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Journal of Bacteriology, November 1998, p. 5660-5667, Vol. 180, No. 21
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Wound-Released Chemical Signals May Elicit Multiple Responses
from an Agrobacterium tumefaciens Strain Containing an
Octopine-Type Ti Plasmid
Virginia S.
Kalogeraki and
Stephen C.
Winans*
Section of Microbiology, Cornell University,
Ithaca, New York 14853
Received 4 June 1998/Accepted 25 August 1998
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ABSTRACT |
The vir regions of octopine-type and nopaline-type Ti
plasmids direct the transfer of oncogenic T-DNA from
Agrobacterium tumefaciens to the nuclei of host plant
cells. Previous studies indicate that at least two genetic loci at the
left ends of these two vir regions are sufficiently
conserved to form heteroduplexes visible in the electron
microscope. To initiate an investigation of these genetic loci, we
determined the DNA sequences of these regions of both Ti plasmids and
identified both conserved loci. One of these is the 2.5-kb
virH locus, which was previously identified on the octopine-type Ti plasmid but thought to be absent from the
nopaline-type Ti plasmid. The virH operon contains two
genes that resemble P-450-type monooxygenases. The other locus encodes
a 0.5-kb gene designated virK. In addition, we identified
other potential genes in this region that are not conserved
between these two plasmids. To determine (i) whether these genes are
members of the vir regulon and, (ii) whether they are
required for tumorigenesis, we used a genetic technique to disrupt each
gene and simultaneously fuse its promoter to lacZ.
Expression of these genes was also measured by nuclease S1 protection
assays. virK and two nonconserved genes, designated virL and virM, were strongly induced by the
vir gene inducer acetosyringone. Disruptions of
virH, virK, virL, or
virM did not affect tumorigenesis of
Kalanchöe diagramontiana leaves or carrot
disks, suggesting that they may play an entirely different role during
pathogenesis.
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INTRODUCTION |
Agrobacterium tumefaciens
has the unique ability to stably modify the genomes of host plants in a
way that is beneficial to the bacterium (45). It does this
by transferring approximately 15 genes from a 200-kb plasmid (the Ti
plasmid) to plant nuclei, where they become covalently integrated into
the host genome. This transfer process requires the products of
approximately 20 known vir genes located on a nontransferred
portion of the Ti plasmid (44), as well as a smaller number
of chromosomally encoded proteins. Most transferred genes can be
categorized into two distinct groups. The first group leads to the
production of opines, compounds that are released by the plant and used
by the bacterium as nutrient sources (8), while the second
group of transferred genes mediates the overproduction of the
phytohormones auxin and cytokinin, which interfere with the plant's
natural phytohormone balance and cause neoplastic growth, resulting in
crown gall tumors (1).
An early event leading to tumorigenesis is the transcriptional
induction of the vir regulon in response to a variety of
chemical signals that are released from plant wounds. The most
important of these signals consist of a family of related
phenolic compounds, including acetosyringone, that act in
combination with extracellular acidity and particular
monosaccharides (16). These signals are perceived by the
chromosomally encoded sugar-binding protein ChvE and the Ti
plasmid-encoded proteins VirA and VirG (2, 18, 40).
VirA is a transmembrance sensory histidine kinase which phosphorylates the response regulator VirG (20, 21, 43). Phospho-VirG binds to binding sites designated vir boxes
found upstream of each vir promoter and coordinately
activates transcription of these promoters (15, 22).
The previously characterized vir regulon includes eight
operons (36, 39). Of these, the virA,
virB (6, 23), virD, and
virG operons are essential for tumorigenesis, while
virC and virE are required for efficient
tumorigenesis, and virF is required for tumorigenesis on
certain plant hosts (17). virH of the
octopine-type Ti plasmid was thought not to be required for
tumorigenesis, although none of the available mutations disrupts both
genes of the virH operon. The tzs gene of
nopaline-type Ti plasmids, which directs the production of a cytokinin
precursor, is also part of the vir regulon, but disruptions
of this gene have not been described.
In an attempt to identify the homologous regions between octopine-type
and nopaline-type Ti plasmids, Engler et al. (11) used the
electron microscope to determine which sequences of the octopine-type
Ti plasmid pTiAch5 were able to form heteroduplexes with the
nopaline-type Ti plasmid pTiC58. This study revealed that these
plasmids share four major regions of homology, consisting of
approximately 30% of their sequences. At that time, genetic analysis
of these regions was extremely rudimentary. In hindsight, we now know
that every conserved locus directs a process of fundamental importance
to the biology of these plasmids. For example, each conserved gene in
one cluster was later shown to encode a vir gene (Fig.
1), while most of the nonconserved
sequences were later shown to encode insertion sequence (IS) elements.
The Engler study identified two conserved genetic loci at the left end
of the known vir region that were not subsequently studied.
The reported positions of these conserved sequences suggested that they
might flank the virH operon, although our analysis
necessitates a reevaluation of these mapping data. Since all the genes
in the vir region that are essential for tumorigenesis are
conserved between the two plasmids, we decided to identify these
conserved genes and evaluate their roles in pathogenesis.
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FIG. 1.
Octopine- and nopaline-type Ti plasmid vir
regions. Solid parallelograms represent the regions of homology
described by Engler and colleagues (11). The dashed
parallelogram indicates the region conserved between these plasmids.
Restriction fragments newly sequenced in this study are indicated with
white boxes.
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MATERIALS AND METHODS |
Bacterial strains, plasmids, and media.
A348 is a widely
used strain that harbors the C58 chromosome and the octopine-type Ti
plasmid pTiA6NC, while R10 is a wild-type strain that carries pTiR10,
an octopine-type Ti plasmid that is virtually identical to pTiA6NC. All
strains and plasmids used in this study are described in Table
1. A. tumefaciens strains were
cultured at 28°C in either LB medium, AB defined medium, or induction
broth (3) supplemented, as needed, with 100 µg of
kanamycin, carbenicillin, or spectinomycin per ml.
Sequence determination and analysis.
Plasmid pCC130 contains
BamHI fragment 15 of pTiA6NC, pVIK182 contains
EcoRI fragment 11 of pTiC58, and pVIK187 contains the partially overlapping BamHI fragment 7 of pTiC58, each
introduced into the corresponding sites of pTZ18R. These plasmids were
used as templates for double-stranded automated sequencing using
Taq DNA polymerase, Dyedeoxy Terminator sequencing kits
(Applied Biosystems), and an ABI model 373A Stretch DNA sequencer.
Sequences were analyzed with the DNASTAR program.
Creation of gene disruptions and lacZ fusions.
A
7.2-kb BamHI-NheI DNA fragment containing the
virH locus was obtained from cosmid pVK219 (27)
by digestion with BamHI and XbaI and ligated to
vector pUC12Cam digested with the same enzymes, creating pVIK193. A
3.0-kb Eco47III fragment containing virH was
deleted and replaced with a 2.1-kb SmaI fragment carrying the spectinomycin resistance gene of plasmid pFP45
(12), creating plasmid pVIK200. A 5.3-kb SalI
fragment of pVIK200 that contains the spectinomycin resistance gene and
flanking pTiA6 plasmid DNA and was introduced into the suicide vector
pWM91 (31), creating pVIK202. pVIK202 was transferred from
Escherichia coli S17-1/
pir into A. tumefaciens
R10 by conjugation. Selection for double recombination between pVIK202
and the Ti plasmid was applied by simultaneous selection for resistance
to spectinomycin and sucrose, resulting in strain VIK28, which has the
virH locus replaced by the spectinomycin resistance
cassette.
Internal fragments of each of open reading frames (ORFs)
orf1 to
orf7 and
virK were created by
PCR amplification and cloned
into suicide vectors pVIK107 or pVIK111
(Table
1), creating an
in-frame translational fusion with
lacZ. The resulting plasmids
were conjugally transferred
from S17-1/
pir into
A. tumefaciens A348 and R10 and
selected by using kanamycin. To confirm that
integration of these
suicide plasmids occurred by Campbell-type
homologous recombination, we
digested the entire genome of these
A. tumefaciens strains
with
EcoRI, circularized the resulting
fragments by using T4
DNA ligase, introduced them into
E. coli S17-1/
pir by electroporation, and analyzed the rescued
plasmids
by restriction endonuclease digestion. In each case, the
restriction
map of the recovered plasmid indicated that a single
homologous
recombination event had occurred at the predicted site (data
not
shown).
RNA isolation and RNase S1 protection assays.
RNA was
purified from strain VIK10 cultured at pH 5.3 in the presence or
absence of acetosyringone and from the virG mutant strain
VIK11 in the presence of acetosyringone (25). RNase S1 protection assays were carried out as previously described
(47), using the following oligonucleotides as probes:
orf1, 5'-CGATTGCGGGGTTAAGATCGTGTCATGCTCGCTTCT-3'; orf2,
5'-CCGCGCACCGATTGCCGGAATGATCTGGGTCTCGAATGC-3';
orf3,
5'-GGGCGAATGCGAAGCTTCGACGGAGCCTCTCCTGGGTA-3'; orf4, 5'-CTATCCGAGGTCATTGTGTCCACAAACTCCCCAAC-3';
orf5,
5'-CGCCATCAATTCTGCAACGGTCGGGGTGCCGGCTCGCAAATA-3'; orf6,
5'-CGCGTCCTACATCTGGGCGTATCGCAGCGGTCTTTCG-3';
orf7,
5'-GAGCTTGAGGACGCGGATAAGCAGGCTGTTTCCATTCATGGG-3'; virK,
5'-CACCGACAAGGATTGTACTGATCCGTTTCATAACTAT-3';
virG, 5'-CACGTGAAGGATGTACAATCATCTCCAGAGCCCG-3'; virB1,
5'-CCCCGATCTCTTAAACATACCTTATCTCCTTAGCTCGCCCTGG-3'; and rpoD,
5'-CCCTTCGCGCGACAGAAGCTCGACGGAA CCCATTTCTATAC-3'. Each
oligonucleotide contained four noncomplementary nucleotides at its 3'
end, and removal of these nucleotides ensured that the resistance of
the remaining part of the oligonucleotide was due to hybridization. Oligonucleotides complementary to virB1 and to
rpoD were used as inducible and constitutive controls,
respectively.
Southern hybridizations.
Southern hybridization analysis
were performed with Zeta-Probe GO blotting membranes according to the
procedures described by the manufacturer. Hybridization temperatures
used were 65°C for high-stringency and 42°C for lower-stringency
conditions. Radiolabeled probes were created by PCR in the presence of
[
-32P]dCTP (30).
Cytochrome P-450 difference spectra.
The virH1
gene was cloned as an Eco47III-EheI fragment into
the SmaI site of pTZ18R, creating pVIK204, while
virH2 was introduced as an
EcoRV-Eco47III fragment into the SmaI
site of pTZ18R, creating pVIK206. Derivatives of strain JM101
containing pVIK204 or pVIK206 were cultured in 100 ml of LB broth
containing carbenicillin (100 µg/ml) to early log phase, treated
with isopropyl-
-D-thiogalactopyranoside (IPTG; 1 mM,
final concentration), and incubated for an additional 3 h. Cells
were collected by centrifugation and disrupted in a French
pressure cell. Cellular debris was removed by centrifugation (65,000 × g for 30 min). The supernatants were treated
with crystalline dithionite and then analyzed across the visible
spectrum in the presence and absence of carbon monoxide
(28).
Tumorigenesis assays.
A. tumefaciens wild-type and
mutant strains were used for tumorigenesis assays in both
Kalanchoë diagremontiana leaves (29) and
carrot disks (19).
Assays of toxicity of aromatic compounds.
Petri dishes
containing AB minimal salts, (pH 5.5) and 100 µM acetosyringone, and
containing or lacking of 0.04% glucose, were spotted individually with
100 µl of 100 µM solutions of acetosyringone, salisylic acid,
4-hydroxybenzoate, 4-hydroxybenzaldehyde, syringic acid,
dimethoxybenzoate, protocatechuate, quinate, ferulate,
acetovallone, vanillin, 2,4-dinitrophenol,
trans-4-hydroxy-3-methoxycinnamic acid,
p-coumaric acid, 4-hydroxyacetophenone,
3,5-dimethoxyacetophenone, phenanthrenequinone,
DL-,
-diaminopimelic acid, and
5,5-dithiobis-2-nitrobenzoic acid (all dissolved in
dimethylformamide). Three milliliters of top agar (0.5%
agar in water) containing approximately 107 bacteria
that were previously cultured in the presence of acetosyringone and
then washed with phosphate buffer (pH 5.5) was poured over these plates
and allowed to solidify. Plates were incubated at 28°C for an
interval of 5 days and scored for a zone of inhibition.
Nucleotide sequence accession numbers.
The sequences
reported were deposited in the GenBank DNA sequence database (accession
no. AF039888 for sequences from pTiA6NC and accession no.
AF034769 for sequences from pTiC58).
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RESULTS |
Sequence analysis of the left ends of the vir regions
of pTiA6NC and pTiC58.
Figure 1 shows all of the genetic loci in
the vir region that were identified by Engler and colleagues
(11) as being conserved between octopine-type and
nopaline-type Ti plasmids (indicated by parallelograms). These
conserved sequences include two regions to the left of
virA, one 2.5 and the other 0.5 kb in length, that had not
previously been genetically described. According to the Engler
analysis, both of these conserved regions are located on EcoRI fragment 11 of pTiC58 (Fig. 1). To begin an analysis
of the left end of the vir region, we sequenced
BamHI fragment 15 of pTiA6NC and EcoRI fragment
11 of pTiC58. In the latter sequence, we found a 0.5-kb ORF that
resembled an ORF that lies between virH and virA
of pTiA6NC. These genes, both designated virK, also strongly
resemble a gene designated y4wH from Rhizobium
sp. strain NGR234 (Table 2).
virK evidently represents the conserved region between
virH and virA (Fig. 1). Surprisingly,
however, the BamHI fragment 15 of pTiA6NC showed no
conservation with EcoRI fragment 11 of pTiC58. This made us
suspect that there could be an error in the Engler analysis, such
that these conserved sequences either do not exist or are located
elsewhere.
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TABLE 2.
Amino acid sequence similarities between ORFs found in
this study and the products of other sequenced genes
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In an effort to identify the conserved region at the extreme left
end of the
vir region, we completed the sequence of
BamHI
fragment 7 of pTiC58 (Fig.
1). Unexpectedly, we
discovered an
operon that strongly resembles the
virH operon
of pTiA6NC (Table
2). This finding was surprising for two reasons.
First,
virH was previously thought to be absent from pTiC58.
Second, since
these conserved sequences almost certainly represent the
remaining
conserved sequence identified by the Engler study, it appears
that an error had been made in interpreting heteroduplexed DNA
fragments (see Discussion).
As might be expected, the newly sequenced regions of both plasmids
contained several nonconserved ORFs. Five ORFs (Fig.
1)
were
identified in
BamHI fragment 15, while three were
identified
in pTiC58 in addition to those described above. We also
analyzed
two additional ORFs (
orf5 and
orf6) that
were previously (
26)
sequenced but not characterized (Fig.
1). Of these, the predicted
amino acid sequences of
orf5,
orf8, and
orf9 resemble genes of
various IS
elements (Table
2). Interestingly, the first 100 deduced
amino acid
residues of the
orf10 product are similar to the
N-terminus
sequence of the VirF proteins of
A. tumefaciens and
A. vitis (Fig.
2 and Table
2). No other ORF resembles
any known protein sequences,
although
orf2 contains a
nucleotide-binding motif (GQQGAGKSTL)
that matches the highly conserved
Walker A consensus sequence
(GXXXXGK[T/S]) found in many
nucleotide-binding proteins (
41).
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FIG. 2.
Sequence similarity between Orf10 of pTiC58 and the VirF
of pTiA6. Straight lines represent polypeptide sequence with no
similarity. Boxes represent conserved regions; black boxes represent
regions of stronger similarity than gray boxes (also see Table 2 for
similarity scores).
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Nuclease S1 protection assays.
It was already known that the
virH operon is strongly induced by the VirA-VirG regulatory
system in response to wound-released chemical signals. We wanted to
determine whether virK or any of the nonconserved ORFs found
on pTiA6NC was regulated in a similar fashion. To do this, we cultured
strain VIK10 (an otherwise wild-type derivative of R10 containing a
virG-lacZ fusion on the Ti plasmid) in the presence or
absence of acetosyringone and cultured strain VIK11 (an isogenic
derivative of VIK10 containing a virG null mutation
[25]) in induction broth containing acetosyringone. We
then isolated total RNA from all three cultures and performed S1
protection assays using 5'-radiolabeled oligonucleotides that would
hybridize to RNA of each gene. Protection of the probes complementary
to virK, orf2, and orf3 was strongly
enhanced by vir gene induction (Fig.
3). In contrast, orf1,
orf4, orf5, orf6, and orf7
either are not expressed or are constitutively expressed at low levels.
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FIG. 3.
Nuclease S1 protection assays. Lane 1, oligonucleotide
without S1 digestion; lane 2, protection by RNA purified from strain
VIK10 cultured without acetosyringone; lane 3, protection by RNA
purified from strain VIK10 cultured with acetosyringone; lane 4, protection by RNA purified from strain VIK11 cultured with
acetosyringone.
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Translational fusions between Ti plasmid ORFs and lacZ.
To provide additional evidence for the transcriptional regulation of
the ORFs described above, we created gene fusions between them and
lacZ. This was done by PCR amplifying an internal fragment of each ORF, cloning each fragment into a suicide vector that carries a
lacZ reporter (25), introducing the vectors into
A. tumefaciens A348 and R10, and selecting for Campbell-type
homologous recombination, which causes integration of the entire
plasmid into the corresponding locus. This procedure results in
simultaneous disruption of the target gene and fusion of its promoter
and translation start site to the lacZ reading frame
(25).
All strains constructed were assayed for
-galactosidase activity
after incubation in the presence and absence of
vir
gene-inducing
conditions. The results indicate that
virK-lacZ,
orf2-lacZ, and
orf3-lacZ
fusions expressed dramatically elevated
-galactosidase
activity
in response to
vir-inducing stimuli, while the remaining
five fusions did not respond to these conditions (Table
3). These
data are in full agreement with
the nuclease S1 protection assays
described above (Fig.
3). On the
basis of these two kinds of evidence,
we conclude that
virK,
orf2, and
orf3 are members of the
vir
regulon,
while the remaining ORFs are not. We therefore renamed the
latter
two ORFs
virM and
virL, respectively, to
indicate that they are
coregulated with other
vir
genes. These designations are not meant
to imply that these genes are
required for tumorigenesis (see
below).
Tumorigenesis assays.
As described above, each of the eight
ORFs described above was disrupted by Campbell-type integration. We
tested each strain for the ability to cause neoplastic growth of
K. diagremontiana leaves or carrot disks. Strains R10 and
A348 were used as positive controls. We observed no significant
differences between wild-type and mutant strains (data not
shown).
One member of the
vir regulon (
virJ) encodes a
function that is essential for tumorigenesis but functionally redundant
with
the chromosomal gene
acvB (
24,
35). It
seemed plausible that
one or more of the three ORFs likewise could
encode a function
that was essential but genetically redundant. To test
this, we
carried out Southern hybridizations under conditions of low
stringency,
probing the genomic DNA of strains A348, A136, C58, NT1,
R10,
and KYC55 (a derivative of R10 lacking a Ti plasmid
[
5]). These
DNAs were probed with internal fragments
of
virK,
virL, and
virM that were generated by PCR amplification. A probe for the redundant
virJ gene was included in these experiments, since this gene
is
known to have a chromosomal homolog (see above). As expected,
virK sequences were found in strains carrying either a
nopaline-type
or octopine-type Ti plasmid, but in each case, only a
single gene
was identified in each strain (Fig.
4). Similarly, only single
copies of the
virL and
virM genes were found on octopine-type
Ti plasmids, and these genes were not detected on the nopaline-type
Ti
plasmid, indicating that these genes are not conserved between
these plasmids. Using a
virJ probe under identical
conditions,
we detected both
virJ and the chromosomal
acvB gene (data not
shown). These data strongly suggest that
virK,
virL, and
virM are each encoded
by single genes and that the lack of an effect
on tumorigenesis is
not due to genetic redundancy.
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FIG. 4.
Low-stringency Southern hybridizations using internal
PCR fragments for virK (top), virL (middle), and
virM (bottom). The last lane contains molecular mass
standards that hybridize to the same probe.
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Construction of an A. tumefaciens strain lacking
virH1 and virH2.
As described above, transposon
insertions in the virH operon were reported to have either
no effect or only subtle effects on tumorigenesis (26,
39). However, sequence analysis of this operon revealed the
presence of two related ORFs, designated virH1 and
virH2, suggesting that they could be functionally
redundant. If so, all previously characterized Tn3HoHo1
mutations in this operon would disrupt one but not the other.
Furthermore, it was not established which insertions disrupted
virH1 and which disrupted virH2. Insertions in
virH1 (if any exist) might not be polar on virH2,
since not all Tn3HoHo1 insertions of exert polar effects (39). To reexamine the possible role of the
virH operon in tumorigenesis, we created a strain carrying a
deletion of the entire operon. Strain VIK28 is a derivative of
R10 in which the entire virH operon has been replaced
with a spectinomycin resistance gene (Fig.
5A; see Materials and Methods). This
deletion also removed the first 104 nucleotides of virK.
Southern hybridization analysis indicated that this deletion has the
predicted physical features (Fig. 5B). We observed no significant
differences between this mutant and its wild-type parent strain
in tumorigenesis of K. diagramontiana leaves (data not
shown).
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FIG. 5.
Replacement of the virH locus by a gene
mediating resistance to spectinomycin. (A) Double recombination
required for this exchange. (B) Southern hybridizations of the parental
strain R10 (wt [wild type]) and two identical mutants (1 and 2). The
first of these was designated strain VIK28. The extreme left lane
contains molecular mass standards that hybridize with the same probe. A
PCR fragment lying within orf6 was used as a probe.
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Spectral analysis of VirH1 and VirH2.
It was previously
reported that VirH1 and VirH2 resemble monooxygenases of the P-450
family (26). This family of proteins generally
hydroxylate diverse substrates at the expense of molecular oxygen
and have varied biological roles in detoxification of toxic compounds,
in the catabolism of growth substrates, and in the biosynthesis of secondary metabolites (32). These
enzymes show characteristic carbon monoxide difference spectra with a
strong peak at 450 nm. Under certain conditions, difference spectra of these enzymes can have a peak at 420 nm rather than 450 nm (34, 42, 46), although such a peak generally indicates that the enzyme
is catalytically inactive. To determine the difference spectra of VirH1
and of VirH2, we expressed each protein in E. coli (see
Materials and Methods). Cleared extracts containing either enzyme
showed a carbon monoxide difference spectrum with a prominent peak at
420 nm (Fig. 6). We were not able to find conditions in which a peak at 450 nm was observed.
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FIG. 6.
Carbon monoxide difference spectra of VirH1 and VirH2.
Bacterial extracts were prepared as described in Materials and Methods
and scanned across the visible spectrum in the presence and absence of
carbon monoxide.
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Since several bacterial P-450 monooxygenases play roles in catabolism
of toxic compounds (
33,
38), and since plant wound
sites are
known to contain many compounds that are toxic to microorganisms
(
9), we wanted to determine whether
virH or any
other member
of the
vir regulon might have a role in
detoxification. To do
this, we pretreated with acetosyringone three
strains, wild-type
A. tumefaciens strain R10, an isogenic
virH deletion mutant (VIK28),
and an isogenic
virG mutant (VIK10), and then plated these cultures
in top
agar on solid medium containing a variety of phenolic compounds
(20 compounds in all). Zones of growth inhibition for each compound
were
identical in size for all three strains, suggesting that
no member of
the
vir regulon is involved in the detoxification
of these
compounds.
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DISCUSSION |
Our long-term interest in the vir regulon of the
octopine-type Ti plasmid drew our attention to two DNA fragments that
are conserved in the vir regions of two widely studied Ti
plasmids (Fig. 1) but were previously uncharacterized. These sequences were thought to have been located within BamHI
fragment 15 of pTiA6NC and within EcoRI fragment
11 of pTiC58. As described above, our sequence analysis indicates
that these regions are not conserved and that one of the conserved loci
is virH itself. The second conserved locus had been
previously sequenced in pTiA6NC (26) but not characterized
and had not previously been found in pTiC58. At the same time, we also
wanted to identify any new members of the vir regulon,
even if not conserved, since any such genes are by definition induced
at the outset of plant infection and are likely to play significant
roles in the initial stages of host-microbe interactions.
The virH and virK loci.
Since it was
widely believed that virH is absent from nopaline-type Ti
plasmids, we were quite surprised to identify this operon in
BamHI fragment 7 of pTiC58. Since we found no conserved genes in the positions predicted by the Engler study (11),
it appears that a mistake had been made in that study in
interpretation of the heteroduplexed DNA. While Engler et al.
showed a short nonconserved region separating the genes now designated
virH and virK on pTiC58 and a longer one on
pTiAch5, we find exactly the opposite and therefore propose
that these single-stranded regions separating virH and
virK in the Engler study had been incorrectly assigned.
The
virH locus consists of two genes that code for proteins
of the P-450 family of monooxygenases (
32). This locus
of pTiA6NC
is a member of the
vir regulon
(
26) and is therefore induced
at the outset of infection.
While induction of the pTiC58
virH region has not been
tested, DNA sequence similarity between these
operons extends into the
5' regulatory region.
virH was previously
believed to
be restricted only to octopine-type Ti plasmids and
in some cases
speculated to account for different properties of
these two Ti plasmids
(
13,
26).
Monooxygenases of the P-450 family acquire their name from their strong
increase in absorption at 450 nm when treated with
carbon monoxide
(
34). Several of these enzymes have been shown
to have a
difference spectrum at 420 nm rather than 450 nm (
42,
46).
Proteins that absorb at this shorter wavelength (termed
P-420 enzymes)
are generally catalytically inactive. The P-450CAM
protein of
Pseudomonas putida shows a difference maximum at 450
nm when
purified from
P. putida but under different conditions
shows
a difference maximum of 420 when expressed in
E. coli
(
46).
E. coli extracts containing VirH1 or
VirH2 showed carbon monoxide
difference spectra at 420 nm.
We were unable to find conditions
where extracts containing either
protein show a peak at 450 nm.
An increase in absorption at 420 nm is
nevertheless a hallmark
of this family of proteins, and we therefore
conclude that VirH1
and VirH2 are probably members of this family and
likely to carry
out hydroxylation or related oxidation reactions.
Transposon insertions in the
virH operon have been
previously described (
26) but not precisely mapped to one or
the other
of these genes. Since
virH1 and
virH2
are homologous to each other,
it seemed plausible that they could be
functionally redundant.
Because the transposon used to mutagenize
this operon (Tn
3HoHo1
[
39]) does
not always exert polar effects on downstream genes,
even an insertion
in
virH1 might not disrupt expression of both
genes.
We therefore created a deletion of the entire
virH
locus
and compared mutant and wild-type strains for the ability to
cause
tumorigenesis. Our results showed that at least for the plant
host tested,
virH is not required for efficient
tumorigenesis.
We also addressed the question of whether these proteins could play a
role in detoxifying compounds found at plant wound sites.
Members of
the family of the P-450 monooxygenases have often been
reported to be
involved in the detoxification of toxic compounds.
For example,
P-450CAM of
P. putida metabolizes the rather
toxic
compound camphor, in the process converting it to a source of
carbon and energy (
32). However, neither
virH nor
any member
of the
vir regulon altered the toxicity of any
the 20 different
phenolic compounds tested.
As described above, the other conserved locus in the Engler study is a
gene that we now designate
virK. This gene was
previously
sequenced in pTiA6NC but not genetically analyzed
(
26). We showed
that this gene is strongly induced by
acetosyringone in a VirG-dependent
(and probably VirA-dependent)
manner. Disruption of
virK did not
affect
tumorigenesis.
virK strongly resembles a gene
designated
y4wH found on pNGR234a of
Rhizobium
sp. strain NGR234 (
14).
Directly upstream of
y4wH is a possible binding site for the NodD
transcriptional
regulator, suggesting that the product of this
gene may play a role at
the outset of the nodulation of host legumes.
The role of
virK and its
Rhizobium homolog in plant-microbe
interactions
will require additional studies.
Other acetosyringone-inducible loci.
We also studied seven
previously uncharacterized ORFs that might encode proteins. RNase S1
protection assays (Fig. 3) and translational -galactosidase fusions
(Table 3) revealed that orf2 and orf3 are
strongly induced by acetosyringone. These ORFs were therefore
renamed virM and virL, respectively, to indicate that the genes are members of the vir regulon. Neither
protein resembles any other sequenced protein, although VirM has a
possible ATP-binding motif (41).
Two different chromosomal backgrounds were used to create single-copy
translational fusions between each
orf and
lacZ.
When
the fusions were created in the wild-type octopine strain
R10,
induction was consistently twofold higher than when
strain A348,
which harbors a nopaline chromosome and the octopine-type
Ti plasmid
pTiA6NC, was used. Since pTiA6NC and pTiR10 are virtually
identical,
these data suggest that the host chromosomal backgrounds
played
a role in induction. One possibility is that the sugar-binding
ChvE proteins are functionally different in these strains. It
was
previously reported that a particular VirA protein interacts
optimally with ChvE from the same chromosomal background
(
10).
Neither
virM nor
virL was required for efficient
tumorigenesis of two plants. Southern hybridizations proved that
the absence
of a tumorigenic phenotype is not due to any homolog
found elsewhere
on the
A. tumefaciens genome. We conclude
that neither protein
plays a critical role in tumorigenesis. They could
play more subtle
roles in tumorigenesis that are difficult to
demonstrate under
laboratory conditions. It seems equally
plausible that these proteins,
as well as VirHI, VirH2, and VirK,
carry out roles in pathogenesis
that are unrelated to T-DNA
transfer. The fact the VirK strongly
resembles the product of a
Rhizobium sp. gene that seems to be
under the control of the
NodD transcriptional regulator strongly
supports this hypothesis. At
this point it is difficult even to
speculate about what these roles
might be. In the case of the
virH-encoded monooxygenases, a
role in phytohormone biosynthesis
could be one possibility, since a
monooxygenase could convert
tryptophan to indoleacetamide (on the
pathway to auxin). A monooxygenase
could also carry out the
hydroxylation step required to convert
isopentenyladenine to zeatin, a
cytokinin. A P-450 type enzyme
has been implicated in cytokinin
biosynthesis in
Rhodococcus fasciens (
7).
| |
ACKNOWLEDGMENTS |
We thank Jeff Scott for providing preparations of insect
microsomal P-450. Many thanks go to J. Shapleigh, C. Fuqua,
J. Zhu, S. Jafri, and R. Akakura for helpful suggestions and
discussions.
This work was supported by General Medical Sciences award GM42893 from
NIH.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Section of
Microbiology, Wong Hall, Cornell University, Ithaca, NY 14853. Phone:
(607) 255-2413. Fax: (607) 255-3904. E-mail:
scw2{at}cornell.edu.
| |
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Journal of Bacteriology, November 1998, p. 5660-5667, Vol. 180, No. 21
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Abstract
Introduction
