Skip to main content
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1989 Oct;171(10):5601–5606. doi: 10.1128/jb.171.10.5601-5606.1989

Regulation of the phosphate regulon of Escherichia coli: analysis of mutant phoB and phoR genes causing different phenotypes.

M Yamada 1, K Makino 1, M Amemura 1, H Shinagawa 1, A Nakata 1
PMCID: PMC210403  PMID: 2676981

Abstract

The phoB gene product of Escherichia coli is the transcriptional activator for the genes in the phosphate regulon as well as for phoB itself, all of which are induced by phosphate starvation. The phoR gene product modulates PhoB function in response to the phosphate concentrations in the medium. We quantitatively compared the levels of expression of the phoA, phoB, phoE, and pstS genes in several phoB mutants with different phenotypes by constructing operon fusions of these genes with the gene for chloramphenicol acetyltransferase. Although all the phoB mutants examined had little activator function for phoA, three among the four mutants showed various levels of the activator function for phoB, pstS, and phoE. To study the functional motifs of the PhoB and PhoR proteins, we cloned and sequenced the four classical phoB and six phoR mutant genes. All of the phoB mutations and one of the phoR mutations were missense mutations, and most of the altered amino acids were in the highly conserved amino acids among the regulatory proteins homologous to PhoB or PhoR protein, such as the OmpR, SfrA, and VirG proteins or the EnvZ, CpxA, and VirA proteins. The other five phoR mutations were nonsense mutations.

Full text

PDF
5601

Selected References

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

  1. Amemura M., Shinagawa H., Makino K., Otsuji N., Nakata A. Cloning of and complementation tests with alkaline phosphatase regulatory genes (phoS and phoT) of Escherichia coli. J Bacteriol. 1982 Nov;152(2):692–701. doi: 10.1128/jb.152.2.692-701.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Birnboim H. C., Doly J. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 1979 Nov 24;7(6):1513–1523. doi: 10.1093/nar/7.6.1513. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bolivar F., Rodriguez R. L., Greene P. J., Betlach M. C., Heyneker H. L., Boyer H. W., Crosa J. H., Falkow S. Construction and characterization of new cloning vehicles. II. A multipurpose cloning system. Gene. 1977;2(2):95–113. [PubMed] [Google Scholar]
  4. Bracha M., Yagil E. A ne type of alkaline phosphatase-negative mutants in Escherichia coli K12. Mol Gen Genet. 1973 Mar 27;122(1):53–60. doi: 10.1007/BF00337973. [DOI] [PubMed] [Google Scholar]
  5. Brosius J. Plasmid vectors for the selection of promoters. Gene. 1984 Feb;27(2):151–160. doi: 10.1016/0378-1119(84)90136-7. [DOI] [PubMed] [Google Scholar]
  6. ECHOLS H., GAREN A., GAREN S., TORRIANI A. Genetic control of repression of alkaline phosphatase in E. coli. J Mol Biol. 1961 Aug;3:425–438. doi: 10.1016/s0022-2836(61)80055-7. [DOI] [PubMed] [Google Scholar]
  7. GAREN A., GAREN S. Genetic evidence on the nature of the repressor for alkaline phosphatase in E. coli. J Mol Biol. 1963 May;6:433–438. doi: 10.1016/s0022-2836(63)80054-6. [DOI] [PubMed] [Google Scholar]
  8. Guan C. D., Wanner B., Inouye H. Analysis of regulation of phoB expression using a phoB-cat fusion. J Bacteriol. 1983 Nov;156(2):710–717. doi: 10.1128/jb.156.2.710-717.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Kimura S., Makino K., Shinagawa H., Amemura M., Nakata A. Regulation of the phosphate regulon of Escherichia coli: characterization of the promoter of the pstS gene. Mol Gen Genet. 1989 Feb;215(3):374–380. doi: 10.1007/BF00427032. [DOI] [PubMed] [Google Scholar]
  10. Kreuzer K., Pratt C., Torriani A. Genetic analysis of regulatory mutants of alkaline phosphatase of E. coli. Genetics. 1975 Nov;81(3):459–468. doi: 10.1093/genetics/81.3.459. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. 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]
  12. 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]
  13. Makino K., Shinagawa H., Amemura M., Nakata A. Nucleotide sequence of the phoR gene, a regulatory gene for the phosphate regulon of Escherichia coli. J Mol Biol. 1986 Dec 5;192(3):549–556. doi: 10.1016/0022-2836(86)90275-5. [DOI] [PubMed] [Google Scholar]
  14. Makino K., Shinagawa H., Nakata A. Cloning and characterization of the alkaline phosphatase positive regulatory gene (phoM) of Escherichia coli. Mol Gen Genet. 1984;195(3):381–390. doi: 10.1007/BF00341438. [DOI] [PubMed] [Google Scholar]
  15. 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]
  16. Nixon B. T., Ronson C. W., Ausubel F. M. Two-component regulatory systems responsive to environmental stimuli share strongly conserved domains with the nitrogen assimilation regulatory genes ntrB and ntrC. Proc Natl Acad Sci U S A. 1986 Oct;83(20):7850–7854. doi: 10.1073/pnas.83.20.7850. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Ohsuye K., Nomura M., Tanaka S., Kubota I., Nakazato H., Shinagawa H., Nakata A., Noguchi T. Expression of chemically synthesized alpha-neo-endorphin gene fused to E. coli alkaline phosphatase. Nucleic Acids Res. 1983 Mar 11;11(5):1283–1294. doi: 10.1093/nar/11.5.1283. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Oishi M., Cosloy S. D. The genetic and biochemical basis of the transformability of Escherichia coli K12. Biochem Biophys Res Commun. 1972 Dec 18;49(6):1568–1572. doi: 10.1016/0006-291x(72)90520-7. [DOI] [PubMed] [Google Scholar]
  19. Overbeeke N., Bergmans H., van Mansfeld F., Lugtenberg B. Complete nucleotide sequence of phoE, the structural gene for the phosphate limitation inducible outer membrane pore protein of Escherichia coli K12. J Mol Biol. 1983 Feb 5;163(4):513–532. doi: 10.1016/0022-2836(83)90110-9. [DOI] [PubMed] [Google Scholar]
  20. Ronson C. W., Astwood P. M., Nixon B. T., Ausubel F. M. Deduced products of C4-dicarboxylate transport regulatory genes of Rhizobium leguminosarum are homologous to nitrogen regulatory gene products. Nucleic Acids Res. 1987 Oct 12;15(19):7921–7934. doi: 10.1093/nar/15.19.7921. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Ronson C. W., Nixon B. T., Ausubel F. M. Conserved domains in bacterial regulatory proteins that respond to environmental stimuli. Cell. 1987 Jun 5;49(5):579–581. doi: 10.1016/0092-8674(87)90530-7. [DOI] [PubMed] [Google Scholar]
  22. Sanger F., Coulson A. R., Barrell B. G., Smith A. J., Roe B. A. Cloning in single-stranded bacteriophage as an aid to rapid DNA sequencing. J Mol Biol. 1980 Oct 25;143(2):161–178. doi: 10.1016/0022-2836(80)90196-5. [DOI] [PubMed] [Google Scholar]
  23. 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]
  24. Shaw W. V. Chloramphenicol acetyltransferase from chloramphenicol-resistant bacteria. Methods Enzymol. 1975;43:737–755. doi: 10.1016/0076-6879(75)43141-x. [DOI] [PubMed] [Google Scholar]
  25. Shinagawa H., Makino K., Nakata A. Regulation of the pho regulon in Escherichia coli K-12. Genetic and physiological regulation of the positive regulatory gene phoB. J Mol Biol. 1983 Aug 15;168(3):477–488. doi: 10.1016/s0022-2836(83)80297-6. [DOI] [PubMed] [Google Scholar]
  26. Southern E. M. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975 Nov 5;98(3):503–517. doi: 10.1016/s0022-2836(75)80083-0. [DOI] [PubMed] [Google Scholar]
  27. Stock A., Chen T., Welsh D., Stock J. CheA protein, a central regulator of bacterial chemotaxis, belongs to a family of proteins that control gene expression in response to changing environmental conditions. Proc Natl Acad Sci U S A. 1988 Mar;85(5):1403–1407. doi: 10.1073/pnas.85.5.1403. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Tommassen J., Koster M., Overduin P. Molecular analysis of the promoter region of the Escherichia coli K-12 phoE gene. Identification of an element, upstream from the promoter, required for efficient expression of phoE protein. J Mol Biol. 1987 Dec 20;198(4):633–641. doi: 10.1016/0022-2836(87)90206-3. [DOI] [PubMed] [Google Scholar]
  29. Tommassen J., de Geus P., Lugtenberg B., Hackett J., Reeves P. Regulation of the pho regulon of Escherichia coli K-12. Cloning of the regulatory genes phoB and phoR and identification of their gene products. J Mol Biol. 1982 May 15;157(2):265–274. doi: 10.1016/0022-2836(82)90233-9. [DOI] [PubMed] [Google Scholar]
  30. Vieira J., Messing J. The pUC plasmids, an M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene. 1982 Oct;19(3):259–268. doi: 10.1016/0378-1119(82)90015-4. [DOI] [PubMed] [Google Scholar]
  31. Wanner B. L., Latterell P. Mutants affected in alkaline phosphatase, expression: evidence for multiple positive regulators of the phosphate regulon in Escherichia coli. Genetics. 1980 Oct;96(2):353–366. doi: 10.1093/genetics/96.2.353. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Willsky G. R., Malamy M. H. Control of the synthesis of alkaline phosphatase and the phosphate-binding protein in Escherichia coli. J Bacteriol. 1976 Jul;127(1):595–609. doi: 10.1128/jb.127.1.595-609.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. 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]
  34. Winans S. C., Elledge S. J., Krueger J. H., Walker G. C. Site-directed insertion and deletion mutagenesis with cloned fragments in Escherichia coli. J Bacteriol. 1985 Mar;161(3):1219–1221. doi: 10.1128/jb.161.3.1219-1221.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. 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]

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

RESOURCES