NusG prevents transcriptional invasion of H-NS-silenced genes

Autoři: Lionello Bossi aff001;  Mathilde Ratel aff001;  Camille Laurent aff001;  Patricia Kerboriou aff001;  Andrew Camilli aff002;  Eric Eveno aff003;  Marc Boudvillain aff003;  Nara Figueroa-Bossi aff001
Působiště autorů: Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS, Université Paris-Sud, Université Paris-Saclay, France aff001;  Department of Molecular Biology and Microbiology, Tufts University, Boston, MA, United States of America aff002;  Centre de Biophysique Moléculaire, CNRS UPR4301, rue Charles Sadron, France aff003
Vyšlo v časopise: NusG prevents transcriptional invasion of H-NS-silenced genes. PLoS Genet 15(10): e32767. doi:10.1371/journal.pgen.1008425
Kategorie: Research Article
doi: 10.1371/journal.pgen.1008425


Evolutionarily conserved NusG protein enhances bacterial RNA polymerase processivity but can also promote transcription termination by binding to, and stimulating the activity of, Rho factor. Rho terminates transcription upon anchoring to cytidine-rich motifs, the so-called Rho utilization sites (Rut) in nascent RNA. Both NusG and Rho have been implicated in the silencing of horizontally-acquired A/T-rich DNA by nucleoid structuring protein H-NS. However, the relative roles of the two proteins in H-NS-mediated gene silencing remain incompletely defined. In the present study, a Salmonella strain carrying the nusG gene under the control of an arabinose-inducible repressor was used to assess the genome-wide response to NusG depletion. Results from two complementary approaches, i) screening lacZ protein fusions generated by random transposition and ii) transcriptomic analysis, converged to show that loss of NusG causes massive upregulation of Salmonella pathogenicity islands (SPIs) and other H-NS-silenced loci. A similar, although not identical, SPI-upregulated profile was observed in a strain with a mutation in the rho gene, Rho K130Q. Surprisingly, Rho mutation Y80C, which affects Rho’s primary RNA binding domain, had either no effect or made H-NS-mediated silencing of SPIs even tighter. Thus, while corroborating the notion that bound H-NS can trigger Rho-dependent transcription termination in vivo, these data suggest that H-NS-elicited termination occurs entirely through a NusG-dependent pathway and is less dependent on Rut site binding by Rho. We provide evidence that through Rho recruitment, and possibly through other still unidentified mechanisms, NusG prevents pervasive transcripts from elongating into H-NS-silenced regions. Failure to perform this function causes the feedforward activation of the entire Salmonella virulence program. These findings provide further insight into NusG/Rho contribution in H-NS-mediated gene silencing and underscore the importance of this contribution for the proper functioning of a global regulatory response in growing bacteria. The complete set of transcriptomic data is freely available for viewing through a user-friendly genome browser interface.

Klíčová slova:

DNA transcription – Genetic loci – Pathogenesis – Salmonella – Arabinose – Transcriptional termination – RNA polymerase


1. Ochman H, Lawrence JG, Groisman EA. Lateral gene transfer and the nature of bacterial innovation. Nature. 2000;405(6784):299–304. doi: 10.1038/35012500 10830951.

2. Diard M, Hardt WD. Evolution of bacterial virulence. FEMS Microbiol Rev. 2017;41(5):679–97. doi: 10.1093/femsre/fux023 28531298.

3. Groisman EA, Ochman H. Pathogenicity islands: bacterial evolution in quantum leaps. Cell. 1996;87(5):791–4. doi: 10.1016/s0092-8674(00)81985-6 8945505.

4. Lucchini S, Rowley G, Goldberg MD, Hurd D, Harrison M, Hinton JC. H-NS mediates the silencing of laterally acquired genes in bacteria. PLoS Pathog. 2006;2(8):e81. doi: 10.1371/journal.ppat.0020081 16933988.

5. Navarre WW, Porwollik S, Wang Y, McClelland M, Rosen H, Libby SJ, et al. Selective silencing of foreign DNA with low GC content by the H-NS protein in Salmonella. Science. 2006;313(5784):236–8. doi: 10.1126/science.1128794 16763111.

6. Cardinale CJ, Washburn RS, Tadigotla VR, Brown LM, Gottesman ME, Nudler E. Termination factor Rho and its cofactors NusA and NusG silence foreign DNA in E. coli. Science. 2008;320(5878):935–8. doi: 10.1126/science.1152763 18487194.

7. Sturm A, Heinemann M, Arnoldini M, Benecke A, Ackermann M, Benz M, et al. The cost of virulence: retarded growth of Salmonella Typhimurium cells expressing type III secretion system 1. PLoS Pathog. 2011;7(7):e1002143. doi: 10.1371/journal.ppat.1002143 21829349.

8. Lamberte LE, Baniulyte G, Singh SS, Stringer AM, Bonocora RP, Stracy M, et al. Horizontally acquired AT-rich genes in Escherichia coli cause toxicity by sequestering RNA polymerase. Nat Microbiol. 2017;2:16249. doi: 10.1038/nmicrobiol.2016.249 28067866.

9. Wade JT, Grainger DC. Pervasive transcription: illuminating the dark matter of bacterial transcriptomes. Nat Rev Microbiol. 2014;12(9):647–53. doi: 10.1038/nrmicro3316 25069631.

10. Ali SS, Xia B, Liu J, Navarre WW. Silencing of foreign DNA in bacteria. Curr Opin Microbiol. 2012;15(2):175–81. doi: 10.1016/j.mib.2011.12.014 22265250.

11. Dorman CJ. H-NS, the genome sentinel. Nat Rev Microbiol. 2007;5(2):157–61. doi: 10.1038/nrmicro1598 17191074.

12. Grainger DC. Structure and function of bacterial H-NS protein. Biochem Soc Trans. 2016;44(6):1561–9. doi: 10.1042/BST20160190 27913665.

13. Singh SS, Singh N, Bonocora RP, Fitzgerald DM, Wade JT, Grainger DC. Widespread suppression of intragenic transcription initiation by H-NS. Genes Dev. 2014;28(3):214–9. doi: 10.1101/gad.234336.113 24449106.

14. Boudvillain M, Figueroa-Bossi N, Bossi L. Terminator still moving forward: expanding roles for Rho factor. Curr Opin Microbiol. 2013;16:118–24. doi: 10.1016/j.mib.2012.12.003 23347833

15. Ray-Soni A, Bellecourt MJ, Landick R. Mechanisms of Bacterial Transcription Termination: All Good Things Must End. Annu Rev Biochem. 2016;85:319–47. doi: 10.1146/annurev-biochem-060815-014844 27023849.

16. Alifano P, Rivellini F, Limauro D, Bruni CB, Carlomagno MS. A consensus motif common to all Rho-dependent prokaryotic transcription terminators. Cell. 1991;64(3):553–63. doi: 10.1016/0092-8674(91)90239-u 1703923.

17. Di Salvo M, Puccio S, Peano C, Lacour S, Alifano P. RhoTermPredict: an algorithm for predicting Rho-dependent transcription terminators based on Escherichia coli, Bacillus subtilis and Salmonella enterica databases. BMC Bioinformatics. 2019;20(1):117. doi: 10.1186/s12859-019-2704-x 30845912.

18. Nadiras C, Eveno E, Schwartz A, Figueroa-Bossi N, Boudvillain M. A multivariate prediction model for Rho-dependent termination of transcription. Nucleic Acids Res. 2018;46(16):8245–60. doi: 10.1093/nar/gky563 29931073.

19. Bossi L, Schwartz A, Guillemardet B, Boudvillain M, Figueroa-Bossi N. A role for Rho-dependent polarity in gene regulation by a noncoding small RNA. Genes Dev. 2012;26(16):1864–73. doi: 10.1101/gad.195412.112 22895254.

20. Richardson JP, Grimley C, Lowery C. Transcription termination factor rho activity is altered in Escherichia coli with suA gene mutations. Proc Natl Acad Sci U S A. 1975;72(5):1725–8. doi: 10.1073/pnas.72.5.1725 1098042.

21. Peters JM, Mooney RA, Kuan PF, Rowland JL, Keles S, Landick R. Rho directs widespread termination of intragenic and stable RNA transcription. Proc Natl Acad Sci U S A. 2009;106(36):15406–11. doi: 10.1073/pnas.0903846106 19706412.

22. Peters JM, Mooney RA, Grass JA, Jessen ED, Tran F, Landick R. Rho and NusG suppress pervasive antisense transcription in Escherichia coli. Genes Dev. 2012;26(23):2621–33. doi: 10.1101/gad.196741.112 23207917.

23. Saxena S, Gowrishankar J. Modulation of Rho-dependent transcription termination in Escherichia coli by the H-NS family of proteins. J Bacteriol. 2011;193(15):3832–41. doi: 10.1128/JB.00220-11 21602341.

24. Chandraprakash D, Seshasayee AS. Inhibition of factor-dependent transcription termination in Escherichia coli might relieve xenogene silencing by abrogating H-NS-DNA interactions in vivo. J Biosci. 2014;39(1):53–61. doi: 10.1007/s12038-014-9413-4 24499790.

25. Boudreau BA, Hron DR, Qin L, van der Valk RA, Kotlajich MV, Dame RT, et al. StpA and Hha stimulate pausing by RNA polymerase by promoting DNA-DNA bridging of H-NS filaments. Nucleic Acids Res. 2018;46(11):5525–46. doi: 10.1093/nar/gky265 29718386.

26. Kotlajich MV, Hron DR, Boudreau BA, Sun Z, Lyubchenko YL, Landick R. Bridged filaments of histone-like nucleoid structuring protein pause RNA polymerase and aid termination in bacteria. Elife. 2015;4. doi: 10.7554/eLife.04970 25594903.

27. Tomar SK, Artsimovitch I. NusG-Spt5 proteins-Universal tools for transcription modification and communication. Chem Rev. 2013;113(11):8604–19. doi: 10.1021/cr400064k 23638618.

28. Herbert KM, Zhou J, Mooney RA, Porta AL, Landick R, Block SM. E. coli NusG inhibits backtracking and accelerates pause-free transcription by promoting forward translocation of RNA polymerase. J Mol Biol. 2010;399(1):17–30. doi: 10.1016/j.jmb.2010.03.051 20381500.

29. Turtola M, Belogurov GA. NusG inhibits RNA polymerase backtracking by stabilizing the minimal transcription bubble. Elife. 2016;5. doi: 10.7554/eLife.18096 27697152.

30. Burns CM, Nowatzke WL, Richardson JP. Activation of Rho-dependent transcription termination by NusG. Dependence on terminator location and acceleration of RNA release. J Biol Chem. 1999;274(8):5245–51. doi: 10.1074/jbc.274.8.5245 9988775.

31. Li J, Mason SW, Greenblatt J. Elongation factor NusG interacts with termination factor rho to regulate termination and antitermination of transcription. Genes Dev. 1993;7(1):161–72. doi: 10.1101/gad.7.1.161 8422985.

32. Sullivan SL, Gottesman ME. Requirement for E. coli NusG protein in factor-dependent transcription termination. Cell. 1992;68(5):989–94. doi: 10.1016/0092-8674(92)90041-a 1547498.

33. Burmann BM, Schweimer K, Luo X, Wahl MC, Stitt BL, Gottesman ME, et al. A NusE:NusG complex links transcription and translation. Science. 2010;328(5977):501–4. doi: 10.1126/science.1184953 20413501.

34. Lawson MR, Ma W, Bellecourt MJ, Artsimovitch I, Martin A, Landick R, et al. Mechanism for the Regulated Control of Bacterial Transcription Termination by a Universal Adaptor Protein. Mol Cell. 2018;71(6):911–22. doi: 10.1016/j.molcel.2018.07.014 30122535.

35. Mooney RA, Schweimer K, Rosch P, Gottesman M, Landick R. Two structurally independent domains of E. coli NusG create regulatory plasticity via distinct interactions with RNA polymerase and regulators. J Mol Biol. 2009;391(2):341–58. doi: 10.1016/j.jmb.2009.05.078 19500594.

36. Saxena S, Myka KK, Washburn R, Costantino N, Court DL, Gottesman ME. Escherichia coli transcription factor NusG binds to 70S ribosomes. Mol Microbiol. 2018;108(5):495–504. doi: 10.1111/mmi.13953 29575154.

37. McGary K, Nudler E. RNA polymerase and the ribosome: the close relationship. Curr Opin Microbiol. 2013;16(2):112–7. doi: 10.1016/j.mib.2013.01.010 23433801; PubMed Central PMCID: PMC4066815.

38. Valabhoju V, Agrawal S, Sen R. Molecular Basis of NusG-mediated Regulation of Rho-dependent Transcription Termination in Bacteria. J Biol Chem. 2016;291(43):22386–403. doi: 10.1074/jbc.M116.745364 27605667.

39. Shashni R, Qayyum MZ, Vishalini V, Dey D, Sen R. Redundancy of primary RNA-binding functions of the bacterial transcription terminator Rho. Nucleic Acids Res. 2014;42(15):9677–90. doi: 10.1093/nar/gku690 25081210.

40. Bustamante VH, Martinez LC, Santana FJ, Knodler LA, Steele-Mortimer O, Puente JL. HilD-mediated transcriptional cross-talk between SPI-1 and SPI-2. Proc Natl Acad Sci U S A. 2008;105(38):14591–6. doi: 10.1073/pnas.0801205105 18799744.

41. Ellermeier CD, Ellermeier JR, Slauch JM. HilD, HilC and RtsA constitute a feed forward loop that controls expression of the SPI1 type three secretion system regulator hilA in Salmonella enterica serovar Typhimurium. Mol Microbiol. 2005;57(3):691–705. doi: 10.1111/j.1365-2958.2005.04737.x 16045614.

42. Golubeva YA, Sadik AY, Ellermeier JR, Slauch JM. Integrating global regulatory input into the Salmonella pathogenicity island 1 type III secretion system. Genetics. 2012;190(1):79–90. doi: 10.1534/genetics.111.132779 22021388.

43. Smith C, Stringer AM, Mao C, Palumbo MJ, Wade JT. Mapping the Regulatory Network for Salmonella enterica Serovar Typhimurium Invasion. MBio. 2016;7(5). doi: 10.1128/mBio.01024-16 27601571.

44. Chalissery J, Banerjee S, Bandey I, Sen R. Transcription termination defective mutants of Rho: role of different functions of Rho in releasing RNA from the elongation complex. J Mol Biol. 2007;371(4):855–72. doi: 10.1016/j.jmb.2007.06.013 17599352.

45. Figueroa-Bossi N, Schwartz A, Guillemardet B, D'Heygere F, Bossi L, Boudvillain M. RNA remodeling by bacterial global regulator CsrA promotes Rho-dependent transcription termination. Genes Dev. 2014;28(11):1239–51. doi: 10.1101/gad.240192.114 24888591.

46. Matsumoto Y, Shigesada K, Hirano M, Imai M. Autogenous regulation of the gene for transcription termination factor rho in Escherichia coli: localization and function of its attenuators. J Bacteriol. 1986;166(3):945–58. doi: 10.1128/jb.166.3.945-958.1986 2423505.

47. Dillon SC, Espinosa E, Hokamp K, Ussery DW, Casadesús J, Dorman CJ. LeuO is a global regulator of gene expression in Salmonella enterica serovar Typhimurium. Mol Microbiol. 2012;85(6):1072–89. doi: 10.1111/j.1365-2958.2012.08162.x 22804842.

48. Espinosa E, Casadesús J. Regulation of Salmonella enterica pathogenicity island 1 (SPI-1) by the LysR-type regulator LeuO. Mol Microbiol. 2014;91(6):1057–69. doi: 10.1111/mmi.12500 24354910.

49. Chen CC, Chou MY, Huang CH, Majumder A, Wu HY. A cis-spreading nucleoprotein filament is responsible for the gene silencing activity found in the promoter relay mechanism. J Biol Chem. 2005;280(6):5101–12. doi: 10.1074/jbc.M411840200 15582999.

50. Fang M, Wu HY. A promoter relay mechanism for sequential gene activation. J Bacteriol. 1998;180(3):626–33. 9457867.

51. Nagarajavel V, Madhusudan S, Dole S, Rahmouni AR, Schnetz K. Repression by binding of H-NS within the transcription unit. J Biol Chem. 2007;282(32):23622–30. doi: 10.1074/jbc.M702753200 17569663.

52. Rangarajan AA, Schnetz K. Interference of transcription across H-NS binding sites and repression by H-NS. Mol Microbiol. 2018;108(3):226–39. doi: 10.1111/mmi.13926 29424946

53. Buels R, Yao E, Diesh CM, Hayes RD, Munoz-Torres M, Helt G, et al. JBrowse: a dynamic web platform for genome visualization and analysis. Genome Biol. 2016;17:66. doi: 10.1186/s13059-016-0924-1 27072794.

54. Lithgow JK, Haider F, Roberts IS, Green J. Alternate SlyA and H-NS nucleoprotein complexes control hlyE expression in Escherichia coli K-12. Mol Microbiol. 2007;66(3):685–98. doi: 10.1111/j.1365-2958.2007.05950.x 17892462.

55. Will WR, Bale DH, Reid PJ, Libby SJ, Fang FC. Evolutionary expansion of a regulatory network by counter-silencing. Nat Commun. 2014;5:5270. doi: 10.1038/ncomms6270 25348042.

56. Bidnenko V, Nicolas P, Grylak-Mielnicka A, Delumeau O, Auger S, Aucouturier A, et al. Termination factor Rho: From the control of pervasive transcription to cell fate determination in Bacillus subtilis. PLoS Genet. 2017;13(7):e1006909. doi: 10.1371/journal.pgen.1006909 28723971.

57. Botella L, Vaubourgeix J, Livny J, Schnappinger D. Depleting Mycobacterium tuberculosis of the transcription termination factor Rho causes pervasive transcription and rapid death. Nat Commun. 2017;8:14731. doi: 10.1038/ncomms14731 28348398.

58. Mäder U, Nicolas P, Depke M, Pane-Farre J, Debarbouille M, van der Kooi-Pol MM, et al. Staphylococcus aureus Transcriptome Architecture: From Laboratory to Infection-Mimicking Conditions. PLoS Genet. 2016;12(4):e1005962. doi: 10.1371/journal.pgen.1005962 27035918.

59. Pani B, Banerjee S, Chalissery J, Muralimohan A, Loganathan RM, Suganthan RB, et al. Mechanism of inhibition of Rho-dependent transcription termination by bacteriophage P4 protein Psu. J Biol Chem. 2006;281(36):26491–500. doi: 10.1074/jbc.M603982200 16829521.

60. Mayer A, Landry HM, Churchman LS. Pause & go: from the discovery of RNA polymerase pausing to its functional implications. Curr Opin Cell Biol. 2017;46:72–80. doi: 10.1016/ 28363125.

61. Jain S, Gupta R, Sen R. Rho-dependent transcription termination in bacteria recycles RNA polymerases stalled at DNA lesions. Nat Commun. 2019;10(1):1207. doi: 10.1038/s41467-019-09146-5 30872584.

62. Kang JY, Mooney RA, Nedialkov Y, Saba J, Mishanina TV, Artsimovitch I, et al. Structural Basis for Transcript Elongation Control by NusG Family Universal Regulators. Cell. 2018;173(7):1650–62. doi: 10.1016/j.cell.2018.05.017 29887376.

63. Lilleengen K. Typing of Salmonella typhimurium by means of bacteriophage. Acta Pathol Microbiol Scand. 1948;77(Suppl):2–125.

64. Figueroa-Bossi N, Coissac E, Netter P, Bossi L. Unsuspected prophage-like elements in Salmonella typhimurium. Mol Microbiol. 1997;25(1):161–73. doi: 10.1046/j.1365-2958.1997.4451807.x 11902718.

65. Bertani G. Lysogeny at mid-twentieth century: P1, P2, and other experimental systems. J Bacteriol. 2004;186(3):595–600. doi: 10.1128/JB.186.3.595-600.2004 14729683.

66. Maloy SR, Roth JR. Regulation of proline utilization in Salmonella typhimurium: characterization of put::Mu d(Ap, lac) operon fusions. J Bacteriol. 1983;154(2):561–8. 6302076.

67. Schmieger H. Phage P22-mutants with increased or decreased transduction abilities. Mol Gen Genet. 1972;119(1):75–88. doi: 10.1007/bf00270447 4564719.

68. Datsenko KA, Wanner BL. One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci U S A. 2000;97(12):6640–5. doi: 10.1073/pnas.120163297 10829079.

69. Uzzau S, Figueroa-Bossi N, Rubino S, Bossi L. Epitope tagging of chromosomal genes in Salmonella. Proc Natl Acad Sci U S A. 2001;98(26):15264–9. doi: 10.1073/pnas.261348198 11742086.

70. Reznikoff WS. Transposon Tn5. Annu Rev Genet. 2008;42:269–86. doi: 10.1146/annurev.genet.42.110807.091656 18680433.

71. Macconkey A. Lactose-Fermenting Bacteria in Faeces. J Hyg (Lond). 1905;5(3):333–79. doi: 10.1017/s002217240000259x 20474229.

72. Duncan MC, Forbes JC, Nguyen Y, Shull LM, Gillette RK, Lazinski DW, et al. Vibrio cholerae motility exerts drag force to impede attack by the bacterial predator Bdellovibrio bacteriovorus. Nat Commun. 2018;9(1):4757. doi: 10.1038/s41467-018-07245-3 30420597.

73. Figueroa-Bossi N, Lemire S, Maloriol D, Balbontín R, Casadesús J, Bossi L. Loss of Hfq activates the σE-dependent envelope stress response in Salmonella enterica. Mol Microbiol. 2006;62(3):838–52. doi: 10.1111/j.1365-2958.2006.05413.x 16999834.

74. Miller JH. A Short Course in Bacterial Genetics. A Laboratory Manual and Handbook for Escherichia coli and Related Bacteria. Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press; 1992.

75. Thomsen ND, Berger JM. Running in reverse: the structural basis for translocation polarity in hexameric helicases. Cell. 2009;139(3):523–34. doi: 10.1016/j.cell.2009.08.043 19879839.

76. Skordalakes E, Berger JM. Structure of the Rho transcription terminator: mechanism of mRNA recognition and helicase loading. Cell. 2003;114(1):135–46. doi: 10.1016/s0092-8674(03)00512-9 12859904.

77. T Robinson JT, Thorvaldsdottir H, Winckler W, Guttman M, Lander ES, Getz G, et al. Integrative genomics viewer. Nat Biotechnol. 2011;29(1):24–6. doi: 10.1038/nbt.1754 21221095.

Genetika Reprodukční medicína

Článek vyšel v časopise

PLOS Genetics

2019 Číslo 10

Nejčtenější v tomto čísle
Kurzy Podcasty Doporučená témata Časopisy
Zapomenuté heslo

Nemáte účet?  Registrujte se

Zapomenuté heslo

Zadejte e-mailovou adresu, se kterou jste vytvářel(a) účet, budou Vám na ni zaslány informace k nastavení nového hesla.


Nemáte účet?  Registrujte se