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Applied and Environmental Microbiology, November 2008, p. 6756-6765, Vol. 74, No. 21
0099-2240/08/$08.00+0     doi:10.1128/AEM.01468-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Construction and Expression of Sugar Kinase Transcriptional Gene Fusions by Using the Sinorhizobium meliloti ORFeome{triangledown}

Jodi L. Humann,1 Brenda K. Schroeder,1 Michael W. Mortimer,1 Brent L. House,1,2 Svetlana N. Yurgel,1 Scott C. Maloney,1 Kristel L. Ward,1 Heather M. Fallquist,2 Hope T. Ziemkiewicz,1 and Michael L. Kahn1,2*

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

Received 30 June 2008/ Accepted 2 September 2008

The Sinorhizobium meliloti ORFeome project cloned 6,314 open reading frames (ORFs) into a modified Gateway entry vector system from which the ORFs could be transferred to destination vectors in vivo via bacterial conjugation. In this work, a reporter gene destination vector, pMK2030, was constructed and used to generate ORF-specific transcriptional fusions to β-glucuronidase (gusA) and green fluorescent protein (gfp) reporter genes. A total of 6,290 ORFs were successfully transferred from the entry vector library into pMK2030. To demonstrate the utility of this system, reporter plasmids corresponding to 30 annotated sugar kinase genes were integrated into the S. meliloti SM1021 and/or SM8530 genome. Expression of these genes was measured using a high-throughput β-glucuronidase assay to track expression on nine different carbon sources. Six ORFs integrated into SM1021 and SM8530 had different basal levels of expression in the two strains. The annotated activities of three other sugar kinases were also confirmed.

* Corresponding author. Mailing address: P.O. Box 646340, Pullman, WA 99164-6340. Phone: (509) 335-8327. Fax: (509) 335-7643. E-mail: kahn{at}wsu.edu

{triangledown} Published ahead of print on 12 September 2008.

Applied and Environmental Microbiology, November 2008, p. 6756-6765, Vol. 74, No. 21
0099-2240/08/$08.00+0     doi:10.1128/AEM.01468-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.





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