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Volume 159, Issue Pt_1

High-resolution detection of DNA binding sites of the global transcriptional regulator GlxR in Free

Abstract

The transcriptional regulator GlxR has been characterized as a global hub within the gene-regulatory network of . Chromatin immunoprecipitation with a specific anti-GlxR antibody and subsequent high-throughput sequencing (ChIP-seq) was applied to to get new insights into the gene composition of the GlxR regulon. In a comparative approach, cells were grown with either glucose or acetate as the sole carbon source prior to immunoprecipitation. High-throughput sequencing resulted in 69 million reads and 2.6 Gb of genomic information. After mapping of these data on the genome sequence of , 107 enriched DNA fragments were detected from cells grown with glucose as carbon source. GlxR binding sites were identified in the sequence of 79 enriched DNA fragments, of which 21 sites were not previously reported. Electrophoretic mobility shift assays with 40-mer oligomers covering the GlxR binding sites were performed for validation of the results. The detection of new binding sites confirmed the role of GlxR as a regulator of carbon source metabolism and energy conversion, but additionally revealed binding of GlxR in front of the 6C non-coding RNA gene and to non-canonical DNA binding sites within protein-coding regions. The present study underlines the dynamics within the GlxR regulon by identifying targets during growth on glucose and contributes to the expansion of knowledge of this important transcriptional regulator.

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  • Accepted:
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2013-01-01
2024-04-19
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References

  1. Agresti A.( 1992). A survey of exact inference for contingency tables. Stat Sci 7:131–153 [View Article]
     [Google Scholar]
  2. Arndt A., Eikmanns B. J.( 2007). The alcohol dehydrogenase gene adhA in Corynebacterium glutamicum is subject to carbon catabolite repression. J Bacteriol 189:7408–7416 [View Article][PubMed]
     [Google Scholar]
  3. Arndt A., Auchter M., Ishige T., Wendisch V. F., Eikmanns B. J.( 2008). Ethanol catabolism in Corynebacterium glutamicum. J Mol Microbiol Biotechnol 15:222–233 [View Article][PubMed]
     [Google Scholar]
  4. Babu M. M., Luscombe N. M., Aravind L., Gerstein M., Teichmann S. A.( 2004). Structure and evolution of transcriptional regulatory networks. Curr Opin Struct Biol 14:283–291 [View Article][PubMed]
     [Google Scholar]
  5. Babu M. M., Lang B., Aravind L.( 2009). Methods to reconstruct and compare transcriptional regulatory networks. Methods Mol Biol 541:163–180 [View Article][PubMed]
     [Google Scholar]
  6. Bailey T. L., Elkan C.( 1994). Fitting a mixture model by expectation maximization to discover motifs in biopolymers. Proc Int Conf Intell Syst Mol Biol 2:28–36[PubMed]
     [Google Scholar]
  7. Baumbach J., Wittkop T., Kleindt C. K., Tauch A.( 2009). Integrated analysis and reconstruction of microbial transcriptional gene regulatory networks using CoryneRegNet. Nat Protoc 4:992–1005 [View Article][PubMed]
     [Google Scholar]
  8. 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 [View Article][PubMed]
     [Google Scholar]
  9. Blasco B., Chen J. M., Hartkoorn R., Sala C., Uplekar S., Rougemont J., Pojer F., Cole S. T.( 2012). Virulence regulator EspR of Mycobacterium tuberculosis is a nucleoid-associated protein. PLoS Pathog 8:e1002621 [View Article][PubMed]
     [Google Scholar]
  10. Blom J., Jakobi T., Doppmeier D., Jaenicke S., Kalinowski J., Stoye J., Goesmann A.( 2011). Exact and complete short-read alignment to microbial genomes using Graphics Processing Unit programming. Bioinformatics 27:1351–1358 [View Article][PubMed]
     [Google Scholar]
  11. Botsford J. L., Harman J. G.( 1992). Cyclic AMP in prokaryotes. Microbiol Rev 56:100–122[PubMed]
     [Google Scholar]
  12. Bussmann M., Emer D., Hasenbein S., Degraf S., Eikmanns B. J., Bott M.( 2009). Transcriptional control of the succinate dehydrogenase operon sdhCAB of Corynebacterium glutamicum by the cAMP-dependent regulator GlxR and the LuxR-type regulator RamA. J Biotechnol 143:173–182 [View Article][PubMed]
     [Google Scholar]
  13. Butcher B. G., Bronstein P. A., Myers C. R., Stodghill P. V., Bolton J. J., Markel E. J., Filiatrault M. J., Swingle B., Gaballa A.& other authors ( 2011). Characterization of the Fur regulon in Pseudomonas syringae pv. tomato DC3000. J Bacteriol 193:4598–4611 [View Article][PubMed]
     [Google Scholar]
  14. Cha P. H., Park S. Y., Moon M. W., Subhadra B., Oh T. K., Kim E., Kim J. F., Lee J. K.( 2010). Characterization of an adenylate cyclase gene (cyaB) deletion mutant of Corynebacterium glutamicum ATCC 13032. Appl Microbiol Biotechnol 85:1061–1068 [View Article][PubMed]
     [Google Scholar]
  15. Davies B. W., Bogard R. W., Mekalanos J. J.( 2011). Mapping the regulon of Vibrio cholerae ferric uptake regulator expands its known network of gene regulation. Proc Natl Acad Sci U S A 108:12467–12472 [View Article][PubMed]
     [Google Scholar]
  16. De Lay N., Gottesman S.( 2009). The Crp-activated small noncoding regulatory RNA CyaR (RyeE) links nutritional status to group behavior. J Bacteriol 191:461–476 [View Article][PubMed]
     [Google Scholar]
  17. Dharmadi Y., Gonzalez R.( 2004). DNA microarrays: experimental issues, data analysis, and application to bacterial systems. Biotechnol Prog 20:1309–1324 [View Article][PubMed]
     [Google Scholar]
  18. Gomelsky M.( 2011). cAMP, c-di-GMP, c-di-AMP and now cGMP: bacteria use them all!. Mol Microbiol 79:562–565 [View Article][PubMed]
     [Google Scholar]
  19. Grainger D. C., Busby S. J.( 2008). Methods for studying global patterns of DNA binding by bacterial transcription factors and RNA polymerase. Biochem Soc Trans 36:754–757 [View Article][PubMed]
     [Google Scholar]
  20. Grainger D. C., Hurd D., Harrison M., Holdstock J., Busby S. J.( 2005). Studies of the distribution of Escherichia coli cAMP-receptor protein and RNA polymerase along the E. coli chromosome. Proc Natl Acad Sci U S A 102:17693–17698 [View Article][PubMed]
     [Google Scholar]
  21. Hackl M., Jakobi T., Blom J., Doppmeier D., Brinkrolf K., Szczepanowski R., Bernhart S. H., Höner Zu Siederdissen C., Bort J. A.& other authors ( 2011). Next-generation sequencing of the Chinese hamster ovary microRNA transcriptome: identification, annotation and profiling of microRNAs as targets for cellular engineering. J Biotechnol 153:62–75 [View Article][PubMed]
     [Google Scholar]
  22. Han S. O., Inui M., Yukawa H.( 2007). Expression of Corynebacterium glutamicum glycolytic genes varies with carbon source and growth phase. Microbiology 153:2190–2202 [View Article][PubMed]
     [Google Scholar]
  23. Håndstad T., Rye M. B., Drabløs F., Sætrom P.( 2011). A ChIP-Seq benchmark shows that sequence conservation mainly improves detection of strong transcription factor binding sites. PLoS ONE 6:e18430 [View Article][PubMed]
     [Google Scholar]
  24. 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 [View Article][PubMed]
     [Google Scholar]
  25. Jutras B. L., Bowman A., Brissette C. A., Adams C. A., Verma A., Chenail A. M., Stevenson B.( 2012). EbfC (YbaB) is a new type of bacterial nucleoid-associated protein and a global regulator of gene expression in the Lyme disease spirochete. J Bacteriol 194:3395–3406 [View Article][PubMed]
     [Google Scholar]
  26. Kahramanoglou C., Seshasayee A. S., Prieto A. I., Ibberson D., Schmidt S., Zimmermann J., Benes V., Fraser G. M., Luscombe N. M.( 2011). Direct and indirect effects of H-NS and Fis on global gene expression control in Escherichia coli. Nucleic Acids Res 39:2073–2091 [View Article][PubMed]
     [Google Scholar]
  27. 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 [View Article][PubMed]
     [Google Scholar]
  28. 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[PubMed]
     [Google Scholar]
  29. Kim H. J., Kim T. H., Kim Y., Lee H. S.( 2004). Identification and characterization of glxR, a gene involved in regulation of glyoxylate bypass in Corynebacterium glutamicum. J Bacteriol 186:3453–3460 [View Article][PubMed]
     [Google Scholar]
  30. Kohl T. A., Tauch A.( 2009). The GlxR regulon of the amino acid producer Corynebacterium glutamicum: detection of the corynebacterial core regulon and integration into the transcriptional regulatory network model. J Biotechnol 143:239–246 [View Article][PubMed]
     [Google Scholar]
  31. Kohl T. A., Baumbach J., Jungwirth B., Pühler A., Tauch A.( 2008). The GlxR regulon of the amino acid producer Corynebacterium glutamicum: in silico and in vitro detection of DNA binding sites of a global transcription regulator. J Biotechnol 135:340–350 [View Article][PubMed]
     [Google Scholar]
  32. Kolb A., Busby S., Buc H., Garges S., Adhya S.( 1993). Transcriptional regulation by cAMP and its receptor protein. Annu Rev Biochem 62:749–797 [View Article][PubMed]
     [Google Scholar]
  33. Kotrbova-Kozak A., Kotrba P., Inui M., Sajdok J., Yukawa H.( 2007). Transcriptionally regulated adhA gene encodes alcohol dehydrogenase required for ethanol and n-propanol utilization in Corynebacterium glutamicum R. Appl Microbiol Biotechnol 76:1347–1356 [View Article][PubMed]
     [Google Scholar]
  34. Krämer R., Lambert C., Hoischen C., Ebbighausen H.( 1990). Uptake of glutamate in Corynebacterium glutamicum. 1. Kinetic properties and regulation by internal pH and potassium. Eur J Biochem 194:929–935 [View Article][PubMed]
     [Google Scholar]
  35. Kronemeyer W., Peekhaus N., Krämer R., Sahm H., Eggeling L.( 1995). Structure of the gluABCD cluster encoding the glutamate uptake system of Corynebacterium glutamicum. J Bacteriol 177:1152–1158[PubMed]
     [Google Scholar]
  36. Laub M. T., Chen S. L., Shapiro L., McAdams H. H.( 2002). Genes directly controlled by CtrA, a master regulator of the Caulobacter cell cycle. Proc Natl Acad Sci U S A 99:4632–4637 [View Article][PubMed]
     [Google Scholar]
  37. Letek M., Valbuena N., Ramos A., Ordóñez E., Gil J. A., Mateos L. M.( 2006). Characterization and use of catabolite-repressed promoters from gluconate genes in Corynebacterium glutamicum. J Bacteriol 188:409–423 [View Article][PubMed]
     [Google Scholar]
  38. Lun D. S., Sherrid A., Weiner B., Sherman D. R., Galagan J. E.( 2009). A blind deconvolution approach to high-resolution mapping of transcription factor binding sites from ChIP-seq data. Genome Biol 10:R142 [View Article][PubMed]
     [Google Scholar]
  39. MacQuarrie K. L., Fong A. P., Morse R. H., Tapscott S. J.( 2011). Genome-wide transcription factor binding: beyond direct target regulation. Trends Genet 27:141–148 [View Article][PubMed]
     [Google Scholar]
  40. Markel E., Maciak C., Butcher B. G., Myers C. R., Stodghill P., Bao Z., Cartinhour S., Swingle B.( 2011). An extracytoplasmic function sigma factor-mediated cell surface signaling system in Pseudomonas syringae pv. tomato DC3000 regulates gene expression in response to heterologous siderophores. J Bacteriol 193:5775–5783 [View Article][PubMed]
     [Google Scholar]
  41. Nishimura T., Teramoto H., Toyoda K., Inui M., Yukawa H.( 2011). Regulation of the nitrate reductase operon narKGHJI by the cAMP-dependent regulator GlxR in Corynebacterium glutamicum. Microbiology 157:21–28 [View Article][PubMed]
     [Google Scholar]
  42. Panhorst M., Sorger-Herrmann U., Wendisch V. F.( 2011). The pstSCAB operon for phosphate uptake is regulated by the global regulator GlxR in Corynebacterium glutamicum. J Biotechnol 154:149–155 [View Article][PubMed]
     [Google Scholar]
  43. Park P. J.( 2009). ChIP-seq: advantages and challenges of a maturing technology. Nat Rev Genet 10:669–680 [View Article][PubMed]
     [Google Scholar]
  44. Park S. Y., Moon M. W., Subhadra B., Lee J. K.( 2010). Functional characterization of the glxR deletion mutant of Corynebacterium glutamicum ATCC 13032: involvement of GlxR in acetate metabolism and carbon catabolite repression. FEMS Microbiol Lett 304:107–115 [View Article][PubMed]
     [Google Scholar]
  45. Pauling J., Röttger R., Tauch A., Azevedo V., Baumbach J.( 2012). CoryneRegNet 6.0 – updated database content, new analysis methods and novel features focusing on community demands. Nucleic Acids Res 40:Database issueD610–D614 [View Article][PubMed]
     [Google Scholar]
  46. Pollack J. R., Iyer V. R.( 2002). Characterizing the physical genome. Nat Genet 32:Suppl.515–521 [View Article][PubMed]
     [Google Scholar]
  47. Rickemberg H. V.( 1974). Cyclic AMP in prokaryotes. Annu Rev Microbiol 28:353–369 [View Article][PubMed]
     [Google Scholar]
  48. Rodionov D. A.( 2007). Comparative genomic reconstruction of transcriptional regulatory networks in bacteria. Chem Rev 107:3467–3497 [View Article][PubMed]
     [Google Scholar]
  49. Rye M. B., Sætrom P., Drabløs F.( 2011). A manually curated ChIP-seq benchmark demonstrates room for improvement in current peak-finder programs. Nucleic Acids Res 39:e25 [View Article][PubMed]
     [Google Scholar]
  50. Sala C., Haouz A., Saul F. A., Miras I., Rosenkrands I., Alzari P. M., Cole S. T.( 2009). Genome-wide regulon and crystal structure of BlaI (Rv1846c) from Mycobacterium tuberculosis. Mol Microbiol 71:1102–1116 [View Article][PubMed]
     [Google Scholar]
  51. Sambrook J., Fritsch E., Maniatis T.( 1989). Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
     [Google Scholar]
  52. Schröder J., Tauch A.( 2010). Transcriptional regulation of gene expression in Corynebacterium glutamicum: the role of global, master and local regulators in the modular and hierarchical gene regulatory network. FEMS Microbiol Rev 34:685–737[PubMed]
     [Google Scholar]
  53. Smollett K. L., Smith K. M., Kahramanoglou C., Arnvig K. B., Buxton R. S., Davis E. O.( 2012). Global analysis of the regulon of the transcriptional repressor LexA, a key component of SOS response in Mycobacterium tuberculosis. J Biol Chem 287:22004–22014 [View Article][PubMed]
     [Google Scholar]
  54. Swiercz J. P., Hindra, Bobek J., Haiser H. J., Di Berardo C., Tjaden B., Elliot M. A.( 2008). Small non-coding RNAs in Streptomyces coelicolor. Nucleic Acids Res 36:7240–7251 [View Article][PubMed]
     [Google Scholar]
  55. Toyoda K., Teramoto H., Inui M., Yukawa H.( 2011). Genome-wide identification of in vivo binding sites of GlxR, a cyclic AMP receptor protein-type regulator in Corynebacterium glutamicum. J Bacteriol 193:4123–4133 [View Article][PubMed]
     [Google Scholar]
  56. Wade J. T., Struhl K., Busby S. J., Grainger D. C.( 2007). Genomic analysis of protein–DNA interactions in bacteria: insights into transcription and chromosome organization. Mol Microbiol 65:21–26 [View Article][PubMed]
     [Google Scholar]
  57. Wilbanks E. G., Facciotti M. T.( 2010). Evaluation of algorithm performance in ChIP-seq peak detection. PLoS ONE 5:e11471 [View Article][PubMed]
     [Google Scholar]
  58. Zhao Z., Ding J. Y., Ma W. H., Zhou N. Y., Liu S. J.( 2012). Identification and characterization of γ-aminobutyric acid uptake system GabPCg (NCgl0464) in Corynebacterium glutamicum. Appl Environ Microbiol 78:2596–2601 [View Article][PubMed]
     [Google Scholar]
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High-resolution detection of DNA binding sites of the global transcriptional regulator GlxR in Corynebacterium glutamicum
Microbiology 159, 12 (2013); https://doi.org/10.1099/mic.0.062059-0
/content/journal/micro/10.1099/mic.0.062059-0
/content/journal/micro/10.1099/mic.0.062059-0
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