Pilus assembly by Agrobacterium T-DNA transfer genes.

Agrobacterium tumefaciens can genetically transform eukaryotic cells. In many bacteria, pili are required for interbacterial DNA transfer. The formation of pili by Agrobacterium required induction of tumor-inducing (Ti) plasmid-encoded virulence genes and growth at low temperature. A genetic analysis demonstrated that virA, virG, virB1 through virB11, and virD4 are the only Ti plasmid genes necessary for pilus assembly. The loss and gain of pili in various mutants correlated with the loss and gain of transferred DNA (T-DNA) transfer functions, which is consistent with the view that Agrobacterium pili are required for transfer of DNA to plant cells in a process similar to that of conjugation.

Plant phenolic compounds induce expression of the Agrobacterium tumefaciens loci needed for virulence.

The virulence loci of Agrobacterium tumefaciens are a set of linked transcriptional units that play an essential role in the early stages of plant tumorigenesis. These loci are induced upon cocultivation of the bacteria with plant cells. Seven phenolic compounds that are widely distributed among the angiosperm plants--catechol, gallic acid, pyrogallic acid, p-hydroxybenzoic acid, protocatechuic acid, beta-resorcylic acid, and vanillin--are able to induce the expression of the virulence loci. These phenolics in combination induce each transcriptional locus of the vir loci. Furthermore, this induction displays similar kinetics and genetic control to that observed during cocultivation of the bacteria with plant cells.

RP4 promotion of transfer of a large Agrobacterium plasmid which confers virulence.

Introduction of RP4 plasmid into Agrobacterium tumefaciens promotes the transfer on solid medium of large virulence-associated plasmids from virulent donor strains to a plasmidless avirulent recipient. Exconjugants were selected for the ability to utilize octopine or nopaline as the sole source of arginine, traits which are coded for by virulence-associated plasmids in the strains employed here. All exconjugants retained the arginine auxotrophy of the recipient strain, and were resistant to ampicillin and kanamycin, drugs to which RP4 confers resistance. Five exconjugant clones from one cross were shown by alkaline sucrose gradient analysis to contain both RP4 plasmid and the large virulence-associated plasmid of the donor strain. All five exconjugants exhibited virulence on carrot, sunflower and kalanchoe plants. These results indicate that virulence and the ability to degrade octopine are plasmid-borne traits in A. tumefaciens strains 15955 and A6, and extend the evidence that large plasmids in A. tumefaciens are vectors of virulence genes.

Identification of the product of an Agrobacterium tumefaciens chromosomal virulence gene.

The chvB operon of Agrobacterium tumefaciens is required for bacterial attachment to plant cells and for efficient crown gall tumor formation. As defined by the virulence phenotypes of mutants with transposon insertions mapping in the region, the operon was previously mapped to a 5-kilobase (kb) stretch of chromosomal DNA. We report here that the operon is actually about 8.5 kb long and that it contains a 7-kb gene coding for a large membrane protein involved in the synthesis of cyclic beta-1,2-glucan. Mutants with transposon insertions within the 5-kb phenotypically defined operon do not synthesize this functional protein, do not synthesize beta-1,2-glucan, and do not form tumors. However, mutants with insertions that map up to 3.5 kb downstream of the phenotypically defined operon synthesize truncated proteins that are active in beta-1,2-glucan synthesis. These mutants form tumors. The truncated proteins correspond closely in size with the map positions of the insertions, suggesting that the insertions truncate the proteins by translational termination. A plasmid that contains only the phenotypically defined chvB operon also codes for a truncated protein. A fusion product between the protein and beta-galactosidase carried on a Tn3-HoHo1 insertion was observed in one mutant. Partial trypsin digestion of wild-type inner membranes generated truncated proteins that were active in beta-1,2-glucan synthesis, demonstrating that a large portion of the protein is not required for beta-1,2-glucan synthesis. The correlation between beta-1,2-glucan synthesis by the truncated proteins and tumorigenesis strongly implicates the polysaccharide product of this protein in tumor formation.

Mutants of the Agrobacterium tumefaciens virA gene exhibiting acetosyringone-independent expression of the vir regulon.

Hydroxylamine-induced mutations in the virA gene of Agrobacterium tumefaciens that do not require the plant phenolic-inducing compound acetosyringone for vir regulon induction were isolated. The isolation was based on the activation of both virB::lacZ and virE::cat fusions by mutant virA loci in the absence of acetosyringone. Three of these virA(Ais) (acetosyringone-independent signaling) mutants were characterized. All three mutants expressed a virB::lacZ fusion at high levels in the absence of acetosyringone. One virA (Ais) mutant, virA112, exhibited vir gene expression in the absence of inducing monosaccharides and acidic growth conditions, both of which are normally required for vir gene induction. The phenotype of the virA112 mutant resulted from a glycine to glutamic acid change near His-474, the site of VirA autophosphorylation.

T-DNA genes responsible for inducing a necrotic response on grape vines.

Agrobacterium tumefaciens supervirulent strain A281 induces a progressive necrotic response, rather than tumor formation, when inoculated on stems of several grape cultivars. The Ti plasmid, and specifically its T-DNA, is required for the process. In the present study, 40 T-DNA insertion mutants of A281 were generated via transposon mutagenesis and tested for their necrosis-inducing ability on grape stems in vitro. Ten mutants were attenuated in inducing necrogenesis. Restriction mapping and DNA sequencing revealed that at least two genes, tms1 and 6b, whose gene products are involved in the synthesis and activity modulation of auxin, are responsible for inducing necrogenesis. Double mutants of tms1 and 6b were totally non-necrogenic. The orientation of grapevine stem explants showed strong effects on the occurrence and progress of necrogenesis. Inoculation of Agrobacterium on physiological basal ends resulted in the greatest degree of necrogenesis. In addition, gene 5 of T-DNA, which modulates auxin responses in plants by the autoregulated synthesis of an auxin antagonist, was found to be separated from other TL-DNA genes by a novel insertion sequence, IS1312. Since a T-DNA borderlike sequence occurs in IS1312, gene 5 might not always be transferred into plants. Based on the accumulated data, we propose that the necrogenesis induced by Agrobacterium results from the sensitivity of grapevine cells to elevated levels of auxin or a precursor of auxin.

Discrete regions of the sensor protein virA determine the strain-specific ability of Agrobacterium to agroinfect maize.

The ability of Agrobacterium strains to infect transformation-recalcitrant maize plants has been shown to be determined mainly by the virA locus, implicating vir gene induction as the major factor influencing maize infection. In this report, we further explore the roles of vir induction-associated bacterial factors in maize infection using the technique of agroinfection. The Ti plasmid and virA source are shown to be important in determining the ability of a strain to infect maize, and the monosaccharide binding protein ChvE is absolutely required for maize agroinfection. The linker domain of VirAC58 from an agroinfection-competent strain, C58, is sufficient to convert VirAA6 of a nonagroinfecting strain, A348,to agroinfection competence. The periplasmic domain of VirAC58 is also able to confer a moderate level of agroinfection competence to VirAA6. In addition, the VirAA6 protein from A348 is agroinfection competent when removed from its cognate Ti plasmid background and placed in a pTiC58 background. The presence of a pTiA6-encoded, VirAA6-specific inhibitor is hypothesized and examined.

Characterization of a putative RND-type efflux system in Agrobacterium tumefaciens.

Sequencing of a 7277 bp fragment adjacent to the chvH locus of Agrobacterium tumefaciens revealed four open reading frames (ORFs), designated ameR, ameA, ameB and ameC, respectively. These ORFs exhibit amino acid similarities to components of Resistance-Nodulation-Cell Division (RND) type efflux systems. AmeA and AmeB show high homology to membrane fusion proteins (MFP) and RND-type transporters, whereas AmeC shows similarity to NodT and other members of outer membrane factor families. Mutations of the ameA and ameB genes did not affect the susceptibility profile of the wild-type strain to several detergents and antibiotics. In contrast, mutations of the ameC gene dramatically affected the susceptibility of the strain to these same inhibitory compounds. RT-PCR analysis demonstrated that the ameABC genes form an operon. In addition, ameC gene has its own promoter gene located in the intergenic region between ameB and ameC. Mapping upstream of ameA is ameR, which encodes a protein that shows similarity especially at its N-terminal end to the TetR family of bacterial transcriptional regulators. AmeR negatively regulates expression of the ameABC operon. A mutation in ameR increased the resistance of the cells to several antimicrobial agents. This regulatory locus appears to be in the same operon as a gene located upstream which codes for a product that has high similarity to numerous 4-(N-succinocarboxamide)-5-aminoimidazole ribonucleotide (SAICAR) synthetases. The possible role of the putative efflux pump coded by the ame genes is discussed.

The sensing of plant signal molecules by Agrobacterium: genetic evidence for direct recognition of phenolic inducers by the VirA protein.

The virulence (vir) genes of Agrobacterium tumefaciens are induced by low-molecular-weight phenolic compounds and monosaccharides through a two-component regulatory system consisting of the VirA and VirG proteins. Although it is clear that the monosaccharides require binding to a periplasmic binding protein before they can interact with the sensor VirA protein, it is not certain whether the phenolic compounds also interact with a binding protein or directly interact with the sensor protein. To shed light on this question, we tested the vir-inducing abilities of several different phenolic compounds using two wild-type strains of A. tumefaciens, KU12 and A6. We found that several compounds such as 4-hydroxyacetophone and p-coumaric acid induced the vir of KU12, but not A6. On the other hand, acetosyringone and several other phenolic compounds induced the vir of A6, but not KU12. By transferring different Ti plasmids into isogenic chromosomal backgrounds, we showed that the phenolic sensing determinant is associated with the Ti plasmid. Subcloning of the Ti plasmid indicated that the virA locus determines which phenolic compounds can function as vir inducers. These results suggest that VirA directly senses the phenolic compounds for vir activation.

Characterization of an unusual sensor gene (virA) of Agrobacterium.

Previous studies have shown that the virulence(vir) genes of Agrobacterium tumefaciens strain KU12 are induced by a unique set of phenolic compounds that are non-functional in most strains of Agrobacterium. Further, strain KU12 is not induced by phenolic compounds that induce the vir genes in other strains. Previous studies have shown that these differences in inducing activity result from differences in the sensor protein for these signal molecules, the VirA protein. To gain some understanding of the basis for these differences in sensing ability, we sequenced the entire virA locus of pTiKU12, including its promoter region and compared this sequence with five different published virA sequences that respond in different ways to inducing compounds. The virA gene of KU12 is composed of an open single reading frame coding for 851 aa. At the aa level, the VirA protein of pTiKU12 is 45, 45, 49, 49 and 64% identical to the VirA proteins from pTiA6, pTi15955, pRiA4, pTiC58 and pTiAg162, respectively. The transcription start sites of pTiKU12 and pTiA6 virA genes differ significantly when mapped by primer extension. Unlike all other vir genes, except the virA gene of pTiAg162, pTiKU12 virA is constitutively expressed, and its synthesis is not induced by phenolic compounds. The lack of induction is accounted for by the fact that the promoter region does not have the conserved VirG-binding dodecadeoxynucleotide sequence (vir-box) that was previously identified in all promoter regions of inducible vir genes.

The binding site of the transcriptional activator VirG from Agrobacterium comprises both conserved and specific nonconserved sequences.

Virulence genes of Agrobacterium tumefaciens are transcriptionally activated in response to phenolic compounds and certain sugars. The transcription activator VirG specifically binds to fragments containing the conserved vir box sequence present in the promoter region of all vir genes. This study shows that both the vir box as well as specific nonconserved sequences downstream of the vir box are required for VirG binding and transcriptional activation. Insertion of the identified VirG binding site into the lac promoter resulted in transcriptional activation of this heterologous promoter in response to the plant phenolic signal molecule acetosyringone.

DNA and protein transfer from bacteria to eukaryotes- the agrobacterium story.

Abstract Agrobacterium is a well-studied plant pathogen, which has the unique ability to transfer DNA and protein into a number of eukaryotes. The DNA is integrated randomly into the plant genome where it is expressed, thereby leading to the disease crown gall. This system is a paradigm for the interaction of a number of plant and animal pathogens which transfer proteins into their host cells. In Agrobacterium, the tumour inducing (Ti) plasmid codes for the functions specifically required for the transfer process. These genes, termed virulence or vir genes, are activated by plant signal molecules acting through a two component regulatory system. A key structure coded by 11 genes of the vir B operon is a pilus, synthesized at 20 degrees C, but poorly at 25 degrees C. How this pilus functions in DNA and protein transfer is unclear, but homologous genes are found in many animal pathogens. In addition to Ti plasmid-encoded vir genes, chromosomal virulence genes have also been identified. However, these mutations are often pleiotropic because they involve both the normal physiology of Agrobacterium as well as the metabolism of Agrobacterium when it is associated with plant cells. Based on 16S ribosomal RNA sequencing, Agrobacterium is closely related to the intracellular pathogen of animals, Brucella. Several chromosomal mutations of Agrobacterium required for virulence in plants are also required for invasion of animal host cells by Brucella.

Cross pathway regulation: effect of histidine on the synthesis and activity of enzymes of aromatic acid biosynthesis in Bacillus subtilis.

l-Histidine and, to a lesser degree, l-phenylalanine at concentrations of 10(-4)m inhibit the growth of leaky mutants (bradytrophs) of Bacillus subtilis that are deficient in the synthesis of p-hydroxyphenylpyruvate, the first intermediate specific to tyrosine synthesis. The inhibition can be overcome by growth factor amounts of l-tyrosine and p-hydroxyphenylpyruvate. Histidine and phenylalanine are capable of inhibiting the synthesis of tyrosine in several ways, and the major physiological effect which results in growth inhibition has not been established. Both l-histidine and l-phenylalanine inhibit the activity of prephenate dehydrogenase at concentrations about 100-fold higher than the inhibitory concentration of l-tyrosine. Histidine also appears to repress the synthesis of prephenate dehydrogenase because a histidine bradytroph growing in histidine-supplemented medium has a twofold lower level of this enzyme than the same cells growing in unsupplemented medium. These same two amino acids also inhibit the growth of a bradytroph deficient in dehydroquinate synthetase, an early enzyme in the pathway of tyrosine, phenylalanine, and tryptophan synthesis. The inhibition is overcome by a combination of tyrosine and phenylalanine. Histidine-resistant derivatives of both the prephenate dehydrogenase and dehydroquinate synthetase-deficient strains, which simultaneously have gained resistance to phenylalanine, have been isolated. Most of these resistant mutants synthesize additional tyrosine compared with the parent strain. One class of resistant mutants excretes tyrosine and has a number of enzymes of aromatic acid synthesis which are no longer repressible by any combination of the aromatic amino acids. Tyrosine inhibits the growth of histidine bradytrophs. Histidine, at growth factor levels, overcomes the inhibition.

Association of the virD2 protein with the 5' end of T strands in Agrobacterium tumefaciens.

The soil bacterium Agrobacterium tumefaciens can incite tumors in many dicotyledonous plants by transferring a portion (T-DNA) of its Ti plasmid into susceptible plant cells. The T-DNA is flanked by border sequences that serve as recognition sites for specific cleavage by an endonuclease that comprises two virD-encoded proteins (VirD1 and VirD2). After cleavage, both double-stranded, nicked T-DNA molecules and single-stranded T-DNA molecules (T strands) were present. We have determined that a protein is tightly associated with, and probably covalently attached to, the 5' end of the T strands. Analysis of deletion derivatives in Escherichia coli, immunoprecipitation, and a procedure combining immunoblot and nucleic acid hybridization data identified this protein as the gene product of virD2.