Skip to main content
PMC home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1993 Oct;175(20):6626–6636. doi: 10.1128/jb.175.20.6626-6636.1993

The chromosomal response regulatory gene chvI of Agrobacterium tumefaciens complements an Escherichia coli phoB mutation and is required for virulence.

N J Mantis 1, S C Winans 1
PMCID: PMC206774  PMID: 8407840

Abstract

In an effort to identify the Agrobacterium tumefaciens phosphate regulatory gene(s), we isolated a clone from an A. tumefaciens cosmid library that restored regulated alkaline phosphatase activity to an Escherichia coli phoB mutant. The gene that complemented phoB was localized by subcloning and deletion analysis, and the DNA sequence was determined. An open reading frame, denoted chvI, was identified that encoded a predicted protein with amino acid similarity to the family of bacterial response regulators and 35% identify to PhoB. Surprisingly, an A. tumefaciens chvI mutant showed normal induction of phosphatase activity and normal virG expression when grown in phosphate-limiting media. However, this mutant was unable to grow in media containing tryptone, peptone, or Casamino Acids and was also more sensitive than the wild type to acidic extracellular pH. This mutant was avirulent on Kalanchoeë diagremontiana and was severely attenuated in vir gene expression. The pH-inducible expression of virG was also abolished. Growth of the chvI mutant was inhibited by K. diagremontiana wound sap, suggesting that avirulence may be due, in part, to the inability of this mutant to survive the plant wound environment.

Full text

PDF
6626

Images in this article

6631Image
on p.6631
6632Image
on p.6632
6634Image
on p.6634

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Aiba H., Nagaya M., Mizuno T. Sensor and regulator proteins from the cyanobacterium Synechococcus species PCC7942 that belong to the bacterial signal-transduction protein families: implication in the adaptive response to phosphate limitation. Mol Microbiol. 1993 Apr;8(1):81–91. doi: 10.1111/j.1365-2958.1993.tb01205.x. [DOI] [PubMed] [Google Scholar]
  2. Anba J., Bidaud M., Vasil M. L., Lazdunski A. Nucleotide sequence of the Pseudomonas aeruginosa phoB gene, the regulatory gene for the phosphate regulon. J Bacteriol. 1990 Aug;172(8):4685–4689. doi: 10.1128/jb.172.8.4685-4689.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Aoyama T., Takanami M., Makino K., Oka A. Cross-talk between the virulence and phosphate regulons of Agrobacterium tumefaciens caused by an unusual interaction of the transcriptional activator with a regulatory DNA element. Mol Gen Genet. 1991 Jul;227(3):385–390. doi: 10.1007/BF00273927. [DOI] [PubMed] [Google Scholar]
  4. Booth I. R. Regulation of cytoplasmic pH in bacteria. Microbiol Rev. 1985 Dec;49(4):359–378. doi: 10.1128/mr.49.4.359-378.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Brickman E., Beckwith J. Analysis of the regulation of Escherichia coli alkaline phosphatase synthesis using deletions and phi80 transducing phages. J Mol Biol. 1975 Aug 5;96(2):307–316. doi: 10.1016/0022-2836(75)90350-2. [DOI] [PubMed] [Google Scholar]
  6. Cangelosi G. A., Ankenbauer R. G., Nester E. W. Sugars induce the Agrobacterium virulence genes through a periplasmic binding protein and a transmembrane signal protein. Proc Natl Acad Sci U S A. 1990 Sep;87(17):6708–6712. doi: 10.1073/pnas.87.17.6708. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Cangelosi G. A., Best E. A., Martinetti G., Nester E. W. Genetic analysis of Agrobacterium. Methods Enzymol. 1991;204:384–397. doi: 10.1016/0076-6879(91)04020-o. [DOI] [PubMed] [Google Scholar]
  8. Chang C. H., Winans S. C. Functional roles assigned to the periplasmic, linker, and receiver domains of the Agrobacterium tumefaciens VirA protein. J Bacteriol. 1992 Nov;174(21):7033–7039. doi: 10.1128/jb.174.21.7033-7039.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Charles T. C., Nester E. W. A chromosomally encoded two-component sensory transduction system is required for virulence of Agrobacterium tumefaciens. J Bacteriol. 1993 Oct;175(20):6614–6625. doi: 10.1128/jb.175.20.6614-6625.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Chen C. Y., Winans S. C. Controlled expression of the transcriptional activator gene virG in Agrobacterium tumefaciens by using the Escherichia coli lac promoter. J Bacteriol. 1991 Feb;173(3):1139–1144. doi: 10.1128/jb.173.3.1139-1144.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Cooley M. B., D'Souza M. R., Kado C. I. The virC and virD operons of the Agrobacterium Ti plasmid are regulated by the ros chromosomal gene: analysis of the cloned ros gene. J Bacteriol. 1991 Apr;173(8):2608–2616. doi: 10.1128/jb.173.8.2608-2616.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Dente L., Cesareni G., Cortese R. pEMBL: a new family of single stranded plasmids. Nucleic Acids Res. 1983 Mar 25;11(6):1645–1655. doi: 10.1093/nar/11.6.1645. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Farrand S. K., O'Morchoe S. P., McCutchan J. Construction of an Agrobacterium tumefaciens C58 recA mutant. J Bacteriol. 1989 Oct;171(10):5314–5321. doi: 10.1128/jb.171.10.5314-5321.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Fields P. I., Groisman E. A., Heffron F. A Salmonella locus that controls resistance to microbicidal proteins from phagocytic cells. Science. 1989 Feb 24;243(4894 Pt 1):1059–1062. doi: 10.1126/science.2646710. [DOI] [PubMed] [Google Scholar]
  15. Foster J. W., Hall H. K. Adaptive acidification tolerance response of Salmonella typhimurium. J Bacteriol. 1990 Feb;172(2):771–778. doi: 10.1128/jb.172.2.771-778.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Garfinkel D. J., Nester E. W. Agrobacterium tumefaciens mutants affected in crown gall tumorigenesis and octopine catabolism. J Bacteriol. 1980 Nov;144(2):732–743. doi: 10.1128/jb.144.2.732-743.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Gay P., Le Coq D., Steinmetz M., Berkelman T., Kado C. I. Positive selection procedure for entrapment of insertion sequence elements in gram-negative bacteria. J Bacteriol. 1985 Nov;164(2):918–921. doi: 10.1128/jb.164.2.918-921.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Gelvin S. B. Crown gall disease and hairy root disease : a sledgehammer and a tackhammer. Plant Physiol. 1990 Feb;92(2):281–285. doi: 10.1104/pp.92.2.281. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Goss T. J., O'Hara G. W., Dilworth M. J., Glenn A. R. Cloning, characterization, and complementation of lesions causing acid sensitivity in Tn5-induced mutants of Rhizobium meliloti WSM419. J Bacteriol. 1990 Sep;172(9):5173–5179. doi: 10.1128/jb.172.9.5173-5179.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Gray J., Wang J., Gelvin S. B. Mutation of the miaA gene of Agrobacterium tumefaciens results in reduced vir gene expression. J Bacteriol. 1992 Feb;174(4):1086–1098. doi: 10.1128/jb.174.4.1086-1098.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Groisman E. A., Chiao E., Lipps C. J., Heffron F. Salmonella typhimurium phoP virulence gene is a transcriptional regulator. Proc Natl Acad Sci U S A. 1989 Sep;86(18):7077–7081. doi: 10.1073/pnas.86.18.7077. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Han D. C., Chen C. Y., Chen Y. F., Winans S. C. Altered-function mutations of the transcriptional regulatory gene virG of Agrobacterium tumefaciens. J Bacteriol. 1992 Nov;174(21):7040–7043. doi: 10.1128/jb.174.21.7040-7043.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Iuchi S., Lin E. C. arcA (dye), a global regulatory gene in Escherichia coli mediating repression of enzymes in aerobic pathways. Proc Natl Acad Sci U S A. 1988 Mar;85(6):1888–1892. doi: 10.1073/pnas.85.6.1888. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Jin S. G., Prusti R. K., Roitsch T., Ankenbauer R. G., Nester E. W. Phosphorylation of the VirG protein of Agrobacterium tumefaciens by the autophosphorylated VirA protein: essential role in biological activity of VirG. J Bacteriol. 1990 Sep;172(9):4945–4950. doi: 10.1128/jb.172.9.4945-4950.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Kaniga K., Delor I., Cornelis G. R. A wide-host-range suicide vector for improving reverse genetics in gram-negative bacteria: inactivation of the blaA gene of Yersinia enterocolitica. Gene. 1991 Dec 20;109(1):137–141. doi: 10.1016/0378-1119(91)90599-7. [DOI] [PubMed] [Google Scholar]
  26. Lamb C. J., Lawton M. A., Dron M., Dixon R. A. Signals and transduction mechanisms for activation of plant defenses against microbial attack. Cell. 1989 Jan 27;56(2):215–224. doi: 10.1016/0092-8674(89)90894-5. [DOI] [PubMed] [Google Scholar]
  27. Leroux B., Yanofsky M. F., Winans S. C., Ward J. E., Ziegler S. F., Nester E. W. Characterization of the virA locus of Agrobacterium tumefaciens: a transcriptional regulator and host range determinant. EMBO J. 1987 Apr;6(4):849–856. doi: 10.1002/j.1460-2075.1987.tb04830.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Lippincott J. A., Lippincott B. B. The genus Agrobacterium and plant tumorigenesis. Annu Rev Microbiol. 1975;29:377–405. doi: 10.1146/annurev.mi.29.100175.002113. [DOI] [PubMed] [Google Scholar]
  29. Makino K., Shinagawa H., Amemura M., Kawamoto T., Yamada M., Nakata A. Signal transduction in the phosphate regulon of Escherichia coli involves phosphotransfer between PhoR and PhoB proteins. J Mol Biol. 1989 Dec 5;210(3):551–559. doi: 10.1016/0022-2836(89)90131-9. [DOI] [PubMed] [Google Scholar]
  30. Makino K., Shinagawa H., Amemura M., Nakata A. Nucleotide sequence of the phoB gene, the positive regulatory gene for the phosphate regulon of Escherichia coli K-12. J Mol Biol. 1986 Jul 5;190(1):37–44. doi: 10.1016/0022-2836(86)90073-2. [DOI] [PubMed] [Google Scholar]
  31. Mantis N. J., Winans S. C. The Agrobacterium tumefaciens vir gene transcriptional activator virG is transcriptionally induced by acid pH and other stress stimuli. J Bacteriol. 1992 Feb;174(4):1189–1196. doi: 10.1128/jb.174.4.1189-1196.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Metts J., West J., Doares S. H., Matthysse A. G. Characterization of three Agrobacterium tumefaciens avirulent mutants with chromosomal mutations that affect induction of vir genes. J Bacteriol. 1991 Feb;173(3):1080–1087. doi: 10.1128/jb.173.3.1080-1087.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Miller S. I., Kukral A. M., Mekalanos J. J. A two-component regulatory system (phoP phoQ) controls Salmonella typhimurium virulence. Proc Natl Acad Sci U S A. 1989 Jul;86(13):5054–5058. doi: 10.1073/pnas.86.13.5054. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Parkinson J. S., Kofoid E. C. Communication modules in bacterial signaling proteins. Annu Rev Genet. 1992;26:71–112. doi: 10.1146/annurev.ge.26.120192.000443. [DOI] [PubMed] [Google Scholar]
  35. 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]
  36. Sciaky D., Montoya A. L., Chilton M. D. Fingerprints of Agrobacterium Ti plasmids. Plasmid. 1978 Feb;1(2):238–253. doi: 10.1016/0147-619x(78)90042-2. [DOI] [PubMed] [Google Scholar]
  37. Seki T., Yoshikawa H., Takahashi H., Saito H. Cloning and nucleotide sequence of phoP, the regulatory gene for alkaline phosphatase and phosphodiesterase in Bacillus subtilis. J Bacteriol. 1987 Jul;169(7):2913–2916. doi: 10.1128/jb.169.7.2913-2916.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Shimoda N., Toyoda-Yamamoto A., Nagamine J., Usami S., Katayama M., Sakagami Y., Machida Y. Control of expression of Agrobacterium vir genes by synergistic actions of phenolic signal molecules and monosaccharides. Proc Natl Acad Sci U S A. 1990 Sep;87(17):6684–6688. doi: 10.1073/pnas.87.17.6684. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. 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]
  40. Stachel S. E., Zambryski P. C. virA and virG control the plant-induced activation of the T-DNA transfer process of A. tumefaciens. Cell. 1986 Aug 1;46(3):325–333. doi: 10.1016/0092-8674(86)90653-7. [DOI] [PubMed] [Google Scholar]
  41. Veluthambi K., Jayaswal R. K., Gelvin S. B. Virulence genes A, G, and D mediate the double-stranded border cleavage of T-DNA from the Agrobacterium Ti plasmid. Proc Natl Acad Sci U S A. 1987 Apr;84(7):1881–1885. doi: 10.1073/pnas.84.7.1881. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Vernade D., Herrera-Estrella A., Wang K., Van Montagu M. Glycine betaine allows enhanced induction of the Agrobacterium tumefaciens vir genes by acetosyringone at low pH. J Bacteriol. 1988 Dec;170(12):5822–5829. doi: 10.1128/jb.170.12.5822-5829.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Wanner B. L., Chang B. D. The phoBR operon in Escherichia coli K-12. J Bacteriol. 1987 Dec;169(12):5569–5574. doi: 10.1128/jb.169.12.5569-5574.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Winans S. C., Ebert P. R., Stachel S. E., Gordon M. P., Nester E. W. A gene essential for Agrobacterium virulence is homologous to a family of positive regulatory loci. Proc Natl Acad Sci U S A. 1986 Nov;83(21):8278–8282. doi: 10.1073/pnas.83.21.8278. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Winans S. C., Kerstetter R. A., Nester E. W. Transcriptional regulation of the virA and virG genes of Agrobacterium tumefaciens. J Bacteriol. 1988 Sep;170(9):4047–4054. doi: 10.1128/jb.170.9.4047-4054.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Winans S. C., Kerstetter R. A., Ward J. E., Nester E. W. A protein required for transcriptional regulation of Agrobacterium virulence genes spans the cytoplasmic membrane. J Bacteriol. 1989 Mar;171(3):1616–1622. doi: 10.1128/jb.171.3.1616-1622.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Winans S. C. Transcriptional induction of an Agrobacterium regulatory gene at tandem promoters by plant-released phenolic compounds, phosphate starvation, and acidic growth media. J Bacteriol. 1990 May;172(5):2433–2438. doi: 10.1128/jb.172.5.2433-2438.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Winans S. C. Two-way chemical signaling in Agrobacterium-plant interactions. Microbiol Rev. 1992 Mar;56(1):12–31. doi: 10.1128/mr.56.1.12-31.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Journal of Bacteriology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES