Inefficient translation of constrains behaviour of the NsrR regulon in Free

Abstract

The NsrR protein of is a transcriptional repressor that contains an [Fe–S] cluster that is the binding site for nitric oxide (NO). Reaction of NsrR with NO leads to de-repression of its target genes, which include those encoding an NO scavenging flavohaemoglobin and the RIC (repair of iron centres) protein involved in the repair of NO-damaged [Fe–S] clusters. The gene is promoter proximal in a transcription unit with , encoding the cold shock-inducible RNase R. Here, we show that is expressed from a strong promoter, but that its translation is extremely inefficient, leading to a low cellular NsrR concentration. Conversion of the start codon from the wild-type GUG to AUG increased the efficiency of translation (which, nevertheless, remained extremely low) and had measurable effects on the expression of some NsrR-regulated genes. We conclude that NsrR abundance in the cell is such that promoters with low-affinity NsrR binding sites may partially escape NsrR-mediated repression. Expression profiling confirmed that genes regulated by NsrR (whether directly or indirectly) tend to express lower mRNA levels when the start codon is AUG than when it is GUG. Transcriptomics data implicated the pyruvate oxidase gene as a novel NsrR target, which we confirmed and showed to be due to read-through transcription from the upstream - genes. We also present evidence to suggest that NsrR is a regulator of the genes, which encode the components of an [Fe–S] cluster biogenesis and repair system.

Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.000151
2015-10-01
2024-03-29
Loading full text...

Full text loading...

/deliver/fulltext/micro/161/10/2029.html?itemId=/content/journal/micro/10.1099/mic.0.000151&mimeType=html&fmt=ahah

References

  1. Baba T., Ara T., Hasegawa M., Takai Y., Okumura Y., Baba M., Datsenko K.A., Tomita M., Wanner B.L., Mori H. 2006; Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection. Mol Syst Biol 2:20060008 [View Article][PubMed]
    [Google Scholar]
  2. Bodenmiller D.M., Spiro S. 2006; The yjeB (nsrR) gene of Escherichia coli encodes a nitric oxide-sensitive transcriptional regulator. J Bacteriol 188:874–881 [View Article][PubMed]
    [Google Scholar]
  3. Branchu P., Matrat S., Vareille M., Garrivier A., Durand A., Crépin S., Harel J., Jubelin G., Gobert A.P. 2014; NsrR, GadE, and GadX interplay in repressing expression of the Escherichia coli O157 : H7 LEE pathogenicity island in response to nitric oxide. PLoS Pathog 10:e1003874 [View Article][PubMed]
    [Google Scholar]
  4. Brandes N., Rinck A., Leichert L.I., Jakob U. 2007; Nitrosative stress treatment of E. coli targets distinct set of thiol-containing proteins. Mol Microbiol 66:901–914 [View Article][PubMed]
    [Google Scholar]
  5. Cairrão F., Cruz A., Mori H., Arraiano C.M. 2003; Cold shock induction of RNase R and its role in the maturation of the quality control mediator SsrA/tmRNA. Mol Microbiol 50:1349–1360 [View Article][PubMed]
    [Google Scholar]
  6. Chang Y.Y., Cronan J.E. Jr 1982; Mapping nonselectable genes of Escherichia coli by using transposon Tn10: location of a gene affecting pyruvate oxidase. J Bacteriol 151:1279–1289[PubMed]
    [Google Scholar]
  7. Chang Y.Y., Wang A.Y., Cronan J.E. Jr 1994; Expression of Escherichia coli pyruvate oxidase (PoxB) depends on the sigma factor encoded by the rpoS (katF) gene. Mol Microbiol 11:1019–1028 [View Article][PubMed]
    [Google Scholar]
  8. Chen Y.-J., Liu P., Nielsen A.A., Brophy J.A., Clancy K., Peterson T., Voigt C.A. 2013; Characterization of 582 natural and synthetic terminators and quantification of their design constraints. Nat Methods 10:659–664 [View Article][PubMed]
    [Google Scholar]
  9. Chismon D.L., Browning D.F., Farrant G.K., Busby S.J. 2010; Unusual organization, complexity and redundancy at the Escherichia coli hcp-hcr operon promoter. Biochem J 430:61–68 [View Article][PubMed]
    [Google Scholar]
  10. Conway T., Creecy J.P., Maddox S.M., Grissom J.E., Conkle T.L., Shadid T.M., Teramoto J., San Miguel P., Shimada T., other authors. 2014; Unprecedented high-resolution view of bacterial operon architecture revealed by RNA sequencing. MBio 5:e01442–e01414 [View Article][PubMed]
    [Google Scholar]
  11. Corker H., Poole R.K. 2003; Nitric oxide formation by Escherichia coli. Dependence on nitrite reductase, the NO-sensing regulator Fnr, and flavohemoglobin Hmp. J Biol Chem 278:31584–31592 [View Article][PubMed]
    [Google Scholar]
  12. Crack J.C., Munnoch J., Dodd E.L., Knowles F., Al Bassam M.M., Kamali S., Holland A.A., Cramer S.P., Hamilton C.J., other authors. 2015; NsrR from Streptomyces coelicolor is a nitric oxide-sensing [4Fe-4S] cluster protein with a specialized regulatory function. J Biol Chem 290:12689–12704 [View Article][PubMed]
    [Google Scholar]
  13. Crane B.R., Sudhamsu J., Patel B.A. 2010; Bacterial nitric oxide synthases. Annu Rev Biochem 79:445–470 [View Article][PubMed]
    [Google Scholar]
  14. D'Autréaux B., Touati D., Bersch B., Latour J.-M., Michaud-Soret I. 2002; Direct inhibition by nitric oxide of the transcriptional ferric uptake regulation protein via nitrosylation of the iron. Proc Natl Acad Sci U S A 99:16619–16624 [View Article][PubMed]
    [Google Scholar]
  15. Datsenko K.A., Wanner B.L. 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 [View Article][PubMed]
    [Google Scholar]
  16. Fang F.C. 2004; Antimicrobial reactive oxygen and nitrogen species: concepts and controversies. Nat Rev Microbiol 2:820–832 [View Article][PubMed]
    [Google Scholar]
  17. Filenko N.A., Browning D.F., Cole J.A. 2005; Transcriptional regulation of a hybrid cluster (prismane) protein. Biochem Soc Trans 33:195–197 [View Article][PubMed]
    [Google Scholar]
  18. Filenko N., Spiro S., Browning D.F., Squire D., Overton T.W., Cole J., Constantinidou C. 2007; The NsrR regulon of Escherichia coli K-12 includes genes encoding the hybrid cluster protein and the periplasmic, respiratory nitrite reductase. J Bacteriol 189:4410–4417 [View Article][PubMed]
    [Google Scholar]
  19. Gardner A.M., Gessner C.R., Gardner P.R. 2003; Regulation of the nitric oxide reduction operon (norRVW) in Escherichia coli. Role of NorR and σ54 in the nitric oxide stress response. J Biol Chem 278:10081–10086 [View Article][PubMed]
    [Google Scholar]
  20. Giel J.L., Rodionov D., Liu M., Blattner F.R., Kiley P.J. 2006; IscR-dependent gene expression links iron-sulphur cluster assembly to the control of O2-regulated genes in Escherichia coli. Mol Microbiol 60:1058–1075 [View Article][PubMed]
    [Google Scholar]
  21. Gomes C.M., Giuffrè A., Forte E., Vicente J.B., Saraiva L.M., Brunori M., Teixeira M. 2002; A novel type of nitric-oxide reductase, Escherichia coli flavorubredoxin. J Biol Chem 277:25273–25276 [View Article][PubMed]
    [Google Scholar]
  22. Hager L.P. 1957; Trypsin activation of a ferricyanide-linked pyruvic acid oxidation. J Biol Chem 229:251–263[PubMed]
    [Google Scholar]
  23. Husseiny M.I., Hensel M. 2005; Rapid method for the construction of Salmonella enterica serovar Typhimurium vaccine carrier strains. Infect Immun 73:1598–1605 [View Article][PubMed]
    [Google Scholar]
  24. Hutchings M.I., Mandhana N., Spiro S. 2002; The NorR protein of Escherichia coli activates expression of the flavorubredoxin gene norV in response to reactive nitrogen species. J Bacteriol 184:4640–4643 [View Article][PubMed]
    [Google Scholar]
  25. Hyduke D.R., Jarboe L.R., Tran L.M., Chou K.J., Liao J.C. 2007; Integrated network analysis identifies nitric oxide response networks and dihydroxyacid dehydratase as a crucial target in Escherichia coli. Proc Natl Acad Sci U S A 104:8484–8489 [View Article][PubMed]
    [Google Scholar]
  26. Ishihama A., Kori A., Koshio E., Yamada K., Maeda H., Shimada T., Makinoshima H., Iwata A., Fujita N. 2014; Intracellular concentrations of 65 species of transcription factors with known regulatory functions in Escherichia coli. J Bacteriol 196:2718–2727 [View Article][PubMed]
    [Google Scholar]
  27. Justino M.C., Vicente J.B., Teixeira M., Saraiva L.M. 2005; New genes implicated in the protection of anaerobically grown Escherichia coli against nitric oxide. J Biol Chem 280:2636–2643 [View Article][PubMed]
    [Google Scholar]
  28. Justino M.C., Baptista J.M., Saraiva L.M. 2009; Di-iron proteins of the Ric family are involved in iron-sulfur cluster repair. Biometals 22:99–108 [View Article][PubMed]
    [Google Scholar]
  29. Khudyakov Yu.E., Neplyueva V.S., Kalinina T.I., Smirnov V.D. 1988; Effect of structure of the initiator codon on translation in E. coli. FEBS Lett 232:369–371 [View Article][PubMed]
    [Google Scholar]
  30. Langley D., Guest J.R. 1977; Biochemical genetics of the α-keto acid dehydrogenase complexes of Escherichia coli K12: isolation and biochemical properties of deletion mutants. J Gen Microbiol 99:263–276 [View Article][PubMed]
    [Google Scholar]
  31. Lee J.-H., Yeo W.-S., Roe J.-H. 2003; Regulation of the sufABCDSE operon by Fur. J Microbiol 41:109–114
    [Google Scholar]
  32. Lee J.-H., Yeo W.-S., Roe J.-H. 2004; Induction of the sufA operon encoding Fe-S assembly proteins by superoxide generators and hydrogen peroxide: involvement of OxyR, IHF and an unidentified oxidant-responsive factor. Mol Microbiol 51:1745–1755 [View Article][PubMed]
    [Google Scholar]
  33. Lee K.-C., Yeo W.-S., Roe J.-H. 2008; Oxidant-responsive induction of the suf operon, encoding a Fe-S assembly system, through Fur and IscR in Escherichia coli. J Bacteriol 190:8244–8247 [View Article][PubMed]
    [Google Scholar]
  34. Lin H.-Y., Bledsoe P.J., Stewart V. 2007; Activation of yeaR-yoaG operon transcription by the nitrate-responsive regulator NarL is independent of oxygen-responsive regulator Fnr in Escherichia coli K-12. J Bacteriol 189:7539–7548 [View Article][PubMed]
    [Google Scholar]
  35. Miller J.H. 1992 A Short Course in Bacterial Genetics NY: Cold Spring Harbor Laboratory Cold Spring Harbor;
    [Google Scholar]
  36. O'Donnell S.M., Janssen G.R. 2001; The initiation codon affects ribosome binding and translational efficiency in Escherichia coli of cI mRNA with or without the 5′ untranslated leader. J Bacteriol 183:1277–1283 [View Article][PubMed]
    [Google Scholar]
  37. Outten F.W., Djaman O., Storz G. 2004; suf operon requirement for Fe-S cluster assembly during iron starvation in Escherichia coli. Mol Microbiol 52:861–872 [View Article][PubMed]
    [Google Scholar]
  38. Partridge J.D., Bodenmiller D.M., Humphrys M.S., Spiro S. 2009; NsrR targets in the Escherichia coli genome: new insights into DNA sequence requirements for binding and a role for NsrR in the regulation of motility. Mol Microbiol 73:680–694 [View Article][PubMed]
    [Google Scholar]
  39. Pullan S.T., Gidley M.D., Jones R.A., Barrett J., Stevanin T.M., Read R.C., Green J., Poole R.K. 2007; Nitric oxide in chemostat-cultured Escherichia coli is sensed by Fnr and other global regulators: unaltered methionine biosynthesis indicates lack of S nitrosation. J Bacteriol 189:1845–1855 [View Article][PubMed]
    [Google Scholar]
  40. Rankin L.D., Bodenmiller D.M., Partridge J.D., Nishino S.F., Spain J.C., Spiro S. 2008; Escherichia coli NsrR regulates a pathway for the oxidation of 3-nitrotyramine to 4-hydroxy-3-nitrophenylacetate. J Bacteriol 190:6170–6177 [View Article][PubMed]
    [Google Scholar]
  41. Reddy P., Peterkofsky A., McKenney K. 1985; Translational efficiency of the Escherichia coli adenylate cyclase gene: mutating the UUG initiation codon to GUG or AUG results in increased gene expression. Proc Natl Acad Sci U S A 82:5656–5660 [View Article][PubMed]
    [Google Scholar]
  42. Ren B., Zhang N., Yang J., Ding H. 2008; Nitric oxide-induced bacteriostasis and modification of iron-sulphur proteins in Escherichia coli. Mol Microbiol 70:953–964[PubMed]
    [Google Scholar]
  43. Rhodius V.A., Suh W.C., Nonaka G., West J., Gross C.A. 2006; Conserved and variable functions of the σE stress response in related genomes. PLoS Biol 4:e2 [View Article][PubMed]
    [Google Scholar]
  44. Richardson A.R., Payne E.C., Younger N., Karlinsey J.E., Thomas V.C., Becker L.A., Navarre W.W., Castor M.E., Libby S.J., Fang F.C. 2011; Multiple targets of nitric oxide in the tricarboxylic acid cycle of Salmonella enterica serovar Typhimurium. Cell Host Microbe 10:33–43 [View Article][PubMed]
    [Google Scholar]
  45. Simons R.W., Houman F., Kleckner N. 1987; Improved single and multicopy lac-based cloning vectors for protein and operon fusions. Gene 53:85–96 [View Article][PubMed]
    [Google Scholar]
  46. Spiro S. 2007; Regulators of bacterial responses to nitric oxide. FEMS Microbiol Rev 31:193–211 [View Article][PubMed]
    [Google Scholar]
  47. Stenström C.M., Holmgren E., Isaksson L.A. 2001; Cooperative effects by the initiation codon and its flanking regions on translation initiation. Gene 273:259–265 [View Article][PubMed]
    [Google Scholar]
  48. Tucker N.P., Hicks M.G., Clarke T.A., Crack J.C., Chandra G., Le Brun N.E., Dixon R., Hutchings M.I. 2008; The transcriptional repressor protein NsrR senses nitric oxide directly via a [2Fe-2S] cluster. PLoS One 3:e3623 [View Article][PubMed]
    [Google Scholar]
  49. van den Berg W.A.M., Hagen W.R., van Dongen W.M.A.M. 2000; The hybrid-cluster protein (‘prismane protein’) from Escherichia coli. Characterization of the hybrid-cluster protein, redox properties of the [2Fe-2S] and [4Fe-2S-2O] clusters and identification of an associated NADH oxidoreductase containing FAD and [2Fe-2S]. Eur J Biochem 267:666–676 [View Article][PubMed]
    [Google Scholar]
  50. Vey J.L., Yang J., Li M., Broderick W.E., Broderick J.B., Drennan C.L. 2008; Structural basis for glycyl radical formation by pyruvate formate-lyase activating enzyme. Proc Natl Acad Sci U S A 105:16137–16141 [View Article][PubMed]
    [Google Scholar]
  51. Yamamoto K., Watanabe H., Ishihama A. 2014; Expression levels of transcription factors in Escherichia coli: growth phase- and growth condition-dependent variation of 90 regulators from six families. Microbiology 160:1903–1913 [View Article][PubMed]
    [Google Scholar]
  52. Yamamoto N., Nakahigashi K., Nakamichi T., Yoshino M., Takai Y., Touda Y., Furubayashi A., Kinjyo S., Dose H., other authors. 2009; Update on the Keio collection of Escherichia coli single-gene deletion mutants. Mol Syst Biol 5:335 [View Article][PubMed]
    [Google Scholar]
  53. Yeo W.S., Lee J.H., Lee K.C., Roe J.H. 2006; IscR acts as an activator in response to oxidative stress for the suf operon encoding Fe-S assembly proteins. Mol Microbiol 61:206–218 [View Article][PubMed]
    [Google Scholar]
  54. Yukl E.T., Elbaz M.A., Nakano M.M., Moënne-Loccoz P. 2008; Transcription factor NsrR from Bacillus subtilis senses nitric oxide with a 4Fe-4S cluster. Biochemistry 47:13084–13092 [View Article][PubMed]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.000151
Loading
/content/journal/micro/10.1099/mic.0.000151
Loading

Data & Media loading...

Most cited Most Cited RSS feed