ChvD, a chromosomally encoded ATP-binding cassette transporter-homologous protein involved in regulation of virulence gene expression in Agrobacterium tumefaciens.
A yeast two-hybrid screen searching for chromosomally encoded proteins that interact with the Agrobacterium tumefaciens VirB8 protein was carried out. This screen identified an interaction candidate homologous to the partial sequence of a gene that had previously been identified in a transposon screen as a potential regulator of virG expression, chvD. In this report, the cloning of the entire chvD gene is described and the gene is sequenced and characterized. Insertion of a promoterless lacZ gene into the chvD locus greatly attenuated virulence and vir gene expression. Compared to that of the wild-type strain, growth of the chvD mutant was reduced in rich, but not minimal, medium. Expression of chvD, as monitored by expression of beta-galactosidase activity from the chvD-lacZ fusion, occurred in both rich and minimal media as well as under conditions that induce virulence gene expression. The ChvD protein is highly homologous to a family of ATP-binding cassette transporters involved in antibiotic export from bacteria and has two complete Walker box motifs. Molecular genetic analysis demonstrated that disruption of either Walker A box, singly, does not inactivate this protein's effect on virulence but that mutations in both Walker A boxes renders it incapable of complementing a chvD mutant strain. Constitutive expression of virG in the chvD mutant strain restored virulence, supporting the hypothesis that ChvD controls virulence through effects on virG expression.
Liu
Z
Z
Plant Science Institute, Department of Biology, University of Pennsylvania, Philadelphia, PA 19104-6018, USA.
Jacobs
M
M
Schaff
D A
DA
McCullen
C A
CA
Binns
A N
AN
D018528:ATP-Binding Cassette Transporters / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D016960:Agrobacterium tumefaciens / Q000235:genetics / Q000378:metabolism / Q000472:pathogenicity*; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D001665:Binding Sites; D003001:Cloning, Molecular; D003470:Culture Media; D004268:DNA-Binding Proteins / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009154:Mutation; D010935:Plant Diseases / Q000382:microbiology; D010947:Plants, Toxic; D017422:Sequence Analysis, DNA; D014026:Nicotiana / Q000382:microbiology*; D014157:Transcription Factors / Q000235:genetics*; D020798:Two-Hybrid System Techniques; D014774:Virulence / Q000235:genetics
D018528:ATP-Binding Cassette Transporters; D001426:Bacterial Proteins; C428118:ChvD protein, Agrobacterium tumefaciens; D003470:Culture Media; D004268:DNA-Binding Proteins; D014157:Transcription Factors; C058483:virG protein, Shigella flexneri; D000255:Adenosine Triphosphate