Genetic analysis of Agrobacterium.
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DNA Transposable Elements↗
Biomedical subjects
E W Nester
Publications and source records attributed to E W Nester.
Correction: characterization of the virB operon from Agrobacterium tumefaciens Ti plasmid.
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Amino Acid Sequence↗
Sugars induce the Agrobacterium virulence genes through a periplasmic binding protein and a transmembrane signal protein.
Phenolic plant metabolites such as acetosyringone induce transcription of the virulence (vir) genes of Agrobacterium tumefaciens through the transmembrane VirA protein. We report here that certain sugars induce the vir genes synergistically with phenolic inducers by way of a distinct regulatory pathway that includes VirA and a chromosomally encoded virulence protein, ChvE. Sequence comparison showed that ChvE is a periplasmic galactose-binding protein corresponding to the GBP1 protein isolated from Agrobacterium radiobacter. Like homologous sugar-binding proteins in Escherichia coli, ChvE was required for chemotaxis toward galactose and several other sugars. These sugars strongly induced vir gene expression in wild-type cells when acetosyringone was absent or present in low concentrations. Mutations in chvE abolished vir gene induction by sugars and resulted in a limited host range for infection but did not affect vir gene induction by acetosyringone. A mutant lacking the periplasmic domain of VirA exhibited the same regulatory phenotype and limited host range as chvE mutants. These data show that the vir genes are regulated by two separate classes of plant-derived inducers by way of distinct regulatory pathways that can be separated by mutation. Induction by sugars is essential for infection of some but not all plant hosts.
Amino Acid Sequence↗
Mutational analysis of the VirG protein, a transcriptional activator of Agrobacterium tumefaciens virulence genes.
The VirG protein of Agrobacterium tumefaciens is required in conjunction with the VirA protein for transcriptional activation of the virulence (vir) genes in response to plant phenolic compounds. These proteins are members of a family of two component regulatory systems. vir genes are activated via a cascade of phosphorylation reactions involving a specific aspartic acid residue of the VirG protein. We have conducted a mutational analysis of the VirG protein. By mutating conserved and nonconserved aspartic acid residues in the N-terminal domain, we demonstrated that two of three conserved aspartic acid residues located in two different regions are important for the phosphorylation of VirG by VirA phosphate. A third conserved N-terminal region was also shown to be critical for the biological function of VirG as a transcriptional activator. The identification of phosphorylatable but biologically inactive mutated VirG proteins suggests that not only phosphorylation but also a conformational change is necessary for its activity. We further demonstrated that phosphorylation is not required for sequence-specific binding to a vir gene regulatory sequence (vir box) and that the C-terminal domain is sufficient for DNA binding. The data support the model of a two-domain structure for the VirG protein and demonstrate that the sequence homologies to other two-component regulatory systems reflect both functional and structural homologies.
Amino Acid Sequence↗
Sugar-mediated induction of Agrobacterium tumefaciens virulence genes: structural specificity and activities of monosaccharides.
The virulence genes of Agrobacterium tumefaciens are induced by specific plant phenolic metabolites and sugars (G. A. Cangelosi, R. G. Ankenbauer, and E. W. Nester, Proc. Natl. Acad. Sci. USA, in press). In this report, monosaccharides, derivatives, and analogs which induce the vir regulon have been identified and the structural requirements for monosaccharide-mediated induction have been determined. Pyranose sugars with equatorial hydroxyls at C-1, C-2, and C-3 displayed strong vir gene-inducing activity; the C-4 hydroxyl could be epimeric and a wide variety of substitutions at C-5 were permissible. The acidic monosaccharide derivatives D-galacturonic acid and D-glucuronic acid were the strongest inducers among the monosaccharides tested. Eight of the 11 inducing compounds are known plant metabolites, and 7 are monomers of major plant cell wall polysaccharides. A role for monosaccharides and plant phenolic compounds as wound-specific plant metabolites which signal the ChvE/VirA/VirG regulatory system is proposed.
Bacterial Proteins↗
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.
Amino Acids↗
The regulatory VirG protein specifically binds to a cis-acting regulatory sequence involved in transcriptional activation of Agrobacterium tumefaciens virulence genes.
Virulence genes of Agrobacterium tumefaciens are induced in parallel in the presence of plant phenolic compounds such as acetosyringone and the two regulatory vir genes virA and virG. In this study we identified a cis-acting regulatory sequence in the 5'-noncoding region of the virE operon that is essential for this activation. To do this, we constructed a series of deletion mutants by using exonuclease Bal 31. Western blot (immunoblot) analysis showed that the 70 base pairs upstream of the transcriptional start site were sufficient for full virE gene induction. A conserved dodecadeoxynucleotide sequence (vir box), which was previously identified in the nontranscribed sequences of all vir genes, was located at 5' end of the minimum required promoter sequence. Deletion of this vir box only completely abolished induction of the virE gene. This demonstrates that the vir box functions as an upstream regulatory sequence. To study the role of the VirG protein in the activation process, we overproduced the native-sized VirG protein in Escherichia coli by fusing the lacZ' start codon ATG with the second virG codon AAA using site-directed mutagenesis. The VirG protein was purified and renatured from E. coli and was shown to bind to a specific sequence in two vir gene promoters. Footprinting analysis of the virE and virB promoters identified the 12-base-pair vir box as the VirG-binding core sequence.
Bacterial Proteins↗
Biochemical characterization of avirulent exoC mutants of Agrobacterium tumefaciens.
The synthesis of periplasmic beta(1-2)glucan is required for crown gall tumor formation by Agrobacterium tumefaciens and for effective nodulation of alfalfa by Rhizobium meliloti. The exoC (pscA) gene is required for this synthesis by both bacteria as well as for the synthesis of capsular polysaccharide and normal lipopolysaccharide. We tested the possibility that the pleiotropic ExoC phenotype is due to a defect in the synthesis of an intermediate common to several polysaccharide biosynthetic pathways. Cytoplasmic extracts from wild-type A. tumefaciens and from exoC mutants of A. tumefaciens containing a cloned wild-type exoC gene synthesized in vitro UDP-glucose from glucose, glucose 1-phosphate, and glucose 6-phosphate. Extracts from exoC mutants synthesized UDP-glucose from glucose 1-phosphate but not from glucose or glucose 6-phosphate. Membranes from exoC mutant cells synthesized beta(1-2)glucan in vitro when exogenous UDP-glucose was added and contained the 235-kilodalton protein, which has been shown to carry out this synthesis in wild-type cells. We conclude that the inability of exoC mutants to synthesize beta(1-2)glucan is due to a deficiency in the activity of the enzyme phosphoglucomutase (EC 2.7.5.1), which in wild-type bacteria converts glucose 6-phosphate to glucose 1-phosphate, an intermediate in the synthesis of UDP-glucose. This interpretation can account for all of the deficiencies in polysaccharide synthesis which have been observed in these mutants.
Chromatography, Gel↗
A chromosomal Agrobacterium tumefaciens gene required for effective plant signal transduction.
The vir gene products of Agrobacterium tumefaciens carry out the transfer of T-DNA to the plant genome. Effective transcriptional induction of the vir genes by plant signal molecules is controlled by two vir gene products, VirA and VirG. In this study we have identified and cloned a chromosomal region which is also required for vir gene induction. Transposon insertions within this region reduce induction significantly and strongly attenuate virulence, resulting in a restricted host range for infection. The reduction in vir gene transcription can be partially overcome by high concentrations of the inducer molecule acetosyringone. Expression of virG at low pH and low phosphate concentrations, which is independent of plant signals, is not affected by these mutations. Sequence analysis of the region revealed two divergent open reading frames, which we have designated chvE and ORF1. Several transposon insertions mapped in chvE; this resulted in attenuated virulence. chvE codes for a putative protein which is homologous to two periplasmic receptor proteins involved in chemotaxis and uptake of sugars. Whether ORF1 is required for virulence is uncertain. One transposon insertion resulting in avirulence maps in or near the 5' end of ORF1, and several which do not affect virulence map in its 3' end. ORF1 codes for a putative protein which is homologous to a family of transcriptional activator proteins.
Acetophenones↗
Osmosensitivity phenotypes of Agrobacterium tumefaciens mutants that lack periplasmic beta-1,2-glucan.
The periplasmic cyclic beta-1,2-glucan of Agrobacterium tumefaciens is believed to maintain high osmolarity in the periplasm during growth of the bacteria on low-osmotic-strength media. Strains with mutations in the chvA or chvB gene do not accumulate beta-1,2-glucan in their periplasm and exhibit pleiotropic phenotypes, including inability to form crown gall tumors on plants. We examined the effects of medium osmolarity to determine whether some or all of these phenotypes result from suboptimal periplasmic osmolarity. The mutants grew more slowly than wild-type cells and exhibited altered periplasmic and cytoplasmic protein content when cultured in low-osmotic-strength media, but not when cultured in high-osmotic-strength media. These observations support a role for periplasmic glucan in osmoadaptation. However, the mutants were avirulent and exhibited reduced motility regardless of the osmolarity of the medium. Therefore, beta-1,2-glucan may play roles in virulence and motility that are unrelated to its role in osmoadaptation.
Bacterial Proteins↗
Nucleotide sequence of the traI (helicase I) gene from the sex factor F.
A 6.9-kilobase region of the Escherichia coli F plasmid containing the 3' half of the traD gene and the entire traI gene (encodes the TraI protein, DNA helicase I and TraI, a polypeptide arising from an internal in-frame translational start in traI) has been sequenced. A previously unidentified open reading frame (tentatively trbH) lies between traD and traI.
Amino Acid Sequence↗
Phosphorylation of the VirG protein of Agrobacterium tumefaciens by the autophosphorylated VirA protein: essential role in biological activity of VirG.
Agrobacterium tumefaciens virulence genes are induced by plant signals through the VirA-VirG two-component regulatory system. The VirA protein is a membrane-spanning sensor molecule that possesses an autophosphorylating activity, and the VirG protein is a sequence-specific DNA-binding protein. In this report, we demonstrate that the VirG protein is phosphorylated by the VirA protein and that the phosphate is directly transferred from the phosphorylated VirA molecule (phosphohistidine) to the VirG protein. The chemical stability of the phospho-VirG bond suggested that the VirG protein was phosphorylated at the aspartate and/or glutamate residue. The phosphorylated VirG protein was reduced with tritiated sodium borohydride and subjected to proteolytic digestion with the Achromobacter protease I enzyme. The resulting peptide fragments were separated by C8 reversed-phase high-pressure liquid chromatography, and the tritium-labeled peptide was sequenced. Amino acid sequence data showed that the aspartate residue at position 52 was the only site phosphorylated. Changing this aspartate into asparagine resulted in a nonphosphorylatable and biologically nonfunctional gene product. As a control, a randomly chosen aspartate was changed into an asparagine (position 72), and no effect on its phosphorylation or biological activity was observed. Unlike its homologs, including CheA-CheY, EnvZ-OmpR, and NtrB-NtrC, the phospho-VirG molecule was very stable in vitro. The possible implications of these observations and the function of VirG phosphorylation in vir gene activation are discussed.
Aspartic Acid↗
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.
Bacterial Proteins↗
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.
Amino Acid Sequence↗
A gene required for transfer of T-DNA to plants encodes an ATPase with autophosphorylating activity.
The virB operon of the Agrobacterium tume-faciens pTiA6NC plasmid likely plays a role in directing T-DNA transfer events at the bacterial membrane, as determined previously by mutagenesis and cellular fractionation studies and by DNA sequence analysis of the approximately 12-kilobase-pair operon. The DNA sequence analysis also revealed consensus mononucleotide binding domains in the deduced virB5 and virB11 gene products, suggesting that one or both of these proteins couple energy, by means of nucleotide triphosphate (NTP) hydrolysis, to T-DNA transport. In this report, the product of virB11, an essential virulence gene, was overproduced in Escherichia coli and purified by using immunoaffinity chromatography. The immunoaffinity purified protein, as well as NaDodSO4/polyacrylamide gel-eluted protein, bound and hydrolyzed ATP in the absence of DNA effectors. VirB11 protein also demonstrated in vitro autophosphorylation activity. VirB11 protein was localized primarily to the cytoplasmic membrane by immunoblot analysis of membrane fractions. The deduced VirB11 protein exhibits sequence similarity to comG ORF1, a protein required for uptake of DNA by competent Bacillus subtilis cells. These findings suggest that phosphorylation may serve to activate a component(s) of the A. tumefaciens T-DNA transport apparatus and may also represent a general activation mechanism of other bacterial DNA transport systems.
Adenosine Triphosphatases↗
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.
Bacterial Proteins↗
Role for [corrected] Agrobacterium tumefaciens ChvA protein in export of beta-1,2-glucan.
Functional chvA and chvB genes are required for attachment of Agrobacterium tumefaciens to plant cells, an early step in crown gall tumor formation. Strains defective in these loci do not secrete normal amounts of cyclic beta-1,2-glucan. Whereas chvB is required for beta-1,2-glucan synthesis, the role of chvA in glucan synthesis or export has not been clearly defined. We found that cultures of chvA mutants contained as much neutral beta-1,2-glucan in the cell pellets as did the wild type, with no detectable accumulation of glucan in the culture supernatant. The cytoplasm of chvA mutant cells contained over three times more soluble beta-1,2-glucan than did the cytoplasm of the wild-type parent. Unlike the wild type, chvA mutants contained no detectable periplasmic glucan. The amino acid sequence of chvA is highly homologous to the sequences of bacterial and eucaryotic export proteins, as observed previously in the case of ndvA, a rhizobial homolog of chvA. Strong sequence homology within this family of export proteins is concentrated in the carboxy-terminal portions of the proteins, but placement of consensus ATP-binding sites, internal signal sequences, and hydrophobic domains are conserved over their entire lengths. These data suggest a model for beta-1,2-glucan synthesis in A. tumefaciens in which glucan is synthesized inside the inner membrane with the participation of ChvB and transported across the inner membrane with the participation of ChvA.
ATP-Binding Cassette Transporters↗
A protein required for transcriptional regulation of Agrobacterium virulence genes spans the cytoplasmic membrane.
The VirA protein is one of two proteins required for transcriptional activation of Agrobacterium tumefaciens virulence genes in response to phenolic compounds released by plants during infection. We describe two experimental approaches which indicate that this protein has a transmembrane topology. First, spheroplasts of Escherichia coli or wild-type A. tumefaciens expressing the VirA protein were treated with proteinase K to digest periplasmic proteins, and the remaining proteins were immunologically stained on Western blots (immunoblots) by using anti-VirA antibody. Second, transposon TnphoA was used to generate translational fusions between virA and phoA, the latter of which is the structural gene for alkaline phosphatase. Both techniques indicated that VirA spans the cytoplasmic membrane, with approximately 275 amino acids near the amino terminus being localized in the periplasmic space and the rest of the protein being localized in the cytoplasm. We also show that overexpression of VirA in E. coli is deleterious to cell growth and that this phenomenon depends on the synthesis of either the second hydrophobic core or some nearby portion of the VirA protein.
Bacterial Proteins↗