Molecular genetic characterization of essential conjugal transfer determinants on the nopaline-type Ti plasmid from Agrobacterium tumefaciens strain C58
Cook, David Michael
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https://hdl.handle.net/2142/21255
Description
Title
Molecular genetic characterization of essential conjugal transfer determinants on the nopaline-type Ti plasmid from Agrobacterium tumefaciens strain C58
Author(s)
Cook, David Michael
Issue Date
1996
Doctoral Committee Chair(s)
Farrand, Stephen K.
Department of Study
Crop Sciences
Discipline
Crop Sciences
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Biology, Molecular
Agriculture, Plant Pathology
Language
eng
Abstract
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.
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