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. 1991 Aug;173(16):5110–5120. doi: 10.1128/jb.173.16.5110-5120.1991

Genetic and molecular analyses of picA, a plant-inducible locus on the Agrobacterium tumefaciens chromosome.

L J Rong 1, S J Karcher 1, S B Gelvin 1
PMCID: PMC208202  PMID: 1860822

Abstract

picA is an Agrobacterium tumefaciens chromosomal locus, identified by Mu d11681 mutagenesis, that is inducible by certain acidic polysaccharides found in carrot root extract. Cloning and genetic analysis of a picA::lacZ fusion defined a region of the picA promoter that is responsible for the induction of this locus. Furthermore, we identified a possible negative regulator of picA expression upstream of the picA locus. This sequence, denoted pgl, has extensive homology to polygalacturonase genes from several organisms and inhibited the induction of the picA promoter when present in multiple copies in A. tumefaciens. DNA sequence analysis indicated at least two long open reading frames (ORFs) in the picA region. S1 nuclease mapping was used to identify the transcription initiation site of picA. Mutation of ORF1, but not ORF2, of the picA locus was responsible for an increased aggregation of A. tumefaciens, forming "ropes" in the presence of pea root cap cells. In addition, a potato tuber disk virulence assay indicated that a preinduced picA mutant was more virulent than was the wild-type control, a further indication that the picA locus regulates the surface properties of the bacterium in the presence of plant cells or plant cell extracts.

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Selected References

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  1. Albright L. M., Huala E., Ausubel F. M. Prokaryotic signal transduction mediated by sensor and regulator protein pairs. Annu Rev Genet. 1989;23:311–336. doi: 10.1146/annurev.ge.23.120189.001523. [DOI] [PubMed] [Google Scholar]
  2. Bolton G. W., Nester E. W., Gordon M. P. Plant phenolic compounds induce expression of the Agrobacterium tumefaciens loci needed for virulence. Science. 1986 May 23;232(4753):983–985. doi: 10.1126/science.3085219. [DOI] [PubMed] [Google Scholar]
  3. Darzins A., Chakrabarty A. M. Cloning of genes controlling alginate biosynthesis from a mucoid cystic fibrosis isolate of Pseudomonas aeruginosa. J Bacteriol. 1984 Jul;159(1):9–18. doi: 10.1128/jb.159.1.9-18.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Ditta G., Stanfield S., Corbin D., Helinski D. R. Broad host range DNA cloning system for gram-negative bacteria: construction of a gene bank of Rhizobium meliloti. Proc Natl Acad Sci U S A. 1980 Dec;77(12):7347–7351. doi: 10.1073/pnas.77.12.7347. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Douglas C. J., Halperin W., Nester E. W. Agrobacterium tumefaciens mutants affected in attachment to plant cells. J Bacteriol. 1982 Dec;152(3):1265–1275. doi: 10.1128/jb.152.3.1265-1275.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Figurski D. H., Helinski D. R. Replication of an origin-containing derivative of plasmid RK2 dependent on a plasmid function provided in trans. Proc Natl Acad Sci U S A. 1979 Apr;76(4):1648–1652. doi: 10.1073/pnas.76.4.1648. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Gelvin S. B., Karcher S. J., DiRita V. J. Methylation of the T-DNA in Agrobacterium tumefaciens and in several crown gall tumors. Nucleic Acids Res. 1983 Jan 11;11(1):159–174. doi: 10.1093/nar/11.1.159. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Genetello C., Van Larebeke N., Holsters M., De Picker A., Van Montagu M., Schell J. Ti plasmids of Agrobacterium as conjugative plasmids. Nature. 1977 Feb 10;265(5594):561–563. doi: 10.1038/265561a0. [DOI] [PubMed] [Google Scholar]
  9. Grierson D., Tucker G. A., Keen J., Ray J., Bird C. R., Schuch W. Sequencing and identification of a cDNA clone for tomato polygalacturonase. Nucleic Acids Res. 1986 Nov 11;14(21):8595–8603. doi: 10.1093/nar/14.21.8595. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Hanahan D. Studies on transformation of Escherichia coli with plasmids. J Mol Biol. 1983 Jun 5;166(4):557–580. doi: 10.1016/s0022-2836(83)80284-8. [DOI] [PubMed] [Google Scholar]
  11. He S. Y., Collmer A. Molecular cloning, nucleotide sequence, and marker exchange mutagenesis of the exo-poly-alpha-D-galacturonosidase-encoding pehX gene of Erwinia chrysanthemi EC16. J Bacteriol. 1990 Sep;172(9):4988–4995. doi: 10.1128/jb.172.9.4988-4995.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Henikoff S. Unidirectional digestion with exonuclease III creates targeted breakpoints for DNA sequencing. Gene. 1984 Jun;28(3):351–359. doi: 10.1016/0378-1119(84)90153-7. [DOI] [PubMed] [Google Scholar]
  13. Hinton J. C., Gill D. R., Lalo D., Plastow G. S., Salmond G. P. Sequence of the peh gene of Erwinia carotovora: homology between Erwinia and plant enzymes. Mol Microbiol. 1990 Jun;4(6):1029–1036. doi: 10.1111/j.1365-2958.1990.tb00675.x. [DOI] [PubMed] [Google Scholar]
  14. Hirsch P. R., Beringer J. E. A physical map of pPH1JI and pJB4JI. Plasmid. 1984 Sep;12(2):139–141. doi: 10.1016/0147-619x(84)90059-3. [DOI] [PubMed] [Google Scholar]
  15. Huang J. H., Schell M. A. DNA sequence analysis of pglA and mechanism of export of its polygalacturonase product from Pseudomonas solanacearum. J Bacteriol. 1990 Jul;172(7):3879–3887. doi: 10.1128/jb.172.7.3879-3887.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Kerr A., Manigault P., Tempé J. Transfer of virulence in vivo and in vitro in Agrobacterium. Nature. 1977 Feb 10;265(5594):560–561. doi: 10.1038/265560a0. [DOI] [PubMed] [Google Scholar]
  17. Klapwijk P. M., Scheulderman T., Schilperoort R. A. Coordinated regulation of octopine degradation and conjugative transfer of Ti plasmids in Agrobacterium tumefaciens: evidence for a common regulatory gene and separate operons. J Bacteriol. 1978 Nov;136(2):775–785. doi: 10.1128/jb.136.2.775-785.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Knauf V. C., Nester E. W. Wide host range cloning vectors: a cosmid clone bank of an Agrobacterium Ti plasmid. Plasmid. 1982 Jul;8(1):45–54. doi: 10.1016/0147-619x(82)90040-3. [DOI] [PubMed] [Google Scholar]
  19. Matthysse A. G. Effect of Plasmid pSa and of Auxin on Attachment of Agrobacterium tumefaciens to Carrot Cells. Appl Environ Microbiol. 1987 Oct;53(10):2574–2582. doi: 10.1128/aem.53.10.2574-2582.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Messing J., Crea R., Seeburg P. H. A system for shotgun DNA sequencing. Nucleic Acids Res. 1981 Jan 24;9(2):309–321. doi: 10.1093/nar/9.2.309. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Metcalf W. W., Steed P. M., Wanner B. L. Identification of phosphate starvation-inducible genes in Escherichia coli K-12 by DNA sequence analysis of psi::lacZ(Mu d1) transcriptional fusions. J Bacteriol. 1990 Jun;172(6):3191–3200. doi: 10.1128/jb.172.6.3191-3200.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Montoya A. L., Moore L. W., Gordon M. P., Nester E. W. Multiple genes coding for octopine-degrading enzymes in Agrobacterium. J Bacteriol. 1978 Dec;136(3):909–915. doi: 10.1128/jb.136.3.909-915.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Overdier D. G., Olson E. R., Erickson B. D., Ederer M. M., Csonka L. N. Nucleotide sequence of the transcriptional control region of the osmotically regulated proU operon of Salmonella typhimurium and identification of the 5' endpoint of the proU mRNA. J Bacteriol. 1989 Sep;171(9):4694–4706. doi: 10.1128/jb.171.9.4694-4706.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Pearson W. R., Lipman D. J. Improved tools for biological sequence comparison. Proc Natl Acad Sci U S A. 1988 Apr;85(8):2444–2448. doi: 10.1073/pnas.85.8.2444. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Rogowsky P. M., Powell B. S., Shirasu K., Lin T. S., Morel P., Zyprian E. M., Steck T. R., Kado C. I. 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. 1990 Mar;23(2):85–106. doi: 10.1016/0147-619x(90)90028-b. [DOI] [PubMed] [Google Scholar]
  26. Rong L., Karcher S. J., O'Neal K., Hawes M. C., Yerkes C. D., Jayaswal R. K., Hallberg C. A., Gelvin S. B. picA, a novel plant-inducible locus on the Agrobacterium tumefaciens chromosome. J Bacteriol. 1990 Oct;172(10):5828–5836. doi: 10.1128/jb.172.10.5828-5836.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Salser W., Gesteland R. F., Bolle A. In vitro synthesis of bacteriophage lysozyme. Nature. 1967 Aug 5;215(5101):588–591. doi: 10.1038/215588a0. [DOI] [PubMed] [Google Scholar]
  28. Sancar A., Hack A. M., Rupp W. D. Simple method for identification of plasmid-coded proteins. J Bacteriol. 1979 Jan;137(1):692–693. doi: 10.1128/jb.137.1.692-693.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Silhavy T. J., Beckwith J. R. Uses of lac fusions for the study of biological problems. Microbiol Rev. 1985 Dec;49(4):398–418. doi: 10.1128/mr.49.4.398-418.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Stachel S. E., Nester E. W., Zambryski P. C. A plant cell factor induces Agrobacterium tumefaciens vir gene expression. Proc Natl Acad Sci U S A. 1986 Jan;83(2):379–383. doi: 10.1073/pnas.83.2.379. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Veluthambi K., Krishnan M., Gould J. H., Smith R. H., Gelvin S. B. Opines stimulate induction of the vir genes of the Agrobacterium tumefaciens Ti plasmid. J Bacteriol. 1989 Jul;171(7):3696–3703. doi: 10.1128/jb.171.7.3696-3703.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Vieira J., Messing J. Production of single-stranded plasmid DNA. Methods Enzymol. 1987;153:3–11. doi: 10.1016/0076-6879(87)53044-0. [DOI] [PubMed] [Google Scholar]
  34. Wang K., Herrera-Estrella A., Van Montagu M. Overexpression of virD1 and virD2 genes in Agrobacterium tumefaciens enhances T-complex formation and plant transformation. J Bacteriol. 1990 Aug;172(8):4432–4440. doi: 10.1128/jb.172.8.4432-4440.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Worcel A., Burgi E. Properties of a membrane-attached form of the folded chromosome of Escherichia coli. J Mol Biol. 1974 Jan 5;82(1):91–105. doi: 10.1016/0022-2836(74)90576-2. [DOI] [PubMed] [Google Scholar]
  36. Yanisch-Perron C., Vieira J., Messing J. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene. 1985;33(1):103–119. doi: 10.1016/0378-1119(85)90120-9. [DOI] [PubMed] [Google Scholar]
  37. Zambryski P. Basic processes underlying Agrobacterium-mediated DNA transfer to plant cells. Annu Rev Genet. 1988;22:1–30. doi: 10.1146/annurev.ge.22.120188.000245. [DOI] [PubMed] [Google Scholar]

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