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Journal of Bacteriology, August 1999, p. 5033-5041, Vol. 181, No. 16
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Essential Components of the Ti Plasmid trb System, a Type IV Macromolecular Transporter

Pei-Li Li,1 Ingyu Hwang,1,dagger Heather Miyagi,2 Heather True,2,Dagger and Stephen K. Farrand1,2,*

Departments of Crop Sciences1 and Microbiology,2 University of Illinois at Urbana-Champaign, Urbana, Illinois 61801

Received 2 April 1999/Accepted 11 June 1999

The trb operon from pTiC58 is one of three loci that are required for conjugal transfer of this Ti plasmid. The operon, which probably codes for the mating bridge responsible for pair formation and DNA transfer, contains 12 genes, 11 of which are related to genes from other members of the type IV secretion system family. The 12th gene, traI, codes for production of Agrobacterium autoinducer (AAI). Insertion mutations were constructed in each of the 12 genes, contained on a full-length clone of the trb region, using antibiotic resistance cassettes or a newly constructed transposon. This transposon, called mini-Tn5Ptrb, was designed to express genes downstream of the insertion site from a promoter regulated by TraR and AAI. Each mutation could trans complement downstream Tn3HoHo1 insertions in the trb operon of full-sized Ti plasmids. When marker-exchanged into the transfer-constitutive Ti plasmid pTiC58Delta accR mutations in trbB, -C, -D, -E, -L, -F, -G, and -H abolished conjugal transfer from strain UIA5, which lacks the 450-kb catabolic plasmid pAtC58. However, these mutants retained residual conjugal transfer activity when tested in strain NT1, which contains this large plasmid. The trbJ mutant failed to transfer at a detectable frequency from either strain, while the trbI mutant transferred at very low but detectable levels from both donors. Only the trbK mutant was unaffected in conjugal transfer from either donor. Transfer of each of the marker-exchange mutants was restored by a clone expressing only the wild-type allele of the corresponding mutant trb gene. An insertion mutation in traI abolished the production of AAI and also conjugal transfer. This defect was restored by culturing the mutant donor in the presence of AAI. We conclude that all of the trb genes except trbI and trbK are essential for conjugal transfer of pTiC58. We also conclude that mutations in any one of the trb genes except traI and trbJ can be complemented by functions coded for by pAtC58.

* Corresponding author. Mailing address: Department of Crop Sciences, University of Illinois at Urbana-Champaign, 240 Edward R. Madigan Laboratory, 1201 West Gregory Dr., Urbana, IL 61801. Phone: (217) 333-1524. Fax: (217) 244-7830. E-mail: stephenf{at}uiuc.edu.

dagger Present address: Plant Protectants Research Unit, Korean Research Institute of Bioscience and Biotechnology, Yusung, Taejon, South Korea 305-600.

Dagger Present address: Howard Hughes Medical Institute, Department of Molecular Genetics and Cell Biology, The University of Chicago, Chicago, IL 60637.

Journal of Bacteriology, August 1999, p. 5033-5041, Vol. 181, No. 16
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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