 |
INTRODUCTION |
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 |
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 |
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.
|
|
Further experimentation revealed that incubation for 20 min at room
temperature in phosphate buffer, even without washing, was sufficient
to cause a loss of VirB10 monomer from Ax42 cells and that depletion of
VirB10 monomer was more pronounced when the cells were incubated for
longer periods of time at room temperature in the phosphate buffer
(Fig. 1, lanes 8 to 10). Curiously, we found that exposure to the
cross-linking agent caused a further decrease in the amount of VirB10
monomer compared to samples incubated at room temperature for the same
length of time (45 min) under otherwise identical conditions (compare
lanes 9 and 10). This observation raised the possibility that some
VirB10 in Ax42 cells was indeed aggregated, perhaps into complexes too
large to enter the resolving gel. However, neither immunoblotting the
stacking gel nor dot blotting the sample revealed any material that
cross-reacted with the anti-VirB10 antisera (5a).
Accumulation of monomeric VirB10 was not affected when cells were
incubated on ice, rather than at room temperature, in phosphate buffer
either without (lane 11) or with (lane 12) the cross-linking agent.
Furthermore, cells of the wild-type strain A348 subjected to the same
incubation conditions, either at room temperature or on ice, did not
exhibit any loss of VirB10 monomer (Fig. 1, lanes 1 to 6). From these data, we conclude that VirB10 is rendered more labile in the absence of
wild-type VirB9, such that incubation in phosphate buffer at room
temperature, but not on ice, leads to substantially diminished steady-state levels of VirB10.
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.
|
|
In contrast, accumulation of the monomeric form of VirB9 (Fig. 2B) was
relatively unaffected by growth temperature, as was the prominent
60-kDa VirB9-containing aggregate. However, we did observe
significantly weaker cross-reactivity in the larger (75- and 130-kDa)
VirB9-containing high-molecular-weight species in cells grown at the
higher temperature. Thus, although the overall levels of VirB9 are not
influenced by temperature, the assembly of VirB9 into large oligomeric
complexes does appear to be compromised.
We also examined the effect of growth temperature on the accumulation
of other VirB proteins. Cell extracts from wild-type cells grown at
either 19 or 28°C were probed with antibodies raised against VirB8 or
VirB11. Only small differences in the levels of VirB8 were observed
(Fig. 3B). However, cells grown at 28°C consistently accumulated
decreased amounts of VirB11 relative to cells grown at 19°C (Fig.
3C). This was true both of cells washed and incubated at room
temperature for 30 min and of cells pelleted and resuspended directly
in SDS-PAGE sample buffer (data not shown).
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.
|
|
We also compared the amounts of other VirB proteins in chvB
and wild-type cells grown at either 19 or 28°C. Levels of VirB11 and
VirB9 were indistinguishable in the two strains (Fig. 4B and C), while
the amount of VirB8 was moderately diminished in the chvB
strain grown at 28°C but not in cells grown at 19°C (Fig. 4D).
Taken together, our results indicate that VirB10, which is already less
abundant in cells grown at 28°C, is particularly unstable at 28°C
in cells exhibiting an apparent defect in osmoadaption; this
instability in chvB cells is not a general attribute of all VirB proteins.
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.
To distinguish between these two possibilities, we grew both Ax42 and
wild-type cells in induction medium (ABIM) containing 200 µM AS,
pelleted the cells, and then washed them three times and incubated them
for 30 min at room temperature either in sodium phosphate buffer, as
before, or in induction medium lacking AS. We reasoned that if removal
of AS was sufficient to cause a loss of VirB10, Ax42 cells exposed to
either phosphate buffer or ABIM lacking AS should exhibit diminished
levels of VirB10; however, if the observed loss was due to an increased
osmotic gradient across the inner membrane, only cells shifted from
ABIM to phosphate buffer would lose VirB10. The results of this
experiment are depicted in Fig. 6. By
comparing lanes 3 and 4 or lanes 7 and 8, it is evident that incubation
in phosphate buffer, but not ABIM lacking AS, resulted in decreased
levels of VirB10; the extent of the loss was comparable in cells grown
at either 19 or 28°C. Thus, we conclude that changes in the
osmolarity of the medium, rather than a requirement for continuous
transcription and translation, can account for the observed loss of
VirB10 in Ax42 cells that are washed prior to cross-linking.
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 |
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.
In this report, we have examined the stability of a subset of VirB
proteins at permissive and nonpermissive temperatures for virulence.
Our data indicate that VirB10, and to a lesser extent VirB11,
accumulates to lower levels in cells grown at 28°C than in cells
grown at 19°C (Fig. 2A and 3C). Steady-state levels of VirB8 and
monomeric VirB9 are relatively unaffected by the growth temperature
(Fig. 2B and 3B). (In some previous studies [see, e.g., reference
6], cross-linked versions of VirB10 were detected in cells that were reportedly grown at elevated temperatures, but ours
is the first study in which direct comparisons have been made between
cells grown at 19 and 28°C, and it seems likely that the growth
temperature in the previous reports was less rigorously controlled.)
Our findings thus suggest that destabilization of particular VirB
proteins may be a key factor in the observed loss of DNA transfer
function at high temperature. Both VirB10 and VirB11 have been
implicated as essential, and perhaps rate-limiting, components of the
T-DNA transfer machinery (77, 84), and VirB10 is known to
assemble into multimeric protein complexes (79). We
postulate that VirB10 cannot contribute to the formation of a
functional transport apparatus because it does not accumulate sufficiently at 28°C or, alternatively, that it is not stable because
it is not incorporated into multiprotein transport complexes. Our data
do not allow us to discriminate unambiguously between these
possibilities, although we favor a model in which assembly of VirB10,
rather than its accumulation per se, is the temperature-dependent process (2a). Furthermore, in cells grown at 28°C, VirB9
appears to be stable in its monomeric form and can assemble into its
apparent dimeric (60-kDa) form, yet it fails to assemble into the
larger-molecular-weight aggregates detectable by chemical cross-linking
(Fig. 2B). If DNA transport complex assembly is nucleated by one or two
proteins, the loss of those proteins at an elevated temperature could
easily destabilize the entire complex (see below). In this context, we note that disruption of energetically favored interactions among VirB
proteins, perhaps due to the loss of only a subset of those interacting
components, could contribute to the magnitude of the inactivation
energy calculated by Braun (12). Finally, although we favor
an explanation in which a loss of protein stability accounts for the
diminished levels of VirB10 and VirB11, we cannot exclude the
possibility that at elevated temperatures, transcription of the
virB polycistronic message does not proceed as efficiently all the way to the 3' end; in the study by Fullner and Nester, transcription was measured from a lacZ fusion located at the
very 5' end of the virB operon (2).
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).
chvA, chvB, and exoC were first
identified as chromosomal loci required for virulence. Originally,
mutations in these genes were found to result in defects in the
attachment of A. tumefaciens to plant cells (28);
chvB mutants exhibit several other pleiotropic phenotypes as
well. Subsequent reports have demonstrated that some of the other
defects associated with chvB mutations, including poor
growth and altered periplasmic protein content, can be suppressed by
growing the cells in a high-osmolarity medium (16).
chvB mutants are also sensitive to the level of calcium in
the growth medium, and Swart et al. showed that defects in motility
seen in chvB mutants could be prevented by decreasing the
calcium concentration in the growth medium from 7 mM to 0.15 mM
(71). However, calcium is required for the activity of the
calcium-binding protein rhicadhesin, which mediates the first step in
the attachment of A. tumefaciens to plant cells
(65). Addition of 100 mM NaCl or 200 mM melezitose to the
calcium-containing medium restored rhicadhesin activity, attachment,
and virulence to chvB mutants, presumably by preempting the
need for cyclic -1,2-glucan as an osmoregulatory molecule in the
periplasm (70). Raising the external osmolarity had a similar effect in Rhizobium meliloti mutants unable to
synthesize -1,2-glucans. In this case, pleiotropic cell surface
defects associated with mutations in the ndv genes, the
Rhizobium homologs of the chv genes, were rescued
by the addition of 100 mM NaCl to the medium (30).
The apparent role of the chvB gene product in osmoadaption
has led to the suggestion (71) that periplasmic
-1,2-glucan may interact with the lipid bilayers of both the inner
and outer membranes to stabilize them against large differences in
osmolarity across the bilayer. Such oligosaccharide effects might in
turn modulate assembly of membrane-associated protein complexes, either by contributing directly to the stability of membrane proteins (13) or, potentially, by influencing the fluidity of the
membrane (23). In this paper, we report that a
chvB mutant exhibits dramatically lowered levels of a
specific protein, VirB10, residing in the membrane system of A. tumefaciens. Significantly, the decrease in VirB10 accumulation in
the chvB mutant strain was observed in cells grown at
28°C, but not in cells grown at 19°C, and occurred in addition to
the temperature-dependent drop in VirB10 pools seen even in wild-type
cells (Fig. 4A). These observations suggest that VirB10 may be both
inherently labile and fairly sensitive to the existence of an osmotic
gradient across the inner membrane. We conclude that in wild-type cells
grown at 19°C, VirB10 is apparently stabilized, perhaps by assembly
into oligomeric complexes, even in chvB mutant cells, which
lack the osmoadaptive -1,2-glucan in the periplasm. In contrast,
when cells are grown at 28°C, pools of VirB10 are smaller and the
protein which does accumulate appears not be stabilized against the
effects of a hypotonic environment. Attempts to rescue the VirB10
instability phenotype by including 200 mM melezitose or 100 mM NaCl in
the growth medium met with limited success; when cells were grown at
28°C in calcium-containing medium plus 200 mM melezitose and then
induced in ABIM, the amount of VirB10 in the chvB cells was
approximately equal to that in the wild-type cells, but these levels
were substantially lower than the VirB10 levels seen in cells grown
without melezitose. Inclusion of 100 mM NaCl caused a similar overall
reduction in the levels of several VirB proteins (2a), and
in fact, the data in Table 1 do not support the claim (70)
that addition of 100 mM NaCl to the growth medium can enhance the
tumorigenicity of a chvB mutant. However, our finding that
virulence can be partially restored to a chvB mutant by
growth and cocultivation at 19°C even in the absence of added osmotic
support (Table 1; Fig. 5) indicates that one, although certainly not
the only, factor contributing to the avirulence of this strain under
typical assay conditions may be a lack of VirB10.
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).
Another recent study also suggests that the abundance of VirB9 can
influence the conformation of VirB10 in the membrane (84). It may be, for example, that in the absence of VirB9, individual VirB10
molecules are free to diffuse within the plane of the lipid bilayer but
that interactions with VirB9 serve to limit movement and thereby
promote clustering of VirB10 monomers. In this scenario, periplasmic
VirB9 might in turn be immobilized by its interactions with the
lipoprotein VirB7, which is anchored in the outer membrane by a fatty
acid modification and may therefore serve to tether other components of
the transport machinery (4, 32). Our data further indicate
that in the absence of interactions with VirB9, VirB10 is rendered more
sensitive to the existence of an osmotic gradient across the membrane,
such as occurs when cells grown in induction medium are washed in
sodium phosphate buffer (Fig. 6). Significantly, accumulation of VirB10
is substantially decreased even in wild-type cells that are not
subjected to washing if the cells are grown at 28°C (see, for
example, Fig. 4A); conversely, in the absence of VirB9, monomeric
VirB10 disappears upon washing in cells grown at both low and high
temperature (Fig. 3A), while in a chvB mutant at 28°C,
even the presence of VirB9 cannot stabilize VirB10 (Fig. 4A). Taken
together, our results suggest a hierarchy of requirements for VirB10
stabilization. In this model, VirB9-dependent assembly of VirB10 into a
multiprotein complex stabilizes the inherently labile VirB10, but
complex assembly cannot occur at elevated temperatures; thus, in cells
grown at 28°C, VirB10 is less abundant even in wild-type cells and
its degradation is exacerbated in a cell lacking the osmoadaptive
function of the ChvB protein. In contrast, in cells grown at 19°C,
VirB10 is insensitive to the absence of the ChvB protein precisely
because it is complexed with other components of the transport apparatus.
Berger and Christie (9) and Fernandez et al. (33)
have previously published data showing greatly diminished levels of VirB4, VirB8, and VirB11, as well as VirB10, in a strain containing a
precise deletion of the virB9 gene. In contrast, our strain Ax42 carries a nonpolar transposon insertion that eliminates only the
last 31 codons of virB9 and replaces them with 39 codons
from the transposon that happen to be in frame, so that Ax42 expresses, albeit at lowered levels, an almost full-length VirB9. This truncated form of VirB9 is, however, completely nonfunctional in promoting tumorigenesis (24). Interestingly, we observed no decrease
in the steady-state levels of VirB8 or VirB11 in strain Ax42 at 19°C (Fig. 7B and C), and the amount of VirB10 was reduced only when Ax42
cells were washed in buffer prior to lysis (Fig. 1 and 6), although the
turnover rate for VirB10 in Ax42 appeared to be accelerated compared to
that in the wild-type strain (Fig. 7A). These data imply that the
truncated version of VirB9 encoded in strain Ax42 may be able to
participate in certain stabilizing interactions, despite the fact that
it does not appear to interact with VirB7 (6). In fact, we
have consistently found significantly more VirB11 in Ax42 than in
wild-type cells (see, for example, Fig. 3C), and turnover of VirB11 at
temperatures of 28°C or higher is less pronounced in Ax42 than in
wild-type strain A348 (data not shown). We cannot rule out the
possibility that the proximity of the virB promoter in the
Tn5virB transposon within virB9 allows for
enhanced expression of virB11 in strain Ax42; however, we do
not see a similar enhancement in the VirB11 levels in strain Ax56,
which carries the same transposon in virB10 (data not
shown). Furthermore, regulated expression of virB7 and
virB8 from the lac promoter results in coordinate
increases in the levels of VirB7-11, but VirB11 accumulation is
actually higher in the absence of induction than in the presence of low
concentrations of inducer (33). Perhaps in the absence of
any VirB9, VirB11 forms aberrant complexes which stabilize it, while
low-level induction of VirB9 results in incorporation of some VirB11
into productive complexes and in turnover of the remaining VirB11. Such
a scenario would be consistent with the suggestion that mutant VirB11
molecules can be sequestered by excessive VirB9, VirB10, or VirB11
(84).
Work in several laboratories has led to the proposal that a critical
step in the assembly of the T-DNA transport apparatus is the
dimerization of VirB9, located in the periplasm, with the lipoprotein
VirB7, which is anchored in the outer membrane (84). In this
model, additional components are recruited via direct or indirect
interactions with this heterodimer (18). An important puzzle
that remains to be solved is how associations are forged across the
peptidoglycan barrier between the inner and outer membranes (27). This issue is especially trenchant if, as has been
proposed, a single multimeric VirB protein complex spans both membranes (18, 83). One attractive model suggests that assembly of the multiprotein DNA transport apparatus may be facilitated by localized lysis of the murein cell wall by VirB1 (5, 57). Furthermore, although the existence of adhesion sites between the inner and outer
membranes is highly controversial (27, 29), there are hints
from electron microscopic analysis of thin sections that F pili may
form at such junctions (35). Fractionation studies suggest
that several VirB proteins may partition with both membranes; in many
cases, the observed partitioning of a given VirB protein varies from
one study to another and may reflect interactions among proteins within
a structure that spans the periplasm and which is sheared when the
membranes are separated on sucrose gradients (4, 5, 32, 34, 45,
63, 74). One candidate for a peptidoglycan-spanning component of
the transport machinery is VirB10. Sequence analysis and studies of
virB-phoA fusions indicate that VirB10 contains a single
membrane-spanning region with a large periplasmic domain and a small
cytoplasmic amino terminus (79). This topology could allow
for interactions with both VirB11, located on the inner face of the
cytoplasmic membrane, and the periplasmic VirB9. In fact, a small
proportion of the VirB10 is found in sucrose gradient fractions with
densities intermediate between those of the inner and the outer
membranes (34). Second, like its homolog, TraB, which is
involved in F pilus assembly, VirB10 has a proline-rich region within
the putative periplasmic domain, immediately adjacent to the membrane
anchor (36). Although the prolines in VirB10 are not
uniformly spaced, it is conceivable that this portion of the
periplasmic domain of VirB10 assumes an elongated conformation; it is
perhaps worth noting that the VirB10 homolog PtlG contains a similar
proline-rich region in which the spacing of the prolines is more
regular. Similar proline-rich features in at least two other bacterial
proteins, TonB from E. coli and the group C streptococcal M
protein, have been proposed to confer on these proteins a rigid,
extended shape which allows them to thread through the peptidoglycan
layer (81). Third, any model for formation of the VirB pore
complex must take into account the observed effect of elevated
temperature on T-DNA transport function (39). In this
regard, it is noteworthy that of the proteins tested in the present
work, it was VirB10 which exhibited the largest decrease in abundance
at 28°C.
It is tempting to speculate that VirB10, positioned correctly through
its interactions with the VirB9-VirB7 heterodimer, acts to nucleate
temperature-dependent assembly of a stable multiprotein complex which
mediates T-DNA transfer and that other proteins, such as VirB2 through
VirB6, are assembled because of their interactions with VirB10. In
fact, a very recent report demonstrates that processed VirB2, the major
pilin of the "T pilus," is exported from A. tumefaciens in a VirB-dependent manner; strikingly, VirB2 accumulates at both 19 and 28°C but appears in the exocellular fraction only at the lower
temperature (51). We hypothesize that under conditions (e.g., in a chvB mutant at 28°C) in which the nucleating
protein is itself unstable, other interacting species might also be
degraded more rapidly. Whether the observed failure of the stable VirB9 monomer to assemble into high-molecular-weight aggregates at 28°C is
a cause or a consequence of the loss of VirB10 remains to be determined. Although such a model for assembly of the T-DNA transport apparatus is highly speculative, there is growing evidence to support
the notion that certain components may be rate limiting. In
particular, Ward et al. showed that T-DNA-mediated virulence is
inhibited in A. tumefaciens cells carrying the
broad-host-range plasmid RSF1010, which can itself move to the
host plant cells in a VirB-dependent manner, but that overexpression of
VirB9, VirB10, and VirB11 is sufficient to overcome this oncogenic
suppression (77). More intriguing yet, overexpression of
either VirB9 or VirB10 alone can suppress the avirulence associated
with dominant mutations in VirB11 (84). Finally, the recent
discovery in Helicobacter pylori of a cag
pathogenicity island consisting of only VirB4, VirB9, VirB10, VirB11,
and VirD4 homologs provides support for the notion that these
components represent the core structure on which the complete VirB
transport machinery is assembled (17, 19, 21, 75).
We are grateful to Andrew Binns, Karl Johnson, Eugene Nester,
Lisa Stahl, and Paul Hyman for helpful conversations and support during
this project.
This work was supported by grants to L.M.B. and to Andrew N. Binns from
the National Science Foundation (MCB9506144 and MCB9513662, respectively) and to J.B. from the Deutsche Forschungsgemeinschaft.
| 1.
|
Anderson, L. B.,
A. V. Hertzel, and A. Das.
1996.
Agrobacterium tumefaciens VirB7 and VirB9 form a disulfide-linked protein complex.
Proc. Natl. Acad. Sci. USA
93:8889-8894[Abstract/Free Full Text].
|
| 2.
|
Ankenbauer, R. G.,
E. A. Best,
C. A. Palanca, and E. W. Nester.
1991.
Mutants of the Agrobacterium tumefaciens virA gene exhibiting acetosyringone-independent expression of the vir regulon.
Mol. Plant-Microbe Interact.
4:400-406[Medline].
|
| 2a.
| Banta, L. M. Unpublished data.
|
| 3.
|
Baron, C., and P. C. Zambryski.
1995.
The plant response in pathogenesis, symbiosis, and wounding: variations on a common theme?
Annu. Rev. Genet.
29:107-129[Medline].
|
| 4.
|
Baron, C.,
Y. R. Thorstenson, and P. C. Zambryski.
1997.
The lipoprotein VirB7 interacts with VirB9 in the membranes of Agrobacterium tumefaciens.
J. Bacteriol.
179:1211-1218[Abstract/Free Full Text].
|
| 5.
|
Baron, C.,
M. Llosa,
S. Zhou, and P. C. Zambryski.
1997.
VirB1, a component of the T-complex transfer machinery of Agrobacterium tumefaciens, is processed to a C-terminal secreted product, VirB1*.
J. Bacteriol.
179:1203-1210[Abstract/Free Full Text].
|
| 5a.
| Beaupre, C., and J. Bohne. Unpublished data.
|
| 6.
|
Beaupre, C. E.,
J. Bohne,
E. M. Dale, and A. N. Binns.
1997.
Interactions between VirB9 and VirB10 membrane proteins involved in movement of DNA from Agrobacterium tumefaciens into plant cells.
J. Bacteriol.
179:78-89[Abstract/Free Full Text].
|
| 7.
|
Beijersbergen, A.,
A. D. Dulk-Ras,
R. A. Schilperoort, and P. J. J. Hooykaas.
1992.
Conjugative transfer by the virulence system of Agrobacterium tumefaciens.
Science
256:1324-1327[Abstract/Free Full Text].
|
| 8.
|
Berger, B. R., and P. J. Christie.
1993.
The Agrobacterium tumefaciens virB4 gene product is an essential virulence protein requiring an intact nucleoside triphosphate-binding domain.
J. Bacteriol.
175:1723-1734[Abstract/Free Full Text].
|
| 9.
|
Berger, B. R., and P. J. Christie.
1994.
Genetic complementation analysis of the Agrobacterium tumefaciens virB operon: virB2 through virB11 are essential virulence genes.
J. Bacteriol.
176:3646-3660[Abstract/Free Full Text].
|
| 9a.
| Bohne, J. Unpublished data.
|
| 10.
|
Bradley, D. E., and J. Whelan.
1985.
Conjugation systems of IncT plasmids.
J. Gen. Microbiol.
131:2665-2671[Medline].
|
| 11.
|
Braun, A. C.
1947.
Thermal studies on the factors responsible for tumor initiation in crown gall.
Am. J. Bot.
34:234-240.
|
| 12.
|
Braun, A. C.
1950.
Thermal inactivation studies on the tumor-inducing principle in crown gall.
Phytopathology
40:3.
|
| 13.
|
Breedveld, M. W., and K. J. Miller.
1994.
Cyclic -glucans of members of the family Rhizobiaceae.
Microbiol. Rev.
58:145-161[Abstract/Free Full Text].
|
| 14.
|
Buchanan-Wollaston, V.,
J. E. Passiatore, and F. Cannon.
1987.
The mob and oriT mobilization functions of a bacterial plasmid promote its transfer to plants.
Nature
328:172-175.
|
| 15.
|
Cangelosi, G. A.,
G. Martinetti,
J. A. Leigh,
C. C. Lee,
C. Theines, and E. W. Nester.
1989.
Role of Agrobacterium tumefaciens ChvA protein in export of -1,2-glucan.
J. Bacteriol.
171:1609-1615[Abstract/Free Full Text].
|
| 16.
|
Cangelosi, G. A.,
G. Martinetti, and E. W. Nester.
1990.
Osmosensitivity phenotypes of Agrobacterium tumefaciens mutants that lack periplasmic -1,2-glucan.
J. Bacteriol.
172:2172-2174[Abstract/Free Full Text].
|
| 17.
|
Censini, S.,
C. Lange,
Z. Xiang,
J. E. Crabtree,
P. Ghiara,
M. Bordovsky,
R. Rappuoli, and A. Covacci.
1996.
cag, a pathogenicity island of Helicobacter pylori, encodes type I-specific and disease-associated virulence factors.
Genetics
93:14648-14653.
|
| 18.
|
Christie, P. J.
1997.
Agrobacterium tumefaciens T-complex transport apparatus: a paradigm for a new family of multifunctional transporters in eubacteria.
J. Bacteriol.
179:3085-3094[Free Full Text].
|
| 19.
|
Christie, P. J.
1997.
The cag pathogenicity island: mechanistic insights.
Trends Microbiol.
5:264-265[Medline].
|
| 20.
|
Christie, P. J.,
J. E. Ward,
M. P. Gordon, and E. W. Nester.
1989.
A gene required for transfer of T-DNA to plants encodes an ATPase with autophosphorylating activity.
Proc. Natl. Acad. Sci. USA
86:9677-9681[Abstract/Free Full Text].
|
| 21.
|
Covacci, A.,
S. Falkow,
D. E. Berg, and R. Rappuoli.
1997.
Did the inheritance of a pathogenicity island modify the virulence of Helicobacter pylori?
Trends Microbiol.
5:205-208[Medline].
|
| 22.
|
Covacci, A., and R. Rappuoli.
1993.
Pertussis toxin export requires accessory genes located downstream from the pertussis toxin operon.
Mol. Microbiol.
8:429-434[Medline].
|
| 23.
|
Crowe, J. H.,
M. A. Whittam,
D. Chapman, and L. M. Crowe.
1984.
Interactions of phospholipid monolayers with carbohydrates.
Biochim. Biophys. Acta
769:151-159[Medline].
|
| 24.
|
Dale, E. M.,
A. N. Binns, and J. E. Ward, Jr.
1993.
Construction and characterization of Tn5virB, a transposon that generates nonpolar mutations, and its use to define virB8 as an essential virulence gene in Agrobacterium tumefaciens.
J. Bacteriol.
175:887-891[Abstract/Free Full Text].
|
| 25.
|
Dang, T. A. T., and P. J. Christie.
1997.
The VirB4 ATPase of Agrobacterium tumefaciens is a cytoplasmic membrane protein exposed at the periplasmic surface.
J. Bacteriol.
179:453-462[Abstract/Free Full Text].
|
| 26.
|
De Vos, G., and P. Zambryski.
1989.
Expression of Agrobacterium nopaline-specific VirD1, VirD2, and VirC1 proteins and their requirement for T-strand production in E. coli.
Mol. Plant-Microbe Interact.
2:43-52[Medline].
|
| 27.
|
Dijkstra, A. J., and W. Keck.
1996.
Peptidoglycan as a barrier to transenvelope transport.
J. Bacteriol.
178:5555-5562[Free Full Text].
|
| 28.
|
Douglas, C. J.,
R. J. Staneloni,
R. A. Rubin, and E. W. Nester.
1985.
Identification and genetic analysis of an Agrobacterium tumefaciens chromosomal virulence region.
J. Bacteriol.
161:850-860[Abstract/Free Full Text].
|
| 29.
|
Durrenberger, M. B.,
W. Villiger, and T. Bachi.
1991.
Conjugational junctions: morphology of specific contacts in conjugating Escherichia coli bacteria.
J. Struct. Biol.
107:146-156[Medline].
|
| 30.
|
Dylan, T.,
D. R. Helinski, and G. S. Ditta.
1990.
Hypoosmotic adaptation in Rhizobium meliloti requires -(1 2)-glucan.
J. Bacteriol.
172:1400-1408[Abstract/Free Full Text].
|
| 31.
|
Farizo, K. M.,
T. G. Cafarella, and D. L. Burns.
1996.
Evidence for a ninth gene, ptlI, in the locus encoding the pertussis toxin secretion system of Bordetella pertussis and formation of a PtlI-PtlF complex.
J. Biol. Chem.
271:31643-31649[Abstract/Free Full Text].
|
| 32.
|
Fernandez, D.,
T. A. T. Dang,
G. M. Spudich,
X.-R. Zhou,
B. R. Berger, and P. J. Christie.
1996.
The Agrobacterium tumefaciens virB7 gene product, a proposed component of the T-complex transport apparatus, is a membrane-associated lipoprotein exposed at the periplasmic surface.
J. Bacteriol.
178:3156-3167[Abstract/Free Full Text].
|
| 33.
|
Fernandez, D.,
G. M. Spudich,
X.-R. Zhou, and P. J. Christie.
1996.
The Agrobacterium tumefaciens VirB7 lipoprotein is required for stabilization of VirB proteins during assembly of the T-complex transport apparatus.
J. Bacteriol.
178:3168-3176[Abstract/Free Full Text].
|
| 34.
|
Finberg, K. E.,
T. R. Muth,
S. P. Young,
J. B. Maken,
S. M. Heitritter,
A. N. Binns, and L. M. Banta.
1995.
Interactions of VirB9, -10, and -11 with the membrane fraction of Agrobacterium tumefaciens: solubility studies provide evidence for tight associations.
J. Bacteriol.
177:4881-4889[Abstract/Free Full Text].
|
| 35.
|
Firth, N.,
K. Ippen-Ihler, and R. A. Skurray.
1996.
Structure and function of the F factor and mechanism of conjugation, p. 2377-2401.
In
F. C. Neidhardt, R. Curtiss III, J. L. Ingraham, E. C. C. Lin, K. B. Low, B. Magasanik, W. S. Reznikoff, M. Riley, M. Schaechter, and H. E. Umbarger (ed.), Escherichia coli and Salmonella: cellular and molecular biology, 2nd ed, vol. 2. ASM Press, Washington, D.C.
|
| 36.
|
Frost, L. S.,
K. Ippen-Ihler, and R. A. Skurray.
1994.
Analysis of the sequence and gene products of the transfer region of the F sex factor.
Microbiol. Rev.
58:162-210[Abstract/Free Full Text].
|
| 37.
|
Fullner, K. J.,
K. M. Stephens, and E. W. Nester.
1994.
An essential virulence protein of Agrobacterium tumefaciens, VirB4, requires an intact mononucleotide binding domain to function in transfer of T-DNA.
Mol. Gen. Genet.
245:704-715[Medline].
|
| 38.
|
Fullner, K. J.,
J. C. Lara, and E. W. Nester.
1996.
Pilus assembly by Agrobacterium T-DNA transfer genes.
Science
273:1107-1109[Abstract].
|
| 39.
|
Fullner, K. J., and E. W. Nester.
1996.
Temperature affects the T-DNA transfer machinery of Agrobacterium tumefaciens.
J. Bacteriol.
178:1498-1504[Abstract/Free Full Text].
|
| 40.
|
Garfinkel, D. J.,
R. B. Simpson,
L. W. Ream,
F. F. White,
M. P. Gordon, and E. W. Nester.
1981.
Genetic analysis of crown gall: fine structure map of the T-DNA by site-directed mutagenesis.
Cell
27:143-153[Medline].
|
| 41.
|
Hess, K. M.,
M. W. Dudley,
D. G. Lynn,
R. D. Joerger, and A. N. Binns.
1991.
Mechanism of phenolic activation of Agrobacterium virulence genes: development of a specific inhibitor of bacterial sensor/response systems.
Proc. Natl. Acad. Sci. USA
88:7854-7858[Abstract/Free Full Text].
|
| 42.
|
Hooykaas, P. J. J., and A. G. M. Beijersbergen.
1994.
The virulence system of Agrobacterium tumefaciens.
Annu. Rev. Phytopathol.
32:157-179.
|
| 43.
|
Jin, S.,
Y.-N. Song,
W.-Y. Deng,
M. P. Gordon, and E. W. Nester.
1993.
The regulatory VirA protein of Agrobacterium tumefaciens does not function at elevated temperatures.
J. Bacteriol.
175:6830-6835[Abstract/Free Full Text].
|
| 44.
|
Jones, A. L.,
K. Shirasu, and C. I. Kado.
1994.
The product of the virB4 gene of Agrobacterium tumefaciens promotes accumulation of VirB3 protein.
J. Bacteriol.
176:5255-5261[Abstract/Free Full Text].
|
| 45.
|
Jones, A. L.,
E.-M. Lai,
K. Shirasu, and C. I. Kado.
1996.
VirB2 is a processed pilin-like protein encoded by the Agrobacterium tumefaciens Ti plasmid.
J. Bacteriol.
178:5706-5711[Abstract/Free Full Text].
|
| 46.
|
Kado, C.
1994.
Genes of the virB operon are involved in the synthesis of a conjugational pilus-like apparatus required for the transfer of the T-DNA from Agrobacterium tumefaciens to plant cells, p. 23-36.
In
S. Furasuki, and A. D. Ryer (ed.), Advances in plant biotechnology. Elsevier, Amsterdam, The Netherlands.
|
| 47.
|
Kado, C. I.
1994.
Promiscuous DNA transfer system of Agrobacterium tumefaciens: role of the virB operon in sex pilus assembly and synthesis.
Mol. Microbiol.
12:17-22[Medline].
|
| 48.
|
Kennedy, E. P.
1982.
Osmotic regulation and the biosynthesis of membrane-derived oligosaccharides in Escherichia coli.
Proc. Natl. Acad. Sci. USA
79:1092-1095[Abstract/Free Full Text].
|
| 49.
|
Kushner, S. R.
1987.
Useful host strains and techniques for recombinant DNA experiments, p. 1225-1230.
In
F. C. Neidhardt, J. L. Ingraham, K. B. Low, B. Magasanik, M. Schaechter, and H. E. Umbarger (ed.), Escherichia coli and Salmonella typhimurium: cellular and molecular biology, vol. 2. American Society for Microbiology, Washington, D.C.
|
| 50.
|
Laemmli, U. K.
1970.
Cleavage of structural proteins during the assembly of the head of bacteriophage T4.
Nature
227:680-685 |
![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
Top
Abstract
Introduction
Present address: Microbiology Department, University of
Würzburg, D-97074 Würzburg, Germany.
