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. 1994 Jul;176(13):4066–4072. doi: 10.1128/jb.176.13.4066-4072.1994

Carboxy-terminal deletion analysis of the major pneumococcal autolysin.

J L Garcia 1, E Diaz 1, A Romero 1, P Garcia 1
PMCID: PMC205605  PMID: 7912694

Abstract

Autolysins are endogenous enzymes that specifically degrade the covalent bonds of the cell walls and eventually can induce bacterial lysis. One of the best-characterized autolysins, the major pneumococcal LytA amidase, has evolved by the fusion of two domains, the N-terminal catalytic domain and the C-terminal domain responsible for the binding to cell walls. The precise biochemical role played by the six repeat units that form the C-terminal domain of the LytA amidase has been investigated by producing serial deletions. Biochemical analyses of the truncated mutants revealed that the LytA amidase must contain at least four units to efficiently recognize the choline residues of pneumococcal cell walls. The loss of an additional unit dramatically reduces its hydrolytic activity as well as the binding affinity, suggesting that the catalytic efficiency of this enzyme can be considerably improved by keeping the protein attached to the cell wall substrate. Truncated proteins lacking one or two repeat units were more sensitive to the inhibition by free choline than the wild-type enzyme, whereas the N-terminal catalytic domain was insensitive to this inhibition. In addition, the truncated proteins were inhibited by deoxycholate (DOC), and the expression of a LytA amidase lacking the last 11 amino acids in Streptococcus pneumoniae M31, a strain having a deletion in the lytA gene, conferred to the cells an atypical phenotype (Lyt+ DOC-) (cells autolysed at the end of the stationary phase but were not sensitive to lysis induced by DOC), which has been previously observed in some clinical isolates of pneumococci. Our results are in agreement with the existence of several choline-binding sites and suggest that the stepwise acquisition of the repeat units and the tail could be considered an evolutionary advantage for the enzyme, since the presence of these motifs increases its hydrolytic activity.

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Selected References

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

  1. Berry A. M., Lock R. A., Hansman D., Paton J. C. Contribution of autolysin to virulence of Streptococcus pneumoniae. Infect Immun. 1989 Aug;57(8):2324–2330. doi: 10.1128/iai.57.8.2324-2330.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Birnboim H. C., Doly J. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 1979 Nov 24;7(6):1513–1523. doi: 10.1093/nar/7.6.1513. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. 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]
  4. Béliveau C., Potvin C., Trudel J., Asselin A., Bellemare G. Cloning, sequencing, and expression in Escherichia coli of a Streptococcus faecalis autolysin. J Bacteriol. 1991 Sep;173(18):5619–5623. doi: 10.1128/jb.173.18.5619-5623.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Chu C. P., Kariyama R., Daneo-Moore L., Shockman G. D. Cloning and sequence analysis of the muramidase-2 gene from Enterococcus hirae. J Bacteriol. 1992 Mar;174(5):1619–1625. doi: 10.1128/jb.174.5.1619-1625.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Croux C., García J. L. Sequence of the lyc gene encoding the autolytic lysozyme of Clostridium acetobutylicum ATCC824: comparison with other lytic enzymes. Gene. 1991 Jul 31;104(1):25–31. doi: 10.1016/0378-1119(91)90460-s. [DOI] [PubMed] [Google Scholar]
  7. Díaz E., García J. L. Characterization of the transcription unit encoding the major pneumococcal autolysin. Gene. 1990 May 31;90(1):157–162. doi: 10.1016/0378-1119(90)90454-y. [DOI] [PubMed] [Google Scholar]
  8. Díaz E., López R., García J. L. Role of the major pneumococcal autolysin in the atypical response of a clinical isolate of Streptococcus pneumoniae. J Bacteriol. 1992 Sep;174(17):5508–5515. doi: 10.1128/jb.174.17.5508-5515.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. García E., García J. L., García P., Arrarás A., Sánchez-Puelles J. M., López R. Molecular evolution of lytic enzymes of Streptococcus pneumoniae and its bacteriophages. Proc Natl Acad Sci U S A. 1988 Feb;85(3):914–918. doi: 10.1073/pnas.85.3.914. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. García E., García J. L., Ronda C., García P., López R. Cloning and expression of the pneumococcal autolysin gene in Escherichia coli. Mol Gen Genet. 1985;201(2):225–230. doi: 10.1007/BF00425663. [DOI] [PubMed] [Google Scholar]
  11. García J. L., García E., López R. Overproduction and rapid purification of the amidase of Streptococcus pneumoniae. Arch Microbiol. 1987;149(1):52–56. doi: 10.1007/BF00423136. [DOI] [PubMed] [Google Scholar]
  12. García P., García J. L., García E., López R. Nucleotide sequence and expression of the pneumococcal autolysin gene from its own promoter in Escherichia coli. Gene. 1986;43(3):265–272. doi: 10.1016/0378-1119(86)90215-5. [DOI] [PubMed] [Google Scholar]
  13. García P., García J. L., García E., López R. Purification and characterization of the autolytic glycosidase of Streptococcus pneumoniae. Biochem Biophys Res Commun. 1989 Jan 16;158(1):251–256. doi: 10.1016/s0006-291x(89)80205-0. [DOI] [PubMed] [Google Scholar]
  14. Gilkes N. R., Henrissat B., Kilburn D. G., Miller R. C., Jr, Warren R. A. Domains in microbial beta-1, 4-glycanases: sequence conservation, function, and enzyme families. Microbiol Rev. 1991 Jun;55(2):303–315. doi: 10.1128/mr.55.2.303-315.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. 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]
  16. Höltje J. V., Tomasz A. Purification of the pneumococcal N-acetylmuramyl-L-alanine amidase to biochemical homogeneity. J Biol Chem. 1976 Jul 25;251(14):4199–4207. [PubMed] [Google Scholar]
  17. Inouye S., Inouye M. Up-promoter mutations in the lpp gene of Escherichia coli. Nucleic Acids Res. 1985 May 10;13(9):3101–3110. doi: 10.1093/nar/13.9.3101. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Kuroda A., Sekiguchi J. Cloning, sequencing and genetic mapping of a Bacillus subtilis cell wall hydrolase gene. J Gen Microbiol. 1990 Nov;136(11):2209–2216. doi: 10.1099/00221287-136-11-2209. [DOI] [PubMed] [Google Scholar]
  19. Lacks S. A., Lopez P., Greenberg B., Espinosa M. Identification and analysis of genes for tetracycline resistance and replication functions in the broad-host-range plasmid pLS1. J Mol Biol. 1986 Dec 20;192(4):753–765. doi: 10.1016/0022-2836(86)90026-4. [DOI] [PubMed] [Google Scholar]
  20. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  21. Mosser J. L., Tomasz A. Choline-containing teichoic acid as a structural component of pneumococcal cell wall and its role in sensitivity to lysis by an autolytic enzyme. J Biol Chem. 1970 Jan 25;245(2):287–298. [PubMed] [Google Scholar]
  22. Ortega S., García J. L., Zazo M., Varela J., Muñoz-Willery I., Cuevas P., Giménez-Gallego G. Single-step purification on DEAE-sephacel of recombinant polypeptides produced in Escherichia coli. Biotechnology (N Y) 1992 Jul;10(7):795–798. doi: 10.1038/nbt0792-795. [DOI] [PubMed] [Google Scholar]
  23. Ronda C., García J. L., García E., Sánchez-Puelles J. M., López R. Biological role of the pneumococcal amidase. Cloning of the lytA gene in Streptococcus pneumoniae. Eur J Biochem. 1987 May 4;164(3):621–624. doi: 10.1111/j.1432-1033.1987.tb11172.x. [DOI] [PubMed] [Google Scholar]
  24. Sanchez-Puelles J. M., Ronda C., Garcia J. L., Garcia P., Lopez R., Garcia E. Searching for autolysin functions. Characterization of a pneumococcal mutant deleted in the lytA gene. Eur J Biochem. 1986 Jul 15;158(2):289–293. doi: 10.1111/j.1432-1033.1986.tb09749.x. [DOI] [PubMed] [Google Scholar]
  25. Sanchez-Puelles J. M., Sanz J. M., Garcia J. L., Garcia E. Immobilization and single-step purification of fusion proteins using DEAE-cellulose. Eur J Biochem. 1992 Jan 15;203(1-2):153–159. doi: 10.1111/j.1432-1033.1992.tb19840.x. [DOI] [PubMed] [Google Scholar]
  26. 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]
  27. Sanz J. M., Díaz E., García J. L. Studies on the structure and function of the N-terminal domain of the pneumococcal murein hydrolases. Mol Microbiol. 1992 Apr;6(7):921–931. doi: 10.1111/j.1365-2958.1992.tb01542.x. [DOI] [PubMed] [Google Scholar]
  28. Sanz J. M., Lopez R., Garcia J. L. Structural requirements of choline derivatives for 'conversion' of pneumococcal amidase. A new single-step procedure for purification of this autolysin. FEBS Lett. 1988 May 23;232(2):308–312. doi: 10.1016/0014-5793(88)80759-2. [DOI] [PubMed] [Google Scholar]
  29. Sánchez-Puelles J. M., García J. L., López R., García E. 3'-end modifications of the Streptococcus pneumoniae lytA gene: role of the carboxy terminus of the pneumococcal autolysin in the process of enzymatic activation (conversion). Gene. 1987;61(1):13–19. doi: 10.1016/0378-1119(87)90360-x. [DOI] [PubMed] [Google Scholar]
  30. Sánchez-Puelles J. M., Sanz J. M., García J. L., García E. Cloning and expression of gene fragments encoding the choline-binding domain of pneumococcal murein hydrolases. Gene. 1990 Apr 30;89(1):69–75. doi: 10.1016/0378-1119(90)90207-8. [DOI] [PubMed] [Google Scholar]
  31. Taylor J. W., Ott J., Eckstein F. The rapid generation of oligonucleotide-directed mutations at high frequency using phosphorothioate-modified DNA. Nucleic Acids Res. 1985 Dec 20;13(24):8765–8785. doi: 10.1093/nar/13.24.8765. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Tomasz A., Westphal M. Abnormal autolytic enzyme in a pneumococus with altered teichoic acid composition. Proc Natl Acad Sci U S A. 1971 Nov;68(11):2627–2630. doi: 10.1073/pnas.68.11.2627. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Wang X., Wilkinson B. J., Jayaswal R. K. Sequence analysis of a Staphylococcus aureus gene encoding a peptidoglycan hydrolase activity. Gene. 1991 Jun 15;102(1):105–109. doi: 10.1016/0378-1119(91)90547-o. [DOI] [PubMed] [Google Scholar]
  34. Wren B. W. A family of clostridial and streptococcal ligand-binding proteins with conserved C-terminal repeat sequences. Mol Microbiol. 1991 Apr;5(4):797–803. doi: 10.1111/j.1365-2958.1991.tb00752.x. [DOI] [PubMed] [Google Scholar]

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