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. 1992 Apr;11(4):1493–1501. doi: 10.1002/j.1460-2075.1992.tb05194.x

Penicillin binding protein 2 is dispensable in Escherichia coli when ppGpp synthesis is induced.

D Vinella 1, R D'Ari 1, A Jaffé 1, P Bouloc 1
PMCID: PMC556598  PMID: 1563353

Abstract

Mecillinam, a beta-lactam antibiotic which specifically inactivates penicillin binding protein 2 (PBP2) in Escherichia coli, prevents lateral cell wall elongation, inducing spherical morphology and cell death. Two mecillinam resistant mutants, lov-1 and lovB, both able to dispense entirely with PBP2, are shown here to be affected in the aminoacyl-tRNA synthetase genes argS and alaS, respectively. Although the argS and alaS mutants grow slowly, we show that there is no correlation between mecillinam resistance and either growth rate or translation speed. A role of the ribosomes in mecillinam sensitivity, suggested by our earlier report that the lov-1 mutation is suppressed by certain rpsL(StrR) alleles affecting ribosomal protein S12, is supported by the present observation that a pseudo-streptomycin dependent mutant is mecillinam resistant in the presence of streptomycin. The argS and alaS mutants have high pools of the nucleotide ppGpp (effector of the stringent response) and the mecillinam resistance of both mutations is suppressed by a relA mutation, inactivating the ribosome-associated ppGpp synthetase and preventing ppGpp synthesis in response to aminoacyl-tRNA starvation. Furthermore, a ptacrelA' multicopy plasmid makes a wild type strain mecillinam resistant. The effect of ppGpp is probably mediated by RNA polymerase, since sublethal doses of the polymerase inhibitor rifampicin suppress mecillinam resistance in argS, alaS and ptacrelA'-bearing strains. We conclude that ppGpp regulates the transcription of a gene whose product is involved in mecillinam sensitivity, possibly as part of a chain of interacting elements which coordinate ribosomal activity with that of the PBPs.

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Selected References

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  1. 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]
  2. Bachmann B. J. Linkage map of Escherichia coli K-12, edition 8. Microbiol Rev. 1990 Jun;54(2):130–197. doi: 10.1128/mr.54.2.130-197.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Barbour A. G., Mayer L. W., Spratt B. G. Mecillinam resistance in Escherichia coli: dissociation of growth inhibition and morphologic change. J Infect Dis. 1981 Jan;143(1):114–121. doi: 10.1093/infdis/143.1.114. [DOI] [PubMed] [Google Scholar]
  4. Blumenthal R. M., Dennis P. P. Gene expression in Escherichia coli B/r during partial rifampicin-mediated restrictions of transcription initiation. Mol Gen Genet. 1978 Sep 20;165(1):79–86. doi: 10.1007/BF00270379. [DOI] [PubMed] [Google Scholar]
  5. Bouloc P., Jaffé A., D'Ari R. Preliminary physiologic characterization and genetic analysis of a new Escherichia coli mutant, lov, resistant to mecillinam. Rev Infect Dis. 1988 Jul-Aug;10(4):905–910. doi: 10.1093/clinids/10.4.905. [DOI] [PubMed] [Google Scholar]
  6. 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]
  7. Bylund J. E., Haines M. A., Walsh K., Bouloc P., D'Ari R., Higgins M. L. Buoyant density studies of several mecillinam-resistant and division mutants of Escherichia coli. J Bacteriol. 1991 Sep;173(17):5396–5402. doi: 10.1128/jb.173.17.5396-5402.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Cooper P. H., Hirshfield I. N., Maas W. K. Map location of arginyl-tRNA synthetase mutations in Escherichia coli K-12. Mol Gen Genet. 1969 Aug 15;104(4):383–390. doi: 10.1007/BF00334238. [DOI] [PubMed] [Google Scholar]
  9. D'Ari R., Jaffé A., Bouloc P., Robin A. Cyclic AMP and cell division in Escherichia coli. J Bacteriol. 1988 Jan;170(1):65–70. doi: 10.1128/jb.170.1.65-70.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Dombou M., Bhide S. V., Mizushima S. Appearance of elongation factor Tu in the outer membrane of sucrose-dependent spectinomycin-resistant mutants of Escherichia coli. Eur J Biochem. 1981 Jan;113(2):397–403. doi: 10.1111/j.1432-1033.1981.tb05079.x. [DOI] [PubMed] [Google Scholar]
  11. Eriani G., Dirheimer G., Gangloff J. Isolation and characterization of the gene coding for Escherichia coli arginyl-tRNA synthetase. Nucleic Acids Res. 1989 Jul 25;17(14):5725–5736. doi: 10.1093/nar/17.14.5725. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Gaal T., Gourse R. L. Guanosine 3'-diphosphate 5'-diphosphate is not required for growth rate-dependent control of rRNA synthesis in Escherichia coli. Proc Natl Acad Sci U S A. 1990 Jul;87(14):5533–5537. doi: 10.1073/pnas.87.14.5533. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Goodell W., Tomasz A. Alteration of Escherichia coli murein during amino acid starvation. J Bacteriol. 1980 Dec;144(3):1009–1016. doi: 10.1128/jb.144.3.1009-1016.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. 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]
  15. Ishiguro E. E., Ramey W. D. Stringent control of peptidoglycan biosynthesis in Escherichia coli K-12. J Bacteriol. 1976 Sep;127(3):1119–1126. doi: 10.1128/jb.127.3.1119-1126.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Ishino F., Park W., Tomioka S., Tamaki S., Takase I., Kunugita K., Matsuzawa H., Asoh S., Ohta T., Spratt B. G. Peptidoglycan synthetic activities in membranes of Escherichia coli caused by overproduction of penicillin-binding protein 2 and rodA protein. J Biol Chem. 1986 May 25;261(15):7024–7031. [PubMed] [Google Scholar]
  17. Iwaya M., Jones C. W., Khorana J., Strominger J. L. Mapping of the mecillinam-resistant, round morphological mutants of Escherichia coli. J Bacteriol. 1978 Jan;133(1):196–202. doi: 10.1128/jb.133.1.196-202.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Jacobson G. R., Rosenbusch J. P. Abundance and membrane association of elongation factor Tu in E. coli. Nature. 1976 May 6;261(5555):23–26. doi: 10.1038/261023a0. [DOI] [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. Jasin M., Regan L., Schimmel P. Dispensable pieces of an aminoacyl tRNA synthetase which activate the catalytic site. Cell. 1984 Apr;36(4):1089–1095. doi: 10.1016/0092-8674(84)90059-x. [DOI] [PubMed] [Google Scholar]
  22. Jensen K. F. Hyper-regulation of pyr gene expression in Escherichia coli cells with slow ribosomes. Evidence for RNA polymerase pausing in vivo? Eur J Biochem. 1988 Aug 15;175(3):587–593. doi: 10.1111/j.1432-1033.1988.tb14232.x. [DOI] [PubMed] [Google Scholar]
  23. Koch A. L. Biophysics of bacterial walls viewed as stress-bearing fabric. Microbiol Rev. 1988 Sep;52(3):337–353. doi: 10.1128/mr.52.3.337-353.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Kohara Y., Akiyama K., Isono K. The physical map of the whole E. coli chromosome: application of a new strategy for rapid analysis and sorting of a large genomic library. Cell. 1987 Jul 31;50(3):495–508. doi: 10.1016/0092-8674(87)90503-4. [DOI] [PubMed] [Google Scholar]
  25. Kusser W., Ishiguro E. E. Suppression of mutations conferring penicillin tolerance by interference with the stringent control mechanism of Escherichia coli. J Bacteriol. 1987 Sep;169(9):4396–4398. doi: 10.1128/jb.169.9.4396-4398.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Lee C. A., Beckwith J. Suppression of growth and protein secretion defects in Escherichia coli secA mutants by decreasing protein synthesis. J Bacteriol. 1986 Jun;166(3):878–883. doi: 10.1128/jb.166.3.878-883.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Little R., Bremer H. Quantitation of guanosine 5',3'-bisdiphosphate in extracts from bacterial cells by ion-pair reverse-phase high-performance liquid chromatography. Anal Biochem. 1982 Nov 1;126(2):381–388. doi: 10.1016/0003-2697(82)90531-0. [DOI] [PubMed] [Google Scholar]
  28. Lleo M. M., Canepari P., Satta G. Bacterial cell shape regulation: testing of additional predictions unique to the two-competing-sites model for peptidoglycan assembly and isolation of conditional rod-shaped mutants from some wild-type cocci. J Bacteriol. 1990 Jul;172(7):3758–3771. doi: 10.1128/jb.172.7.3758-3771.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Low B., Gates F., Goldstein T., Söll D. Isolation and partial characterization of temperature-sensitive Escherichia coli mutants with altered leucyl- and seryl-transfer ribonucleic acid synthetases. J Bacteriol. 1971 Nov;108(2):742–750. doi: 10.1128/jb.108.2.742-750.1971. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. 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]
  31. 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]
  32. Miyoshi Y., Yamagata H. Sucrose-dependent spectinomycin-resistant mutants of Escherichia coli. J Bacteriol. 1976 Jan;125(1):142–148. doi: 10.1128/jb.125.1.142-148.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Mizuno T., Yamada H., Yamagata H., Mizushima S. Coordinated alterations in ribosomes and cytoplasmic membrane in sucrose-dependent, spectinomycin-resistant mutants of Escherichia coli. J Bacteriol. 1976 Feb;125(2):524–530. doi: 10.1128/jb.125.2.524-530.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. 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]
  35. 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]
  36. Park J. T., Burman L. FL-1060: a new penicillin with a unique mode of action. Biochem Biophys Res Commun. 1973 Apr 16;51(4):863–868. doi: 10.1016/0006-291x(73)90006-5. [DOI] [PubMed] [Google Scholar]
  37. Payne S. H., Ames B. N. A procedure for rapid extraction and high-pressure liquid chromatographic separation of the nucleotides and other small molecules from bacterial cells. Anal Biochem. 1982 Jun;123(1):151–161. doi: 10.1016/0003-2697(82)90636-4. [DOI] [PubMed] [Google Scholar]
  38. Putney S. D., Royal N. J., Neuman de Vegvar H., Herlihy W. C., Biemann K., Schimmel P. Primary structure of a large aminoacyl-tRNA synthetase. Science. 1981 Sep 25;213(4515):1497–1501. doi: 10.1126/science.7025207. [DOI] [PubMed] [Google Scholar]
  39. Ruffler D., Buckel P., Piepersberg W., Böck A. Alanyl-tRNA synthetase of Escherichia coli: genetic analysis of the structural gene and of suppressor mutations. Mol Gen Genet. 1974;134(4):313–323. doi: 10.1007/BF00337466. [DOI] [PubMed] [Google Scholar]
  40. 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]
  41. Sarubbi E., Rudd K. E., Cashel M. Basal ppGpp level adjustment shown by new spoT mutants affect steady state growth rates and rrnA ribosomal promoter regulation in Escherichia coli. Mol Gen Genet. 1988 Aug;213(2-3):214–222. doi: 10.1007/BF00339584. [DOI] [PubMed] [Google Scholar]
  42. Satta G., Pardee A. B. Inhibition of Escherichia coli division by protein X. J Bacteriol. 1978 Mar;133(3):1492–1500. doi: 10.1128/jb.133.3.1492-1500.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Schreiber G., Metzger S., Aizenman E., Roza S., Cashel M., Glaser G. Overexpression of the relA gene in Escherichia coli. J Biol Chem. 1991 Feb 25;266(6):3760–3767. [PubMed] [Google Scholar]
  44. 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]
  45. Spratt B. G., Boyd A., Stoker N. Defective and plaque-forming lambda transducing bacteriophage carrying penicillin-binding protein-cell shape genes: genetic and physical mapping and identification of gene products from the lip-dacA-rodA-pbpA-leuS region of the Escherichia coli chromosome. J Bacteriol. 1980 Aug;143(2):569–581. doi: 10.1128/jb.143.2.569-581.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Spratt B. G. Comparison of the binding properties of two 6 beta-amidinopenicillanic acid derivatives that differ in their physiological effects on Escherichia coli. Antimicrob Agents Chemother. 1977 Jan;11(1):161–166. doi: 10.1128/aac.11.1.161. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. 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]
  48. 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]
  49. 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]
  50. Stoker N. G., Fairweather N. F., Spratt B. G. Versatile low-copy-number plasmid vectors for cloning in Escherichia coli. Gene. 1982 Jun;18(3):335–341. doi: 10.1016/0378-1119(82)90172-x. [DOI] [PubMed] [Google Scholar]
  51. 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]
  52. Tamaki S., Matsuzawa H., Matsuhashi M. Cluster of mrdA and mrdB genes responsible for the rod shape and mecillinam sensitivity of Escherichia coli. J Bacteriol. 1980 Jan;141(1):52–57. doi: 10.1128/jb.141.1.52-57.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Trieu-Cuot P., Courvalin P. Nucleotide sequence of the Streptococcus faecalis plasmid gene encoding the 3'5"-aminoglycoside phosphotransferase type III. Gene. 1983 Sep;23(3):331–341. doi: 10.1016/0378-1119(83)90022-7. [DOI] [PubMed] [Google Scholar]
  54. Tuomanen E. Phenotypic tolerance: the search for beta-lactam antibiotics that kill nongrowing bacteria. Rev Infect Dis. 1986 Jul-Aug;8 (Suppl 3):S279–S291. doi: 10.1093/clinids/8.supplement_3.s279. [DOI] [PubMed] [Google Scholar]
  55. Tuomanen E., Tomasz A. Mechanism of phenotypic tolerance of nongrowing pneumococci to beta-lactam antibiotics. Scand J Infect Dis Suppl. 1990;74:102–112. [PubMed] [Google Scholar]
  56. Vanderwel D., Ishiguro E. E. Properties of cell wall peptidoglycan synthesized by amino acid deprived re1A mutants of Escherichia coli. Can J Microbiol. 1984 Oct;30(10):1239–1246. doi: 10.1139/m84-196. [DOI] [PubMed] [Google Scholar]
  57. Vieira J., Messing J. Production of single-stranded plasmid DNA. Methods Enzymol. 1987;153:3–11. doi: 10.1016/0076-6879(87)53044-0. [DOI] [PubMed] [Google Scholar]
  58. 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]
  59. 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]
  60. Waxman D. J., Strominger J. L. Penicillin-binding proteins and the mechanism of action of beta-lactam antibiotics. Annu Rev Biochem. 1983;52:825–869. doi: 10.1146/annurev.bi.52.070183.004141. [DOI] [PubMed] [Google Scholar]
  61. Westling-Häggström B., Normark S. Genetic and physiological analysis of an envB spherelike mutant of Escherichia coli K-12 and characterization of its transductants. J Bacteriol. 1975 Jul;123(1):75–82. doi: 10.1128/jb.123.1.75-82.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  62. 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]
  63. Young C. C., Bernlohr R. W. Elongation factor Tu is methylated in response to nutrient deprivation in Escherichia coli. J Bacteriol. 1991 May;173(10):3096–3100. doi: 10.1128/jb.173.10.3096-3100.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  64. de Boer P. A., Crossley R. E., Rothfield L. I. A division inhibitor and a topological specificity factor coded for by the minicell locus determine proper placement of the division septum in E. coli. Cell. 1989 Feb 24;56(4):641–649. doi: 10.1016/0092-8674(89)90586-2. [DOI] [PubMed] [Google Scholar]

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