Molecular and General Genetics MGG

, Volume 204, Issue 2, pp 237–242 | Cite as

Isolation, characterization and physiological properties of an autolytic-deficient mutant of Streptococcus pneumoniae

  • Rubens López
  • Jose M. Sánchez-Puelles
  • Ernesto García
  • Jose Luís García
  • Concepción Ronda
  • Pedro García


A spontaneous mutation in the gene lyt encoding the pneumococcal autolysin has been characterized. This mutation, named lyt-32, which behaves as a high-efficiency marker in pneumococcal transformation, is a single base pair GC deletion causing the appearance of two consecutive termination codons in the amino terminal part of the sequence of the autolysin gene. The mutant lyt gene did not code for a polypeptide of relative molecular mass corresponding to the pneumococcal E form amidase in Escherichia coli maxicells. Pneumococcal cells containing the lyt-32 mutation (M32) were fully transformable, multiplied at a normal growth rate forming small chains and showed a tolerant response when treated with beta-lactam antibiotics. Strain M32 represents the first example of a mutant of Streptococcus pneumoniae completely lacking amidase as a consequence of an alteration in the structural gene coding for the pneumococcal autolysin.

Key words

Autolysins Pneumococcal amidase Frame shift mutation Recombinant plasmids maxicells 


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  1. Barany F, Tomasz A (1980) Genetic transformation of Streptococcus pneumoniae by heterologous plasmid deoxyribonucleic acid. J Bacteriol 144:698–709Google Scholar
  2. Birnboim HC, Doly J (1979) A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res 7:1513–1523Google Scholar
  3. Bolívar F, Rodríguez RL, Greene PJ, Betlach MC, Heynecker HL, Boyer HW, Crosa JH, Falkow S (1977) Construction and characterization of new cloning vehicles. II. A multipurpose cloning system. Gene 2:95–113Google Scholar
  4. Boyer HW, Roulland-Dussoix D (1969) A complementation analysis of the restriction and modification of DNA in Escherichia coli. J Mol Biol 41:459–474Google Scholar
  5. Briese T, Hakenbeck R (1985) Interaction of the pneumococcal amidase with lipoteichoic acid and choline. Eur J Biochem 146:417–427Google Scholar
  6. Fischer H, Tomasz A (1984) Production and release of peptidoglycan and wall teichoic acid polymers in pneumococci treated with beta-lactam antibiotics. J Bacteriol 157:507–513Google Scholar
  7. García E, Rojo JM, García P, Ronda C, López R, Tomasz A (1982) Preparation of antiserum against the pneumococcal autolysin-inhibition of autolysin activity and some autolytic processes by the antibody. FEMS Microbiol Lett 14:133–136Google Scholar
  8. García P, García E, Ronda C, Tomasz A, López R (1983) Inhibition of lysis by antibody against phage-associated lysin and requirement of choline residues in the cell wall for progeny phage release in Streptococcus pneumoniae. Curr Microbiol 8:137–140Google Scholar
  9. García P, Méndez E, García E, Ronda C, López R (1984) Biochemical characterization of a murein hydrolase induced by bacteriophage Dp-1 in Streptococcus pneumoniae: comparative study between bacteriophage-associated lysin and the host amidase. J Bacteriol 159:793–796Google Scholar
  10. García E, Ronda C, García JL, López R (1985a) A rapid procedure to detect the autolysin phenotype in Streptococcus pneumoniae. FEMS Microbiol Lett 29:77–81Google Scholar
  11. García E, García JL, Ronda C, García P, López R (1985b) Cloning and expression of the pneumococcal autolysin gene in Escherichia coli. Mol Gen Genet 201:225–230Google Scholar
  12. García P, García JL, García E, López R (1986) Nucleotide sequence and expression of the pneumococcal autolysin gene from its own promoter in Escherichia coli. Gene (in press)Google Scholar
  13. Höltje JV, Tomasz A (1975) Specific recognition of choline residues in the cell wall teichoic acid by the N-acetylmuramyl-l-alanine amidase of pneumococcus. J Biol Chem 250:6072–6076Google Scholar
  14. Höltje JV, Tomasz A (1976) Purification of the pneumococcal N-acetylmuramyl-l-alanine amidase to biochemical homogeneity. J Biol Chem 251:4199–4207Google Scholar
  15. Howard LV, Gooder H (1974) Specificity of the autolysin of Streptococcus (Diplococcus) pneumoniae. J Bacteriol 117:796–804Google Scholar
  16. Huet J, Sentenac A, Fromageot P (1982) Spot-immunodetection of conserved determinants in eukaryotic RNA polymerases. J Biol Chem 257:2613–2618Google Scholar
  17. Lacks S (1970) Mutants of Diplococcus pneumoniae that lack deoxyribonucleases and other activities possibly pertinent to genetic transformation. J Bacteriol 101:373–383Google Scholar
  18. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227:680–685Google Scholar
  19. López R, Ronda C, García P, Escarmis C, García E (1984) Restriction cleavage maps of the DNAs of Streptococcus pneumoniae bacteriophages containing protein covalently bound to their 5′ ends. Mol Gen Genet 197:67–74Google Scholar
  20. Maniatis T, Fritsch EF, Sambrook J (1982) Molecular cloning. A laboratory manual. Cold Spring Harbor Laboratory Press, New YorkGoogle Scholar
  21. Messing J (1983) New M13 vectors for cloning. In: Wu R, Grossman L, Moldave K (eds) Methods in enzymology, vol 101. Academic Press, New York, pp 20–78Google Scholar
  22. Messing J, Crea R, Seeburg PH (1981) A system for shotgun DNA sequencing. Nucleic Acids Res 9:309–321Google Scholar
  23. Mosser JL, Tomasz A (1970) Choline-containing teichoic acid as a structural component of pneumococcal cell wall and its role in sensitivity of lysis by an autolytic enzyme. J Biol Chem 245:287–298Google Scholar
  24. Rogers HJ, Perkins HR, Ward JB (1980) Microbial cell walls and membranes. Chapman and Hall, LondonGoogle Scholar
  25. Sancar A, Hack AM, Rupp WD (1979) Simple method for identification of plasmid-coded proteins. J Bacteriol 137:692–693Google Scholar
  26. Sanger F, Nicklen S, Coulson AR (1977) DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 74:5463–5467Google Scholar
  27. Seto H, López R, Tomasz A (1975) Cell surface-located deoxyribonucleic acid receptors in transformable pneumococci. J Bacteriol 122:1339–1350Google Scholar
  28. Tiraby G, Sicard AM (1973) Integration efficiencies of spontaneous mutant alleles of amiA locus in pneumococcal transformation. J Bacteriol 116:1130–1135Google Scholar
  29. Tomasz A (1984) Building and breaking of bonds in the cell wall of bacteria—The role for autolysins. In: Nombela C (ed) Microbial cell wall synthesis and autolysis. Elsevier Science Publishers, Amsterdam, pp 3–12Google Scholar
  30. Tomasz A, Westphal M (1971) Abnormal autolytic enzyme in a pneumococcus with altered teichoic acid composition. Proc Natl Acad Sci USA 68:2627–2630Google Scholar
  31. Tomasz A, Westphal M, Briles EB, Fletcher P (1975) On the physiological functions of teichoic acids. J Supramol Struct 3:1–16Google Scholar

Copyright information

© Springer-Verlag 1986

Authors and Affiliations

  • Rubens López
    • 1
  • Jose M. Sánchez-Puelles
    • 1
  • Ernesto García
    • 1
  • Jose Luís García
    • 1
  • Concepción Ronda
    • 1
  • Pedro García
    • 1
  1. 1.Centro de Investigaciones Biológicas C.S.I.C.MadridSpain

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