The 5.67-megabase genome of the plant pathogen Agrobacterium tumefaciens C58 consists of a circular chromosome, a linear chromosome, and two plasmids. Extensive orthology and nucleotide colinearity between the genomes of A. tumefaciens and the plant symbiont Sinorhizobium meliloti suggest a recent evolutionary divergence. Their similarities include metabolic, transport, and regulatory systems that promote survival in the highly competitive rhizosphere; differences are apparent in their genome structure and virulence gene complement. Availability of the A. tumefaciens sequence will facilitate investigations into the molecular basis of pathogenesis and the evolutionary divergence of pathogenic and symbiotic lifestyles.

The genetic transformation of plant cells by Agrobacterium tumefaciens is mediated by the genes of the Ti plasmid vir region. To determine the genetic and transcriptional organization of the vir region of pTiA6, vir plasmid clones were saturated with insertion mutations of a Tn3‐lacZ transposon. This element is both an insertion mutagen and a reporter for the expression of the sequences into which it has inserted. One hundred and twenty‐four vir::Tn3‐lac insertions were analyzed for their mutagenic effect on Agrobacterium virulence, and for their expression of beta‐galactosidase activity, the lacZ gene product, in vegetative bacteria and in bacteria cocultivated with plant cells. These data in conjunction with genetic complementation results show that the pTiA6 vir region contains six distinct vir complementation groups: virA, virB, virC, virD, virE and virG. Mutations in these loci eliminate (virA, virB, virD and virG) or significantly restrict (virC and virE) the ability of Agrobacterium to transform plant cells. Each of the vir loci corresponds to a single vir transcription unit: virA is constitutively expressed and non‐inducible; virB, virC, virD and virE are expressed only upon activation by plant cells; and virG is both constitutively expressed and plant‐inducible. The two largest vir operons, virB and virD, are probably polycistronic. The pTiA6 vir region also contains plant‐inducible loci (pin) which are non‐essential for virulence.

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The construction and use of a Tn3‐lac transposon, Tn3‐HoHo1, is described. Tn3‐HoHo1 can serve as a transposon mutagen and provides a new and useful system for the random generation of both transcriptional and translational lacZ gene fusions. In these fusions the production of beta‐galactosidase, the lacZ gene product, is placed under the control of the gene into which Tn3‐HoHo1 has inserted. The expression of the gene can thus be analyzed by monitoring beta‐galactosidase activity. Tn3‐HoHo1 carries a non‐functional transposase gene; consequently, it can transpose only if transposase activity is supplied in trans, and is stable in the absence of this activity. A system for the insertion of Tn3‐HoHo1 into sequences specifically contained within plasmids is described. The applicability of Tn3‐HoHo1 was demonstrated studying three functional regions of the Agrobacterium tumefaciens A6 Ti plasmid. These regions code for octopine catabolism, virulence and plant tumor phenotype. The regulated expression of genes contained within each of these regions was analyzed in Agrobacterium employing Tn3‐HoHo1 generated lac fusions.

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Renaturation kinetics of labeled Agrobacterium tumefaciens DNA are not influenced by addition of 104-fold excess of crown gall tumor DNA. Reconstruction experiments demonstrated that 0.01% added bacterial DNA produces a detectable increase in rate of renaturation of labeled DNA. Crown gall tumor DNA therefore cannot contain as much as 0.01% A. tumefaciens DNA, (one entire bacterial genome per three diploid tumor cells).By estimated levels range from 0.2% (6, 7) to 0.9% (8). DNA from A. tumefaciens bacteriophage PS8 has been reported present in tumor DNA at a level of 1.8% (8), despite the fact that neither viable PS8 phage nor PS8 DNA could be detected in the bacterial strain which incited the tumor examined. Unfortunately, most of these nucleic acid hybridization studies (5-7, 9) failed to include thermal dissociation profiles of the duplexes, an essential control to support the conclusion that authentic phage or bacterial nucleic acids were detected. Thermal dissociation profiles of bacterial or phage cRNA hybridized to filter-bound tumor DNA (8) have been reported (18)

Phenolic plant metabolites such as acetosyringone induce transcription of the virulence (vir) genes of Agrobactrium 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.Ti plasmids in Agrobacterium tumefaciens strains carry >20 virulence (vir) genes that code for most of the proteins involved in crown gall infection of wound sites on dicotyledonous plants. The biology of crown gall tumorigenesis, which involves transfer and integration of a piece of bacterial DNA into the host plant genome, has been reviewed recently (1,2). The vir genes are induced by phenolic plant metabolites such as acetosyringone. Two Ti plasmid gene products, the

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