Inhibition ofAgrobacterium tumefaciens Oncogenicity - Journal of

0 downloads 0 Views 1MB Size Report
Mar 21, 1994 - ofAgrobacterium tumefaciens when it is resident in this organism. Oncogenic inhibition ... deletions into or-3 caused the loss of oncogenic inhibition. Deletion analysis ... gella flexneri (46), encodes resistanceto streptomycin, chlor- amphenicol ... Escherichia coli strains were cultured in Luria-Bertani medium.
Vol. 176, No. 18

JOURNAL OF BACTERIOLOGY, Sept. 1994, p. 5697-5703

0021-9193/94/$04.00+0 Copyright © 1994, American Society for Microbiology

Inhibition of Agrobacterium tumefaciens Oncogenicity by the osa Gene of pSa CHAO-YING CHENt ANm CLARENCE I. KADO* Department of Plant Pathology, University of Califomia, Davis, California 95616 Received 21 March 1994/Accepted 13 July 1994

The IncW plasmid pSa originally derived from Shigelaflexneri completely inhibits the tumor-inducing ability of Agrobacterium tumefaciens when it is resident in this organism. Oncogenic inhibition is mediated through the expression of the osa gene on pSa. This gene is part of a 3.1-kb DNA segment of pSa that contains four open reading frames revealed by sequencing. Specific deletions and TnCAT insertions within this segment localized the oncogenic inhibitory activity to the last open reading frame, orf-4, designated osa (for oncogenic suppression activity). No promoter exists immediately upstream of the coding sequence of osa since TnCAT insertions or deletions into or-3 caused the loss of oncogenic inhibition. Deletion analysis showed that the promoter of or-i is required for osa transcription. The first three orfs have no role in oncogenic inhibition, since osa alone placed under the control of a constitutive Pkm promoter completely inhibited A. tumefaciens oncogenicity. This inhibition of oncogenicity by osa is not limited to a specific host plant but appears to show broad host specificity. Because the osa-encoded product has close homologies to the fiwA-encoded product of the IncP plasmid RP1, osa may be involved in fertility inhibition that would prevent or reduce the formation of stable mating pairs and T-DNA transfer between A. tumefaciens and plants.

to carrot cells as did the LPS derived from the same strain without pSa (31). Further insights on the mechanism of oncogenic suppression, the location of osa with respect to neighboring genes, and comparative analysis of the amino acid sequence of the Osa protein might provide important clues to the nature of Osa activity. The osa gene is located between the region containing conjugative transfer genes (trw genes) and the region containing the repA and recA genes and the origin of plasmid DNA replication (orn) (6, 35). There are three orfs upstream of osa in the 3.1-kb pSa segment conferring oncogenic suppression, the most distal being orf-1 (630 bp), which encodes a 23.2-kDa polypeptide, followed by the 525-bp nuc gene, which encodes a 19.7-kDa DNase (7), and orf-3 (603 bp), which encodes a 22.8-kDa polypeptide of unknown function. orf-3 is followed by the 570-bp osa gene. Although previous studies have indicated that osa plays a major part in oncogenic inhibition (6), the contribution of each upstream gene, such as nuc, to this activity has remained unclear. In the present study, the contribution of nuc and the two orfs to overall oncogenic inhibitory activity in A. tumefaciens was examined. These studies indicate that osa alone is sufficient for completely inhibiting the oncogenicity of A. tumefaciens and that osa is part of an operon, consisting of at least nuc, the two orfs, and osa, whose transcription is dependent on the promoter of orf-1.

The 39-kb IncW plasmid pSa, originally isolated from Shigella flexneri (46), encodes resistance to streptomycin, chloramphenicol, kanamycin, spectinomycin, and sulfonamides and has a broad host specificity that allows for self-mobilization and replication in a wide range of gram-negative bacteria (45). The introduction of pSa into Agrobacterium tumefaciens results in the intriguing capacity to completely inhibit the ability of this organism to incite crown gall tumors (30). Tumor pathogenesis is mediated during infection by the transfer of a specific sector (T-DNA) of the resident Ti plasmid from A. tumefaciens to plant cells. T-DNA is integrated into the plant genome and expressed to form growth-promoting hormones, culminating in the formation of tumors (4, 14). Attempts to elucidate the nature of pSa-mediated oncogenic inhibition have eliminated a number of possibilities. For example, pSa does not affect the stability or conjugal self-transfer of the resident Ti plasmid between A. tumefaciens strains (10). Furthermore, the induction of the Ti plasmid virulence (vir) genes and the processing of T-DNA into T intermediates remain operational in the presence of pSa (5). Since T-DNA is processed in the presence of pSa, other possible mechanisms have been explored. For instance, it had been claimed that pSa-mediated oncogenic inhibition was due to a decrease in auxin synthesis in A. tumefaciens (3), but this notion was not verified, since A. tumefaciens made to produce auxin in abundance was still completely suppressed for oncogenicity by pSa (17). It had also been thought that pSa altered an exocellular lipopolysaccharide (LPS) sufficiently to affect the plant cell-binding capacity of A. tumefaciens (34), but it was later shown that there were no alterations in LPS in A. tumefaciens carrying pSa and that the LPS derived from such strains still blocked the binding of virulent A. tumefaciens cells

MATERIALS AND METHODS Bacterial strains, plasmids, and growth conditions. The bacterial strains and plasmids used are listed in Table 1. Escherichia coli strains were cultured in Luria-Bertani medium (32) at 370C. A. tumefaciens strains were grown in 523 medium (20) and minimal 925 medium (25) at 290C. Antibiotics were added at the following concentrations (in micrograms per milliliter) shown in parentheses: ampicillin (50), chloramphenicol (20), gentamicin (3), kanamycin (15), neomycin (30), tetracycline (10), and spectinomycin (50) for E. coli; and chloramphenicol (3), gentamicin (15), kanamycin (20), neomy-

* Corresponding author. Mailing address: Department of Plant Pathology, University of California, Davis, CA 95616. Phone: (916) 752-0325. Fax: (916) 752-5674. t Present address: Department of Plant Pathology and Entomology, National Taiwan University, Taipei, Taiwan, Republic of China. 5697

5698

CHEN AND KADO

J. BAcrEFRIOL.

TABLE 1. Bacterial strains and plasmids used in this study Strain or plasmid

Relevant characteristics

Strains A. tumefaciens LBA4301 Rmr Cmr Rec E. coli DH5a supE44 lacU169(f80 lacZ M15) hsdR17 recA1 endA1 gyrA96 thi-1 rel41 E. coli HB101 supE44 hsdS20(rB-mB ) recA13 ara-14 proA2 lacYl galK2 rpsL20 xyl-S mtl-1 Plasmids pTiCS8Trac Transfer constitutive derivative of pTi58 pUCD105 pTAR on parA; pBR322 on; Apr KMr Gmr Spr pUCD3960 650-bp fragment containing osa cloned in the KpnI site of pUCD105 pUCD3962 1,250-bp fragment containing osa and orf-2 cloned in the KpnI site of pUCD105 pUCD3964 1,970-bp fragment containing osa, orf-2, and nuc cloned in the KpnI site of pUCD105 pUCD3965 2,600-bp fragment containing osa, orf-2, nuc, and orf-1 cloned in the KInI site of pUCD105 pUCD3968 580-bp fragment, with the first 60 bp of osa deleted, cloned in the KpnI site of pUCD105 pUCD1311 3.1-kb pSa segment cloned in pUCD105 pRK2013::TnCAT TnCAT in RK2013 pUCD3940-1 TnCAT inserted 658 bp upstream of the 5' end of osa; KMr Gmr Tcr Cmr pUCD3940-10 TnCAT inserted 563 bp downstream of the 5' end of osa on the BglII site of osa TnCAT inserted 489 bp upstream of the 5' end of osa pUCD3940-13 pUCD3940-15 TnCAT inserted 58 bp downstream of the 5' end of osa pUCD3940-26 TnCAT inserted 56 bp upstream of the 5' end of osa TnCAT inserted 2,025 bp upstream of the 5' end of osa pUCD3940-30 pTZ19R Commercial cloning vector 5-kb Asp718-SalI fragment of pUCD3991 cloned in pTZ19R pUCD3991 pUCD3991-2 900-bp deletion of the 3' end of pUCD3940-15 pUCD3991-5 2,080-bp deletion of the 3' end of pUCD3940-15 pUCD3991-7 700-bp deletion of the 3' end of pUCD3940-15

cin (30), rifampin (25), spectinomycin (50), and tetracycline (5) for A. tumefaciens. DNA manipulation. Plasmid DNA was isolated by an alkaline lysis protocol (21) and cleaved with appropriate restriction endonucleases, and the resulting DNA fragments were separated by agarose gel electrophoresis for molecular cloning (38). DNA fragments isolated from the agarose gel were purified with GeneClean according to the instructions of the manufacturer (Bio 101, San Diego, Calif.). The transformation of A. tumefaciens was performed by electroporation (9), and that of E. coli was performed by calcium chloride treatment (33). Deletion analysis. Plasmid pUCD105 (45), constructed by David Zaitlin in our laboratory, was maintained in A. tumefaciens. This vector contains oniV of A. tumefaciens plasmid pTAR (13) and the aminoglycoside resistance operon of pSa which encodes resistance to kanamycin and spectinomycin (44). Kanamycin and spectinomycin resistance genes are transcribed together with the Pkm promoter upstream of the kanamycin resistance gene (44). The osa-containing fragments bearing deletions of individual upstream orfs were generated by PCR with pUCD1311 as the template. PCR was performed with a Gene Amp kit in a DNA thermal cycler according to the manufacturer's instructions (Perkin-Elmer Cetus, Norwalk, Conn.). A total of 25 reaction cycles were carried out under the following conditions: 2 min of denaturation at 92°C, 2 min of annealing at 50°C, and 2 min of extension at 72°C. The primers for PCR were as follows: primer 1 (5'-CCAAGGTACCATOI GAGGCTAACGATGCTGATGTTGCTAC-3'), primer 2 (5'-

CCAAGGTACCTGGiAGGAAAGACCATGACCGAC-3'),

primer 3 (5'-CCAAGGTACCAGGAGGCAAACAATGAAA AGCG-3'), primer 4 (5'-CCAAGGTACCAGLGAGGCCCGC AAGAATGGCAATC-3'), primer 1#60 (5'-CCAAGGTAC

CATGGAGGCTAACGATGCTTGCGCAGTCGAGC-3'), and primer R (5'-CCAAGGTACCAGGTCAAACGGAATA GAGCAC-3'). Primers 1 to 4 and primer 1#60 included a purine-rich sequence (GGAGG) (each underlined sequence)

Reference or source

22 38 38 24 45 This paper This paper This paper This paper

This paper 6 23 This paper This paper This paper This paper This paper This paper U.S. Biochemical Corp. This paper This paper This paper This paper

upstream of the first open reading frame in each PCRgenerated DNA fragment. Furthermore, a KjpnI recognition sequence (GGTACC) was added to both ends of each primer. This allowed each PCR-generated fragment to be inserted into the KpnI site within the aminoglycoside resistance operon of pUCD1O5. Clones which had inserts oriented in the direction of transcription of the aminoglycoside resistance operon were confirmed by spectinomycin resistance and BglII-HindIII double digestion. TnCAT mutagenesis and DNA sequencing. TnCAT was transposed into pUCD1311 containing the 3.1-kb pSa DNA fragment cloned in pUCD1OS according to the method of Kamoun et al. (23). A. tumefaciens LBA4301 containing pUCD 1311 was mated with E. coli HB1O1(pRK2013::TnCAT) at 300C. LBA4301 transconjugants were selected on 1.5% 523 medium agar plates containing rifampin, tetracycline, and gentamicin and subsequently screened for chloramphenicol resistance before miniscreening the plasmids for TnCAT insertions. The transposition of TnCAT allows transcriptional and translational fusions with a promoterless chloramphenicol acetyltransferase (CAT) gene. Isolated plasmid DNAs were transferred into E. coli DH5ot by transformation, and the resulting transformants were selected on 1.5% Luria-Bertani agar containing tetracycline and gentamicin. The insertion sites and orientations of the TnCAT inserts were determined by EcoRI-SalI restriction enzyme mapping and verified by nucleotide sequence analyses. DNA sequences were determined by the dideoxy chain termination method with doublestranded DNA as the template (39). For sequencing, the primer 5'-TGGTGGCAACTATATAGGG-3', located at the 5' end of the CAT gene (1) of TnCAT, was used. CAT activity was measured spectrophotometrically at 412 nm on cell extracts (8) prepared from log-phase cells of E. coli and A. tumefaciens strains as follows. Cells were harvested by centrifugation (5,000 x g for 10 min at 4°C), resuspended in 0.5 ml of assay buffer (50 mM Tris-Cl [pH 7.8]-50 mM dithiothreitol),

ONCOGENIC INHIBITION BY osa

VOL. 176, 1994

3

2

1

L

lI

11

RA

RD

111 3.1 kb

I

1

A

D

PB

nuc 630 bp

Oncogenic suppression

osa

orf 1 >52 63

5699

-

5

603 bp Pim rKm

pUCD3965 v

BLN --

+

.................pUCD3964 4.

.-

,........

Pt

UCD3960}

J-km

pUCD3%8

FIG. 1. Promoter fusions of the open reading frames and designated genes in the 3.1-kb cloned DNA fragment of pSa. Constructs bearing the constitutive Pkm promoter fused to orf-1, nuc, orf-3, and osa were tested for oncogenic suppressive activity, and the results are indicated by a plus or minus at the right of each plasmid. Tumor formation on the stems of D. stramonium plants was assessed 7 weeks after inoculation. The restriction endonuclease sites are as follows: A, AviII; B, BglII; P, PmlI; R, RsaI; D, DdeI.

and lysed by sonication (80% output with two 20-s bursts) with Microson ultrasonic cell disrupter (Heat Systems-Ultrasonics Inc., Farmingdale, N.Y.). The lysed mixture was centrifuged (10,000 X g for 10 min at 40C) to remove cell debris, and the supernatant was used in the assay. Virulence assays. Plasmids with deletions and TnCAT insertions were introduced into A. tumefaciens LBA4301 (pTiC58Trac) by electroporation. The resulting transformants were verified for the presence of the Ti plasmid by plasmid miniscreening (21) and growth on basal minimum 925 medium (25) containing 50 pug of nopaline per ml. Virulence was assayed by inoculating plant stems with A. tumefaciens strains that were grown for 2 days at 290C on 523 medium agar (1.5%) containing the appropriate antibiotics. Cells were scraped from the agar surface with sterile toothpicks and transferred by puncturing the plant stems or leaves (as for Kalanchoe daigremontiana). Approximately 109 cells per ml were deposited at the punctured site. Apium graveolens (celery), Chenopodium amaranticolor, Datura stramonium (jimsonweed), K daigremontiana, Nicotiana tabacum (tobacco), Helianthus annuus (sunflower), and Lycopersicon esculentum (tomato) were used. Three plants of each host were inoculated. The development of tumors at the inoculated sites was recorded at 7-day intervals. a

RESULTS The neighboring genes of osa are not required for oncogenic inhibition. Along with orf-1, nuc, and orf-3, the osa gene is transcribed toward the BglII site in the 3.1-kb pSa fragment (Fig. 1). Five individual clones containing these orfs in vector pUCD105 were prepared by using specific primers for PCR amplification. pUCD3960 containing only osa (650 bp) was generated with primer 1 and primer R; pUCD3964 containing orf-2 and osa (1,250 bp) was generated with primer 2 and primer R; pUCD3964 containing nuc, orf-2, and osa (1,970 bp) was generated with primer 3 and primer R; pUCD3965 containing orf-1, nuc, orf-2, and osa (2,600 bp) was generated with primer 4 and primer R; pUCD3968 containing osa with 60 bp deleted from its 5' end was generated with primer 1#60 and primer R. Each clone, under the control of the constitutive

was tested in A. tumefaciens LBA4301 (pTiC58Trac) for the loss of tumorigenicity on celery, C. amaranticolor, jimsonweed, K daigremontiana, tobacco, sunflower, and tomato plants. As shown in Fig. 1, all clones except pUCD3968 completely inhibited oncogenicity. pUCD3968 contains an osa gene truncated at the 5' end by 60 bp and, therefore, was not expected to inhibit oncogenicity. The presence of each clone and pTiC58Trac was verified by plasmid isolation and growth on nopaline medium. The expression of osa by each clone in A. tumefaciens was verified by Western blot (immunoblot) analysis with antibody to the Osa protein (data not shown). In every instance, clones containing osa and expressing Osa protein caused the loss of oncogenicity for A. tumefaciens. Orderly truncations from the 5' end of the 3.1-kb DNA fragment that removed orf-1, nuc, and orf-3 systematically showed that oncogenic inhibition remained with pUCD3960 containing solely osa (Fig. 1). These results suggest that orf-J, nuc, and orf-3 do not play a direct role in the oncogenic inhibition of A. tumefaciens and that osa appears to be the only gene within the 3.1-kb DNA segment that is required for this inhibition. Although apparent, we have not ruled out the possibility that the plasmid-encoded nuclease function might still play a role in oncogenic inhibition because a nuclease function could be provided by a chromosomally encoded gene in lieu of the nuc gene product. However, assays of pSa-free A. tumefaciens did not reveal any exocellular nuclease activity (7). Further truncation (60 bp) into the Osa coding sequence itself caused the loss of oncogenic inhibition activity as shown by clone pUCD3968. Thus, only plasmids containing an intact osa gene can suppress oncogenicity (Fig. 2). Expression of osa requires upstream sequences. The results described above implicate osa as the only gene of pSa involved in oncogenic inhibition and thus rule out the possibility that genes other than osa within the 3.1-kb DNA fragment are required for inhibitory activity. To test the possibility that osa is not expressed because a promoter upstream and distal to osa might be required, TnCAT insertions were made in the 3.1-kb DNA fragment. Three TnCAT insertions at positions -658,

promoter Pkm,

CHEN AND KADO

5700

J. BAC7ERIOL.

I

(A)

pUCD105 pUCD3960

(B)

pUCD1O5

-489, and -56 (determined by nucleotide sequencing) of the osa start codon (ATG) prevented oncogenic inhibitory activity when clones pUCD3940-1, pUCD3940-13, and pUCD3940-26 bearing the TnCAT insertions were tested in A. tumefaciens LBA4301(pTiC58Trac) (Fig. 3). Because TnCAT contains a stop codon, its polar effect suggests that transcription is initiated upstream of the most distal insertion site of -658, at which an insertion blocked oncogenic inhibition. An insertion at position -2025 and upstream of orfi1 was tested in clone pUCD3940-30. This insertion did not inhibit oncogenic suppressive activity because the insertion is 65 bp upstream of orff1 and S bp beyond the apparent -35 consensus promoter of this orf described previously (6). A mutant clone, pUCD394015, with TnCAT located in osa at position +58 abolished oncogenic inhibitory activity as expected. TnCAT inserted at position +563, which is 4 bp before the 3' end of osa, did not inhibit this activity as shown for clone pUCD3940-10 (Fig. 3). The last and perhaps the penultimate amino acid residues of Osa protein are therefore not absolutely essential for oncogenic inhibition. To verify further that transcription is initiated some distance from the Osa coding sequence, the CAT activities of each TnCAT insertional mutant in both E. coli and A. tumefaciens were determined. As shown in Table 2, each insertion (except mutant plasmids pUCD3940-10 and pUCD3940-30) showed CAT activity. The activity in E. coli was approximately 40 times greater than that in A. tumefaciens. This large difference may be a reflection of the higher efficiency of the promoter in E. coli than that in A. tumefaciens. Because CAT is produced by TnCAT mutants (except for clones pUCD3940-10 and pUCD 3940-30) and TnCAT insertions upstream of osa prevent oncogenic suppression, transcription must initiate from a promoter which is situated 659 bp upstream of osa and downstream of the most distal upstream TnCAT insertion.

pUCD3940-15 pUCD3940-30

I

pUCD3960 pUCD3940-15 pUCD3940-30

FIG. 2. Oncogenic suppressive activities of TnCAT insertional mutants. D. stramonium plant stems were inoculated with A. tumefaciens LBA4301(pTiC58Trac) containing the TnCAT insertions illustrated in Fig. 3. Tumor formation was scored 3 (A) and 5 (B) weeks after inoculation.

1

i

0

II

II

RA

oif 1>

nuc

1 l

RD

I

1

I

D

A

1

l

t(-8)

*(+58)

*(-56)

* (-20M ...r 5

.

1

F -

pUCD1311

PB

osa

3

*(-658)

4

Oncogenic suppression

3 kb

2

- pUCD3940-1 (+563) - pUCD394O-1O

f

3]

pUCD3940-13

3-

pUCD3940-15

}-

pUCD3940-26

C

}- pUCD3940-30

FIG. 3. TnCAT insertional mutations of the 3.1-kb DNA segment of pSa bearing the osa locus. The bold vertical lines represent the locations of putative promoters in this DNA segment cloned in pUCD1311. The genes and orfs are shown by horizontal arrows which also indicate the direction of transcription. TnCAT insertion sites are shown by vertical arrowheads. The number in parentheses beside each arrowhead indicates the number of base pairs between the insertion and the initiation codon of osa, either upstream of osa as indicated by a positive number or downstream of the translational start of osa as indicated by a negative number. The stippled arrowhead represents a TnCAT insertion in the Osa protein coding region. The designation of each plasmid bearing a TnCAT insertion is listed to the right of each illustration. The plus and minus signs indicate the positive and negative oncogenic activity, respectively, of each A. tumefaciens strain containing the designated TnCAT plasmid. The restriction endonuclease sites are identified in the legend to Fig. 1.

ONCOGENIC INHIBITION BY osa

VOL. 176, 1994

plasmid pUCD3960 (Fig. 1) was tested on jimsonweed, no tumors developed. Subsequent oncogenicity assays with celery, C. amaranticolor, K daigremontiana, tobacco, sunflower, and tomato plants also showed the inability of A. tumefaciens to form tumors when osa is present. The control strain, A. tumefaciens LBA4301(pTiC58Trac) containing the vector plasmid pUCD105, continued to induce tumors on these plants. These results indicate that oncogenic inhibition by osa is not restricted to a particular plant species, but the tumor-inducing ability of A. tumefaciens on many species of susceptible plant hosts tested herein is inhibited by osa.

TABLE 2. Transcription of osa as measured by CAT activity in E. coli DH5ot and A. tumefaciens LBA4301(pTiC58Trac) CAT activity (nmol/min/mg)b E. coli A. tumefaciens

TnCAT Plasmid

insertion site (bp)a

pUCD3940-1 pUCD3940-10 pUCD3940-13 pUCD3940-15 pUCD3940-26 pUCD3940-30 pUCD1311

A412/min E. coli A. tumefaciens

-658 4.58 0.41 +563 -489 8.30 5.32 +58 2.36 -56 -2025 12.50 None 0.00

122.8