The virB operon of Agrobacterium tumefaciens pTiC58 encodes 11 open reading frames

Abstract

Agrobacterium tumefaciens genetically transforms plant cells by transferring a copy of its T-DNA to the plant where it is integrated and stably maintained. In the presence of wounded plant cells this process is activated and mediated by the products of the vir genes which are grouped into six distinct loci. The largest is the virB locus spanning 9.5 kb. Transposon mutagenesis studies have shown that virB gene products are required for virulence but their functions remain largely unknown. To provide information relevant to understanding the function of VirB polypeptides, the nucleotide sequence of the virB operon from a nopaline plasmid, pTiC58, is presented here. Eleven open reading frames (ORFs) are predicted from this sequence. The predicted sizes of 10 of the 11 VirB polypeptides are verified by specific expression in Escherichia coli. Only the product of the smallest ORF potentially encoding a 5.8 kDa polypeptide has not been detected. The initiation of translation of five virB ORFs occurs at codons that overlap the termination codons of the ORF immediately upstream; thus, translational coupling may be an important mechanism for efficient translation of the large virB polycistronic mRNA. Based on hydropathy plot analysis nine of the virB ORFs encode proteins that may interact with membranes; these data support the earlier hypothesis that virB gene products may form a membrane pore or channel to mediate exit of the T-DNA copy (T-strands) from Agrobacterium into the plant cell. A comparison of the two published octopine virB sequences with the nopaline sequence presented here is made.

Complementation analysis of Agrobacterium tumefaciens Ti plasmid virB genes by use of a vir promoter expression vector: virB9, virB10, and virB11 are essential virulence genes

“…3B), and the observed protein size closely matches the molecular mass of 87,325 daltons predicted by the revised pTiA6 virB nucleotide sequence (49). The nopaline type plasmid pTiC58 virB4 gene product has been identified in E. coli maxicells as an 80-kDa protein (30), and thus our data show that the octopine type plasmid pTiA6 VirB4 protein is similar in size. Our results also indicate that the 88-kDa pTiA6 virB protein previously identified by Engstrom et al (15) (Fig.…”

“…In addition, the virBlll gene encodes an inner membrane-associated ATPase that shares amino acid similarity with comG open reading frame 1, a protein required for DNA uptake during transformation of Bacillus subtilis (1,7). These results, along with the functional similarities observed between the processes of T-DNA transfer and bacterial conjugation (43), have led us and others (30,45,48,59) to suggest that the virB gene products might form a membrane-associated T-complex transport apparatus analogous to the multicomponent membrane pore proposed to mediate DNA transfer in both plasmid conjugal (51) and bacterial (1) transformation systems.…”

“…However, the virB proteins are likely to play a central role in this process, because transposon insertions within virB abolish virulence (40), even though virB gene products are not required for T-DNA processing (42), and since most of the 11 predicted virB gene products are likely to be membrane associated (30,45,48,49). Cellular fractionation studies have confirmed a membrane localization for several VirB proteins (15), including the virBJO gene product, which we have shown to be a component of an inner membrane protein complex in A. tumefaciens (50).…”

The transfer of DNA from Agrobacterium tumefaciens into plants: a feast of fundamental insights

“…Sequence analysis has identi®ed signal peptidase I and II sites in several VirB proteins (Kuldau et al, 1990;Ward et al, 1988), which implies constitutive secretion pathways are involved in the assembly of the transporter. More speci®c activities are also indicated.…”

Common components in the assembly of type 4 fimbriae, DNA transfer systems, filamentous phage and protein‐secretion apparatus: a general system for the formation of surface‐associated protein complexes

“…, 1992;Lessl et al, 1992). The Ti plasmid virB operon is essential for virulence , and includes a gene (ORF-11) which like kilB is homologous to pilB (Ward et al, 1988;Kuldau et al, 1990;Shirasu et aI. , 1990).…”