Mutational analysis of the input domain of the VirA protein of Agrobacterium tumefaciens.
The transmembrane sensor protein VirA activates VirG in response to high levels of acetosyringone (AS). In order to respond to low levels of AS, VirA requires the periplasmic sugar-binding protein ChvE and monosaccharides released from plant wound sites. To better understand how VirA senses these inducers, the C58 virA gene was randomly mutagenized, and 14 mutants defective in vir gene induction and containing mutations which mapped to the input domain of VirA were isolated. Six mutants had single missense mutatiions in three widely separated areas of the periplasmic domain. Eight mutants had mutations in or near an amphipathic helix, TM1, or TM2. Four of the mutations in the periplasmic domain, when introduced into the corresponding A6 virA sequence, caused a specific defect in the vir gene response to glucose. This suggests that most of the periplasmic domain is required for the interaction with, or response to, ChvE. Three of the mutations from outside the periplasmic domain, one from each transmembrane domain and one from the amphiphathic helix, were made in A6 virA. These mutants were defective in the vir gene response to AS. These mutations did not affect the stability or topology of VirA or prevent dimerization; therefore, they may interfere with detection of AS or transmission of the signals to the kinase domain. Characterization of C58 chvE mutants revealed that, unlike A6 VirA, C58 VirA requires ChvE for activation of the vir genes.
Doty
S L
SL
Department of Microbiology, University of Washington, Seattle 98195, USA.
Yu
M C
MC
Lundin
J I
JI
Heath
J D
JD
Nester
E W
EW
D000098:Acetophenones / Q000494:pharmacology; D016960:Agrobacterium tumefaciens / Q000235:genetics*; D000469:Alkaline Phosphatase / Q000096:biosynthesis / Q000235:genetics; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D015153:Blotting, Western; D002874:Chromosome Mapping; D004252:DNA Mutational Analysis; D015964:Gene Expression Regulation, Bacterial; D005947:Glucose / Q000494:pharmacology; D008565:Membrane Proteins / Q000235:genetics*; D026901:Membrane Transport Proteins; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D033902:Periplasmic Binding Proteins; D010944:Plants / Q000382:microbiology; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins; D017422:Sequence Analysis, DNA; D015398:Signal Transduction; D015533:Transcriptional Activation; D037521:Virulence Factors; D001616:beta-Galactosidase / Q000096:biosynthesis / Q000235:genetics
D000098:Acetophenones; D001426:Bacterial Proteins; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D033902:Periplasmic Binding Proteins; D011993:Recombinant Fusion Proteins; D037521:Virulence Factors; C051667:acetosyringone; D000469:Alkaline Phosphatase; D001616:beta-Galactosidase; D005947:Glucose