INTRODUCTION

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The soil bacterium Agrobacterium tumefaciens induces the formation of crown gall tumors on a number of susceptible plants, including most dicotyledenous species. Infection, which occurs at a wound site, requires the presence in the bacterium of a 200-kb tumor-inducing (Ti) plasmid. In a mechanism analogous to conjugal transfer, a single-stranded portion of the Ti plasmid, the T-DNA, is excised and localized to the host plant cell nucleus, where it is stably integrated into the host genome. Expression of genes on the T-DNA encoding biosynthetic enzymes results in the unregulated production of plant growth hormones and uncontrolled plant cell division. Bacterial genes required for transformation include the products of the vir regulon, located on a nontransferred region of the Ti plasmid (for reviews, see references 3, 42, and 61).

Movement of the T-DNA out of the bacterial cell is mediated by the products of the virB operon (for a review, see reference 18). The 11 virB open reading frames exhibit sequence similarity to genes required for the conjugal transfer of plasmids from a number of incompatibility groups (47, 52, 59). Furthermore, the virB genes show considerable conservation in both nucleotide sequence and gene order with the ptl operon, required for the secretion of pertussis toxin from the mammalian pathogen Bordetella pertussis (22, 31, 80). Ten of the 11 virB genes are required for tumorigenesis, although not for the generation of the T strand (8, 9, 24, 26, 37, 64, 78). Almost all of the VirB proteins examined are found in association with the membrane system of A. tumefaciens (8, 20, 25, 33, 34, 44, 60, 63, 68, 73, 74, 79). The recent observation of pili on the surface of A. tumefaciens (38, 46, 51) lends credence to a model in which T-DNA transfer is mediated by a sex pilus similar to those mediating conjugation of a variety of mobilizable plasmids. Further support for the notion that one or more virB gene products are responsible for the elaboration of the pilus comes from the finding that pilus formation requires only virA, virG, virB1 to 11, and one more gene, virD4, which also encodes a membrane-associated protein (58) implicated in T-DNA transfer per se (38, 53).

Several lines of evidence suggest that interactions among VirB proteins are crucial to the assembly of a T-DNA transport apparatus in the bacterial cell membrane. Disulfide bonding between VirB9, located in the periplasm, and VirB7, an outer-membrane-associated lipoprotein (4, 32), stabilizes VirB9, and both VirB7 and VirB9 can also form what are likely to be homomeric complexes (1, 4, 6, 33, 66). Formation of the VirB7-VirB9 dimer appears to play a central role in promoting assembly of the putative VirB-protein transport complex, since both virulence and accumulation of VirB4, VirB5, and VirB8 to -11 are correlated with the modulation of VirB7 levels (33). VirB9 is also required for the formation of VirB10-containing high-molecular-weight aggregates, although VirB9 and VirB10 are not components of the same cross-linkable complexes, and random mutagenesis has been used to identify specific residues in VirB9 that are necessary for VirB10 assembly into complexes (6). VirB11 also appears to interact with VirB9 and VirB10, as evidenced by the recent report that overexpression of virB9, virB10, and/or virB11 (but not virB8) can suppress the avirulent phenotype associated with dominant mutations in virB11 (84). Finally, it has been proposed that VirB9, -10, and -11 may act in a rate-limiting capacity in the assembly of the T-DNA transport machinery; VirB-dependent movement of an RSF1010 derivative into plants (14) suppresses tumor formation by the T-DNA, but overexpression of virB9 to vir11 restores tumorigenicity (77).

It has been known for some time that tumor formation on several host species is optimal at 22°C and does not occur at temperatures above 29°C (62). Loss of virulence at high temperatures is not due to the effects of temperature on the plants (11). Temperature-dependent suppression of tumorigenesis is also not attributable to alterations in the levels of vir gene transcription, which were found to be comparable at 19 and 28°C and only slightly less than the maximal transcriptional induction observed at 22°C (39). Inhibition of vir gene induction does occur at temperatures above 32°C and is likely due to an inactivating conformational change in VirA (43). However, even in a virG mutant strain constitutively expressing the vir genes at 32°C, tumor formation does not occur (43). Using the virB-dependent conjugal transfer of an RSF1010 derivative (7) as a measure of DNA movement across the bacterial membrane, Fullner and Nester recently demonstrated that the avirulence observed at temperatures above 28°C could be attributed to a nonfunctional DNA transfer machinery (39). However, the specific basis for this lack of transfer was not determined.

In A. tumefaciens, growth in hypoosmotic medium results in the accumulation of cyclic -1,2-glucan in the periplasm (55). Since synthesis of this polysaccharide is reduced in cells growing under conditions of high osmolarity, it is believed that regulated synthesis and export of the glucan to the periplasm provides a mechanism by which the cell can minimize the osmotic gradient across the inner membrane (55). One of the chromosomally encoded gene products required for the periplasmic accumulation of -1,2-glucan is ChvB, which catalyzes the synthesis of the oligosaccharide from UDP-glucose (85). It has been proposed that the presence of the oligosaccharide in the periplasm may stabilize outer membrane proteins against improper assembly or disassembly (71). By the same token, it seems likely that an excessive osmotic gradient across the inner membrane, such as in a chvB mutant grown in low-osmolarity medium, might well compromise the stability of membrane-associated protein complexes and could contribute to the observed avirulence associated with mutations in chvB.

In this study, we have investigated the role of growth temperature and, indirectly, osmoregulatory mechanisms in the stability of components of the T-DNA transport apparatus. Our findings demonstrate that accumulation of VirB10 is significantly decreased in cells grown at 28°C, compared to that in cells grown at 19°C, and is further diminished in cells lacking a functional ChvB protein. We have also found that VirB9 protects VirB10 from degradation under conditions of osmotic stress. Our data suggest a model in which VirB9-dependent assembly of VirB10 stabilizes the inherently labile protein against turnover. We propose that a lack of VirB10 stabilization at 28°C contributes to the previously documented defect in the T-DNA transfer process at high temperature.

	MATERIALS AND METHODS

Top Abstract Introduction Materials & Methods Results Discussion References

Bacterial strains and media. The A. tumefaciens wild-type strain used was A348, which has the C58 chromosomal background and carries the pTiA6NC plasmid (40). The mutant strains used were Ax42 (A348 containing pTiA6 virB9::Tn5virB) (24), 358mx (A348 containing pTiA6 virE::Tn3HoHo1) (67), and A1020, which is A348 chvB::Tn5 (28). Cells were grown on MG/L, and ABIM induction medium was prepared as described previously (34). TYC medium is 0.5% tryptone-0.3% yeast extract supplemented with 7 mM CaCl₂ (70).

Chemicals and reagents. Acetosyringone (AS; 3',5'-dimethoxy-4'-hydroxyacetophenone) was purchased from Aldrich (Milwaukee, Wis.). Chemical cross-linking agents disuccinimidyl suberate (DSS) and bis(sulfosuccinimidyl suberate) (BS³) were obtained from Pierce (Rockford, Ill.). Timentin was the product of SmithKline Beecham (Philadelphia, Pa.). The ECL Western blotting analysis kit, secondary antibody (donkey anti-rabbit antibody conjugated to horseradish peroxidase), and prestained molecular weight markers were purchased from Amersham (Arlington Heights, Ill.). Acrylamide solutions were obtained from Bio-Rad Laboratories (Hercules, Calif.). Nitrocellulose was the product of Schleicher and Schuell (Keene, N.H.). All other chemicals were the products of Sigma Chemical Co. (St. Louis, Mo.).

Cross-linking and immunoblotting. Whole cells were treated with either BS³ or DSS as described previously (6), except that 6 ml of cells was pelleted as the starting material. Cross-linking reactions were quenched with 20 mM N-ethylmaleimide in 50 mM Tris-HCl (pH 7.5) and incubated for 5 min at room temperature unless otherwise noted. Where indicated, the washing steps were omitted and the cross-linking reaction was performed on ice. Samples were processed for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) by pelleting the cells, either after the washing and cross-linking steps or directly out of the induction medium, and resuspending them in sample buffer (0.1 M Tris-Cl [pH 6.8], 1.2 M -mercaptoethanol, 4.3% SDS, 0.13% bromophenol blue, 9.8% glycerol). The volumes of sample buffer were adjusted so that each sample represented an equivalent number of cells per milliliter. Samples were denatured by boiling for 5 min and subjected to electrophoresis on 7 or 10% acrylamide gels (50). Proteins were transferred to nitrocellulose in a Tris-glycine-methanol transfer buffer, using a Hofer (San Francisco, Calif.) transblot apparatus. Nitrocellulose filters were exposed to primary antisera raised in rabbits and secondary donkey anti-rabbit antisera. Detection was performed with an ECL enhanced chemiluminescence kit as described by the manufacturer (Amersham) Antisera recognizing VirB5 (34), VirB8 (34), VirB9 (24), VirB10 (79), and VirB11 (20) have been previously characterized. The more rapidly migrating bands seen in several of the immunoblotting figures (particularly with anti-VirB10 and anti-VirB11) are presumed to be proteolytic products and, at least for VirB10, have been previously reported (79, 84).

Virulence assays. Leaf square transformation assays were performed on Nicotiana tabacum cv. Havana 425 basically as described previously (6). Bacterial cultures to be tested for virulence were grown at either 19 or 28°C. Where indicated, the bacterial strains were subcultured to ABIM containing 200 µM AS and induced overnight at either 19 or 28°C prior to inoculation. Leaf square explants were infected with a suspension of agrobacteria at an A₆₀₀ of 0.3 to 0.5. After 2 days of cocultivation at 19 or 28°C on hormone-free Murashige and Skoog (MS) medium (56) supplemented with 100 µM AS, the leaf pieces were washed and then placed on hormone-free MS plates containing 200 µg of timentin per ml to kill the bacteria. After 3 more days of incubation at either 19 or 28°C, the leaf pieces were transferred to 22°C, and they were incubated at that temperature until they were scored.

	RESULTS

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VirB9 is required for retention of VirB10 in cells washed in buffer. VirB10 exists as a component of several high-molecular-weight complexes that can be visualized by exposing whole cells to chemical cross-linking agents prior to preparation for SDS-PAGE and immunoblotting analysis (79). Beaupre et al. (6) have recently demonstrated that wild-type VirB9 is required for the assembly of VirB10 into cross-linkable aggregates; in strain Ax42, which contains a nonpolar Tn5virB transposon insertion in the virB9 gene (24), no cross-linked complexes are seen. Furthermore, the monomeric form of VirB10 was also almost undetectable in Ax42 cells that had been treated with the cross-linking agent BS³. However, in that study, the cells were washed three times in sodium phosphate buffer (pH 7.6) prior to exposure to BS³ for 30 min at room temperature (6). When we repeated these cross-linking experiments on strain Ax42, omitting the washing steps and incubating with BS³ on ice rather than at room temperature, we obtained a significantly different result (Fig. 1, compare lanes 10 and 12). In particular, the monomeric form of VirB10 (which migrates with an apparent molecular mass of approximately 48 kDa) was far more abundant in cells that had been cross-linked on ice without washing (lane 12); by comparing this sample with a sample pelleted directly from the induction medium and lysed by boiling in SDS-PAGE sample buffer (lane 7), we concluded that in unwashed cells incubated on ice, little loss of VirB10 monomer had occurred. Significantly, however, even under these experimental conditions, at which the monomer levels in strain Ax42 were maintained, no VirB10-containing high-molecular-weight aggregates were observed.

View larger version (51K): [in this window] [in a new window]   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.

Steady-state levels of VirB10 are greatly diminished in cells grown at 28°C relative to those in cells grown at 19°C. In light of the apparent role of VirB proteins in the formation of a membrane-associated T-DNA transport apparatus, it seems likely that environmental conditions which affect the stability of VirB proteins or impede proper assembly of VirB-containing complexes would result in attenuated virulence. Indeed, experiments by Fullner and Nester (39) demonstrated that conjugal transfer of an RSF1010 derivative via the VirB delivery mechanism occurs at 19°C but not at 28°C; VirB-dependent secretion of the pilin VirB2 (51), and hence formation of pili (38), is also inhibited in cells grown at 28°C. If the defect in DNA transfer at 28°C observed by Fullner and Nester stems from either incomplete assembly or instability of the transport machinery (39), one might predict that the sizes or abundance of VirB9- and/or VirB10-containing complexes identifiable by cross-linking (6, 79) would be altered in cells grown at elevated temperatures. To test this hypothesis, we grew cells of the wild-type strain A348 at either 19 or 28°C and then treated them with one of two related chemical cross-linking agents, BS³ or DSS, before subjecting them to SDS-PAGE and immunoblotting analysis. This experiment revealed that the steady-state levels of VirB10 were much lower in cells grown at 28°C than in cells grown at 19°C (Fig. 2A). Although the cell extracts used in the experiment shown in Fig. 2A were from cells that had been washed in phosphate buffer and incubated at room temperature during the cross-linking step, a significant decrease in the amount of VirB10 was also seen in cells grown at 28°C, pelleted, and lysed directly from induction medium (data not shown) (see Fig. 4A [cf. lanes 1 and 3]). Significantly, growth at 19°C was not sufficient to stabilize the monomeric VirB10 against the loss seen in Ax42 cells washed and incubated at room temperature (Fig. 3A, compare lanes 1 and 3). Thus, steady-state levels of VirB10 are diminished in cells grown at 28°C regardless of whether the cells are washed and incubated at room temperature, and they are decreased in Ax42 cells incubated in phosphate buffer regardless of whether the cells were grown at 19 or 28°C.

View larger version (54K): [in this window] [in a new window]   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.

View larger version (29K): [in this window] [in a new window]   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.

Instability of VirB10 at 28°C is exacerbated in a chvB mutant. Accumulation of cyclic -1,2 glucan in the periplasm may function to minimize the osmotic gradient across the inner membrane that would otherwise form, particularly in cells grown in a low-osmolarity medium (55). We postulated that an excessive difference in osmolarity across the membrane in a chvB mutant strain might influence the stability of one or more VirB proteins that are localized in the membrane system or periplasm of agrobacteria. To determine whether this was the case, we prepared whole-cell extracts from wild-type and chvB strains grown at 19 or 28°C and subjected them to immunoblotting analysis. As depicted in Fig. 4A, the amount of VirB10 present in the chvB strain A1020 was comparable to the amount in the wild-type strain A348 when the cells were grown at 19°C (lanes 1 and 2). However, when cells were grown at 28°C, a striking decrease in VirB10 levels was seen in chvB cells relative to wild-type cells (Fig. 4A, lanes 3 and 4).

View larger version (22K): [in this window] [in a new window]   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.

Incubation at 19°C restores partial virulence to chvB mutants. The data described above suggested that chvB mutants, routinely characterized as avirulent in most media, might in fact exhibit some ability to incite tumors if grown and inoculated at 19°C. We tested this hypothesis with tobacco leaf explants and the chvB mutant strain A1020 grown at either 19 or 28°C. Cocultivation was also performed at 19 or 28°C, but all leaf explants were shifted to 22°C 3 days after the elimination of the bacteria. As shown in Fig. 5, a limited number of tumors formed, albeit slowly, on leaf explants infected with strain A1020 at 19°C. In contrast, almost no tumors were seen on explants inoculated with the same strain at 28°C. Pursuant to a previous report (70) suggesting that the virulence of a chvB mutant could be influenced by the osmolarity of the medium in which the cells were grown, we occasionally observed a small enhancement of tumorigenicity when chvB cells were grown at 19°C in medium supplemented with 100 mM NaCl, but in most assays we found no significant effect of inclusion of 100 mM NaCl on the number of tumors incited by strain A1020 (Table 1).

View larger version (73K): [in this window] [in a new window]   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.

VirB9 stabilizes VirB10 against differences in osmolarity across the inner membrane. Transcription of the virB operon in response to the inducer AS is dependent on the concerted activities of the phenolic sensor VirA and the transcriptional activator VirG (82). Experiments using bromoacetosyringone, which acts as an inhibitor of AS induction, suggest that vir expression requires continuous stimulation by AS (41). The loss of VirB10 observed when Ax42 cells were washed in sodium phosphate buffer, described above, might therefore be attributable to the removal of AS; if turnover of VirB10 is particularly rapid in the absence of wild-type VirB9, continuous protein synthesis would be required to maintain VirB10 levels, and since prokaryotic regulation is exerted primarily at the level of transcription (49), continuous synthesis might in turn require that levels of the polycistronic virB transcript be continually replenished. On the other hand, the observed instability of VirB10 in a chvB mutant grown at 28°C suggested an alternative explanation, namely, that it is the change in osmotic strength of the medium, rather than the removal of AS, that causes VirB10 to disappear from Ax42 cells upon incubation in phosphate buffer at room temperature.

View larger version (34K): [in this window] [in a new window]   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.

Turnover of VirB10, VirB11, and VirB8 in the absence of functional VirB9. The results described thus far suggest that a large osmotic gradient across the inner membrane can destabilize the VirB10 protein in a strain grown at 28°C or in a strain lacking wild-type VirB9. However, these data do not preclude the possibility that VirB10 has a shorter half-life in Ax42 cells even when they are maintained in ABIM under inducing conditions. To test this more directly, we examined the effect of using chloramphenicol to block protein synthesis in cells growing in induction medium. Strains A348 and Ax42 were induced overnight at 19°C in the presence of 200 µM AS prior to the addition of 100 µg of chloramphenicol/ml, a concentration of antibiotic that we determined to be effective in preventing growth of these strains on both liquid and solid media. Aliquots of cells were removed at 0, 1, 3, and 6 h after the addition of chloramphenicol and processed for SDS-PAGE and immunoblotting analysis. The results depicted in Fig. 7A reveal that as the length of exposure to the protein synthesis inhibitor increased, the amount of VirB10 in strain Ax42 decreased (lanes 5 to 8). In contrast, no such decrease was observed in wild-type cells (Fig. 7A, lanes 1 to 4). Figures 7B and C indicate that unlike VirB10, VirB8 and VirB11 were relatively stable in both A348 and Ax42 cells at 19°C, at least over the 6-h duration of this experiment, and in fact VirB11 appeared to be slightly more stable in Ax42 than in A348. On the other hand, levels of VirB5 diminished rapidly in both A348 and Ax42 cells (Fig. 7D). When this experiment was performed on cells grown at 28°C, some loss of VirB10 was seen even in wild-type cells (Fig. 7E). Likewise, turnover of VirB11 was apparent in wild-type cells at 28°C but not in strain Ax42 (data not shown).

View larger version (32K): [in this window] [in a new window]   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.

	DISCUSSION

Top Abstract Introduction Materials & Methods Results Discussion References

Effect of temperature-dependent VirB10 and VirB11 accumulation on virulence. Early work on the Agrobacterium-induced formation of crown galls on tomato plants revealed that the efficiency of the infection process was strikingly influenced by temperature. Optimal tumor formation on tomato occurred at 22°C, and the sizes of the tumors were equivalent at 18 and 26°C but decreased as the temperature was raised to 28 to 30°C, above which no tumors were observed (62). Despite the fact that these observations were published more than 7 decades ago, the physiological basis for this temperature dependence has been the subject of relatively few studies in the intervening years. One set of experiments, published in 1950, led to the postulate that it was denaturation of a complex protein structure that was responsible for thermal inactivation of the "tumor inducing principle" (12). In this study, Braun used measurements of the size and weight of tumors and the delay in inception period over a narrow range of temperatures to calculate an energy of inactivation for tumorigenesis. More recently, Fullner and Nester have used the VirB-dependent movement of an RSF1010 derivative between agrobacteria to explore the possibility that the loss of virulence reflects a decrease in the function of the VirB transport machinery at high temperature. In their experiments, these authors found that RSF1010 conjugation was optimal at 19°C and did not occur at temperatures higher than 28°C. Although a decrease in viability of the bacteria in these conjugation assays could account for some of the decrease in transfer frequency between 22 and 25°C, it could not account for the drastic reduction between 25 and 28°C. Additional experiments demonstrated that the genetic requirements for conjugation are identical at 19 and 28°C and that conjugation via virB-independent mechanisms can occur at 28°C, implying that the temperature effect measured was specific to the use of the VirB transport apparatus (39). Interestingly, however, conjugal transfer of the Ti plasmid (which is not mediated by VirB) is also thermosensitive (72). One known consequence of growth at 28°C is a dramatic decrease in the VirB-dependent export of the major pilin VirB2 (51) and hence in the number of pili observed on A. tumefaciens (38). Also apparent are alterations in the number of mating pair aggregates (54) and the morphology of pili (10) elaborated by Escherichia coli carrying certain self-transmissible plasmids when the cells are grown at 37°C rather than at 26 to 30°C. However, these reports leave unresolved the underlying differences in the cell that lead to changes in the abundance or structure of pili at high temperature.

Role of osmoadaption in stability of VirB proteins. The membrane systems of gram-negative bacteria enclose and segregate two aqueous compartments, the cytoplasm and the periplasm. In a classic study, Stock et al. demonstrated that for E. coli, the osmolarity of periplasm is similar to that of the cytoplasm, approximately 300 mosM for cells grown in standard media (69). It follows that a cell exposed to a hypotonic environment must have a mechanism to maintain a periplasmic osmolarity that is substantially higher than that of the surrounding milieu. For E. coli, it is generally believed that this differential is achieved by the accumulation in the periplasm of membrane-derived oligosaccharides, which with molecular weights of 2,200 to 2,600 are too large to pass through the outer membrane (48). In E. coli, the oligosaccharides that are synthesized in cells shifted to low-osmolarity medium are highly branched -1,2 glucans that are multiply substituted with anionic residues (48). The negative charges contribute to a Donnan potential of 30 mV (negative inside) across the outer membrane (69). In A. tumefaciens, accumulation of neutral cyclic -1,2 glucans in the periplasm is similarly osmoregulated, and concentrations of anionic oligosaccharides also rise in cells grown in hypotonic media (55). Periplasmic accumulation of these osmoadaptive cyclic oligosaccharides requires the function of at least two other genes, chvA and exoC, in addition to chvB. The exoC gene encodes the enzyme phosphoglucomutase, which is required for the biosynthesis of UDP-glucose (76). Mutants lacking a functional ChvA protein accumulate -1,2-glucan in the cytoplasm but do not export it to the periplasm (15).

VirB9-dependent assembly of VirB10-containing complexes. It has previously been reported that VirB9 is required for both the stabilization of VirB10 (9, 33) and VirB10 assembly into high-molecular-weight complexes (6). Our results confirm and extend these findings to provide a more nuanced view of the role of VirB9 in promoting the accumulation and proper assembly of VirB10. Beaupre et al. have shown that in strain Ax42, which carries a nonpolar transposon insertion in the virB9 gene (24), no VirB10-containing complexes are detected (6). However, under the conditions used for that cross-linking experiment, the monomeric form of VirB10 was also almost undetectable. This disappearance of VirB10 from strain Ax42 could be the result of enhanced VirB10 degradation, but it is also possible that the absence of VirB9 causes the release of VirB10 from the membrane. Under the cross-linking conditions used by Beaupre et al., we could not detect VirB10 in the culture medium or in the buffer washes (9a), but by omitting the buffer washes and performing the cross-linking on ice, we obtained nearly complete recovery of monomeric VirB10 (Fig. 1). Furthermore, in the present study, we have demonstrated that even under cross-linking conditions in which the monomeric form of VirB10 is present, no cross-linked complexes are seen (Fig. 1). From these data, we conclude that VirB9 facilitates the formation of VirB10-containing aggregates, perhaps by positioning VirB10 so that it can engage in productive interactions, most likely with other VirB10 molecules (6).