title: VirA of Agrobacterium tumefaciens is an intradimer transphosphorylase and can actively block vir gene expression in the absence of phenolic signals.
abstract: The VirA-VirG two-component system regulates the 30-gene vir regulon in response to host-released chemical signals. VirA is a homodimeric membrane-spanning histidine protein kinase. Here, we show that mutations in two essential VirA residues, His-474 and Gly-657, can be complemented by the formation of mixed heterodimers, indicating that each subunit of a VirA dimer transphosphorylates the opposite subunit. VirA contains a receiver domain that inhibits kinase activity. We use the forced heterodimer system to show that the two receiver domains of a VirA dimer act independently and that each inhibits the phosphoacceptor subdomain of the opposite subunit. We also demonstrate that merodiploid strains co-expressing constitutive VirA mutants and wild-type VirA show levels of vir gene expression far lower than haploid strains expressing just the constitutive alleles. The fact that wild-type VirA can actively block vir gene expression in the absence of phenolic signals suggests that it might have a phospho-VirG phosphatase activity. The receiver domain of VirA is essential for this activity, whereas residues H474 and G657 of the kinase domain are not required. Merodiploid strains co-expressing a constitutive VirA allele and an allele that is kinase inactive but proficient in the inhibitory activity show strongly inducible vir gene expression, indicating that the inhibitory activity is modulated by environmental signals.
authors.affiliation: Department of Microbiology, 360A Wing Hall, Cornell University, Ithaca, NY 14853, USA.
authors.forename: Anja
authors.initials: A
authors.lastname: Brencic
authors.forename: Qi
authors.initials: Q
authors.lastname: Xia
authors.forename: Stephen C
authors.initials: SC
authors.lastname: Winans
mesh_terms: D016960:Agrobacterium tumefaciens / Q000201:enzymology* / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D019281:Dimerization; D015964:Gene Expression Regulation, Bacterial; D005816:Genetic Complementation Test; D010770:Phosphotransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011487:Protein Conformation; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism; D018085:Regulon; D015398:Signal Transduction; D037521:Virulence Factors / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D021122:Protein Subunits; D037521:Virulence Factors; D010770:Phosphotransferases