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. 1986 Nov;168(2):590–594. doi: 10.1128/jb.168.2.590-594.1986

Proline transport in Salmonella typhimurium: putP permease mutants with altered substrate specificity.

D K Dila, S R Maloy
PMCID: PMC213521  PMID: 3536852

Abstract

The putP gene encodes a proline permease required for Salmonella typhimurium LT2 to grow on proline as the sole source of nitrogen. The wild-type strain is sensitive to two toxic proline analogs (azetidine-2-carboxylic acid and 3,4-dehydroproline) also transported by the putP permease. Most mutations in putP prevent transport of all three substrates. Such mutants are unable to grow on proline and are resistant to both of the analogs. To define domains of the putP gene that specify the substrate binding site, we used localized mutagenesis to isolate rare mutants with altered substrate specificity. The position of the mutations in the putP gene was determined by deletion mapping. Most of the mutations are located in three small (approximately 100-base-pair) deletion intervals of the putP gene. The sensitivity of the mutants to the proline analogs was quantitated by radial streaking to determine the affinity of the mutant permeases for the substrates. Some of the mutants showed apparent changes in the kinetics of the substrates transported. These results indicate that the substrate specificity mutations are probably due to amino acid substitutions at or near the active site of proline permease.

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Selected References

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

  1. Berkowitz D., Hushon J. M., Whitfield H. J., Jr, Roth J., Ames B. N. Procedure for identifying nonsense mutations. J Bacteriol. 1968 Jul;96(1):215–220. doi: 10.1128/jb.96.1.215-220.1968. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Brooker R. J., Wilson T. H. Isolation and nucleotide sequencing of lactose carrier mutants that transport maltose. Proc Natl Acad Sci U S A. 1985 Jun;82(12):3959–3963. doi: 10.1073/pnas.82.12.3959. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Cairney J., Booth I. R., Higgins C. F. Osmoregulation of gene expression in Salmonella typhimurium: proU encodes an osmotically induced betaine transport system. J Bacteriol. 1985 Dec;164(3):1224–1232. doi: 10.1128/jb.164.3.1224-1232.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Cairney J., Booth I. R., Higgins C. F. Salmonella typhimurium proP gene encodes a transport system for the osmoprotectant betaine. J Bacteriol. 1985 Dec;164(3):1218–1223. doi: 10.1128/jb.164.3.1218-1223.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Cairney J., Higgins C. F., Booth I. R. Proline uptake through the major transport system of Salmonella typhimurium is coupled to sodium ions. J Bacteriol. 1984 Oct;160(1):22–27. doi: 10.1128/jb.160.1.22-27.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Chen C. C., Tsuchiya T., Yamane Y., Wood J. M., Wilson T. H. Na+ (Li+)-proline cotransport in Escherichia coli. J Membr Biol. 1985;84(2):157–164. doi: 10.1007/BF01872213. [DOI] [PubMed] [Google Scholar]
  7. Csonka L. N. A third L-proline permease in Salmonella typhimurium which functions in media of elevated osmotic strength. J Bacteriol. 1982 Sep;151(3):1433–1443. doi: 10.1128/jb.151.3.1433-1443.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Gutnick D., Calvo J. M., Klopotowski T., Ames B. N. Compounds which serve as the sole source of carbon or nitrogen for Salmonella typhimurium LT-2. J Bacteriol. 1969 Oct;100(1):215–219. doi: 10.1128/jb.100.1.215-219.1969. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Hobson A. C., Gho D., Müller-Hill B. Isolation, genetic analysis, and characterization of Escherichia coli mutants with defects in the lacY gene. J Bacteriol. 1977 Sep;131(3):830–838. doi: 10.1128/jb.131.3.830-838.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Hong J. S., Ames B. N. Localized mutagenesis of any specific small region of the bacterial chromosome. Proc Natl Acad Sci U S A. 1971 Dec;68(12):3158–3162. doi: 10.1073/pnas.68.12.3158. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Kaback H. R. The lac carrier protein in Escherichia coli. J Membr Biol. 1983;76(2):95–112. doi: 10.1007/BF02000610. [DOI] [PubMed] [Google Scholar]
  12. Maloy S. R., Roth J. R. Regulation of proline utilization in Salmonella typhimurium: characterization of put::Mu d(Ap, lac) operon fusions. J Bacteriol. 1983 May;154(2):561–568. doi: 10.1128/jb.154.2.561-568.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Markgraf M., Bocklage H., Müller-Hill B. A change of threonine 266 to isoleucine in the lac permease of Escherichia coli diminishes the transport of lactose and increases the transport of maltose. Mol Gen Genet. 1985;198(3):473–475. doi: 10.1007/BF00332941. [DOI] [PubMed] [Google Scholar]
  14. Menzel R., Roth J. Identification and mapping of a second proline permease Salmonella typhimurium. J Bacteriol. 1980 Mar;141(3):1064–1070. doi: 10.1128/jb.141.3.1064-1070.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Menzel R., Roth J. Regulation of the genes for proline utilization in Salmonella typhimurium: autogenous repression by the putA gene product. J Mol Biol. 1981 May 5;148(1):21–44. doi: 10.1016/0022-2836(81)90233-3. [DOI] [PubMed] [Google Scholar]
  16. Mieschendahl M., Büchel D., Bocklage H., Müller-Hill B. Mutations in the lacY gene of Escherichia coli define functional organization of lactose permease. Proc Natl Acad Sci U S A. 1981 Dec;78(12):7652–7656. doi: 10.1073/pnas.78.12.7652. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Niiya S., Yamasaki K., Wilson T. H., Tsuchiya T. Altered cation coupling to melibiose transport in mutants of Escherichia coli. J Biol Chem. 1982 Aug 10;257(15):8902–8906. [PubMed] [Google Scholar]
  18. Ratzkin B., Roth J. Cluster of genes controlling proline degradation in Salmonella typhimurium. J Bacteriol. 1978 Feb;133(2):744–754. doi: 10.1128/jb.133.2.744-754.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Sanderson K. E., Roth J. R. Linkage map of Salmonella typhimurium, Edition VI. Microbiol Rev. 1983 Sep;47(3):410–453. doi: 10.1128/mr.47.3.410-453.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Schmieger H. Phage P22-mutants with increased or decreased transduction abilities. Mol Gen Genet. 1972;119(1):75–88. doi: 10.1007/BF00270447. [DOI] [PubMed] [Google Scholar]
  21. Shiota S., Yamane Y., Futai M., Tsuchiya T. Escherichia coli mutants possessing an Li+-resistant melibiose carrier. J Bacteriol. 1985 Apr;162(1):106–109. doi: 10.1128/jb.162.1.106-109.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Shuman H. A., Beckwith J. Escherichia coli K-12 mutants that allow transport of maltose via the beta-galactoside transport system. J Bacteriol. 1979 Jan;137(1):365–373. doi: 10.1128/jb.137.1.365-373.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Tristram H., Neale S. The activity and specificity of the proline permease in wild-type and analogue-resistant strains of Escherichia coli. J Gen Microbiol. 1968 Jan;50(1):121–137. doi: 10.1099/00221287-50-1-121. [DOI] [PubMed] [Google Scholar]
  24. Tsuchiya T., Wilson T. H. Cation-sugar cotransport in the melibiose transport system of Escherichia coli. Membr Biochem. 1978;2(1):63–79. doi: 10.3109/09687687809063858. [DOI] [PubMed] [Google Scholar]
  25. Wilson D. B. Cellular transport mechanisms. Annu Rev Biochem. 1978;47:933–965. doi: 10.1146/annurev.bi.47.070178.004441. [DOI] [PubMed] [Google Scholar]
  26. Wilson T. H., Kusch M., Kashket E. R. A mutant in Escherichia coli energy-uncoupled for lactose transporta defect in the lactose-operon. Biochem Biophys Res Commun. 1970 Sep 30;40(6):1409–1414. doi: 10.1016/0006-291x(70)90024-0. [DOI] [PubMed] [Google Scholar]
  27. Wong P. T., Kashket E. R., Wilson T. H. Energy coupling in the lactose transport system of Escherichia coli. Proc Natl Acad Sci U S A. 1970 Jan;65(1):63–69. doi: 10.1073/pnas.65.1.63. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Wood J. M., Zadworny D. Amplification of the put genes and identification of the put gene products in Escherichia coli K12. Can J Biochem. 1980 Oct;58(10):787–796. doi: 10.1139/o80-110. [DOI] [PubMed] [Google Scholar]
  29. Wood J. M., Zadworny D. Characterization of an inducible porter required for L-proline catabolism by Escherichia coli K12. Can J Biochem. 1979 Oct;57(10):1191–1199. doi: 10.1139/o79-155. [DOI] [PubMed] [Google Scholar]
  30. Wright J. K., Seckler R. The lactose/H+ carrier of Escherichia coli: lac YUN mutation decreases the rate of active transport and mimics an energy-uncoupled phenotype. Biochem J. 1985 Apr 1;227(1):287–297. doi: 10.1042/bj2270287. [DOI] [PMC free article] [PubMed] [Google Scholar]

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