The Agrobacterium tumefaciens VirB7 lipoprotein is required for stabilization of VirB proteins during assembly of the T-complex transport apparatus.

The Agrobacterium tumefaciens virB7 gene product is a lipoprotein whose function is required for the transmission of oncogenic T-DNA to susceptible plant cells. Three lines of study provided evidence that VirB7 interacts with and stabilizes other VirB proteins during the assembly of the putative T-complex transport apparatus. First, a precise deletion of virB7 from the pTiA6NC plasmid of wild-type strain A348 was correlated with significant reductions in the steady-state levels of several VirB proteins, including VirB4, VirB9, VirB10, and VirB11; trans expression of virB7 in the delta virB7 mutant partially restored the levels of these proteins, and trans coexpression of virB7 and virB8 fully restored the levels of these proteins to wild-type levels. Second, modulation of VirB7 levels resulted in corresponding changes in the levels of other VirB proteins in the following cell types: (i) a delta virB7 mutant expressing virB7 and virB8 from isopropyl-beta-D-thiogalactopyranoside (IPTG)-inducible Plac and other virB genes from acetosyringone (AS)-inducible PvirB; (ii) a delta virB operon mutant expressing virB7 and virB8 from Plac and virB9, virB10, and virB11 from PvirB; and (iii) a delta virB operon mutant expressing virB7 from IPTG-inducible Pklac and virB9 from an AS-inducible PvirB. Third, the synthesis of a VirB7::PhoA fusion protein in strain A348 was correlated with a significant reduction in the steady-state levels of VirB4, VirB5, and VirB7 through VirB11; these cells also exhibited a severely attenuated virulence phenotype, indicating that synthesis of the fusion protein perturbs the assembly of VirB proteins into a stabilized protein complex required for T-complex transport. Extracts of AS-induced cells electrophoresed under nonreducing conditions possessed undetectable levels of the 32-kDa VirB9 and 4.5-kDa VirB7 monomers and instead possessed a 36-kDa complex that cross-reacted with both VirB7 and VirB9 antisera and accumulated as a function of virB7 expression. Our results are consistent with a model in which VirB7 stabilizes VirB9 by formation of a covalent intermolecular cross-link; in turn, the VirB7-VirB9 heterodimer promotes the assembly of a functional T-complex transport machinery.     

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

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.     

VirB1, a component of the T-complex transfer machinery of Agrobacterium tumefaciens, is processed to a C-terminal secreted product, VirB1.

During genetic transformation of plant cells by Agrobacterium tumefaciens, 11 VirB proteins and VirD4 are proposed to form a transmembrane bridge to transfer a DNA-protein complex (T-complex) into the plant cytoplasm. In this study, the localization of the first product of the virB operon, VirB1, was studied in detail. While full-length VirB1 localized mostly to the inner membrane, an immunoreactive VirB1 product was found as soluble processed form, designated VirB1*. Equal amounts of VirB1* could be detected in concentrated culture supernatants versus associated with the cell. VirB1* was purified from the supernatant of vir-induced cells by ammonium sulfate precipitation and Q-Sepharose chromatography. Sequence analysis of the N terminus of VirB1* localized the processing site after amino acid 172 of VirB1. Cell-associated VirB1* was partly removed by vortexing, suggesting a loose association with the cell or active secretion. However, cross-linking and coimmunoprecipitation showed a close association of cell-bound VirB1* with the VirB9-VirB7 heterodimer, a membrane-associated component of the T-complex transfer machinery. Homologies of the N-terminal part of VirB1 to bacterial transglycosylases suggest that it may assist T-complex transfer by local lysis of the bacterial cell wall, whereas the exposed localization of the C-terminal processing product VirB1* predicts direct interaction with the plant. Thus, VirB1 may be a bifunctional protein where both parts have different functions in T-complex transfer from Agrobacterium to plant cells.     

The product of the virB4 gene of Agrobacterium tumefaciens promotes accumulation of VirB3 protein.

The process of T-DNA transfer from Agrobacterium tumefaciens to plant cells is thought to involve passage of a DNA-protein complex through a specialized structure in the bacterial membrane. The virB operon of A. tumefaciens encodes 11 proteins, of which 9 are known to be located in the membranes and 10 have been shown to be essential for virulence. Sequence comparisons between proteins encoded by the virB operon and those encoded by operons from conjugative plasmids indicated that VirB proteins may form a structure similar to a conjugative pilus. Here, we examine the effects of mutations in virB4 on the accumulation and localization of other VirB proteins. VirB4 shares amino acid sequence similarity with the TraC protein of plasmid F, which is essential for pilus formation in Escherichia coli, and with the PtlC protein of Bordetella pertussis, which is required for toxin secretion. Polar and nonpolar virB4 mutants were examined, and all were shown to be unable to accumulate VirB3 protein to wild-type levels. A low level of VirB3 protein which was present in induced NT1RE cells harboring virB4 nonpolar mutant pBM1130 was found to associate with the inner membrane fraction only, whereas in wild-type cells VirB3 associated with both inner and outer membranes. The results indicate that for VirB3 to accumulate in the outer membrane, VirB4 must also be present, and it is possible that one role of VirB4 is in the correct assembly of a VirB protein membrane structure.     

The Agrobacterium tumefaciens virE2 gene product is a single-stranded-DNA-binding protein that associates with T-DNA.

Agrobacterium tumefaciens transfers T-DNA into the plant genome by a process mediated by Ti plasmid-encoded vir genes. Cleavage at T-DNA border sequences by the VirD endonuclease generates linear, single-stranded T-DNA molecules. In the work described in this report, we used electrophoretic mobility shift assays to show that the purified virE2 gene product binds to single-stranded DNA. VirE2 protein associates with T-DNA as shown by immunoprecipitation studies with VirE2-specific antiserum. The VirE2 protein was detected primarily in the cytoplasm, but also in the inner and outer membrane and periplasmic fractions. Virulence of a virE2 mutant was restored by mixed infection with strains carrying an intact vir region, but not with virA, virB, virD, virE, or virG mutants or chvA, chvB, or exoC mutants. We propose that the VirE2 protein is involved in the processing of T-DNA and in T-strand protection during transfer to the plant cell.     

The Agrobacterium tumefaciens virulence gene chvE is part of a putative ABC-type sugar transport operon.

The Agrobacterium tumefaciens virulence determinant ChvE is a periplasmic binding protein which participates in chemotaxis and virulence gene induction in response to monosaccharides which occur in the plant wound environment. The region downstream of the A. tumefaciens chvE gene was cloned and sequenced for nucleotide and expression analysis. Three open reading frames transcribed in the same direction as chvE were revealed. The first two, together with chvE, encode putative proteins of a periplasmic binding protein-dependent sugar uptake system, or ABC-type (ATP binding cassette) transporter. The third open reading frame encodes a protein of unknown function. The deduced transporter gene products are related on the amino acid level to bacterial sugar transporters and probably function in glucose and galactose uptake. We have named these genes gguA, -B, and -C, for glucose galactose uptake. Mutations in gguA, gguB, or gguC do not affect virulence of A. tumefaciens on Kalanchoe diagremontiana; growth on 1 mM galactose, glucose, xylose, ribose, arabinose, fucose, or sucrose; or chemotaxis toward glucose, galactose, xylose, or arabinose.     

The Agrobacterium tumefaciens virC1 gene product binds to overdrive, a T-DNA transfer enhancer.

In Agrobacterium tumefaciens, a cis-active 24-base-pair sequence adjacent to the right border of the T-DNA, called overdrive, stimulates tumor formation by increasing the level of T-DNA processing. Recent results from our laboratory have suggested that the virC operon which enhances T-DNA processing probably does so because the VirC1 protein interacts with overdrive (N. Toro, A. Datta, M. Yanofsky, and E. W. Nester, Proc. Natl. Acad. Sci. USA 85:8558-8562, 1988). We report here the purification of the VirC1 protein from cells of Escherichia coli harboring a plasmid containing the coding sequences of the virC locus of the octopine Ti plasmid. By gel mobility shift and DNase I footprinting assays, we showed that this purified virC1 gene product binds to overdrive but not to the right border of T-DNA.     

The lipoprotein VirB7 interacts with VirB9 in the membranes of Agrobacterium tumefaciens.

VirB9 and VirB7 are essential components of the putative VirB membrane channel required for transfer of the T-complex from Agrobacterium tumefaciens into plants. In this report, we present a biochemical analysis of their interaction and cellular localization. A comparison of relative electrophoretic mobilities under nonreducing and reducing conditions suggested that they form thiol-sensitive complexes with other proteins. Two-dimensional gel electrophoresis identified one complex as a heterodimer of VirB9 and VirB7 covalently linked by a disulfide bond, as well as VirB7 homodimers and monomers. Immunoprecipitation with VirB9-specific antiserum isolated the heterodimeric VirB9-VirB7 complex. Incubation with reducing agent split the complex into its constituent VirB9 and VirB7, which further confirmed linkage via cysteine residues. The interaction between VirB9 and VirB7 also was observed in the yeast two-hybrid system. Membrane attachment of VirB9-VirB7 may be conferred by lipoprotein modification, since labeling with [3H]palmitic acid in A. tumefaciens verified that VirB7 is a lipoprotein associated with VirB9. VirB9 and VirB7 showed equal distribution between inner and outer membranes, in accord with their proposed association with the transmembrane VirB complex.     

Identification of a virB10 protein aggregate in the inner membrane of Agrobacterium tumefaciens.

Products of the virB operon are proposed components of a membrane-associated T-DNA transport apparatus in Agrobacterium tumefaciens. Here we identified the virB10 gene product and raised specific antiserum to the protein. While the virB10 reading frame contains two potential ATG translation start sites located 32 codons apart, we found that only the downstream ATG was required for efficient VirB10 synthesis. Cellular localization studies and analysis of translational fusions with the Escherichia coli alkaline phosphatase gene (phoA) indicated that VirB10 was anchored in the inner membrane and contained a periplasmic domain. This work also demonstrated the utility of alkaline phosphatase as a reporter for secreted proteins in A. tumefaciens. Several high-molecular-weight forms of VirB10 were observed after treatment of A. tumefaciens whole cells or inner membranes with protein cross-linking agents, suggesting that VirB10 exists as a native oligomer or forms an aggregate with other membrane proteins. These results provide the first biochemical evidence that a VirB protein complex is membrane associated in A. tumefaciens.     

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.

The virB gene products of the Agrobacterium tumefaciens tumor-inducing (Ti) plasmid have been proposed to mediate T-DNA transport through the bacterial cell wall into plant cells. Previous genetic analysis of the approximately 9.5-kilobase-pair virB operon has been limited to transposon insertion mutagenesis. Due to the polarity of the transposon insertions, only the last gene in the operon, virB11, is known to provide an essential virulence function. We have now begun to assess the contribution of the other virB genes to virulence. First, several previously isolated Tn3-HoHo1 insertions in the 3' end of the virB operon were precisely mapped by nucleotide sequence analysis. Protein extracts from A. tumefaciens strains harboring these insertions on the Ti plasmid were subjected to immunostaining analysis with VirB4-, VirB10-, and VirB11-specific antisera to determine the effect of the insertion on virB gene expression. In this manner, avirulent mutants containing polar insertions in the virB9 and virB10 genes were identified. To carry out a complementation analysis with these virB mutants, expression vectors were constructed that allow cloned genes to be expressed from the virB promoter in A. tumefaciens. These plasmids were used to express combinations of the virB9, virB10, and virB11 genes in trans in the virB insertion mutants, thereby creating strains lacking only one of these three virB gene products. Virulence assays on Kalanchoe daigremontiana demonstrated that in addition to virB11, the virB9 and virB10 genes are required for tumorigenicity.     

The product of the F plasmid transfer operon gene, traF, is a periplasmic protein.

The products of clones carrying the F plasmid transfer operon gene, traF, were analyzed. Proteins expressed in maxicells were labeled with [35S]methionine and examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Clones carrying the wild-type traF gene expressed two polypeptide products that were not products of clones containing the traF13 amber mutation. These migrated with apparent molecular weights (Ma) of 27,000 and 25,000. A pulse-chase experiment suggested that the larger product was a precursor of the smaller one. In the presence of ethanol, the Ma-27,000 polypeptide accumulated and the Ma-25,000 product was not expressed. These results indicated that the traF protein undergoes proteolytic processing associated with export. Cell fractionation experiments further indicated that the greatest concentration of the mature (Ma 25,000) TraF protein was located in the periplasm. The DNA sequence of traF and the position of the transition mutation in traF13 DNA were also determined. Sequence analysis suggested that traF would be expressed as a 247-amino-acid, Mr-28,006 polypeptide. The 19 amino acids at the amino terminus of this polypeptide appear to constitute a typical membrane leader peptide, while the remainder of the molecule (Mr 25,942) is predicted to be primarily hydrophilic in character.     

Construction and characterization of Tn5virB, a transposon that generates nonpolar mutations, and its use to define virB8 as an essential virulence gene in Agrobacterium tumefaciens.

Transfer of the T-DNA from the Ti plasmid of Agrobacterium tumefaciens into plant cells involves movement of a single-stranded DNA-protein intermediate across several membrane and cell wall barriers. The 11 VirB proteins encoded by the Ti plasmid are hypothesized to form at least part of a membrane-localized T-DNA transport apparatus. Although available genetic and biochemical analyses support this hypothesis, detailed study of this transport apparatus is hindered by the fact that most available mutations in the virB operon are in the form of transposon insertions that have polar effects. In this study we constructed a transposon, Tn5virB, that can be used to generate nonpolar insertions in operons of A. tumefaciens and used it to demonstrate that virB8 is an essential virulence gene.     

The VirA protein of Agrobacterium tumefaciens is autophosphorylated and is essential for vir gene regulation.

The virA and virG gene products are required for the regulation of the vir regulon on the tumor-inducing (Ti) plasmid of Agrobacterium tumefaciens. VirA is a membrane-associated protein which is homologous to the sensor molecules of other two-component regulatory systems. We overproduced truncated VirA proteins in Escherichia coli by deleting different lengths of the 5'-coding region of the virA gene and placing these genes under lacZ control. These proteins were purified from polyacrylamide gels and renatured. The renatured proteins became radiolabeled when they were incubated with [gamma-32P]ATP but not with [gamma-32P]GTP or [alpha-32P]ATP, which suggests an ATP gamma-phosphate-specific autophosphorylation. The smallest VirA protein, which retained only the C-terminal half of the protein, gave the strongest autophosphorylation signal, which demonstrates that the C-terminal domain has the autophosphorylation site. The phosphorylated amino acid was identified as phosphohistidine, and a highly conserved histidine was found in all of the VirA homologs. When this histidine was changed to glutamine, which cannot be phosphorylated, the resulting VirA protein lost both its ability to autophosphorylate and its biological function as a vir gene regulator. Results of this study indicate that VirA autophosphorylation is required for the induction of the vir regulon and subsequent tumor induction on plants by A. tumefaciens.     

Functional roles assigned to the periplasmic, linker, and receiver domains of the Agrobacterium tumefaciens VirA protein.

VirA and VirG activate the Agrobacterium tumefaciens vir regulon in response to phenolic compounds, monosaccharides, and acidity released from plant wound sites. VirA contains an amino-terminal periplasmic domain and three cytoplasmic domains: a linker, a protein kinase, and a phosphoryl receiver. We constructed internal deletions of virA that truncate one or more domains and tested the ability of the resulting proteins to mediate environmentally responsive vir gene activation in vivo. The periplasmic domain is required for sensing of monosaccharides (in agreement with earlier results), while the linker domain is required for sensing of phenolic compounds and acidity. The phosphoryl receiver domain of VirA plays an inhibitory role in signal transduction that may be modulated by phosphorylation. The carboxy terminus of the protein was also dispensable for tumorigenesis, while the periplasmic domain was required.     

The hupC gene product is a component of the electron transport system for hydrogen oxidation in Pseudomonas hydrogenovora

The hydrogenase gene cluster containing nine genes (hupSLCDFGHIJ) was identified by sequencing of an 8.8-kb DNA region from Pseudomonas hydrogenovora. To investigate the function of the hupC gene product, we isolated a hupC-null mutant (HID3) of P. hydrogenovora by introducing an in-frame deletion into the hupC. The mutant, HID3, could not grow autotrophically but retained half the level of hydrogenase activity of the wild-type strain. Results of the oxygen consumption test and Western blot analysis revealed that the hupC gene product is a b-type cytochrome but not involved in the hydrogenase maturation process.     

The Escherichia coli amidase AmiC is a periplasmic septal ring component exported via the twin-arginine transport pathway

The N-acetylmuramoyl-l-alanine amidases of Escherichia coli (AmiA, B and C) are periplasmic enzymes that remove murein cross-links by cleaving the peptide moiety from N-acetylmuramic acid. Ami- cells form chains, indicating that the amidases help to split the septal murein. Interestingly, cells defective in the twin-arginine protein transport (Tat) pathway show a similar division defect. We find that both AmiA and AmiC are routed to the periplasm via Tat, providing an explanation for the Tat- division phenotype. Taking advantage of the ability of Tat to export prefolded (fluorescent) green fluorescent protein (GFP) to the periplasm, we sublocalized AmiA and AmiC in live cells using functional fusions to GFP. Interestingly, the periplasmic localization of the fusions differed markedly. AmiA-GFP appeared to be dispersed throughout the periplasm in all cells. AmiC-GFP similarly appeared throughout the periplasm in small cells, but was concentrated almost exclusively at the septal ring in constricting cells. Recruitment of AmiC to the ring was mediated by an N-terminal non-amidase targeting domain and required the septal ring component FtsN. AmiC therefore replaces FtsN as the latest known recruit to the septal ring and is the first entirely periplasmic component to be localized.