Stability of the Agrobacterium tumefaciens VirB10 protein is modulated by growth temperature and periplasmic osmoadaption

Abstract

Export of oncogenic T-DNA from the phytopathogen Agrobacterium tumefaciens is mediated by the products of the virB operon. It has recently been reported (K. J. Fullner and E. W. Nester, J. Bacteriol. 178:1498-1504, 1996) that DNA transfer does not occur at elevated temperatures; these observations correlate well with much earlier studies on the temperature sensitivity of crown gall tumor development on plants. In testing the hypothesis that this loss of DNA movement reflects a defect in assembly or maintenance of a stable DNA transfer machinery at high temperature, we have found that steady-state levels of VirB10 are sensitive to growth temperature while levels of several other VirB proteins are considerably less affected. This temperature-dependent failure to accumulate VirB10 is exacerbated in an attachment-deficient mutant strain (chvB) which exhibits pleiotropic defects in periplasmic osmoadaption, and virulence of a chvB mutant can be partially restored by lowering the temperature at which the bacteria and the plant tissue are cocultivated. Furthermore, the stability of VirB10 is diminished in cells lacking functional VirB9, but only under conditions of low osmolarity. We propose that newly synthesized VirB10 is inherently labile in the presence of a large osmotic gradient across the inner membrane and is rapidly degraded unless it is stabilized by VirB9-dependent assembly into oligomeric complexes. The possibility that VirB10-containing complexes are not assembled properly at elevated temperatures suggests an explanation for the decades-old observation that tumor formation is exquisitely sensitive to ambient temperature.

FIG. 1

Cross-linking conditions influence the stability of VirB10 in Agrobacterium cells lacking wild-type VirB9. Cells of wild-type strain A348 (lanes 1 to 6) or strain Ax42 (virB9::Tn5virB) were induced overnight at 25°C in the presence of 200 μM AS. Cells were pelleted and incubated in 50 mM sodium phosphate buffer, pH 7.6, either at room temperature (RT) (lanes 1 to 4 and 7 to 10) or on ice (lanes 5 to 6 and 11 to 12) for the indicated lengths of time. For the samples in lanes 4, 6, 10, and 12, the cross-linking agent BS³ was included in the incubation at a final concentration of 500 μM (X). At the end of the incubation, cells were quenched with an equal volume of 50 mM Tris-Cl, pH 7.5, and processed for SDS-PAGE and immunoblotting analysis with anti-VirB10 antibodies, as described in Materials and Methods. The migration positions of proteins of known molecular mass (in kilodaltons) are shown at the left.

FIG. 2

Effect of growth temperature on the accumulation of VirB9 and VirB10. A. tumefaciens A348 was induced overnight in ABIM containing 200 μM AS at either 19 or 28°C, as indicated. Cells were washed three times in sodium phosphate buffer (pH 7.6), cross-linked with 200 μM BS³ (lanes 3 and 4) or DSS (lanes 5 and 6), and processed for electrophoresis on an SDS–7% (A) or –10% (B) polyacrylamide gel and immunoblotting as described in Materials and Methods. (A) Probed with anti-VirB10; (B) probed with anti-VirB9. The migration positions of proteins of known molecular mass (in kilodaltons) are shown at the right.

FIG. 3

Accumulation of VirB proteins at 19 and at 28°C in the presence and absence of functional VirB9. A. tumefaciens A348 (wild type) (lanes 1 and 2) and Ax42 (virB9::Tn5virB) (lanes 3 and 4) were grown and induced in ABIM containing 200 μM AS at either 19°C (odd-numbered lanes) or 28°C (even-numbered lanes). Cells were washed in sodium phosphate buffer and cross-linked with BS³, as described in Materials and Methods, prior to being processed for immunoblotting. (A) Probed with anti-VirB10; (B) probed with anti-VirB8; (C) probed with anti-VirB11. The migration positions of proteins of known molecular mass (in kilodaltons) are shown at the left.

FIG. 4

Accumulation of VirB proteins in wild-type and chvB mutant cells at 19 and 28°C. A. tumefaciens A348 (wild type) (lanes 1 and 3) and A1020 (chvB::Tn5) (lanes 2 and 4) were induced overnight at either 19 or 28°C, as indicated, in ABIM containing 200 μM AS. Cells were pelleted, and total crude extracts were subjected to electrophoresis on a 10% gel and immunoblot analysis as described in Materials and Methods. (A) Probed with anti-VirB10; (B) probed with anti-VirB11; (C) probed with anti-VirB9; (D) probed with anti-VirB8. The migration positions of proteins of known molecular mass (in kilodaltons) are shown at the left.

FIG. 5

Tumor formation by a chvB mutant strain at 19°C but not at 28°C. Tobacco leaf explants were cocultivated at either 19 or 28°C, as described in Materials and Methods, with A. tumefaciens A348 (wild type) or A1020 (chvB::Tn5) which had been grown overnight in TYC at the same temperature. The explants were transferred to selection medium containing hormone-free MS supplemented with 200 μg of timentin per ml and incubated at the same temperature for 3 days and then at 22°C for 21 days.

FIG. 6

Comparison of VirB10 levels in a virB9 nonpolar mutant washed in sodium phosphate buffer or in ABIM. A. tumefaciens A348 (wild-type) and Ax42 (virB9::Tn5virB) were induced overnight in ABIM containing 200 μM AS at either 19°C (lanes 1 to 4) or 28°C (lanes 5 to 8). Cells were pelleted and washed three times in either 50 mM sodium phosphate buffer, pH 7.6 (odd-numbered lanes), or ABIM without AS (even-numbered lanes). After incubation for 30 min at room temperature in the same solutions, cells were processed for SDS-PAGE and immunoblotting, using anti-VirB10, as described in Materials and Methods. The migration positions of proteins of known molecular mass (in kilodaltons) are shown at the left.

FIG. 7

Analysis of VirB protein stability in the presence or absence of functional VirB9. A. tumefaciens A348 (wild type) and Ax42 (virB9::Tn5virB) were induced at 19°C (A to D) or 28°C (E) in ABIM containing 200 μM AS to an optical density at 600 nm of approximately 0.5. Chloramphenicol was added to a final concentration of 100 μg/ml, and incubation was continued at the same temperature for the indicated number of hours. Total crude extracts were analyzed by SDS-PAGE and immunoblotting as described in Materials and Methods. (A and E) Probed with anti-VirB10; (B) probed with anti-VirB8; (C) probed with anti-VirB11; (D) probed with anti-VirB5. The migration positions of proteins of known molecular mass (in kilodaltons) are shown at the left.