Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 2001 Jun 15;356(Pt 3):851–858. doi: 10.1042/0264-6021:3560851

Overproduction, purification and novel redox properties of the dihaem cytochrome c, NapB, from Haemophilus influenzae.

A Brigé 1, J A Cole 1, W R Hagen 1, Y Guisez 1, J J Van Beeumen 1
PMCID: PMC1221913  PMID: 11389694

Abstract

The napB gene of the pathogenic bacterium Haemophilus influenzae encodes a dihaem cytochrome c, the small subunit of a heterodimeric periplasmic nitrate reductase similar to those found in other bacteria. In order to obtain sufficient protein for biophysical studies, we aimed to overproduce the recombinant dihaem protein in Escherichia coli. Initial expression experiments indicated that the NapB signal peptide was not cleaved by the leader peptidase of the host organism. Apocytochrome was formed under aerobic, semi-aerobic and anaerobic growth conditions in either Luria--Bertani or minimal salts medium. The highest amounts of apo-NapB were produced in the latter medium, and the bulk was inserted into the cytoplasmic membrane. The two haem groups were covalently attached to the pre-apocytochrome only under anaerobic growth conditions, and with 2.5 mM nitrite or at least 10 mM nitrate supplemented to the minimal salts growth medium. In order to obtain holocytochrome, the gene sequence encoding mature NapB was cloned in-frame with the E. coli ompA (outer membrane protein A) signal sequence. Under anaerobic conditions, NapB was secreted into the periplasmic space, with the OmpA signal peptide being correctly processed and with both haem c groups attached covalently. Unless expressed in the DegP-protease-deficient strain HM125, some of the recombinant NapB polypeptides were N-terminally truncated as a result of proteolytic activity. Under aerobic growth conditions, co-expression with the E. coli ccm (cytochrome c maturation) genes resulted in a higher yield of holocytochrome c. The pure recombinant NapB protein showed absorption maxima at 419, 522 and 550 nm in the reduced form. The midpoint reduction potentials of the two haem groups were determined to be -25 mV and -175 mV. These results support our hypothesis that the Nap system fulfils a nitrate-scavenging role in H. influenzae.

Full Text

The Full Text of this article is available as a PDF (173.2 KB).

Selected References

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

  1. Arslan E., Schulz H., Zufferey R., Künzler P., Thöny-Meyer L. Overproduction of the Bradyrhizobium japonicum c-type cytochrome subunits of the cbb3 oxidase in Escherichia coli. Biochem Biophys Res Commun. 1998 Oct 29;251(3):744–747. doi: 10.1006/bbrc.1998.9549. [DOI] [PubMed] [Google Scholar]
  2. Bell L. C., Richardson D. J., Ferguson S. J. Periplasmic and membrane-bound respiratory nitrate reductases in Thiosphaera pantotropha. The periplasmic enzyme catalyzes the first step in aerobic denitrification. FEBS Lett. 1990 Jun 4;265(1-2):85–87. doi: 10.1016/0014-5793(90)80889-q. [DOI] [PubMed] [Google Scholar]
  3. Berks B. C., Richardson D. J., Reilly A., Willis A. C., Ferguson S. J. The napEDABC gene cluster encoding the periplasmic nitrate reductase system of Thiosphaera pantotropha. Biochem J. 1995 Aug 1;309(Pt 3):983–992. doi: 10.1042/bj3090983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Berks B. C., Richardson D. J., Robinson C., Reilly A., Aplin R. T., Ferguson S. J. Purification and characterization of the periplasmic nitrate reductase from Thiosphaera pantotropha. Eur J Biochem. 1994 Feb 15;220(1):117–124. doi: 10.1111/j.1432-1033.1994.tb18605.x. [DOI] [PubMed] [Google Scholar]
  5. Bursakov S. A., Carneiro C., Almendra M. J., Duarte R. O., Caldeira J., Moura I., Moura J. J. Enzymatic properties and effect of ionic strength on periplasmic nitrate reductase (NAP) from Desulfovibrio desulfuricans ATCC 27774. Biochem Biophys Res Commun. 1997 Oct 29;239(3):816–822. doi: 10.1006/bbrc.1997.7560. [DOI] [PubMed] [Google Scholar]
  6. Carter J. P., Hsaio Y. H., Spiro S., Richardson D. J. Soil and sediment bacteria capable of aerobic nitrate respiration. Appl Environ Microbiol. 1995 Aug;61(8):2852–2858. doi: 10.1128/aem.61.8.2852-2858.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Carter J. P., Richardson D. J., Spiro S. Isolation and characterisation of a strain of Pseudomonas putida that can express a periplasmic nitrate reductase. Arch Microbiol. 1995 Mar;163(3):159–166. doi: 10.1007/BF00305348. [DOI] [PubMed] [Google Scholar]
  8. Choe M., Reznikoff W. S. Identification of the regulatory sequence of anaerobically expressed locus aeg-46.5. J Bacteriol. 1993 Feb;175(4):1165–1172. doi: 10.1128/jb.175.4.1165-1172.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Darwin A., Tormay P., Page L., Griffiths L., Cole J. Identification of the formate dehydrogenases and genetic determinants of formate-dependent nitrite reduction by Escherichia coli K12. J Gen Microbiol. 1993 Aug;139(8):1829–1840. doi: 10.1099/00221287-139-8-1829. [DOI] [PubMed] [Google Scholar]
  10. David P. S., Morrison M. R., Wong S. L., Hill B. C. Expression, purification, and characterization of recombinant forms of membrane-bound cytochrome c-550nm from Bacillus subtilis. Protein Expr Purif. 1999 Feb;15(1):69–76. doi: 10.1006/prep.1998.1001. [DOI] [PubMed] [Google Scholar]
  11. De Sutter K., Hostens K., Vandekerckhove J., Fiers W. Production of enzymatically active rat protein disulfide isomerase in Escherichia coli. Gene. 1994 Apr 20;141(2):163–170. doi: 10.1016/0378-1119(94)90566-5. [DOI] [PubMed] [Google Scholar]
  12. Dias J. M., Than M. E., Humm A., Huber R., Bourenkov G. P., Bartunik H. D., Bursakov S., Calvete J., Caldeira J., Carneiro C. Crystal structure of the first dissimilatory nitrate reductase at 1.9 A solved by MAD methods. Structure. 1999 Jan 15;7(1):65–79. doi: 10.1016/s0969-2126(99)80010-0. [DOI] [PubMed] [Google Scholar]
  13. Fleischmann R. D., Adams M. D., White O., Clayton R. A., Kirkness E. F., Kerlavage A. R., Bult C. J., Tomb J. F., Dougherty B. A., Merrick J. M. Whole-genome random sequencing and assembly of Haemophilus influenzae Rd. Science. 1995 Jul 28;269(5223):496–512. doi: 10.1126/science.7542800. [DOI] [PubMed] [Google Scholar]
  14. Grant S. G., Jessee J., Bloom F. R., Hanahan D. Differential plasmid rescue from transgenic mouse DNAs into Escherichia coli methylation-restriction mutants. Proc Natl Acad Sci U S A. 1990 Jun;87(12):4645–4649. doi: 10.1073/pnas.87.12.4645. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Guisez Y., Faché I., Campfield L. A., Smith F. J., Farid A., Plaetinck G., Van der Heyden J., Tavernier J., Fiers W., Burn P. Efficient secretion of biologically active recombinant OB protein (leptin) in Escherichia coli, purification from the periplasm and characterization. Protein Expr Purif. 1998 Mar;12(2):249–258. doi: 10.1006/prep.1997.0836. [DOI] [PubMed] [Google Scholar]
  16. Hagen W. R. Direct electron transfer of redox proteins at the bare glassy carbon electrode. Eur J Biochem. 1989 Jul 1;182(3):523–530. doi: 10.1111/j.1432-1033.1989.tb14859.x. [DOI] [PubMed] [Google Scholar]
  17. Herbaud M. L., Aubert C., Durand M. C., Guerlesquin F., Thöny-Meyer L., Dolla A. Escherichia coli is able to produce heterologous tetraheme cytochrome c(3) when the ccm genes are co-expressed. Biochim Biophys Acta. 2000 Aug 31;1481(1):18–24. doi: 10.1016/s0167-4838(00)00117-5. [DOI] [PubMed] [Google Scholar]
  18. Iobbi-Nivol C., Crooke H., Griffiths L., Grove J., Hussain H., Pommier J., Mejean V., Cole J. A. A reassessment of the range of c-type cytochromes synthesized by Escherichia coli K-12. FEMS Microbiol Lett. 1994 Jun 1;119(1-2):89–94. doi: 10.1111/j.1574-6968.1994.tb06872.x. [DOI] [PubMed] [Google Scholar]
  19. Kostanjevecki V., Leys D., Van Driessche G., Meyer T. E., Cusanovich M. A., Fischer U., Guisez Y., Van Beeumen J. Structure and characterization of Ectothiorhodospira vacuolata cytochrome b(558), a prokaryotic homologue of cytochrome b(5). J Biol Chem. 1999 Dec 10;274(50):35614–35620. doi: 10.1074/jbc.274.50.35614. [DOI] [PubMed] [Google Scholar]
  20. Meerman H. J., Georgiou G. Construction and characterization of a set of E. coli strains deficient in all known loci affecting the proteolytic stability of secreted recombinant proteins. Biotechnology (N Y) 1994 Nov;12(11):1107–1110. doi: 10.1038/nbt1194-1107. [DOI] [PubMed] [Google Scholar]
  21. Meissner P. S., Sisk W. P., Berman M. L. Bacteriophage lambda cloning system for the construction of directional cDNA libraries. Proc Natl Acad Sci U S A. 1987 Jun;84(12):4171–4175. doi: 10.1073/pnas.84.12.4171. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Moreno-Vivián C., Cabello P., Martínez-Luque M., Blasco R., Castillo F. Prokaryotic nitrate reduction: molecular properties and functional distinction among bacterial nitrate reductases. J Bacteriol. 1999 Nov;181(21):6573–6584. doi: 10.1128/jb.181.21.6573-6584.1999. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Nielsen H., Engelbrecht J., Brunak S., von Heijne G. Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites. Protein Eng. 1997 Jan;10(1):1–6. doi: 10.1093/protein/10.1.1. [DOI] [PubMed] [Google Scholar]
  24. Page L., Griffiths L., Cole J. A. Different physiological roles of two independent pathways for nitrite reduction to ammonia by enteric bacteria. Arch Microbiol. 1990;154(4):349–354. doi: 10.1007/BF00276530. [DOI] [PubMed] [Google Scholar]
  25. Potter L. C., Cole J. A. Essential roles for the products of the napABCD genes, but not napFGH, in periplasmic nitrate reduction by Escherichia coli K-12. Biochem J. 1999 Nov 15;344(Pt 1):69–76. doi: 10.1042/0264-6021:3440069. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Potter L. C., Millington P., Griffiths L., Thomas G. H., Cole J. A. Competition between Escherichia coli strains expressing either a periplasmic or a membrane-bound nitrate reductase: does Nap confer a selective advantage during nitrate-limited growth? Biochem J. 1999 Nov 15;344(Pt 1):77–84. [PMC free article] [PubMed] [Google Scholar]
  27. Price N. J., Brennan L., Faria T. Q., Vijgenboom E., Canters G. W., Turner D. L., Santos H. High yield of Methylophilus methylotrophus cytochrome c by coexpression with cytochrome c maturation gene cluster from Escherichia coli. Protein Expr Purif. 2000 Dec;20(3):444–450. doi: 10.1006/prep.2000.1318. [DOI] [PubMed] [Google Scholar]
  28. Reyes F., Gavira M., Castillo F., Moreno-Vivián C. Periplasmic nitrate-reducing system of the phototrophic bacterium Rhodobacter sphaeroides DSM 158: transcriptional and mutational analysis of the napKEFDABC gene cluster. Biochem J. 1998 May 1;331(Pt 3):897–904. doi: 10.1042/bj3310897. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Reyes F., Roldán M. D., Klipp W., Castillo F., Moreno-Vivián C. Isolation of periplasmic nitrate reductase genes from Rhodobacter sphaeroides DSM 158: structural and functional differences among prokaryotic nitrate reductases. Mol Microbiol. 1996 Mar;19(6):1307–1318. doi: 10.1111/j.1365-2958.1996.tb02475.x. [DOI] [PubMed] [Google Scholar]
  30. Richardson D. J., McEwan A. G., Page M. D., Jackson J. B., Ferguson S. J. The identification of cytochromes involved in the transfer of electrons to the periplasmic NO3- reductase of Rhodobacter capsulatus and resolution of a soluble NO3(-)-reductase--cytochrome-c552 redox complex. Eur J Biochem. 1990 Nov 26;194(1):263–270. doi: 10.1111/j.1432-1033.1990.tb19452.x. [DOI] [PubMed] [Google Scholar]
  31. Roldán M. D., Sears H. J., Cheesman M. R., Ferguson S. J., Thomson A. J., Berks B. C., Richardson D. J. Spectroscopic characterization of a novel multiheme c-type cytochrome widely implicated in bacterial electron transport. J Biol Chem. 1998 Oct 30;273(44):28785–28790. doi: 10.1074/jbc.273.44.28785. [DOI] [PubMed] [Google Scholar]
  32. Sabaty M., Gagnon J., Verméglio A. Induction by nitrate of cytoplasmic and periplasmic proteins in the photodenitrifier Rhodobacter sphaeroides forma sp. denitrificans under anaerobic or aerobic condition. Arch Microbiol. 1994;162(5):335–343. doi: 10.1007/BF00263781. [DOI] [PubMed] [Google Scholar]
  33. Siddiqui R. A., Warnecke-Eberz U., Hengsberger A., Schneider B., Kostka S., Friedrich B. Structure and function of a periplasmic nitrate reductase in Alcaligenes eutrophus H16. J Bacteriol. 1993 Sep;175(18):5867–5876. doi: 10.1128/jb.175.18.5867-5876.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Simon J., Gross R., Einsle O., Kroneck P. M., Kröger A., Klimmek O. A NapC/NirT-type cytochrome c (NrfH) is the mediator between the quinone pool and the cytochrome c nitrite reductase of Wolinella succinogenes. Mol Microbiol. 2000 Feb;35(3):686–696. doi: 10.1046/j.1365-2958.2000.01742.x. [DOI] [PubMed] [Google Scholar]
  35. Stellwagen E., Wilgus H. Relationship of protein thermostability to accessible surface area. Nature. 1978 Sep 28;275(5678):342–343. doi: 10.1038/275342a0. [DOI] [PubMed] [Google Scholar]
  36. Thomas P. E., Ryan D., Levin W. An improved staining procedure for the detection of the peroxidase activity of cytochrome P-450 on sodium dodecyl sulfate polyacrylamide gels. Anal Biochem. 1976 Sep;75(1):168–176. doi: 10.1016/0003-2697(76)90067-1. [DOI] [PubMed] [Google Scholar]
  37. Thöny-Meyer L., Künzler P. Translocation to the periplasm and signal sequence cleavage of preapocytochrome c depend on sec and lep, but not on the ccm gene products. Eur J Biochem. 1997 Jun 15;246(3):794–799. doi: 10.1111/j.1432-1033.1997.t01-1-00794.x. [DOI] [PubMed] [Google Scholar]
  38. Vollack K. U., Xie J., Härtig E., Römling U., Zumft W. G. Localization of denitrification genes on the chromosomal map of Pseudomonas aeruginosa. Microbiology. 1998 Feb;144(Pt 2):441–448. doi: 10.1099/00221287-144-2-441. [DOI] [PubMed] [Google Scholar]
  39. Yanisch-Perron C., Vieira J., Messing J. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene. 1985;33(1):103–119. doi: 10.1016/0378-1119(85)90120-9. [DOI] [PubMed] [Google Scholar]
  40. da Costa P. N., Conte C., Saraiva L. M. Expression of a Desulfovibrio tetraheme cytochrome c in Escherichia coli. Biochem Biophys Res Commun. 2000 Feb 24;268(3):688–691. doi: 10.1006/bbrc.2000.2198. [DOI] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES