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.

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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)

Growth of Alcaligenes eutrophus JMP134 on 2,4-dichlorophenoxyacetate requires a 2,4-dichlorophenol hydroxylase encoded by gene tfdB. Catabolism of either 2,4-dichlorophenoxyacetate or 3-chlorobenzoate involves enzymes encoded by the chlorocatechol oxidative operon consisting of tfdCDEF, which converts 3-chloro-and 3,5-dichlorocatechol to maleylacetate and chloromaleylacetate, respectively. Transposon mutagenesis has localized tfdB and t.fdCDEF to EcoRI fragment B of plasmid pJP4 (R. H. Don, A. J. Wieghtman, H.-J. Knackmuss, and K. N. Timmis, J. Bacteriol. 161:85-90, 1985). We present the complete nucleotide sequence of tfdB and tfdCDEF contained within a 7,954-base-pair HindIII-SstI fragment from EcoRI fragment B. Sequence and expression analysis of tfdB in Escherichia coli suggested that 2,4-dichlorophenol hydroxylase consists of a single subunit of 65 kilodaltons. The amino acid sequences of proteins encoded by tfdD and tfdE were found to be 63 and 53% identical to those of functionally similar enzymes encoded by clcB and ckD, respectively, from plasmid pAC27 of Pseudomonas putida. P. putida(pAC27) can utilize 3-chlorocatechol but not dichlorinated catechols. A region of DNA adjacent to clcD in pAC27 was found to be 47% identical in amino acid sequence to tfdiF, a gene important in catabolizing dichlorocatechols. The region in pAC27 does not appear to encode a protein, suggesting that the absence of a functional trans-chlorodienelactone isomerase may prevent P. putida(pAC27) from utilizing 3,5-dichlorocatechol.Bacteria play a crucial role in the dissimilation of environmental pollutants. In general, the catabolism of aromatic compounds is channeled through catechol intermediates (33). Hence, the ability of an organism to form and catabolize catechols or their derivatives can play a major part in the utilization of an aromatic compound. Comparison of the degradative pathways of different aromatics may help in understanding the evolution of growth substrate specificity.Chlorinated aromatics, such as 2,4-dichlorophenoxyacetic acid (2,4D) and 3-chlorobenzoate (3CBA), are catabolized via a modified ortho pathway (11,12,20,28,34,35; for a review, see reference 33). Pseudomonas putida plasmid pAC27, a deletion derivative of pAC25, confers catabolism of 3CBA (5), whereas Alcaligenes eutrophus JMP134 plasmid pJP4 enables degradation of both 2,4D and 3CBA (10, 11). In both cases, 3CBA appears to be degraded to 3-chlorocatechol by enzymes encoded by chromosomal genes. 2,4D is catabolized by A. eutrophus JMP134 to 2,4-dichlorophenol (2,4DCP) by 2,4D monooxygenase. 2,4DCP is then hydroxylated by 2,4DCP hydroxylase to form 3,5-dichlorocatechol (11; Fig. 1).3-Chlorocatechol is oxidized to maleylacetic acid by enzymes encoded by the clcABD operon in pAC27 (13) and by the tfdCDEF operon of pJP4 (11). The maleylacetic acid is then catabolized by chromosomally encoded enzymes (21). Enzymes involved in this transformation are chlorocatechol 1,2-dioxygenase (catechol 1,2-dioxygenase II or pyrocatechase II; EC 1.13.11.1), encoded b...

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Phytohormone overproduction in crown gall tumors is due to the expression of several T-DNA genes. The Infection of a wound site by Agrobacterium tumefaciens strains containing Ti (tumor-inducing) plasmids results in the neoplastic plant disease known as crown gall (1-5). In the plant genome, the integrated region (T-DNA) of the Ti plasmid expresses two phenotypic traits characteristic of transformed cells: phytohormone autonomy and opine synthesis. The T-DNA in tumors incited by octopine-type Ti plasmids encodes eight transcripts (6). Five of these map within three loci (tmr, tms, tml), which affect tumor morphology (7-11). Transposon inactivation of the tms locus (transcripts 1 and 2) or the tmr locus (transcript 4) results in tumors that proliferate shoots or roots, respectively. Inactivation of the tml locus (transcripts 6a and 6b) results in larger tumors on certain plant species. The region of T-DNA containing these loci is shown to be highly conserved in octopine and nopaline plasmids (12)(13)(14). The nucleotide sequences of the tmr (15,16) and tms (15,(17)(18)(19) genes have been recently determined and have been shown to be highly conserved. The synthesis of several proteins from the T-DNA region has been demonstrated in Escherichia coli minicells and in coupled transcription/translation systems of E. coli and A. tumefaciens (20). Three of these proteins had molecular weights identical to the predicted sizes of the tmr and tms gene products, as determined from the nucleotide sequence.Previous studies have also shown that the tms and tmr loci are involved in auxin and cytokinin metabolism, respectively (9-11, 21, 22). Tumors incited by tms mutants have decreased endogenous auxin levels (21) and are auxin dependent in culture (23,24

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