#PAGE_PARAMS# #ADS_HEAD_SCRIPTS# #MICRODATA#

Comprehensive Mapping of the Flagellar Regulatory Network


Flagella are surface-associated appendages that propel bacteria and are involved in diverse functions such as chemotaxis, surface attachment, and host cell invasion. Flagella are incredibly complex macromolecular machines that are energetically costly to produce, assemble, and power. Flagellar production is tightly regulated and flagellar components are only synthesized when flagellar motility is advantageous. Regulation also ensures that flagellar components are produced in roughly the same order in which they are needed, increasing efficiency of the assembly process. The transcriptional regulation of flagellar genes has been studied extensively in the model organism Escherichia coli; however, many questions remain. We have used an unbiased, genome-wide approach to comprehensively identify all of the binding sites and regulatory targets for the two key regulators of flagellar synthesis, FlhDC and FliA. Our results redefine the flagellar regulatory network, and suggest that FliA binds many sites that are not associated with productive transcription. This work is important because it suggests possible new functions for FliA outside of the transcription of canonical mRNAs, and it provides new insight into the temporal orchestration of gene expression that coordinates the flagellar assembly process.


Vyšlo v časopise: Comprehensive Mapping of the Flagellar Regulatory Network. PLoS Genet 10(10): e32767. doi:10.1371/journal.pgen.1004649
Kategorie: Research Article
prolekare.web.journal.doi_sk: https://doi.org/10.1371/journal.pgen.1004649

Souhrn

Flagella are surface-associated appendages that propel bacteria and are involved in diverse functions such as chemotaxis, surface attachment, and host cell invasion. Flagella are incredibly complex macromolecular machines that are energetically costly to produce, assemble, and power. Flagellar production is tightly regulated and flagellar components are only synthesized when flagellar motility is advantageous. Regulation also ensures that flagellar components are produced in roughly the same order in which they are needed, increasing efficiency of the assembly process. The transcriptional regulation of flagellar genes has been studied extensively in the model organism Escherichia coli; however, many questions remain. We have used an unbiased, genome-wide approach to comprehensively identify all of the binding sites and regulatory targets for the two key regulators of flagellar synthesis, FlhDC and FliA. Our results redefine the flagellar regulatory network, and suggest that FliA binds many sites that are not associated with productive transcription. This work is important because it suggests possible new functions for FliA outside of the transcription of canonical mRNAs, and it provides new insight into the temporal orchestration of gene expression that coordinates the flagellar assembly process.


Zdroje

1. MacnabRM (1992) Genetics and biogenesis of bacterial flagella. Annu Rev Genet 26: 131–158 doi:10.1146/annurev.ge.26.120192.001023

2. ChilcottGS, HughesKT (2000) Coupling of flagellar gene expression to flagellar assembly in Salmonella enterica serovar typhimurium and Escherichia coli. Microbiol Mol Biol Rev 64: 694–708 doi:10.1128/MMBR.64.4.694-708.2000.Updated

3. Chevance FFV, HughesKT (2008) Coordinating assembly of a bacterial macromolecular machine. Nat Rev Microbiol 6: 455–465 doi:10.1038/nrmicro1887

4. SoutourinaOA, BertinPN (2003) Regulation cascade of flagellar expression in Gram-negative bacteria. FEMS Microbiol Rev 27: 505–523 doi:10.1016/S0168-6445(03)00064-0

5. ShinS, ParkC (1995) Modulation of flagellar expression in Escherichia coli by acetyl phosphate and the osmoregulator OmpR. J Bacteriol 177: 4696–4702.

6. LehnenD, BlumerC, PolenT (2002) LrhA as a new transcriptional key regulator of flagella, motility and chemotaxis genes in Escherichia coli. Mol Microbiol 45: 521–532.

7. SperandioV, TorresA, KaperJ (2002) Quorum sensing Escherichia coli regulators B and C (QseBC): a novel two-component regulatory system involved in the regulation of flagella and motility by. Mol Microbiol 43: 809–821.

8. SoutourinaO, KolbA (1999) Multiple control of flagellum biosynthesis in Escherichia coli: role of H-NS protein and the cyclic AMP-catabolite activator protein complex in transcription of the flhDC. J Bacteriol 181: 7500–7508.

9. YakhninA, BakerC (2013) CsrA activates flhDC expression by protecting flhDC mRNA from RNase E-mediated cleavage. Mol Microbiol 87: 851–866 doi:10.1111/mmi.12136

10. ThomasonM, FontaineF (2012) A small RNA that regulates motility and biofilm formation in response to changes in nutrient availability in Escherichia coli. Mol Microbiol 84: 17–35 doi:10.1111/j.1365-2958.2012.07965.x

11. Lay NDe, GottesmanS (2012) A complex network of small non-coding RNAs regulate motility in Escherichia coli. Mol Microbiol 86: 524–538 doi:10.1111/j.1365-2958.2012.08209.x

12. LiuX, FujitaN, IshihamaA, MatsumuraP (1995) The C-terminal region of the alpha subunit of Escherichia coli RNA polymerase is required for transcriptional activation of the flagellar level II operons by the FlhD/FlhC complex. J Bacteriol 177: 5186–5188.

13. HelmannJD, ChamberlinMJ (1987) DNA sequence analysis suggests that expression of flagellar and chemotaxis genes in Escherichia coli and Salmonella typhimurium is controlled by an alternative sigma factor. Proc Natl Acad Sci U S A 84: 6422–6424.

14. ArnostiDN, ChamberlinMJ (1989) Secondary sigma factor controls transcription of flagellar and chemotaxis genes in Escherichia coli. Proc Natl Acad Sci U S A 86: 830–834.

15. ArnostiD (1990) Regulation of Escherichia coli sigma F RNA Polymerase by flhD and flhC Flagellar Regulatory Genes. J Bacteriol 172: 4106–4108.

16. OhnishiK, KutsukakeK, SuzukiH, LinoT (1992) A novel transcriptional regulation mechanism in the flagellar regulon of Salmonella typhimurium: an anti-sigma factor inhibits the activity of the flagellum-specific Sigma factor, σF. Mol Microbiol 6: 3149–3157 doi:10.1111/j.1365-2958.1992.tb01771.x

17. ZaslaverA, MayoAE, RosenbergR, BashkinP, SberroH, et al. (2004) Just-in-time transcription program in metabolic pathways. Nat Genet 36: 486–491 doi:10.1038/ng1348

18. HollandsK, LeeDJ, LloydGS, BusbySJW (2010) Activation of sigma 28-dependent transcription in Escherichia coli by the cyclic AMP receptor protein requires an unusual promoter organization. Mol Microbiol 75: 1098–1111 doi:10.1111/j.1365-2958.2009.06913.x

19. KunduTK, KusanoS, IshihamaA (1997) Promoter selectivity of Escherichia coli RNA polymerase sigmaF holoenzyme involved in transcription of flagellar and chemotaxis genes. J Bacteriol 179: 4264–9.

20. ZhaoK, LiuM, BurgessRR (2007) Adaptation in bacterial flagellar and motility systems: from regulon members to “foraging”-like behavior in E. coli. Nucleic Acids Res 35: 4441–4452 doi:10.1093/nar/gkm456

21. KoM, ParkC (2000) Two novel flagellar components and H-NS are involved in the motor function of Escherichia coli. J Mol Biol 303: 371–382.

22. LiuX, MatsumuraP (1996) Differential regulation of multiple overlapping promoters in flagellar class II operons in Escherichia coii. Mol Microbiol 21: 613–620.

23. KalirS, AlonU (2004) Using a quantitative blueprint to reprogram the dynamics of the flagella gene network. Cell 117: 713–720 doi:10.1016/j.cell.2004.05.010

24. StaffordGP, OgiT, HughesC (2005) Binding and transcriptional activation of non-flagellar genes by the Escherichia coli flagellar master regulator FlhD2C2. Microbiology 151: 1779–1788 doi:10.1099/mic.0.27879-0

25. PrüßB, CampbellJW, Van DykTK, ZhuC, KoganY, et al. (2003) FlhD/FlhC is a regulator of anaerobic respiration and the Entner-Doudoroff pathway through induction of the methyl-accepting chemotaxis protein Aer. J Bacteriol 185: 534–543.

26. PrüßB, MatsumuraP (1996) A regulator of the flagellar regulon of Escherichia coli, flhD, also affects cell division. J Bacteriol 178: 668–674.

27. PrüßB, MarkovicD, MatsumuraP (1997) The Escherichia coli flagellar transcriptional activator flhD regulates cell division through induction of the acid response gene cadA. J Bacteriol 179: 3818–3821.

28. PrüßB, LiuX, HendricksonW, MatsumuraP, PruBM (2001) FlhD/FlhC-regulated promoters analyzed by gene array and lacZ gene fusions. FEMS Microbiol Lett 197: 91–97.

29. SiegeleDA, BainS, MaoW (2010) Mutations in the flhD gene of Escherichia coli K-12 do not cause the reported effect on cell division. FEMS Microbiol Lett 309: 94–99 doi:10.1111/j.1574-6968.2010.02021.x

30. YuHHY, KiblerD, TanM (2006) In Silico Prediction and Functional Validation of 28-Regulated Genes in Chlamydia and Escherichia coli. J Bacteriol 188: 8206–8212 doi:10.1128/JB.01082-06

31. IdeN, KutsukakeK (1997) Identification of a novel Escherichia coli gene whose expression is dependent on the flagellum-specific sigma factor, FliA, but dispensable for motility development. Gene 199: 19–23.

32. ParkK, ChoiS, KoM, ParkC (2001) Novel sigmaF-dependent genes of Escherichia coli found using a specified promoter consensus. FEMS Microbiol Lett 202: 243–250.

33. HuertaAM, Collado-VidesJ (2003) Sigma70 Promoters in Escherichia coli: Specific Transcription in Dense Regions of Overlapping Promoter-like Signals. J Mol Biol 333: 261–278 doi:10.1016/j.jmb.2003.07.017

34. GalaganJ, LyubetskayaA, GomesA (2013) ChIP-Seq and the Complexity of Bacterial Transcriptional Regulation. Curr Top Microbiol Immunol 363: 43–68 doi:10.1007/82

35. WadeJT, StruhlK, BusbySJW, GraingerDC (2007) Genomic analysis of protein-DNA interactions in bacteria: insights into transcription and chromosome organization. Mol Microbiol 65: 21–26 doi:10.1111/j.1365-2958.2007.05781.x

36. McClureR, BalasubramanianD, SunY, BobrovskyyM, SumbyP, et al. (2013) Computational analysis of bacterial RNA-Seq data. Nucleic Acids Res 41: e140 doi:10.1093/nar/gkt444

37. StringerAM, CurrentiS, BonocoraRP, BaranowskiC, PetroneBL, et al. (2014) Genome-Scale Analyses of Escherichia coli and Salmonella enterica AraC Reveal Noncanonical Targets and an Expanded Core Regulon. J Bacteriol 196: 660–671 doi:10.1128/JB.01007-13

38. GalaganJ, MinchK, PetersonM (2013) The Mycobacterium tuberculosis regulatory network and hypoxia. Nature 499: 178–183 doi:10.1038/nature12337

39. MyersK, YanH, OngI, ChungD, LiangK (2013) Genome-scale analysis of Escherichia coli FNR reveals complex features of transcription factor binding. PLoS Genet 9: e1003565 doi:10.1371/journal.pgen.1003565

40. DongT, MekalanosJ (2012) Characterization of the RpoN regulon reveals differential regulation of T6SS and new flagellar operons in Vibrio cholerae O37 strain V52. Nucleic Acids Res 40: 7766–7775 doi:10.1093/nar/gks567

41. BlankaA, SchulzS, EckweilerD, BieleckaA, NicolaiT, et al. (2014) Identification of the Alternative Sigma Factor SigX Regulon and Its Implications for Pseudomonas aeruginosa Pathogenicity. J Bacteriol 196: 345–356 doi:10.1128/JB.01034-13

42. CamposA, MatsumuraP (2001) Extensive alanine scanning reveals protein-protein and protein-DNA interaction surfaces in the global regulator FlhD from Escherichia coli. Mol Microbiol 39: 581–594.

43. WangS, FlemingRT, WestbrookEM, MatsumuraP, McKayDB (2006) Structure of the Escherichia coli FlhDC complex, a prokaryotic heteromeric regulator of transcription. J Mol Biol 355: 798–808 doi:10.1016/j.jmb.2005.11.020

44. BarkerCS, PrüßBM, MatsumuraP (2004) Increased Motility of Escherichia coli by Insertion Sequence Element Integration into the Regulatory Region of the flhD Operon. J Bacteriol 186: 7529–7537 doi:10.1128/JB.186.22.7529

45. WangX, WoodTK (2011) IS 5 inserts upstream of the master motility operon flhDC in a quasi-Lamarckian way. ISME J 5: 1517–1525 doi:10.1038/ismej.2011.27

46. LeeC, ParkC (2013) Mutations Upregulating the flhDC Operon in Escherichia coli K-12. J Microbiol 51: 140–144.

47. KimD, HongJS, QiuY, NagarajanH, SeoJ, et al. (2012) Comparative Analysis of Regulatory Elements between Escherichia coli and Klebsiella pneumoniae by Genome- Wide Transcription Start Site Profiling. PLoS Genet 8: e1002867 doi:10.1371/journal.pgen.1002867

48. TeytelmanL, ThurtleDM, RineJ, OudenaardenA Van (2013) Highly expressed loci are vulnerable to misleading ChIP localization of multiple unrelated proteins. Proc Natl Acad Sci U S A 110: 18602–18607 doi:10.1073/pnas.1316064110/-/DCSupplemental.www.pnas.org/cgi/doi/10.1073/pnas.1316064110

49. LiuX, BrutlagD, LiuJ (2001) BioProspector: discovering conserved DNA motifs in upstream regulatory regions of co-expressed genes. Pac Symp Biocomput 127–138.

50. LeeY, BarkerCS, MatsumuraP, BelasR (2011) Refining the binding of the Escherichia coli flagellar master regulator, FlhD4C2, on a base-specific level. J Bacteriol 193: 4057–4068 doi:10.1128/JB.00442-11

51. SalgadoH, Peralta-GilM, Gama-CastroS, Santos-ZavaletaA, Muñiz-RascadoL, et al. (2013) RegulonDB v8.0: omics data sets, evolutionary conservation, regulatory phrases, cross-validated gold standards and more. Nucleic Acids Res 41: D203–13 doi:10.1093/nar/gks1201

52. BaileyTL, BodenM, BuskeFA, FrithM, GrantCE, et al. (2009) MEME SUITE: tools for motif discovery and searching. Nucleic Acids Res 37: W202–8 doi:10.1093/nar/gkp335

53. BaileyTL, MachanickP (2012) Inferring direct DNA binding from ChIP-seq. Nucleic Acids Res 40: 1–10 doi:10.1093/nar/gks433

54. LiuX, MatsumuraP (1994) The FlhD/FlhC complex, a transcriptional activator of the Escherichia coli flagellar class II operons. J Bacteriol 176: 7345–7351.

55. ChoB-K, KimD, KnightEM, ZenglerK, PalssonBO (2014) Genome-scale reconstruction of the sigma factor network in Escherichia coli: topology and functional states. BMC Biol 12: 4 doi:10.1186/1741-7007-12-4

56. KutsukakeK, IdeN (1995) Transcriptional analysis of the flgK and fliD operons of Salmonella typhimurium which encode flagellar hook-associated proteins. Mol Gen Genet 247: 275–281.

57. WozniakCE, Chevance FFV, HughesKT (2010) Multiple promoters contribute to swarming and the coordination of transcription with flagellar assembly in Salmonella. J Bacteriol 192: 4752–4762 doi:10.1128/JB.00093-10

58. PesaventoC, HenggeR (2012) The global repressor FliZ antagonizes gene expression by σS-containing RNA polymerase due to overlapping DNA binding specificity. Nucleic Acids Res 40: 4783–4793 doi:10.1093/nar/gks055

59. KalirS, McClureJ, PabbarajuK, SouthwardC, RonenM, et al. (2001) Ordering genes in a flagella pathway by analysis of expression kinetics from living bacteria. Science 292: 2080–2083 doi:10.1126/science.1058758

60. AlonU (2007) Network motifs: theory and experimental approaches. Nat Rev Genet 8: 450–461 doi:10.1038/nrg2102

61. HartkoornRC, SalaC, UplekarS, BussoP, RougemontJ, et al. (2012) Genome-wide definition of the SigF regulon in Mycobacterium tuberculosis. J Bacteriol 194: 2001–2009 doi:10.1128/JB.06692-11

62. WadeJT, Castro RoaD, GraingerDC, HurdD, BusbySJW, et al. (2006) Extensive functional overlap between sigma factors in Escherichia coli. Nat Struct Mol Biol 13: 806–814 doi:10.1038/nsmb1130

63. SinghSS, SinghN, BonocoraRP, FitzgeraldDM, WadeJT, et al. (2014) Widespread suppression of intragenic transcription initiation by H-NS. Genes Dev 28: 214–219 doi:10.1101/gad.234336.113

64. IdeN, IkebeT, KutsukakeK (1999) Reevaluation of the promoter structure of the class 3 flagellar operons of Escherichia coli and Salmonella. Genes Genet Syst 74: 113–116.

65. PetersJM, MooneyRA, GrassJA, JessenED, TranF, et al. (2012) Rho and NusG suppress pervasive antisense transcription in Escherichia coli. Genes Dev 26: 2621–2633 doi:10.1101/gad.196741.112

66. BrewsterRC, WeinertFM, GarciaHG, SongD, RydenfeltM, et al. (2014) The Transcription Factor Titration Effect Dictates Level of Gene Expression. Cell 156: 1312–1323 doi:10.1016/j.cell.2014.02.022

67. GöpelY, GörkeB (2014) Lies and deception in bacterial gene regulation: the roles of nucleic acid decoys. Mol Microbiol 92: 641–647 doi:10.1111/mmi.12604

68. ManganS, AlonU (2003) Structure and function of the feed-forward loop network motif. Proc Natl Acad Sci U S A 100: 11980–11985 doi:10.1073/pnas.2133841100

69. SeshasayeeASN, BertoneP, FraserGM, LuscombeNM (2006) Transcriptional regulatory networks in bacteria: from input signals to output responses. Curr Opin Microbiol 9: 511–519 doi:10.1016/j.mib.2006.08.007

70. StringerAM, SinghN, YermakovaA, PetroneBL, AmarasingheJJ, et al. (2012) FRUIT, a scar-free system for targeted chromosomal mutagenesis, epitope tagging, and promoter replacement in Escherichia coli and Salmonella enterica. PLoS One 7: e44841 doi:10.1371/journal.pone.0044841

71. BabaT, AraT, HasegawaM, TakaiY, OkumuraY, et al. (2006) Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection. Mol Syst Biol 2: 2006.0008 doi:10.1038/msb4100050

72. DatsenkoKA, WannerBL (2000) One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci U S A 97: 6640–6645 doi:10.1073/pnas.120163297

73. GuzmanL, BelinD, CarsonMJ, BeckwithJ (1995) Tight regulation, modulation, and high-level expression by vectors containing the arabinose PBAD promoter. J Bacteriol 177: 4121–4130.

74. BonocoraRP, FitzgeraldDM, StringerAM, WadeJT (2013) Non-canonical protein-DNA interactions identified by ChIP are not artifacts. BMC Genomics 14: 254 doi:10.1186/1471-2164-14-254

75. SchmittgenT, LivakK (2008) Analyzing real-time PCR data by the comparative Ct method. Nat Protoc 3: 1101–1108.

76. CrooksG, HonG (2004) WebLogo: a sequence logo generator. Genome Res 14: 1188–1190 doi:10.1101/gr.849004.1

Štítky
Genetika Reprodukčná medicína

Článok vyšiel v časopise

PLOS Genetics


2014 Číslo 10
Najčítanejšie tento týždeň
Najčítanejšie v tomto čísle
Kurzy

Zvýšte si kvalifikáciu online z pohodlia domova

Získaná hemofilie - Povědomí o nemoci a její diagnostika
nový kurz

Eozinofilní granulomatóza s polyangiitidou
Autori: doc. MUDr. Martina Doubková, Ph.D.

Všetky kurzy
Prihlásenie
Zabudnuté heslo

Zadajte e-mailovú adresu, s ktorou ste vytvárali účet. Budú Vám na ňu zasielané informácie k nastaveniu nového hesla.

Prihlásenie

Nemáte účet?  Registrujte sa

#ADS_BOTTOM_SCRIPTS#