1887

Microbiology Society logo

Volume 155, Issue 5

Genetic makeup of the LexA regulon deduced from comparative transcriptomics and DNA band shift assays Free

Abstract

The gene of ATCC 13032 was deleted to create the mutant strain NJ2114, which has an elongated cell morphology and an increased doubling time. To characterize the SOS regulon in , the transcriptomes of NJ2114 and a DNA-damage-induced wild-type strain were compared with that of a wild-type control using DNA microarray hybridization. The expression data were combined with bioinformatic pattern searches for LexA binding sites, leading to the detection of 46 potential SOS boxes located upstream of differentially expressed transcription units. Binding of a hexahistidyl-tagged LexA protein to 40 double-stranded oligonucleotides containing the potential SOS boxes was demonstrated by DNA band shift assays. It turned out that LexA binds not only to SOS boxes in the promoter–operator region of upregulated genes, but also to SOS boxes detected upstream of downregulated genes. These results demonstrated that LexA controls directly the expression of at least 48 SOS genes organized in 36 transcription units. The deduced genes encode a variety of physiological functions, many of them involved in DNA repair and survival after DNA damage, but nearly half of them have hitherto unknown functions. Alignment of the LexA binding sites allowed the corynebacterial SOS box consensus sequence TcGAA(a/c)AnnTGTtCGA to be deduced. Furthermore, the common intergenic region of and the differentially expressed - operon, encoding a cell division suppressor and a regulator of deoxyribonucleotide biosynthesis, was characterized in detail. Promoter mapping revealed differences in - expression during SOS response and normal growth conditions. One of the four LexA binding sites detected in the intergenic region is involved in regulating - transcription, whereas the other sites are apparently used for negative autoregulation of expression.

  • Received:
  • Accepted:
  • Revised:
  • Published Online:
Keyword(s): AFM, atomic force microscopy , Cy3, indocarbocyanine , HMM, hidden Markov model , PLP, pyridoxal 5′-phosphate and RACE, rapid amplification of cDNA ends
SGM
Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.025841-0
2009-05-01
2024-04-16
Loading full text...

Full text loading...

/deliver/fulltext/micro/155/5/1459.html?itemId=/content/journal/micro/10.1099/mic.0.025841-0&mimeType=html&fmt=ahah

References

  1. Altschul S. F., Madden T. L., Schäffer A. A., Zhang J., Zhang Z., Miller W., Lipman D. J. 1997; Gapped blast and psi-blast: a new generation of protein database search programs. Nucleic Acids Res 25:3389–3402
     [Google Scholar]
  2. Aravind L., Wolf Y. I., Koonin E. V. 2000; The ATP-cone: an evolutionarily mobile, ATP-binding regulatory domain. J Mol Microbiol Biotechnol 2:191–194
     [Google Scholar]
  3. Baumbach J., Apeltsin L. 2008; Linking Cytoscape and the corynebacterial reference database CoryneRegNet. BMC Genomics 9:184
     [Google Scholar]
  4. Baumbach J., Wittkop T., Rademacher K., Rahmann S., Brinkrolf K., Tauch A. 2007; CoryneRegNet 3.0 – an interactive systems biology platform for the analysis of gene regulatory networks in corynebacteria and Escherichia coli . J Biotechnol 129:279–289
     [Google Scholar]
  5. Beckstette M., Homann R., Giegerich R., Kurtz S. 2006; Fast index based algorithms and software for matching position specific scoring matrices. BMC Bioinformatics 7:389
     [Google Scholar]
  6. Bigot S., Sivanathan V., Possoz C., Barre F. X., Cornet F. 2007; FtsK, a literate chromosome segregation machine. Mol Microbiol 64:1434–1441
     [Google Scholar]
  7. Borovok I., Gorovitz B., Yanku M., Schreiber R., Gust B., Chater K., Aharonowitz Y., Cohen G. 2004; Alternative oxygen-dependent and oxygen-independent ribonucleotide reductases in Streptomyces : cross-regulation and physiological role in response to oxygen limitation. Mol Microbiol 54:1022–1035
     [Google Scholar]
  8. Boshoff H. I., Reed M. B., Barry C. E. III, Mizrahi V. 2003; DnaE2 polymerase contributes to in vivo survival and the emergence of drug resistance in Mycobacterium tuberculosis . Cell 113:183–193
     [Google Scholar]
  9. Brent R., Ptashne M. 1981; Mechanism of action of the lexA gene product. Proc Natl Acad Sci U S A 78:4204–4208
     [Google Scholar]
  10. Brinkrolf K., Brune I., Tauch A. 2007; The transcriptional regulatory network of the amino acid producer Corynebacterium glutamicum . J Biotechnol 129:191–211
     [Google Scholar]
  11. Brooks P. C., Movahedzadeh F., Davis E. O. 2001; Identification of some DNA damage-inducible genes of Mycobacterium tuberculosis : apparent lack of correlation with LexA binding. J Bacteriol 183:4459–4467
     [Google Scholar]
  12. Brune I., Brinkrolf K., Kalinowski J., Pühler A., Tauch A. 2005; The individual and common repertoire of DNA-binding transcriptional regulators of Corynebacterium glutamicum , Corynebacterium efficiens , Corynebacterium diphtheriae and Corynebacterium jeikeium deduced from the complete genome sequences. BMC Genomics 6:86
     [Google Scholar]
  13. Brune I., Werner H., Hüser A. T., Kalinowski J., Pühler A., Tauch A. 2006; The DtxR protein acting as dual transcriptional regulator directs a global regulatory network involved in iron metabolism of Corynebacterium glutamicum . BMC Genomics 7:21
     [Google Scholar]
  14. Brune I., Jochmann N., Brinkrolf K., Hüser A. T., Gerstmeir R., Eikmanns B. J., Kalinowski J., Pühler A., Tauch A. 2007; The IclR-type transcriptional repressor LtbR regulates the expression of leucine and tryptophan biosynthesis genes in the amino acid producer Corynebacterium glutamicum . J Bacteriol 189:2720–2733
     [Google Scholar]
  15. Butala M., Zgur-Bertok D., Busby S. J. 2009; The bacterial LexA transcriptional repressor. Cell Mol Life Sci 66:82–93
     [Google Scholar]
  16. Crooks G. E., Hon G., Chandonia J. M., Brenner S. E. 2004; WebLogo: a sequence logo generator. Genome Res 14:1188–1190
     [Google Scholar]
  17. da Rocha R. P., Paquola A. C., Marques M. do V., Menck C. F., Galhardo R. S. 2008; Characterization of the SOS regulon of Caulobacter crescentus . J Bacteriol 190:1209–1218
     [Google Scholar]
  18. Davis E. O., Dullaghan E. M., Rand L. 2002; Definition of the mycobacterial SOS box and use to identify LexA-regulated genes in Mycobacterium tuberculosis . J Bacteriol 184:3287–3295
     [Google Scholar]
  19. Dondrup M., Goesmann A., Bartels D., Kalinowski J., Krause L., Linke B., Rupp O., Sczyrba A., Pühler A., Meyer F. 2003; EMMA: a platform for consistent storage and efficient analysis of microarray data. J Biotechnol 106:135–146
     [Google Scholar]
  20. Dullaghan E. M., Brooks P. C., Davis E. O. 2002; The role of multiple SOS boxes upstream of the Mycobacterium tuberculosis lexA gene – identification of a novel DNA-damage-inducible gene. Microbiology 148:3609–3615
     [Google Scholar]
  21. Eddy S. R. 1998; Profile hidden Markov models. Bioinformatics 14:755–763
     [Google Scholar]
  22. Erill I., Campoy S., Barbé J. 2007; Aeons of distress: an evolutionary perspective on the bacterial SOS response. FEMS Microbiol Rev 31:637–656
     [Google Scholar]
  23. Fernández de Henestrosa A. R., Ogi T., Aoyagi S., Chafin D., Hayes J. J., Ohmori H., Woodgate R. 2000; Identification of additional genes belonging to the LexA regulon in Escherichia coli . Mol Microbiol 35:1560–1572
     [Google Scholar]
  24. Finn R. D., Tate J., Mistry J., Coggill P. C., Sammut S. J., Hotz H. R., Ceric G., Forslund K., Eddy S. R. other authors 2008; The Pfam protein families database. Nucleic Acids Res 36:D281–D288
     [Google Scholar]
  25. Fitzpatrick T. B., Amrhein N., Kappes B., Macheroux P., Tews I., Raschle T. 2007; Two independent routes of de novo vitamin B6 biosynthesis: not that different after all. Biochem J 407:1–13
     [Google Scholar]
  26. Friedberg E. C., Walker G. C., Siede W., Wood R. D., Schultz R. A., Ellenberger T. 2005 DNA Repair and Mutagenesis Washington, DC: American Society for Microbiology;
  27. Galhardo R. S., Rocha R. P., Marques M. V., Menck C. F. 2005; An SOS-regulated operon involved in damage-inducible mutagenesis in Caulobacter crescentus . Nucleic Acids Res 33:2603–2614
     [Google Scholar]
  28. Gamulin V., Cetkovic H., Ahel I. 2004; Identification of a promoter motif regulating the major DNA damage response mechanism of Mycobacterium tuberculosis . FEMS Microbiol Lett 238:57–63
     [Google Scholar]
  29. Gibb E. A., Edgell D. R. 2007; Multiple controls regulate the expression of mobE , an HNH homing endonuclease gene embedded within a ribonucleotide reductase gene of phage Aeh1. J Bacteriol 189:4648–4661
     [Google Scholar]
  30. Gough J., Karplus K., Hughey R., Chothia C. 2001; Assignment of homology to genome sequences using a library of hidden Markov models that represent all proteins of known structure. J Mol Biol 313:903–919
     [Google Scholar]
  31. Grant S. G., Jessee J., Bloom F. R., Hanahan D. 1990; Differential plasmid rescue from transgenic mouse DNAs into Escherichia coli methylation-restriction mutants. Proc Natl Acad Sci U S A 87:4645–4649
     [Google Scholar]
  32. Grinberg I., Shteinberg T., Gorovitz B., Aharonowitz Y., Cohen G., Borovok I. 2006; The Streptomyces NrdR transcriptional regulator is a Zn ribbon/ATP cone protein that binds to the promoter regions of class Ia and class II ribonucleotide reductase operons. J Bacteriol 188:7635–7644
     [Google Scholar]
  33. Gutekunst K., Phunpruch S., Schwarz C., Schuchardt S., Schulz-Friedrich R., Appel J. 2005; LexA regulates the bidirectional hydrogenase in the cyanobacterium Synechocystis sp. PCC 6803 as a transcription activator. Mol Microbiol 58:810–823
     [Google Scholar]
  34. Horton R. M., Hunt H. D., Ho S. N., Pullen J. K., Pease L. R. 1989; Engineering hybrid genes without the use of restriction enzymes: gene splicing by overlap extension. Gene 77:61–68
     [Google Scholar]
  35. Hüser A. T., Becker A., Brune I., Dondrup M., Kalinowski J., Plassmeier J., Pühler A., Wiegrabe I., Tauch A. 2003; Development of a Corynebacterium glutamicum DNA microarray and validation by genome-wide expression profiling during growth with propionate as carbon source. J Biotechnol 106:269–286
     [Google Scholar]
  36. Iyer V. N., Szybalski W. 1963; A molecular mechanism of mitomycin action: linking of complementary DNA strands. Proc Natl Acad Sci U S A 50:355–362
     [Google Scholar]
  37. Jungwirth B., Emer D., Brune I., Hansmeier N., Pühler A., Eikmanns B. J., Tauch A. 2008; Triple transcriptional control of the resuscitation promoting factor 2 ( rpf2 ) gene of Corynebacterium glutamicum by the regulators of acetate metabolism RamA and RamB and the cAMP-dependent regulator GlxR. FEMS Microbiol Lett 281:190–197
     [Google Scholar]
  38. Justice S. S., Garcia-Lara J., Rothfield L. I. 2000; Cell division inhibitors SulA and MinC/MinD block septum formation at different steps in the assembly of the Escherichia coli division machinery. Mol Microbiol 37:410–423
     [Google Scholar]
  39. Kalinowski J., Bathe B., Bartels D., Bischoff N., Bott M., Burkovski A., Dusch N., Eggeling L., Eikmanns B. J. other authors 2003; The complete Corynebacterium glutamicum ATCC 13032 genome sequence and its impact on the production of l-aspartate-derived amino acids and vitamins. J Biotechnol 104:5–25
     [Google Scholar]
  40. Keilhauer C., Eggeling L., Sahm H. 1993; Isoleucine synthesis in Corynebacterium glutamicum : molecular analysis of the ilvB-ilvN-ilvC operon. J Bacteriol 175:5595–5603
     [Google Scholar]
  41. Kirchner O., Tauch A. 2003; Tools for genetic engineering in the amino acid-producing bacterium Corynebacterium glutamicum . J Biotechnol 104:287–299
     [Google Scholar]
  42. Lehmann A. R. 2006; New functions for Y family polymerases. Mol Cell 24:493–495
     [Google Scholar]
  43. Little J. W. 1991; Mechanism of specific LexA cleavage: autodigestion and the role of RecA coprotease. Biochimie 73:411–421
     [Google Scholar]
  44. Little J. W., Mount D. W. 1982; The SOS regulatory system of Escherichia coli . Cell 29:11–22
     [Google Scholar]
  45. Little J. W., Mount D. W., Yanisch-Perron C. R. 1981; Purified LexA protein is a repressor of the recA and lexA genes. Proc Natl Acad Sci U S A 78:4199–4203
     [Google Scholar]
  46. Mazón G., Erill I., Campoy S., Cortés P., Forano E., Barbé J. 2004; Reconstruction of the evolutionary history of the LexA-binding sequence. Microbiology 150:3783–3795
     [Google Scholar]
  47. McHardy A. C., Tauch A., Rückert C., Pühler A., Kalinowski J. 2003; Genome-based analysis of biosynthetic aminotransferase genes of Corynebacterium glutamicum . J Biotechnol 104:229–240
     [Google Scholar]
  48. Möller C., Allen M., Elings V., Engel A., Müller D. J. 1999; Tapping-mode atomic force microscopy produces faithful high-resolution images of protein surfaces. Biophys J 77:1150–1158
     [Google Scholar]
  49. Movahedzadeh F., Colston M. J., Davis E. O. 1997; Determination of DNA sequences required for regulated Mycobacterium tuberculosis RecA expression in response to DNA-damaging agents suggests that two modes of regulation exist. J Bacteriol 179:3509–3518
     [Google Scholar]
  50. Mukherjee A., Cao C., Lutkenhaus J. 1998; Inhibition of FtsZ polymerization by SulA, an inhibitor of septation in Escherichia coli . Proc Natl Acad Sci U S A 95:2885–2890
     [Google Scholar]
  51. Napolitano R., Janel-Bintz R., Wagner J., Fuchs R. P. 2000; All three SOS-inducible DNA polymerases (Pol II, Pol IV and Pol V) are involved in induced mutagenesis. EMBO J 19:6259–6265
     [Google Scholar]
  52. Ogino H., Teramoto H., Inui M., Yukawa H. 2008; DivS, a novel SOS-inducible cell-division suppressor in Corynebacterium glutamicum . Mol Microbiol 67:597–608
     [Google Scholar]
  53. Ohmori H., Friedberg E. C., Fuchs R. P., Goodman M. F., Hanaoka F., Hinkle D., Kunkel T. A., Lawrence C. W., Livneh Z. & other authors; 2001; The Y-family of DNA polymerases. Mol Cell 8:7–8
     [Google Scholar]
  54. Pages V., Koffel-Schwartz N., Fuchs R. P. 2003; recX , a new SOS gene that is co-transcribed with the recA gene in Escherichia coli . DNA Repair (Amst 2:273–284
     [Google Scholar]
  55. Pátek M., Nesvera J., Guyonvarch A., Reyes O., Leblon G. 2003; Promoters of Corynebacterium glutamicum . J Biotechnol 104:311–323
     [Google Scholar]
  56. Peter H., Bader A., Burkovski A., Lambert C., Krämer R. 1997; Isolation of the putP gene of Corynebacterium glutamicum and characterization of a low-affinity uptake system for compatible solutes. Arch Microbiol 168:143–151
     [Google Scholar]
  57. Price M. N., Huang K. H., Alm E. J., Arkin A. P. 2005; A novel method for accurate operon predictions in all sequenced prokaryotes. Nucleic Acids Res 33:880–892
     [Google Scholar]
  58. Rand L., Hinds J., Springer B., Sander P., Buxton R. S., Davis E. O. 2003; The majority of inducible DNA repair genes in Mycobacterium tuberculosis are induced independently of RecA. Mol Microbiol 50:1031–1042
     [Google Scholar]
  59. Rigali S., Derouaux A., Giannotta F., Dusart J. 2002; Subdivision of the helix-turn-helix GntR family of bacterial regulators in the FadR, HutC, MocR, and YtrA subfamilies. J Biol Chem 277:12507–12515
     [Google Scholar]
  60. Rodionov D. A., Gelfand M. S. 2005; Identification of a bacterial regulatory system for ribonucleotide reductases by phylogenetic profiling. Trends Genet 21:385–389
     [Google Scholar]
  61. Sambrook J., Fritsch E. F., Maniatis T. 1989 Molecular Cloning: a Laboratory Manual , 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
  62. Sassanfar M., Roberts J. W. 1990; Nature of the SOS-inducing signal in Escherichia coli . The involvement of DNA replication. J Mol Biol 212:79–96
     [Google Scholar]
  63. Schäfer A., Tauch A., Jäger W., Kalinowski J., Thierbach G., Pühler A. 1994; Small mobilizable multi-purpose cloning vectors derived from the Escherichia coli plasmids pK18 and pK19: selection of defined deletions in the chromosome of Corynebacterium glutamicum . Gene 145:69–73
     [Google Scholar]
  64. Schnarr M., Oertel-Buchheit P., Kazmaier M., Granger-Schnarr M. 1991; DNA binding properties of the LexA repressor. Biochimie 73:423–431
     [Google Scholar]
  65. Shen-Orr S. S., Milo R., Mangan S., Alon U. 2002; Network motifs in the transcriptional regulation network of Escherichia coli . Nat Genet 31:64–68
     [Google Scholar]
  66. Slilaty S. N., Little J. W. 1987; Lysine-156 and serine-119 are required for LexA repressor cleavage: a possible mechanism. Proc Natl Acad Sci U S A 84:3987–3991
     [Google Scholar]
  67. Stoddard B. L. 2005; Homing endonuclease structure and function. Q Rev Biophys 38:49–95
     [Google Scholar]
  68. Stohl E. A., Brockman J. P., Burkle K. L., Morimatsu K., Kowalczykowski S. C., Seifert H. S. 2003; Escherichia coli RecX inhibits RecA recombinase and coprotease activities in vitro and in vivo . J Biol Chem 278:2278–2285
     [Google Scholar]
  69. Studier F. W., Rosenberg A. H., Dunn J. J., Dubendorff J. W. 1990; Use of T7 RNA polymerase to direct expression of cloned genes. Methods Enzymol 185:60–89
     [Google Scholar]
  70. Tapias A., Fernández S., Alonso J. C., Barbé J. 2002; Rhodobacter sphaeroides LexA has dual activity: optimising and repressing recA gene transcription. Nucleic Acids Res 30:1539–1546
     [Google Scholar]
  71. Tauch A., Kirchner O., Wehmeier L., Kalinowski J., Pühler A. 1994; Corynebacterium glutamicum DNA is subjected to methylation-restriction in Escherichia coli . FEMS Microbiol Lett 123:343–347
     [Google Scholar]
  72. Tauch A., Kassing F., Kalinowski J., Pühler A. 1995; The Corynebacterium xerosis composite transposon Tn 5432 consists of two identical insertion sequences, designated IS 1249 , flanking the erythromycin resistance gene ermCX . Plasmid 34:119–131
     [Google Scholar]
  73. Tauch A., Kirchner O., Löffler B., Götker S., Pühler A., Kalinowski J. 2002; Efficient electrotransformation of Corynebacterium diphtheriae with a mini-replicon derived from the Corynebacterium glutamicum plasmid pGA1. Curr Microbiol 45:362–367
     [Google Scholar]
  74. Tauch A., Schneider J., Szczepanowski R., Tilker A., Viehoever P., Gartemann K. H., Arnold W., Blom J., Brinkrolf K. other authors 2008; Ultrafast pyrosequencing of Corynebacterium kroppenstedtii DSM44385 revealed insights into the physiology of a lipophilic corynebacterium that lacks mycolic acids. J Biotechnol 136:22–30
     [Google Scholar]
  75. Thompson J. D., Gibson T. J., Plewniak F., Jeanmougin F., Higgins D. G. 1997; The clustal_x Windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 25:4876–4882
     [Google Scholar]
  76. Torrents E., Grinberg I., Gorovitz-Harris B., Lundstrom H., Borovok I., Aharonowitz Y., Sjoberg B. M., Cohen G. 2007; NrdR controls differential expression of the Escherichia coli ribonucleotide reductase genes. J Bacteriol 189:5012–5021
     [Google Scholar]
  77. Wade J. T., Reppas N. B., Church G. M., Struhl K. 2005; Genomic analysis of LexA binding reveals the permissive nature of the Escherichia coli genome and identifies unconventional target sites. Genes Dev 19:2619–2630
     [Google Scholar]
  78. Walker G. C. 1984; Mutagenesis and inducible responses to deoxyribonucleic acid damage in Escherichia coli . Microbiol Rev 48:60–93
     [Google Scholar]
  79. Walker G. C. 1996; The SOS response of Escherichia coli . In Escherichia coli and Salmonella: Cellular and Molecular Biology Edited by Neidhardt F. C., Curtis R. III, Ingraham J. L., Lin E. C. C., Low K. B., Magasanik B., Reznikoff W. S., Riley M., Schaecheter M., Umbarger H. E. Washington, DC: American Society for Microbiology;
     [Google Scholar]
  80. Wittkop T., Baumbach J., Lobo F. P., Rahmann S. 2007; Large scale clustering of protein sequences with FORCE – a layout based heuristic for weighted cluster editing. BMC Bioinformatics 8:396
     [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.025841-0
Loading
Genetic makeup of the Corynebacterium glutamicum LexA regulon deduced from comparative transcriptomics and in vitro DNA band shift assays
Microbiology 155, 1459 (2009); https://doi.org/10.1099/mic.0.025841-0
/content/journal/micro/10.1099/mic.0.025841-0
/content/journal/micro/10.1099/mic.0.025841-0
Loading

Data & Media loading...

Supplements

Supplementary material 1

PDF

Most cited Most Cited RSS feed

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error