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. 1996 Aug;178(15):4548–4554. doi: 10.1128/jb.178.15.4548-4554.1996

Identification and characterization of phoN-Sf, a gene on the large plasmid of Shigella flexneri 2a encoding a nonspecific phosphatase.

K I Uchiya 1, M Tohsuji 1, T Nikai 1, H Sugihara 1, C Sasakawa 1
PMCID: PMC178222  PMID: 8755883

Abstract

A gene encoding a nonspecific phosphatase, named PhoN-Sf, was identified on the large virulence plasmid (pMYSH6000) of Shigella flexneri 2a YSH6000. The phosphatase activity in YSH6000 was observed under high-phosphate conditions. However, it was found that low-phosphate conditions induced a slightly higher level of activity. The nucleotide sequence of the phoN-Sf region cloned from pMYSH6000 possessing the phoN-Sf gene encoded 249 amino acids with a typical signal sequence at the N terminus. The deduced amino acid sequence of the PhoN-Sf protein revealed significant homology to sequences of nonspecific acid phosphatases of other bacteria, such as Providencia stuartii (PhoN, 83.2%), Morganella morganii (PhoC, 80.6%), Salmonella typhimurium (PhoN, 47.8%), and Zymomonas mobilis (PhoC, 34.8%). The PhoN-Sf protein was purified, and its biochemical properties were characterized. The apparent molecular mass of the protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis was calculated to be 27 kDa. The 20 amino acids at the N terminus corresponded to the 20 amino acid residues following the putative signal sequence of PhoN-Sf protein deduced from the nucleotide sequence. The PhoN-Sf activity had a pH optimum of 6.6, and the optimum temperature was 37 degrees C. The enzymatic activity was inhibited by diisopropyl fluorophosphate, N-bromosuccinimide, or dithiothreitol but not by EDTA. The subcellular localization of the PhoN-Sf protein in YSH6000 revealed that the protein was found predominantly in the periplasm. Examination of Shigella and enteroinvasive Escherichia coli strains for PhoN-Sf production by immunoblotting with the PhoN-specific antibody and for the presence of phoN-Sf DNA by using a phoN-Sf probe indicated that approximately one-half of the strains possessed the phoN-Sf gene on the large plasmid and expressed the PhoN-Sf protein. The Tn5 insertion mutants of YSH6000 possessing phoN-Sf::Tn5 still retained wild-type levels of invasiveness, as well as the subsequent spreading capacity in MK2 epithelial cell monolayers, thus suggesting that the PhoN-Sf activity is not involved in expression of the virulence phenotypes of Shigella strains under in vitro conditions.

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Selected References

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

  1. Adler B., Sasakawa C., Tobe T., Makino S., Komatsu K., Yoshikawa M. A dual transcriptional activation system for the 230 kb plasmid genes coding for virulence-associated antigens of Shigella flexneri. Mol Microbiol. 1989 May;3(5):627–635. doi: 10.1111/j.1365-2958.1989.tb00210.x. [DOI] [PubMed] [Google Scholar]
  2. Allaoui A., Mounier J., Prévost M. C., Sansonetti P. J., Parsot C. icsB: a Shigella flexneri virulence gene necessary for the lysis of protrusions during intercellular spread. Mol Microbiol. 1992 Jun;6(12):1605–1616. doi: 10.1111/j.1365-2958.1992.tb00885.x. [DOI] [PubMed] [Google Scholar]
  3. Allaoui A., Sansonetti P. J., Parsot C. MxiJ, a lipoprotein involved in secretion of Shigella Ipa invasins, is homologous to YscJ, a secretion factor of the Yersinia Yop proteins. J Bacteriol. 1992 Dec;174(23):7661–7669. doi: 10.1128/jb.174.23.7661-7669.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Andrews G. P., Hromockyj A. E., Coker C., Maurelli A. T. Two novel virulence loci, mxiA and mxiB, in Shigella flexneri 2a facilitate excretion of invasion plasmid antigens. Infect Immun. 1991 Jun;59(6):1997–2005. doi: 10.1128/iai.59.6.1997-2005.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Argast M., Boos W. Co-regulation in Escherichia coli of a novel transport system for sn-glycerol-3-phosphate and outer membrane protein Ic (e, E) with alkaline phosphatase and phosphate-binding protein. J Bacteriol. 1980 Jul;143(1):142–150. doi: 10.1128/jb.143.1.142-150.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Baudry B., Kaczorek M., Sansonetti P. J. Nucleotide sequence of the invasion plasmid antigen B and C genes (ipaB and ipaC) of Shigella flexneri. Microb Pathog. 1988 May;4(5):345–357. doi: 10.1016/0882-4010(88)90062-9. [DOI] [PubMed] [Google Scholar]
  7. Benjelloun-Touimi Z., Sansonetti P. J., Parsot C. SepA, the major extracellular protein of Shigella flexneri: autonomous secretion and involvement in tissue invasion. Mol Microbiol. 1995 Jul;17(1):123–135. doi: 10.1111/j.1365-2958.1995.mmi_17010123.x. [DOI] [PubMed] [Google Scholar]
  8. Buysse J. M., Stover C. K., Oaks E. V., Venkatesan M., Kopecko D. J. Molecular cloning of invasion plasmid antigen (ipa) genes from Shigella flexneri: analysis of ipa gene products and genetic mapping. J Bacteriol. 1987 Jun;169(6):2561–2569. doi: 10.1128/jb.169.6.2561-2569.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Chang C. N., Kuang W. J., Chen E. Y. Nucleotide sequence of the alkaline phosphatase gene of Escherichia coli. Gene. 1986;44(1):121–125. doi: 10.1016/0378-1119(86)90050-8. [DOI] [PubMed] [Google Scholar]
  10. Dassa E., Cahu M., Desjoyaux-Cherel B., Boquet P. L. The acid phosphatase with optimum pH of 2.5 of Escherichia coli. Physiological and Biochemical study. J Biol Chem. 1982 Jun 25;257(12):6669–6676. [PubMed] [Google Scholar]
  11. FALKOW S., SCHNEIDER H., BARON L. S., FORMAL S. B. VIRULENCE OF ESCHERICHIA-SHIGELLA GENETIC HYBRIDS FOR THE GUINEA PIG. J Bacteriol. 1963 Dec;86:1251–1258. doi: 10.1128/jb.86.6.1251-1258.1963. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Groisman E. A., Ochman H. Cognate gene clusters govern invasion of host epithelial cells by Salmonella typhimurium and Shigella flexneri. EMBO J. 1993 Oct;12(10):3779–3787. doi: 10.1002/j.1460-2075.1993.tb06056.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Hartman A. B., Venkatesan M., Oaks E. V., Buysse J. M. Sequence and molecular characterization of a multicopy invasion plasmid antigen gene, ipaH, of Shigella flexneri. J Bacteriol. 1990 Apr;172(4):1905–1915. doi: 10.1128/jb.172.4.1905-1915.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Inouye H., Beckwith J. Synthesis and processing of an Escherichia coli alkaline phosphatase precursor in vitro. Proc Natl Acad Sci U S A. 1977 Apr;74(4):1440–1444. doi: 10.1073/pnas.74.4.1440. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Kasahara M., Nakata A., Shinagawa H. Molecular analysis of the Salmonella typhimurium phoN gene, which encodes nonspecific acid phosphatase. J Bacteriol. 1991 Nov;173(21):6760–6765. doi: 10.1128/jb.173.21.6760-6765.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. LEVINTHAL C., SIGNER E. R., FETHEROLF K. Reactivation and hybridization of reduced alkaline phosphatase. Proc Natl Acad Sci U S A. 1962 Jul 15;48:1230–1237. doi: 10.1073/pnas.48.7.1230. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  18. Lett M. C., Sasakawa C., Okada N., Sakai T., Makino S., Yamada M., Komatsu K., Yoshikawa M. virG, a plasmid-coded virulence gene of Shigella flexneri: identification of the virG protein and determination of the complete coding sequence. J Bacteriol. 1989 Jan;171(1):353–359. doi: 10.1128/jb.171.1.353-359.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. 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]
  20. Makino K., Shinagawa H., Amemura M., Kimura S., Nakata A., Ishihama A. Regulation of the phosphate regulon of Escherichia coli. Activation of pstS transcription by PhoB protein in vitro. J Mol Biol. 1988 Sep 5;203(1):85–95. doi: 10.1016/0022-2836(88)90093-9. [DOI] [PubMed] [Google Scholar]
  21. Makino S., Sasakawa C., Kamata K., Kurata T., Yoshikawa M. A genetic determinant required for continuous reinfection of adjacent cells on large plasmid in S. flexneri 2a. Cell. 1986 Aug 15;46(4):551–555. doi: 10.1016/0092-8674(86)90880-9. [DOI] [PubMed] [Google Scholar]
  22. Makino S., Sasakawa C., Yoshikawa M. Genetic relatedness of the basic replicon of the virulence plasmid in shigellae and enteroinvasive Escherichia coli. Microb Pathog. 1988 Oct;5(4):267–274. doi: 10.1016/0882-4010(88)90099-x. [DOI] [PubMed] [Google Scholar]
  23. Matsudaira P. Sequence from picomole quantities of proteins electroblotted onto polyvinylidene difluoride membranes. J Biol Chem. 1987 Jul 25;262(21):10035–10038. [PubMed] [Google Scholar]
  24. Nakata N., Sasakawa C., Okada N., Tobe T., Fukuda I., Suzuki T., Komatsu K., Yoshikawa M. Identification and characterization of virK, a virulence-associated large plasmid gene essential for intercellular spreading of Shigella flexneri. Mol Microbiol. 1992 Aug;6(16):2387–2395. doi: 10.1111/j.1365-2958.1992.tb01413.x. [DOI] [PubMed] [Google Scholar]
  25. Pond J. L., Eddy C. K., Mackenzie K. F., Conway T., Borecky D. J., Ingram L. O. Cloning, sequencing, and characterization of the principal acid phosphatase, the phoC+ product, from Zymomonas mobilis. J Bacteriol. 1989 Feb;171(2):767–774. doi: 10.1128/jb.171.2.767-774.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Sakai T., Sasakawa C., Makino S., Kamata K., Yoshikawa M. Molecular cloning of a genetic determinant for Congo red binding ability which is essential for the virulence of Shigella flexneri. Infect Immun. 1986 Feb;51(2):476–482. doi: 10.1128/iai.51.2.476-482.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Sakai T., Sasakawa C., Makino S., Yoshikawa M. DNA sequence and product analysis of the virF locus responsible for congo red binding and cell invasion in Shigella flexneri 2a. Infect Immun. 1986 Nov;54(2):395–402. doi: 10.1128/iai.54.2.395-402.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. 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]
  29. Sansonetti P. J., Kopecko D. J., Formal S. B. Involvement of a plasmid in the invasive ability of Shigella flexneri. Infect Immun. 1982 Mar;35(3):852–860. doi: 10.1128/iai.35.3.852-860.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Sansonetti P. J., Kopecko D. J., Formal S. B. Shigella sonnei plasmids: evidence that a large plasmid is necessary for virulence. Infect Immun. 1981 Oct;34(1):75–83. doi: 10.1128/iai.34.1.75-83.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Sasakawa C., Adler B., Tobe T., Okada N., Nagai S., Komatsu K., Yoshikawa M. Functional organization and nucleotide sequence of virulence Region-2 on the large virulence plasmid in Shigella flexneri 2a. Mol Microbiol. 1989 Sep;3(9):1191–1201. doi: 10.1111/j.1365-2958.1989.tb00269.x. [DOI] [PubMed] [Google Scholar]
  32. Sasakawa C., Berg D. E. IS50-mediated inverse transposition. Discrimination between the two ends of an IS element. J Mol Biol. 1982 Aug 5;159(2):257–271. doi: 10.1016/0022-2836(82)90495-8. [DOI] [PubMed] [Google Scholar]
  33. Sasakawa C., Kamata K., Sakai T., Murayama S. Y., Makino S., Yoshikawa M. Molecular alteration of the 140-megadalton plasmid associated with loss of virulence and Congo red binding activity in Shigella flexneri. Infect Immun. 1986 Feb;51(2):470–475. doi: 10.1128/iai.51.2.470-475.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Sasakawa C., Komatsu K., Tobe T., Suzuki T., Yoshikawa M. Eight genes in region 5 that form an operon are essential for invasion of epithelial cells by Shigella flexneri 2a. J Bacteriol. 1993 Apr;175(8):2334–2346. doi: 10.1128/jb.175.8.2334-2346.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Sasakawa C., Makino S., Kamata K., Yoshikawa M. Isolation, characterization, and mapping of Tn5 insertions into the 140-megadalton invasion plasmid defective in the mouse Sereny test in Shigella flexneri 2a. Infect Immun. 1986 Oct;54(1):32–36. doi: 10.1128/iai.54.1.32-36.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Scholten M., Janssen R., Bogaarts C., van Strien J., Tommassen J. The pho regulon of Shigella flexneri. Mol Microbiol. 1995 Jan;15(2):247–254. doi: 10.1111/j.1365-2958.1995.tb02239.x. [DOI] [PubMed] [Google Scholar]
  37. Silva R. M., Saadi S., Maas W. K. A basic replicon of virulence-associated plasmids of Shigella spp. and enteroinvasive Escherichia coli is homologous with a basic replicon in plasmids of IncF groups. Infect Immun. 1988 Apr;56(4):836–842. doi: 10.1128/iai.56.4.836-842.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Thaller M. C., Berlutti F., Schippa S., Lombardi G., Rossolini G. M. Characterization and sequence of PhoC, the principal phosphate-irrepressible acid phosphatase of Morganella morganii. Microbiology. 1994 Jun;140(Pt 6):1341–1350. doi: 10.1099/00221287-140-6-1341. [DOI] [PubMed] [Google Scholar]
  39. Tobe T., Nagai S., Okada N., Adler B., Yoshikawa M., Sasakawa C. Temperature-regulated expression of invasion genes in Shigella flexneri is controlled through the transcriptional activation of the virB gene on the large plasmid. Mol Microbiol. 1991 Apr;5(4):887–893. doi: 10.1111/j.1365-2958.1991.tb00762.x. [DOI] [PubMed] [Google Scholar]
  40. Tommassen J., Lugtenberg B. Outer membrane protein e of Escherichia coli K-12 is co-regulated with alkaline phosphatase. J Bacteriol. 1980 Jul;143(1):151–157. doi: 10.1128/jb.143.1.151-157.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Uchiya K., Tobe T., Komatsu K., Suzuki T., Watarai M., Fukuda I., Yoshikawa M., Sasakawa C. Identification of a novel virulence gene, virA, on the large plasmid of Shigella, involved in invasion and intercellular spreading. Mol Microbiol. 1995 Jul;17(2):241–250. doi: 10.1111/j.1365-2958.1995.mmi_17020241.x. [DOI] [PubMed] [Google Scholar]
  43. Venkatesan M. M., Buysse J. M., Kopecko D. J. Characterization of invasion plasmid antigen genes (ipaBCD) from Shigella flexneri. Proc Natl Acad Sci U S A. 1988 Dec;85(23):9317–9321. doi: 10.1073/pnas.85.23.9317. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Venkatesan M. M., Buysse J. M., Oaks E. V. Surface presentation of Shigella flexneri invasion plasmid antigens requires the products of the spa locus. J Bacteriol. 1992 Mar;174(6):1990–2001. doi: 10.1128/jb.174.6.1990-2001.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]

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