Cell Wall Hydrolases

Rubens López; Ernesto García; Pedro García; José Luis García

doi:10.1128/9781555816537.ch6

Chapter 6 : Cell Wall Hydrolases

Authors: Rubens López¹, Ernesto García¹, Pedro García¹, José Luis García¹

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Affiliations: 1: Departamento de Microbiología Molecular, Centro de Investigaciones Biológicas, CSIC, E-28040 Madrid, Spain

Content Type: Monograph

Publication Year: 2004

Category: Bacterial Pathogenesis; Clinical Microbiology

Book DOI: 10.1128/9781555816537

Chapter DOI: 10.1128/9781555816537.ch6

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Abstract:

Many bacterial species possess more than one enzyme that hydrolyzes the same bond, a fact that complicates the determination of their biological role(s). This redundancy also contributes to the common thought of assigning a basic role to lytic enzymes in the biology of bacteria. The activity of some pneumococcal murein hydrolases (MHs) appears to be constrained by the membrane lipoteichoic acid (LTA) at the posttranslational level. Cell wall hydrolases (CWHs) of Streptococcus pneumoniae show both substrate and bond specificities. The lytA gene encodes the major S. pneumoniae autolysin (amidase) and represents the first example of a bacterial autolytic gene that was cloned and expressed. LytB is most probably a glucosaminidase capable of degrading Ch-containing cell walls. All the pneumococcal CWHs described have been shown to possess an absolute requirement for the presence of Ch for activity. The cloning of lytA has facilitated the isolation of the genes encoding the cell wall lytic enzymes from pneumococcal bacteriophages based on sequence homologies. This global analysis led the authors to propose that pneumococcal cell wall lytic enzymes could be the result of the fusion of two independent functional domains. The construction of active chimeric proteins between lysins of phage and bacteria led to new enzymes exhibiting novel properties that were, as expected, a combination of those showed by the parental enzymes.

Citation: López R, García E, García P, García J. 2004. Cell Wall Hydrolases, p 75-88. In Tuomanen E, Mitchell T, Morrison D, Spratt B (ed), The Pneumococcus. ASM Press, Washington, DC. doi: 10.1128/9781555816537.ch6

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Cell Wall Hydrolases

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/content/10.1128/9781555816537.chap6.fig6-1

FIGURE 1

Diagrammatic sketch of the cell wall of pneumococci, indicating the chemical bonds cleaved by different CWHs. The peptidoglycan chains and a repeat unit of TA are shown. Abbreviations: G and M, N-acetylglucosamine and N-acetylmuramic acid residues, respectively; Rib, ribitol-5-phosphate; GalNAc, N-acetyl-D-galactosamine; AATGal, 2-acetamido- 4-amino,2,4,6-trideoxy-D-galactose; Glc, Dglucose; ChP, phosphorylcholine.

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FIGURE 1

/content/10.1128/9781555816537.chap6.fig6-2

FIGURE 2

Schematic representation of the structures of lytA, lytB, lytC, and pce. (A) The genes are labeled as in references 32 and 65 above and below the arrows, respectively. Arrows indicate the direction of transcription of the ORFs. (B) The CWHs are drawn from the NH2 end to the COOH end. Black and dotted bars indicate the parts of the proteins corresponding to the signal peptide and the active center, respectively. Shaded rectangles correspond to the CBRs. aa, amino acids.

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FIGURE 2

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FIGURE 3

Localization of GFP-LytB in the surface of S. pneumoniae. A culture of a lytB mutant of the R6 strain received fusion protein (9.5 μg/ml) and was incubated for 1 min at 37°C. Pictures were taken with a Nikon Eclipse inverted microscope in phase-contrast (a) and using fluorescence (b). Bars, 4 μm. (Reprinted from the Journal of Bacteriology [8] with permission of the publisher.)

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FIGURE 3

References

/content/book/10.1128/9781555816537.chap6

1. Balachandran, P.,, S. K. Hollingshead,, J. C. Paton,, and D. E. Briles. 2001. The autolytic enzyme LytA of Streptococcus pneumoniae is not responsible for releasing pneumolysin. J. Bacteriol. 183: 3108– 3116.

2. Bateman, A.,, E. Birney,, L. Cerruti,, R. Durbin,, L. Etwiller,, S. R. Eddy,, S. Griffiths- Jones,, K. L. Howe,, M. Marshall,, and E. L. L. Sonnhammer. 2002. The Pfam protein families database. Nucleic Acids Res. 30: 276– 280.

3. Bergé, M.,, P. García,, F. Iannelli,, M. F. Prere,, C. Granadel,, A. Polissi,, and J. P. Claverys. 2001. The puzzle of zmpB and extensive chain formation, autolysis defect and nontranslocation of choline-binding proteins in Streptococcus pneumoniae. Mol. Microbiol. 39: 1651– 1660.

4. Charpentier, E.,, R. Novak,, and E. Tuomanen. 2000. Regulation of growth inhibition at high temperature, autolysis, transformation and adherence in Streptococcus pneumoniae by clpC. Mol. Microbiol. 37: 717– 726.

5. Chastanet, A.,, M. Prudhomme,, J. P. Claverys,, and T. Msadek. 2001. Regulation of Streptococcus pneumoniae clp genes and their role in competence development and stress survival. J. Bacteriol. 183: 7295– 7307.

6. de las Rivas, B. 2002. Isolation and characterization of new choline-binding proteins from Streptococcus pneumoniae. Ph.D. thesis. Universidad Complutense de Madrid, Madrid, Spain.

7. de las Rivas, B.,, J. L. García,, R. López,, and P. García. 2001. Molecular characterization of the pneumococcal teichoic acid phosphorylcholine esterase. Microb. Drug Resist. 7: 213– 222.

8. de las Rivas, B.,, J. L. García,, R. López,, and P. García. 2002. Purification and polar localization of pneumococcal LytB, a putative endo-β- N-acetylglucosaminidase: the chain-dispersing murein hydrolase. J. Bacteriol. 184: 4988– 5000.

9. de Saizieu, A.,, U. Certa,, J. Warrington,, C. Gray,, W. Keck,, and J. Mous. 1998. Bacterial transcripts imaging by hybridization of total RNA by oligonucleotide arrays. Nat. Biotechnol. 16: 45– 48.

10. Díaz, E.,, E. García,, C. Ascaso,, E. Méndez,, R. López,, and J. L. García. 1989. Subcellular localization of the major pneumococcal autolysin: a peculiar mechanism of secretion in Escherichia coli. J. Biol. Chem. 264: 1238– 1244.

11. Díaz, E.,, and J. L. García. 1990. Characterization of the transcription unit encoding the major pneumococcal autolysin. Gene 90: 157– 162.

12. Díaz, E.,, R. López,, and J. L. García. 1990. Chimeric phage-bacterial enzymes: a clue to the modular evolution of genes. Proc. Natl. Acad. Sci. USA 87: 8125– 8129.

13. Díaz, E.,, R. López,, and J. L. García. 1992. Role of the major pneumococcal autolysin in the atypical response of a clinical isolate of Streptococcus pneumoniae. J. Bacteriol. 174: 5508– 5515.

14. Fernández-Tornero, C.,, E. Garcia,, R. López,, G. Giménez-Gallego,, and A. Romero. 2002. Two new crystal forms of the choline-binding domain of the major pneumococcal autolysin: insights into the dynamics of the active homodimer. J. Mol. Biol. 321: 163– 173.

15. Fernández-Tornero, C.,, R. López,, E. García,, G. Giménez-Gallego,, and A. Romero. 2001. A novel solenoid fold in the cell wall anchoring domain of the pneumococcal virulence factor LytA. Nat. Struct. Biol. 8: 1020– 1024.

16. Fernández-Tornero, C.,, A. Ramón,, C. Fernández- Cabrera,, G. Giménez-Gallego,, and A. Romero. 2002. Expression, crystallization and preliminary X-ray diffraction studies on the complete choline-binding domain of the major pneumococcal autolysin. Acta Crystallogr. D Biol. Crystallogr. 58: 556– 558.

17. García, E.,, J. L. García,, P. García,, A. Arrarás,, J. M. Sánchez-Puelles,, and R. López. 1988. Molecular evolution of lytic enzymes of Streptococcus pneumoniae and its bacteriophages. Proc. Natl. Acad. Sci. USA 85: 914– 918.

18. García, E.,, J. L. García,, P. García,, C. Ronda,, J. M. Sánchez-Puelles,, and R. López,. 1987. Molecular genetics of the pneumococcal amidase: characterization of lytA mutants, p. 189– 192. In J. J. Ferretti, and R. Curtiss III (ed.), Streptococcal Genetics. ASM Press, Washington, D.C.

19. García, E.,, J. L. García,, C. Ronda,, P. García,, and R. López. 1985. Cloning and expression of the pneumococcal autolysin gene in Escherichia coli. Mol. Gen. Genet. 201: 225– 230.

20. García, J. L.,, E. Díaz,, A. Romero,, and P. García. 1994. Carboxy-terminal deletion analysis of the major pneumococcal autolysin. J. Bacteriol. 176: 4066– 4072.

21. García, P.,, J. L. García,, E. García,, J. M. Sánchez-Puelles,, and R. López. 1990. Modular organization of the lytic enzymes of Streptococcus pneumoniae and its bacteriophages. Gene 86: 81– 88.

21a.. García, P.,, M. P. González,, E. García,, J. L. García,, and R. López. 1999. The molecular characterization of the first autolytic lysozyme of Streptococcus pneumoniae reveals evolutionary mobile domains. Mol. Microbiol. 33: 128– 138.

22. García, P.,, M. P. González,, E. García,, R. López,, and J. L. García. 1999. LytB, a novel pneumococcal murein hydrolase essential for cell separation. Mol. Microbiol. 31: 1275– 1277.

23. Gerber, J.,, H. Eiffert,, H. Fleischer,, A. Wellmer,, U. Munzel,, and R. Nau. 2001. Reduced release of DNA from Streptococcus pneumoniae after treatment with rifampin in comparison to spontaneous growth and ceftriaxone treatment. Eur. J. Clin. Microbiol. Infect. Dis. 20: 490– 493.

24. Ghigo, J. M. 2003. Are there biofilm-specific physiological pathways beyond a reasonable doubt? Res. Microbiol. 154: 1– 8.

25. Ginsburg, I. 2002. The role of bacteriolysis in the pathophysiology of inflammation, infection and post-infectious sequelae. APMIS 110: 753– 770.

26. Gosink, K. K.,, E. R. Mann,, C. Guglielmo,, E. I. Tuomanen,, and H. R. Masure. 2000. Role of novel choline binding proteins in virulence of Streptococcus pneumoniae. Infect. Immun. 68: 5690– 5695.

27. Heidrich, C.,, A. Ursinus,, J. Berger,, H. Schwarz,, and J.-V. Höltje. 2002. Effects of multiple deletions of murein hydrolases on viability, septum cleavage, and sensitivity to large toxic molecules in Escherichia coli. J. Bacteriol. 184: 6093– 6099.

28. Holmes, A. R.,, R. McNab,, K. W. Millsap,, M. Rohde,, S. Hammerschmidt,, J. L. Mawdsley,, and H. F. Jenkinson. 2001. The pavA gene of Streptococcus pneumoniae encodes a fibronectin- binding protein that is essential for virulence. Mol. Microbiol. 41: 1395– 1408.

29. Höltje, J.-V.,, and A. Tomasz. 1975. Lipoteichoic acid: a specific inhibitor of autolysin activity in pneumococcus. Proc. Natl. Acad. Sci. USA 72: 1690– 1694.

30. Höltje, J.-V.,, and A. Tomasz. 1974. Teichoic acid phosphorylcholine esterase. A novel enzyme activity in pneumococcus. J. Biol. Chem. 249: 7032– 7034.

31. Höltje, J.-V.,, and A. Tomasz. 1975. Specific recognition of choline residues in the cell wall teichoic acid by N-acetylmuramic acid L-alanine amidase of pneumococcus. J. Biol. Chem. 250: 6072– 6076.

32. Hoskins, J.,, W. E. Alborn,, J. Arnold,, L. C. Blaszczak,, S. Burgett,, B. S. DeHoff,, S. T. Estrem,, L. Fritz,, D.-J. Fu,, W. Fuller,, C. Geringer,, R. Gilmour,, J. S. Glass,, H. Khoje,, A. R. Kraft,, R. E. Lagace,, D. J. LeBlanc,, L. N. Lee,, E. J. Lefkowitz,, J. Lu,, P. Matsushima,, S. M. McAhren,, M. McHenney,, K. McLeaster,, C. W. Mundy,, T. I. Nicas,, F. H. Norris,, M. O’Gara,, R. B. Peery,, G. T. Robertson,, P. Rockey,, P.-M. Sun,, M. E. Winkler,, Y. Yang,, M. Young-Bellido,, G. Zhao,, C. A. Zook,, R. H. Baltz,, R. Jaskunas,, P. R. J. Rosteck,, P. L. Skatrud,, and J. I. Glass. 2001. Genome of the bacterium Streptococcus pneumoniae strain R6. J. Bacteriol. 183: 5709– 5717.

33. Jedrzejas, M. J. 2001. Pneumococcal virulence factors: structure and function. Microbiol. Mol. Biol. Rev. 65: 187– 207.

34. Kwon, H.-Y.,, S.-W. Kim,, M.-H. Choi,, A. D. Ogunniyi,, J. C. Paton,, S.-H. Park,, S.-N. Pyo,, and D.-K. Rhee. 2003. Effect of heat shock and mutations in ClpL and ClpP on virulence gene expression in Streptococcus pneumoniae. Infect. Immun. 71: 3757– 3765.

35. Lee, C. J.,, T. R. Wang,, and C. E. Frasch. 2001. Immunogenicity in mice of pneumococcal glycoconjugate vaccines using pneumococcal protein carriers. Vaccine 19: 3216– 3225.

36. López, R.,, E. García,, P. García,, and J. L. García. 1997. The pneumococcal cell wall degrading enzymes: a modular design to create new lysins? Microb. Drug Resist. 3: 199– 211.

37. López, R.,, E. García,, P. García,, and J. L. García. 2000. The pneumococcal cell wall degrading enzymes: a modular design to create new lysins? p. 197– 209. In A. Tomasz (ed.), Streptococcus pneumoniae—Molecular Biology & Mechanisms of Disease. Mary Ann Liebert, Inc., Larchmont, N.Y.

38. Majcherczyk, P. A.,, H. Langen,, D. Heumann,, M. Fountoulakis,, M. P. Glauser,, and P. Moreillon. 1999. Digestion of Streptococcus pneumoniae cell walls with its major peptidoglycan hydrolase releases branched stem peptides carrying proinflammatory activity. J. Biol. Chem. 274: 12537– 12543.

39. Majcherczyk, P. A.,, E. Rubli,, D. Heumann,, M. P. Glauser,, and P. Moreillon. 2003. Teichoic acids are not required for Streptococcus pneumoniae and Staphylococcus aureus cell walls to trigger the release of tumor necrosis factor by peripheral blood monocytes. Infect. Immun. 71: 3707– 3713.

40. Medrano, F. J.,, M. Gasset,, C. López- Zúmel,, P. Usobiaga,, J. L. García,, and M. Menéndez. 1996. Structural characterization of the unligated and choline-bound forms of the major pneumococcal autolysin LytA amidase. Conformational transitions induced by temperature. J. Biol. Chem. 271: 29152– 29161.

41. Mitchell, T. J.,, J. E. Alexander,, P. J. Morgan,, and P. W. Andrew. 1997. Molecular analysis of virulence factors of Streptococcus pneumoniae. Soc. Appl. Bacteriol. Symp. Ser. 26: 62S– 71S.

42. Mortier-Barrière, I.,, A. de Saizieu,, J. P. Claverys,, and B. Martin. 1998. Competence-specific induction of recA is required for full recombination proficiency during transformation in Streptococcus pneumoniae. Mol. Microbiol. 27: 159– 170.

43. Novak, R.,, E. Charpentier,, J. S. Braun,, E. Park,, S. Murti,, E. Tuomanen,, and R. Masure. 2000. Extracellular targeting of choline-binding proteins in Streptococcus pneumoniae by a zinc metalloprotease. Mol. Microbiol. 36: 366– 376.

44. Obregón, V.,, P. García,, E. García,, A. Fenoll,, R. López,, and J. L. García. 2002. Molecular peculiarities of the lytA gene isolated from clinical pneumococcal strains that are bile insoluble. J. Clin. Microbiol. 40: 2545– 2554.

45. Ottolenghi, E.,, and R. D. Hotchkiss. 1960. Appearance of genetic transforming activity in pneumococcal cultures. Science 132: 1257– 1258.

46. Ottolenghi, E.,, and R. D. Hotchkiss. 1962. Release of genetic transforming agent from pneumococcal cultures during growth and disintegration. J. Exp. Med. 116: 491– 519.

47. Peterson, S.,, R. T. Cline,, H. Tettelin,, V. Sharov,, and D. A. Morrison. 2000. Gene expression analysis of the Streptococcus pneumoniae competence regulons by use of DNA microarrays. J. Bacteriol. 182: 6192– 6202.

48. Ramirez, M. 1998. DNA exchange in natural populations of Streptococcus pneumoniae. Ph.D. thesis. Universidade Nova de Lisboa, Lisbon, Portugal.

49. Rice, K. C.,, B. A. Firek,, J. B. Nelson,, S. J. Yang,, T. G. Patton,, and K. W. Bayles. 2003. The Staphylococcus aureus cidAB operon: evaluation of its role in regulation of murein hydrolase activity and penicillin tolerance. J. Bacteriol. 185: 2635– 2643.

50. Rimini, R.,, B. Jansson,, G. Feger,, T. C. Roberts,, M. de Francesco,, A. Gozzi,, F. Faggioni,, E. Domenici,, D. M. Wallace,, N. Frandsen,, and A. Polissi. 2000. Global analysis of transcription kinetics during competence development in Streptococcus pneumoniae using high density DNA arrays. Mol. Microbiol. 36: 1279– 1292.

51. Robertson, G. T.,, W. L. Ng,, J. Foley,, R. Gilmour,, and M. E. Winkler. 2002. Global transcriptional analysis of clpP mutations of type 2 Streptococcus pneumoniae and their effects on physiology and virulence. J. Bacteriol. 184: 3508– 3520.

52. Robertson, G. T.,, W.-L. Ng,, R. Gilmour,, and M. E. Winckler. 2003. Essentiality of clpX, but not clpP, clpL, clpC, or clpE, in Streptococcus pneumoniae R6. J. Bacteriol. 185: 2961– 2966.

53. Romero, A.,, R. López,, and P. García. 1992. The insertion site of the temperate phage HB-746 is located near the phage remnant in the pneumococcal host chromosome. J. Virol. 66: 2860– 2864.

54. Ronda, C.,, J. L. García,, E. García,, J. M. Sánchez-Puelles,, and R. López. 1987. Biological role of the pneumococcal amidase. Cloning of the lytA gene in Streptococcus pneumoniae. Eur. J. Biochem. 164: 621– 624.

55. Sánchez-Puelles, J. M.,, J. L. García,, R. López,, and E. García. 1987. 3′-end modifications of the Streptococcus pneumoniae lytA gene: role of the carboxy terminus of the pneumococcal autolysin in the presence of enzymatic activation (conversion). Gene 61: 13– 19.

56. Sánchez-Puelles, J. M.,, C. Ronda,, E. García,, E. Méndez,, J. L. García,, and R. López. 1986. A new peptidoglycan hydrolase in Streptococcus pneumoniae. FEMS Microbiol. Lett. 35: 163– 166.

57. Sánchez-Puelles, J. M.,, C. Ronda,, J. L. García,, P. García,, R. López,, and E. García. 1986. Searching for autolysin functions. Characterization of a pneumococcal mutant deleted in the lytA gene. Eur. J. Biochem. 158: 289– 293.

58. Sánchez-Puelles, J. M.,, J. M. Sanz,, J. L. García,, and E. García. 1990. Cloning and expression of gene fragments encoding the choline-binding domain of pneumococcal murein hydrolases. Gene 89: 69– 75.

59. Sanz, J. M.,, E. Díaz,, and J. L. García. 1992. Studies on the structure and function of the N-terminal domain of the pneumococcal murein hydrolases. Mol. Microbiol. 6: 921– 931.

60. Seto, H.,, and A. Tomasz. 1975. Protoplast formation and leakage of intramembrane cell components: induction by the competence activator substance of pneumococci. J. Bacteriol. 121: 344– 353.

61. Severin, A.,, D. Horne,, and A. Tomasz. 1997. Autolysis and cell wall degradation in a choline-independent strain of Streptococcus pneumoniae. Microb. Drug Resist. 3: 391– 400.

62. Severin, A.,, and A. Tomasz,. 2000. The peptidoglycan of Streptococcus pneumoniae, p. 179– 195. In A. Tomasz (ed.), Streptococcus pneumoniae— Molecular Biology & Mechanisms of Disease. Mary Ann Liebert, Inc., Larchmont, N.Y.

63. Shockman, G. D.,, and J.-V. Höltje,. 1994. Microbial peptidoglycan (murein) hydrolases, p. 131– 166. In J.-M. Ghuysen, and R. Hakenbeck (ed.), Bacterial Cell Wall. Elsevier, Amsterdam, The Netherlands.

64. Steinmoen, H.,, E. Knutsen,, and L. S. Håvarstein. 2002. Induction of natural competence in Streptococcus pneumoniae triggers lysis and DNA release from a subfraction of the cell population. Proc. Natl. Acad. Sci. USA 99: 7681– 7686.

65. Tettelin, H.,, K. E. Nelson,, I. T. Paulsen,, J. A. Eisen,, T. D. Read,, S. Peterson,, J. Heidelberg,, R. T. DeBoy,, D. H. Haft,, R. J. Dodson,, A. S. Durkin,, M. Gwinn,, J. F. Kolonay,, W. C. Nelson,, J. D. Peterson,, L. A. Umayam,, O. White,, S. L. Salzberg,, M. R. Lewis,, D. Radune,, E. Holtzapple,, H. Khouri,, A. M. Wolf,, T. R. Utterback,, C. L. Hansen,, L. A. McDonald,, T. V. Feldblyum,, S. Angiuoli,, T. Dickinson,, E. K. Hickey,, I. E. Holt,, B. J. Loftus,, F. Yang,, H. O. Smith,, J. C. Venter,, B. A. Dougherty,, D. A. Morrison,, S. K. Hollingshead,, and C. M. Fraser. 2001. Complete genome sequence of a virulent isolate of Streptococcus pneumoniae. Science 293: 498– 506.

66. Tomasz, A. 1968. Biological consequences of the replacement of choline by ethanolamine in the cell wall of Pneumococcus: chain formation, loss of transformability, and loss of autolysis. Proc. Natl. Acad. Sci. USA 59: 86– 93.

67. Tomasz, A.,, and W. Fischer,. 2000. The cell wall of Streptococcus pneumoniae, p. 191– 200. In V. A. Fischetti,, R. P. Novick,, J. J. Ferretti,, D. A. Portnoy,, and J. I. Rood (ed.), Gram-Positive Pathogens. ASM Press, Washington, D.C.

68. Tomasz, A.,, and M. Westphal. 1971. Abnormal autolytic enzyme in a pneumococcus with altered teichoic acid composition. Proc. Natl. Acad. Sci. USA 68: 2627– 2630.

69. Usobiaga, P.,, F. J. Medrano,, M. Gasset,, J. L. Garcia,, J. L. Saiz,, G. Rivas,, J. Laynez,, and M. Menendez. 1996. Structural organization of the major autolysin from Streptococcus pneumoniae. J. Biol. Chem. 271: 6832– 6838.

70. Varea, J.,, J. Saiz,, C. López-Zumel,, B. Monterroso,, F. J. Medrano,, J. L. Arrondo,, I. Iloro,, J. Laynez,, J. L. García,, and M. Menéndez. 2000. Do sequence repeats play an equivalent role in the choline-binding module of pneumococcal LytA amidase? J. Biol. Chem. 275: 26842– 26855.

71. Vollmer, W.,, and A. Tomasz. 2001. Identification of the teichoic acid phosphocholine esterase in Streptococcus pneumoniae. Mol. Microbiol. 39: 1610– 1622.

72. Waite, R. D.,, D. W. Penfold,, J. K. Struthers,, and C. G. Dowson. 2003. Spontaneous sequence duplications within capsule genes cap8E and tts control phase variation in Streptococcus pneumoniae serotypes 8 and 37. Microbiology 149: 497– 504.

73. Whatmore, A. M.,, and C. G. Dowson. 1999. The autolysin-encoding gene ( lytA) of Streptococcus pneumoniae displays restricted allelic variation despite localized recombination events with genes of pneumococcal bacteriophage encoding cell wall lytic enzymes. Infect. Immun. 67: 4551– 4556.

74. Yother, J.,, K. Leopold,, J. White,, and W. Fischer. 1998. Generation and properties of a Streptococcus pneumoniae mutant which does not require choline or analogs for growth. J. Bacteriol. 180: 2093– 2101.

75. Young, R. 2002. Bacteriophage holins: deadly diversity. J. Mol. Microbiol. Biotechnol. 4: 21– 36.