The VirB4 ATPase of Agrobacterium tumefaciens is a cytoplasmic membrane protein exposed at the periplasmic surface

Abstract

The VirB4 ATPase of Agrobacterium tumefaciens, a putative component of the T-complex transport apparatus, associates with the cytoplasmic membrane independently of other products of the Ti plasmid. VirB4 was resistant to extraction from membranes of wild-type strain A348 or a Ti-plasmidless strain expressing virB4 from an IncP replicon. To evaluate the membrane topology of VirB4, a nested deletion method was used to generate a high frequency of random fusions between virB4 and phoA, which encodes a periplasmically active alkaline phosphatase (AP) deleted of its signal sequence. VirB4::PhoA hybrid proteins exhibiting AP activity in Escherichia coli and A. tumefaciens had junction sites that mapped to two regions, between residues 58 and 84 (region 1) and between residues 450 and 514 (region 2). Conversely, VirB4::␤-galactosidase hybrid proteins with junction sites mapping to regions 1 and 2 exhibited low ␤-galactosidase activities and hybrid proteins with junction sites elsewhere exhibited high ␤-galactosidase activities. Enzymatically active VirB5::PhoA hybrid proteins had junction sites that were distributed throughout the length of the protein. Proteinase K treatment of A. tumefaciens spheroplasts resulted in the disappearance of the 87-kDa VirB4 protein and the concomitant appearance of two immunoreactive species of ϳ35 and ϳ45 kDa. Taken together, our data support a model in which VirB4 is topologically configured as an integral cytoplasmic membrane protein with two periplasmic domains.

Topology of the VirB4 C Terminus in the Agrobacterium tumefaciens VirB/D4 Type IV Secretion System

“…In addition, studies with detergent-solubilized Brucella suis T4SSs demonstrate that the VirB8 periplasmic domain and a VirB4 hexamer exist in the same complex (9). Our VirB4 C-terminal periplasmic placement is further supported by degradation of VirB4 in Agrobacterium spheroplasts treated with proteinase K (14,15). Finally, an independent and elegant transfer DNA immunoprecipitation assay, which defines the order of interaction between the T strand and components of the T4SS, suggests that the T strand first interacts with VirD4 and VirB11 in the cytoplasm (14); these authors have additional genetic data that place the requirement for VirB4 downstream of these initial contacts.…”

“…VirB4 topology based on sequence analyses predicts from zero (8) to four transmembrane domains (38), whereas alkaline phosphatase studies report either no alkaline phosphatase transposon insertions in VirB4 (31,32) or periplasmic exposure of the region around residue 450 (15). Our VirB4 two-hybrid interactions with the periplasmic domains of VirB8 and VirB10 and our fractionation and Western blot data strongly indicate periplasmic exposure of the VirB4 C-terminal hexameric domain.…”

“…Proteinase K susceptibil- ity studies demonstrate that VirB11 is not exposed to the periplasm (15). Our two-hybrid data superimposed onto our VirB4 and VirB11 structural models show one face of the VirB4 hexameric domain interacts with the N-terminal face (residues 1-106) of the VirB11 hexamer.…”

“…Early work suggested a possible periplasmic location of a small N-terminal region of VirB4 (residues 76 -84) predominantly based on findings in E. coli (15); however, the same authors demonstrate that a region around amino acid 237 is cytoplasmic in Agrobacterium. We favor an N-terminal cytoplasmic assignment in our model described below (Fig.…”

Agrobacterium tumefaciens VirB6 Domains Direct the Ordered Export of a DNA Substrate Through a Type IV Secretion System

“…However, multiple isolates bearing transposon insertions into only these few sites were recovered, prompting the construction of additional in-frame lacZ insertions by cloning. A ′lacZ gene lacking its first eight codons was isolated as a ~3.2 kb NotI-KpnI restriction fragment from pTAD250 58 and inserted into each of ten similarly digested pSJ4xxx plasmids. 33 As described below, the pSJ4xxx series of plasmids each carry a unique NotI restriction site inserted at ~30 codon intervals along the length of virB6.…”