THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1988 by The American Society for Biochemistry and Molecular Binlog., Inc. Vol .263, No. Issue of April 25, pp. 580445814,1988 Printed in U. S. A. Characterization of the uirB Operon from an Agrobucterium tumefuciens Ti Plasmid* (Received for publication, August 17, 1987) John E. Ward& Donna E. Akiyoshis, Dean Regierg, Asis Dattaq, Milton P. Gordon, and Eugene W. Nester (1 From the Department of Microbiology and Immunology, University of Washington, Seattle, Washington 98195 The virulence genes of the Agrobacterium tumefa- ciens Ti plasmid are grouped into six transcription units and direct the transfer of T-DNA into plant cells. We report here the nucleotide sequence of the largest vir operon, virB, from the Ti plasmid pTiA6NC. This operon contains 11 open reading frames, 7 of which show evidence of translational coupling. trpE:virB gene fusions were used to confirm the reading frames of genes virB2, 4, 5, 6, 7, 8, 10, and 11. In addition, the native gene products of virB6 and virB9 were identified using maxicell and in vitro transcription- translation techniques, and the VirB9 protein was found to be proteolytically processed. The codon usage of the predicted virB genes is very similar to the other pTiA6 vir genes and is much less biased than Escher- ichia coli. Since many of the virB gene products have secretion signals common to exported bacterial pro- teins, it is likely that they will be membrane-associ- ated. We propose that the VirB proteins are involved in the formation of a transmembrane structure which mediates the passage of the transferred T-DNA mole- cule through the bacterial and plant cell membranes. The soil phytopathogen Agrobucterium tumefaciens trans- forms susceptible plants by the transfer and integration of a piece of DNA (T-DNA) from a large tumor-inducing bacterial plasmid (Ti plasmid) into the plant cell genome (for recent reviews see Nester et al., 1984; Stachel andZambryski, 1986a; Koukolikova-Nicola et al., 1987; Powell and Gordon, 1987). Genes on the T-DNA code for biosynthesis of the plant growth regulators auxin and cytokinin, and the overexpression of these phytohormones results in loss of division control and a tumorous phenotype (Nester et al., 1984). Genetic studies have identified several regions outside the T-DNA which are required for T-DNA transfer. A number of constitutively expressed chromosomal loci are required for the binding of Agrobacterium to plant cells (Douglas et al., * This work was supported by National Institutes of Health Grant 5 ROI GM 32618-14 and National Science Foundation Grant DMB- 8315826 The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) 50321 6. $ Supported by American Cancer Society Postdoctoral Fellowship J Current address: Serogene, Hopkinton, MA 07148. ll Current address: School of Life Sciences, Jawaharlal Nehru Uni- 11 To whom correspondence should be addressed. PF-2796. versity, New Delhi, India 110 067. 1985; Cangelosi et al., 1987; Thomashow et al., 1987). Muta- tions in these loci result ina reduced ability of Agrobacterium to attach to plant cells and an attenuated virulence. Another virulence region (uir) spans 35 kbp’ of the pTiA6 plasmid and contains at least six transcriptional loci (uirA, B, C, D, E, and G), designated the uir genes. Mutations in these loci result in either avirulence (uir A, B, D, and G) or attenuated virulence (uirC, E) (Hille et al., 1984; Klee et al., 1983; Stachel et al., 198513; Stachel and Nester, 1986). The uir genes are organized as a single regulon which is inducible by plant cell phenolic compounds (Stachel et al., 1985a; Bolton et al., 1986) via a positive regulatorysystem consisting of uirA and uirG (Stachel and Zambryski, 1986b; Winans et al., 1986). Many of the molecular reactions necessary for processing and transfer of the T-DNA molecule are specified by the uir genes, but the exact functions of individual uir genes are for the most part unknown. The molecular characterization of these genes should facilitate their functional analysis. Toward this end, the nucleotide sequences of the uirA (Leroux et al., 1987), uirC (Yanofsky and Nester, 1986), uirD (Yanofsky et al., 1986; Porter et al., 1987), uirE (Winans et al., 1987), and uirG operons (Winans et al., 1986) from the pTiA6 plasmid recently have all been determined in our laboratory. Herewe extend this analysis to include the nucleotide sequence of the largest uir operon, uiri?. Our resultsshow that the pTiA6 uirB operon contains 11 potential genes, most of which appear to code for membrane-associated proteins. Their functions are discussed in relation to our current understanding of the T- DNA transferprocess. EXPERIMENTAL PROCEDURES Materials-Restriction endonucleases were purchased from New England Biolabs or Bethesda Research Laboratories and used as suggested by the supplier. DNA polymerase I, Klenow fragment, was obtained from either Bethesda Research Laboratories or Boehringer Mannheim; S1 nuclease was from New England Biolabs, and Esch- erichia coli exonuclease 111 and T4 ligase was from Bethesda Research Laboratories. M13 sequencing primer, unlabeled nucleotides, and dideoxynucleotides were purchased from Pharmacia LKB Biotech- nology Inc. 35S-LabeleddATP was from Du Pont-New England Nuclear. Bacterial Strains and Plasmid-E. coli strains JMlOl and MV1193 were used for plasmid maintenance and for the production of ssDNA template and were propagated on either L agar or M9 minimal agar (Miller, 1972) supplemented with the appropriate antibiotic (carben- icillin, 100 rg/ml; kanamycin, 50 pg/ml). DNA fragments from the uirB region of Ti plasmid pTiA6NC were obtained from the cosmid pVK257 (Knauf and Nester, 1982) and subcloned into the PATH vectors of Spindler et al. (1984) as described in the text. Plasmid pJW207 was constructed by cloning the 1.3-kbp EcoRI fragment 30 into pUC119. The 1.8-kbp EcoRI-BamHI fragment from Sal1 frag- The abbreviations used are: kbp, kilobase pair; bp, base pair; ORF, open reading frame; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; ssDNA, single-stranded DNA. 5804
Transcript
THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1988 by The American Society for Biochemistry and Molecular Binlog., Inc.
Vol .263, No. Issue of April 25, pp. 580445814,1988 Printed in U. S. A.
Characterization of the uirB Operon from an Agrobucterium tumefuciens Ti Plasmid*
(Received for publication, August 17, 1987)
John E. Ward& Donna E. Akiyoshis, Dean Regierg, Asis Dattaq, Milton P. Gordon, and Eugene W. Nester (1 From the Department of Microbiology and Immunology, University of Washington, Seattle, Washington 98195
The virulence genes of the Agrobacterium tumefa- ciens Ti plasmid are grouped into six transcription units and direct the transfer of T-DNA into plant cells. We report here the nucleotide sequence of the largest vir operon, virB, from the Ti plasmid pTiA6NC. This operon contains 11 open reading frames, 7 of which show evidence of translational coupling. trpE:virB gene fusions were used to confirm the reading frames of genes virB2, 4, 5, 6, 7, 8, 10, and 11. In addition, the native gene products of virB6 and virB9 were identified using maxicell and in vitro transcription- translation techniques, and the VirB9 protein was found to be proteolytically processed. The codon usage of the predicted virB genes is very similar to the other pTiA6 vir genes and is much less biased than Escher- ichia coli. Since many of the virB gene products have secretion signals common to exported bacterial pro- teins, it is likely that they will be membrane-associ- ated. We propose that the VirB proteins are involved in the formation of a transmembrane structure which mediates the passage of the transferred T-DNA mole- cule through the bacterial and plant cell membranes.
The soil phytopathogen Agrobucterium tumefaciens trans- forms susceptible plants by the transfer and integration of a piece of DNA (T-DNA) from a large tumor-inducing bacterial plasmid (Ti plasmid) into the plant cell genome (for recent reviews see Nester et al., 1984; Stachel and Zambryski, 1986a; Koukolikova-Nicola et al., 1987; Powell and Gordon, 1987). Genes on the T-DNA code for biosynthesis of the plant growth regulators auxin and cytokinin, and the overexpression of these phytohormones results in loss of division control and a tumorous phenotype (Nester et al., 1984).
Genetic studies have identified several regions outside the T-DNA which are required for T-DNA transfer. A number of constitutively expressed chromosomal loci are required for the binding of Agrobacterium to plant cells (Douglas et al.,
* This work was supported by National Institutes of Health Grant 5 ROI GM 32618-14 and National Science Foundation Grant DMB- 8315826 The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) 50321 6.
$ Supported by American Cancer Society Postdoctoral Fellowship
J Current address: Serogene, Hopkinton, MA 07148. ll Current address: School of Life Sciences, Jawaharlal Nehru Uni-
11 To whom correspondence should be addressed.
PF-2796.
versity, New Delhi, India 110 067.
1985; Cangelosi et al., 1987; Thomashow et al., 1987). Muta- tions in these loci result in a reduced ability of Agrobacterium to attach to plant cells and an attenuated virulence. Another virulence region (uir) spans 35 kbp’ of the pTiA6 plasmid and contains at least six transcriptional loci (uirA, B, C, D, E , and G), designated the uir genes. Mutations in these loci result in either avirulence (uir A, B , D, and G) or attenuated virulence (uirC, E ) (Hille et al., 1984; Klee et al., 1983; Stachel et al., 198513; Stachel and Nester, 1986). The uir genes are organized as a single regulon which is inducible by plant cell phenolic compounds (Stachel et al., 1985a; Bolton et al., 1986) via a positive regulatory system consisting of uirA and uirG (Stachel and Zambryski, 1986b; Winans et al., 1986).
Many of the molecular reactions necessary for processing and transfer of the T-DNA molecule are specified by the uir genes, but the exact functions of individual uir genes are for the most part unknown. The molecular characterization of these genes should facilitate their functional analysis. Toward this end, the nucleotide sequences of the uirA (Leroux et al., 1987), uirC (Yanofsky and Nester, 1986), uirD (Yanofsky et al., 1986; Porter et al., 1987), uirE (Winans et al., 1987), and uirG operons (Winans et al., 1986) from the pTiA6 plasmid recently have all been determined in our laboratory. Here we extend this analysis to include the nucleotide sequence of the largest uir operon, uiri?. Our results show that the pTiA6 uirB operon contains 11 potential genes, most of which appear to code for membrane-associated proteins. Their functions are discussed in relation to our current understanding of the T- DNA transfer process.
EXPERIMENTAL PROCEDURES
Materials-Restriction endonucleases were purchased from New England Biolabs or Bethesda Research Laboratories and used as suggested by the supplier. DNA polymerase I, Klenow fragment, was obtained from either Bethesda Research Laboratories or Boehringer Mannheim; S1 nuclease was from New England Biolabs, and Esch- erichia coli exonuclease 111 and T4 ligase was from Bethesda Research Laboratories. M13 sequencing primer, unlabeled nucleotides, and dideoxynucleotides were purchased from Pharmacia LKB Biotech- nology Inc. 35S-Labeled dATP was from Du Pont-New England Nuclear.
Bacterial Strains and Plasmid-E. coli strains JMlOl and MV1193 were used for plasmid maintenance and for the production of ssDNA template and were propagated on either L agar or M9 minimal agar (Miller, 1972) supplemented with the appropriate antibiotic (carben- icillin, 100 rg/ml; kanamycin, 50 pg/ml). DNA fragments from the uirB region of Ti plasmid pTiA6NC were obtained from the cosmid pVK257 (Knauf and Nester, 1982) and subcloned into the PATH vectors of Spindler et al. (1984) as described in the text. Plasmid pJW207 was constructed by cloning the 1.3-kbp EcoRI fragment 30 into pUC119. The 1.8-kbp EcoRI-BamHI fragment from Sal1 frag-
The abbreviations used are: kbp, kilobase pair; bp, base pair; ORF, open reading frame; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; ssDNA, single-stranded DNA.
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5805
FIG. 1. The pTiA6 uirB operon. A, physical map of uirB. The vertical arrows indicate the positions of Vir- or Vir' Tn3HoHol insertions based upon the mapping data of Stachel and Nester (1986). Note that the previously identified Sal1 fragment 35 actually consists of two SalI fragments, here designated 35a and 35b. The 11 uirB ORFs are shown by the solid lines, and the arrowheads represent the promoters of both uirB and the adjacent uirG operon. B, sequencing strategy of the uirB operon. The horizontal arrows indicate the extent of DNA sequence determined from individual clones. The restriction endonuclease sites used in generating templates for DNA sequence analysis are indicated on the lower horizontal line as follows: B, BamHI; Bg, BglII; E, EcoRI; and S, SalI. The nucleotide sequence of SalI fragment 13a was determined from overlapping clones generated by a random shotgun-cloning procedure and is described under "Experimental Procedures."
ment 12 was inserted into the tac promoter expression vector pKK223-3 (obtained from Pharmacia LKB Biotechnology Inc.) to give pJW301. Plasmid DNA was isolated by the procedure of Birn- boim and Doly (1979).
DNA Sequencing and Protein Analysis-The nucleotide sequence of the uirB operon was determined by the chain termination method of Sanger et al. (1977). Templates were generated by two methods: the random shotgun cloning procedure of Deininger (1983), or by progressive deletions of restriction fragments cloned into pUC118 and pUC119 (kindly provided by J . Vieira, Rutgers University) as described by Guo and Wu (1983) and by Dale et al. (1985). SalI fragment 13a was sequenced using templates generated by the random shotgun cloning procedure. Briefly, Sal1 fragment 13a DNA was gel purified, sheared by sonication, and fragments 300-600 bp in size were eluted from a preparative agarose gel. These DNA fragments were then blunt-end ligated into the SmaI site of M13mp18 (Yanisch- Perron et al., 1985), introduced into JM101, and ssDNA templates were isolated and used for sequencing. The DNA sequence of overlap- ping clones was merged using software from BIONET. The uirB nucleotide sequence from the left end of EcoRI fragment 29 to the BglII site 194 bp upstream of uirG was determined using templates obtained from progressive deletions of uirB restriction fragments. In some cases synthetic oligonucleotide primers were used to prime sequencing reactions. The nucleotide sequences of both strands were determined over the entire 10,200-bp region. SDS-PAGE, maxicell, and in vitro transcription-translation procedures for the analysis of plasmid-encoded proteins were as described previously (Yanofsky et al., 1986; Leroux et al., 1987).
RESULTS AND DISCUSSION
DNA Sequence Analysis of the pTiA6NC uirB Operon- Based upon genetic analysis of Tn3HoHol transposon inser- tion mutants, Stachel and Nester (1986) showed that the pTiA6 uirB complementation group was a single transcrip- tional unit of approximately 9.5 kbp, bordered on the left by insertion 204 within SalI fragment 13a, and on the right by insertion 363 in SalI fragment 13b (Fig. 1). Transcription of uirB is plant-inducible and proceeds in the same direction as the adjacent vi& gene (Stachel and Nester, 1986) (Fig. 1). In addition, the uirB promoter and transcriptional start site have been mapped to the left end of the SalI fragment 13a (Das et al., 1986), supporting the Tn3HoHol mapping data. Since insertion 363 is located in the 5'-end of uirG' (Winans et al., 1986), the uirB and uirG loci are likely to be closely linked. We determined the nucleotide sequence of a 10.2-kbp region from pTiA6 extending from the left SalI site of SalI fragment 13a to the BglII site present 194 bp upstream of the proposed translational start site of uirG (Winans et al., 1986). The sequencing strategy used is outlined in Fig. 1 and described under "Experimental Procedures."
The nucleotide sequence of the pTiA6 uirB operon and the uirB-uirG intergenic region (Winans et al., 1986) are shown
TABLE I Predicted uirB protein molecular weights and charge values
in Fig. 2. The region of 5"nontranscribed uirB sequence previously determined by Das et al. (1986) was identical to ours, including the presumptive -35 (base pairs 644-649, TTTTCG) and -10 (base pairs 666-671, GATAAT) regions of the uirB promoter (underlined in Fig. 2). The two uirB transcriptional start sites previously defined by S1 nuclease protection experiments (Das et al., 1986) are located at the A residues of positions 679 and 681, respectively. The uirB sequence downstream of the transcriptional start sites re- vealed 11 potential ORFs (Fig. 2). The predicted uirB protein molecular weights and charge values are given in Table I.
All of the uirB ORFs are preceded by potential ribosome binding sequences with homology to the consensus ribosome binding site of E. coli (TAAGGAGGTG . . . 5-9 bp . . . ATG) (Shine and Dalgarno, 1974) as shown by the ouerlined bases in Fig. 2. In 7 out of 10 cases either the ribosome binding site or the initiation codon of a uirB gene overlaps the termination codon of the preceeding uirB gene (Fig. 2) , suggesting that these genes are translationally coupled. In three cases, how- ever, the spacing between the punctuation codons of adjacent uirB genes is much greater. The uirB6 and uirB7 genes are 96 base pairs apart, while the spacing between the genes uirB7- uirB8 and uirB10-uirB11 is 33 and 36 base pairs, respectively. We do not know whether the efficiency of transcription through these untranslated regions is affected, especially through the uirB6 and uirB7 intergenic region. There do not appear to be any sequences with dyad symmetry capable of forming a p-independent terminator in these regions (Platt, 1986), although a reduction in transcription by a p-dependent mechanism cannot be ruled out. The uirB6 and uirB7 genes of the nopaline-type Ti plasmid pTiC58 are separated by a similar distance,' suggesting that the spacing between these
R. C. Tait, personal communication.
5806 The Ti Plasmid virB Operon
S a l I GTCGACGATCCGTCTTGCGCTGACAGCGIGTCMGCCCATGACCGCCGCATGGCGAAACATTTTTACAAA
C M T C G C T A A G A G T G C G T T C C T G C T G G C G G A C A A G G C A T G A G C G T T T G C A A A
GCTCGCAAGTGTGCCCCATCAGTTGCGACATCTACACTTGCGGCGATAGCTAAGGTGGAGAGTCGCTTTG A R K C A P S V A T S T L A A I A K V E S R F D
ATCCTTTAGCGATTCATGACAACACCACCGGCGMACGCTTCACTGGCAAGATCACACCCAAGCAACCCA P L R I H D N T T G E T L H U Q D H T Q A T Q
AGTCGTCAGGCACCGTCTCGATGCACGGCATTCGCTGGATGTTGGCCTCATGCAMTAAACTCTCGGAAT Y V R H R L D A R H S L D V G L M Q I N S R N
TTTTCCATGCTCGGTCTGACACCTGACGGTGCGCTCAAGGCGTGCCCATCATTATCTGCCGCTGCAAACA F S M L G L I P D G A L K A C P S L S A A A N M
TGTTGAAI\I\GTCGTTATGCAGGCGGCGAAACGATTGACGAGAAGCAAATTGCACTTCGTCGGGCGATCTC L K S R Y A G G E T I D E K Q I A L R R A I S
C G C T T A C A A C A C C G G T A A T T T C A T C C G C G G T T T T G C A A C A G T T G A A A C A G C T G C T A Y N T G N F I R G F A N G Y V R K V E T A A
CAATCGCTGGTGCCCGCGTTAATCGAGCCTCCGCAAGACGATCACAAGGCACTAAAATCGGAAGATACGl Q S L V P A L I E P P Q D D H K A L K S E D T U
GGGACG7TTGGGGGlCTTATCAGCGCCGCTCGCAAGAGGATGGGGTTGGTGGTTCAATCGCTCCGCAACC D V U G S Y Q R R S Q E D G V G G S I A P Q P
GCCAGACCAGGACMCGGCAAATCCGCAGACGACAATCAAGTCTTATTCGACTTATACIAAGGAGGTCCG P D Q D N G K S A D D N ~ V L F D L Y
CATlGATGCGATGCTTlGAGAGATATCGTTTACATCTAMlCGCCTCTCGCTCTCGAATGCGATGATGCG M R C F E R Y R L H L N R L S L S N A M M R
orf2
CGTGATATCGAGCTGCGCCCCAAGCTTGGGCGGTGCAATGGCATGGAGCATTTCCTCATGCGGACCCGCC V I S S C A P S L G G A M A U S I S S C G P A
GCAGCGCAATCTGCGGGTGGCGGCACCGACCCCGCCACAATGGTTAACAATATATGCACGlTTATCCTTG A A Q S A G G G T D P A T H Y N N I C T F I L G
GTCCGTTCGGCCAGTCACTCGCCGTTCTCGGCATTGTCGCTATCGGGATCTCCTGGATGTTCGGGCGGCG P F G Q S L A V L G I V A I G I S U M F G R R
TTCGCTTGGGCTGGTTGCCGGCGTCGTCGGCGGCATTGTTATCATGTTTGGGGCGAGCTTCCTCGGCCAA S L G L V A G V V G G I V I M F G A S F L G Q
CCTGGCGCAATCACTTCACGCAATlTCGCGGGGlTTGTCTCTTTCGAAAACTTTCCAGAGGGCGCCAGCT P G A I T S R N F A G F V S F E N F P E G A S S
CAGGCCACTGGGGCACCGCGATTGCCCGATTTCGTACCAATGGCGGAACGCCTTTCGACTATATCCCGCA G H W G T A I A R F R T N G G T P F D Y I P H
TGAGCACGATGTTGGCATGACGGCAATATTCGGGCCTATCGGGAGGGGTAAGACAACGCTCATGATGTTT E H D V G M T A I F G P I G R G K T T L M M F
Sal I
V L A M L E Q S M V D R A G T V V F F D K D R G GTTTTAGCCATGCTCGMCAGAGCATGGTCGACCGTGCAGGTACGGTCGTGTTCTTCGACMGGACCGGG
GTGGCGAATTGCTGGTTCGCGCCACAGGAGGAACATATTTGGCACTTCGCAGAGGCACACCCAGCGGGTT G E L L V R A T i G T Y L A L R R G T P S G L
GGCGCCGTTGCGTGGCCTAGAAMCACAGCAGCCTCACACGATTTTCTGCGCGAATGGATCGTGGCTCTC A P L R G L E N T A A S H D F L R E Y I V A L
ATAGAGAGTGATGGTCGGGGTGGGATTTCTCCAGAAGAGAACCGCCGTCTGGTCCGGGGTATCCATCGTC I E S D G R G G I S P E E N R R L V R G I H R Q
AGCTCTCGTlTGATCCACAAATGCGTTCAATCGCGGGGTTACGTGAAlTTTlGTTGCATGGCCCTGCCGA L S F D P Q M R S I A G L R E F L L H G P A E
ACGGCAGGAGCGGCTCCAACGCTGGTGCCGGGGCCATGCGCTTGGTTGGGCATTTGACGGCGMGTTGAC R Q E R L Q R W C R G H A L G U A F D G E V D
GAAGTAAAGTTAGATCCGTCGATTACCGGCTTCGACATGACGCATCTTCTCGMTACGAGGMGTATGCG E V K L D P S I T G F D M T H L L E Y E E V C A
CTCCCGCTGCAGCATATCTCCTGCATCGGATTGGCGCCATGATCGACGGCCGCCGTTTTGTGATGAGCIG P A A A Y L L H R I G A M I D G R R F V H S C
S a l I CGATGAGTTTCGCGCCTATTTGTlAAACCCTAAATTTTCGGCCGTCGTCGACAPJlTlCCTCCTGACCGTT
D E F R A Y L L N P K F S A V V D K F L L T V
CGAAAAAACAACGGGATGCTAATACTGGCAACGCAGCAACCAGAGCATGTTCTGGMTCGCCACTAGGAG R K N N G M L I L A T Q Q P E H V L E S P L G A
CCAGCTTGGTTGCGCAATGTATGACGAAGATTTTCTATCCATCACCAACCGCAGATCGATCAGCTTATAT S L V A Q C M T K I F Y P S P T A D R S A Y I
CGATGGACTGAAATGCACCGAAAAGGAATTTCAGGCGATCCGIGAAGACATGACGGTCGGCAGCCGTMG D G L K C T E K E F Q A I R E D M T V G S R K
TTTCTTCTTAAACGAGAAAGTGGAAGCGTCATCTGCGMTTTGATCTGCGGGATATGCGTGMTATGTCG F L L K R E S G S V I C E F D L R D M R E Y V A
FIG. 2. Nucleotide sequence of the uirB operon and the uirB-uirG intergenic region. The beginning of each uirB ORF is indicated along with the predicted amino acid sequence of the gene. The putative uirB promoter sequences are underlined while potential ribosome binding sites are indicated by lines drawn over the DNA sequence. The nucleotide sequence of the extreme uirB 3’-end and the uirB-uirG intergenic region, beginning at the BglII site at nucleotide 10,200, is from Winans et al. (1986).
2380
2450
2520
2590
2660
2730
2800
2870
2940
3010
3080
3150
3220
3290
3360
3430
3500
3570
3640
3710
3780
3850
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3990
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4410
genes has been conserved. It is possible, therefore, that the uirB locus of pTiA6 is transcribed as an operon from the intergenic space between uirB6 and uirB7 has a functional single 5“plant-inducible promoter shown in Fig. 2. Plant- significance, perhaps modulating transcription through this inducible expression of lac2 from the Tn3HoHol transposon region and thus decreasing expression of the distal virB genes. insertion 368, which our sequence data appears to map in the
Stachel and Nester (1986) have presented evidence that the virBll gene, required all of the 5”upstream virB sequences
T h e T i Plasmid virB Operon 5807
C C G T A C I T I C G G G G C G T G C C A A C A C G G T G C G C ~ ~ T G C A G C T C G A C T A C G C G A G G C A C A A G A A G G C A A C T C v L S ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ a ~ ~ ~ ~
~ ~ C T G G C ~ G G C T C A G C G A A T l C A T G G C C C G l C A C C A C 4 P G G C A G A A G A T l G A i ~ A ~ A A A C G A T G ~ M K
A ~ A C G A ~ G C A A C T T A T I G C A A C G G T ~ T T G A C C T G C A G C T T T C T A T A T A ~ T C A G C C C G C G C G G G C G C A G ~ T ~ ~ ~ L I A T v L I C S F L Y I ~ P A R A O P
l ~ ~ ~ G ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ c ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ G C T C G C G A C G G C G C T C G C G A C T G C G G A G A A l C T C A C ~
C A ~ A C ~ A ~ A G C G A T G G T I A C G A T G T T G A C G T C G G C C ~ A C G G C G ~ T A C ~ G G A C T A C T G A C ~ T C G C T C A A C C
EcoRl
S G ~ L S E F H A R H H E A E O ” orf6
~ ~ s D ~ A T E A E T L A T A L A T A E N L T
~ ~ I A ~ V T H L T S A Y G V T G L L T S L N Q
A G A A A A A I C A G T A T C C T T C A A C G A A G G A C C I A G A C M T G A A A T G T T T T C G C C G C G A A ~ G C C A A T G T C G A C K N Q ~ P S T K D L D N L M F S P R ~ P ~ ~ ~
CACGGCACGTGCGATCACCAGCGATACAGATCGTGCCG~CGTGGGTAGCGACGCTGAAGCGGATC~GT~G ~ ~ R A ~ ~ S D T D R A V V G S O A E ~ D L L
GCTTGACGGCGAATGCTGATACGTCTGCACAGCTT~CCCGATCTCGCAATATCATGCAGGCAACCGTGAC L T A N A D T S A ~ L S R S R N I H Q A ~ V ~
C A A T G G T T T G C T T C T C A A G C A G A I C C A T G A C G C A A T G A T T C A A A A C G T A C A G G C G A C A A A C C ~ A T ~ A A C G N G L L L K Q I H D A P I ~ N V Q A T N L L ~
ATGGCTACCGCGCACGCCGGCCTTCACGAGGCGGAAGAGGCGGCCGC~CAACG~AAGGAGCATCAGAAGA M A T A Q A G L H E A E E A A A O R K E H ~ K T
C C G C T G T C A T C T T T G G T G C C C ~ C C C C ~ A A G G C ~ G G G C G A T T T G T ~ C A T C C G C C C A ~ A T C C ~ C A C C G A A T G
C G A G C I C A T T G T A T C C G A C A T T C T G C G A C A A G C C A G C C A A G T T C ~ C C A A T C G A T G A A ~ ~ T C A C G A T ~
C C G G C G C C G T T T A C G G C C A T T C A ~ A C G A T C ~ ~ C G A T G T A G C C T T C A C G A C A G G C T T G G A C ~ C G A T G C T T G
AGACTAlCCAGGAGGCGGTGAGTGCGCCATTGATCGCCTGTGTCACTCTlTGGATTATTGTlCAGGGTAT ~ I ~ E A V S A P L I A C V T L Y I I V O G I
S a i I
R s ~ I T G S A N S A G I A A O N L E T M D K R
A V I F G A L P
M N F T I orf?
P A P F T A I H T I ~ O V A F T I G L D S M L E
l T T A G T C A l A C G C G G C G A A G l C G A l A C C C G C A G C G G T A T C A C T C G G G T G A T C A C G G l C A C C A T C G l T G T T L V I R C L V D T R S G I T R V I T V T I V V
GCTCTAAlTGTTGGGCAGGCTAAClACCAAGACTATGTGGTTTCCAlCTTCGAAAAGACGGTCCCAATCT A L I V G ~ A N Y ~ D Y V V S I F E K T V P I F
P s t I T T G I T C A G C A G T T l A G l G l A A C A G G C T T G C C T C l G C A G A C l G T T C C G G C A C A G T T G G A T A C A A l T T T C G C
V ~ ~ F S V T G L P L ~ ~ V P A ~ L D ~ I F A
V T ~ A V F ~ K I A S E I G P M N D Q D I ~ A C G T G A C C C A G G C C G T l T T l C A G A A A A T C G C A T C C G A A A l C G G T C C G A l G A A C G A C C A G G A C A T C C l T G C l
T l C C A A G G G G C A C A G l G G G T C C T l l A C G G C A C G C T C T G G l C T G C C T T C G G A G T C T A C G A C G C C G T T G G A A E c o R I
F Q G A ~ U V L Y G T L Y S A F G V Y D A V G I
T T C T C A C G R A A G T G C T T C T C G C G A T C G G G C C T C T G A T l C T T G l C G G A l A T A T l T T l G A l C G C A C G C G G G A L T K V L L A l G P L l L V G Y l F D R l R D
CATCGCAGCGAAGTGGATCGGGCAACTTATCACCATCGGTClCllGCTTCTCCTCTTAAACCTCGTGGCA ~ A A K U I G ~ ~ I ~ I G ~ L L L L L N L V A
ACGAlCGTCAlCCTAACCGAAGCGACTGCGCTCACCCTCATGClTGGTGTAATCACCTTlGCCGGTACGA T l V l L T E A T A L T L P L G V I T F A G T T
C C G C G G C C A A G A T C A T T G G T C l T T A C G ~ C T C G A T A T G l T T T T T C l G A C A G G G G A l G C G C T C A T l G T C G C A A K I I G L Y E L D M F F L T G D A L I V A
T l T G C C G G C A A T C G C C G G C M ~ A l T G G A G G C A G T l A C T G G A G C G G C G C A A C C C A A l C T G C C A G C A G C T T G ~ P A I A G N I G G S Y U S G A T ~ S A S S L
T A C C G T C G C T T C G C T C A G G T T G A A C G G G G C T A G G T C G C G C A A A A A T T C G C C T C A A T ~ G A A T T C T A I G A Y R R F A Q V E R G ’ M K
Y C L L C L V V A L S G C ~ T N D T I A S C K
G P I F P L N V G R Y ~ P T P S D L Q L R N S
G G T G G A C G C T A T G A C G G G G C C ~ ~ T A ~ G C C A T G C T A G T G G C G C G C G A A A G C C T G G C C G A G C A C T A T A A G
EcoRl
A A T A l T G C C T G C T G T G C C l A G T l G l C G C T l l G A G C G G C T G C G A G C T G C A A orf8
CGGClAlCGCGATllTGGGGAAlGTTGCTCAAGCGTTCGCTAlAGClACCATGGlCCCGTlGAGCAGGCT A I A I L G N V A Q A F A I A T M V P L S R L
T G T G C C C G T A l A T C T A l G G A l A C G C G C G G A C G G C A C C G T T G A C A G C G A G G T G T C T A l C l C G C G A T l G C C T V P V Y L Y I R A D G I V D S I V S I S R L P
GCAACTCAAGAGGAGGCCGTCGlTAACGCTTCAlTGTGGGAGTACGlTCGCClGCGlGAGAGllATCATG A I Q E E A V V N A S L Y E Y V R L R E S Y D A
CCGATACCGCTCAGTACGCCTACGACCTAGTATCGAACTTCAGIGCCCCAACAGIGCGCCAGGATTACCA D T A ~ Y A Y D L V S N F S A P T V R ~ D Y O
GCAATTClTCAACTATCCCAATCCCAGTTCGCClCAAGlCATTCTTGGCAAACGCGGCAGGGTGGAGGTC Q F f N Y P N P S S P O V I L G K R G R V E V
GAGCACATCGCTTCAAATGATGlAACTCCAAGCACGCAGCAAATTCGClATAAAAGGACCCTCGlCGTlG E H I A ~ N D V ~ P ~ T Q ~ I R Y K R ~ L V V D
A C G G C A A A A T G C C l G l G G T G A G T A C G T G G A C C G C G A C A G l T C G C T A C G A A A A G G T G A C C A G C l l G C C C G G G K M P V V S T Y T A T V R Y E K V T S L P G
CAGATlGAGAllGACCAACCCGGCAGGTCTGGlTGTCACCTCClAlCAGACClCGGAAGATACCGlTTCA R L R L T N P A G L V V T S Y O T S E D T V S
A A C G T A G G C C ~ G C G C A C C A T G A C G A G A A A A G C A C T T T T C A T T T T A G C A T G T T T A T T T G C C G C T G C G A C
N V G Q G A P M * T R K A L F I L A C L F A A A l
orf10 l G G T G C G G A G G C T G A A G A C A C T C C A A l G G C C G G C A A G C l A G A T C C A C G C A T G C G l T A T T l G G C l T A C A A l
G A E A E D T P M A G K L D P R M R Y L A Y N
CCCGATCAAGTCGTGCGCCTCTCCACGACGGCGGTTGGAGCTACITTGGTCGTCACATTCGCCACGAACGAAA P D ~ V V R L S T A V G A T L V V I F A T N E T
C G G T G A C A l C G G T l G C C G T T T C A A A T A G C A A A G A T C l A G C A G C C C T A C C G C G G G G A A A T T A T C T A T T C l T V T S V A V S N S K D L A A L P R G N Y L F F
CAAGGCAAGCCAGGlCClCACGCClCAGCCAGTAATCGlGClAACCCAAGCGAClCCGGGATGCGCCGTT K A S Q V L T P Q P V I V L T Q A I P G C A V
A T G T l T C A G T A T A A G T C C A A G A C G C l G T C A C A C C l C G A T A A A G A G C A G C C C G A T C T C T A T T A C A G C G l C C
B g i I I
M F ~ Y K S K T L S H L D K E Q P D ~ Y Y S V ~
AATTCGCCTACCCCGCCGATGCGGCTCGGCGAAGGGAGGCACAACAGAGGGCTGlTGlGGACAGAClGCA F A Y P A D A A R R R E A Q Q R A V V D R L n
CGCGGAACGAACAAATAATCAACGGAAAGCTGAGGATllATTGGATCAGCCTGlCACAGCCCTTGGTGCG A E R T N N Q R K A E D L L D ~ P V T A L G A
T D S N Y H Y V A ~ G D R S L L P L E V F D N G ACGGACAGlAATTGGCACTACGICGCCCAAGGCGAlCGTlCGClGTTGCCACTCGAAGTCTlCGACAACG
G A T l T A C G A C G G T A l T C C A C l T l C C G G G C M l G l A C G C A l A C C C l C C A l C T A C A C C A T C A A T C C T G A C G G F T T V F H F P G N V R I P S I Y T I N P D G
C A A G G A A G C T G T C G C C A A C l A T T C A G l C A A A G G G A G C G A C G l C G A ~ A T T l C l l C G G l T T C A C G A G G T l G G K E A V A N Y S V K G S O V E I S S V S R G U
C G T C T G A G G G A T G G C C A C A C A G T A C T A l G T A T C T G G M l G C C G C l l A C G A T C C C G T T C G G C C A A A G G C C G R L R O G H T V L C I Y N A A Y D P V R P K A A
N G H R E A R C E T R P E G G K G H N N D S Q C A A A C G G G C A C C G l G A G G C C C G A T G T G A A A C G C G T C C l G A A G G G G G C A A A G G G A T G A A T A A C G A l A G l C A
8amHI GCAAGCGGCACATGAGGTTGAlGCATClGGAlCCCTGGlCTCCGACAMCATCGCCGGCGlCT l lCGGGG
O A A H E V O A S G S L V S D K H R R R L S G
S ~ K L I V G G V V L A L S L S L I Y L G G R ~
K K V N D N A S P S T L I A A N T K P F H P A
T C T C A G A A A T T G A T C G T C G G A G G T G I C G T T C T C G C G T l A T C G T T A A G C C T C A T T T G G C l A G G l G G G C G T C
A A A A G A A G G T G A A T G A C A A C G C A T C G C C G T C A A C T T T G A T C G C A G C A A A C A C T A A G C C A T T T C A l C C A G C
l C C C A l T G A G G l G C C G C C G G A T A C l C C A G C G G T T C A A G A G G C l G l T C A G C C T A C l G T T C C T C A A C C G C C A P I E V P P D T P A V Q E A V ~ P T V P Q P P
AGGGGCGAGCCGGAGCGCATGAGCCACGGCCGGAAGAAACACCGATTlTTGCAlATAGCAGTGGCGAlCA R G E P E R P S H G R K K H R F L H I A V A I K
AGGGGTCAGCAAGCGCGCCAGTCAGGGCGACATGGGCCGAAGACAAGAAGACAAGCGTGACGACAACTCC G S A S A P V R A T Y A L D K K T S V T T T P
l T G C C G A A T G C G A A G 7 G T C C G G C G A G A A C G A T T l G l C G A T A C G T A T G A A A C C C A C C G A G C l G C A G C C C A G C R M R S Y R R E R F V D T Y E T H R A A A Q
CAGGGCACGCTCllGCCGCACCCCGATTTTATGGlAACGCAAGGGACAAlAATlCCGlGCAlCCTGCAAA Q G ~ L L P H P D F M V T Q G T I I P C I ~ Q T
C C G C A A T C G A C A C A A A T l T G G C A G G C l A l G T A A A G T G l G T C T T G C C T C A G G A T A T l C G T G G A A C A A C G A A A I D T N L A G Y V K C V L P Q D I R G I T N
CAATATCGTGCllCTlGATCGlGGCACCACCGTlGTlGGCGAAAlACAGCGTGGCllGCAACAGGGAGAl N I V L L D R G I I V V G E I ~ R G L ~ ~ G D
6580
6650
6720
6790
6860
6930
1000
7070
7140
?210
7280
7350
7420
7490
7560
7630
7700
7770
7840
7910
7980
8050
8120
8190
8260
8330
8400
8470
8540
8610
8680
FIG. 2-continued
present in cis. Thus, there do not appear to be any internal promoters within the pTiA6 uirB operon. I t is interesting that the termination codon of the uirBll gene is located only 48 base pairs upstream of the predicted -35 sequence (base pairs 10,313 to 10,319, TTGTCAT) of the uirG promoter (Das et al., 1986) and 131 base pairs upstream of the predicted uirG initiation codon (Winans et al., 1986) (Fig. 2). There are no obvious transcriptional terminators in this intergenic region, and thus it is possible that transcription initiating at the uirB promoter might also contribute to uirG expression.
In their genetic analysis of the pTiA6 uirB locus, Stachel
and Nester (1986) found that a strain with a Tn3HoHol transposon insertion in the 3’-end of uirB (insertion 41) was still virulent on Kalanchoe daigremontiana. However, three additional avirulent uirB mutants contained transposon in- sertions which mapped to the right of insertion 41. The authors postulated that insertion 41 was present within an untranslated intercistronic region of virB and was nonpolar for downstream uirB gene transcription. In support of this interpretation, we used our virB sequence data, along with restriction mapping of the virB 3’-region DNA from the insertion 41 mutant, to localize the transposon insertion to
5808 The Ti Plasmid virB Operon
GAGCGCGTTTTTGTGTTGTGGGATCGCGCCGAGACCCCTGACCATGCGATGATCTCGTTAACATCGCCAP E R V F V L Y D R A E T P D H A M I S L T S P S
Sal I GCGCGGACGAACTCGGTCGCCCAGGATTGCCGGGCTCGGTCGACAGCCACTTCTGGCAGCGTTTTAGCGG
A D E L G R P G L P G S V D S H F U Q R F S G
AGCCATGCTCTTGAGTGCTGTTCAAGGCGCCTTCCAGGCAGCTAGCACCTACGCTGGCAGCTCGGGTGGC A H L L S A V Q C A F Q A A S T Y A G S S G G
GGGATGAGCTTCIV\CAGCTTTCAAAATIV\CGGTGAACAAACAACTGAGACAGCCCTTMGGCGACCATCA G H S F N S F P N N G E Q T T E T A L K A T I N
ACATACCGCCAACCCTGAAGAAGAATCAGGGTGACACGGTTTCCATTTTCGTGGCACGGGACCTCGATTT I P P T L K K N Q G D T V S I F V A R D L D F
within either the extreme 3'-end of the uirBl0 gene or the intergenic region between uirBl0 and uirBll (data not shown).
uirB Protein Analysis-In order to confirm the ORFs pre- dicted by the uirB nucleotide sequence, a series of plasmids was constructed, each containing a segment of uirB coding sequence joined in frame to the NHz-terminal 35 kDa of the E. coli trpE gene, using the PATH vectors of Spindler et al. (1984). These vectors contain the strong inducible trp pro- moter and a modified trpE gene containing a polylinker cloning region in the 3'-end, which allows the insertion of DNA fragments in the same reading frame as trpE. This allows one to predict the size of the corresponding protein encoded by the gene fusion. Various restriction fragments from the uirB region were subcloned (Fig. 3A), and the trp promoter was induced which resulted in the overexpression of the trpE:uirB fusion proteins in E. coli. The similarity of the predicted and actual fusion protein molecular weight as determined by SDS-PAGE confirmed the uirB ORFs.
Fig. 3B shows the results of this analysis. Proteins from cells containing the PATH1 vector alone showed a 37-kDa TrpE protein (translation of the trpE gene up to the poly- linker site of the vector gives a 35-kDa protein) only upon induction with 3P-indole acrylic acid (Fig. 3B, lanes 1 and 2). Plasmid pJW243 contains a 0.93-kbp Hind111 fragment cloned into the pATH2 vector to create a trpE.:uirB2 fusion. Upon induction, this plasmid resulted in an estimated 42-kDa fusion protein (lane 3) , which compares well with the predicted M, of about 43,000 (35,000 for TrpE' + 7,900 for 'VirB2) and supports the sequence data for the uirB2 gene. Similarly, pJW291 contains a fusion gene consisting of trpE joined near to the amino terminus of uirB4 but missing the uirB4 car- boxyl-terminal end. A polypeptide close to the predicted size of 51 kDa (lane 4 ) was produced. The slightly smaller protein also present within induced pJW291-containing cells may be a breakdown product of the TrpE-VirB4 peptide. Lane 5 shows the predicted 96-kDa protein produced by pJW223, which contains trpE fused to the amino-terminal end of the
The Ti Plasmid virB Operon 5809
pJW223-
pJw275 pATHlO
pJW279 pATH11 -
p J W259
pJW269
pJW293
pATH2
p A T H 3
pJ W207 puc119 Plat*
pJW301 pKK223-3 P,&
n
-.a 4 hl C . .
- 18 ' - C U
FIG. 3. Analysis of VirB proteins. A, the plasmids used to analyze the uirB protein products. The location of the uirB genes and the restriction endonuclease sites used to subclone the various uirB fragments are indicated on the top line as: B, BamHI; Bg, BglII; E, EcoRI; H, HindIII; P, PstI; and S, SalI. The open rectangles represent uirB sequences, the lines represent vector sequences, and the trpE gene of the PATH vectors is shown by a heavy solid line. The arrowheads indicate direction of transcription. B, proteins from uninduced pATH1-containing cells (lane 1 ) and 3B-indole acrylic acid-induced cells containing either the pATHl vector (lane 2 ) or the trpE::uirB plasmid pJW243 (uirB2, lane 3), pJW291 (uirB4, lone 4 , pJW223 (virB5, lane 5), pJW275 (uirB6, lane 6), pJW279 (uirB10, lane 7), or pJW259 (uirB11, lane 8) were separated by SDS-PAGE with a 10% gel and stained with Coomassie Brilliant Blue. Lysates from uninduced (lane IO) and induced (lane 11) pJW269 (trpE::uirB7)-containing cells were prepared by boiling for 5 min in SDS sample buffer and analyzed by SDS-PAGE with a 12.5% gel. Lane 12 shows an induced pJW269 cell lysate prepared by first heating the cells a t 65 "C for 5 min prior to boiling in SDS sample buffer for 5 min. Lanes 9 and 13 correspond to molecular weight markers. C, plasmid-encoded proteins were labeled with ["S]methionine using a coupled in uitro transcription-translation system, separated by SDS- PAGE through a 12.5% gel, and visualized by autoradiography. The plasmids analyzed were PATH1 (lanes 2 and 5), pJW223 (lane 3), pJW293 (trpE::uirBB, lane 4 , pUC119 (lane 6 ) , and pJW207 (lane 7). A no plasmid DNA control reaction is shown in lane 1. Proteins expressed by plasmid pJW301 (lanes 10 and 11) and by its parental vector pKK223-3 (lanes 8 and 9) were [''S]methionine-labeled in E. coli maxicells with (lanes 9 and 11) and without (lanes 8 and 10) isopropyl-1-thio-P-D-galactopyranoside-induction of the tac promoter and analyzed by SDS-PAGE using a 15% gel. The various TrpE-VirB fusion proteins are designated by small dots, and the 8-lactamase, VirB6, and VirB9 proteins are indicated. The molecular weight standards (in kDa) used were myosin (H chain) (200), phosphorylase b (97.4), bovine serum albumin (68), ovalbumin (43). a-chymotrypsinogen (25.5). 0-lactoglobulin (18.4). and lysozyme (14.3).
5810 The Ti Plasmid virB Operon
uirB5 gene. Plasmid pJW275 contains a fusion to uirB6 that encoded the expected 49-kDa protein (lane 6). The predicted 70,000-dalton fusion protein containing most of the carboxyl half of VirBlO was produced in cells containing pJW279 (lane 7). In addition, a lac2::uirBlO fusion was constructed in pUC8, which consisted of the lac promoter and amino-terminal end of lac2 from the vector joined in frame to the carboxyl end of the uirBl0 gene at the SalI site. Upon isopropyl-1-thio$-& galactopyranoside induction, this plasmid overproduced a 12- kDa fusion protein (data not shown), in close agreement with the size predicted from the nucleotide sequence of virB10. Together with the data from pJW279, this confirmed the last half of the uirBl0 ORF. A fusion of trpE to the fourth codon of the virBll gene in pJW259 encoded a protein with the predicted size of 70 kDa (lane 8), confirming the last virB ORF.
Plasmid pJW269 contains the carboxyl two-thirds of virB7 (186 codons) joined to trpE at the PstI site of the vector. However, following electrophoresis of induced pJW269-con- taining cell lysates, prepared by boiling the cells in SDS sample buffer for 5 min, the expected 55-kDa fusion protein was not observed (Fig. 3B, lanes 10 and 11). Instead, a large amount of Coomassie Blue staining material was present at the top of the gel (lane 1 I ) , suggesting the formation of protein aggregates. However, we found that if the cells were first heated a t 65 "C for 5 min in SDS sample buffer containing 6 M urea and then boiled, the aggregates were replaced by a new protein band, which migrated at approximately 50 kDa (lane 12), somewhat smaller than the predicted fusion protein size. Similar results were obtained if the induced pJW269-contain- ing cells were lysed in SDS sample buffer but not boiled prior to electrophoresis (data not shown). Other bacterial proteins have been observed with similar properties (see below). Fi- nally, plasmid pJW293 contains trpE fused to the virB8 gene at the EcoRI site overlapping the uirB8 ribosome binding site (Fig. 2), thus adding two amino acids to the virB8 reading frame and predicting a fusion protein of about 41 kDa. This TrpE-VirB8 peptide was observed when a coupled in vitro transcription-translation system was used to analyze pJW293-encoded proteins (Fig. 3C, lane 4 ) but was difficult to visualize from whole cell extracts, possibly due to the pJW293-containing cells rapidly losing viability following in- duction. All of the other TrpE-VirB fusion proteins identified from whole cell lysates were also visualized when plasmid- encoded proteins were analyzed using either E. coli maxicells or in the in vitro transcription-translation system (data not shown).
When pJW223-encoded proteins were examined using the in vitro transcription-translation system, a band migrating at about 24 kDa was observed in addition to the 96-kDa TrpE- VirB5 fusion peptide (Fig. 3C, lane 3). This new band probably represents the native VirB6 protein which has a predicted M , of 23,569 (Table I). This possibility was confirmed by placing the virB6 gene downstream of the lac promoter in the vector pUC119 to give plasmid pJW207. E. coli maxicells containing pJW207 synthesized the 24-kDa VirB6 protein following Lac induction (Fig. 3C, lane 7).
In summary, the trpE::virB gene fusions confirmed most of the reading frames and the end points of the predicted uirB2, 5,6, 7,8,10, and 11 genes, and also confirmed a large internal portion of the predicted virB4 ORF. In addition, the native VirB6 protein visualized in E. coli maxicells was close to the size predicted from the nucleotide sequence of the gene. Identification of the native VirB9 protein is described below. So far only the VirBl and VirB3 protefns have not been visualized.
virB Codon Usage Analysis-The nucleotide sequence of the virB locus doubles the number of pTiA6 vir gene sequences which have been determined to date. Thus, it was of interest to examine the codon usage of the virB genes with the other vir genes and with E. coli genes. A smaller number of vir genes was recently similarly analyzed (Winans et al., 1987). Table I1 lists the frequency at which all 64 codons were utilized, comparing the 11 virB genes to (i) 19 pTiA6 vir genes (the virB genes; uirA, Leroux et al., 1987; uirG, Winans et al., 1986; virCl and virC2, Yanofsky and Nester, 1986; virDl and uirD2, Yanofsky et al., 1986; virEl and uirE2, Winans et al., 1987) plus the virA gene from pTiAgl62 (Leroux et al., 1987) and (ii) a published group of 25 E. coli nonregulatory proteins (Konigsberg and Godson, 1983). Overall, the codon usage of the virB genes was very similar to that of the other Agrobac- terium uir genes (Winans et al., 1987) and was much less biased than observed in E. coli.
Hydrophobicity and Signal Sequence Analysis of the virB Proteins-The functions of the uirB gene products are un- known. We searched the Protein Information Resource pro- tein data base of the National Biomedical Research Founda- tion for proteins homologous to the uirB proteins using the FASTP program (Lipman and Pearson, 1985), but no signif- icant homologies were detected. An increasing amount of evidence suggests that the vir genes are functionally analogous to the tra genes of conjugative plasmids (for review see Stachel and Zambryski, 1986a), for which the F plasmid transfer system is the paradigm (reviewed in Ippen-Ihler and Minkley, 1986; and Willetts and Skurry, 1987). Most of the F plasmid tra proteins are localized in the E. coli cell envelope (Willetts and Skurry, 1987), and some of the uirB proteins might also be membrane-associated. We used the algorithm of Kyte and Doolittle (1982) to search for regions of hydrophobicity within the predicted VirB proteins indicative of signal sequences common to many exported proteins. The VirB1, VirB5, VirB6, VirB9, and VirBlO protein sequences all contain a region of hydrophobicity at their amino-terminal ends followed by a rather even distribution of hydrophobic and hydrophilic se- quences (Fig. 2 and data not shown). The two small proteins VirB2 and VirB3 both contain a hydrophilic amino terminus but are very hydrophobic in nature overall, similar to the VirB7 protein, which is strikingly hydrophobic throughout. The smallest virB protein, VirB8, contains a very hydrophobic domain at the amino-terminal end and a very hydrophilic domain at the carboxyl-terminal end. Finally, both the VirB4 and VirBll proteins appear to be somewhat hydrophilic in nature with no obvious hydrophobic regions indicative of a signal sequence.
The above data suggest that many of the virB proteins contain regions which might specify a signal sequence or membrane-spanning domain. Therefore, we examined the predicted amino acid sequences in these regions to see if they resemble the secretion signals of other exported proteins. Bacterial signal sequences usually consist of three general domains (Oliver, 1985; Briggs and Gierasch, 1986; Sjostrom et al., 1987): (i) a short hydrophilic amino terminus containing one or more basic amino acids; (ii) a middle hydrophobic core containing 12-20 uncharged amino acids; and (iii) a more polar carboxyl terminus of 4-6 amino acids which are involved in signal peptidase recognition. The -3 and -1 positions relative to the cleavage site are conserved and most frequently contain alanine residues, although other hydrophobic amino acids with small uncharged side chains may be found (Perl- man and Halvorson, 1983; von Heijne, 1986). There appears to be less specificity at the -2 position, and a helix-breaking amino acid, usually a proline or glycine residue, is often
T h e T i Plasm id virB Operon 5811
present at the end of the hydrophobic core at -4 or -5 (Perlman and Halvorson, 1983). These observations have been used to develop statistical “rules” for identifying poten- tial signal sequences and predicting cleavage sites (von Heijne, 1986). Using these criteria we examined the predicted VirB proteins for secretion signals.
Fig. 4 shows that signal peptide-like sequences are present at the NH2-terminal ends of the VirB1, VirB5, VirB6, VirB8, and VirBlO proteins. The amino-terminal sequence of VirB1 consists of two basic residues followed by an 18-amino acid hydrophobic core, which ends with a proline residue located at -5 relative to the cleavage sequence Ser-Ser-AlaZ7 (Perlman and Halvorson, 1983). However, the statistical method of von Heijne (1986) predicts that cleavage would more likely occur 5 residues upstream at the sequence Ser-Leu-Ala’l. Both of these potential leader peptides are typical in size to those of many E. coli outer membrane proteins (Sjostrom et al., 1987). The method of von Heijne (1986) supports the predicted VirBlO amino-terminal signal sequence and predicts that cleavage of the leader peptide would occur after the sequence Ala-Glu-Ala”, as shown in Fig. 4. The VirBlO protein also contains an internal hydrophobic sequence followed by a highly positively charged region (Fig. 4). Such sequences are believed to function as “stop-transfer” signals which, after initiation of secretion by an amino-terminal signal sequence, halt passage of the polypeptide through the cytoplasmic mem- brane (Wickner and Lodish, 1985; Briggs and Gierasch, 1986). The resulting transmembrane protein contains both cyto-
Viral M F K R C S L S L A L M S S F C S S S L A T P L 3 3 A A E tl
+ t
V i r B Z R V I S S C A P S L G G A M A W S I S S C C P A A A , Q t23 t 4 8
t
+ I 3 * 9 2 S L A V L C I V A I C I S W M F G R R
* +
V i r B 3 R P A L F L G V P L T L A G L F M M F A G F V I V I V Q N t 1 6
t
P L Y E V L A P L F G A R t
t 5 9
V i r B S M T G R F T P V P V V S C S L C A S I Y T D R V I C G K R t t + +
t 1
VirBG M K T T Q L I A T V L T C S F L Y I Q P A R A Q + I
+ t
V i r B 8 M K Y C L L C L V V A L S G A C + 1
t
t31 V i r B 9 K I I A A V A A I A I L G N V A Q A A F
t 4 9
Viral0 H T R K A L F I L A C L F A A A T G A E A A E t l t22
+ + “
t285 K H R R R L S ~ S Q K - + * t + t t
t319 W L G G R Q K K
t + + FIG. 4. Potential secretion signals of the VirB proteins. The
location of signal sequence-like regions and possible stop-transfer sequences in eight VirB proteins are shown. The underlined residues represent a hydrophobic core, and charged residues are indicated. The arrowheads indicate predicted signal peptidase cleavage sites (see text).
5812 The Ti Plasmid virB Operon
plasmic and periplasmic domains which are connected and anchored in the inner membrane by the stop-transfer hydro- phobic region. Preliminary results using antisera to the VirBlO protein suggest that VirBlO is indeed localized to the bacterial inner membrane.3
The method of von Heijne (1986) predicts that neither the VirB5 or VirB6 potential leader peptide would be processed. The VirB5 sequence does not contain an obvious cleavage site while the arginine residue present at the -2 position of the possible VirB6 cleavage sequence Ala-Arg-AlaZ3 (Fig. 4) is not found in over 40 published bacterial signal sequences (Mi- chaelis and Beckwith, 1982; Watson, 1984; von Heijne, 1986; Oliver, 1985). Furthermore, we did not observe processing of VirB6 in E. coli maxicells (data not shown). Likewise, even though the VirB3 protein contains an amino-terminal seg- ment of 28 hydrophobic residues, there is no obvious cleavage site (Fig. 4). Many intrinsic membrane proteins of E. coli do not contain a processed leader peptide. Instead, hydrophobic domains are thought to enter the lipid bilayer and anchor these proteins in the membrane (Owen and Kaback, 1979; Wolfe et al., 1983; Dalbey and Wickner, 1986; Briggs and Gierasch, 1986). Thus, even if they lack a processing site, the amino-terminal hydrophobic regions of the VirB3, VirB5, and VirB6 proteins might secure the protein in the inner mem- brane. Alternatively, processing of these signal peptides might require an additional uir protein function similar to the traQ- mediated processing of the t r d gene product in the F system (Ippen-Ihler and Minkley, 1986).
The predicted amino acid sequence homologies between the VirB5 and VirB6 proteins from the octopine plasmid pTiA6 and the nopaline plasmid pTiC58 are both 86% overall,' suggesting that these proteins have been functionally con- served. Fig. 5 shows this homology includes the hydrophobic cores at the amino-terminal ends of the two VirB5 proteins (14 identical residues out of 17). On the other hand, the two VirB6 proteins show a much greater divergence (5 identical residues out of 17) through the potential leader peptide, even though the overall hydrophobic nature of the core is retained. Signal peptides of both prokaryotic and eukaryotic proteins usually lack sequence homology, even among closely related proteins (Watson, 1984; Briggs and Gierasch, 1986). The divergence of the VirB6 amino-terminal sequence reflects the less stringent sequence conservation characteristic of a signal peptide, suggesting this region may function as a membrane anchor. In contrast, the conservation of the VirB5 amino- terminal sequence may reflect the constraints of a specific functional requirement and suggests it may not be used simply as a membrane-spanning domain.
The predicted signal sequence of the VirB8 protein ends with the residues Ala-Leu-Ser-Gly-Cy~'~ (Fig. 4). Interest- ingly, this sequence is similar to Leu-Leu-Ala-Gly-Cys, the E. coli consensus recognition sequence for signal peptidase 11, the enzyme involved in lipoprotein recognition and processing (Yu et al., 1986). In fact, the pentapeptide present in VirB8 is identical to that found in the signal sequence of the traT genes from the plasmids RlOO (Ogata et al., 1982) and F, both of which code for an outer membrane lipoprotein (Perumal and Minkley, 1984). The presence of this conserved processing site in the uirB8 signal sequence strongly suggests that VirB8 is a membrane-associated lipoprotein, and we are currently investigating this possibility. Aside from the conserved mod- ification/processing sequences, no other amino acid sequence homology was detected between VirB8 and the TraT proteins. Yu et al. (1986) have suggested that the amino-terminal lipid modification signal is probably the only common feature
FIG. 5. Comparison of the amino-terminal ends of the VirB5 and VirB6 proteins from pTiA6NC and pTiC58. The VirB5 and VirB6 protein sequences are indicated. The upper line of each pair shows the first 60 amino acid residues from the pTiA6 octopine plasmid proteins, while the bottom line represents the corresponding sequences from the nopaline plasmid pTiC58 proteins. Two dots indicate sequence identity, and one dot indicates a conservative amino acid change. The hydrophobic cores of the pTiA6 proteins are indi- cated by the overlined residues.
among all bacterial lipoproteins. These authors also proposed that the hydrophobic lipid-modified cysteine residue is suffi- cient to target the lipoprotein to the outer membrane, unless an additional hydrophobic stop-transfer sequence is present in the protein. Because of its very hydrophilic carboxyl ter- minus (Fig. 2), it seems likely that the VirB8 protein will be localized to the outer membrane. The significance of a plant- inducible uirB lipoprotein in the A. turnefaciens envelope is unknown.
Both the VirB2 and VirB9 proteins contain an internal stretch of hydrophobic amino acids beginning 23 and 31 residues away from the amino terminus, respectively (Fig. 4). Both potential leader peptides contain possible cleavage sites, Ala-Ala-Ala47 in VirB2 and Ala-Gln-Ala4' in VirB9. A similar internal hydrophobic region is present within the amino- terminal end of the predicted VirB3 protein, beginning after the charged arginine residue at position 16 and extending to about residue 59 (Fig. 4), but no obvious cleavage site is present within this sequence. While the spacing of these signal sequences is unusual, the leader peptides in the precursors of the exported pili proteins from F and related plasmids also begin about 30 residues from the amino-terminal end (Frost et al., 1984; Finlay et al., 1986). In addition, proper precursor processing and secretion of other bacterial proteins containing internalized signal sequences has been reported (Briggs and Gierasch, 1986). To determine whether one of these uirB internal signal sequences could be processed, the uirB9 gene was placed downstream of the inducible tac promoter on the vector pKK223-3 to give plasmid pJW301 (Fig. 3A). Cleavage at the Ala-Gln-Ala4' sequence in VirB9 should remove 48 amino acids from the predicted 26.1-kDa VirB9 protein, giving a mature protein of about 21 kDa. Two pJW301-encoded proteins were visualized in E. coli maxicells migrating at about 33 and 21 kDa, respectively, plus a minor protein of about 26 kDa (Fig. 3C, lane 11). The synthesis of all three proteins depended upon induction of the tac promoter (Fig. 3C, lanes 10 and I I ) , and pulse-chase labeling showed that the 21-kDa protein was derived from the 26-kDa protein (data not shown). The additional 33-kDa protein produced in induced pJW301-containing maxicells (lane 11) may be the product of the truncated uirBl0 gene (containing 307 codons), which is also present in pJW301. These data indicate that the VirB9 protein is synthesized as a 26-kDa precursor which is proc- essed at or near the predicted signal sequence shown in Fig. 4 to yield the mature 21-kDa protein. Furthermore, this finding suggests that the internal signal peptides of VirB2 and VirB9 can be processed normally and may function to
export these proteins from the cytoplasm. In addition, the VirB2 protein contains a potential stop-transfer sequence specified by residues 73-92 (Fig. 4) which might serve to localize the protein in the inner membrane.
The predicted amino acid sequence of the uirB7 gene prod- uct does not reveal a n obvious signal sequence (Fig. a), al- though seven regions contain 13 or more hydrophobic amino acids. These hydrophobic segments consist of residues 3-15, 34-55, 68-80, 103-117, 173-187, 201-241, and 258-273 (Fig. 2). This structure gives the predicted VirB7 protein a hydro- pathic profile which is similar to that of the E. coli integral membrane proteins Lacy and SecY (Foster et al., 1983; Cer- retti et al., 1983), in that it contains many hydrophobic domains capable of directing the spontaneous insertion of the protein into the cytoplasmic membrane. In addition, the pJW269-encoded TrpE-VirB7 fusion protein containing the carboxy two-thirds of VirB7 displayed properties character- istic of certain E. coli integral membrane proteins including Lacy and SecY (Teather et al., 1978; Ito, 1984). First, the TrpE-VirB7 protein appeared to aggregate if boiled in SDS sample buffer (Fig. 3B, lane 1 I ) and second, the fusion protein migrated faster during SDS-gel electrophoresis than expected from the predicted molecular weight (Fig. 3B, lane 12). The TrpE protein synthesized by the vector control did not display any of these characteristics, suggesting that these unusual properties were due to the VirB7 protein sequence. Based upon these similarities, it seems likely that the VirB7 protein is an integral inner membrane protein.
Finally, some membrane-associated proteins from E. coli do not possess any obvious export signals. In the case of the inner membrane protein PBP5, which requires a hydrophilic carboxyl-terminal region for membrane attachment (Pratt et al., 1986), it is believed that interactions with other membrane proteins or the formation of a membrane-anchoring domain due to protein folding may be involved. Thus, the VirB4 and VirBll proteins, which do not resemble exported proteins, might still be membrane-associated. It seems reasonable to predict that, based upon their hydrophobicity profiles and the presence of potential secretion signals (Fig. 4), many of the virB proteins are membrane-associated.
The Role of uirB Gene Products in T-DNA Transfer-It has been proposed that T-DNA transfer represents the process of bacterial conjugation applied to plant cells (Stachel and Zam- bryski, 1986a; Albright et al., 1987; Koukolikova-Nicola et al., 1987). Additional support for this analogy comes from the recent finding that the mob functions of a small mobilizable plasmid can substitute for uirD nicking at a T-DNA border to generate a DNA molecule capable of being transferred to plants by the uir genes (Buchanan-Wollaston et al., 1987). These data suggest that the mechanisms of DNA processing and transfer which occur during conjugal DNA transfer be- tween bacteria are similar to the mechanisms involved in T - DNA transfer from A. tumefaciens to plant cells. This provides a useful framework for examining and predicting uir gene function.
Instead of the F pili-mediated cell-cell contact which occurs during F-mediated conjugation, the products of the A. tume- faciens chromosomal loci chuA, chuB (Douglas et al., 1985), exoC (Cangelosi et al., 1987), and pscA (Thomashow et al., 1987) mediate binding of the bacterium to a wounded plant cell. Whether binding involves attachment to the plant cell wall or cytoplasmic membrane has not yet been resolved conclusively (Matthysse et al., 1982; Kerns et al., 1985). Plant cell wound-induced phenolic compounds (Stachel et al., 1985a) may then enter the periplasmic space and be recog- nized by the inner membrane-associated VirA protein (Leroux
et al., 1987), which in turn transforms the VirG protein into a transcriptional activator of both itself and the remaining uir genes (Winans et al., 1986; Stachel and Zambryski, 1986b). Analogous to the nicking which occurs at oriT by the TraY- Z endonuclease in F plasmid transfer, the VirD1 and VirD2 proteins together introduce a site-specific nick on the bottom strand of each T-DNA border (Yanofsky et al., 1986). Nicking is followed by the generation of a single-stranded T-DNA molecule termed the T-strand, which corresponds to the bot- tom strand of the Ti plasmid DNA present between the borders (Stachel et al., 1986; Wang et al., 1987; Albright et al., 1987; Stachel et al., 1987). The T-strand is proposed to be the molecule transferred to the plant cell. In F conjugal transfer, these events are analogous to the mating signal thought to be transmitted to the origin region via the TraM protein follow- ing donor-recipient pairing, triggering the subsequent steps of DNA unwinding, transfer, and replacement strand synthe- sis (Ippen-Ihler and Minkley, 1986; Willetts and Skurry, 1987). Similar to the polarity of conjugal ssDNA exchange, T-strand transfer is believed to occur in a specific 5’ to 3‘ direction that is dictated by the orientation of the border repeats (Wang et al., 1984; Peralta and Ream, 1985). Recent results from our laboratory suggest that in addition to uirD, the uirC gene products are also involved in early T-DNA processing events.I Mutations in uirC or in virE2, which encodes an ssDNA-binding p r ~ t e i n , ~ result in attenuated vir- ulence (Koukolikova-Nicola et al., 1987), indicating that these gene products affect the efficiency of T-DNA transfer. In summary, several chromosomal loci mediate binding, uirA and uirG regulate expression of the uir regulon, at least two of the uirD gene products are involved in T-DNA processing, and the uirC and uirE loci appear to provide accessory transfer functions.
What transfer functions are the uirB gene products likely to perform? Although a possible role for virB proteins in T- DNA processing cannot be ruled out, the finding of Stachel et al. (1987) that only the 5’-end of the uirD locus is required for T-strand formation in A. turnefaciens makes this seem unlikely. In the F system, pilus formation requires a t least 13 tra genes, some of which are believed to form a membrane- spanning complex necessary for F pili assembly and disassem- bly, as well as for conjugal DNA transfer (Ippen-Ihler and Minkley, 1986). It is thought that following pilus binding and retraction an interaction takes place between proteins in the juxtaposed donor and recipient cell membranes. This inter- action is believed to involve the tra protein pilus-assembly complex and results in the formation of a transmembrane pore through which DNA is transferred. By analogy with the F transfer system and based upon their predicted membrane localization, we propose that the uirB gene products are involved in forming a transmembrane complex capable of delivering the T-DNA into plant cells. Since the uir genes do not appear to specify binding functions (Douglas et al., 1982; Douglas et al., 1985), it seems unlikely that uirB encodes the formation of a pilus-like structure. Rather, following the attachment of A. tumefaciens to damaged plant cells, uirB proteins in the bacterial envelope may interact with proteins present in the exposed plant cytoplasmic membrane to sta- bilize bacterial-plant cell membrane surface-surface contact, similar to the proposed role of the traG and traN membrane proteins of F (Manning et al., 1981). This could result in a localized membrane fusion at the point of binding followed by T-strand DNA transfer through a uirB protein membrane
N. Tor0 and E. Nester, unpublished observation. P. Christie, J. E. Ward, S. C . Winans, and E. W. Nester, J.
Bacterial., submitted for publication.
5814 The Ti Plasmid virB Operon
"pore" into the plant cell cytoplasm. By analogy with bacterial conjugation, DNA transfer may involve interactions between virB membrane proteins and a pilot protein covalently bound to the DNA (Willetts and Skurry, 1987; Merryweather et al., 1986). Evidence from our laboratory indicates that the T- strand does indeed have a protein attached to the 5'-end.6 Alternatively, one or more uirB proteins in the bacterial membrane might package and export the T-strand from the cell and then transport it through the membrane of an adja- cent plant cell in a process similar to that used by ssDNA phage such as M13 (Wickner, 1976). However, due to the large number of uirB genes apparently required and the anal- ogies to bacterial conjugation discussed above, we favor a model involving the formation of a uirB transmembrane pro- tein pore. Experiments currently in progress should provide additional evidence for or against this model.
Acknowledgments-We thank Lin Wong for excellent technical assistance, Dale Parkhurst for synthesis of oligonucleotides, and Drs. Steve Winans and Peter Christie for their critical reading of the manuscript. Special thanks to S. Winans for advice on DNA sequence analysis. We also thank Dr. R. C. Tait for sharing data prior to publication.
Note Added in Proof-Our data suggesting that many VirB proteins are membrane-associated is supported by the recent findings of Engstrom e t al. (Engstrom, P., Zambryski, P., Van Montagu, M., and Stachel, S. (1987) J. Mol. Biol. 197, 635-645), who identified three pTiA6 uirB gene products and and showed that all three proteins are localized in the bacterial envelope.
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