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PMID: 12426353 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't

Regulation of the hydrogenase-4 operon of Escherichia coli by the sigma(54)-dependent transcriptional activators FhlA and HyfR.

Journal of bacteriology ·Vol. 184 ·No. 23 ·2002-12-00 ·Pages 6642-53

Skibinski DA, Golby P, Chang YS, Sargent F, Hoffman R, Harper R, Guest JR, Attwood MM, Berks BC, Andrews SC

Abstract

The hyf locus (hyfABCDEFGHIJ-hyfR-focB) of Escherichia coli encodes a putative 10-subunit hydrogenase complex (hydrogenase-4 [Hyf]); a potential sigma(54)-dependent transcriptional activator, HyfR (related to FhlA); and a putative formate transporter, FocB (related to FocA). In order to gain insight into the physiological role of the Hyf system, we investigated hyf expression by using a hyfA-lacZ transcriptional fusion. This work revealed that hyf is induced under fermentative conditions by formate at a low pH and in an FhlA-dependent fashion. Expression was sigma(54) dependent and was inhibited by HycA, the negative transcriptional regulator of the formate regulon. Thus, hyf expression resembles that of the hyc operon. Primer extension analysis identified a transcriptional start site 30 bp upstream of the hyfA structural gene, with appropriately located -24 and -12 boxes indicative of a sigma(54)-dependent promoter. No reverse transcriptase PCR product could be detected for hyfJ-hyfR, suggesting that hyfR-focB may be independently transcribed from the rest of the hyf operon. Expression of hyf was strongly induced ( approximately 1,000-fold) in the presence of a multicopy plasmid expressing hyfR from a heterologous promoter. This induction was dependent on low pH, anaerobiosis, and postexponential growth and was weakly enhanced by formate. The hyfR-expressing plasmid increased fdhF-lacZ transcription just twofold but did not influence the expression of hycB-lacZ. Interestingly, inactivation of the chromosomal hyfR gene had no effect on hyfA-lacZ expression. Purified HyfR was found to specifically interact with the hyf promoter/operator region. Inactivation of the hyf operon had no discernible effect on growth under the range of conditions tested. No Hyf-derived hydrogenase or formate dehydrogenase activity could be detected, and no Ni-containing protein corresponding to HyfG was observed.

MeSH Terms
Anaerobiosis DNA-Binding Proteins DNA-Directed RNA Polymerases/metabolism Escherichia coli/enzymology,genetics,growth & development Escherichia coli Proteins/genetics,metabolism Formates/metabolism Gene Expression Regulation, Bacterial Hydrogen/metabolism Hydrogen-Ion Concentration Hydrogenase/genetics,metabolism Operon RNA Polymerase Sigma 54 Sigma Factor/metabolism Trans-Activators/genetics,metabolism Transcription, Genetic
Chemicals
DNA-Binding Proteins Escherichia coli Proteins Formates Sigma Factor Trans-Activators rpoN protein, E coli formic acid fhlA protein, E coli Hydrogen Hydrogenase hydrogenase 4, E coli DNA-Directed RNA Polymerases RNA Polymerase Sigma 54
Authors & Affiliations
10 authors, click to expand affiliations / ORCID
Skibinski David A G
School of Animal and Microbial Sciences, The University of Reading, Reading RG6 6AJ, United Kingdom.
Golby Paul
Chang Yung-Sheng
Sargent Frank
Hoffman Ralf
Harper R
Guest John R
Attwood Margaret M
Berks Ben C
Andrews Simon C
References (44)
44 references, click to expand
  1. Sec-independent protein translocation in Escherichia coli. A distinct and pivotal role for the TatB protein.
    J Biol Chem. 1999 Dec 17;274(51):36073-82 PMID: 10593889
  2. Escherichia coli formate-hydrogen lyase. Purification and properties of the selenium-dependent formate dehydrogenase component.
    J Biol Chem. 1990 Oct 25;265(30):18213-8 PMID: 2211698
  3. Analysis of the domain structure and the DNA binding site of the transcriptional activator FhlA.
    Eur J Biochem. 2000 Jun;267(12):3672-84 PMID: 10848985
  4. N-terminal truncations in the FhlA protein result in formate- and MoeA-independent expression of the hyc (formate hydrogenlyase) operon of Escherichia coli.
    Microbiology. 2001 Nov;147(Pt 11):3093-104 PMID: 11700359
  5. The roles of hydrogenases 3 and 4, and the F0F1-ATPase, in H2 production by Escherichia coli at alkaline and acidic pH.
    FEBS Lett. 2002 Apr 10;516(1-3):172-8 PMID: 11959127
  6. The regulation of metabolism in facultative bacteria. 3. The effect of nitrate.
    Biochim Biophys Acta. 1967 Oct 9;148(1):233-42 PMID: 4864932
  7. A colony bank containing synthetic Col El hybrid plasmids representative of the entire E. coli genome.
    Cell. 1976 Sep;9(1):91-9 PMID: 788919
  8. Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from the P15A cryptic miniplasmid.
    J Bacteriol. 1978 Jun;134(3):1141-56 PMID: 149110
  9. Identification and sequence analysis of the gene encoding the transcriptional activator of the formate hydrogenlyase system of Escherichia coli.
    Mol Microbiol. 1990 Aug;4(8):1319-27 PMID: 2280686
  10. Molecular characterization of an operon (hyp) necessary for the activity of the three hydrogenase isoenzymes in Escherichia coli.
    Mol Microbiol. 1991 Jan;5(1):123-35 PMID: 1849603
  11. Construction and properties of a family of pACYC184-derived cloning vectors compatible with pBR322 and its derivatives.
    Gene. 1991 Jun 15;102(1):75-8 PMID: 1840539
  12. Mechanism of regulation of the formate-hydrogenlyase pathway by oxygen, nitrate, and pH: definition of the formate regulon.
    Mol Microbiol. 1991 Nov;5(11):2807-14 PMID: 1779767
  13. Mutational analysis of the operon (hyc) determining hydrogenase 3 formation in Escherichia coli.
    Mol Microbiol. 1992 Jun;6(11):1523-32 PMID: 1625581
  14. Isolation and characterization of hypophosphite--resistant mutants of Escherichia coli: identification of the FocA protein, encoded by the pfl operon, as a putative formate transporter.
    Mol Microbiol. 1994 Mar;11(5):965-82 PMID: 8022272
  15. Purification and DNA-binding properties of FHLA, the transcriptional activator of the formate hydrogenlyase system from Escherichia coli.
    J Biol Chem. 1994 Jul 29;269(30):19590-6 PMID: 8034727
  16. Characterisation of a protease from Escherichia coli involved in hydrogenase maturation.
    Eur J Biochem. 1995 Jan 15;227(1-2):545-50 PMID: 7851435
  17. The hydrogenases and formate dehydrogenases of Escherichia coli.
    Antonie Van Leeuwenhoek. 1994;66(1-3):57-88 PMID: 7747941
  18. Characterization of fhlA mutations resulting in ligand-independent transcriptional activation and ATP hydrolysis.
    J Bacteriol. 1997 Jan;179(1):41-5 PMID: 8981978
  19. A 12-cistron Escherichia coli operon (hyf) encoding a putative proton-translocating formate hydrogenlyase system.
    Microbiology. 1997 Nov;143 ( Pt 11):3633-47 PMID: 9387241
  20. Topological analysis of DcuA, an anaerobic C4-dicarboxylate transporter of Escherichia coli.
    J Bacteriol. 1998 Sep;180(18):4821-7 PMID: 9733683
  21. Transcriptional regulation and organization of the dcuA and dcuB genes, encoding homologous anaerobic C4-dicarboxylate transporters in Escherichia coli.
    J Bacteriol. 1998 Dec;180(24):6586-96 PMID: 9852003
  22. Formic dehydrogenase and the hydrogenlyase enzyme complex in coli-aerogenes bacteria.
    J Bacteriol. 1957 Jun;73(6):706-21 PMID: 13449036
  23. The thiobacilli.
    Bacteriol Rev. 1957 Sep;21(3):195-213 PMID: 13471458
  24. Lactose genes fused to exogenous promoters in one step using a Mu-lac bacteriophage: in vivo probe for transcriptional control sequences.
    Proc Natl Acad Sci U S A. 1979 Sep;76(9):4530-3 PMID: 159458
  25. Studies on transformation of Escherichia coli with plasmids.
    J Mol Biol. 1983 Jun 5;166(4):557-80 PMID: 6345791
  26. On the redox control of synthesis of anaerobically induced enzymes in enterobacteriaceae.
    Arch Microbiol. 1983 Nov;136(2):131-6 PMID: 6360066
  27. In vitro insertional mutagenesis with a selectable DNA fragment.
    Gene. 1984 Sep;29(3):303-13 PMID: 6237955
  28. The seleno-polypeptide of formic dehydrogenase (formate hydrogen-lyase linked) from Escherichia coli: genetic analysis.
    Arch Microbiol. 1985 May;141(4):359-63 PMID: 3160320
  29. Differential expression of hydrogenase isoenzymes in Escherichia coli K-12: evidence for a third isoenzyme.
    J Bacteriol. 1985 Dec;164(3):1324-31 PMID: 3905769
  30. Isolation and characterisation of a soluble active fragment of hydrogenase isoenzyme 2 from the membranes of anaerobically grown Escherichia coli.
    Eur J Biochem. 1986 Apr 15;156(2):277-84 PMID: 3516690
  31. Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes.
    J Mol Biol. 1986 May 5;189(1):113-30 PMID: 3537305
  32. Improved single and multicopy lac-based cloning vectors for protein and operon fusions.
    Gene. 1987;53(1):85-96 PMID: 3596251
  33. Factors affecting transcriptional regulation of the formate-hydrogen-lyase pathway of Escherichia coli.
    Arch Microbiol. 1987 Jun;148(1):44-51 PMID: 2443100
  34. Production of single-stranded plasmid DNA.
    Methods Enzymol. 1987;153:3-11 PMID: 3323803
  35. Involvement of the ntrA gene product in the anaerobic metabolism of Escherichia coli.
    Mol Gen Genet. 1987 Dec;210(3):535-42 PMID: 3323848
  36. Construction of chimaeric promoter regions by exchange of the upstream regulatory sequences from fdhF and nif genes.
    Mol Microbiol. 1989 Jun;3(6):697-703 PMID: 2664422
  37. Initial cloning and sequencing of hydHG, an operon homologous to ntrBC and regulating the labile hydrogenase activity in Escherichia coli K-12.
    J Bacteriol. 1989 Aug;171(8):4448-56 PMID: 2666400
  38. Characterization of a cis regulatory DNA element necessary for formate induction of the formate dehydrogenase gene (fdhF) of Escherichia coli.
    Mol Microbiol. 1989 Feb;3(2):187-95 PMID: 2668685
  39. Mutations in trans which affect the anaerobic expression of a formate dehydrogenase (fdhF) structural gene.
    Arch Microbiol. 1989;152(1):83-9 PMID: 2669674
  40. New method for generating deletions and gene replacements in Escherichia coli.
    J Bacteriol. 1989 Sep;171(9):4617-22 PMID: 2548993
  41. Characterization of divergent NtrA-dependent promoters in the anaerobically expressed gene cluster coding for hydrogenase 3 components of Escherichia coli.
    Mol Microbiol. 1990 Jan;4(1):13-20 PMID: 2181234
  42. Nucleotide sequence and expression of an operon in Escherichia coli coding for formate hydrogenlyase components.
    Mol Microbiol. 1990 Feb;4(2):231-43 PMID: 2187144
  43. Genetic regulation of formate hydrogenlyase of Escherichia coli: role of the fhlA gene product as a transcriptional activator for a new regulatory gene, fhlB.
    J Bacteriol. 1990 Sep;172(9):4798-806 PMID: 2118503
  44. Isolation and characterization of mutated FhlA proteins which activate transcription of the hyc operon (formate hydrogenlyase) of Escherichia coli in the absence of molybdate(1).
    FEMS Microbiol Lett. 2000 Mar 1;184(1):47-52 PMID: 10689164
Article Info
Journal
Journal of bacteriology
Abbr.
J Bacteriol
ISSN
0021-9193
DOI
10.1128/JB.184.23.6642-6653.2002
Published
2002-12-00
Pages
6642-53
Language
English
Region
United States
NLM ID
2985120R
PMCID
PMC135417
Subset
IM
External Links
PubMed source DOI: 10.1128/JB.184.23.6642-6653.2002 PMC Full Text View journal details
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