A novel cytology-based, two-hybrid screen for bacteria applied to protein-protein interaction studies of a type IV secretion system.
DivIVA of Bacillus subtilis and FtsZ of Escherichia coli were used to target heterologous protein complexes to cell division sites of E. coli and Agrobacterium tumefaciens. DivIVA and FtsZ that were fused to the dimerizing leucine zipper (LZ) domain of the yeast transcription activator GCN4 directed the green fluorescent protein (GFP) that was fused to an LZ domain to E. coli division sites, resulting in fluorescence patterns identical to those observed with DivIVA::GFP and FtsZ::GFP. These cell division proteins also targeted the VirE1 chaperone and VirE2 secretion substrate complex to division sites of E. coli and A. tumefaciens. Coproduction of the native VirE1 or VirE2 proteins inhibited the dihybrid interaction in both species, as judged by loss of GFP targeting to division sites. The VirE1 chaperone bound independently to N- and C-terminal regions of VirE2, with a requirement for residues 84 to 147 and 331 to 405 for these interactions, as shown by dihybrid studies with VirE1::GFP and DivIVA fused to N- and C-terminal VirE2 fragments. DivIVA also targeted homo- and heterotypic complexes of VirB8 and VirB10, two bitopic inner membrane subunits of the A. tumefaciens T-DNA transfer system, in E. coli and homotypic complexes of VirB10 in A. tumefaciens. VirB10 self-association in bacteria was mediated by the C-terminal periplasmic domain, as shown by dihybrid studies with fusions to VirB10 truncation derivatives. Together, our findings establish a proof-of-concept for the use of cell-location-specific proteins for studies of interactions among cytosolic and membrane proteins in diverse bacterial species.
Ding
Zhiyong
Z
Department of Microbiology and Molecular Genetics, The University of Texas--Houston Medical School, Houston, Texas 77030, USA.
Zhao
Zhenming
Z
Jakubowski
Simon J
SJ
Krishnamohan
Atmakuri
A
Margolin
William
W
Christie
Peter J
PJ
D016960:Agrobacterium tumefaciens / Q000166:cytology* / Q000235:genetics / Q000378:metabolism; D001412:Bacillus subtilis / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism; D002455:Cell Division; D003598:Cytoskeletal Proteins; D004269:DNA, Bacterial / Q000378:metabolism; D004268:DNA-Binding Proteins; D004926:Escherichia coli / Q000166:cytology* / Q000235:genetics / Q000378:metabolism; D016350:Leucine Zippers / Q000235:genetics; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism; D020798:Two-Hybrid System Techniques
D001426:Bacterial Proteins; D018797:Cell Cycle Proteins; D003598:Cytoskeletal Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; C104636:DivIVA protein, bacteria; C071525:FtsZ protein, Bacteria; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; C036483:T-DNA; D011494:Protein Kinases