Journal of Experimental Botany, Vol. 51, No. 347, pp. 1167-1169,
June 2000
© 2000 Oxford University Press
Gene Note |
Sequence analysis of the vir-region from Agrobacterium tumefaciens octopine Ti plasmid pTi15955
1 Institute of Molecular Plant Sciences, Clusius Laboratory, Leiden University, Wassenaarseweg 64, 2333 AL Leiden, The Netherlands
2 Agrigenetics Corporation, Advanced Research Division, 5649 East Buckeye Road, Madison, WI 53716, USA
Received 29 February 2000; Accepted 20 March 2000
Abstract
The nucleotide sequence of 42 775 bp of the vir-region from the Agrobacterium tumefaciens octopine Ti plasmid pTi15955 is reported here. Although the nucleotide sequences of several parts of this region from this or closely related plasmids have been published previously, the present work establishes for the first time the complete arrangement of all the essential virulence genes and their intergenic regions of an octopine Ti plasmid. The disruption of some of the intergenic areas by insertion (IS) elements is typical for the octopine Ti plasmids. Several new ORFs were identified, including ORFs immediately downstream of virD4 and virE2, which probably represent new genes involved in virulence.
Key words: Agrobacterium tumefaciens, sequence, virulence genes.
The plant pathogenic bacterium Agrobacterium tumefaciens induces tumours, called crown galls, on plants. Tumour induction is due to transformation by an oncogenic DNA segment, the T-DNA, which is derived from a tumour-inducing (Ti) plasmid that is present in the bacterium (for a review see Hooykaas and Beijersbergen, 1994
). Transfer of the T-region is mediated by gene products encoded by the virulence (vir) genes, which are located in the vir-region of the Ti plasmid. vir-gene expression is mediated by the VirA/VirG two component regulatory system which reacts to the presence of plant phenolic compounds such as acetosyringone. The phosphorylated transcriptional regulator VirG activates vir-gene expression after binding to a consensus sequence, the vir-box, which is present in front of each of the vir-operons.
The sequenced vir-region of the octopine Ti plasmid pTi15955 presented here (Table 1; Fig. 1) embraces all the essential vir operons virA to virG and ends at the 3' end of the virH (left end) and virF (right end) operons, respectively. The sequences of some of the genes from pTi15955 have been published previously by this team: virG (Melchers et al., 1986), virA (Melchers et al., 1987), virF (Melchers et al., 1990), and the virB operon (Thompson et al., 1988, later corrected for the insertion of the nucleotide ‘G’ between base 3275 and 3276 giving two G residues instead of one). For the first time, the sequences of the remaining vir operons including the virC, virD and virE operons of pTi15955, as well as the regions between the vir operons, are reported here.
|
|
Several complete or partial insertion (IS) elements were found in the intergenic areas between certain vir operons. An insert of 250 bp was found between the virK and virA operons which showed 85% identity to an internal region of IS66, an IS element found previously in the TL-region of the octopine Ti plasmid of mutant A66 (Machida et al., 1984
). This insert was named IS66-p. At 484 bp downstream of the virJ stop codon an insert of 834 bp with 15 bp imperfect terminal inverted repeats (IRs) was identified, which is flanked by 4 bp direct repeats (DRs) of the target sequence (with one mismatch at one site). This insert was named IS869–1, because of its 85% identity to IS869, an IS element previously identified in the TA-region of Ti plasmid pTiAB3 (Paulus et al., 1991). Furthermore, 53 bp downstream from the virG operon a 4950 bp insert was found with approximately 20 bp terminal IRs (8 mismatches) and flanked by 8 bp perfect DRs. No similarity of this putative IS element, provisionally called IS71, was found with other known IS elements. However, within this IS71 element, another IS element of 2556 bp with 20 bp terminal IRs (2 mismatches) and 8 bp DRs was found. This IS element turned out to be 94% identical to IS66 (Machida et al., 1984). This 2556 bp IS element has been given the name, IS66-1. Sequencing of the virD operon led to the identification of a fifth ORF, ORFD5 located 92 bp downstream from the virD4 stop codon. This ORFD5 has a putative RBS 5 bp upstream of its ATG start codon and codes for a protein of 833 AA having a molecular mass of 93.0 kDa. The ProfileScan program of the ISREC Bioinformatics Group determined two bipartite nuclear localization sequences (NLSs) at amino acid positions 324 to 341 and 766 to 783. Additionally, two negatively charged domains, two helix-turn-helix motifs, one helix-loop-helix motif, and a helical wheel are present suggesting that this hypothetical ORFD5 protein may function as a transcription factor within the plant cell. Downstream of the virE operon an ORF named ORFE3 was found, which encodes a putative hydrophilic protein of 672 AA with a molecular mass of 75.7 kDa. Putative RBS and -10 and -35 promoter sequences are present which partly overlap the 3' end of the virE2 gene. Putative vir-boxes were found in virD4 (position 1898 to 1911) and virE2 (position 842 to 855). However, whether the expression of ORFD5 and ORFE3 is indeed under the control of the VirA/VirG two component regulatory system has yet to be determined.
Further downstream of ORFE3, two ORFs, ORF5 (102 AA) and ORF4 (100 AA), were identified which showed moderate similarity to the beta-subunit of integration host factor (IHF-beta) and cold shock proteins, respectively. Putative RBS and -10 and -35 promoter sequences are present. The expression of ORF4 might be regulated by the VirA/VirG two component regulatory system since a putative vir-box is present upstream of ORF4 at position 37 279 to 37 292. Sequence comparison revealed the presence of a conserved cold shock domain in ORF4. This domain enables proteins to bind to RNA-molecules (Sommerville, 1999). Many cold shock proteins are involved in the control of mRNA translation by their binding capacity to specific mRNAs. Whether ORF4 is involved in the control of vir-mRNA translation has yet to be determined.
Upstream of the virF gene, four ORFs were found, named ORF1', ORF1'', ORF2, and ORF3, which encode putative proteins with homology to the TraA, TraF and TraB conjugation proteins from A. tumefaciens (Melchers et al., 1990; this work). ORF1' and ORF1'' together resemble the 3' half of the traA gene. The two ORFs are not in frame because of one base pair deletion at the 3' end of ORF1'. Furthermore, ORF1'' contains two frame shifts at the 5' end. ORF2 has strong similarity to traF, while ORF3 resembles the 5' end of the traB gene. This region is apparently copied from the pTi tra-region and has become scrambled and deleted during the recombination process. Therefore, it is questionable whether functional proteins are expressed from this region.
The present sequence analysis shows the complete arrangement of vir-genes, other ORFs and IS elements in the vir-region of the octopine Ti plasmid pTi15955. When the predicted amino acid sequences of the Vir-proteins were used for codon usage analysis, it was found that the codon usage was similar to that of proteins encoded by the T-region of the Ti plasmid, thus suggesting a common origin. The arrangement of vir-genes in the pTi15955 plasmid is similar or identical to that in other octopine Ti plasmids. However, when compared to the 28 kbp core of the nopaline Ti vir-region, which was sequenced previously (Rogowsky et al., 1990), it becomes apparent that the core of the octopine Ti vir-region is very similar in gene content and gene order, but that, in contrast to that of the nopaline Ti plasmid, it is interrupted by several IS-elements. Thus these transposition events must have occurred after the evolution of Ti plasmids into nopaline and octopine types.
Acknowledgments
We thank Werner Pansegrau for helping with the sequence analysis. This work was supported by the Netherlands Foundation for Chemical Research (SON) with financial aid from the Netherlands Organization for Scientific Research (NWO).
Notes
3 Present address: Unilever Research Laboratory Vlaardingen, Olivier van Noortlaan 120, 3133 AT Vlaardingen, The Netherlands. 
4 Present address: Abbott Laboratories, Abbott Park, IL 60064, USA.
5 Present address: Promega Corporation, 2800 Woods Hollow Road, Madison, WI 53711–5399, USA.
6 Present address: Zeneca Mogen, Einsteinweg 97, 2333 CB Leiden, The Netherlands.
7 To whom correspondence should be addressed. Fax: +31 71 5274 999. E-mail: genetica{at}rulbim.leidenuniv.nl
References
Hooykaas PJJ, Beijersbergen AGM.1994. The virulence system of Agrobacterium tumefaciens. Annual Review of Phytopathology 32, 157–179.[ISI]
Machida Y, Sakurai M, Kiyokawa S, Ubasawa A, Suzuki Y, Ikeda J-E.1984. Nucleotide sequence of the insertion sequence found in the T-DNA region of mutant Ti plasmid pTiA66 and distribution of its homologues in octopine Ti plasmid. Proceedings of the National Academy of Sciences, USA 81, 7495–7499.
Melchers LS, Maroney MJ, Den Dulk-Ras A, Thompson DV, Van Vuuren HAJ, Schilperoort RA, Hooykaas PJJ.1990. Octopine and nopaline strains of Agrobacterium tumefaciens differ in virulence; molecular characterization of the virF-locus. Plant Molecular Biology 14, 249–259.[ISI][Medline]
Melchers LS, Thompson DV, Idler KB, Neuteboom STC, De Maagd RA, Schilperoort RA, Hooykaas PJJ.1987. Molecular characterization of the virulence gene vir\A of the Agrobacterium tumefaciens octopine Ti plasmid. Plant Molecular Biology 9, 635–645.
Melchers LS, Thompson DV, Idler KB, Schilperoort RA, Hooykaas PJJ.1986. Nucleotide sequence of the virulence gene virG of the Agrobacterium tumefaciens octopine Ti plasmid: significant homology between virG and the regulatory genes ompR, phoB and dye of E. coli. Nucleic Acids Research 14, 9933–9942.
Paulus F, Canaday J, Vincent F, Bonnard G, Kares C, Otten L.1991. Sequence of the iaa and ipt region of different Agrobacterium tumefaciens biotype III octopine strains: reconstruction of octopine Ti plasmid evolution. Plant Molecular Biology 16, 601–614.[ISI][Medline]
Rogowsky PM, Powell BS, Shirasu K, Lin T-S, Morel P, Zyprian EM, Steck TR, Kado CI.1990. Molecular characterization of the vir regulon of Agrobacterium tumefaciens: complete nucleotide sequence and gene organization of the 28.63-kbp regulon cloned as a single unit. Plasmid 23, 85–106.[ISI][Medline]
Sommerville J.1999. Activities of the cold-shock domain proteins in translation control. BioEssays 21, 319–325.[ISI][Medline]
Thompson DV, Melchers LS, Idler KB, Schilperoort RA, Hooykaas PJJ.1988. Analysis of the complete nucleotide sequence of the Agrobacterium tumefaciens virB operon. Nucleic Acids Research 16, 4621–4636.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  A. C. Vergunst, M. C. M. van Lier, A. den Dulk-Ras, T. A. Grosse Stuve, A. Ouwehand, and P. J. J. Hooykaas Positive charge is an important feature of the C-terminal transport signal of the VirB/D4-translocated proteins of Agrobacterium PNAS, January 18, 2005; 102(3): 832 - 837. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. Schrammeijer, A. d. Dulk-Ras, A. C. Vergunst, E. Jurado Jacome, and P. J. J. Hooykaas Analysis of Vir protein translocation from Agrobacterium tumefaciens using Saccharomyces cerevisiae as a model: evidence for transport of a novel effector protein VirE3 Nucleic Acids Res., February 1, 2003; 31(3): 860 - 868. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||