Chapter 30 : Mutagenesis

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

Preview this chapter:
Zoom in

Mutagenesis, Page 1 of 2

| /docserver/preview/fulltext/10.1128/9781555818005/9781555812133_Chap30-1.gif /docserver/preview/fulltext/10.1128/9781555818005/9781555812133_Chap30-2.gif


The first part of this chapter gives a general background on mutagenesis, discusses how the appearance of spontaneous mutations within the chromosome of is important for the dissemination of factors important in virulence and antibiotic resistance, and presents recent evidence for functional DNA repair systems thought to be absent in this organism. Induced mutagenesis has been pivotal in unravelling the pathophysiology of . The second part of the chapter describes the techniques that have been employed, starting from the first reports of chemically induced mutations to the modern systematic approaches that will allow genome-wide analysis of the organism. There are three main categories of mutations: substitutions, frameshifts, and genomic rearrangements. Any bacterial cell contains various DNA repair systems, which protect against both spontaneous errors of replication and the mutagenic effects of chemical and physical agents generated from their own metabolism or the environment. Resistance to metronidazole is associated with loss of oxygen-insensitive NADPH nitroreductase activity, an enzyme that reduces metronidazole to active metabolites that are directly toxic to the bacterium. The genetic polymorphism of was exemplified by a study that found that the nucleotide sequence of a 294-bp fragment of (a housekeeping gene encoding a phosphoglucosamine mutase present in all strains) was unique for each of the 29 strains studied. In conclusion, the relatively recent development of molecular tools that allow site-directed mutagenesis, random mutagenesis, and systematic gene inactivation will greatly facilitate the genetic analysis of .

Citation: Labigne A, Jenks P. 2001. Mutagenesis, p 335-344. In Mobley H, Mendz G, Hazell S (ed), . ASM Press, Washington, DC. doi: 10.1128/9781555818005.ch30

Key Concept Ranking

Genetic Recombination
Genetic Elements
Random Amplified Polymorphic DNA
Microbial Genetics
Highlighted Text: Show | Hide
Loading full text...

Full text loading...


1. Achtman, M.,, T. Azuma,, D. E. Berg,, Y. Ito,, G. Morelli,, Z. J. Pan,, S. Suerbaum,, S. A. Thompson,, A. van der Ende,, and L. J. van Doom. 1999. Recombination and clonal groupings within Helicobacter pylori from different geographical regions. Mol. Microbiol. 32:459470.
2. Akopyants, N. S.,, K. A. Eaton,, and D. E. Berg. 1995. Adaptive mutation and cocolonization during Helicobacter pylori infection of gnotobiotic piglets. Infect. Immun. 63:116121.
3. Allan, E.,, P. Mullany,, and S. Tabaqchali. 1998. Construction and characterization of a Helicobacter pylori clpB mutant and role of the gene in the stress response. J. Bacterial. 180: 426429.
4. Aim, R. A.,, L. S. Ling,, D. T. Moir,, B. L. King,, E. D. Brown,, P. C. Doig,, D. R. Smith,, B. Noonan,, B. C. Guild,, B. L. dejonge,, G. Carmel,, P. J. Tummino,, A. Caruso,, M. Uria-Nickelsen,, D. M. Mills,, C. Ives,, R. Gibson,, D. Merberg,, S. D. Mills,, Q. Jiang,, D. E. Taylor,, G. F. Vovis,, and T. J. Trust. 1999. Genomic-sequence comparison of two unrelated isolates of the human gastric pathogen Helicobacter pylori. Nature 397:176180. (Erratum, 397:719.)
5. Alm, R. A.,, and T. J. Trust. 1999. Analysis of the genetic diversity of Helicobacter pylori: the tale of two genomes. J. Mol. Med. 77:834846.
6. Appelmelk, B. J.,, M. C. Martino,, E. Veenhof,, M. A. Monteiro,, J. J. Masskant,, R. Negrini,, F. Lindh,, M. Perry,, G. Del Giudice,, and C. M. J. E. Vandenbroucke-Grauls. 2000. Phase variation in H type I and Lewis a epitopes of Helicobacter pylori lipopolysaccharide. Infect. Immun. 68:59285932.
7. Bauerfeind, P.,, R. M. Garner,, and L. T. Mobley. 1996. Allelic exchange mutagenesis of nixA in Helicobacter pylori results in reduced nickel transport and urease activity. Infect. Immun. 64:28772880.
8. Bayle, D.,, S. Wangler,, T. Weitzenegger,, W. Steinhilber,, J. Volz,, M. Przybylski,, K. P. Schafer,, G. Sachs,, and K. Melchers. 1998. Properties of the P-type ATPases encoded by the copAP Operons of Helicobacter pylori and Helicobacter felis. J. Bacteriol. 180:317329.
9. Bijlsma, J. J.,, C. M. Vandenbroucke-Grauls,, S. H. Phadnis,, and J. G. Küsters. 1999. Identification of virulence genes of Helicobacter pylori by random insertion mutagenesis. Infect. Immun. 67:24332440.
10. Bina, J. E.,, F. Nano,, and R. E. Hancock. 1997. Utilization of alkaline phosphatase fusions to identify secreted proteins, including potential efflux proteins and virulence factors from Helicobacter pylori. FEMS Microbiol. Lett. 148:6368.
11. Censini, S.,, C. Lange,, Z. Xiang,, J. E. Crabtree,, P. Ghiara,, M. Borodovsky,, R. Rappuoli,, and A. Covacci. 1996. cag, a pathogenicity island of Helicobacter pylori, encodes type I-specific and disease-associated virulence factors. Proc. Natl. Acad. Sci. USA 93:1464814653.
12. Copass, M.,, G. Grandi,, and R. Rappuoli. 1997. Introduction of unmarked mutations in the Helicobacter pylori vacA gene with a sucrose sensitivity marker. Infect. Immun. 65:19491952.
13. Curtiss, E. H.,, D. E. Nix,, and J. J. Schentag. 1990. In vitro selection of resistant Helicobacter pylori. Antimicrob. Agents Chemother. 34:16371641.
14. Dean Rupp, W., 1996. DNA repair, p. 22772294. In F. C. Neidhardt,, R. Curtiss III,, J. L. Ingraham,, E. C. C. Lin,, K. B. Low,, B. Magasanik,, W. S. Reznikoff,, M. Riley,, M. Schaechter,, and H. E. Umbarger (ed.), Escherichia coli and Salmonella: Cellular and Molecular Biology, 2nd ed., vol. 2. ASM Press, Washington, D.C.
15. Eaton, K. A.,, C. L. Brooks,, D. R. Morgan,, and S. Krakowka. 1991. Essential role of urease in pathogenesis of gastritis induced by Helicobacter pylori in gnotobiotic piglets. Infect. Immun. 59:24702475.
16. Eaton, K. A.,, D. R. Morgan,, and S. Krakowka. 1992. Motility as a factor in the colonisation of gnotobiotic piglets by Helicobacter pylori. J. Med. Microbiol. 37:123127.
17. Ferrero, R. L.,, V. Cussac,, P. Courcoux,, and A. Labigne. 1992. Construction of isogenic urease-negative mutants of Helicobacter pylori by allelic exchange. J. Bacteriol. 174:42124217.
18. Ge, Z.,, and D. E. Taylor. 1997. The Helicobacter pylori gene encoding phosphatidylserine synthase: sequence, expression, and insertional mutagenesis. J. Bacteriol. 179:49704976.
19. Goodwin, A.,, D. Kersulyte,, G. Sisson,, S. J. Veldhuyzen van Zanten,, D. E. Berg,, and P. S. Hoffman. 1998. Metronidazole resistance in Helicobacter pylori is due to null mutations in a gene (rdxA) that encodes an oxygen-insensitive NADPH nitroreductase. Mol. Microbiol. 28:383393.
20. Haas, R.,, T. F. Meyer,, and J. P. van Putten. 1993. Aflagellated mutants of Helicobacter pylori generated by genetic transformation of naturally competent strains using transposon shuttle mutagenesis. Mol. Microbiol. 8:753760.
21. Hendricks, J. K.,, and H. L. Mobley. 1997. Helicobacter pylori ABC transporter: effect of allelic exchange mutagenesis on urease activity. J. Bacteriol. 179:58925902.
22. Hofreuter, D.,, S. Odenbreit,, G. Henke,, and R. Haas. 1998. Natural competence for DNA transformation in Helicobacter pylori: identification and genetic characterization of the comB locus. Mol. Microbiol. 28:10271038.
23. Hulten, K.,, A. Gibreel,, O. Skold,, and L. Engstrand. 1997. Macrolide resistance in Helicobacter pylori: mechanism and stability in strains from clarithromycin-treated patients. Anti-microb. Agents Chemother. 41:25502553.
24. Hurtado, A.,, B. Chahal,, R. J. Owen,, and A. W. Smith. 1994. Genetic diversity of the Helicobacter pylori haemagglutinin/ protease (hap) gene. FEMS Microbiol. Lett. 123:173178.
25. Hutchinson, F., 1996. Mutagenesis, p. 22182235. In F. C. Neidhardt,, R. Curtiss III,, J. L. Ingraham,, E. C. C. Lin,, K. B. Low,, B. Magasanik,, W. S. Reznikoff,, M. Riley,, M. Schaechter,, and H. E. Umbarger (ed.), Escherichia coli and Salmonella: Cellular and Molecular Biology, 2nd ed., vol. 2. ASM Press, Washington, D.C.
26. Jenks, P. J.,, R. L. Ferrero,, and A. Labigne. 1999. The role of the rdxA gene in the evolution of metronidazole resistance in Helicobacter pylori. J. Antimicrob. Chemother. 43:753758.
27. Jenks, P. J.,, A. Labigne,, and R. L. Ferrero. 1999. Exposure to metronidazole in vivo readily induces resistance in Helicobacter pylori and reduces the efficacy of eradication therapy in mice. Antimicrob. Agents Chemother. 43:777781.
28. Jeong, J. Y.,, A. K. Mukhopadhyay,, D. Dailidiene,, Y. Wang,, B. Velapatino,, R. H. Gilman,, A. J. Parkinson,, G. B. Nair,, B. C. Wong,, S. K. Lam,, R. Mistry,, I. Segal,, Y. Yuan,, H. Gao,, T. Alarcon,, M. L. Brea,, Y. Ito,, D. Kersulyte,, H. K. Lee,, Y. Gong,, A. Goodwin,, P. S. Hoffman,, and D. E. Berg. 2000. Sequential inactivation of rdxA (HP0954) and frxA (HP0642) nitroreductase genes causes moderate and high-level metronidazole resistance in Helicobacter pylori. J. Bacteriol. 182:50825090.
29. Josenhans, C.,, K. A. Eaton,, T. Thevenot,, and S. Suerbaum. 2000. Switching of flagellar motility in Helicobacter pylori by reversible length variation of a short homopolymeric sequence repeat in fliP, a gene encoding a basal body protein. Infect. Immun. 68:45984603.
30. Josenhans, C.,, S. Friedrich,, and S. Suerbaum. 1998. Green fluorescent protein as a novel marker and reporter system in Helicobacter sp. FEMS Microbiol. Lett. 161:263273.
31. Kansau, I.,, J. Raymond,, E. Bingen,, P. Courcoux,, N. Kalach,, M. Bergeret,, N. Braimi,, C. Dupont,, and A. Labigne. 1996. Genotyping of Helicobacter pylori isolates by sequencing of PCR products and comparison with the RAPD technique. Res. Microbiol. 147:661669.
32. Karita, M.,, M. L. Etterbeek,, M. H. Forsyth,, M. K. Tummuru,, and M. J. Blaser. 1997. Characterization of Helicobacter pylori dapE and construction of a conditionally lethal dapE mutant. Infect. Immun. 65:41584164.
33. Kim, J. S.,, J. H. Chang,, S. I. Chung,, and J. S. Yum. 1999. Molecular cloning and characterization of the Helicobacter pylori fliD gene, an essential factor in flagellar structure and motility. J. Bacteriol. 181:69696976.
34. Kwon, D. H.,, F. A. El-Zaatari,, M. Kato,, M. S. Osato,, R. Reddy,, Y. Yamaoka,, and D. Y. Graham. 2000. Analysis of rdxA and involvement of additional genes encoding NAD(P)H flavin oxidoreductase (FrxA) and ferredoxin-like protein (FdxB) in metronidazole resistance of Helicobacter pylori. Antimicrob. Agents Chemother. 44:21332142.
35. Kwon, D. H.,, M. Kato,, F. A. El-Zaatari,, M. S. Osato,, and D. Y. Graham. 2000. Frame-shift mutations in NAD(P)H flavin oxidoreductase encoding gene (frxA) from metronidazole resistant Helicobacter pylori ATCC43504 and its involvement in metronidazole resistance. FEMS Microbiol. Lett. 188: 197202.
36. Labigne, A., 1997. Random mutagenesis of the H. pylori genome, p. 153163. In C. L. Clayton, and H. M. T. Mobley (ed.), Helicobacter pylori Protocols. Humana Press, Totowa, N.J.
37. Labigne, A.,, P. Courcoux,, and L. Tompkins. 1992. Cloning of Campylobacter jejuni genes required for leucine biosynthesis, and construction of leu-negative mutant of C. jejuni by shuttle transposon mutagenesis. Res. Microbiol. 143:1526.
38. Lee, A.,, J. O'Rourke,, M. Corazon De Ungria,, B. Robertson,, G. Daskalopoulos,, and M. F. Dixon. 1997. A standardized mouse model of Helicobacter pylori infection: introducing the Sydney strain. Gastroenterology 112:13861397.
39. Levinson, G.,, and G. A. Gutman. 1987. Slipped-strand mispairing: a major mechanism for DNA sequence evolution. Molec. Biol. Evol. 4:203221.
40. Marshall, D. G.,, W. G. Dundon,, S. M. Beesley,, and C. J. Smyth. 1998. Helicobacter pylori—a conundrum of genetic diversity. Microbiology 144:29252939.
41. Menard, R.,, P. J. Sansonetti,, and C. Parsot. 1993. Nonpolar mutagenesis of the ipa genes defines IpaB, IpaC, and IpaD as effectors of Shigella flexneri entry into epithelial cells. J. Bacteriol. 175:58995906.
42. Moore, R. A.,, B. Beckthold,, S. Wong,, A. Kureishi,, and L. E. Bryan. 1995. Nucleotide sequence of the gyrA gene and characterization of ciprofloxacin-resistant mutants of Helicobacter pylori. Antimicrob. Agents Chemother. 39:107111.
43. Occhialini, A.,, M. Urdaci,, F. Doucet-Populaire,, C. M. Bebear,, H. Lamouliatte,, and F. Megraud. 1997. Macrolide resistance in Helicobacter pylori: rapid detection of point mutations and assays of macrolide binding to ribosomes. Antimicrob. Agents Chemother. 41:27242728.
44. Odenbreit, S.,, M. Till,, and R. Haas. 1996. Optimized BlaM-transposon shuttle mutagenesis of Helicobacter pylori allows the identification of novel genetic loci involved in bacterial virulence. Mol. Microbiol. 20:361373.
45. Odenbreit, S.,, M. Till,, D. Hofreuter,, G. Faller,, and R. Haas. 1999. Genetic and functional characterization of the alpAB gene locus essential for the adhesion of Helicobacter pylori to human gastric tissue. Mol. Microbiol. 31:15371548.
46. Odenbreit, S.,, B. Wieland,, and R. Haas. 1996. Cloning and genetic characterization of Helicobacter pylori catalase and construction of a catalase-deficient mutant strain. J. Bacteriol. 178:69606967.
47. O'Rourke, E. J.,, C. Chevalier,, S. Boiteux,, A. Labigne,, L. Ielpi,, and J. P. Radicella. 2000. A novel 3-methyladenine DNA glycosylase from Helicobacter pylori defines a new class within the endonuclease III family of base excision repair glycosylases. J. Biol. Chem. 275:2007720083.
48. Owen, R. J.,, E. R. Slater,, J. Xerry,, T. M. Peters,, E. L. Teare,, and A. Grant. 1998. Development of a scheme for genotyping Helicobacter pylori based on allelic variation in urease subunit genes. J. Clin. Microbiol. 36:37103712.
49. Reyrat, J.-M.,, V. Pelicic,, B. Gicquel,, and R. Rappuoli. 1998. Counterselectable markers: untapped tools for bacterial genetics and pathogenesis. Infect. Immun. 66:40114017.
50. Schmitz, A.,, C. Josenhans,, and S. Suerbaum. 1997. Cloning and characterization of the Helicobacter pylori flbA gene, which codes for a membrane protein involved in coordinated expression of flagellar genes. J. Bacteriol. 179:987997.
51. Segal, E. D.,, J. Shon,, and L. S. Tompkins. 1992. Characterization of Helicobacter pylori urease mutants. Infect. Immun. 60:18831889.
52. Seifert, H. S.,, M. So,, and F. Heffron,. 1985. Shuttle mutagenesis: a method for introducing transposons into transformable organisms, p. 123134. In J. Setlow, and A. Hollaeinder (ed.), Genetic Engineering, Principles and Methods, vol. 8. Plenum Press, New York, N.Y.
53. Skouloubris, S.,, A. Labigne,, and H. De Reuse. 1997. Identification and characterization of an aliphatic amidase in Helicobacter pylori. Mol. Microbiol. 25:989998.
54. Skouloubris, S.,, J. M. Thiberge,, A. Labigne,, and H. De Reuse. 1998. The Helicobacter pylori Urel protein is not involved in urease activity but is essential for bacterial survival in vivo. Infect. Immun. 66:45174521.
55. Solca, N. M.,, M. V. Bernasconi,, and J.-C. Piffaretti. 2000. Mechanism of metronidazole resistance in Helicobacter pylori: comparison of the rdxA gene sequences in 30 strains. Antimicrob. Agents Chemother. 44:22072210.
56. Stone, G. G.,, D. Shortridge,, J. Versalovic,, J. Beyer,, R. K. Flamm,, D. Y. Graham,, A. T. Ghoneim,, and S. K. Tanaka. 1997. A PCR-oligonucleotide ligation assay to determine the prevalence of 23S rRNA gene mutations in clarithromycin-resistant Helicobacter pylori. Antimicrob Agents Chemother. 41:712714.
57. Suerbaum, S. 2000. Genetic variability within Helicobacter pylori. Int. J. Med. Microbiol. 290:175181.
58. Suerbaum, S.,, J. M. Smith,, K. Bapumia,, G. Morelli,, N. H. Sith,, E. K. Kunstamun,, M. Dyrek,, and M. Achtman. 1998. Free recombination within Helicobacter pylori. Proc. Natl. Acad. Sei. USA 95:1261912624.
59. Takeuchi, H.,, M. Shirai,, J. K. Akada,, M. Tsuda,, and T. Nakazawa. 1998. Nucleotide sequence and characterization of cdrA, a cell division-related gene of Helicobacter pylori. J. Bacteriol. 180:52635268.
60. Tankovic, J.,, D. Lamarque,, J. C. Delchier,, C. J. Soussy,, A. Labigne,, and P. J. Jenks. 2000. Frequent association between alteration of the rdxA gene and metronidazole resistance in French and North African isolates of Helicobacter pylori. Antimicrob. Agents Chemother. 44:608613.
61. Tomb, J.-F.,, O. White,, A. R. Kerlavage,, R. A. Clayton,, G. G. Sutton,, R. D. Fleischmann,, K. A. Ketchum,, H. P. Klenk,, S. Gill,, B. A. Dougherty,, K. Nelson,, J. Quackenbush,, L. Zhou,, E. F. Kirkness,, S. Peterson,, B. Loftus,, D. Richardson,, R. Dodson,, H. G. Khalak,, A. Globek,, K. McKenney,, L. M. Fitzegerald,, N. Lee,, M. D. Adams,, E. K. Hickey,, D. E. Berg,, J. D. Gocayne,, T. R. Utterback,, J. D. Peterson,, J. M. Kelley,, M. D. Cotton,, J. M. Weidman,, C. Fuji,, C. Bowman,, L. Watthey,, E. Wallin,, W. S. Hayes,, M. Borodovsky,, P. D. Karp,, H. O. Smith,, C. M. Fraser,, and J. C. Venter. 1997. The complete genome sequence of the gastric pathogen Helicobacter pylori. Nature 388:539547.
62. Trieu-Cuot, P.,, G. Gerbaud,, T. Lambert,, and P. Courvalin. 1985. In vivo transfer of genetic information between gram-positive and gram-negative bacteria. EMBO J. 4:35833587.
63. Van Doom, L. J.,, C. Figueiredo,, F. Megraud,, S. Pena,, P. Midolo,, D. M. Queiroz,, F. Carneiro,, B. Vanderborght,, M. D. Pegado,, R. Sanna,, W. De Boer,, P. M. Schneeberger,, P. Correa,, E. K. Ng,, J. Atherton,, M. J. Blaser,, and W. G. Quint. 1999. Geographic distribution of vacA allelic types of Helicobacter pylori. Gastroenterology 116:823830.
64. van Zwet, A. A.,, J. C. Thijs,, W. Schievink-de Vries,, J. Schiphuis,, and J. A. Snijder. 1994. In vitro studies on stability and development of metronidazole resistance in Helicobacter pylori. Antimicrob. Agents Chemother. 38:360362.
65. Versalovic, J.,, D. Shortridge,, K. Kibler,, M. V. Griffy,, J. Beyer,, R. K. Flamm,, S. K. Tanaka,, D. Y. Graham,, and M. F. Go. 1996. Mutations in 23S rRNA are associated with clarithromycin resistance in Helicobacter pylori. Antimicrob. Agents Chemother. 40:477480.
66. Wang, G.,, M. Z. Humayun,, and D. E. Taylor. 1999. Mutation as an origin of genetic variability in Helicobacter pylori. Trends Microbiol. 7:488493.
67. Wang, G.,, and D. E. Taylor. 1998. Site-specific mutations in the 23S rRNA gene of Helicobacter pylori confer two types of resistance to macrolide-lincosamide-streptogramin B antibiotics. Antimicrob. Agents Chemother. 42:19521958.
68. Wang, Y.,, K. P. Roos,, and D. E. Taylor. 1993. Transformation of Helicobacter pylori by chromosomal metronidazole resistance and by a plasmid with a selectable chloramphenicol resistance marker. J. Gen. Microbiol. 139(Pt. 10):24852493.


Generic image for table
Table 1

Major genes and functions involved in DNA repair in ( ) and possibly in ( )

Citation: Labigne A, Jenks P. 2001. Mutagenesis, p 335-344. In Mobley H, Mendz G, Hazell S (ed), . ASM Press, Washington, DC. doi: 10.1128/9781555818005.ch30

This is a required field
Please enter a valid email address
Please check the format of the address you have entered.
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error