University, Pullman, Washington 99164-63402

Received 14 February 2005/ Accepted 26 April 2005

The nitrogen-fixing, symbiotic bacterium Sinorhizobium meliloti reduces molecular dinitrogen to ammonia in a specific symbiotic context, supporting the nitrogen requirements of various forage legumes, including alfalfa. Determining the DNA sequence of the S. meliloti genome was an important step in plant-microbe interaction research, adding to the considerable information already available about this bacterium by suggesting possible functions for many of the >6,200 annotated open reading frames (ORFs). However, the predictive power of bioinformatic analysis is limited, and putting the role of these genes into a biological context will require more definitive functional approaches. We present here a strategy for genetic analysis of S. meliloti on a genomic scale and report the successful implementation of the first step of this strategy by constructing a set of plasmids representing 100% of the 6,317 annotated ORFs cloned into a mobilizable plasmid by using efficient PCR and recombination protocols. By using integrase recombination to insert these ORFs into other plasmids in vitro or in vivo (B. L. House et al., Appl. Environ. Microbiol. 70:2806-2815, 2004), this ORFeome can be used to generate various specialized genetic materials for functional analysis of S. meliloti, such as operon fusions, mutants, and protein expression plasmids. The strategy can be generalized to many other genome projects, and the S. meliloti clones should be useful for investigators wanting an accessible source of cloned genes encoding specific enzymes.

* Corresponding author. Mailing address: Institute of Biological Chemistry, Washington State University, Pullman, WA 99164-6340. Phone: (509) 335-8327. Fax: (509) 335-7643. E-mail: kahn{at}wsu.edu.

Applied and Environmental Microbiology, October 2005, p. 5858-5864, Vol. 71, No. 10
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.10.5858-5864.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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