lungs with multiple air-ffuid levels, pulmonary hypertension, and localized right pneumothorax. From March 1996 to Feb- ruary 1999, she was readmitted seven ...
JOURNAL
OF
BACTERIOLOGY, Feb. 1990, p. 1142-1144
Vol. 172, No. 2
0021-9193/90/021142-03$02.00/0 Copyright C) 1990, American Society for Microbiology
VirA, a Coregulator of Ti-Specified Virulence Genes, Is Phosphorylated In Vitro YONG HUANG, PATRICE MOREL, BRADFORD POWELL, AND CLARENCE I. KADO* Department of Plant Pathology, University of California, Davis, California 95616 Received 28 June 1989/Accepted 1 November 1989 High-level expression of a chimeric virA gene was obtained by replacing the first 524 codons of virA with the first half of trpE. The encoded fusion protein was isolated and found to exhibit autokinase activity. Therefore, a kinase domain is in the C-terminal portion of VirA, and protein phosphorylation may be an important feature of VirA function.
Agrobacterium tumefaciens causes crown gall disease on a wide variety of plants by the transmission of infectious DNA (T-DNA) from a large tumor-inducing (Ti) plasmid to the plant cell genome (for recent reviews, see references 10 and 15). The T-DNA contains phytohormone genes whose expression in the plant cell causes uncontrolled growth resulting in the formation of an overgrowth or tumor. A second sector of this plasmid called the virulence (vir) region contains a number of genes that are not transferred but nevertheless are essential for tumorigenicity (8, 13). These genes are simultaneously induced (21) when the bacteria sense the proximity of a susceptible host by detecting the presence of phenolic compounds elaborated from plant wound tissue (1, 22). The products of two of these genes, virA and virG, are required for this induction (10, 15, 21). VirA is a transmembrane protein that is thought to act as the environmental sensor of inducing compounds (12, 24). Its probable function is to transmit this signal to VirG, a DNA-binding protein (20). VirG is then thought to become capable of enhancing transcription of other vir operons whose encoded proteins, such as VirDl and VirD2 (25), are directly involved in processing T-DNA. Defining the mechanism by which VirA activates VirG is central to understanding the regulation of virulence genes in A. tumefaciens. DNA sequence analysis of virA (1Sa) and virG (19) from the plasmid pTiC58 has revealed that the products of these genes share a common ancestry with a family of twocomponent regulatory proteins that control diverse sets of genes (18). One member of the pair of regulatory proteins detects (directly or indirectly) and transmits environmental signals to the second member, which functions as the immediate effector of gene expression (or of the flagellar motor in one case). Studies on homologous proteins have revealed that the response to nitrogen limitation by NtrBNtrC (7, 16), the control of chemotaxis by CheA-CheBCheY (5), and the response to changes in medium osmolarity by EnvZ-OmpR (6) all involve protein phosphorylation during signal transduction by the sensor component and activation of the regulator component. To determine whether the VirA-VirG regulatory pair also uses this mechanism, we tested whether a chimeric protein containing a portion of the VirA polypeptide could be phosphorylated in vitro. Previous results have shown that overproduction of native VirA in Escherichia coli is deleterious to cell growth (unpublished data) and that this phenomenon depends on the *
synthesis of either the second hydrophobic core or some nearby portion of the VirA protein (24). This severely hampers efforts to obtain sufficient quantities of native VirA for biochemical studies. We reasoned that the removal of this hydrophobic domain might result in high-level synthesis of a truncated protein exhibiting partial VirA activity. A gene fusion between the E. coli trpE gene and the A. tumefaciens virA gene was constructed such that the encoded fusion protein (VirA 2203) contained the first 334 amino acids of TrpE in place of the first 524 amino acids of VirA and was expressed from the tryptophan promoter (Fig. 1). Therefore, VirA 2203 lacked all of the periplasmic region of the native VirA protein and part of its cytoplasmic region (24). However, this protein retained most of the original carboxy terminus, including the consensus nucleotidebinding sequence, Gly-X-Gly-X-X-Gly (23), at amino acids 658 to 663 of VirA (1Sa), and most of the amino acid sequences similar to the proposed transmitter domain of homologous proteins (9). The chimeric gene coding this fusion protein was constructed by inserting the 1.9-kilobasepair BglII-BamHI fragment of pUCD1105 (21) containing the last 928 base pairs of virA into the BamHI site of the pATH1 vector carrying the tryptophan promoter and half of the trpE gene (2). TrpE-containing proteins were prepared as follows: DH5a cells containing either pUCD2203 or pATH1 (control) were grown to stationary phase (overnight) at 28°C in 1 ml of M9CA (14) containing ampicillin (100 ,ug/ml) and tryptophan (20 ,ug/ml) and then divided equally into two tubes of 5 ml of M9CA with ampicillin and shaken at 28°C. After 2 h, 3-p-indoleacrylic acid (20 p.g/ml) was added to one of each of the duplicate cultures to induce the tryptophan promoter. The cultures were grown for another 2 h before harvesting by pelleting cells from 1 ml in a microfuge. Cells were suspended in 100 ,ul of buffer A (50 mM Tris hydrochloride [pH 8.0], 0.5 mM EDTA, 50 mM NaCi, 1 mM dithiothreitol) and then lysed by incubation with 1 mg of lysozyme per ml (final concentration) on ice for 20 min, followed by sonication on ice for 45 to 60 s, 50% duty, 80% full power (Heat Systems-Ultrasonics, Farmingdale, N.Y.). Aliquots (5 RI) of these suspensions were each dissolved in 5 RI1 of Laemmli sample buffer (11) to generate whole-cell extracts for analysis later. Cell debris and insoluble proteins were removed by centrifugation at 15,000 x g for 15 min. Although much of the fusion protein produced by trpE'-'virA recombinant plasmid partitioned with the pellet fraction, plasmid-specific protein was also detected in the supernatant fraction. One of these proteins exhibited a relative mass of 37 kilodaltons
Corresponding author. 1142
VOL. 172, 1990
NOTES Pst
Hpa
Sal
Sal
Pvu 11
Sph
Bgl 11
\\\i\\\ trpE'
|
Eco RV
M~~~~~~0.0'~\\\M /
trpE'
Pvu 11
/
pUCD2201
\\\\\\'A
pUCD2203
//X//XM\I
pUCD2824
/
1143
0.5 Kb
FIG. 1. Restriction map and gene organization of the original virA subclone, pUCD2201 (14), and the trpE'-'virA gene fusions, pUCD2203 and pUCD2824, used in this study. Nontransmitter regions ( M ), membrane-spanning regions (_), transmitter domain ( M ), and trpE' coding sequence (Eli) are shown. Locations of the nucleotide-binding sequence (@) and inclusive histidine (*) are indicated immediately below the full-length virA gene. Kb, Kilobase pairs.
(kDa), a size expected for the TrpE' fragme nt (Fig. 2A). After subsequent fractionation (see below), strain DH5a (pUCD2203) was found to contain soluble pro tein having a relative mass of 70 kDa, in good agreement with the predicted size of the VirA 2203 protein (37 kDa for^TrpE' and 33 kDa for VirA). VirA 2203 was further purifi ed by affinity chromatography by using a 1.5-ml bed volume of Affi-gel Blue (Bio-Rad Laboratories, Richmond, Calif.). B(cund proteins were washed with 3 ml of buffer A, then eiluted in two consecutive steps using 3 ml each of 300 mM and 500 mM NaCl in buffer A. Since most of the activity wats found in the high-salt fraction, this fraction was used for furrther analysis. Salts were diluted 10-fold by successive conc-entration and dilution on Centricon 30 microconcentrators (^Amicon, Danvers, Mass.), and the final retentates (100 to 200 ,u1) were brought to 500 ,ul with the same buffer. VirA 22()3 protein was enriched from this preparation in comparison to whole-cell extracts (data not shown) and appeared to be greater than 50% pure (Fig. 2A). The identities of TrpE'-c4 ontaining proteins were confirmed by Western blot (immunc)blot) analysis (Fig. 2B) by using anti-TrpE' immunoglobulin a prepared by
1
MW
92.5 66.2
2
_a
.-
C
B
A
3
-.4
1
2
3
1 2 w
3 4
_L.
45 31
_
FIG. 2. Analysis of the VirA 2203, VirA 2824, and TrpE' proteins. Shown are proteins from 3-j3-indoleacrylic ac.id-induced cultures of E. coli DH5a containing the pATH1 vect or (lane 1), the trpE'-'virA plasmid pUCD2824 (lane 2), and the trpEE'-'virA plasmid pUCD2203 (lane 3), resolved by electrophoresis on a 12% sodium dodecyl sulfate-polyacrylamide gel. (A) Coomassi Le brilliant blue stain. (B) Immunoblot using anti-TrpE' antibodies. graph of the samples treated by the phosphorylat described in the text. Lane 4 in panel C contains VizrA 2203 protein treated with [a-32P]ATP. The protein positions of the 70- (VirA 2203) (A), 65- (VirA 2824) (A), and 37-kDa (TrpE') (< proteins are shown. Positions of molecular weight standards (1013) are indicated on the left.
i(C) Autoradno7)
standard protocols (4) and alkaline phosphatase-conjugated goat anti-rabbit immunoglobulin G according to the manufacturer (Bio-Rad). Phosphorylation was tested by incubating the partially purified proteins with 5 mM MgCl and 0.02 mM of either [at-32P]ATP or [-y-32P]ATP (Amersham, Arlington Heights, Ill.) in a final reaction volume of 10 ,u1 for 5 min at room temperature. Reactions were terminated by the addition of Laemmli sample buffer and analyzed by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (11). After electrophoresis, the gel was dried under vacuum and autoradiographed with X-OMAT AR film (Eastman Kodak, Rochester, N.Y.) for-12 h at -80°C and an intensifying screen. Result showed that VirA 2203 protein was labeled in the presence of [y-32P]ATP, while no specific protein was labeled in the presence of [a-32P]ATP (Fig. 2C). The truncated TrpE protein encoded by the pATH1 vector was not labeled under the same conditions (Fig. 2C). Since radioactivity appeared on VirA 2203 only with gamma-labeled ATP and because labeling did not require the presence of TrpE', these data strongly suggest that VirA amino acid sequences distal to residue 524 contribute to the kinase activity. To further define the extent of VirA primary structure necessary for autokinase activity, a second trpE'-'virA fusion was constructed. The 2.0-kilobase-pair PvuII fragment of pUCD2206 (1Sa) carrying the last 632 base pairs of virA was inserted into the SmaI site of pATH11 (2). The resulting plasmid, pUCD2824, directed the synthesis of a chimeric VirA protein (VirA 2824) having a relative mass of 65 kDa (Fig. 2B and C). In contrast to the previous fusion protein, this second TrpE'-'VirA protein was not phosphorylated under the same conditions (Fig. 2C, lane 2). This suggests that a sequence important for the autokinase reaction is localized on the VirA protein between amino acids 524 and 623, situated at the amino-terminal end of the proposed transmitter domain and 35 amino acids before the consensus nucleotide-binding sequence. By comparison to aligned sequences of CheA and EnvZ (3, 5), we found that the critical region described here encompasses a histidine residue which may be the phosphorylated site of VirA (Fig. 1). By inference, histidine 618 of VirA likely serves a similar function during signal transduction by VirA. In conclusion, we have shown that a TrpE'-'VirA fusion protein exhibits kinase activity and that the carboxy-terminal portion of VirA is responsible for this activity. On the basis of the similarities between VirA, CheA, NtrB, and
NOTES
1144
EnvZ, we suggest that the native VirA protein also catalyzes autophosphorylation and that VirA-P may function as a protein kinase using the transcriptional activator, VirG, as its principal target. The simple methods described here should be useful in studying other putative sensors of this large family of proteins. These results provide an important clue to the mechanism of signal transduction involved in the induction of virulence genes of A. tumefaciens and further strengthens the view that the two-component regulatory systems share a common mechanism based on phosphate transfer (17). This work was supported by Public Health Service grants CA11526 from the National Cancer Institute and Public Health Service training grant fellowship GM-07377 from the National Institutes of Health awarded to B.P.
LITERATURE CITED 1. Bolton, G. W., E. W. Nester, and M. P. Gordon. 1986. Plant phenolic compounds induce expression of the Agrobacterium tumefaciens loci needed for virulence. Science 232:983-985. 2. Dieckmann, C., and A. Tzagoloff. 1985. Assembly of the mitochondrial membrane system: CBP6, a yeast nuclear gene necessary for synthesis of cytochrome b*. J. Biol. Chem. 260: 1513-1520. 3. Forst, S., J. Delgado, and M. Inouye. 1989. Phosphorylation of OmpR by osmosensor EnvZ modulates expression of the ompF and ompC genes in Escherichia coli. Proc. Natl. Acad. Sci. USA 86:6052-6056. 4. Harlow, E., and D. Lane (ed.). 1988. Antibodies: a laboratory manual, p. 725. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 5. Hess, J. F., R. B. Bourret, and M. I. Simon. 1988. H-istidine phosphorylation and phosphoryl group transfer in bacterial
chemotaxis. Nature (London) 336:139-143. 6. Igo, M. M., and T. J. Silhavy. 1988. EnvZ, a transmembrane environmental sensor of Escherichia coli K-12, is phosphory-
lated in vitro. J. Bacteriol. 170:5971-5973. 7. Keener, J., and S. Kustu. 1988. Protein kinase and phosphoprotein phosphatase activities of nitrogen regulatory proteins NtrB and NtrC of enteric bacteria: roles of the conserved aminoterminal domain of NtrC. Proc. Natl. Acad. Sci. USA 85:
4976-4980. 8. Klee, H. J., F. F. White, V. N. Iyer, M. P. Gordon, and E. W. Nester. 1983. Mutational analysis of the virulence region of an
Agrobacterium tumefaciens Ti plasmid. J. Bacteriol. 153:878883.
9. Kofoid, E. C., and J. S. Parkinson. 1988. Transmitter and receiver modules in bacterial signaling proteins. Proc. Natl.
Acad. Sci. USA 85:49814985. 10. Koukolikova-Nicola, Z., L. Albright, and B. Hohn. 1987. The mechanism of T-DNA transfer from Agrobacterium tumefaciens to plant cell, p. 108-148. In T. Hohn and J. Schell (ed.), Plant gene research, vol. IV. DNA infectious agents. SpringerVerlag, New York. 11. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London)
J. BACTERIOL.
227:680-685. 12. Leroux, B., M. F. Yanofsky, S. C. Winans, J. E. Ward, S. F. Ziegler, and E. W. Nester. 1987. Characterization of the VirA locus of Agrobacterium tumefaciens: a transcriptional regulator and host-range determinant. EMBO J. 6:849456. 13. Lundquist, R. C., T. J. Close, and C. 1. Kado. 1984. Genetic complementation of Agrobacterium tumefaciens Ti plasmid mutants in the virulence region. Mol. Gen. Genet. 193:1-7. 14. Maniatis, T., E. F. Fritsch, and J. Sambrook. 1982. Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 15. Melchers, L. S., and P. J. J. Hooykaas. 1987. Virulence of Agrobacterium, p. 167-220. In B. J. Milfin (ed.), Oxford surveys of plant molecular and cell biology, vol. 4. Oxford University Press, London. 15a.Morel, P., B. S. Powell, P. M. Rogowsky, and C. I. Kado. 1989. Characterization of the virA virulence gene of the nopaline plasmid, pTiC58, of Agrobacterium tumefaciens. Mol. Microbiol. 3:1237-1246. 16. Ninfa, A. J., and B. Magasanik. 1986. Covalent modification of the gInG product, NRI, by the glnL product, NRII, regulates the transcription of the glnALC operon in Escherichia coli. Proc. Natl. Acad. Sci. USA 83:5909-5913. 17. Ninfa, A. J., E. G. Ninfa, A. N. Lupas, A. Stock, B. Magasanik, and J. Stock. 1988. Crosstalk between bacterial chemotaxis signal tranduction proteins and regulators of transcription of the Ntr regulon: evidence that nitrogen assimilation and chemotaxis are controlled by a common phosphotransfer mechanism. Proc. Natl. Acad. Sci. USA 85:5492-5496. 18. Nixon, B. T., C. W. Ronson, and F. M. Ausubel. 1986. Two component regulatory systems responsive to environmental stimuli share strongly conserved domains with the nitrogen assimilation regulatory genes ntrB and ntrC. Proc. Natl. Acad. Sci. USA 83:7850-7854. 19. Powell, B. S., G. K. Powell, R. 0. Morris, P. M. Rogowsky, and C. I. Kado. 1987. Nucleotide sequence of the virG locus of Agrobacterium tumefaciens plasmid pTiC58. Mol. Microbiol. 1:309-316. 20. Powell, B. S., P. M. Rogowsky, and C. I. Kado. 1989. VirG of Agrobacterium tumefaciens plasmid pTiC58 encodes a DNAbinding protein. Mol. Microbiol. 3:411-419. 21. Rogowsky, P. M., T. J. Close, J. A. Chimera, J. J. Shaw, and C. I. Kado. 1987. Regulation of the vir genes of Agrobacterium tumefaciens plasmid pTiC58. J. Bacteriol. 169:5101-5112. 22. Stachel, S. E., E. W. Nester, and P. C. Zambryski. 1986. A plant cell factor induces Agrobacterium tumefaciens Vir gene expression. Proc. Natl. Acad. Sci. USA 83:379-383. 23. Wierenga, R. K., and W. G. J. Hol. 1983. Predicted nucleotidebinding properties of p21 protein and its cancer-associated variants. Nature (London) 302:842-844. 24. Winans, S. C., R. A. Kerstetter, J. E. Ward, and E. W. Nester. 1989. A protein required for transcriptional regulation of Agrobacterium virulence genes spans the cytoplasmic membrane. J. Bacteriol. 171:1616-1622. 25. Yanofsky, M. F., S. G. Porter, C. Young, L. M. Albright, M. P. Gordon, and E. W. Nester. 1986. The virD operon of Agrobacterium tumefaciens encodes a site-specific endonuclease. Cell 47:471-477.
