Article Figures & Data
Figures
- FIG. 1.
The nar::kat mutants. A) Scheme showing the structure of the nar operon, the approximate location of the inserted kat gene inside narC, narI, and narJ, and the narp promoter location (curved arrow). B) Proposed topology for the NarI and NarC proteins. Amino acid positions that define the five membrane-spanning helices (I to V) in NarI and NarC are labeled. Conserved histidines involved in heme B coordination are indicated on helices II and V. The white arrow indicates the position where the NarI protein is interrupted in the narI::kat mutant. Diamonds indicate the presence of heme C groups in NarC. The cytoplasmic (in) and periplasmic (out) faces of the membrane are indicated.
- FIG. 2.
Phenotype of nar::kat mutants. A) Western blot for detecting NarG in soluble (S) and particulate (P) fractions of nitrate/anoxia-induced cultures of the wild-type strain (Wt) and its three nar::kat derivatives (narC, narJ, narI). B) The presence of NarG is analyzed in the soluble (S) and particulate (P) fractions from a narC::kat mutant (narC) and from a derived strain complemented with a wild-type narC gene (narC/pIWnarC) by transformation with plasmid pIWnarC. The enzyme activities (nmol of nitrite/min and OD550 unit) from each fraction are indicated.
- FIG. 3.
Two-hybrid assays. Bacterial two-hybrid assays were developed, and the mean values of the β-galactosidase activity corresponding to six samples in two independent experiments were represented. The whole coding regions of narI (I), narG (G), narH (H), and narJ(J) and deletion derivatives of narC coding for a signal peptide-defective protein (ΔPs) or for its cytoplasmic C-terminal (Cd) domain were cloned into plasmid pT18 and/or pT25 to generate the appropriate X-T18 and T25-X protein fusions, as indicated. Pairs of pT18 and pT25 derivatives were assayed for β-galactosidase expression in E. coli BTH101 cells. Controls for negative (Z) and positive (Gdh/Gdh) interactions were used.
- FIG. 4.
Sensitivity of NarG to trypsin in narI, narC, and narJ mutants. The sensitivities to trypsin of NarG and NarJ from the soluble fraction of the wild-type strain (Wtc) and its three nar::kat derivatives were compared. The sensitivity of NarG was also assayed in membrane fractions of the wild type (Wtm). The samples were incubated with the protease for the indicated times (min).
- FIG. 5.
Model for the synthesis of nitrate reductase in T. thermophilus. A membrane NarCI and a soluble NarGHJ complex are initially formed. In the soluble complex, the NarG protein is inactive (squares). After attachment to the membrane, an inactive pentameric apo-nitrate reductase is formed. The enzyme is subsequently activated through the insertion of the Mo-bis-MGD cofactor (MGD) and the concomitant exit of the NarJ chaperone to render an active, heterotetrameric enzyme (circled NarG). Eventually, the active NarGH complex irreversibly separates from the membrane, giving rise to soluble forms that are active in nitrate reduction with artificial electron donors but not functional in nitrate respiration.
Tables
- TABLE 1.
Strains and plasmids used
Strain or plasmid Description or use Reference or source E. coli DH5α supE44 ΔlacU169 (Φ80 lacZΔM15) hsdR17 recA endA1 gyrA96 thi-1 relA1 10 E. coli BTH101 F′ cya-99 araD139 galE15 galK16 rpsL1(Strr) hsdR2 mcrA1 mcrB1 12 T. thermophilus HB8 Wild-type strain 19; ATCC 27634 T. thermophilus HB8narC HB8 narC::kat 29 T. thermophilus HB8narI HB8 narI::kat This work T. thermophilus HB8narJ HB8 narJ::kat This work T. thermophilus HB27nar T. thermophilus HB27::nar 21 T. thermophilus L T. thermophilus HB27::nar ΔleuB This work T. thermophilus LnarC T. thermophilus HB27::nar ΔleuB narC::kat This work pUC118/119 E. coli cloning plasmids; Ampr 28 pUP3B pUC119 derivative; clone carrying the whole nrc operon; Ampr 7 pKT1 E. coli plasmid carrying the kanamycin thermostable resistance gene (kat); Ampr, Kanr 15 pTH3ΔEcoRV Plasmid for the isolation of ΔleuB mutants 26 pIW Suicidal vector; selection for leucine prototrophy 26 pIWnarC Insertion of the wild-type narC gene into the leuB locus This work pT18 pUC19 derivative expressing T18 fragment of CyaA; Ampr 12 pT25 pACYC184 derivative expressing T25 fragment of Cya, Cmr 12 pT18zip pT18, leucine zipper fused to T18 fragment 12 pT25zip pT25, leucine zipper fused to T25 fragment 12 pT25gdh pT25 derivative, T25-Gdh fusion protein, Cmr 7 pT18gdh pT18 derivative, Gdh-T18 fusion protein, Ampr 7 pT25narJ pT25 derivative, T25-NarJ fusion protein, Cmr 7 pT18narJ pT18 derivative, NarJ-T18 fusion protein, Ampr 7 pT25narG pT25 derivative, T25-NarG fusion protein, Cmr 7 pT18narG pT18 derivative, NarG-T18 fusion protein, Ampr 7 pT25narH pT25 derivative, T25-NarH fusion protein, Cmr This work pT18narI pT18 derivative, NarI-T18 fusion protein, Ampr This work pT25narC-ΔPs pT25 derivative T25-NarCΔPs fusion protein, Cmr This work pT18narC-ΔPs pT18 derivative, NarCΔPs-T18 fusion protein, Ampr This work pT25narC-Cd pT25 derivative, T25-NarCCd fusion protein, Cmr This work pT18narC-Cd pT18 derivative, NarCCd-T18 fusion protein, Ampr This work - TABLE 2.
Oligonucleotides used
Name Sequence Purpose H1 5′-CTGGGTACCTCAGGACGGGAAGGCCCTCTACGG-3′ pT18narC-ΔSP H2 5′-ATTCGATATCCTCCTAAGCTGGGCGCGGATC-3′ pT18narC-ΔSP/Cd H4 5′-CTGGGTACCTGTCCTCTGGCAGCTTCGGCCGAG-3′ pT18narC-Cd H7 5′-AAAACTGCAGGGATGAAGGTTAGAGCCCACATGTCC-3′ pT25narH H8 5′-TTTAGGTACCTCGCCCCGCAAGGGGGGCTGGATG-3′ pT25narH H17 5′-AAAACTGCAGGGCAGGACGGGAAGGCCCTCTACGG-3′ pT25narC-ΔSP H18 5′-ATTCGATATCCCTCCTAAGCTGGGCGCGGATC-3′ pT25narC-ΔSP/Cd H20 5′-AAAACTGCAGGGGTCCTCTGGCAGCTTCGGCCGAG-3′ pT25narC-Cd H24 5′-ACGCGTCGACGATGAAGGTTAGAGCCCACAT-3′ pT18narH H25 5′-ATTCGATATCTCGCCCCGCAAGGGGGGCTGGA-3′ pT18narH H26 5′-ACGCGTCGACGATGAAGTGGAACGCCGCGCTCTTC-3′ pT18narI H27 5′-ATTCGATATCAGCTGACCTCGCCAGTTGCG-3′ pT18narI o27-35 5′-CGCAGTACCAGTCGTAG-3′ Presence of narC::kat okat2 5′-GAAACTTCTGGAATCGC-3′ Presence of narC::kat okat3 5′-GGAACGAATATTGGATA-3′ Presence of kat okat4 5′-AGAAATTCTCTAGCGAT-3′ Presence of kat
