title: Construction and expression of sugar kinase transcriptional gene fusions by using the Sinorhizobium meliloti ORFeome.
abstract: 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 beta-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 beta-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.
authors.affiliation: Institute of Biological Chemistry, Washington State University, Pullman, Washington 99164-6340, USA.
authors.forename: Jodi L
authors.initials: JL
authors.lastname: Humann
authors.forename: Brenda K
authors.initials: BK
authors.lastname: Schroeder
authors.forename: Michael W
authors.initials: MW
authors.lastname: Mortimer
authors.forename: Brent L
authors.initials: BL
authors.lastname: House
authors.forename: Svetlana N
authors.initials: SN
authors.lastname: Yurgel
authors.forename: Scott C
authors.initials: SC
authors.lastname: Maloney
authors.forename: Kristel L
authors.initials: KL
authors.lastname: Ward
authors.forename: Heather M
authors.initials: HM
authors.lastname: Fallquist
authors.forename: Hope T
authors.initials: HT
authors.lastname: Ziemkiewicz
authors.forename: Michael L
authors.initials: ML
authors.lastname: Kahn
mesh_terms: D019677:Artificial Gene Fusion; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D004269:DNA, Bacterial; D020869:Gene Expression Profiling; D017930:Genes, Reporter; D005822:Genetic Vectors; D005966:Glucuronidase / Q000235:genetics / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D010957:Plasmids; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D011995:Recombination, Genetic; D017422:Sequence Analysis, DNA; D016962:Sinorhizobium meliloti / Q000235:genetics* / Q000378:metabolism*; D013194:Staining and Labeling / Q000379:methods*
chemical_list: D001426:Bacterial Proteins; D004269:DNA, Bacterial; D011993:Recombinant Fusion Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D005966:Glucuronidase
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