Mutational analysis of TraR. Correlating function with molecular structure of a quorum-sensing transcriptional activator

TraR, the quorum-sensing activator of the Agrobacterium tumefaciens Ti plasmid conjugation system, induces gene expression in response to its quormone, N-(3-oxooctanoyl)-L-homoserine lactone. Ligand binding results in dimerization of TraR and is required for its activity. Analysis of N- and C-terminal deletion mutants of TraR localized the quormone-binding domain to a region between residues 39 and 140 and the primary dimerization domain to a region between residues 119 and 156. The dominant-negative properties of these mutants predicted a second dimerization domain at the C terminus of the protein. Analysis of fusions of N-terminal fragments of TraR to lambda cI' confirmed the dimerization activity of these two domains. Fifteen single amino acid substitution mutants of TraR defective in dimerization were isolated. According to the analysis of these mutants, Asp-70 and Gly-113 are essential for quormone binding, whereas Ala-38 and Ala-105 are important, but not essential. Additional residues located within the N-terminal half of TraR, including three located in alpha-helix 9, contribute to dimerization, but are not required for ligand binding. These results and the recently reported crystal structure of TraR are consistent with and complement each other and together define some of the structural and functional relationships of this quorum-sensing activator.     

Dimerization of the Agrobacterium tumefaciens VirB4 ATPase and the effect of ATP-binding cassette mutations on the assembly and function of the T-DNA transporter

The Agrobacterium tumefaciens VirB4 ATPase functions with other VirB proteins to export T-DNA to susceptible plant cells and other DNA substrates to a variety of prokaryotic and eukaryotic cells. Previous studies have demonstrated that VirB4 mutants with defects in the Walker A nucleotide-binding motif are non-functional and exert a dominant negative phenotype when synthesized in wild-type cells. This study characterized the oligomeric structure of VirB4 and examined the effects of Walker A sequence mutations on complex formation and transporter activity. VirB4 directed dimer formation when fused to the amino-terminal portion of cI repressor protein, as shown by immunity of Escherichia coli cells to lambda phage infection. VirB4 also dimerized in Agrobacterium tumefaciens, as demonstrated by the recovery of a detergent-resistant complex of native protein and a functional, histidine-tagged derivative by precipitation with anti-His6 antibodies and by Co2+ affinity chromatography. Walker A sequence mutants directed repressor dimerization in E. coli and interacted with His-VirB4 in A. tumefaciens, indicating that ATP binding is not required for self-association. A dimerization domain was localized to a proposed N-terminal membrane-spanning region of VirB4, as shown by the dominance of an allele coding for the N-terminal 312 residues and phage immunity of host cells expressing cI repressor fusions to alleles for the first 237 or 312 residues. A recent study reported that the synthesis of a subset of VirB proteins, including VirB4, in agrobacterial recipients has a pronounced stimulatory effect on the virB-dependent conjugal transfer of plasmid RSF1010 by agrobacterial donors. VirB4'312 suppressed the stimulatory effect of VirB proteins for DNA uptake when synthesized in recipient cells. In striking contrast, Walker A sequence mutants contributed to the stimulatory effect of VirB proteins to the same extent as native VirB4. These findings indicate that the oligomeric structure of VirB4, but not its capacity to bind ATP, is important for the assembly of VirB proteins as a DNA uptake system. The results of these studies support a model in which VirB4 dimers or homomultimers contribute structural information for the assembly of a transenvelope channel competent for bidirectional DNA transfer, whereas an ATP-dependent activity is required for configuring this channel as a dedicated export machine.