Construction and expression of sugar kinase transcriptional gene fusions by using the Sinorhizobium meliloti ORFeome.
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.
Humann
Jodi L
JL
Institute of Biological Chemistry, Washington State University, Pullman, Washington 99164-6340, USA.
Schroeder
Brenda K
BK
Mortimer
Michael W
MW
House
Brent L
BL
Yurgel
Svetlana N
SN
Maloney
Scott C
SC
Ward
Kristel L
KL
Fallquist
Heather M
HM
Ziemkiewicz
Hope T
HT
Kahn
Michael L
ML
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*
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D011993:Recombinant Fusion Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D005966:Glucuronidase
Appl Environ Microbiol. 2004 May;70(5):2806-15
15128536
J Bacteriol. 2002 Sep;184(18):5067-76
12193623
Proc Natl Acad Sci U S A. 1979 Apr;76(4):1648-52
377280
Proc Natl Acad Sci U S A. 2001 Aug 14;98(17):9883-8
11481432
J Bacteriol. 1983 Oct;156(1):168-76
6137474
J Bacteriol. 2004 Aug;186(16):5460-72
15292148
EMBO J. 1987 Dec 20;6(13):3901-7
3327686
Proc Natl Acad Sci U S A. 2001 Aug 14;98(17):9889-94
11481431
Cell. 1989 Feb 24;56(4):661-72
2537152
Appl Environ Microbiol. 2005 Oct;71(10):5858-64
16204497
Biotechniques. 1999 May;26(5):824-6, 828
10337469
Plasmid. 2000 Jan;43(1):12-23
10610816
J Biotechnol. 2002 Aug 28;97(3):243-52
12084480
J Biol Chem. 1982 Aug 10;257(15):8724-30
7096330
Appl Environ Microbiol. 2006 Nov;72(11):7156-67
16963549
J Bacteriol. 2002 Oct;184(19):5385-92
12218025
Proc Natl Acad Sci U S A. 2004 Nov 23;101(47):16636-41
15542588
J Mol Biol. 1993 Dec 5;234(3):534-41
8254658
J Mol Biol. 1969 May 14;41(3):459-72
4896022
J Bacteriol. 1982 Jan;149(1):114-22
6274841
Proteomics. 2004 Jul;4(7):1859-72
15221743
J Bacteriol. 2005 Feb;187(3):1161-72
15659691
J Mol Biol. 1983 Jun 5;166(4):557-80
6345791
J Bacteriol. 2005 Dec;187(23):7931-44
16291666
Mol Plant Microbe Interact. 2004 Mar;17(3):292-303
15000396
Proc Natl Acad Sci U S A. 2001 Aug 14;98(17):9877-82
11481430
Nat Rev Microbiol. 2007 Aug;5(8):619-33
17632573