A Protein Required for Transcriptional Regulation of Agrobacterium ...

Vol. 171, No. 3

JOURNAL OF BACTERIOLOGY, Mar. 1989, p. 1616-1622

0021-9193/89/031616-07$02.00/0 Copyright C 1989, American Society for Microbiology

A Protein Required for Transcriptional Regulation of Agrobacterium Virulence Genes Spans the Cytoplasmic Membrane STEPHEN C. WINANS,t RANDALL A. KERSTETTER, JOHN E. WARD, AND EUGENE W. NESTER* Department of Microbiology, University of Washington, Seattle, Washington 98195 Received 16 September 1988/Accepted 22 December 1988

The VirA protein is one of two proteins required for transcriptional activation of Agrobacterium tumefaciens virulence genes in response to phenolic compounds released by plants during infection. We describe two experimental approaches which indicate that this protein has a transmembrane topology. First, spheroplasts of Escherichia coli or wild-type A. tumefaciens expressing the VirA protein were treated with proteinase K to digest periplasmic proteins, and the remaining proteins were immunologically stained on Western blots (immunoblots) by using anti-VirA antibody. Second, transposon TnphoA was used to generate translational fusions between virA and phoA, the latter of which is the structural gene for alkaline phosphatase. Both techniques indicated that VirA spans the cytoplasmic membrane, with approximately 275 amino acids near the amino terminus being localized in the periplasmic space and the rest of the protein being localized in the cytoplasm. We also show that overexpression of VirA in E. coli is deleterious to cell growth and that this phenomenon depends on the synthesis of either the second hydrophobic core or some nearby portion of the VirA protein.

Several genera of bacteria which establish either antagonistic or mutualistic associations with higher plants have recently been shown to sense the proximity of susceptible plants and to respond to this information by increasing the transcription of genes that are required for infection. An example is found in Agrobacterium tumefaciens, which causes crown gall disease in a wide variety of dicotyledonous plants (for reviews, see references 17 and 27) and which requires the expression of a set of plasmid-encoded genes (vir genes) to incite this disease. These genes are strongly and coordinately induced by exposing the bacteria to any of a class of phenolic compounds, some of which are released from plant wounds (25). Two of these genes, virA and virG, are required for this induction (26). Similarly, the nod genes of several Rhizobium species are induced by certain isoflavonoids that are released by root hairs of legumes (22), and 14 genes of Xanthomonas campestris have been identified which are transcribed when bacteria are grown on the surface of plant leaves but not on agar medium (21). The nucleic acid sequences of virA and virG indicate that the products of these genes share a common ancestry with a family of two component regulatory proteins that control diverse sets of genes (12, 19, 24, 31). Based partly on the properties of other members of this gene family (6, 18), we have proposed that VirA may be a two-domain transmembrane protein with a periplasmically localized amino terminus that is capable of binding phenolic inducers and a cytoplasmically localized carboxy terminus with protein kinase activity. We have proposed that VirA can phosphorylate VirG, thereby converting it from an inactive form to a form that is capable of binding to vir promoters, activating their transcription (31). This model is supported by the hydropathy profile of VirA (12, 15). The amino terminus of the predicted protein contains a sequence of amino acids which is strongly similar to a class of so-called signal sequences that are thought to guide

the secretion of proteins across the cytoplasmic membrane (30). Between amino acids 260 and 290 lies a region of hydrophobic and positively charged residues similar to the so-called stop transfer sequences found in several transmembrane proteins (9). If both of these domains function as proposed, then the amino-terminal 260 amino acids of the mature protein would reside in the periplasmic space, while amino acids 290 to 829 would lie in the cytoplasm. In this report we describe the overproduction of VirA in Escherichia coli and then use two techniques to learn more about the topology of the protein. First, we used anti-VirA antibodies to detect VirA protein on Western immunoblots of protein extracts from bacteria which were converted to spheroplasts and then treated with protease to digest periplasmic proteins. Second, the transposon TnphoA (13, 14) was used to create a series of translational fusions between VirA and alkaline phosphatase (AP). TnphoA generated translational fusions between a target gene and phoA, the structural gene for AP. Since this protein has enzymatic activity only if it is secreted, such fusions have been used to determine whether the product of the target gene is secreted by measuring the level of AP of the fusion proteins (for examples, see references 1, 3, 5, 7, and 28). MATERIALS AND METHODS Enzymes and reagents. Restriction endonucleases were purchased from either New England BioLabs, Inc. (Beverly, Mass.) or Bethesda Research Laboratories (Gaithersburg, Md.). The DNA sequencing kit (Sequenase) was purchased from U.S. Biochemical Corp. Isopropyl-p-D-thiogalactopyranoside (IPTG), phenylmethylsulfonyl fluoride (PMSF), carbenicillin, kanamycin, Triton X-100, Coomassie brilliant blue G, 5-bromo-4-chloro-3-indoyl phosphate (X-P), and p-nitrophenyl phosphate were purchased from Sigma Chemical Co. (St. Louis, Mo.). Tween 20, 5-bromo-4-chloro3-indoyl phosphate, p-Nitro Blue Tetrazolium, alkaline phosphatase-conjugated goat anti-rabbit and goat anti-mouse immunoglobulin G, mouse monoclonal anti-p-galactosidase, and nitrocellulose paper for immunostaining of protein gels were purchased from Bio-Rad Laboratories (Richmond,

* Corresponding author. t Present address: Department of Microbiology, Cornell University, Ithaca, NY 14853.

1616

VOL. 171, 1989

TRANSMEMBRANE TOPOLOGY OF AGROBACTERIUM VirA PROTEIN

Calif.). Oligodeoxynucleotides were synthesized with a DNA synthesizer (Biosearch 8600). [35S]dATP was purchased from Dupont, NEN Research Products (Boston, Mass.). Plasmids and strains. Plasmid pTZ18R was purchased from U.S. Biochemical Corp. Strain RZ1032 (dut ung) was obtained from E. Davie (Department of Biochemistry, University of Washington, Seattle, Wash.). Bacteriophage X:: TnphoA and strain CC159 (AphoA, F' lac lacIq pro) were obtained from C. Manoil (Department of Genetics, University of Washington). Overproduction of VirA. A 4.4-kilobase KpnI fragment containing the virA gene was cloned into the KpnI site of vector pTZ18R, such that the cloned gene was oriented in the same direction as the lac promoter, with 1.4 kilobases of DNA separating the two. The resulting clone, pSW169, was introduced into strain RZ1032 by transformation, and singlestranded circular DNA containing uracil residues was isolated by methods described by Kunkel et al. (10). The synthetic oligodeoxynucleotide CAGGAAACAGCTATGA ACGGAAGATATTCA was used to prime the in vitro synthesis of the complementary strand. After ligation, the plasmid DNA was first digested with EcoRI (which digests the parent plasmid but not the desired deletion derivative) and was then used to transform JM101 to Cbr. The DNA sequence of the fusion junction was determined on one strand by using as a primer the synthetic oligonucleotide TTATGCTTCCGGCTC, which is complementary to the packaged strand of pTZ18R from 54 to 70 base pairs upstream of the fusion junction. Immunoblotting. Proteins were size-fractionated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) by the method of Laemmli (11) and were immunostained with polyclonal anti-VirA antisera and APconjugated goat anti-rabbit antisera (20). Anti-VirA antisera were raised against the TrpE-VirA fusion protein encoded by pVA1 (12) in white rabbits by conventional techniques

(8).

Isolation of virA::phoA translational fusions. Strain JM101(pSW191A) was infected with bacteriophage X:: TnphoA at an approximate multiplicity of infection of 1 and plated onto L plates containing 100 p.g of kanamycin per ml and 100 ,ug of carbenicillin per ml. Colonies were pooled, and the DNA that was isolated from them was used to transform the phoA mutant strain CC159 to Knr Cbr. The resulting colonies all contained derivatives of pSW191A containing TnphoA insertions. Liquid AP assays were performed as described by Manoil and Beckwith (13). Nucleotide sequence analysis. The insertion loci of TnphoA in several pSW191A insertion derivatives were determined by sequencing the DNA at the point of insertion. Doublestranded plasmid DNA was purified and denatured by the method of Murphy and Kevanagh (16) and sequenced on one strand by procedures described in the Sequenase kit (U.S. Biochemical Corp.). DNA sequencing reactions were primed with either the reverse primer supplied with the kit or primers that were complementary to sequences within the virA gene.

RESULTS Overproduction of VirA. The VirA protein was overproduced in E. coli by fusing the virA coding sequence to the lacZ ribosome-binding site of pTZ18R. We made use of a synthetic oligodeoxynucleotide to delete all sequences separating the lacZ ribosome-binding site from the initiation

A

12

3

2

3

4

5

1617

6

200O 9710-

68

43).-

B

1

4

5 6

200L97

1-

68I-

FIG. 1. Expression of VirA protein from VirA-overproducing E. coli strains. Strains were grown to the early log phase in rich broth and, for cultures shown in lanes 1 to 4, incubated with 5 mM IPTG for 1 h (IPTG was omitted from the cultures shown in lanes 5 and 6). After the addition of PMSF to 10 mM, cultures were lysed with the sample buffer described by Laemmli (11). (A) Profile of total protein stained with Coomassie brilliant blue G; (B) protein immunostained with anti-VirA antisera. Lanes 1, JM101(pTZ18R); lanes 2, JM101(pSW169); lanes 3 and 5, JM101(pSW191A); lanes 4 and 6, JM101(pSW191B). Numbers indicate molecular weights (in thousands).

codon of virA. This procedure resulted in the positioning of the virA initiation codon directly downstream of the lacZ ribosome-binding site. The DNA sequence of the fusion junction of two such clones was determined. pSW191B had the expected sequence (CAGGAAACAGCT/A[GAACGG, in which the first set of underlined nucleotides indicate the lacZ ribosome-binding site, the slash indicates the deletion junction, and the second set of underlined nucleotides indicate the virA initiation codon). pSW191A was similar, but this clone was missing a C residue between the ribosomebinding site and the ATG codon (resulting in the sequence

CAGGAAACAGT/ATGAACGG). Visualization of the VirA protein. To determine whether pSW191A and pSW191B overproduced the VirA protein, strain JM101 containing pTZ18R, pSW169, pSW191A, or pSW191B was cultured in L broth in the presence of 5 mM IPTG for 1 h and lysed, and total proteins were sizefractionated by SDS-PAGE. Neither JM101(pSW191A) nor JM101(pSW191B) showed any protein bands that were not found in the control lanes (Fig. 1A). When identical protein extracts were transferred to nitrocellulose and immunologically stained with anti-VirA antisera, strains containing either pSW191A or pSW191B showed two bands that were not seen in strains containing either pTZ18R or pSW169 (Fig. 1B). The two bands had molecular weights of approximately 91,000 and 88,000, which were both in good agreement with the predicted molecular weight of the VirA protein (91,797). The larger of the two proteins may represent a precursor form of the protein, while the smaller protein may have been produced from the larger one by proteolytic cleavage of the proposed N-terminal signal sequence (12). The smaller band comigrated with the VirA

1618

J. BACTERIOL.

WINANS ET AL. A

1.0

` I. (. Im.-

:, 2 I

f.441*.

wer

:. :.

7 Q-

0.5

_:

68

_.w

f-

43 l-

0

B

1-J

1

2

3

4

-9

6

''

w

C., 9 Il-

0.1

0

1

2

3

TIME (HOURS)

FIG. 2. Impairment of cell division by induction of virA with IPTG. Cultures were grown to the mid-log phase, subcultured to an optical density at 600 nm (ODwo,) of 0.05, and cultured with or without 5 mM IPTG. Symbols: 0, JM101(pTZ18R) without IPTG; A, JM101(pSW191A) without IPTG; 0, JM101(pTZ18R) with IPTG; A, JM101(pSW191A) with IPTG.

protein that was expressed in wild-type A. tumefaciens (data not shown). pSW191A reproducibly expressed VirA more strongly than pSW191B did (compare lanes 3 and 4 in Fig. 1B). This was somewhat surprising, since pSW191B contained a perfect fusion of the lacZ ribosome-binding site to the ATG codon of virA, while pSW191A was missing 1 base. pSW191A was used in all subsequent experiments. Also surprising was the observation that IPTG had only a modest effect on the expression of VirA (compare lanes 3 and 5 or lanes 4 and 6 in Fig. 1B). The addition of IPTG, however, strongly impaired the growth rate of strains containing pSW191A (Fig. 2) and slightly impaired the growth rate of a strain containing pSW191B (data not shown). It is possible that the impaired growth could in some way limit the synthesis of the protein, preventing strong overproduction. The portion of the VirA protein responsible for growth impairment is discussed below. The VirA protein expressed in wild-type A. tumefaciens is associated exclusively with the cytoplasmic membrane (12). To determine whether the same protein that was overproduced in E. coli had similar properties, cells were grown to the late log phase in L broth containing 5 mM IPTG, suspended in 10% glycerol-20 mM Tris (pH 8.0)-20 mM EDTA-1 mM PMSF, and disrupted with a French pressure cell. The resulting suspension was centrifuged at 12,000 rpm for 15 min in a rotor (SW55.1; Beckman Instruments, Inc., Fullerton, Calif.). The supernatant was recentrifuged at 50,000 rpm in the same rotor for 90 min. The pellets obtained from low-speed and high-speed centrifugation and the supernatant of the high-speed centrifugation were assayed for

FIG. 3. Fractionation of an E. coli culture expressing VirA. Cultures of 5 ml of JM101(pTZ18R) (lanes 1, 3, 5, and 7) or JM101(pSW191A) (lanes 2, 4, 6, and 8) were grown to the early log phase and incubated with IPTG for 1 h. They were then suspended in 1 ml of 10o glycerol-10 mM Tris-10 mM EDTA-PMSF (10 mM), and cells were disrupted with a French pressure cell. Lanes 1 and 2, Whole-cell protein; lanes 3 and 4, pellet after centrifugation (SW55.1 centrifuge; Beckman) at 12,000 rpm for 15 min; lanes 5 and 6, pellet after centrifugation at 50,000 rpm for 90 min; lanes 7 and 8, supernatant after centrifugation at 50,000 rpm for 90 min. (A) Profile of total protein stained with Coomassie brilliant blue G; (B) protein immunostained with anti-VirA antisera. Numbers indicate molecular weights (in thousands).

VirA protein by immunoblotting. A small amount of the VirA protein was pelleted by low-speed centrifugation, while all of the remaining VirA protein was pelleted by high-speed centrifugation (Fig. 3B). A similar gel in which the extracts shown in lanes 5 and 6 of Fig. 3A were loaded more heavily shows the VirA protein that was determined by Coomassie brilliant blue G staining (data not shown). Thus, the VirA protein that was overproduced in E. coli, like the VirA protein that was expressed in A. tumefaciens, is associated with the particulate fraction. VirA could be solubilized by the addition of Triton X-100 to a final concentration of 0.1%. Susceptibility of VirA to protease digestion in E. coli spheroplasts. Cultures of JM101(pSW191A) were cultured without the induction of VirA, converted to spheroplasts by treatment with lysozyme and EDTA, and treated with proteinase K. Cellular protein was size fractionated by SDS-PAGE, transferred to nitrocellulose and probed with anti-VirA antibody. Spheroplasts that were not treated with protease showed the 91,000- and 88,000-dalton proteins. Treatment with proteinase K caused the loss of these bands, while a new band appeared at 60,000 daltons (Fig. 4B, lane 3). We were concerned that three alternative interpretations were possible. (i) The entire VirA protein was localized periplasmically and contained a 60,000-dalton protease-resistant fragment; (ii) the entire protein was cytoplasmic, the spheroplasts were actually lysed, and the 60,000-dalton fragment was protease resistant; and (iii) the protein was cytoplasmic and the creation of the 60,000-dalton fragment occurred after the lysis step. All three of these interpretations require that the 60,000-dalton fragment be resistant to


VOL. 171, 1989

C

B

A

2

*

1619

3

4

1

-K200 lu

2

3

4

200

.:

-M

:::.

...

.:-

97 ]W

..

. . . .: . . .:

_:

97 7

1m4

:::

;-NW --

68li

43

)W

~68~ -W 6843 43

-.......... ........ ........

FIG. 4. Susceptibility of VirA to protease digestion in E. coli spheroplasts. Strains were incubated in rich broth to the late log phase without IPTG and suspended in 20o sucrose-10 mM Tris (pH 7.5). EDTA and lysozyme were then added to 25 mM and 10 mg/ml, respectively. Where indicated, proteinase K was added to 100 pug/ml. Lysis of spheroplasts was done by centrifugation and suspension in 0.05% Triton X-100, followed by brief sonication. Lanes 1, JM101(pTZ18R) without protease; lanes 2, JM101(pSW191) without protease; lanes 3, JM101(pSW191) with protease; lanes 4, JM101(pSW191) with protease after cell lysis. (A) Total protein staining with Coomassie brilliant blue G; (B) immunostaining with anti-VirA antisera; (C) immunostaining with anti-,B-galactosidase antisera. Numbers indicate molecular weights (in thousands).

protease. To show that this was not the case, a portion of the spheroplasts were lysed by resuspension and brief sonication in water containing 0.05% Triton X-100 and were then treated with proteinase K. Under these conditions, no protein was stained with the antibody (Fig. 4B, lane 4). This indicates that the 60,000-dalton fragment is not intrinsically protease resistant; rather, it indicates that its resistance in spheroplasts requires the integrity of the cytoplasmic membrane. To determine that our preparation of spheroplasts did not contain lysed cells, each sample was probed with a monoclonal anti-,-galactosidase antibody to visualize WBgalactosidase, a control protein that is known to lie in the cytoplasm. This antiserum stained six proteins, the largest two of which were 87,000 and 85,000 daltons. This is approximately the estimated size of the truncated ,-galactosidase enzyme produced by the lacZ M15 allele of lacZ carried by these strains. The smaller proteins may be ,galactosidase degradation products. None of these proteins was susceptible to proteolysis by proteinase K in spheroplasts (Fig. 4C, lane 3), but all were degraded when cells were lysed (Fig. 4C, lane 4). We conclude that approximately 28,000 daltons of the protein that is present in the spheroplasts is accessible to proteinase K digestion, indicating an extracellular localization. The remaining 60,000 daltons that was inaccessible to protease is therefore localized either within the inner membrane or in the cytoplasm. TnphoA insertions in the virA gene. To confirm these findings and to determine which end of the protein was periplasmically localized, we created a random series of transposon insertions in pSW191A using transposon TnphoA (13, 14). In the first of two screenings, 36 mutants were isolated. Each of these was streaked onto plates containing the AP indicator X-P; 32 clones formed white colonies, while 3 clones formed light blue colonies and 1 clone formed a dark blue colony. These clones were mapped with restriction endonucleases, and it was found that the insertion which gave rise to dark blue colonies was localized in the interval between the two hydrophobic cores of the virA gene, while two insertions which formed light blue colonies were localized after the second hydrophobic region of the gene. The third insertion forming light blue colonies mapped outside virA. Clones forming white colonies contained insertions

from all parts of pSW191A and were presumed not to have resulted in translational fusions. In a second screen for insertion mutations, strain CC159 was transformed with the same pools of plasmid DNA containing insertion derivatives of pSW191A and was plated directly onto medium containing X-P. Most of the clones that were picked were those which formed light blue or dark blue colonies. An additional 24 independent mutants were isolated in virA. With three exceptions, all insertions between the two hydrophobic cores formed dark blue colonies, while all insertions in the latter part of the gene formed light blue colonies. The three clones showing anomalous phenotypes are described below. The loci of all insertions are shown in Table 1. Liquid assays of these insertions (Table 1) indicated that insertions in the amino-terminal one-third of the protein caused the synthesis of about 50-fold higher levels of AP than did insertions in the carboxy-terminal two-thirds of the protein. We found three insertions which expressed somewhat anomalous levels of AP. These were clone 56, which formed light blue colonies and carried an insertion of TnphoA at the extreme amino terminus, and clones 64 and 57, which formed extremely light blue colonies and carried insertions that were localized in the putative secreted region of the protein. The DNA sequences of these clones were determined. Clone 56 had an insertion which caused a translational fusion to amino acid 22 of VirA. This insertion presumably disrupted the signal sequence and resulted in a cytoplasmically localized fusion protein. The DNA sequence of insertions 64 and 57 indicated that the transposon was inserted into the wrong reading frames, and therefore translational fusions were not created. Presumably, AP was expressed by these plasmids at low levels because of occasional frameshift errors in either the transcription or the translation of the gene fusion. Such errors would give rise to trace amounts of a secreted fusion protein. We also isolated an insertion in a region downstream of virA which expressed 58 U of AP activity. This suggests the existence of a gene lying 3' to virA which encodes a secreted protein. The DNA sequence of this region has been published recently (15), and an open reading frame encoding a protein with a probable signal sequence was found (15). The

WINANS ET AL.

1620

J. BACTERIOL.

TABLE 1. Insertions of TnphoA in the virA gene

A

Insertion'

Positionb

Orientation'

Color"

activity'

Colony morphology/

56 54 74 71 76 1 75 58 16 70 59 64 57

22 23 83 ca. 107 ca. 117 ca. 117 ca. 123 ca. 133 ca. 140 ca. 180 189 191 ca. 230 244 ca. 323 ca. 350 ca. 397 ca. 413 ca. 457 ca. 543 ca. 550 ca. 590 ca.597 ca. 607 ca. 607 ca. 623 ca. 660 ca. 697 ca. 700 ca. 707 ca. 710 ca. 740 ca. 790 ca. 790 ca. 810 ca. 823 ca. 943

I I I I I I I II II I I I I II I I I I I I I I I I II I I II II I I I I I I I I

LB W DB DB DB DB DB W W DB DB VLB VLB W W LB LB LB LB LB LB LB LB LB W LB W W W W W NDTM LB LB W LB W

6 6 370 577 315 708 827 3 1 465 349 2 9 1 1 16 9 9 5 9 7 3 9 7 1 7 1 1 1 1 1 2 4 4 1 3 ND

L L S S S S S L L S S M M L S S S S S S S S S S S S S S S S S S S S S S S

23 22 61 77 49 67 78 47 65 50 80 12 9 66 63 13 20 2 53 15 81 32 62 17

AP

"The first hydrophobic core includes amino acids 21 to 36 and the second hydrophobic core includes amino acids 269 to 278. Insertions 74 through 23 are between the two hydrophobic regions. b Position is expressed as the number of base pairs downstream of the initiation codon. ca. indicates the approximate position based on restriction endonuclease mapping. The positions of all others were determined by DNA sequencing. ' Abbreviations: I, virA and phoA of the TnphoA insertion were in the same orientation; II, virA and phoA were in opposite orientations. d Strain CC159 containing the indicated plasmids was streaked onto Lcarbenicillin-IPTG-X-P plates and scored after 2 days. Abbreviations: W, white colonies; VLB, very light blue colonies; LB, light blue colonies; DB, dark blue colonies. e Strain CC159 containing the indicated plasmids was grown to the mid-log phase with 200 ,ug of carbenicillin per ml, 100 ,ug of kanamycin per ml, and 2 mM IPTG and was assayed by the method of Manoil and Beckwith (13, 14). f Strain JM101 containing the indicated plasmid was streaked for single colonies on L-carbenicillin-IPTG plates. The colony size was scored after 1 day of growth. S, Small colonies; M, medium-sized colonies; L, large colonies. 9 ND, Not determined.

existence of an insertion in this region which expresses AP strongly suggests that this open reading frame does indeed encode a protein and that this protein is secreted. Effect of TnphoA insertions on impaired growth rate. We described above that a strain containing pSW191A was severely impaired in cell division when cultured in the presence of IPTG. In addition to impairing growth in broth cultures, IPTG also caused bacteria containing pSW191A to form small colonies on solid culture media. We sought to use the set of insertion derivatives of this plasmid to determine

B -q' .N

20D 1097

]OW

-_668

-_43

:-

FIG. 5. Susceptibility of VirA to protease digestion in A. tumefaciens spheroplasts. Strain A348 was induced for 18 h in minimal medium (pH 5.5) with 100 ,M acetosyringone and was then washed three times with 20 mM Tris (pH 7.5) and suspended in 20% sucrose-50 mM Tris (pH 7.4)-2 mM EDTA-200 ,uM lysozyme. A total of 100 ,ug of proteinase K per ml was added where indicated. Lanes 1, A348 total protein; lanes 2, A348 spheroplasts without protease; lanes 3, A348 spheroplasts with protease. (A) Proteins stained with Coomassie brilliant blue G; (B) immunostaining with anti-VirA antisera. Numbers indicate molecular weights (in thousands).

which part of VirA causes this phenotype. Strains containing the insertion derivatives described in Table 1 were streaked for single colonies on L agar containing 5 mM ITPG, 200 jig of carbenicillin per ml, and 50 ,ug of kanamycin per ml. The results are indicated in Table 1. All but seven of the insertion derivatives formed small colonies. However, it has been observed by others that high-level expression of several AP fusion proteins can impair the growth rate (C. Manoil, personal communication). This phenomenon is unrelated to the impaired growth caused by overproduction of the native VirA protein. Therefore, we should direct our attention to insertions which do not result in AP fusions. Two such insertions deserve special consideration. Insertion 23 abolished the small colony morphology of the parent, while insertion 22, which was only about 90 amino acid residues downstream, did not affect this phenomenon. It is significant that the putative stop-transfer region of VirA was within this 90-amino-acid interval. That is, the VirA fragment made by the pSW191A insertion 22 contained the stop-transfer signal, while the VirA fragment made by pSW191A insertion 23 lacked the stop-transfer signal. Therefore, it is the second hydrophobic core of VirA (or, less likely, a nearby part of the protein) which is responsible for impaired growth. VirA topology in A. tumefaciens. To confirm that the VirA protein expressed in A. tumefaciens was similar in its localization and topology to that of the overproduced protein in E. coli, the susceptibility of VirA to protease digestion in A. tumefaciens spheroplasts was tested. Cultures of strain A348 were induced with acetosyringone and were then converted to spheroplasts with lysozyme and EDTA. After cellular proteins were treated with proteinase K, they were size fractionated by SDS-PAGE, transferred to nitrocellulose, and probed with anti-VirA antibody. Spheroplasts that were not treated with protease showed the 88,000-dalton protein (Fig. 5B, lane 2). Treatment with protease resulted in the loss of this band and the appearance of the 60,000-dalton fragment (Fig. 5B, lane 3). The 28,000 daltons of the protein digested by proteinase K indicated an extracellular localization. The remaining 60,000 daltons was therefore localized

VOL. 171, 1989


either within the inner membrane or in the cytoplasm, just as was found with the overproduced protein in E. coli. DISCUSSION Results of previous studies (12, 26, 31, 32) have indicated that VirA plays a central role in the ability of A. tumefaciens to relay information about the presence of phenolic inducers to the level of gene expression. In the present study, we provided two types of evidence that this protein spans the cytoplasmic membrane. First, approximately 28,000 daltons of the protein was susceptible to proteinase K in cells which were converted to spheroplasts, while the remainder of the protein (60,000 daltons) was protease resistant. When the cytoplasmic membrane was disrupted, the VirA protein was totally digested, indicating that protease resistance in spheroplasts depends on the integrity of the cytoplasmic membrane. Second, TnphoA insertions in the 5' end of the gene gave rise to VirA-AP fusions with high AP activity, while insertions at the 3' end of the gene gave rise to fusions with low AP activity. This indicates that the amino-terminal part of the protein is secreted into the periplasm, while the remainder of the protein is cytoplasmically localized. This transmembrane topology is well suited to a protein whose proposed role is to sense the presence of environmental signaling molecules and to transmit this information to the cytoplasm, where it results ultimately in altered patterns of gene expression. We noted earlier (31) that VirA has amino acid homology to at least seven proteins which are part of two-component regulatory systems. At least two members of this family have protein kinase activity (6, 18), and it is likely that other members of this family may also be kinases. The region of VirA which is homologous to these other proteins lies in the interval between amino acids 420 and 650; therefore, it is confined to the cytoplasmic portion of the

protein. The hydropathy profile of VirA, with one hydrophobic region at the amino terminus and a second hydrophobic region from amino acids 260 to 280, is reminiscent of the profiles of a number of methyl-accepting chemoreceptors. At least one of these proteins has indeed been shown, like VirA, to span the cytoplasmic membrane (13). Some of the members of the family of proteins that are homologous to VirA also have a hydropathy profile similar to that of VirA (for example, EnvZ, DctB, and CpxA; see references 4, 23, and 1, respectively). Various investigators have proposed that these proteins may have a topology similar to the one we described here for VirA. Recently, methods similar to those used in this study were described which indicate that CpxA does indeed have a transmembrane topology (2, 29). ACKNOWLEDGMENTS We thank C. Manoil for many helpful discussions, G. Cangelosi and P. Christie for critical appraisal of this manuscript, and D. Parkhurst for oligonucleotide synthesis. This work was supported by Public Health Service grant 5 ROI GM 32618-14 from the National Institutes of Health and grant DMB-8315826 from the National Science Foundation. S.C.W. was supported by fellowship DRG-800 from the Damon Runyon-Walter Winchell Cancer Fund. LITERATURE CITED 1. Akiyama, Y., and K. Ito. 1987. Topology analysis of the SecY protein, an integral membrane protein involved in protein export in Escherichia coli. EMBO J. 6:3465-3470. 2. Albin, R., R. Weber, and P. M. Silverman. 1986. The Cpx proteins of Escherichia coli K12. Immunologic detection of the

1621

chromosomal cpxA gene product. J. Biol. Chem. 261:46984705. 3. Chun, S. Y., and J. S. Parkinson. 1988. Bacterial motility: membrane topology of the Escherichia coli MotB protein. Science 239:276-278. 4. Comeau, D. E., K. Ikenaka, K. Tsung, and M. Inouye. 1986. Primary characterization of the protein products of the Escherichia coli ompB locus: structure and regulation of synthesis of the OmpR and EnvZ proteins. J. Bacteriol. 164:578-584. 5. Gutierrez, C., J. Barondess, C. Manoil, and J. Beckwith. 1987. The use of transposon TnphoA to detect genes for cell envelope proteins subject to a common regulatory stimulus. Analysis of osmotically regulated genes in Escherichia coli. J. Mol. Biol. 195:289-297. 6. Hess, J. F., K. Oosawa, N. Kaplan, and M. I. Simon. 1988. Phosphorylation of three proteins in the signaling pathway of bacterial chemotaxis. Cell 53:79-87. 7. Hinton, J. C., and G. P. Salmond. 1987. Use of TnphoA to enrich for extracellular enzyme mutants of Erwinia carotovora subspecies carotovora. Mol. Microbiol. 3:381-386. 8. Hurn, B. A. L., and S. M. Chantler. 1980. Production of reagent antibodies. Methods Enzymol. 70:104-142. 9. Kuhn, A., W. Wickner, and G. Kreil. 1986. The cytoplasmic carboxy terminus of M13 procoat is required for the membrane insertion of its central domain. Nature (London) 322:335-339. 10. Kunkel, T. A., J. D. Roberts, and R. A. Zakoor. 1986. Rapid and effecient site-directed mutagenesis without phenotypic selection. Methods Enzymol. 154:367-382. 11. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227:680-685. 12. Leroux, B., M. F. Yanofsky, S. C. Winans, J. E. Ward, S. F. Zeigler, and E. W. Nester. 1987. Characterization of the virA locus of Agrobacterium tumefaciens: a transcriptional regulator and host range determinant. EMBO J. 6:849-856. 13. Manoil, C., and J. Beckwith. 1986. A genetic approach to analyzing membrane protein topology. Science 233:1403-1408. 14. Manoil, C., and J. Beckwith. 1986. TnphoA: a transposon probe for protein export signals. Proc. Natl. Acad. Sci. USA 82: 8129-8133. 15. Melchers, L. S., D. V. Thompson, K. B. Idler, T. C. Neuteboom, R. A. deMaagd, R. A. Schilperoort, and P. J. J. Hooykaas. 1987. Molecular characterization of the virulence gene virA of the Agrobacterium tumefaciens Ti plasmid. Plant Mol. Biol. 9:

635-645. 16. Murphy, G., and T. Kevanagh. 1988. Speeding-up the sequencing of double-stranded DNA. Nucleic Acids Res. 16:5198. 17. Nester, E. W., M. P. Gordon, R. M. Amasino, and M. F. Yanofsky. 1984. Crown gall: a molecular and physiological analysis. Annu. Rev. Plant Physiol. 35:387-413. 18. Ninfa, A. J., and B. Magasanik. 1986. Covalent modification of the glnG product, NRI by the glnL product, NRII, regulates the transcription of the glnALG operon in Escherichia coli. Proc. Natl. Acad. Sci. USA 83:5909-5913. 19. Nixon, B. T., C. W. Ronson, and F. M. Ausubel. 1986. Twocomponent 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. 20. Osborn, M. J., J. E. Gander, E. Parisi, and J. Carson. 1972. Mechanism of assembly of the outer membrane of Salmonella typhimurium. Isolation and characterization of cytoplasmic and outer membrane. J. Biol. Chem. 247:3962-3972. 21. Osbourn, A. E., C. E. Barber, and M. J. Daniels. 1987. Identification of plant-induced genes of the bacterial pathogen Xanthomonas campestris pathovar campestris using a promoterprobe plasmid. EMBO J. 6:23-28. 22. Peters, N. K., J. W. Frost, and S. R. Long. 1986. A plant flavone, luteolin, induces expression of Rhizobium meliloti nodulation genes. Science 233:917-1008. 23. Ronson, C. W., P. M. Astwood, B. T. Nixon, and F. M. Ausubel. 1987. Deduced products of C4-dicarboxylate transport regulatory genes of Rhizobium leguminosarum are homologous to

1622

WINANS ET AL.

nitrogen regulatory

7901-7934.

gene

J. BACTERIOL. products. Nucleic Acids Res. 15:

24. Ronson, C. W., B. T. Nixon, and F. M. Ausubel. 1987. Conserved domains in bacterial regulatory proteins that respond to environmental stimuli. Cell 49:579-581. 25. Stachel, S. E., E. Messens, M. Van Montague, and P. Zambryski. 1985. Identification of the signal molecules produced by wounded plant cells that activate T-DNA transfer in Agrobacterium tumefaciens. Nature (London) 318:624-629. 26. Stachel, S.E., and P. C. Zambryski. 1986. virA and virG control the plant-induced activation of the T-DNA transfer process of A. tumefaciens. Cell 46:325-333. 27. Stachel, S. E., and P. C. Zhmbryski. 1986. Agrobacterium tumefaciens and the susceptible plant cell: a novel adaptation of extracellular recognition and DNA conjugation. Cell 47:155157. 28. Taylor, R. K., V. L. Miller, D. B. Furlong, and J. J. Mekalanos.

29. 30. 31.

32.

1987. Use of phoA gene fusions to identify a pilus colonization factor coordinately regulated with cholera toxin. Proc. Natl. Acad. Sci. USA 84:2833-2837. Weber, R. F., and P. M. Silverman. The Cpx proteins of Escherichia coli K12. Structure of the CpxA polypeptide as an inner membrane component. J. Mol. Biol. 203:467-478. Wickner, W. T., and H. F. Lodish. 1985. Multiple mechanisms of protein insertion into and across membranes. Science 230: 400-407. Winans, S. C., P. R. Ebert, S. E. Stachel, M. P. Gordon, and E. W. Nester. 1986. A gene essential for Agrobacterium virulence is homologous to a family of positive regulatory loci. Proc. Natl. Acad. Sci. USA 83:8278-8282. Winans, S. C., R. A. Kerstetter, and E. W. Nester. 1988. Transcriptional regulation of the virA and virG genes of Agrobacterium tumefaciens. J. Bacteriol. 170:4047-4054.