Mol Gen Genet (1991) 230:302-309 © Springer-Verlag 1991

Characterization of the supervirulent virG gene of the Agrobacterium tumefaciens plasmid pTiBo542 Chin-Yi Chen, Lu Wang, and Stephen C. Winans Section of Microbiology,Cornell University,Ithaca, NY 14853, USA Received June 24, 1991

Summary. The virG gene of the Agrobacterium tumefaciens Ti plasmid pTiBo542 has previously been reported to elicit stronger vir gene expression than its counterpart in the pTiA6 plasmid, a property we call the "superactivator" phenotype. The DNA sequence of the pTiBo542 virG gene was determined and compared to that of the pTiA6 gene. The DNA sequences of these genes differ at 16 positions: two differences are in the promoter regions, 12 are in the coding regions, and two are in the 3' untranslated regions. The 3' end of the pTiA6 virG gene also contains a probable insertion sequence that is not found downstream of the pTiBo542 gene. The base pair differences in the two coding regions result in only two amino acid differences, both in the aminoterminal halves of the proteins. Five hybrid virG genes were constructed and used to activate the expression of a virB." .'lacZ gene fusion. Differences in the coding regions of these genes accounted for most of the superactivator phenotype, while differences at the promoter and 3' untranslated regions also contributed. These findings suggest that the properties of these VirG proteins and their quantities are important for vir gene induction, and also suggest a long-term selective pressure for mutations contributing to differences between these two genes. Key words: VirG Agrobacterium pervirulence pTiBo542

vir activation

Su-

Introduction Agrobacterium tumefaciens incites the crown gall disease

of dicotyledonous plants by transferring a discrete fragment of plasmid-encoded DNA to the nuclei of plant cells (reviewed by Ream 1989; Zambryski 1988; Kado 1991). This DNA becomes integrated into plant genomic DNA and directs the overproduction of phytohormones, which causes tumorous cell proliferation, and of opines, which are carbon and nitrogen compounds that serve Offprint requests to ." S.C. Winans

as nutrient sources for the infecting bacteria. Transfer of this DNA requires the products of about 25 virulence (vir) genes arranged in seven operons (Stachel and Nester 1986). These genes, like the transferred DNA, are localized on the Ti (tumor-inducing) plasmid. The vir operons are transcriptionally induced during infection by the synergistic action of phenolic compounds, monosaccharides, and low pH (Stachel et al. 1985; Stachel and Zambryski 1986; Winans et al. 1988; Melchers et al. 1989; Rogowsky et al. 1987; Cangelosi et al. 1990; Alt-M6rbe et al. 1989). Induction requires the products of the virA and virG genes, two members of the family of "twocomponent" transcriptional regulatory proteins (Winans et al. 1986; Powell et al. 1987; Leroux et al. 1987; Melchers et al. 1986, 1987; Morel et al. 1989; Stock et al. 1989 b). VirA is an environmental sensor and transmembrane protein kinase which phosphorylates VirG (Winans et al. 1989; Jin et al. 1990a, b; Huang et al. 1990). VirG protein binds to specific sites found upstream of all vir promoters to activate their transcription (Jin et al. 1990c; Pazour and Das 1990; Powell and Kado 1990). A number of different Ti plasmids have been characterized, including the '° supervirulent" plasmid pTiBo542 (Hood et al. 1984, 1986a, b; 1987; Jin et al. 1987). A strain containing this plasmid incited tumors that developed faster than those incited by an isogenic strain containing any other Ti plasmid tested (An etal. 1985; Hood et al. 1986b). Cosmids containing the virB and virG operons from the pTiBo542 plasmid were able to confer supervirulence to a strain containing the pTiA6 plasmid (Jin et al. 1987). Transposon insertions in the virG gene of a representative cosmid completely abolished this property, while insertions in the 3' end of virB attenuated it. Insertions in the 5' end of virB did not affect the phenotype (Jin et al. 1987). A strain containing pTiA6 and the virG gene from pTiBo542 cloned onto a multicopy plasmid was also supervirulent, although full supervirulence required that virG be expressed from an exogenous promoter supplied by the plasmid vector. This finding indicates that the pTiBo542 virG gene is dominant in merodiploids, and suggests that it may be the major determinant of the supervirulent phenotype.

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Fig. 1. Physical and genetic maps of the virG loci of the pTiBo542 and pTiA6 Ti plasmids. Thick horizontal lines represent DNA fragments cloned into the plasmids indicated at the left

Strains containing various vir.':lac gene fusions expressed /%galactosidase at higher levels when the pTiBo542 virG was present than in the presence of the pTiA6 virG gene (Jin et al. 1987). This suggested that the "supervirulent" phenotype of pTiBo542 was attributable to the ability of the pTiBo542 virG gene to activate vir gene transcription more strongly than the pTiA6 allele, a property we refer to as the "superactivator" phenotype. In this paper, we compare the pTiBo542 and pTiA6 virG genes at the nucleotide level. We find that the two genes are 98% identical, and that differences between their promoters, coding sequences, and 3' untranslated regions all contribute to the superactivator phenotype.

Materials and methods Strains, plasmids, and reagents. Agrobacterium strains A136(pSM243) and A348 and Escherichia coli strain JM101 were obtained from E. Nester (University of Washington). Plasmid pToK9 (Jin et al. 1987) is a derivative of p V K I 0 2 containing the virG gene of pTiBo542 as a 5.2 kb SalI fragment (Fig. 1). Broad host-range plasmid vector p U C D 2 (Close et al. 1984) was obtained from C. Kado (University of California, Davis). Plasraids p T Z 1 8 R and p T Z 1 9 R and the Sequenase Version 2.0 D N A Sequencing Kits were purchased from United States Biochemical Corp. (Cleveland, OH). Synthetic oligonucleotides used for D N A sequencing were obtained from the Cornell Nucleotide Synthesis Center. L-Nitrophenyl-/%D-galactoside (ONPG), carbenicillin, tetracycline, and 2-(N-morpholino)ethanesulfonic acid (MES) were purchased from Sigma (St Louis, MO). Restriction endonucleases and T4 D N A ligase were purchased from Bethesda Research Laboratories (Gaithersburg, MD), and were handled using standard recombinant D N A techniques as recommended by the manufacturers. Acetosyringone was purchased from Aldrich Co. (Milwaukee, WI). Adenosine 5'-~-[35S]thiotriphosphate was purchased from Amersham (Arlington Heights, IL). D N A sequencing. Plasmid D N A to be sequenced was isolated in single-stranded form using helper phage

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Fig. 2. Construction of hybrid alleles of virG containing DNA from the pTiA6 and pTiBo542 plasmids (see the Materials and methods). pSWI67 and pCC108 contain the pTiA6 and pTiBo542 virG genes, respectively. The central boxes represent virG coding sequences. All plasmids contain a small amount of the virBll gene at their left ends (not shown), pCCI08 contains a small amount of the 3' end of virC, shown as a cross-hatched box at the entreme right, while pSWI67 contains a small amount of insertion sequence IS66vl (see text), shown as a black box at the extreme right. Thin boxes represent non-coding sequences. All hatched boxes are derived from pTiBo542 DNA M13KO7 (Dente et al. 1983), and was sequenced using a Sequenase kit according to the recommendations of the manufacturer. Most of the oligonucleotides used to prime D N A synthesis were custom-made according to the known sequences of the pTiA6 virG or virC genes. Sequencing of the top strand was done using the oligonucleotides 5 ' - T T A T G C T T C C G G C T C G T - 3 ' (complementary to vector DNA), 5 ' - T G A A A G G T G A G C C G T TG-3', 5 ' - C T T G A G G A G A C G G A T A A - 3 ' , and 5'-ACTT C T C A T T G C C A G T C - 3 ' . Sequencing of the bottom strand was done using oligonucleotides 5'-CAACGGCTCACCTTTCA-3', 5'-TTATCCGTCTCCTCAAG-3', 5'GACTGGCAATGAGAAGT-3', 5'-GCTGCCATCGTCCCCCC-3', and 5 ' - G A G C T T T C G G C C A C A A G C - 3 ' (complementary to the 3' end of virC). D N A sequences were analyzed using the computer programs Gene-Pro (Riverside Scientific Co.) and F A S T A (Pearson and Lipman 1988). Plasmid constructions. To subclone the pTiBo542 virG gene, plasmid pToK9 was digested with BglII and PstI, and products of this digestion were ligated to p T Z 1 8 R digested with B a m H I and PstI. From this procedure plasmid pSW254 was isolated (Fig. 1), which contains the virG gene oriented the same direction as P~,c- To clone virG in the opposite orientation, pSW254 and pTZ19R were digested with EcoRI and Pstl, and ligated together. From the resulting transformants, plasmid pCC106 was isolated. pCC108 (Fig. 1) was constructed by deleting the smaller of two SphI fragments of pSW254, pSW272, p C C I I 6 , pCC109, p C C l l 0 , and p C C l l 7 were constructed from pCC108 and pSW167 (Jin et al. 1990c) and contain regions of the pTiA6 virG and the pTiBo542 virG genes as indicated in Fig. 2. Plasmids pSW167, pCC108, pSW272, pCCl16, pCC109, pCCl10, and p C C l 1 7 were converted to a linear form by digestion with EcoRI and then ligated with the large EcoRI frag-

304 A gggGATCTGGCTCGCGGCGGACGCACGACGCCGGGGCGAGACCATAGGCGATCTCCTTAA .... I W L A A D A R R R G E T I G D L L N T TCAATAGTAGCTGTAACCTCGAAGCGTTTCACTTGTAACAACGATTGAGAACTTTTGTCA Q * * -35 T TAAAATTGAAATACTTGGTI'CGCATTTTCGTCATCCGCGGTCAGCCGCAATTCTGACGAA -10 I P1 BstXI CTGCCCATTTAGCTGGAGATGATTGTACATCCTTCACGTGAAAATTTCTCAAGCGCTGTG -10 T P2 G AACAAGGGTTCAGATTTTAGATTGAAAGGTGAGCCGTTGAAACACGTTCTTCTTATCGAT M K H V L L I D V C T GACGATGTCGCTATGCGGCATCTTATTATCGAATACCTTACGATCCACGCCTTCAAAGTG D D V A M R H L I I E Y L T I H A F K V

A ACCGCGGTAGCCGACAGCACCCAGTTCACTAGAGTACTCTCTTCCGCGACGGTCGATGTC T A V A D S T Q F T R V L S S A T V D V

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300

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Fig. 4A and B. Sequence of an IS element between the virG and virC genes of pTiA6. A Top line, sequences 3' of the pTiA6 virG gene (Winans et al. 1986); bottom line, sequences 3' of the convergently transcribed pTiA6 virC gene (Yanofsky and Nester 1986); middle line, sequences between virG and virC of pTiBo542. Lower case letters represent insertion sequence IS66vl DNA present in pTiA6. Dyad symmetry at the termini of IS66vl are denoted by single dots. B Similarity between the ends of IS66vl and the ends of other Agrobacterium IS elements IS66 (Machida et al. 1984); IS866 (Bonnard et al. 1989), IS867 (L. Otten, personal communication), and IS868 (Paulus et al. 1991). Bases identical to IS66vl are underlined

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m e n t of p U C D 2 , thus g e n e r a t i n g pSW194, p C C l 1 3 , pSW301, p C C l l 9 , p C l l 4 , p C C l 1 5 , a n d p C C I 2 0 , respectively (Fig. 2). These plasmids c o n t a i n the tet r gene a n d the b r o a d h o s t - r a n g e rep region of p U C D 2 . T h e y were introduced into Agrobacterium strain

A136(pSM243) by electroporation (Mattenovich et al. 1989) using a Gene Pulser apparatus (Bio-Rad, Richmond, CA). Bacteria were cultured and assayed for/~galactosidase as described previously ( C h e n a n d W i n a n s

GAAGCTGGCGGTGAGGTGAAACTTACGGCAGGTGAGTTCAATCTTCTCCTCGCGTTTTTA 780 E A G G E V K L T A G E F N L L L A F L

GAGAAACCCCGCGACGTTCTATCGCGCGAGCAACTTCTCATTGCCAGTCGAGTACGCGAC 840 E K P R D V L S R E Q L L I A S R V R D T A GAGGAGGTTTACGACAGGAGTATAGATGTTCTCATTTTGCGGCTGCGCCGCAAACTTGAG 900 E E V Y D R S I D V L I L R L R R K k E BamHI GCGGATCCGTCAAGCCCTCAACTGATAAAAACAGCAAGAGGTGCCGGTTATTTCTTTGAC A D P S S P Q L I K T A R G A G Y F F D

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G T XX TTAATTATCTGGCTCAAAAGGTGACTGAGGACGCGGCCAGCGGCCTCAAACCTACACTCA

1080

G C T C T ATATTTGGTGAGGGGTTCCGATAGGTCCCTCTTCACCAATTGCTCGATGGCTTCTCTCCA 1140 * W N S S P K E G R R G A GCAAAGAATGACGCGAGCG 1159 A F F S A L....

Fig. 3. Nucleotide and protein sequence of the virG gene of pTiBo542. Differences at the nucleotide level between this gene and the pTiA6 virG gene are indicated above the DNA sequence, while differences at the protein level are indicated below the protein sequence. Two "XXs" above the bases " A C " at bases 1074-1075 represent a 2 bp deletion in the pTiA6 gene. The - 1 0 and - 3 5 sites of promoter Pl and the - 1 0 site of promoter P2 are underlined, and transcription initiation sites are indicated with arrows. The indicated BstXI and BamHI sites were used to construct hybrid

1991). Results S e q u e n c e analysis o f p T i B o 5 4 2 v i r G gene

The elevated level of tumorigenesis of an Agrobacterium strain containing the pTiBo542 Ti plasmid compared to a strain containing pTiA6 is due at least in part to differences at their respective virG loci (Jin et al. 1987). To begin a more thorough analysis of the virG gene of pTiBo542, we sequenced this gene and compared the sequence to that of the pTiA6 virG gene. Figure 3 shows the DNA and protein sequences of the pTiBo542 virG gene, the 3' end of virBll, and the 3' end of the convergently transcribed virC2 gene. Perhaps the most striking difference between these two sequences is the small size of the intergenic region between virG and virC (120 bp) in pTiBo542 compared to the corresponding intergenic region in pTiA6 (approximately 5000 bp; see Fig. 1). The small size of this intergenic region in pTiBo542 is genes. Inverted arrows near the virG termination codon represent an inverted repeat, which could provide a transcription termination site. The sequence "ACTGAGGA" found at bases 1044-1051 (underlined) is present in only one copy in pTiBo542 but found in two copies in pTiA6. Lower ease letters (bases 1-3) represent vector sequences

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5A-D. Induction of a virB:.'lacZ gene fusion in strains containing the pTiA6, pTiBo542, or hybrid virG genes cultured at an initial pH of 5.3. Isogenic strains contained plasmids pSW194 (o), pCC113 (e), pSW301 (A), pCCl19 (A), pCCtl4 (v), pCCll5 (v), or pCC 120 (+). Hybrid gene expression induced with acetosyr-

ingone at: A, 1 gM; B 5 gM; C, 20 gM; D, 100 gM;. All data represent averages of duplicate measurements, fl-Galactosidase specific activity was measured according to the method of Miller (1972)

consistent with the data of Komari et al. (1986). The extra D N A in pTiA6 is flanked by two copies of the sequence A C T G A G G A , while the pTiBo542 plasmid has only one copy o f this sequence (Fig. 3, bases 10441051; see also Fig. 4). This suggests that the 5000 bp of D N A found in pTiA6 is an insertion sequence, a possibility that will be considered further in the Discussion section. There are a total of 26 additional differences between the pTiBo542 sequence and the corresponding region of the pTiA6 plasmid; 25 of these are base substitutions, while the remaining difference is a 2 bp insertion in pTiBo542 (bases 1074-1075). O f these 26 differences, one lies in virBll, while nine differences lie in or close to virC2. There are 16 differences between the two virG genes in or near their coding sequences. Two of these differences are found in the promoter region (bases 112 and 149). Twelve differences between these genes lie in their coding regions; nine of these do not result in amino acid differences, while the remaining three differences alter two amino acids: the seventh amino acid is an isoleucine in the pTiBo542 gene and a valine in the pTiA6 gene, while the 106th amino acid is a threonine in the pTiBo542 gene and an isoleucine in the pTiA6 gene. The remaining two differences lie 3' to the termination codon of the genes. One of these lies in a region of dyad symmetry which could provide a transcriptional termination signal. In pTiBo542, a guanine residue is found at position 1017, while a cytosine residue is found

at this position in pTiA6. This difference creates a stronger dyad symmetry in pTiBo542 than in pTiA6.

Construction and characterization of hybrid virG genes To determine which of the differences between these two virG genes contributes to the superactivator phenotype of the pTiBo542 VirG protein, we constructed five hybrid virG genes, using two conserved restriction endonuclease cleavage sites, BstXI and BamHI (Fig. 2). These hybrid genes and the two parental genes were cloned into the broad host-range plasmid p U C D 2 and introduced into Agrobacterium strain A136(pSM243). This strain does not contain a Ti plasmid, and pSM243 contains the pTiA6 virA gene and a pTiA6 virB::lacZ gene fusion (Stachel and Nester 1986). Seven strains carrying different alleles of virG were incubated under a variety of conditions, including those considered optimal for vir gene induction (pH 5.3 with high levels of acetosyringone, AS) and several suboptimal conditions (pH 6.0 with lower levels of acetosyringone). This was done in an effort to find conditions in which differences between these virG genes were maximized. Several conclusions can be drawn from these experiments. First, induction under suboptimal conditions (for example, in broth pH 6.0, with l, 5, or 20 gM acetosyringone; Fig. 6A C), causes a distinct lag in induction, with little if any fl-galactosidase synthesized before 12 h

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Fig. 6A-D. Induction of a virB.'.'lacZ gene fusion in strains containing the pTiA6, pTiBo542, or hybrid virG genes cultured at an initial pH of 6.0. For further explanation of symbolsand methods see the legend to Fig. 5 of incubation. In contrast, under more optimal conditions (for example, in broth at pH 5.3 containing 100 gM acetosyringone; Fig. 5 D), strong induction was observed after only 8 h. Second, differences in transcriptional efficiency between these genes are most apparent under suboptimal induction conditions. For example, after induction under optimal conditions (pH 5.3 with 100gM acetosyringone; Fig. 5D), the most efficient protein functioned less than two times more efficiently than the least efficient protein. In contrast, after induction at pH 6.0 with I gM or 5 gM acetosyringone (Fig. 6A and B), the most efficient protein functioned about 10-fold more efficiently than the least efficient protein. The third conclusion concerns the relative importance of the promoter, the coding region, and the 3' end of the pTiBo542 virG gene in the superactivator phenotype. Under most conditions, all hybrid virG genes containing the pTiBo542 coding region (plasmids pCC119, pCC114, and pCC120) showed levels of virB expression that were virtually the same as the pTiBo542 parental gene (plasmid pCC113), and higher than genes containing the virG coding region of pTiA6 (plasmids pSW194, pSW30a, and pCC115). This was true in all eight conditions tested and indicates that the pTiBo542 structural gene is sufficient under most conditions to confer virtually full levels of the superactivator phenotype. However, when hybrid gene expression was induced at pH 6.0 with 1 IxM AS (Fig. 6A) or 5 gM AS (Fig. 6B), the promoter and 3' end were necessary for full superactivator phenotype. Therefore, under suboptimal conditions, all three parts of the pTiBo542 gene contributed to superactivation.

Further evidence that the pTiBo542 promoter and 3' end contribute to superactivation was obtained by fusing either of them to the pTiA6 gene. Under some but not all conditions, hybrid virG genes containing just the pTiBo542 promoter (pSW301) or containing just the pTiBo542 3' end (pCC115) caused higher levels of virB expression than the pTiA6 gene (pSW194). The superactivator phenotype of these two hybrid genes depended upon the induction conditions used; pSW301 was stronger than pSW194 only at pH 5.3 (Fig. 5A-D), while pCC115 was stronger than pSW194 only at pH 6.0 (Fig. 6A-D). We conclude that the coding sequence of the pTiBo542 virG gene is the most important determinant of the superactivator phenotype, and that both the promoter and 3' end of this gene also contribute to superactivation. Comparison of pTiBo542 and pTiA6 virG promoters

Since sequences upstream of the BstXI site appeared to play a role in the elevated ability of the pTiBo542 virG gene to elicit virB expression, we tested the hypothesis that this promoter might be stronger than its pTiA6 counterpart. To do this, we fused these two promoters to lacZ, cloned these gene fusions into plasmid pUCD2, introduced them into Agrobacterium strain A348, and tested their relative promoter strengths. Strain A348 contains pTiA6. The pTiA6 virG gene contains two promoters: P1 is induced by acetosyringone in a VirA, VirGdependent fashion, while P2 is induced by chromosomal genes in response to acidic extracellular pH (Winans

307 1990). When induction by both of these stimuli was tested under a variety of conditions, no differences between these two promoters were detectable (data not shown). Discussion

In this study, we have determined the nucleotide sequence of the pTiBo542 virG gene and compared it to the corresponding pTiA6 gene. The DNA sequences of these two genes are about 98% identical, and are much more similar to each other than they are to the virG genes of pTiC58 (Powell et al. 1987) or pRiA4 (Aoyama et al. 1989). However, a 5 kb D N A sequence found in pTiA6 is absent in pTiBo542 (Figs. i, 4A). The 3' ends of the two virG sequences diverge sharply starting about 50 bp downstream from the virG stop codon (Fig. 3). Comparison of the pTiA6 and pTiBo542 sequences in this region suggested strongly that this divergence was due to an insertion sequence in pTiA6. This is shown most clearly in Fig. 4A, in which sequences of the 3' end of the pTiA6 plasmid (line 1), the 3' end of the convergently trasncribed virC gene (line 3) and the corresponding regions of the pTiBo542 plasmid are given. The pTiBo542 plasmid has an 8 bp sequence (ACTGAGGA) which is present in two copies in the pTiA6 plasmid. Directly to the right of the first copy and to the left of the second copy is an imperfect inverted repeat, indicated by dots above the first line and below the third line. Both these features are diagnostic of an IS element (Craig and Kleckner 1987). An IS element in this region was first described by Machida et al. (1984), and we have adopted their designation for this putative IS element: IS66vl. No homologous sequences were revealed in a search of the Genebank and EMBL DNA databases; however, the extreme ends of this element show some similarity to those of other IS elements of Agrobacterium (Machida et al. 1984; Bonnard et al. 1989; L. Otten, personal communication), as shown in Fig. 4 B. Hybrid virG genes were constructed containing D N A from the pTiBo542 and pTiA6 plasmids. Agrobacterium strains containing these genes were incubated under a variety of conditions and the induction of a virB: .'lacZ fusion was monitored over a period of 16 h. We did not test other vir promoters, although in previous studies all vir promoters were more strongly transcribed when the pTiBo542 VirG gene product is expressed than when the pTiA6 gene product is present (Jin et al. 1987). The differences in activation efficiency between these two virG products were most pronounced when bacteria were incubated with low concentrations of inducer and especially at pH 6.0 rather than pH 5.3. Furthermore, under suboptimal induction conditions, a long lag in induction was observed. The latter observation may have been due to the complex transcriptional regulation of the virG genes themselves. The pTiA6 virG gene, and probably also the pTiBo542 gene, is expressed from two promoters. One of these (P1) is inducible by acetosyringone in a VirA, VirG-dependent fashion, while the

other (P2) is inducible by acidic pH using chromosomal regulatory proteins (Winans 1990). The positive autoregulation of promoter P1 could cause the lag in induction that is seen under suboptimal conditions, since the concentration of VirG, which limits the efficiency of vir gene induction, would increase slowly to a concentration high enough for efficient induction of promoter P1 and of the virB promoter. The fact that little if any lag is seen in inductions conducted at pH 5.3 could be due to the rapid, VirG-independent induction of P2 at this pH. Induction at pH 6.0 with high levels of inducer also occurs with only a short lag. This suggests that, if the small amount of VirG present in the cells can be efficiently phosphorylated by VirA, then it can activate Pl sufficiently to increase significantly the pool size of VirG protein early in the incubation period. Several differences between these two genes have been identified which contribute to the superactivator phenotype of the pTiBo542 gene. The most important determinant of the superactivator phenotype lies in the coding region of the pTiBo542 virG gene. We believe that the two amino acid differences are responsible, although we have not ruled out the possibility that one or more silent base changes could also play some role. The promoter and 3' end of the pTiBo542 virG gene appear to play ancillary roles in superactivation, since under at least some conditions (i) replacement of either of these regions of the pTiBo542 gene with its pTiA6 counterpart results in a somewhat weaker activator than the pTiBo542 gene, and (ii) replacement of either of these regions of the pTiA6 virG gene with its pTiBo542 counterpart results in stronger activator than the pTiA6 gene. The two amino acid differences between the two VirG proteins, Val(7) and Ile(106) in the pTiA6 gene versus Ile(7) and Thr(106) in the pTiBo542 gene, are both found in the "receiver" domain of the proteins, that is, the amino-terminal half of the protein which is phosphorylated by VirA. The three-dimensional structure has been determined for CheY, a protein that is homologous to the receiver domain of VirG (Stock et al. 1989a); alignment of the VirG protein to this structure reveals that these residues are located near an "acidic pocket" which contains the phosphorylated aspartate residue. We propose that these changes affect some aspect of phosphorylation of VirG by VirA. They might cause VirG to be more readily phosphorylated, or less readily dephosphorylated, or they might cause phosphorylation to result in a more profound conformational change, causing the pTiBo542 VirG protein to bind promoters more avidly than the pTiA6 protein. A final resolution of this problem will require biochemical characterization. Seemingly contradictory evidence was obtained concerning the role of the pTiBo542 promoter. When the promoters of the two genes were used to make hybrid virG genes, the pTiBo542 virG promoter contributed to the superactivator properties of the hybrid genes (Figs. 5, 6) and these effects were highly reproducible. Since the concentration of VirG protein limits the efficiency of activation of other vir genes (Chen and Winans 1991), it seemed possible that the promoter of the pTi-

308 Bo542 gene could contribute to the superactivator phenotype simply by being a stronger promoter. However, when the strengths of these promoters were compared directly by fusing them to lacZ, no differences were detected. One possible explanation is that the pTiBo542 promoter is a very slightly stronger promoter than its pTiA6 counterpart, but that these differences are too small to be measured directly, using virG:: lacZ fusions. On the other hand, small differences between the promoters might be amplified by the positive autoregulation of virG, to the point that they are detectable in measurements of virB expression. The differences between the two promoters are found in striking locations. The pTiA6 virG promoter contains the sequence A T T T T T G T C A T in two copies, one at the - 3 5 sequence of promoter P1, and the other near the P1 transcription initiation site. In pTiBo542, both copies of this sequence are altered, to the sequences A C T T T T G T C A T and A T T T T C G T C A T , respectively. The role, if any, that these sequences play in the regulation of these promoters is not known; however, if they do play some role, it would appear that they have been disrupted in the pTiBo542 gene. These sequences are similar to a binding site for the integration host factor (IHF) protein (Tsui and Freundlich 1990) and I H F expression decreases transcription of two promoters that are activated by a protein homologous to VirG (Tsui et al. 1988; Huang et al. 1990). We are currently testing whether these sequences could provide binding sites for IHF. Those differences between the two genes that lie 3' to the coding sequence consist of single nucleotide differences 15 and 37 bases downstream from the stop codon, as well as the IS66vl element in the pTiA6 plasmid located 49 bases downstream. We do not know which of these differences causes the pTiBo542 3' end to contribute to superactivation. It is noteworthy that the first of these differences lies in a region of dyad symmetry which could play a role in the termination of virG transcripts. This dyad symmetry is stronger in the pTiBo542 virG gene than in the pTiA6 gene, and this difference might result in a stronger termination signal. Perhaps more important, the stronger putative hairpin loop in the pTiBo542 m R N A might be more resistant to exonucleolytic digestion than the corresponding hairpin of the pTiA6 m R N A (see Mott et al. 1985). This might increase the half-life of the m R N A , and could thereby increase the pool of virG m R N A , and ultimately increase the pool of VirG protein. It is tempting to speculate about the evolution of these two genes, although it is not possible to know which (if either) of these genes is the ancestor of the other. However, if the pTiA6 virG gene is the ancestor, then it would appear that continual selection for increased efficiency of vir gene transcription has occurred. Conversely, if the pTiBo542 gene is the ancestor, then selection for decreased efficiency has occured. Either way, the result has been an accumulation of multiple mutations, each of which contributes to this phenotype. It is interesting that the supervirulent phenotype of pTiBo542, which is due to its superactivating virG allele (Jin et al. 1987), is influenced strongly by one or more

genes found on the chromosome or cryptic plasmids. The supervirulent strain A281 was created by conjugal transfer of pTiBo542 from strain Bo542 into strain A136 (Sciaky et al. 1978). Bo542 was isolated from a dahlia gall and contains three cryptic plasmids in addition to its Ti plasmid (Sciaky et al. 1978). A136 contains the C58 chromosome and one cryptic plasmid. A281 incites larger tumors on tomato and tobacco than isogenic strains containing other Ti plasmids (Hood et al. 1986b) and is virulent on soybeans and peas (Hood et al. 1987). Significantly, strain Bo542 is weakly virulent on tomato and tobacco, and avirulent on soybeans and peas (Hood et al. 1986b, 1987). Evidently some difference in the chromosomes or cryptic plasmids between A281 and Bo542 plays a central role in supervirulence. It is possible that the weak virulence associated with the Bo542 chromosome may have caused the compensatory evolution of the superactivating virG of pTiBo542. Alternatively, the stronger virulence associated with the C58 chromosome may have caused the evolution of the weaker pTiA6 virG gene from a more strongly activating ancestor. Acknowledgements. We thank L. Otten for sharing unpublished data and N. Mantis for helpful discussions. This work was supported by N.I.H. grant no. 1R29 GM2893-01. This work was also supported by a grant from the Cornell Biotechnology Program which is sponsored by the New York State Science and Technology Foundation, a consortium of industries, the US Army Research Office, and the National Science Foundation. C.Y.C. was supported by the Cornell/National Science Foundation Plant Science Center, which is a unit in the U.S. Department of Agriculture/ D.O.E./N.S.F Plant Science Center Program, a unit of the Cornell Biotechnology Program. References

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