Molecular genetic characterization of essential conjugal transfer determinants on the nopaline-type Ti plasmid from Agrobacterium tumefaciens strain C58

Ti plasmids of Agrobacterium tumefaciens encode a conjugal transfer system. I characterized a region, called tra, that is necessary for transfer of the nopaline-type Ti plasmid, pTiC58. tra contains the origin of conjugal transfer (oriT) and a cluster of genes the products of which are predicted to function as DNA processing and transfer components. The oriT is contained within a 260-bp intergenic region flanked by transfer genes. Analysis of Tn3HoHo1-generated mutations that abolish transfer of the Ti plasmid defined an essential region of approximately six kilobases surrounding the oriT site. Expression from lacZ fusions in the tra region indicated that there is divergent transcriptional activity on both sides of the oriT site. I determined the nucleotide sequence of 8755-bp that spans this region on pTiC58. Eight ORFs, traA-D, traF, traG, and orfX, were predicted from the sequence. Directed expression of gene products from traA, traC, traD, and traG in E. coli, confirmed that these ORFs encode proteins of the predicted sizes. Deduced amino acid sequences from tra genes showed that proteins encoded by tra are similar to other transfer proteins. I also examined the regulation of gene expression within the tra locus and found that in addition to the transcriptional activator, TraR and its coinducer, the N-acyl homoserine lactone molecule, AAI, control of gene activation in tra requires two other determinants. One regulatory determinant is the cis-acting promoter sequence, called the tra box, found in the intergenic region. This DNA sequence is required for expression of the divergent tra operons that is mediated by TraR and AAI. The second regulatory determinant is the product of the traM gene which modulates activation of tra genes mediated by TraR and AAI. Studies on activation of expression of tra genes led to the localization of the gene, traI, which is responsible for production of AAI in Agrobacterium. Finally, I used the information gathered from the characterization of the tra locus and its regulation to reconstitute the transfer regions from pTiC58 on two independently replicating plasmids as a binary conjugal transfer system.

Name thesis:degree_name

Ph.D.

Level thesis:degree_level

Dissertation

Discipline thesis:degree_discipline

Crop Sciences

Grantor

University of Illinois at Urbana-Champaign

Year dc:date

1996

Author dc:creator

• Cook, David Michael

Contributors dc:contributor

• Farrand, Stephen K.

• Biology, Molecular
• Agriculture, Plant Pathology

Statement dc:rights

• Copyright 1996 Cook, David Michael

Language dc:language

eng

Identifier

AAI9625125
(UMI)AAI9625125

OAI identifier oai:identifier

oai:www.ideals.illinois.edu:2142/21255