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Journal of Bacteriology, December 2001, p. 6852-6861, Vol. 183, No. 23
0021-9193/01/$04.00+0   DOI: 10.1128/JB.183.23.6852-6861.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Elevated Temperature Differentially Affects Virulence, VirB Protein Accumulation, and T-Pilus Formation in Different Agrobacterium tumefaciens and Agrobacterium vitis Strains

Christian Baron,* Natalie Domke, Michael Beinhofer, and Siegfried Hapfelmeierdagger

Institut für Genetik und Mikrobiologie der Universität München, Lehrstuhl für Mikrobiologie, D-80638 Munich, Germany

Received 2 July 2001/Accepted 11 September 2001

That gene transfer to plant cells is a temperature-sensitive process has been known for more than 50 years. Previous work indicated that this sensitivity results from the inability to assemble a functional T pilus required for T-DNA and protein transfer to recipient cells. The studies reported here extend these observations and more clearly define the molecular basis of this assembly and transfer defect. T-pilus assembly and virulence protein accumulation were monitored in Agrobacterium tumefaciens strain C58 at different temperatures ranging from 20°C to growth-inhibitory 37°C. Incubation at 28°C but not at 26°C strongly inhibited extracellular assembly of the major T-pilus component VirB2 as well as of pilus-associated protein VirB5, and the highest amounts of T pili were detected at 20°C. Analysis of temperature effects on the cell-bound virulence machinery revealed three classes of virulence proteins. Whereas class I proteins (VirB2, VirB7, VirB9, and VirB10) were readily detected at 28°C, class II proteins (VirB1, VirB4, VirB5, VirB6, VirB8, VirB11, VirD2, and VirE2) were only detected after cell growth below 26°C. Significant levels of class III proteins (VirB3 and VirD4) were only detected at 20°C and not at higher temperatures. Shift of virulence-induced agrobacteria from 20 to 28 or 37°C had no immediate effect on cell-bound T pili or on stability of most virulence proteins. However, the temperature shift caused a rapid decrease in the amount of cell-bound VirB3 and VirD4, and VirB4 and VirB11 levels decreased next. To assess whether destabilization of virulence proteins constitutes a general phenomenon, levels of virulence proteins and of extracellular T pili were monitored in different A. tumefaciens and Agrobacterium vitis strains grown at 20 and 28°C. Levels of many virulence proteins were strongly reduced at 28°C compared to 20°C, and T-pilus assembly did not occur in all strains except "temperature-resistant" Ach5 and Chry5. Virulence protein levels correlated well with bacterial virulence at elevated temperature, suggesting that degradation of a limited set of virulence proteins accounts for the temperature sensitivity of gene transfer to plants.

* Corresponding author. Mailing address: Institut für Genetik und Mikrobiologie der Universität München, Lehrstuhl für Mikrobiologie, Maria-Ward-Str. 1a, D-80638 München, Germany. Phone: 49-89-2180-6138. Fax: 49-89-2180-996138. E-mail: cbaron{at}lrz.uni-muenchen.de.

dagger Present address: Institut für Mikrobiologie der ETH Zürich, CH-8092 Zürich, Switzerland.

Journal of Bacteriology, December 2001, p. 6852-6861, Vol. 183, No. 23
0021-9193/01/$04.00+0   DOI: 10.1128/JB.183.23.6852-6861.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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