Impact of Phages on Evolution of Bacterial Pathogenicity

Harald Brüssow

doi:10.1128/9781555815530.ch11

Chapter 11 : Impact of Phages on Evolution of Bacterial Pathogenicity

VIEW AFFILIATIONS HIDE AFFILIATIONS

Affiliations: 1: Nestlé Research Centre, Nutrition and Health Department/Food and Health Microbiology, CH-1000 Lausanne 26, Vers-chez-les-Blanc, Switzerland

Content Type: Monograph

Publication Year: 2007

Category: Genomics and Bioinformatics; Bacterial Pathogenesis

Book DOI: 10.1128/9781555815530

Chapter DOI: 10.1128/9781555815530.ch11

MyBook is a cheap paperback edition of the original book and will be sold at uniform, low price.

Preview this chapter:

Impact of Phages on Evolution of Bacterial Pathogenicity, Page 1 of 2

< Previous page | Next page > /docserver/preview/fulltext/10.1128/9781555815530/9781555814519_Chap11-1.gif /docserver/preview/fulltext/10.1128/9781555815530/9781555814519_Chap11-2.gif

Abstract:

An interesting facet of Streptococcus pyogenes and Staphylococcus aureus, is their double role as commensals and pathogens. In this context, certain aspects of bacterial pathogenicity can be interpreted as a recall of antipredation strategies that bacteria evolved against phagocytosis by protozoan grazers. Many bacteria contain multiple genomes of bacterial viruses in their chromosomes. Prophage DNA can constitute a sizable part of the total bacterial DNA. When genomes from closely related bacteria were compared in a dot plot analysis, prophage sequences frequently accounted for a substantial, if not major, part of the differences between the genomes. Prophages can be present in many different forms, ranging from inducible prophages via prophages showing deletions, insertion, and rearrangements, to prophage remnants that lost most of the phage genome. In prophages from gram-negative bacteria, “extra” genes were identified near both prophage DNA ends. Prophages seem to be only transient passengers on the bacterial chromosomes, at least when seen on an evolutionary timescale. In an appealing model, the emergence of new, unusually virulent subclones of M3 strains is explained by the sequential acquisition of prophages, suggesting bacterial pathogenicity evolution in the fast lane. The virulence genes have certainly not evolved in phages but are the result of close bacterial interaction with the eukaryotic cell. Phages are perhaps only the handy gene carriers efficiently shuttling genes around in the bacterial world.

Citation: Brüssow H. 2007. Impact of Phages on Evolution of Bacterial Pathogenicity, p 267-300. In Pallen M, Nelson K, Preston G (ed), Bacterial Pathogenomics. ASM Press, Washington, DC. doi: 10.1128/9781555815530.ch11

Key Concept Ranking

Bacteria and Archaea: 0.58253855
Mobile Genetic Elements: 0.58085704
Gene Expression and Regulation: 0.517497
Bacterial Proteins: 0.4586461
Bacterial Pathogenesis: 0.44812995

0.58253855

Highlighted Text: Show | Hide

Full text loading...

Figures

Impact of Phages on Evolution of Bacterial Pathogenicity

[com.pub2web.rdf.cci.facet.ContentItem[id=http://asm.metastore.ingenta.com/content/author/10.1128/9781555815530.ch11-1,webId=/content/author/10.1128/9781555815530.ch11-1,properties={foaf_givenname=Harald, foaf_name=Harald Brüssow, foaf_surname=Brüssow, pub_isAffiliatedWith=[com.pub2web.rdf.cci.facet.ContentItem[id=http://asm.metastore.ingenta.com/content/affiliation/10.1128/9781555815530.ch11-aff1]]}]]

/content/10.1128/9781555815530.ch11.ch11fig01

FIGURE 1

Genome alignment of S. pyogenes serotype M18 (vertical) against serotype M3 (horizontal) shows that practically all diversity is prophage induced. Regions of nonalignment are circled, and prophages are marked with rectangles.

10.1128/9781555815530/f0268-01_thmb.gif

10.1128/9781555815530/f0268-01.gif

FIGURE 1

/content/10.1128/9781555815530.ch11.ch11fig02

FIGURE 2

S. pyogenes prophage inactivation by point mutations in DNA replication or DNA assembly proteins (vertical black arrow). Virulence genes are circled.

10.1128/9781555815530/f0270-01_thmb.gif

10.1128/9781555815530/f0270-01.gif

FIGURE 2

S. pyogenes prophage inactivation by point mutations in DNA replication or DNA assembly proteins (vertical black arrow). Virulence genes are circled.

/content/10.1128/9781555815530.ch11.ch11fig03

FIGURE 3

Dot plot matrix of S. pyogenes prophages identified in S. pyogenes sequencing projects.

10.1128/9781555815530/f0271-01_thmb.gif

10.1128/9781555815530/f0271-01.gif

FIGURE 3

Dot plot matrix of S. pyogenes prophages identified in S. pyogenes sequencing projects.

/content/10.1128/9781555815530.ch11.ch11fig04

FIGURE 4

Selective list of virulence genes encoded on prophages.

10.1128/9781555815530/f0272-01_thmb.gif

10.1128/9781555815530/f0272-01.gif

FIGURE 4

Selective list of virulence genes encoded on prophages.

/content/10.1128/9781555815530.ch11.ch11fig05

FIGURE 5

Genome organization of a typical S. pyogenes prophage encoding two virulence factors (circled).

10.1128/9781555815530/f0273-01_thmb.gif

10.1128/9781555815530/f0273-01.gif

FIGURE 5

Genome organization of a typical S. pyogenes prophage encoding two virulence factors (circled).

References

/content/book/10.1128/9781555815530.ch11

1. Aziz, R. K.,, R. A. Edwards,, W. W. Taylor,, D. E. Low,, A. McGeer, and, M. Kotb. 2005. Mosaic prophages with horizontally acquired genes account for the emergence and diversification of the globally disseminated M1T1 clone of Streptococcus pyogenes. J. Bacteriol. 187: 3311– 3318.

2. Aziz, R. K.,, S. A. Ismail,, H. W. Park, and, M. Kotb. 2004. Post-proteomic identification of a novel phage-encoded streptodornase, Sda1, in invasive M1T1 Streptococcus pyogenes. Mol. Microbiol. 54: 184– 197.

3. Aziz, R. K.,, M. J. Pabst,, A. Jeng,, R. Kansal,, D. E. Low,, V. Nizet, and, M. Kotb. 2004. Invasive M1T1 group A Streptococcus undergoes a phase-shift in vivo to prevent proteolytic degradation of multiple virulence factors by SpeB. Mol. Microbiol. 51: 123– 134.

4. Baba, T.,, F. Takeuchi,, M. Kuroda,, H. Yuzawa,, K. Aoki,, A. Oguchi,, Y. Nagai,, N. Iwama,, K. Asano,, T. Naimi,, H. Kuroda,, L. Cui,, K. Yamamoto, and, K. Hiramatsu. 2002. Genome and virulence determinants of high virulence community-acquired MRSA. Lancet 359: 1819– 1827.

5. Baggett, H. C.,, T. W. Hennessy,, K. Rudolph,, D. Bruden,, A. Reasonover,, A. Parkinson,, R. Sparks,, R. M. Donlan,, P. Martinez,, K. Mongkolrattanothai, and, J. C. Butler. 2004. Community-onset methicillin-resistant Staphylococcus aureus associated with antibiotic use and the cytotoxin Panton-Valentine leukocidin during a furunculosis outbreak in rural Alaska. J. Infect. Dis. 189: 1565– 1573.

6. Banks, D. J.,, S. B. Beres, and, J. M. Musser. 2002. The fundamental contribution of phages to GAS evolution, genome diversification and strain emergence. Trends Microbiol. 10: 515– 521.

7. Banks, D. J.,, B. Lei, and, J. M. Musser. 2003. Prophage induction and expression of prophage-encoded virulence factors in group A Streptococcus serotype M3 strain MGAS315. Infect. Immun. 71: 7079– 7086.

8. Banks, D. J.,, S. F. Porcella,, K. D. Barbian,, S. B. Beres,, L. E. Philips,, J. M. Voyich,, F. R. DeLeo,, J. M. Martin,, G. A. Somerville, and, J. M. Musser. 2004. Progress toward characterization of the group A Streptococcus metagenome: complete genome sequence of a macrolide-resistant serotype M6 strain. J. Infect. Dis. 190: 727– 738.

9. Banks, D. J.,, S. F. Porcella,, K. D. Barbian,, J. M. Martin, and, J. M. Musser. 2003. Structure and distribution of an unusual chimeric genetic element encoding macrolide resistance in phylo-genetically diverse clones of group A Streptococcus. J. Infect. Dis. 188: 1898– 1908.

10. Barondess, J. J., and, J. Beckwith. 1990. A bacterial virulence determinant encoded by lysogenic coliphage lambda. Nature 346: 871– 874.

11. Bensing, B. A.,, I. R. Siboo, and, P. M. Sullam. 2001. Proteins PblA and PblB of Streptococcus mitis, which promote binding to human platelets, are encoded within a lysogenic bacteriophage. Infect. Immun. 69: 6186– 6192.

12. Beres, S. B.,, G. L. Sylva,, D. E. Sturdevant,, C. N. Granville,, M. Liu,, S. M. Ricklefs,, A. R. Whitney,, L. D. Parkins,, N. P. Hoe,, G. J. Adams,, D. E. Low,, F. R. DeLeo,, A. McGeer, and, J. M. Musser. 2004. Genome-wide molecular dissection of serotype M3 group A Streptococcus strains causing two epidemics of invasive infections. Proc. Natl. Acad. Sci. USA 101: 11833– 11838.

13. Beres, S. B.,, G. L. Sylva,, K. D. Barbian,, B. Lei,, J. S. Hoff,, N. D. Mammarella,, M. Y. Liu,, J. C. Smoot,, S. F. Porcella,, L. D. Parkins,, D. S. Campbell,, T. M. Smith,, J. K. Mc-Cormick,, D. Y. Leung,, P. M. Schlievert, and, J. M. Musser. 2002. Genome sequence of a serotype M3 strain of group A Streptococcus: phage-encoded toxins, the high-virulence phenotype, and clone emergence. Proc. Natl. Acad. Sci. USA 99: 10078– 10083.

14. Betley, M. J., and, J. J. Mekalanos. 1985. Staphylococcal enterotoxin A is encoded by phage. Science 229: 185– 187.

15. Blair, J. E., and, M. Carr. 1961. Lysogeny in staphylococci. J. Bacteriol. 82: 984– 993.

16. Borst, D. W., and, M. J. Betley. 1994. Promoter analysis of the staphylococcal enterotoxin A gene. J. Biol. Chem. 269: 1883– 1888.

17. Bossi, L.,, J. A. Fuentes,, G. Mora, and, N. Figueroa-Bossi. 2003. Prophage contribution to bacterial population dynamics. J. Bacteriol. 185: 6467– 6471.

18. Botstein, D. 1980. A theory of modular evolution for bacteriophages. Ann. N. Y. Acad. Sci. 354: 484– 490.

19. Boyd, E. F., and, H. Brüssow. 2002. Common themes among bacteriophage-encoded virulence factors and diversity among the bacteriophages involved. Trends Microbiol. 10: 521– 529.

20. Breitbart, M.,, P. Salamon,, B. Andresen,, J. M. Mahaffy,, A. M. Segall,, D. Mead,, F. Azam, and, F. Rohwer. 2002. Genomic analysis of uncultured marine viral communities. Proc. Natl. Acad. Sci. USA 99: 14250– 14255.

21. Brinkmann, V.,, U. Reichard,, C. Goosmann,, B. Fauler,, Y. Uhlemann,, D. S. Weiss,, Y. Weinrauch, and, A. Zychlinsky. 2004. Neutrophil extracellular traps kill bacteria. Science 303: 1532– 1535.

22. Broudy, T. B., and, V.A. Fischetti. 2003. In vivo lysogenic conversion of Tox(-) Streptococcus pyo-genes to Tox(+) with lysogenic streptococci or free phage. Infect. Immun. 71: 3782– 3786.

23. Broudy, T. B.,, V. Pancholi, and, V. A. Fischetti. 2001. Induction of lysogenic bacteriophage and phage-associated toxin from group A streptococci during coculture with human pharyngeal cells. Infect. Immun. 69: 1440– 1443.

24. Broudy, T. B.,, V. Pancholi, and, V. A. Fischetti. 2002. The in vitro interaction of Streptococcus pyo-genes with human pharyngeal cells induces a phage-encoded extracellular DNase. Infect. Immun. 70: 2805– 2811.

25. Brussow, H. 2007. The Quest for Food: a Natural History of Eating. Springer Publisher, New York, NY.

26. Brüssow, H., and, F. Desiere. 2001. Comparative phage genomics and the evolution of Siphoviridae: insights from dairy phages. Mol. Microbiol. 39: 213– 222.

27. Brüssow, H.,, C. Canchaya, and, W.-D. Hardt. 2004. Phages and the evolution of bacterial pathogens: from genomic rearrangements to lysogenic conversion. Microbiol. Mol. Biol. Rev. 68: 560– 602.

28. Brüssow, H., and, R. W. Hendrix. 2002. Phage genomics: small is beautiful. Cell 108: 13– 16.

29. Bushman, F. 2002. Lateral DNA Transfer. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

30. Canchaya, C.,, F. Desiere,, W. M. McShan,, J. J. Ferretti,, J. Parkhill, and, H. Brussow. 2002. Genome analysis of an inducible prophage and prophage remnants integrated in the Streptococcus pyogenes strain SF370. Virology 302: 245– 258.

31. Canchaya, C.,, G. Fournous, and, H. Brussow. 2004. The impact of prophages on bacterial chromosomes. Mol. Microbiol. 53: 9– 18.

32. Canchaya, C.,, C. Proux,, G. Fournous,, A. Bruttin, and, H. Brussow. 2003. Prophage genomics. Microbiol. Mol. Biol. Rev. 67: 238– 276.

33. Casjens, S. 2003. Prophages and bacterial genomics: what have we learned so far? Mol. Microbiol. 49: 277– 300.

34. Casjens, S.,, G. F. Hatfull, and, R.W. Hendrix. 1992. Evolution of dsDNA tailed-bacteriophage genomes. Semin.Virol. 3: 383– 397.

35. Chatellier, S.,, N. Ihendyane,, R. G. Kansal,, F. Khambaty,, H. Basma,, A. Norrby-Teglund,, D. E. Low,, A. McGeer, and, M. Kotb. 2000. Genetic relatedness and superantigen expression in group A streptococcus serotype M1 isolates from patients with severe and nonsevere invasive diseases. Infect. Immun. 68: 3523– 3534.

36. Chaussee, M. S.,, G. L. Sylva,, D. E. Sturdevant,, L. M. Smoot,, M. R. Graham,, R. O. Watson, and, J. M. Musser. 2002. Rgg influences the expression of multiple regulatory loci to coregulate virulence factor expression in Streptococcus pyogenes. Infect. Immun. 70: 762– 770.

37. Chickering, H. T., and, J. H. Park. 1919. Staphylococcus aureus pneumonia. JAMA 72: 617– 626.

38. Clark, A. J.,, W. Inwood,, T. Cloutier, and, T. S. Dhillon. 2001. Nucleotide sequence of coliphage HK620 and the evolution of lambdoid phages. J. Mol. Biol. 311: 657– 679.

39. Coleman, D. C.,, D. J. Sullivan,, R. J. Russell,, J. P. Arbuthnott,, B. F. Carey, and, H. M. Pomeroy. 1989. Staphylococcus aureus bacteriophages mediating the simultaneous lysogenic conversion of beta-lysin, staphylokinase and enterotoxin A: molecular mechanism of triple conversion. J. Gen. Microbiol. 135: 1679– 1697.

40. Davies, H. D.,, A. McGeer,, B. Schwartz,, K. Green,, D. Cann,, A. E. Simor, and, D. E. Low. 1996. Invasive group A streptococcal infections in Ontario, Canada. Ontario Group A Streptococcal Study Group. N. Engl. J. Med. 335: 547– 554.

41. de Haas, C. J.,, K. E. Veldkamp,, A. Peschel,, F. Weerkamp,, W. J. van Wamel,, E. C. Heezius,, M. J. Poppelier,, K. P. van Kessel, and, J. A. van Strijp. 2004. Chemotaxis inhibitory protein of Staphylococcus aureus, a bacterial antiinflammatory agent. J. Exp. Med. 199: 687– 695.

42. Dempsey, R. M.,, D. Carroll,, H. Kong,, L. Higgins,, C. T. Keane, and, D. C. Coleman. 2005. Sau42I, a BcgI-like restriction-modification system encoded by the Staphylococcus aureus quadruple-converting phage Phi42. Microbiology 151: 1301– 1311.

43. Desiere, F.,, W. M. McShan,, D. van Sinderen,, J. J. Ferretti, and, H. Brussow. 2001. Comparative genomics reveals close genetic relationships between phages from dairy bacteria and pathogenic streptococci: evolutionary implications for prophage-host interactions. Virology 288: 325– 341.

44. Diep, B. A.,, S. R. Gill,, R. F. Chang,, T. H. Phan,, J. H. Chen,, M. G. Davidson,, F. Lin,, J. Lin,, H. A. Carleton,, E. F. Mongodin,, G. F. Sensabaugh, and, F. Perdreau-Remington. 2006. Complete genome sequence of USA300, an epidemic clone of community-acquired meticillin-resistant Staphylococcus aureus. Lancet 367: 731– 739.

45. Diep, B. A.,, G. F. Sensabaugh,, N. S. Somboona,, H. A. Carleton, and, F. Perdreau-Remington. 2004. Widespread skin and soft-tissue infections due to two methicillin-resistant Staphylococcus aureus strains harboring the genes for Panton-Valentine leucocidin. J. Clin. Microbiol. 42: 2080– 2084.

46. Dozois, C. M.,, F. Daigle, and, R. Curtiss III. 2003. Identification of pathogen-specific and conserved genes expressed in vivo by an avian pathogenic Escherichia coli strain. Proc. Natl. Acad. Sci. USA 100: 247– 252.

47. Ferretti, J. J.,, W. M. McShan,, D. Ajdic,, D. J. Savic,, G. Savic,, K. Lyon,, C. Primeaux,, S. Sezate,, A. N. Suvorov,, S. Kenton,, H. S. Lai,, S. P. Lin,, Y. Qian,, H. G. Jia,, F. Z. Najar,, Q. Ren,, H. Zhu,, L. Song,, J. White,, X. Yuan,, S. W. Clifton,, B. A. Roe, and, R. McLaughlin. 2001. Complete genome sequence of an M1 strain of Streptococcus pyogenes. Proc. Natl. Acad. Sci. USA 98: 4658– 4663.

48. Finck-Barbancon, V.,, G. Duportail,, O. Meunier, and, D. A. Colin. 1993. Pore formation by a two-component leukocidin from Staphylococcus aureus within the membrane of human polymorphonuclear leukocytes. Biochim. Biophys. Acta 1182: 275– 282.

49. Fitzgerald, J. R.,, D. E. Sturdevant,, S. M. Mackie,, S. R. Gill, and, J. M. Musser. 2001. Evolutionary genomics of Staphylococcus aureus: insights into the origin of methicillin-resistant strains and the toxic shock syndrome epidemic. Proc. Natl. Acad. Sci. USA 98: 8821– 8826.

50. Genestier, A. L.,, M. C. Michallet,, G. Prevost,, G. Bellot,, L. Chalabreysse,, S. Peyrol,, F. Thivolet,, J. Etienne,, G. Lina,, F. M. Vallette,, F. Vandenesch, and, L. Genestier. 2005. Staphylococcus aureus Panton-Valentine leukocidin directly targets mitochondria and induces Bax-independent apoptosis of human neutrophils. J. Clin. Invest. 115: 3117– 3127.

51. Gillet, Y.,, B. Issartel,, P. Vanhems,, J. C. Four-net,, G. Lina,, M. Bes,, F. Vandenesch,, Y. Piemont,, N. Brousse,, D. Floret, and, J. Etienne. 2002. Association between Staphylococcus aureus strains carrying gene for Panton-Valentine leukocidin and highly lethal necrotising pneumonia in young immunocompetent patients. Lancet 359: 753– 759.

52. Goerke, C.,, S. Papenberg,, S. Dasbach,, K. Dietz,, R. Ziebach,, B. C. Kahl, and, C. Wolz. 2004. Increased frequency of genomic alterations in Staphylococcus aureus during chronic infection is in part due to phage mobilization. J. Infect. Dis. 189: 724– 734.

53. Graham, M. R.,, L. M. Smoot,, C. A. Migliaccio,, K. Virtaneva,, D. E. Sturdevant,, S. F. Porcella,, M. J. Federle,, G. J. Adams,, J. R. Scott, and, J. M. Musser. 2002. Virulence control in group A Streptococcus by a two-component gene regulatory system: global expression profiling and in vivo infection modeling. Proc. Natl. Acad. Sci. USA 99: 13855– 13860.

54. Green, N. M.,, S. B. Beres,, E. A. Graviss,, J. E. Allison,, A. J. McGeer,, J. Vuopio-Varkila,, R. B. LeFebvre, and, J. M. Musser. 2005. Genetic diversity among type emm28 group A Streptococcus strains causing invasive infections and pharyngitis. J. Clin. Microbiol. 43: 4083– 4091.

55. Green, N. M.,, S. Zhang,, S. F. Porcella,, M. J. Nagiec,, K. D. Barbian,, S. B. Beres,, R. B. LeFebvre, and, J. M. Musser. 2005. Genome sequence of a serotype M28 strain of group a streptococcus: potential new insights into puerperal sepsis and bacterial disease specificity. J. Infect. Dis. 192: 760– 770.

56. Haas, P. J.,, C. J. de Haas,, W. Kleibeuker,, M. J. Poppelier,, K. P. van Kessel,, J. A. Kruijtzer,, R. M. Liskamp, and, J. A. van Strijp. 2004. N-terminal residues of the chemotaxis inhibitory protein of Staphylococcus aureus are essential for blocking formylated peptide receptor but not C5a receptor. J. Immunol. 173: 5704– 5711.

57. Halperin, S. A.,, P. Ferrieri,, E. D. Gray,, E. L. Kaplan, and, L. W. Wannamaker. 1987. Antibody response to bacteriophage hyaluronidase in acute glomerulonephritis after group A streptococcal infection. J. Infect. Dis. 155: 253– 261.

58. Hendrix, R. W.,, J. G. Lawrence,, G. F. Hatfull, and, S. Casjens. 2000. The origins and ongoing evolution of viruses. Trends Microbiol. 8: 504– 508.

59. Holden, M. T.,, E. J. Feil,, J. A. Lindsay,, S. J. Peacock,, N. P. Day,, M. C. Enright,, T. J. Foster,, C. E. Moore,, L. Hurst,, R. Atkin,, A. Barron,, N. Bason,, S. D. Bentley,, C. Chilling-worth,, T. Chillingworth,, C. Churcher,, L. Clark,, C. Corton,, A. Cronin,, J. Doggett,, L. Dowd,, T. Feltwell,, Z. Hance,, B. Harris,, H. Hauser,, S. Holroyd,, K. Jagels,, K. D. James,, N. Lennard,, A. Line,, R. Mayes,, S. Moule,, K. Mungall,, D. Ormond,, M. A. Quail,, E. Rabbi-nowitsch,, K. Rutherford,, M. Sanders,, S. Sharp,, M. Simmonds,, K. Stevens,, S. White-head,, B. G. Barrell,, B. G. Spratt, and, J. Parkhill. 2004. Complete genomes of two clinical Staphylococcus aureus strains: evidence for the rapid evolution of virulence and drug resistance. Proc. Natl. Acad. Sci. USA 101: 9786– 9791.

60. Ikebe, T.,, A. Wada,, Y. Inagaki,, K. Sugama,, R. Suzuki,, D. Tanaka,, A. Tamaru,, Y. Fujinaga,, Y. Abe,, Y. Shimizu, and, H. Watanabe. 2002. Dissemination of the phage-associated novel super-antigen gene speL in recent invasive and noninvasive Streptococcus pyogenes M3/T3 isolates in Japan. Infect. Immun. 70: 3227– 3233.

61. Inagaki, Y.,, F. Myouga,, H. Kawabata,, S. Yamai, and, H. Watanabe. 2000. Genomic differences in Streptococcus pyogenes serotype M3 between recent isolates associated with toxic shock– like syndrome and past clinical isolates. J. Infect. Dis. 181: 975– 983.

62. Ingrey, K. T.,, J. Ren, and, J. F. Prescott. 2003. A fluoroquinolone induces a novel mitogen-encoding bacteriophage in Streptococcus canis. Infect. Immun. 71: 3028– 3033.

63. Jin, T.,, M. Bokarewa,, T. Foster,, J. Mitchell,, J. Higgins, and, A. Tarkowski. 2004. Staphylococcus aureus resists human defensins by production of staphylokinase, a novel bacterial evasion mechanism. J. Immunol. 172: 1169– 1176.

64. Juhala, R. J.,, M. E. Ford,, R. L. Duda,, A. Youlton,, G. F. Hatfull, and, R. W. Hendrix. 2000. Genomic sequences of bacteriophages HK97 and HK022: pervasive genetic mosaicism in the lamb-doid bacteriophages. J. Mol. Biol. 299: 27– 51.

65. Kaneko, J.,, T. Kimura,, S. Narita,, T. Tomita, and, Y. Kamio. 1998. Complete nucleotide sequence and molecular characterization of the temperate staphylococcal bacteriophage phiPVL carrying Panton-Valentine leukocidin genes. Gene 215: 57– 67.

66. Kansal, R. G.,, A. McGeer,, D. E. Low,, A. Norrby-Teglund, and, M. Kotb. 2000. Inverse relation between disease severity and expression of the streptococcal cysteine protease, SpeB, among clonal M1T1 isolates recovered from invasive group A streptococcal infection cases. Infect. Immun. 68: 6362– 6369.

67. Kazmi, S. U.,, R. Kansal,, R. K. Aziz,, M. Hooshdaran,, A. Norrby-Teglund,, D. E. Low,, A. B. Halim, and, M. Kotb. 2001. Reciprocal, temporal expression of SpeA and SpeB by invasive M1T1 group a streptococcal isolates in vivo. Infect. Immun. 69: 4988– 4995.

68. Kuroda, M.,, T. Ohta,, I. Uchiyama,, T. Baba,, H. Yuzawa,, I. Kobayashi,, L. Cui,, A. Oguchi,, K. Aoki,, Y. Nagai,, J. Lian,, T. Ito,, M. Kanamori,, H. Matsumaru,, A. Maruyama,, H. Murakami,, A. Hosoyama,, Y. Mizutani-Ui,, N. K. Takahashi,, T. Sawano,, R. Inoue,, C. Kaito,, K. Sekimizu,, H. Hirakawa,, S. Kuhara,, S. Goto,, J. Yabuzaki,, M. Kanehisa,, A. Yamashita,, K. Oshima,, K. Furuya,, C. Yoshino,, T. Shiba,, M. Hattori,, N. Ogasawara,, H. Hayashi, and, K. Hiramatsu. 2001. Whole genome sequencing of meticillin-resistant Staphylococcus aureus. Lancet 357: 1225– 1240.

69. Kwan, T.,, J. Liu,, M. DuBow,, P. Gros, and, J. Pelletier. 2005. The complete genomes and proteomes of 27 Staphylococcus aureus bacteriophages. Proc. Natl. Acad. Sci. USA 102: 5174– 5179.

70. Ladhani, S.,, C. L. Joannou,, D. P. Lochrie,, R. W. Evans, and, S. M. Poston. 1999. Clinical, microbial, and biochemical aspects of the exfoliative toxins causing staphylococcal scalded-skin syndrome. Clin. Microbiol. Rev. 12: 224– 242.

71. Lawrence, J. G.,, R. W. Hendrix, and, S. Casjens. 2003. Where are the pseudogenes in bacterial genomes? Trends Microbiol. a: 535– 540.

72. Lindsay, J. A.,, A. Ruzin,, H. F. Ross,, N. Kurepina, and, R. P. Novick. 1998. The gene for toxic shock toxin is carried by a family of mobile pathogenicity islands in Staphylococcus aureus. Mol. Microbiol. 29: 527– 543.

73. Luria, S. E., and, M. Delbrück. 1943. Mutations of bacteria from virus sensitivity to virus resistance. Genetics 28: 491– 511.

74. Matsumoto, M.,, N. P. Hoe,, M. Liu,, S. B. Beres,, G. L. Sylva,, C. M. Brandt,, G. Haase, and, J. M. Musser. 2003. Intrahost sequence variation in the streptococcal inhibitor of complement gene in patients with human pharyngitis. J. Infect. Dis. 187: 604– 612.

75. Matz, C.,, P. Deines,, J. Boenigk,, H. Arndt,, L. Eberl,, S. Kjelleberg, and, K. Jurgens. 2004. Impact of violacein-producing bacteria on survival and feeding of bacteriovorous nano. Appl. Environ. Microbiol. 70: 1593– 1599.

76. Matz, C., and, S. Kjelleberg. 2005. Off the hook-how bacteria survive protozoan grazing. Trends Microbiol. 13: 302– 307.

77. McClelland, M.,, K. E. Sanderson,, J. Spieth,, S. W. Clifton,, P. Latreille,, L. Courtney,, S. Porwollik,, J. Ali,, M. Dante,, F. Du,, S. Hou,, D. Layman,, S. Leonard,, C. Nguyen,, K. Scott,, A. Holmes,, N. Grewal,, E. Mulvaney,, E. Ryan,, H. Sun,, L. Florea,, W. Miller,, T. Stoneking,, M. Nhan,, R. Waterston, and, R. K. Wilson. 2001. Complete genome sequence of Salmonella enterica serovar Typhimurium LT2. Nature 413: 852– 856.

78. McGrath, S.,, G. F. Fitzgerald, and, D. van Sinderen. 2002. Identification and characterization of phage-resistance genes in temperate lactococcal bacteriophages. Mol. Microbiol. 43: 509– 520.

79. Miller, A. A.,, N. C. Engleberg, and, V. J. DiRita. 2001. Repression of virulence genes by phosphorylation-dependent oligomerization of CsrR at target promoters in S. pyogenes. Mol. Microbiol. 40: 976– 990.

80. Miller, L. G.,, F. Perdreau-Remington,, G. Rieg,, S. Mehdi,, J. Perlroth,, A. S. Bayer,, A. W. Tang,, T. O. Phung, and, B. Spellberg. 2005. Necrotizing fasciitis caused by community-associated methicillin-resistant Staphylococcus aureus in Los Angeles. N. Engl. J. Med. 352: 1445– 1453.

81. Mirold, S.,, K. Ehrbar,, A. Weissmüller,, R. Prager,, H. Tschäpe,, H. Rüssmann, and, W. D. Hardt. 2001. Salmonella host cell invasion emerged by acquisition of a mosaic of separate genetic elements, including Salmonella pathogenicity island 1 (SPI1), SPI5, and sopE2. J. Bacteriol. 183: 2348– 2358.

82. Mirold, S.,, W. Rabsch,, H. Tschape, and, W. D. Hardt. 2001. Transfer of the Salmonella type III effector sopE between unrelated phage families. J. Mol. Biol. 312: 7– 16.

83. Morgan, G. J.,, G. F. Hatfull,, S. Casjens, and, R. W. Hendrix. 2002. Bacteriophage Mu genome sequence: analysis and comparison with Mu-like prophages in Haemophilus, Neisseria and Deinococcus. J. Mol. Biol. 317: 337– 359.

84. Nagiec, M. J.,, B. Lei,, S. K. Parker,, M. L. Vasil,, M. Matsumoto,, R. M. Ireland,, S. B. Beres,, N. P. Hoe, and, J. M. Musser. 2004. Analysis of a novel prophage-encoded group A Streptococcus extracellular phospholipase A(2). J. Biol. Chem. 279: 45909– 45918.

85. Nakagawa, I.,, K. Kurokawa,, A. Yamashita,, M. Nakata,, Y. Tomiyasu,, N. Okahashi,, S. Kawabata,, K. Yamazaki,, T. Shiba,, T. Yasunaga,, H. Hayashi,, M. Hattori, and, S. Hamada. 2003. Genome sequence of an M3 strain of Streptococcus pyogenes reveals a large-scale genomic rearrangement in invasive strains and new insights into phage evolution. Genome Res. 13: 1042– 1055.

86. Nakayama, K.,, S. Kanaya,, M. Ohnishi,, Y. Terawaki, and, T. Hayashi. 1999. The complete nucleotide sequence of phi CTX, a cytotoxin-converting phage of Pseudomonas aeruginosa: implications for phage evolution and horizontal gene transfer via bacteriophages. Mol. Microbiol. 31: 399– 419.

87. Narita, S.,, J. Kaneko,, J. Chiba,, Y. Piemont,, S. Jarraud,, J. Etienne, and, Y. Kamio. 2001. Phage conversion of Panton-Valentine leukocidin in Staphylococcus aureus: molecular analysis of a PVL-converting phage, phiSLT. Gene 268: 195– 206.

88. Nariya, H.,, A. Nishiyama, and, Y. Kamio. 1997. Identification of the minimum segment in which the threonine246 residue is a potential phosphorylated site by protein kinase A for the LukS-specific function of staphylococcal leukocidin. FEBS Lett. 415: 96– 100.

89. Nilsson, A. S.,, J. L. Karlsson, and, E. Haggard-Ljungquist. 2003. Site-specific recombination links the evolution of P2-like coliphages and pathogenic enterobacteria. Mol. Biol. Evol. 21: 1– 13.

90. Ohnishi, M.,, K. Kurokawa, and, T. Hayashi. 2001. Diversification of Escherichia coli genomes: are bacteriophages the major contributors? Trends Microbiol. 9: 481– 485.

91. Ohnishi, M.,, J. Terajima,, K. Kurokawa,, K. Nakayama,, T. Murata,, K. Tamura,, Y. Ogura,, H. Watanabe, and, T. Hayashi. 2002. Genomic diversity of enterohemorrhagic Escherichia coli O157 revealed by whole genome PCR scanning. Proc. Natl. Acad. Sci. USA 99: 17043– 17048.

92. Okamoto, K.,, J. A. Mudd, and, J. Marmur. 1968. Conversion of Bacillus subtilis DNA to phage DNA following mitomycin C induction. J. Mol. Biol. 34: 429– 437.

93. Pan, E. S.,, B. A. Diep,, E. D. Charlebois,, C. Auerswald,, H. A. Carleton,, G. F. Sensabaugh, and, F. Perdreau-Remington. 2005. Population dynamics of nasal strains of methicillin-resistant Staphylococcus aureus and their relation to community-associated disease activity. J. Infect. Dis. 192: 811– 818.

94. Pedulla, M. L.,, M. E. Ford,, J. M. Houtz,, T. Karthikeyan,, C. Wadsworth,, J. A. Lewis,, D. Jacobs-Sera,, J. Falbo,, J. Gross,, N. R. Pannunzio,, W. Brucker,, V. Kumar,, J. Kandasamy,, L. Keenan,, S. Bardarov,, J. Kriakov,, J. G. Lawrence,, W. R. Jacobs, Jr.,, R. W. Hendrix, and, G. F. Hatfull. 2003. Origins of highly mosaic mycobacteriophage genomes. Cell 113: 171– 182.

95. Pelludat, C.,, S. Mirold, and, W. D. Hardt. 2003. The SopEPhi phage integrates into the ssrA gene of Salmonella enterica serovar Typhimurium A36 and is closely related to the Fels-2 prophage. J. Bacteriol. 185: 5182– 5191.

96. Perna, N. T.,, G. Plunkett III,, V. Burland,, B. Mau,, J. D. Glasner,, D. J. Rose,, G. F. Mayhew,, P. S. Evans,, J. Gregor,, H. A. Kirkpatrick,, G. Posfai,, J. Hackett,, S. Klink,, A. Boutin,, Y. Shao,, L. Miller,, E. J. Grotbeck,, N. W. Davis,, A. Lim,, E. T. Dimalanta,, K. D. Potamousis,, J. Apodaca,, T. S. Anantharaman,, J. Lin,, G. Yen,, D. C. Schwartz,, R. A. Welch, and, F. R. Blattner. 2001. Genome sequence of enterohaemorrhagic Escherichia coli O157:H7. Nature 409: 529– 533.

97. Porwollik, S.,, R. M. Wong, and, M. McClel-land. 2002. Evolutionary genomics of Salmonella: gene acquisitions revealed by microarray analysis. Proc. Natl. Acad. Sci. USA 99: 8956– 8961.

98. Postma, B.,, M. J. Poppelier,, J. C. van Galen,, E. R. Prossnitz,, J. A. van Strijp,, C. J. de Haas, and, K. P. van Kessel. 2004. Chemotaxis in-hibitory protein of Staphylococcus aureus binds specifically to the C5a and formylated peptide receptor. J. Immunol. 172: 6994– 7001.

99. Prescott, J. F.,, C. W. Miller,, K. A. Mathews,, J. A. Yager, and, L. DeWinter. 1997. Update on canine streptococcal toxic shock syndrome and necrotizing fasciitis. Can. Vet. J. 38: 241– 242.

100. Proft, T.,, S. L. Moffatt,, C. J. Berkahn, and, J. D. Fraser. 1999. Identification and characterization of novel superantigens from Streptococcus pyogenes. J. Exp. Med. 189: 89– 102.

101. Proux, C.,, D. van Sinderen,, J. Suarez,, P. Garcia,, V. Ladero,, G. F. Fitzgerald,, F. Desiere, and, H. Brussow. 2002. The dilemma of phage taxonomy illustrated by comparative genomics of Sfi21-like Siphoviridae in lactic acid bacteria. J. Bacteriol. 184: 6026– 6036.

102. Rahimpour, R.,, G. Mitchell,, M. H. Khandaker,, C. Kong,, B. Singh,, L. Xu,, A. Ochi,, R. D. Feldman,, J. G. Pickering,, B. M. Gill, and, D. J. Kelvin. 1999. Bacterial superantigens induce down-modulation of CC chemokine responsiveness in human monocytes via an alternative chemokine ligand-independent mechanism. J. Immunol. 162: 2299– 2307.

103. Rohwer, F. 2003. Global phage diversity. Cell 113: 141.

104. Rooijakkers, S. H.,, M. Ruyken,, A. Roos,, M. R. Daha,, J. S. Presanis,, R. B. Sim,, W. J. van Wamel,, K. P. van Kessel, and, J. A. van Strijp. 2005. Immune evasion by a staphylococcal complement inhibitor that acts on C3 convertases. Nat. Immunol. 6: 920– 927.

105. Ruzin, A.,, J. Lindsay, and, R. P. Novick. 2001. Molecular genetics of SaPI1—a mobile pathogenicity island in Staphylococcus aureus. Mol. Microbiol. 41: 365– 377.

106. Sakaguchi, Y.,, T. Hayashi,, K. Kurokawa,, K. Nakayama,, K. Oshima,, Y. Fujinaga,, M. Ohnishi,, E. Ohtsubo,, M. Hattori, and, K. Oguma. 2005. The genome sequence of Clostridium botulinum type C neurotoxin-converting phage and the molecular mechanisms of unstable lysogeny. Proc. Natl. Acad. Sci. USA 102: 17472– 17477.

107. Shelburne, S. A.,, III, C. Granville,, M. Tokuyama,, I. Sitkiewicz,, P. Patel, and, J. M. Musser. 2005. Growth characteristics of and virulence factor production by group A Streptococcus during cultivation in human saliva. Infect. Immun. 73: 4723– 4731.

108. Smith, N. L.,, E. J. Taylor,, A. M. Lindsay,, S. J. Charnock,, J. P. Turkenburg,, E. J. Dodson,, G. J. Davies, and, G.W. Black. 2005. Structure of a group A streptococcal phage-encoded virulence factor reveals a catalytically active triple-stranded beta-helix. Proc. Natl. Acad. Sci. USA 102: 17652– 17657.

109. Smoot, J. C.,, K. D. Barbian,, J. J. Van Gompel,, L. M. Smoot,, M. S. Chaussee,, G. L. Sylva,, D. E. Sturdevant,, S. M. Ricklefs,, S. F. Porcella,, L. D. Parkins,, S. B. Beres,, D. S. Campbell,, T. M. Smith,, Q. Zhang,, V. Kapur,, J. A. Daly,, L. G. Veasy, and, J. M. Musser. 2002. Genome sequence and comparative microarray analysis of serotype M18 group A Streptococcus strains associated with acute rheumatic fever outbreaks. Proc. Natl. Acad. Sci. USA 99: 4668– 4673.

110. Smoot, L. M.,, J. K. McCormick,, J. C. Smoot,, N. P. Hoe,, I. Strickland,, R. L. Cole,, K. D. Barbian,, C. A. Earhart,, D. H. Ohlendorf,, L. G. Veasy,, H. R. Hill,, D. Y. Leung,, P. M. Schlievert, and, J. M. Musser. 2002. Characterization of two novel pyrogenic toxin superantigens made by an acute rheumatic fever clone of Streptococcus pyogenes associated with multiple disease outbreaks. Infect. Immun. 70: 7095– 7104.

111. Smoot, L. M.,, J. C. Smoot,, M. R. Graham,, G. A. Somerville,, D. E. Sturdevant,, C. A. Migliaccio,, G. L. Sylva, and, J. M. Musser. 2001. Global differential gene expression in response to growth temperature alteration in group A Streptococcus. Proc. Natl. Acad. Sci. USA 98: 10416– 10421.

112. Sriskandan, S.,, M. Unnikrishnan,, T. Krausz, and, J. Cohen. 2000. Mitogenic factor (MF) is the major DNase of serotype M89 Streptococcus pyogenes. Microbiology 146: 2785– 2792.

113. Stover, C. K.,, X. Q. Pham,, A. L. Erwin,, S. D. Mizoguchi,, P. Warrener,, M. J. Hickey,, F. S. Brinkman,, W. O. Hufnagle,, D. J. Kowalik,, M. Lagrou,, R. L. Garber,, L. Goltry,, E. Tolentino,, S. Westbrock-Wadman,, Y. Yuan,, L. L. Brody,, S. N. Coulter,, K. R. Folger,, A. Kas,, K. Larbig,, R. Lim,, K. Smith,, D. Spencer,, G. K. Wong,, Z. Wu,, I. T. Paulsen,, J. Reizer,, M. H. Saier,, R. E. Hancock,, S. Lory, and, M.V. Olson. 2000. Complete genome sequence of Pseudomonas aeruginosa PA01, an opportunistic pathogen. Nature 406: 959– 964.

114. Sumby, P.,, K. D. Barbian,, D. J. Gardner,, A. R. Whitney,, D. M. Welty,, R. D. Long,, J. R. Bailey,, M. J. Parnell,, N. P. Hoe,, G. G. Adams,, F. R. DeLeo, and, J. M. Musser. 2005. Extracellular deoxyribonuclease made by group A Streptococcus assists pathogenesis by enhancing evasion of the innate immune response. Proc. Natl. Acad. Sci. USA 102: 1679– 1684.

115. Sumby, P.,, S. F. Porcella,, A. G. Madrigal,, K. D. Barbian,, K. Virtaneva,, S. M. Ricklefs,, D. E. Sturdevant,, M. R. Graham,, J. Vuopio-Varkila,, N. P. Hoe, and, J. M. Musser. 2005. Evolutionary origin and emergence of a highly successful clone of serotype M1 group a Streptococcus involved multiple horizontal gene transfer events. J. Infect. Dis. 192: 771– 782.

116. Sumby, P., and, M. K. Waldor. 2003. Transcription of the toxin genes present within the Staphylococcal phage phiSa3ms is intimately linked with the phage’s life cycle. J. Bacteriol. 185: 6841– 6851.

117. Susskind, M. M., and, D. Botstein. 1978. Molecular genetics of bacteriophage P22. Micro-biol. Rev. 42: 385– 413.

118. Tobe, T.,, S. A. Beatson,, H. Taniguchi,, H. Abe,, C. M. Bailey,, A. Fivian,, R. Younis,, S. Matthews,, O. Marches,, G. Frankel,, T. Hayashi, and, M. J. Pallen. 2006. An extensive repertoire of type III secretion effectors in Escherichia coli O157 and the role of lambdoid phages in their dissemination. Proc. Natl. Acad. Sci. USA 103: 14941– 14946.

119. Ubeda, C.,, E. Maiques,, E. Knecht,, I. Lasa,, R. P. Novick, and, J. R. Penades. 2005. Antibiotic-induced SOS response promotes horizontal dissemination of pathogenicity island-encoded virulence factors in staphylococci. Mol. Microbiol. 56: 836– 844.

120. van Wamel, W. J.,, S. H. Rooijakkers,, M. Ruyken,, K. P. van Kessel, and, J. A. van Strijp. 2006. The innate immune modulators staphylococcal complement inhibitor and chemotaxis inhibitory protein of Staphylococcus aureus are located on beta-hemolysin-converting bacteriophages. J. Bacteriol. 188: 1310– 1315.

121. Ventura, M.,, A. Bruttin,, C. Canchaya, and, H. Brüssow. 2002. Transcription analysis of Streptococcus thermophilus phages in the lysogenic state. Virology 302: 21– 32.

122. Virtaneva, K.,, M. R. Graham,, S. F. Porcella,, N. P. Hoe,, H. Su,, E. A. Graviss,, T. J. Gardner,, J. E. Allison,, W. J. Lemon,, J. R. Bailey,, M. J. Parnell, and, J. M. Musser. 2003. Group A Streptococcus gene expression in humans and cynomolgus macaques with acute pharyngitis. Infect. Immun. 71: 2199– 2207.

123. Virtaneva, K.,, S. F. Porcella,, M. R. Graham,, R. M. Ireland,, C. A. Johnson,, S. M. Ricklefs,, I. Babar,, L. D. Parkins,, R. A. Romero,, G. J. Corn,, D. J. Gardner,, J. R. Bailey,, M. J. Parnell, and, J. M. Musser. 2005. Longitudinal analysis of the group A Streptococcus transcriptome in experimental pharyngitis in cynomolgus macaques. Proc. Natl. Acad. Sci. USA 102: 9014– 9019.

124. von Eiff, C.,, K. Becker,, K. Machka,, H. Stammer, and, G. Peters. 2001. Nasal carriage as a source of Staphylococcus aureus bacteremia. Study Group. N. Engl. J. Med. 344: 11– 16.

125. Voyich, J. M.,, K. R. Braughton,, D. E. Sturdevant,, S. D. Kobayashi,, B. Lei,, K. Virtaneva,, D. W. Dorward,, J. M. Musser, and, F. R. DeLeo. 2003. Genome-wide protective response used by group A Streptococcus to evade destruction by human polymorphonuclear leukocytes. Proc. Natl. Acad. Sci. USA 100: 1996– 2001.

126. Voyich, J. M.,, K. R. Braughton,, D. E. Sturdevant,, A. R. Whitney,, B. Said-Salim,, S. F. Porcella,, R. D. Long,, D. W. Dorward,, D. J. Gardner,, B. N. Kreiswirth,, J. M. Musser, and, F. R. DeLeo. 2005. Insights into mechanisms used by Staphylococcus aureus to avoid destruction by human neutrophils. J. Immunol. 175: 3907– 3919.

127. Wagner, P. L., and, M. K. Waldor. 2002. Bacteriophage control of bacterial virulence. Infect. Immun. 70: 3985– 3993.

128. Waldor, M. K. 1998. Bacteriophage biology and bacterial virulence. Trends Microbiol. 6: 295– 297.

129. Waldor, M. K., and, J. J. Mekalanos. 1996. Lysogenic conversion by a filamentous phage encoding cholera toxin. Science 272: 1910– 1914.

130. Westmoreland, B. C.,, W. Szybalski, and, H. Ris. 1969. Mapping of deletions and substitutions in heteroduplex DNA molecules of bacteriophage lambda by electron microscopy. Science 163: 1343– 1348.

131. Whiteley, M.,, M. G. Bangera,, R. E. Bum-garner,, M. R. Parsek,, G. M. Teitzel,, S. Lory, and, E. P. Greenberg. 2001. Gene expression in Pseudomonas aeruginosa biofilms. Nature 413: 860– 864.

132. Wildschutte, H.,, D. M. Wolfe,, A. Tamewitz, and, J. G. Lawrence. 2004. Protozoan predation, diversifying selection, and the evolution of antigenic diversity in Salmonella. Proc. Natl. Acad. Sci. USA 101: 10644– 10649.

133. Winkler, K. C.,, J. de Waart, and, C. Groot-sen. 1965. Lysogenic conversion of staphylococci to loss of beta-toxin. J. Gen. Microbiol. 39: 321– 333.

134. Wommack, K. E., and, R. R. Colwell. 2000. Virioplankton: viruses in aquatic ecosystems. Microbiol. Mol. Biol. Rev. 64: 69– 114.

135. Yamaguchi, T.,, T. Hayashi,, H. Takami,, K. Nakasone,, M. Ohnishi,, K. Nakayama,, S. Yamada,, H. Komatsuzawa, and, M. Sugai. 2000. Phage conversion of exfoliative toxin A production in Staphylococcus aureus. Mol. Micro-biol. 38: 694– 705.

136. Zou, D.,, J. Kaneko,, S. Narita, and, Y. Kamio. 2000. Prophage, phiPV83-pro, carrying panton-valentine leukocidin genes, on the Staphylococcus aureus P83 chromosome: comparative analysis of the genome structures of phiPV83-pro, phiPVL, phi11, and other phages. Biosci. Biotechnol. Biochem. 64: 2631– 2643.