1.5.5
MOLECULAR EVOLUTION OF TI PLASMIDS: NEW INSIGHTS INTO AN OLD DILEMMA

PM OGER1 and SK FARRAND1, 2

Departments of 1Crop Sciences and 2Microbiology, University of Illinois at Urbana-Champaign, Urbana, Il 61801, USA

Background and objectives
Agrobacterium tumefaciens is a plant pathogen responsible for the induction of crown gall tumours. In the course of infection the bacterium transfers to the plant nucleus a piece of DNA, which harbours hormone synthesis genes whose expression leads to the tumorigenic growth of the plant cell. Upon induction, the transformed plant cells synthesize novel compounds, called opines, specifically catabolized by the inciting bacterium. The pathogenicity traits of Agrobacterium are localized on a plasmid, the Ti plasmid, which is composed of four major functional regions: the T-DNA, the virulence region (vir), the opine catabolic loci and the conjugal transfer regions (tra). These plasmids are an unusual mosaic since they harbour eukaryotic and bacterial genes, and also two different DNA transfer systems.

The conjugal transfer apparatus of Agrobacterium Ti plasmids is composed of four operons, with strong homologies to the transfer apparatus of the plasmid RP4. The first region contains two divergent operons flanking the oriT and encoding all the DNA metabolic functions, while the second contains the genes necessary for the mating bridge formation. The last operon encodes the regulatory protein TraR. The regulation of the conjugal transfer apparatus is entirely dependent upon the function of this positive regulator. However, TraR-mediated activation of conjugation is itself regulated at three different levels. Firstly, traR expression is regulated by the opines present in the tumours. This is due to the positioning of the traR gene within an opine-regulated operon [1, 2]. Secondly, activation by TraR is modulated by the antiactivator TraM. Lastly, TraR is functional only when a threshold concentration of the signal molecule 3-oxo-C8 homo serine lactone is present through a mechanism known as auto-induction.

We have been examining the mechanisms which lead to the regulation of the conjugal transfer by the opines, with emphasis on nopaline, agrocinopines A and C and mannopine.

Results and conclusions
We have examined our collection for strains of Agrobacterium harbouring functional copies of traR, whose expression is dependent upon the presence of one of these opines. A restricted number of eight strains is currently being fully characterized. All but two of these strains gave a hybridization signal with a probe consisting of the traR ORF. Moreover, among the six positive strains, five contain two copies of traR. With the exception of one plasmid, both copies of the gene are functional, and are regulated by different opines. Each of these six alleles of traR from four plasmids have been cloned and sequenced.

Two copies of the traR gene are regulated by the agrocinopines A and B, two are regulated by nopaline, one is regulated by the agrocinopines C and D, and one is a non-functional copy of traR. The protein sequences of TraR are strongly conserved among the new alleles isolated. However, in most cases the sequence relatedness drops rapidly a few base pairs from either end of the traR ORF. Only in the case of the two genes regulated by agrocinopines A and B did the homology extend to more than 2 kb upstream of the traR ORF. These results suggest that in this latter case, the origin of the genes must be common. However, all of the other placements of traR within an opine-regulated operon originated from independent events. As a consequence, we postulate a strong selective pressure for the regulation of traR by the opines.

The deficient allele that we identified by hybridization presents some interesting features. This gene, present in pTiC58, is immediately downstream of a truncated copy of the agrocinopine catabolic locus. This locus lacks the first two-thirds of the first catabolic gene, while the traR ORF is truncated in its C-terminal domain. The entire locus is flanked on the right by the T-DNA and on the left by the vir region, and must therefore be a remnant of the events leading to the insertion in this region of the pathogenic functions of this Ti plasmid.

References
1. Fuqua C, Winans SC, 1994. Journal of Bacteriology 176, 2796-2806.
2. Oger PM, KIM KS, Sackett RL et al., 1998. Molecular Microbiology 27, in press.