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. 1992 Sep;174(18):5961–5970. doi: 10.1128/jb.174.18.5961-5970.1992

Cloning, sequencing, and enhanced expression of the dihydropteroate synthase gene of Escherichia coli MC4100.

W S Dallas 1, J E Gowen 1, P H Ray 1, M J Cox 1, I K Dev 1
PMCID: PMC207134  PMID: 1522070

Abstract

The Escherichia coli gene coding for dihydropteroate synthase (DHPS) has been cloned and sequenced. The protein has 282 amino acids and a compositional molecular mass of 30,314 daltons. Increased expression of the enzyme was realized by using a T7 expression system. The enzyme was purified and crystallized. A temperature-sensitive mutant was isolated and found to express a DHPS with a lower specific activity and lower affinities for para-aminobenzoic acid and sulfathiazole. The allele had a point mutation that changed a phenylalanine codon to a leucine codon, and the mutation was in a codon that is conserved among published DHPS sequences.

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Selected References

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  1. Ames G. F. Resolution of bacterial proteins by polyacrylamide gel electrophoresis on slabs. Membrane, soluble, and periplasmic fractions. J Biol Chem. 1974 Jan 25;249(2):634–644. [PubMed] [Google Scholar]
  2. BROWN G. M. The biosynthesis of folic acid. II. Inhibition by sulfonamides. J Biol Chem. 1962 Feb;237:536–540. [PubMed] [Google Scholar]
  3. Beck E., Ludwig G., Auerswald E. A., Reiss B., Schaller H. Nucleotide sequence and exact localization of the neomycin phosphotransferase gene from transposon Tn5. Gene. 1982 Oct;19(3):327–336. doi: 10.1016/0378-1119(82)90023-3. [DOI] [PubMed] [Google Scholar]
  4. Bock L., Miller G. H., Schaper K. J., Seydel J. K. Sulfonamide structure-activity relationships in a cell-free system. 2. Proof for the formation of a sulfonamide-containing folate analog. J Med Chem. 1974 Jan;17(1):23–28. doi: 10.1021/jm00247a006. [DOI] [PubMed] [Google Scholar]
  5. Bolivar F. Construction and characterization of new cloning vehicles. III. Derivatives of plasmid pBR322 carrying unique Eco RI sites for selection of Eco RI generated recombinant DNA molecules. Gene. 1978 Oct;4(2):121–136. doi: 10.1016/0378-1119(78)90025-2. [DOI] [PubMed] [Google Scholar]
  6. 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]
  7. Casadaban M. J. Transposition and fusion of the lac genes to selected promoters in Escherichia coli using bacteriophage lambda and Mu. J Mol Biol. 1976 Jul 5;104(3):541–555. doi: 10.1016/0022-2836(76)90119-4. [DOI] [PubMed] [Google Scholar]
  8. Chang A. C., Cohen S. N. Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from the P15A cryptic miniplasmid. J Bacteriol. 1978 Jun;134(3):1141–1156. doi: 10.1128/jb.134.3.1141-1156.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Cohen S. N., Chang A. C. Revised interpretation of the origin of the pSC101 plasmid. J Bacteriol. 1977 Nov;132(2):734–737. doi: 10.1128/jb.132.2.734-737.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Enquist L. W., Skalka A. Replication of bacteriophage lambda DNA dependent on the function of host and viral genes. I. Interaction of red, gam and rec. J Mol Biol. 1973 Apr 5;75(2):185–212. doi: 10.1016/0022-2836(73)90016-8. [DOI] [PubMed] [Google Scholar]
  11. Ferone R. The enzymic synthesis of dihydropteroate and dihydrofolate by Plasmodium berghei. J Protozool. 1973 Aug;20(3):459–464. doi: 10.1111/j.1550-7408.1973.tb00926.x. [DOI] [PubMed] [Google Scholar]
  12. Friden P., Newman T., Freundlich M. Nucleotide sequence of the ilvB promoter-regulatory region: a biosynthetic operon controlled by attenuation and cyclic AMP. Proc Natl Acad Sci U S A. 1982 Oct;79(20):6156–6160. doi: 10.1073/pnas.79.20.6156. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Guerry P., van Embden J., Falkow S. Molecular nature of two nonconjugative plasmids carrying drug resistance genes. J Bacteriol. 1974 Feb;117(2):619–630. doi: 10.1128/jb.117.2.619-630.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. HOTCHKISS R. D., EVANS A. H. Fine structure of a genetically modified enzyme as revealed by relative affinities for modified substrate. Fed Proc. 1960 Dec;19:912–925. [PubMed] [Google Scholar]
  15. Harley C. B., Reynolds R. P. Analysis of E. coli promoter sequences. Nucleic Acids Res. 1987 Mar 11;15(5):2343–2361. doi: 10.1093/nar/15.5.2343. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Harvey R. J. Growth and initiation of protein synthesis in Escherichia coli in the presence of trimethoprim. J Bacteriol. 1973 Apr;114(1):309–322. doi: 10.1128/jb.114.1.309-322.1973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Heffron F., Bedinger P., Champoux J. J., Falkow S. Deletions affecting the transposition of an antibiotic resistance gene. Proc Natl Acad Sci U S A. 1977 Feb;74(2):702–706. doi: 10.1073/pnas.74.2.702. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Henikoff S. Unidirectional digestion with exonuclease III creates targeted breakpoints for DNA sequencing. Gene. 1984 Jun;28(3):351–359. doi: 10.1016/0378-1119(84)90153-7. [DOI] [PubMed] [Google Scholar]
  19. Hsu L. M., Klee H. J., Zagorski J., Fournier M. J. Structure of an Escherichia coli tRNA operon containing linked genes for arginine, histidine, leucine, and proline tRNAs. J Bacteriol. 1984 Jun;158(3):934–942. doi: 10.1128/jb.158.3.934-942.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Hutchison C. A., 3rd, Phillips S., Edgell M. H., Gillam S., Jahnke P., Smith M. Mutagenesis at a specific position in a DNA sequence. J Biol Chem. 1978 Sep 25;253(18):6551–6560. [PubMed] [Google Scholar]
  21. Ish-Horowicz D., Burke J. F. Rapid and efficient cosmid cloning. Nucleic Acids Res. 1981 Jul 10;9(13):2989–2998. doi: 10.1093/nar/9.13.2989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Ishii S., Kuroki K., Imamoto F. tRNAMetf2 gene in the leader region of the nusA operon in Escherichia coli. Proc Natl Acad Sci U S A. 1984 Jan;81(2):409–413. doi: 10.1073/pnas.81.2.409. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Jacobs W. R., Barrett J. F., Clark-Curtiss J. E., Curtiss R., 3rd In vivo repackaging of recombinant cosmid molecules for analyses of Salmonella typhimurium, Streptococcus mutans, and mycobacterial genomic libraries. Infect Immun. 1986 Apr;52(1):101–109. doi: 10.1128/iai.52.1.101-109.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  25. Lee J. S., An G., Friesen J. D., Isono K. Cloning and the nucleotide sequence of the genes for Escherichia coli ribosomal proteins L28 (rpmB) and L33 (rpmG). Mol Gen Genet. 1981;184(2):218–223. doi: 10.1007/BF00272908. [DOI] [PubMed] [Google Scholar]
  26. Lopez P., Espinosa M., Greenberg B., Lacks S. A. Sulfonamide resistance in Streptococcus pneumoniae: DNA sequence of the gene encoding dihydropteroate synthase and characterization of the enzyme. J Bacteriol. 1987 Sep;169(9):4320–4326. doi: 10.1128/jb.169.9.4320-4326.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Mullis K. B., Faloona F. A. Specific synthesis of DNA in vitro via a polymerase-catalyzed chain reaction. Methods Enzymol. 1987;155:335–350. doi: 10.1016/0076-6879(87)55023-6. [DOI] [PubMed] [Google Scholar]
  28. Murray V. Improved double-stranded DNA sequencing using the linear polymerase chain reaction. Nucleic Acids Res. 1989 Nov 11;17(21):8889–8889. doi: 10.1093/nar/17.21.8889. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Nagase T., Ishii S., Imamoto F. Differential transcriptional control of the two tRNA(fMet) genes of Escherichia coli K-12. Gene. 1988 Jul 15;67(1):49–57. doi: 10.1016/0378-1119(88)90007-8. [DOI] [PubMed] [Google Scholar]
  30. Nichols B. P., Guay G. G. Gene amplification contributes to sulfonamide resistance in Escherichia coli. Antimicrob Agents Chemother. 1989 Dec;33(12):2042–2048. doi: 10.1128/aac.33.12.2042. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. O'Farrell P. H., Kutter E., Nakanishi M. A restriction map of the bacteriophage T4 genome. Mol Gen Genet. 1980;179(2):421–435. doi: 10.1007/BF00425473. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. PATO M. L., BROWN G. M. MECHANISMS OF RESISTANCE OF ESCHERICHIA COLI TO SULFONAMIDES. Arch Biochem Biophys. 1963 Dec;103:443–448. doi: 10.1016/0003-9861(63)90435-1. [DOI] [PubMed] [Google Scholar]
  33. Richey D. P., Brown G. M. The biosynthesis of folic acid. IX. Purification and properties of the enzymes required for the formation of dihydropteroic acid. J Biol Chem. 1969 Mar 25;244(6):1582–1592. [PubMed] [Google Scholar]
  34. Roland S., Ferone R., Harvey R. J., Styles V. L., Morrison R. W. The characteristics and significance of sulfonamides as substrates for Escherichia coli dihydropteroate synthase. J Biol Chem. 1979 Oct 25;254(20):10337–10345. [PubMed] [Google Scholar]
  35. Rose R. E. The nucleotide sequence of pACYC184. Nucleic Acids Res. 1988 Jan 11;16(1):355–355. doi: 10.1093/nar/16.1.355. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Rosenberg A. H., Lade B. N., Chui D. S., Lin S. W., Dunn J. J., Studier F. W. Vectors for selective expression of cloned DNAs by T7 RNA polymerase. Gene. 1987;56(1):125–135. doi: 10.1016/0378-1119(87)90165-x. [DOI] [PubMed] [Google Scholar]
  37. Rosenberg M., Court D. Regulatory sequences involved in the promotion and termination of RNA transcription. Annu Rev Genet. 1979;13:319–353. doi: 10.1146/annurev.ge.13.120179.001535. [DOI] [PubMed] [Google Scholar]
  38. Rådström P., Swedberg G. RSF1010 and a conjugative plasmid contain sulII, one of two known genes for plasmid-borne sulfonamide resistance dihydropteroate synthase. Antimicrob Agents Chemother. 1988 Nov;32(11):1684–1692. doi: 10.1128/aac.32.11.1684. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. SHIOTA T., DISRAELY M. N., MCCANN M. P. THE ENZYMATIC SYNTHESIS OF FOLATE-LIKE COMPOUNDS FROM HYDROXYMETHYLDIHYDROPTERIDINE PYROPHOSPHATE. J Biol Chem. 1964 Jul;239:2259–2266. [PubMed] [Google Scholar]
  40. Shine J., Dalgarno L. The 3'-terminal sequence of Escherichia coli 16S ribosomal RNA: complementarity to nonsense triplets and ribosome binding sites. Proc Natl Acad Sci U S A. 1974 Apr;71(4):1342–1346. doi: 10.1073/pnas.71.4.1342. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Shiota T., Baugh C. M., Jackson R., Dillard R. The enzymatic synthesis of hydroxymethyldihydropteridine pyrophosphate and dihydrofolate. Biochemistry. 1969 Dec;8(12):5022–5028. doi: 10.1021/bi00840a052. [DOI] [PubMed] [Google Scholar]
  42. Short J. M., Fernandez J. M., Sorge J. A., Huse W. D. Lambda ZAP: a bacteriophage lambda expression vector with in vivo excision properties. Nucleic Acids Res. 1988 Aug 11;16(15):7583–7600. doi: 10.1093/nar/16.15.7583. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Sköld O. R-factor-mediated resistance to sulfonamides by a plasmid-borne, drug-resistant dihydropteroate synthase. Antimicrob Agents Chemother. 1976 Jan;9(1):49–54. doi: 10.1128/aac.9.1.49. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Spector T., Hajian G. Statistical methods to distinguish competitive, noncompetitive, and uncompetitive enzyme inhibitors. Anal Biochem. 1981 Aug;115(2):403–409. doi: 10.1016/0003-2697(81)90025-7. [DOI] [PubMed] [Google Scholar]
  45. Stassi D. L., Lopez P., Espinosa M., Lacks S. A. Cloning of chromosomal genes in Streptococcus pneumoniae. Proc Natl Acad Sci U S A. 1981 Nov;78(11):7028–7032. doi: 10.1073/pnas.78.11.7028. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Studier F. W., Moffatt B. A. Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. J Mol Biol. 1986 May 5;189(1):113–130. doi: 10.1016/0022-2836(86)90385-2. [DOI] [PubMed] [Google Scholar]
  47. Sundström L., Rådström P., Swedberg G., Sköld O. Site-specific recombination promotes linkage between trimethoprim- and sulfonamide resistance genes. Sequence characterization of dhfrV and sulI and a recombination active locus of Tn21. Mol Gen Genet. 1988 Aug;213(2-3):191–201. doi: 10.1007/BF00339581. [DOI] [PubMed] [Google Scholar]
  48. Swedberg G., Sköld O. Characterization of different plasmid-borne dihydropteroate synthases mediating bacterial resistance to sulfonamides. J Bacteriol. 1980 Apr;142(1):1–7. doi: 10.1128/jb.142.1.1-7.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Talarico T. L., Dev I. K., Dallas W. S., Ferone R., Ray P. H. Purification and partial characterization of 7,8-dihydro-6-hydroxymethylpterin-pyrophosphokinase and 7,8-dihydropteroate synthase from Escherichia coli MC4100. J Bacteriol. 1991 Nov;173(21):7029–7032. doi: 10.1128/jb.173.21.7029-7032.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Then R., Angehrn P. Sulphonamide-induced 'thymineless death' in Escherichia coli. J Gen Microbiol. 1973 Jun;76(2):255–263. doi: 10.1099/00221287-76-2-255. [DOI] [PubMed] [Google Scholar]
  51. Wise E. M., Jr, Abou-Donia M. M. Sulfonamide resistance mechanism in Escherichia coli: R plasmids can determine sulfonamide-resistant dihydropteroate synthases. Proc Natl Acad Sci U S A. 1975 Jul;72(7):2621–2625. doi: 10.1073/pnas.72.7.2621. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Zagursky R. J., Berman M. L. Cloning vectors that yield high levels of single-stranded DNA for rapid DNA sequencing. Gene. 1984 Feb;27(2):183–191. doi: 10.1016/0378-1119(84)90139-2. [DOI] [PubMed] [Google Scholar]

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