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PMID: 8990298 Published · ppublish English Journal Article Research Support, U.S. Gov't, P.H.S.

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

Journal of bacteriology ·Vol. 179 ·No. 2 ·1997-01-00 ·Pages 453-62

Dang TA, Christie PJ

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::beta-galactosidase hybrid proteins with junction sites mapping to regions 1 and 2 exhibited low beta-galactosidase activities and hybrid proteins with junction sites elsewhere exhibited high beta-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 approximately 35 and approximately 45 kDa. Taken together, our data support a model in which VirB4 is topologically configured as an integral cytoplasmic membrane protein with two periplasmic domains.

MeSH Terms
Adenosine Triphosphatases/genetics,metabolism Agrobacterium tumefaciens/enzymology Alkaline Phosphatase/genetics Bacterial Proteins/genetics,metabolism Cloning, Molecular Endopeptidase K/metabolism Exodeoxyribonucleases/metabolism Gene Deletion Lac Operon Membrane Proteins/genetics,metabolism Recombinant Fusion Proteins/genetics,metabolism Virulence Factors
Chemicals
Bacterial Proteins Membrane Proteins Recombinant Fusion Proteins Virulence Factors Exodeoxyribonucleases exodeoxyribonuclease III Alkaline Phosphatase Endopeptidase K Adenosine Triphosphatases
Authors & Affiliations
2 authors, click to expand affiliations / ORCID
Dang T A
Department of Microbiology and Molecular Genetics, The University of Texas Health Science Center at Houston, 77030, USA.
Christie P J
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Article Info
Journal
Journal of bacteriology
Abbr.
J Bacteriol
ISSN
0021-9193
DOI
10.1128/jb.179.2.453-462.1997
Published
1997-01-00
Pages
453-62
Language
English
Region
United States
NLM ID
2985120R
PMCID
PMC178716
Subset
IM
Grants
NIGMS NIH HHS · R01 GM048746 · United States
NIGMS NIH HHS · GM48746 · United States
External Links
PubMed source DOI: 10.1128/jb.179.2.453-462.1997 PMC Full Text View journal details
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