Skip to main content
NCBI home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1993 Nov;175(21):6797–6809. doi: 10.1128/jb.175.21.6797-6809.1993

Use of the rep technique for allele replacement to construct mutants with deletions of the pstSCAB-phoU operon: evidence of a new role for the PhoU protein in the phosphate regulon.

P M Steed 1, B L Wanner 1
PMCID: PMC206803  PMID: 8226621

Abstract

The phosphate regulon is negatively regulated by the PstSCAB transporter and PhoU protein by a mechanism that may involve protein-protein interaction(s) between them and the Pi sensor protein, PhoR. In order to study such presumed interaction(s), mutants with defined deletions of the pstSCAB-phoU operon were made. This was done by construction of M13 recombinant phage carrying these mutations and by recombination of them onto the chromosome by using a rep host (which cannot replicate M13) for allele replacement. These mutants were used to show that delta (pstSCAB-phoU) and delta (pstB-phoU) mutations abolished Pi uptake by the PstSCAB transporter, as expected, and that delta phoU mutations had no effect on uptake. Unexpectedly, delta phoU mutations had a severe growth defect, and this growth defect was (largely) alleviated by a compensatory mutation in the pstSCAB genes or in the phoBR operon, whose gene products positively regulate expression of the pstSCAB-phoU operon. Because delta phoU mutants that synthesize a functional PstSCAB transporter constitutively grew extremely poorly, the PhoU protein must have a new role, in addition to its role as a negative regulator. A role for the PhoU protein in intracellular Pi metabolism is proposed. Further, our results contradict those of M. Muda, N. N. Rao, and A. Torriani (J. Bacteriol. 174:8057-8064, 1992), who reported that the PhoU protein was required for Pi uptake.

Full text

PDF
6797

Images in this article

6805Image
on p.6805
6807Image
on p.6807

Selected References

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

  1. Agrawal D. K., Wanner B. L. A phoA structural gene mutation that conditionally affects formation of the enzyme bacterial alkaline phosphatase. J Bacteriol. 1990 Jun;172(6):3180–3190. doi: 10.1128/jb.172.6.3180-3190.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Amemura M., Makino K., Shinagawa H., Kobayashi A., Nakata A. Nucleotide sequence of the genes involved in phosphate transport and regulation of the phosphate regulon in Escherichia coli. J Mol Biol. 1985 Jul 20;184(2):241–250. doi: 10.1016/0022-2836(85)90377-8. [DOI] [PubMed] [Google Scholar]
  3. 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]
  4. Bebenek K., Kunkel T. A. The use of native T7 DNA polymerase for site-directed mutagenesis. Nucleic Acids Res. 1989 Jul 11;17(13):5408–5408. doi: 10.1093/nar/17.13.5408. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Blum P., Holzschu D., Kwan H. S., Riggs D., Artz S. Gene replacement and retrieval with recombinant M13mp bacteriophages. J Bacteriol. 1989 Jan;171(1):538–546. doi: 10.1128/jb.171.1.538-546.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Cox G. B., Webb D., Godovac-Zimmermann J., Rosenberg H. Arg-220 of the PstA protein is required for phosphate transport through the phosphate-specific transport system in Escherichia coli but not for alkaline phosphatase repression. J Bacteriol. 1988 May;170(5):2283–2286. doi: 10.1128/jb.170.5.2283-2286.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Cox G. B., Webb D., Rosenberg H. Specific amino acid residues in both the PstB and PstC proteins are required for phosphate transport by the Escherichia coli Pst system. J Bacteriol. 1989 Mar;171(3):1531–1534. doi: 10.1128/jb.171.3.1531-1534.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Elvin C. M., Hardy C. M., Rosenberg H. Pi exchange mediated by the GlpT-dependent sn-glycerol-3-phosphate transport system in Escherichia coli. J Bacteriol. 1985 Mar;161(3):1054–1058. doi: 10.1128/jb.161.3.1054-1058.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Kunkel T. A., Bebenek K., McClary J. Efficient site-directed mutagenesis using uracil-containing DNA. Methods Enzymol. 1991;204:125–139. doi: 10.1016/0076-6879(91)04008-c. [DOI] [PubMed] [Google Scholar]
  10. 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]
  11. Messing J. Cloning in M13 phage or how to use biology at its best. Gene. 1991 Apr;100:3–12. doi: 10.1016/0378-1119(91)90344-b. [DOI] [PubMed] [Google Scholar]
  12. 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]
  13. Metcalf W. W., Wanner B. L. Construction of new beta-glucuronidase cassettes for making transcriptional fusions and their use with new methods for allele replacement. Gene. 1993 Jul 15;129(1):17–25. doi: 10.1016/0378-1119(93)90691-u. [DOI] [PubMed] [Google Scholar]
  14. Metcalf W. W., Wanner B. L. Mutational analysis of an Escherichia coli fourteen-gene operon for phosphonate degradation, using TnphoA' elements. J Bacteriol. 1993 Jun;175(11):3430–3442. doi: 10.1128/jb.175.11.3430-3442.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Muda M., Rao N. N., Torriani A. Role of PhoU in phosphate transport and alkaline phosphatase regulation. J Bacteriol. 1992 Dec;174(24):8057–8064. doi: 10.1128/jb.174.24.8057-8064.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Prentki P., Krisch H. M. In vitro insertional mutagenesis with a selectable DNA fragment. Gene. 1984 Sep;29(3):303–313. doi: 10.1016/0378-1119(84)90059-3. [DOI] [PubMed] [Google Scholar]
  17. Shapira S. K., Chou J., Richaud F. V., Casadaban M. J. New versatile plasmid vectors for expression of hybrid proteins coded by a cloned gene fused to lacZ gene sequences encoding an enzymatically active carboxy-terminal portion of beta-galactosidase. Gene. 1983 Nov;25(1):71–82. doi: 10.1016/0378-1119(83)90169-5. [DOI] [PubMed] [Google Scholar]
  18. Simons R. W., Houman F., Kleckner N. Improved single and multicopy lac-based cloning vectors for protein and operon fusions. Gene. 1987;53(1):85–96. doi: 10.1016/0378-1119(87)90095-3. [DOI] [PubMed] [Google Scholar]
  19. Surin B. P., Rosenberg H., Cox G. B. Phosphate-specific transport system of Escherichia coli: nucleotide sequence and gene-polypeptide relationships. J Bacteriol. 1985 Jan;161(1):189–198. doi: 10.1128/jb.161.1.189-198.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Tessman E. S., Peterson P. K. Bacterial rep- mutations that block development of small DNA bacteriophages late in infection. J Virol. 1976 Nov;20(2):400–412. doi: 10.1128/jvi.20.2.400-412.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Wanner B. L., Boline J. A. Mapping and molecular cloning of the phn (psiD) locus for phosphonate utilization in Escherichia coli. J Bacteriol. 1990 Mar;172(3):1186–1196. doi: 10.1128/jb.172.3.1186-1196.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Wanner B. L. Gene regulation by phosphate in enteric bacteria. J Cell Biochem. 1993 Jan;51(1):47–54. doi: 10.1002/jcb.240510110. [DOI] [PubMed] [Google Scholar]
  23. Wanner B. L. Is cross regulation by phosphorylation of two-component response regulator proteins important in bacteria? J Bacteriol. 1992 Apr;174(7):2053–2058. doi: 10.1128/jb.174.7.2053-2058.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Wanner B. L., McSharry R. Phosphate-controlled gene expression in Escherichia coli K12 using Mudl-directed lacZ fusions. J Mol Biol. 1982 Jul 5;158(3):347–363. doi: 10.1016/0022-2836(82)90202-9. [DOI] [PubMed] [Google Scholar]
  25. Wanner B. L. Molecular cloning of Mu d(bla lacZ) transcriptional and translational fusions. J Bacteriol. 1987 May;169(5):2026–2030. doi: 10.1128/jb.169.5.2026-2030.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Wanner B. L. Novel regulatory mutants of the phosphate regulon in Escherichia coli K-12. J Mol Biol. 1986 Sep 5;191(1):39–58. doi: 10.1016/0022-2836(86)90421-3. [DOI] [PubMed] [Google Scholar]
  27. Wanner B. L. Overlapping and separate controls on the phosphate regulon in Escherichia coli K12. J Mol Biol. 1983 May 25;166(3):283–308. doi: 10.1016/s0022-2836(83)80086-2. [DOI] [PubMed] [Google Scholar]
  28. Wanner B. L., Sarthy A., Beckwith J. Escherichia coli pleiotropic mutant that reduces amounts of several periplasmic and outer membrane proteins. J Bacteriol. 1979 Oct;140(1):229–239. doi: 10.1128/jb.140.1.229-239.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Wanner B. L., Wilmes-Riesenberg M. R. Involvement of phosphotransacetylase, acetate kinase, and acetyl phosphate synthesis in control of the phosphate regulon in Escherichia coli. J Bacteriol. 1992 Apr;174(7):2124–2130. doi: 10.1128/jb.174.7.2124-2130.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Wanner B. L., Wilmes M. R., Young D. C. Control of bacterial alkaline phosphatase synthesis and variation in an Escherichia coli K-12 phoR mutant by adenyl cyclase, the cyclic AMP receptor protein, and the phoM operon. J Bacteriol. 1988 Mar;170(3):1092–1102. doi: 10.1128/jb.170.3.1092-1102.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Webb D. C., Rosenberg H., Cox G. B. Mutational analysis of the Escherichia coli phosphate-specific transport system, a member of the traffic ATPase (or ABC) family of membrane transporters. A role for proline residues in transmembrane helices. J Biol Chem. 1992 Dec 5;267(34):24661–24668. [PubMed] [Google Scholar]
  32. Wilmes-Riesenberg M. R., Wanner B. L. TnphoA and TnphoA' elements for making and switching fusions for study of transcription, translation, and cell surface localization. J Bacteriol. 1992 Jul;174(14):4558–4575. doi: 10.1128/jb.174.14.4558-4575.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES