Membrane topology and functional analysis of the sensory protein VirA of Agrobacterium tumefaciens.
The virA gene of Agrobacterium tumefaciens encodes an inner membrane that mediates the transcriptional activation of virulence genes in response to plant signal molecules. We report here a functional analysis of the N-terminal, C-terminal and periplasmic domains of VirA in transmembrane signalling. First, we show that VirA has a transmembrane topology by analysis of the alkaline phosphatase activities, determined by several virA-phoA gene fusions. Second, we report here the construction of several virA-tar chimeric genes, in which the 3'-coding region of virA is conserved to study transmembrane signalling, as well as the construction of a set of virA deletion mutations. Results of analyses of vir induction behaviour and tumour inducing abilities of agrobacteria carrying these mutant genes do not support existing models for the chemoreceptor function of the VirA periplasmic domain. We demonstrate that the periplasmic domain of VirA can be either replaced by a corresponding region of the E.coli chemosensory protein Tar or even totally deleted from VirA without a loss of function. Here, we present a model of VirA which involves a receptor function for the second membrane-spanning domain and an intracellular signalling function for the cytoplasmic domain of VirA. In addition, we show that VirA plays a role in determining the sensitivity for pH and temperature in acetosyringone-mediated vir induction, and we propose a role for the VirA periplasmic domain in detection of the external pH conditions.
Melchers
L S
LS
Department of Plant Molecular Biology, Leiden University, The Netherlands.
Regensburg-Tuïnk
T J
TJ
Bourret
R B
RB
Sedee
N J
NJ
Schilperoort
R A
RA
Hooykaas
P J
PJ
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D002872:Chromosome Deletion; D005796:Genes; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D009154:Mutation; D015345:Oligonucleotide Probes; D010957:Plasmids; D011487:Protein Conformation; D012231:Rhizobium / Q000235:genetics*; D037521:Virulence Factors
D001426:Bacterial Proteins; D015345:Oligonucleotide Probes; D037521:Virulence Factors