Skip to main content
NCBI home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1996 Sep;178(18):5487–5492. doi: 10.1128/jb.178.18.5487-5492.1996

Nitric oxide, nitrite, and Fnr regulation of hmp (flavohemoglobin) gene expression in Escherichia coli K-12.

R K Poole 1, M F Anjum 1, J Membrillo-Hernández 1, S O Kim 1, M N Hughes 1, V Stewart 1
PMCID: PMC178372  PMID: 8808940

Abstract

Escherichia coli possesses a soluble flavohemoglobin, with an unknown function, encoded by the hmp gene. A monolysogen containing an hmp-lacZ operon fusion was constructed to determine how the hmp promoter is regulated in response to heme ligands (O2, NO) or the presence of anaerobically utilized electron acceptors (nitrate, nitrite). Expression of the phi (hmp-lacZ)1 fusion was similar during aerobic growth in minimal medium containing glucose, glycerol, maltose, or sorbitol as a carbon source. Mutations in cya (encoding adenylate cyclase) or changes in medium pH between 5 and 9 were without effect on aerobic expression. Levels of aerobic and anaerobic expression in glucose-containing minimal media were similar; both were unaffected by an arcA mutation. Anaerobic, but not aerobic, expression of phi (hmp-lacZ)1 was stimulated three- to four-fold by an fnr mutation; an apparent Fnr-binding site is present in the hmp promoter. Iron depletion of rich broth medium by the chelator 2'2'-dipyridyl (0.1 mM) enhanced hmp expression 40-fold under anaerobic conditions, tentatively attributed to effects on Fnr. At a higher chelator concentration (0.4 mM), hmp expression was also stimulated aerobically. Anaerobic expression was stimulated 6-fold by the presence of nitrate and 25-fold by the presence of nitrite. Induction by nitrate or nitrite was unaffected by narL and/or narP mutations, demonstrating regulation of hmp by these ions via mechanisms alternative to those implicated in the regulation of other respiratory genes. Nitric oxide (10 to 20 microM) stimulated aerobic phi (hmp-lacZ)1 activity by up to 19-fold; soxS and soxR mutations only slightly reduced the NO effect. We conclude that hmp expression is negatively regulated by Fnr under anaerobic conditions and that additional regulatory mechanisms are involved in the responses to oxygen, nitrogen compounds, and iron availability. Hmp is implicated in reactions with small nitrogen compounds.

Full Text

The Full Text of this article is available as a PDF (226.1 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Andrews S. C., Shipley D., Keen J. N., Findlay J. B., Harrison P. M., Guest J. R. The haemoglobin-like protein (HMP) of Escherichia coli has ferrisiderophore reductase activity and its C-terminal domain shares homology with ferredoxin NADP+ reductases. FEBS Lett. 1992 May 18;302(3):247–252. doi: 10.1016/0014-5793(92)80452-m. [DOI] [PubMed] [Google Scholar]
  2. Bonner F. T., Hughes M. N., Poole R. K., Scott R. I. Kinetics of the reactions of trioxodinitrate and nitrite ions with cytochrome d in Escherichia coli. Biochim Biophys Acta. 1991 Jan 22;1056(2):133–138. doi: 10.1016/s0005-2728(05)80279-8. [DOI] [PubMed] [Google Scholar]
  3. Chung C. T., Niemela S. L., Miller R. H. One-step preparation of competent Escherichia coli: transformation and storage of bacterial cells in the same solution. Proc Natl Acad Sci U S A. 1989 Apr;86(7):2172–2175. doi: 10.1073/pnas.86.7.2172. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Cotter P. A., Chepuri V., Gennis R. B., Gunsalus R. P. Cytochrome o (cyoABCDE) and d (cydAB) oxidase gene expression in Escherichia coli is regulated by oxygen, pH, and the fnr gene product. J Bacteriol. 1990 Nov;172(11):6333–6338. doi: 10.1128/jb.172.11.6333-6338.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Cramm R., Siddiqui R. A., Friedrich B. Primary sequence and evidence for a physiological function of the flavohemoprotein of Alcaligenes eutrophus. J Biol Chem. 1994 Mar 11;269(10):7349–7354. [PubMed] [Google Scholar]
  6. Crawford M. J., Sherman D. R., Goldberg D. E. Regulation of Saccharomyces cerevisiae flavohemoglobin gene expression. J Biol Chem. 1995 Mar 24;270(12):6991–6996. doi: 10.1074/jbc.270.12.6991. [DOI] [PubMed] [Google Scholar]
  7. D'Mello R., Hill S., Poole R. K. The oxygen affinity of cytochrome bo' in Escherichia coli determined by the deoxygenation of oxyleghemoglobin and oxymyoglobin: Km values for oxygen are in the submicromolar range. J Bacteriol. 1995 Feb;177(3):867–870. doi: 10.1128/jb.177.3.867-870.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Dikshit K. L., Dikshit R. P., Webster D. A. Study of Vitreoscilla globin (vgb) gene expression and promoter activity in E. coli through transcriptional fusion. Nucleic Acids Res. 1990 Jul 25;18(14):4149–4155. doi: 10.1093/nar/18.14.4149. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Dikshit K. L., Spaulding D., Braun A., Webster D. A. Oxygen inhibition of globin gene transcription and bacterial haemoglobin synthesis in Vitreoscilla. J Gen Microbiol. 1989 Oct;135(10):2601–2609. doi: 10.1099/00221287-135-10-2601. [DOI] [PubMed] [Google Scholar]
  10. Eschenbrenner M., Coves J., Fontecave M. Ferric reductases in Escherichia coli: the contribution of the haemoglobin-like protein. Biochem Biophys Res Commun. 1994 Jan 14;198(1):127–131. doi: 10.1006/bbrc.1994.1018. [DOI] [PubMed] [Google Scholar]
  11. Fu H. A., Iuchi S., Lin E. C. The requirement of ArcA and Fnr for peak expression of the cyd operon in Escherichia coli under microaerobic conditions. Mol Gen Genet. 1991 Apr;226(1-2):209–213. doi: 10.1007/BF00273605. [DOI] [PubMed] [Google Scholar]
  12. Green J., Guest J. R. Activation of FNR-dependent transcription by iron: an in vitro switch for FNR. FEMS Microbiol Lett. 1993 Oct 15;113(2):219–222. doi: 10.1111/j.1574-6968.1993.tb06517.x. [DOI] [PubMed] [Google Scholar]
  13. Green J., Guest J. R. Regulation of transcription at the ndh promoter of Escherichia coli by FNR and novel factors. Mol Microbiol. 1994 May;12(3):433–444. doi: 10.1111/j.1365-2958.1994.tb01032.x. [DOI] [PubMed] [Google Scholar]
  14. Guest J. R. Oxygen-regulated gene expression in Escherichia coli. The 1992 Marjory Stephenson Prize Lecture. J Gen Microbiol. 1992 Nov;138(11):2253–2263. doi: 10.1099/00221287-138-11-2253. [DOI] [PubMed] [Google Scholar]
  15. Hill J. J., Alben J. O., Gennis R. B. Spectroscopic evidence for a heme-heme binuclear center in the cytochrome bd ubiquinol oxidase from Escherichia coli. Proc Natl Acad Sci U S A. 1993 Jun 15;90(12):5863–5867. doi: 10.1073/pnas.90.12.5863. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Ioannidis N., Cooper C. E., Poole R. K. Spectroscopic studies on an oxygen-binding haemoglobin-like flavohaemoprotein from Escherichia coli. Biochem J. 1992 Dec 1;288(Pt 2):649–655. doi: 10.1042/bj2880649. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Iuchi S., Chepuri V., Fu H. A., Gennis R. B., Lin E. C. Requirement for terminal cytochromes in generation of the aerobic signal for the arc regulatory system in Escherichia coli: study utilizing deletions and lac fusions of cyo and cyd. J Bacteriol. 1990 Oct;172(10):6020–6025. doi: 10.1128/jb.172.10.6020-6025.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Iuchi S., Lin E. C. Adaptation of Escherichia coli to redox environments by gene expression. Mol Microbiol. 1993 Jul;9(1):9–15. doi: 10.1111/j.1365-2958.1993.tb01664.x. [DOI] [PubMed] [Google Scholar]
  19. Iuchi S., Lin E. C. arcA (dye), a global regulatory gene in Escherichia coli mediating repression of enzymes in aerobic pathways. Proc Natl Acad Sci U S A. 1988 Mar;85(6):1888–1892. doi: 10.1073/pnas.85.6.1888. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Joshi M., Dikshit K. L. Oxygen dependent regulation of Vitreoscilla globin gene: evidence for positive regulation by FNR. Biochem Biophys Res Commun. 1994 Jul 15;202(1):535–542. doi: 10.1006/bbrc.1994.1961. [DOI] [PubMed] [Google Scholar]
  21. Kaldorf M., Linne von Berg K. H., Meier U., Servos U., Bothe H. The reduction of nitrous oxide to dinitrogen by Escherichia coli. Arch Microbiol. 1993;160(6):432–439. doi: 10.1007/BF00245303. [DOI] [PubMed] [Google Scholar]
  22. Kallio P. T., Kim D. J., Tsai P. S., Bailey J. E. Intracellular expression of Vitreoscilla hemoglobin alters Escherichia coli energy metabolism under oxygen-limited conditions. Eur J Biochem. 1994 Jan 15;219(1-2):201–208. doi: 10.1111/j.1432-1033.1994.tb19931.x. [DOI] [PubMed] [Google Scholar]
  23. Kawamukai M., Utsumi R., Takeda K., Higashi A., Matsuda H., Choi Y. L., Komano T. Nucleotide sequence and characterization of the sfs1 gene: sfs1 is involved in CRP*-dependent mal gene expression in Escherichia coli. J Bacteriol. 1991 Apr;173(8):2644–2648. doi: 10.1128/jb.173.8.2644-2648.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Khoroshilova N., Beinert H., Kiley P. J. Association of a polynuclear iron-sulfur center with a mutant FNR protein enhances DNA binding. Proc Natl Acad Sci U S A. 1995 Mar 28;92(7):2499–2503. doi: 10.1073/pnas.92.7.2499. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. LaCelle M., Kumano M., Kurita K., Yamane K., Zuber P., Nakano M. M. Oxygen-controlled regulation of the flavohemoglobin gene in Bacillus subtilis. J Bacteriol. 1996 Jul;178(13):3803–3808. doi: 10.1128/jb.178.13.3803-3808.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Membrillo-Hernández J., Ioannidis N., Poole R. K. The flavohaemoglobin (HMP) of Escherichia coli generates superoxide in vitro and causes oxidative stress in vivo. FEBS Lett. 1996 Mar 11;382(1-2):141–144. doi: 10.1016/0014-5793(96)00154-8. [DOI] [PubMed] [Google Scholar]
  27. Niehaus F., Hantke K., Unden G. Iron content and FNR-dependent gene regulation in Escherichia coli. FEMS Microbiol Lett. 1991 Dec 1;68(3):319–323. doi: 10.1016/0378-1097(91)90376-l. [DOI] [PubMed] [Google Scholar]
  28. Nunoshiba T., deRojas-Walker T., Wishnok J. S., Tannenbaum S. R., Demple B. Activation by nitric oxide of an oxidative-stress response that defends Escherichia coli against activated macrophages. Proc Natl Acad Sci U S A. 1993 Nov 1;90(21):9993–9997. doi: 10.1073/pnas.90.21.9993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Poole R. K., Ioannidis N., Orii Y. Reactions of the Escherichia coli flavohaemoglobin (Hmp) with oxygen and reduced nicotinamide adenine dinucleotide: evidence for oxygen switching of flavin oxidoreduction and a mechanism for oxygen sensing. Proc Biol Sci. 1994 Mar 22;255(1344):251–258. doi: 10.1098/rspb.1994.0036. [DOI] [PubMed] [Google Scholar]
  30. Poole R. K., Kumar C., Salmon I., Chance B. The 650 and chromophore in Escherichia coli is an 'oxy-' or oxygenated compound, not the oxidized form of cytochrome oxidase d: an hypothesis. J Gen Microbiol. 1983 May;129(5):1335–1344. doi: 10.1099/00221287-129-5-1335. [DOI] [PubMed] [Google Scholar]
  31. Poole R. K. Oxygen reactions with bacterial oxidases and globins: binding, reduction and regulation. Antonie Van Leeuwenhoek. 1994;65(4):289–310. doi: 10.1007/BF00872215. [DOI] [PubMed] [Google Scholar]
  32. Privalle C. T., Kong S. E., Fridovich I. Induction of manganese-containing superoxide dismutase in anaerobic Escherichia coli by diamide and 1,10-phenanthroline: sites of transcriptional regulation. Proc Natl Acad Sci U S A. 1993 Mar 15;90(6):2310–2314. doi: 10.1073/pnas.90.6.2310. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Probst I., Wolf G., Schlegel H. G. An oxygen-binding flavohemoprotein from Alcaligenes eutrophus. Biochim Biophys Acta. 1979 Feb 26;576(2):471–478. doi: 10.1016/0005-2795(79)90422-7. [DOI] [PubMed] [Google Scholar]
  34. Puustinen A., Finel M., Haltia T., Gennis R. B., Wikström M. Properties of the two terminal oxidases of Escherichia coli. Biochemistry. 1991 Apr 23;30(16):3936–3942. doi: 10.1021/bi00230a019. [DOI] [PubMed] [Google Scholar]
  35. Rabin R. S., Stewart V. Dual response regulators (NarL and NarP) interact with dual sensors (NarX and NarQ) to control nitrate- and nitrite-regulated gene expression in Escherichia coli K-12. J Bacteriol. 1993 Jun;175(11):3259–3268. doi: 10.1128/jb.175.11.3259-3268.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Simons R. W., Houman F., Kleckner N. Improved single and multicopy lac-based cloning vectors for protein and operon fusions. Gene. 1987;53(1):85–96. doi: 10.1016/0378-1119(87)90095-3. [DOI] [PubMed] [Google Scholar]
  37. Smith M. S. Nitrous oxide production by Escherichia coli is correlated with nitrate reductase activity. Appl Environ Microbiol. 1983 May;45(5):1545–1547. doi: 10.1128/aem.45.5.1545-1547.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Stewart V., Parales J., Jr Identification and expression of genes narL and narX of the nar (nitrate reductase) locus in Escherichia coli K-12. J Bacteriol. 1988 Apr;170(4):1589–1597. doi: 10.1128/jb.170.4.1589-1597.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Stewart V. Requirement of Fnr and NarL functions for nitrate reductase expression in Escherichia coli K-12. J Bacteriol. 1982 Sep;151(3):1320–1325. doi: 10.1128/jb.151.3.1320-1325.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Stewart V., Yanofsky C. Role of leader peptide synthesis in tryptophanase operon expression in Escherichia coli K-12. J Bacteriol. 1986 Jul;167(1):383–386. doi: 10.1128/jb.167.1.383-386.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Sturr M. G., Krulwich T. A., Hicks D. B. Purification of a cytochrome bd terminal oxidase encoded by the Escherichia coli app locus from a delta cyo delta cyd strain complemented by genes from Bacillus firmus OF4. J Bacteriol. 1996 Mar;178(6):1742–1749. doi: 10.1128/jb.178.6.1742-1749.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Tsaneva I. R., Weiss B. soxR, a locus governing a superoxide response regulon in Escherichia coli K-12. J Bacteriol. 1990 Aug;172(8):4197–4205. doi: 10.1128/jb.172.8.4197-4205.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Valenzuela J. G., Walker F. A., Ribeiro J. M. A salivary nitrophorin (nitric-oxide-carrying hemoprotein) in the bedbug Cimex lectularius. J Exp Biol. 1995 Jul;198(Pt 7):1519–1526. doi: 10.1242/jeb.198.7.1519. [DOI] [PubMed] [Google Scholar]
  44. Vasudevan S. G., Armarego W. L., Shaw D. C., Lilley P. E., Dixon N. E., Poole R. K. Isolation and nucleotide sequence of the hmp gene that encodes a haemoglobin-like protein in Escherichia coli K-12. Mol Gen Genet. 1991 Apr;226(1-2):49–58. doi: 10.1007/BF00273586. [DOI] [PubMed] [Google Scholar]
  45. WALTERS C. L., TAYLOR A. M. THE REDUCTION OF NITRITE BY SKELETAL-MUSCLE MITOCHONDRIA. Biochim Biophys Acta. 1965 Mar 22;96:522–524. doi: 10.1016/0005-2787(65)90570-8. [DOI] [PubMed] [Google Scholar]
  46. Webster D. A. Structure and function of bacterial hemoglobin and related proteins. Adv Inorg Biochem. 1988;7:245–265. [PubMed] [Google Scholar]
  47. Zweier J. L., Wang P., Samouilov A., Kuppusamy P. Enzyme-independent formation of nitric oxide in biological tissues. Nat Med. 1995 Aug;1(8):804–809. doi: 10.1038/nm0895-804. [DOI] [PubMed] [Google Scholar]
  48. de Lorenzo V., Wee S., Herrero M., Neilands J. B. Operator sequences of the aerobactin operon of plasmid ColV-K30 binding the ferric uptake regulation (fur) repressor. J Bacteriol. 1987 Jun;169(6):2624–2630. doi: 10.1128/jb.169.6.2624-2630.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Journal of Bacteriology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES