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. 1996 Jul;178(13):3818–3828. doi: 10.1128/jb.178.13.3818-3828.1996

Mecillinam resistance in Escherichia coli is conferred by loss of a second activity of the AroK protein.

D Vinella 1, B Gagny 1, D Joseleau-Petit 1, R D'Ari 1, M Cashel 1
PMCID: PMC232642  PMID: 8682786

Abstract

Mecillinam, a beta-lactam antibiotic specific to penicillin-binding protein 2 (PBP 2) in Escherichia coli, blocks cell wall elongation and, indirectly, cell division, but its lethality can be overcome by increased levels of ppGpp, the nucleotide effector of the stringent response. We have subjected an E. coli K-12 strain to random insertional mutagenesis with a mini-Tn10 element. One insertion, which was found to confer resistance to mecillinam in relA+ and relA strains, was mapped at 75.5 min on the E. coli map and was located between the promoters and the coding sequence of the aroK gene, which codes for shikimate kinase 1, one of two E. coli shikimate kinases, both of which are involved in aromatic amino acid biosynthesis. The mecillinam resistance conferred by the insertion was abolished in a delta relA delta spoT strain completely lacking ppGpp, and it thus depends on the presence of ppGpp. Furthermore, the insertion increased the ppGpp pool approximately twofold in a relA+ strain. However, this increase was not observed in relA strains, although the insertion still conferred mecillinam resistance in these backgrounds, showing that mecillinam resistance is not due to an increased ppGpp pool. The resistance was also abolished in an ftsZ84(Ts) strain under semipermissive conditions, and the aroK::mini-Tn10 allele partially suppressed ftsZ84(Ts); however, it did not increase the concentration of the FtsZ cell division protein. The insertion greatly decreased or abolished the shikimate kinase activity of AroK in vivo and in vitro. The two shikimate kinases of E. coli are not equivalent; the loss of AroK confers mecillinam resistance, whereas the loss of Arol, does not. Furthermore, the ability of the aroK mutation to confer mecillinam resistance is shown to be independent of polar effects on operon expression and of effects on the availability of aromatic amino acids or shikimic acid. Instead, we conclude that the AroK protein has a second activity, possibly related to cell division regulation, which confers mecillinam sensitivity. We were able to separate the AroK activities mutationally with an aroK mutant allele lacking shikimate kinase activity but still able to confer mecillinam sensitivity.

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Selected References

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  1. An G., Justesen J., Watson R. J., Friesen J. D. Cloning the spoT gene of Escherichia coli: identification of the spoT gene product. J Bacteriol. 1979 Mar;137(3):1100–1110. doi: 10.1128/jb.137.3.1100-1110.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Aono R., Yamasaki M., Tamura G. High and selective resistance to mecillinam in adenylate cyclase-deficient or cyclic adenosine 3',5'-monophosphate receptor protein-deficient mutants of Escherichia coli. J Bacteriol. 1979 Feb;137(2):839–845. doi: 10.1128/jb.137.2.839-845.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Begg K. J., Donachie W. D. Cell shape and division in Escherichia coli: experiments with shape and division mutants. J Bacteriol. 1985 Aug;163(2):615–622. doi: 10.1128/jb.163.2.615-622.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bi E. F., Lutkenhaus J. FtsZ ring structure associated with division in Escherichia coli. Nature. 1991 Nov 14;354(6349):161–164. doi: 10.1038/354161a0. [DOI] [PubMed] [Google Scholar]
  5. Bi E., Lutkenhaus J. Cell division inhibitors SulA and MinCD prevent formation of the FtsZ ring. J Bacteriol. 1993 Feb;175(4):1118–1125. doi: 10.1128/jb.175.4.1118-1125.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bochner B. R., Ames B. N. Complete analysis of cellular nucleotides by two-dimensional thin layer chromatography. J Biol Chem. 1982 Aug 25;257(16):9759–9769. [PubMed] [Google Scholar]
  7. Bouloc P., Jaffé A., D'Ari R. The Escherichia coli lov gene product connects peptidoglycan synthesis, ribosomes and growth rate. EMBO J. 1989 Jan;8(1):317–323. doi: 10.1002/j.1460-2075.1989.tb03379.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Bouloc P., Vinella D., D'Ari R. Leucine and serine induce mecillinam resistance in Escherichia coli. Mol Gen Genet. 1992 Nov;235(2-3):242–246. doi: 10.1007/BF00279366. [DOI] [PubMed] [Google Scholar]
  9. Bramhill D., Thompson C. M. GTP-dependent polymerization of Escherichia coli FtsZ protein to form tubules. Proc Natl Acad Sci U S A. 1994 Jun 21;91(13):5813–5817. doi: 10.1073/pnas.91.13.5813. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Cashel M., Gallant J. Two compounds implicated in the function of the RC gene of Escherichia coli. Nature. 1969 Mar 1;221(5183):838–841. doi: 10.1038/221838a0. [DOI] [PubMed] [Google Scholar]
  11. Cashel M. The control of ribonucleic acid synthesis in Escherichia coli. IV. Relevance of unusual phosphorylated compounds from amino acid-starved stringent strains. J Biol Chem. 1969 Jun 25;244(12):3133–3141. [PubMed] [Google Scholar]
  12. Dai K., Lutkenhaus J. ftsZ is an essential cell division gene in Escherichia coli. J Bacteriol. 1991 Jun;173(11):3500–3506. doi: 10.1128/jb.173.11.3500-3506.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. DeFeyter R. C., Pittard J. Genetic and molecular analysis of aroL, the gene for shikimate kinase II in Escherichia coli K-12. J Bacteriol. 1986 Jan;165(1):226–232. doi: 10.1128/jb.165.1.226-232.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. DeFeyter R. C., Pittard J. Purification and properties of shikimate kinase II from Escherichia coli K-12. J Bacteriol. 1986 Jan;165(1):331–333. doi: 10.1128/jb.165.1.331-333.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Doolittle W. F., Yanofsky C. Mutants of Escherichia coli with an altered tryptophanyl-transfer ribonucleic acid synthetase. J Bacteriol. 1968 Apr;95(4):1283–1294. doi: 10.1128/jb.95.4.1283-1294.1968. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Erickson H. P. FtsZ, a prokaryotic homolog of tubulin? Cell. 1995 Feb 10;80(3):367–370. doi: 10.1016/0092-8674(95)90486-7. [DOI] [PubMed] [Google Scholar]
  17. Hattman S., Brooks J. E., Masurekar M. Sequence specificity of the P1 modification methylase (M.Eco P1) and the DNA methylase (M.Eco dam) controlled by the Escherichia coli dam gene. J Mol Biol. 1978 Dec 15;126(3):367–380. doi: 10.1016/0022-2836(78)90046-3. [DOI] [PubMed] [Google Scholar]
  18. Hernandez V. J., Bremer H. Escherichia coli ppGpp synthetase II activity requires spoT. J Biol Chem. 1991 Mar 25;266(9):5991–5999. [PubMed] [Google Scholar]
  19. Jaffé A., Chabbert Y. A., Derlot E. Selection and characterization of beta-lactam-resistant Escherichia coli K-12 mutants. Antimicrob Agents Chemother. 1983 Apr;23(4):622–625. doi: 10.1128/aac.23.4.622. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. James R., Haga J. Y., Pardee A. B. Inhibition of an early event in the cell division cycle of Escherichia coli by FL1060, an amidinopenicillanic acid. J Bacteriol. 1975 Jun;122(3):1283–1292. doi: 10.1128/jb.122.3.1283-1292.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Jonczyk P., Hines R., Smith D. W. The Escherichia coli dam gene is expressed as a distal gene of a new operon. Mol Gen Genet. 1989 May;217(1):85–96. doi: 10.1007/BF00330946. [DOI] [PubMed] [Google Scholar]
  22. Joseleau-Petit D., Thévenet D., D'Ari R. ppGpp concentration, growth without PBP2 activity, and growth-rate control in Escherichia coli. Mol Microbiol. 1994 Sep;13(5):911–917. doi: 10.1111/j.1365-2958.1994.tb00482.x. [DOI] [PubMed] [Google Scholar]
  23. Kleckner N., Bender J., Gottesman S. Uses of transposons with emphasis on Tn10. Methods Enzymol. 1991;204:139–180. doi: 10.1016/0076-6879(91)04009-d. [DOI] [PubMed] [Google Scholar]
  24. Lacks S., Greenberg B. Complementary specificity of restriction endonucleases of Diplococcus pneumoniae with respect to DNA methylation. J Mol Biol. 1977 Jul;114(1):153–168. doi: 10.1016/0022-2836(77)90289-3. [DOI] [PubMed] [Google Scholar]
  25. Lange R., Hengge-Aronis R. Identification of a central regulator of stationary-phase gene expression in Escherichia coli. Mol Microbiol. 1991 Jan;5(1):49–59. doi: 10.1111/j.1365-2958.1991.tb01825.x. [DOI] [PubMed] [Google Scholar]
  26. Lerner C. G., Inouye M. Low copy number plasmids for regulated low-level expression of cloned genes in Escherichia coli with blue/white insert screening capability. Nucleic Acids Res. 1990 Aug 11;18(15):4631–4631. doi: 10.1093/nar/18.15.4631. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Lund F., Tybring L. 6 -amidinopenicillanic acids--a new group of antibiotics. Nat New Biol. 1972 Apr 5;236(66):135–137. doi: 10.1038/newbio236135a0. [DOI] [PubMed] [Google Scholar]
  28. Lutkenhaus J. Regulation of cell division in E. coli. Trends Genet. 1990 Jan;6(1):22–25. doi: 10.1016/0168-9525(90)90045-8. [DOI] [PubMed] [Google Scholar]
  29. Lyngstadaas A., Løbner-Olesen A., Boye E. Characterization of three genes in the dam-containing operon of Escherichia coli. Mol Gen Genet. 1995 Jun 10;247(5):546–554. doi: 10.1007/BF00290345. [DOI] [PubMed] [Google Scholar]
  30. Løbner-Olesen A., Boye E., Marinus M. G. Expression of the Escherichia coli dam gene. Mol Microbiol. 1992 Jul;6(13):1841–1851. doi: 10.1111/j.1365-2958.1992.tb01356.x. [DOI] [PubMed] [Google Scholar]
  31. Løbner-Olesen A., Marinus M. G. Identification of the gene (aroK) encoding shikimic acid kinase I of Escherichia coli. J Bacteriol. 1992 Jan;174(2):525–529. doi: 10.1128/jb.174.2.525-529.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Matsuhashi S., Kamiryo T., Blumberg P. M., Linnett P., Willoughby E., Strominger J. L. Mechanism of action and development of resistance to a new amidino penicillin. J Bacteriol. 1974 Feb;117(2):578–587. doi: 10.1128/jb.117.2.578-587.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Metzger S., Schreiber G., Aizenman E., Cashel M., Glaser G. Characterization of the relA1 mutation and a comparison of relA1 with new relA null alleles in Escherichia coli. J Biol Chem. 1989 Dec 15;264(35):21146–21152. [PubMed] [Google Scholar]
  34. Mukherjee A., Dai K., Lutkenhaus J. Escherichia coli cell division protein FtsZ is a guanine nucleotide binding protein. Proc Natl Acad Sci U S A. 1993 Feb 1;90(3):1053–1057. doi: 10.1073/pnas.90.3.1053. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Mukherjee A., Lutkenhaus J. Guanine nucleotide-dependent assembly of FtsZ into filaments. J Bacteriol. 1994 May;176(9):2754–2758. doi: 10.1128/jb.176.9.2754-2758.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Nanninga N. Cell division and peptidoglycan assembly in Escherichia coli. Mol Microbiol. 1991 Apr;5(4):791–795. doi: 10.1111/j.1365-2958.1991.tb00751.x. [DOI] [PubMed] [Google Scholar]
  37. Neidhardt F. C., Bloch P. L., Smith D. F. Culture medium for enterobacteria. J Bacteriol. 1974 Sep;119(3):736–747. doi: 10.1128/jb.119.3.736-747.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Ogura T., Bouloc P., Niki H., D'Ari R., Hiraga S., Jaffé A. Penicillin-binding protein 2 is essential in wild-type Escherichia coli but not in lov or cya mutants. J Bacteriol. 1989 Jun;171(6):3025–3030. doi: 10.1128/jb.171.6.3025-3030.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Phoenix P., Drapeau G. R. Cell division control in Escherichia coli K-12: some properties of the ftsZ84 mutation and suppression of this mutation by the product of a newly identified gene. J Bacteriol. 1988 Sep;170(9):4338–4342. doi: 10.1128/jb.170.9.4338-4342.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. RayChaudhuri D., Park J. T. Escherichia coli cell-division gene ftsZ encodes a novel GTP-binding protein. Nature. 1992 Sep 17;359(6392):251–254. doi: 10.1038/359251a0. [DOI] [PubMed] [Google Scholar]
  41. Robin A., Joseleau-Petit D., D'Ari R. Transcription of the ftsZ gene and cell division in Escherichia coli. J Bacteriol. 1990 Mar;172(3):1392–1399. doi: 10.1128/jb.172.3.1392-1399.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Rojiani M. V., Jakubowski H., Goldman E. Effect of variation of charged and uncharged tRNA(Trp) levels on ppGpp synthesis in Escherichia coli. J Bacteriol. 1989 Dec;171(12):6493–6502. doi: 10.1128/jb.171.12.6493-6502.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Singer M., Baker T. A., Schnitzler G., Deischel S. M., Goel M., Dove W., Jaacks K. J., Grossman A. D., Erickson J. W., Gross C. A. A collection of strains containing genetically linked alternating antibiotic resistance elements for genetic mapping of Escherichia coli. Microbiol Rev. 1989 Mar;53(1):1–24. doi: 10.1128/mr.53.1.1-24.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Spratt B. G. Distinct penicillin binding proteins involved in the division, elongation, and shape of Escherichia coli K12. Proc Natl Acad Sci U S A. 1975 Aug;72(8):2999–3003. doi: 10.1073/pnas.72.8.2999. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Spratt B. G., Pardee A. B. Penicillin-binding proteins and cell shape in E. coli. Nature. 1975 Apr 10;254(5500):516–517. doi: 10.1038/254516a0. [DOI] [PubMed] [Google Scholar]
  46. Spratt B. G. The mechanism of action of mecillinam. J Antimicrob Chemother. 1977 Jul;3 (Suppl B):13–19. doi: 10.1093/jac/3.suppl_b.13. [DOI] [PubMed] [Google Scholar]
  47. Sánchez M., Valencia A., Ferrándiz M. J., Sander C., Vicente M. Correlation between the structure and biochemical activities of FtsA, an essential cell division protein of the actin family. EMBO J. 1994 Oct 17;13(20):4919–4925. doi: 10.1002/j.1460-2075.1994.tb06819.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Takeshita S., Sato M., Toba M., Masahashi W., Hashimoto-Gotoh T. High-copy-number and low-copy-number plasmid vectors for lacZ alpha-complementation and chloramphenicol- or kanamycin-resistance selection. Gene. 1987;61(1):63–74. doi: 10.1016/0378-1119(87)90365-9. [DOI] [PubMed] [Google Scholar]
  49. Tétart F., Bouché J. P. Regulation of the expression of the cell-cycle gene ftsZ by DicF antisense RNA. Division does not require a fixed number of FtsZ molecules. Mol Microbiol. 1992 Mar;6(5):615–620. doi: 10.1111/j.1365-2958.1992.tb01508.x. [DOI] [PubMed] [Google Scholar]
  50. Uzan M., Danchin A. A rapid test for the rel A mutation in E. coli. Biochem Biophys Res Commun. 1976 Apr 5;69(3):751–758. doi: 10.1016/0006-291x(76)90939-6. [DOI] [PubMed] [Google Scholar]
  51. Vinella D., D'Ari R., Jaffé A., Bouloc P. Penicillin binding protein 2 is dispensable in Escherichia coli when ppGpp synthesis is induced. EMBO J. 1992 Apr;11(4):1493–1501. doi: 10.1002/j.1460-2075.1992.tb05194.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Vinella D., D'Ari R. Overview of controls in the Escherichia coli cell cycle. Bioessays. 1995 Jun;17(6):527–536. doi: 10.1002/bies.950170609. [DOI] [PubMed] [Google Scholar]
  53. Vinella D., D'Ari R. Thermoinducible filamentation in Escherichia coli due to an altered RNA polymerase beta subunit is suppressed by high levels of ppGpp. J Bacteriol. 1994 Feb;176(4):966–972. doi: 10.1128/jb.176.4.966-972.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Vinella D., Jaffé A., D'Ari R., Kohiyama M., Hughes P. Chromosome partitioning in Escherichia coli in the absence of dam-directed methylation. J Bacteriol. 1992 Apr;174(7):2388–2390. doi: 10.1128/jb.174.7.2388-2390.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Vinella D., Joseleau-Petit D., Thévenet D., Bouloc P., D'Ari R. Penicillin-binding protein 2 inactivation in Escherichia coli results in cell division inhibition, which is relieved by FtsZ overexpression. J Bacteriol. 1993 Oct;175(20):6704–6710. doi: 10.1128/jb.175.20.6704-6710.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Wachi M., Doi M., Okada Y., Matsuhashi M. New mre genes mreC and mreD, responsible for formation of the rod shape of Escherichia coli cells. J Bacteriol. 1989 Dec;171(12):6511–6516. doi: 10.1128/jb.171.12.6511-6516.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Wachi M., Doi M., Tamaki S., Park W., Nakajima-Iijima S., Matsuhashi M. Mutant isolation and molecular cloning of mre genes, which determine cell shape, sensitivity to mecillinam, and amount of penicillin-binding proteins in Escherichia coli. J Bacteriol. 1987 Nov;169(11):4935–4940. doi: 10.1128/jb.169.11.4935-4940.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Wachi M., Matsuhashi M. Negative control of cell division by mreB, a gene that functions in determining the rod shape of Escherichia coli cells. J Bacteriol. 1989 Jun;171(6):3123–3127. doi: 10.1128/jb.171.6.3123-3127.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. Wandersman C., Moreno F., Schwartz M. Pleiotropic mutations rendering Escherichia coli K-12 resistant to bacteriophage TP1. J Bacteriol. 1980 Sep;143(3):1374–1383. doi: 10.1128/jb.143.3.1374-1383.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. Wang X. D., de Boer P. A., Rothfield L. I. A factor that positively regulates cell division by activating transcription of the major cluster of essential cell division genes of Escherichia coli. EMBO J. 1991 Nov;10(11):3363–3372. doi: 10.1002/j.1460-2075.1991.tb04900.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  61. 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]
  62. Ward J. E., Jr, Lutkenhaus J. Overproduction of FtsZ induces minicell formation in E. coli. Cell. 1985 Oct;42(3):941–949. doi: 10.1016/0092-8674(85)90290-9. [DOI] [PubMed] [Google Scholar]
  63. Whipp M. J., Pittard A. J. A reassessment of the relationship between aroK- and aroL-encoded shikimate kinase enzymes of Escherichia coli. J Bacteriol. 1995 Mar;177(6):1627–1629. doi: 10.1128/jb.177.6.1627-1629.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  64. Xiao H., Kalman M., Ikehara K., Zemel S., Glaser G., Cashel M. Residual guanosine 3',5'-bispyrophosphate synthetic activity of relA null mutants can be eliminated by spoT null mutations. J Biol Chem. 1991 Mar 25;266(9):5980–5990. [PubMed] [Google Scholar]
  65. de Boer P., Crossley R., Rothfield L. The essential bacterial cell-division protein FtsZ is a GTPase. Nature. 1992 Sep 17;359(6392):254–256. doi: 10.1038/359254a0. [DOI] [PubMed] [Google Scholar]

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