Construction of an efficient expression system for Agrobacterium tumefaciens based on the coliphage T5 promoter.
A versatile expression vector utilizing a promoter of coliphage T5, P(N25) (Gentz and Bujard, 1985. J. Bacteriol. 164, 70-77) and a derivative of the IncW broad-host-range plasmid pJB20 (Beaupré et al., 1997. J. Bacteriol. 179, 78-89) has been developed. This vector successfully expresses virulence proteins of Agrobacterium tumefaciens encoded by virG and a mutant allele of virA, virA (delta1-284, G665D) in Escherichia coli as well as in A. tumefaciens. The signal transduction proteins VirA (delta1-284, G665D) and VirG are fully functional when expressed in Agrobacterium, and the P(N25) driven expression overrides the complex transcriptional regulation present with the native promoters. This expression system will enable a more detailed analysis of the activation events in signal transduction in A. tumefaciens, and we expect it to be useful in other prokaryotes.
Wang
Y
Y
Department of Chemistry, The University of Chicago, IL 60637, USA.
Mukhopadhyay
A
A
Howitz
V R
VR
Binns
A N
AN
Lynn
D G
DG
D000098:Acetophenones / Q000494:pharmacology; D016960:Agrobacterium tumefaciens / Q000235:genetics*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D004274:DNA, Recombinant; D004279:DNA, Viral / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005786:Gene Expression Regulation / Q000187:drug effects; D005822:Genetic Vectors / Q000235:genetics*; D006639:Histidine / Q000235:genetics; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013604:T-Phages / Q000235:genetics*; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D037521:Virulence Factors
D000098:Acetophenones; D001426:Bacterial Proteins; D004274:DNA, Recombinant; D004279:DNA, Viral; D004268:DNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D037521:Virulence Factors; C058483:virG protein, Shigella flexneri; D006639:Histidine; C051667:acetosyringone