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. 2017 Jul 14;292(28):11937-11950.
doi: 10.1074/jbc.M117.789164. Epub 2017 May 30.

The UbiK protein is an accessory factor necessary for bacterial ubiquinone (UQ) biosynthesis and forms a complex with the UQ biogenesis factor UbiJ

Laurent Loiseau  1 , Cameron Fyfe  2 , Laurent Aussel  1 , Mahmoud Hajj Chehade  3 , Sara B Hernández  4 , Bruno Faivre  2 , Djemel Hamdane  2 , Caroline Mellot-Draznieks  2 , Bérengère Rascalou  3 , Ludovic Pelosi  3 , Christophe Velours  5 , David Cornu  6 , Murielle Lombard  2 , Josep Casadesús  4 , Fabien Pierrel  7 , Marc Fontecave  8 , Frédéric Barras  9
Affiliations

The UbiK protein is an accessory factor necessary for bacterial ubiquinone (UQ) biosynthesis and forms a complex with the UQ biogenesis factor UbiJ

Laurent Loiseau et al. J Biol Chem. .

Abstract

Ubiquinone (UQ), also referred to as coenzyme Q, is a widespread lipophilic molecule in both prokaryotes and eukaryotes in which it primarily acts as an electron carrier. Eleven proteins are known to participate in UQ biosynthesis in Escherichia coli, and we recently demonstrated that UQ biosynthesis requires additional, nonenzymatic factors, some of which are still unknown. Here, we report on the identification of a bacterial gene, yqiC, which is required for efficient UQ biosynthesis, and which we have renamed ubiK Using several methods, we demonstrated that the UbiK protein forms a complex with the C-terminal part of UbiJ, another UQ biogenesis factor we previously identified. We found that both proteins are likely to contribute to global UQ biosynthesis rather than to a specific biosynthetic step, because both ubiK and ubiJ mutants accumulated octaprenylphenol, an early intermediate of the UQ biosynthetic pathway. Interestingly, we found that both proteins are dispensable for UQ biosynthesis under anaerobiosis, even though they were expressed in the absence of oxygen. We also provide evidence that the UbiK-UbiJ complex interacts with palmitoleic acid, a major lipid in E. coli Last, in Salmonella enterica, ubiK was required for proliferation in macrophages and virulence in mice. We conclude that although the role of the UbiK-UbiJ complex remains unknown, our results support the hypothesis that UbiK is an accessory factor of Ubi enzymes and facilitates UQ biosynthesis by acting as an assembly factor, a targeting factor, or both.

Keywords: Escherichia coli (E. coli); bioenergetics; coenzyme Q10 (CoQ10); electron transfer; microbiology; ubiquinone.

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Conflict of interest statement

The authors declare that they have no conflicts of interest with the contents of this article

Figures

Figure 1.
Figure 1.
A, HPLC ECD analyses of lipid extracts from 1 mg of E. coli WT or ΔubiK cells transformed with either the empty pK vector (vec) or the pK vector carrying the ubiK gene grown in LB medium in aerobic conditions. The chromatograms are representative of three independent experiments. The peaks corresponding to UQ8, DMK8, OPP, and the coenzyme Q10 (UQ10) standard are indicated. B, quantification of cellular UQ8 content of the E. coli strains described in A based on ECD signal (peak area). C and D, quantification of cellular DMK8 and MK8 from chromatograms at 247 nm (see supplemental Fig. S1B). E, quantification (peak area) of OPP from chromatograms at 275 nm (see supplemental Fig. S1D). B–E, mean ± S.E.; n = 3–5; ***, p < 0.001; ****, p < 0.0001, one-way analysis of variance. F, ECD quantification of cellular UQ8 content of the E. coli WT and ΔubiK cells grown in anaerobic conditions in LB medium; n = 3. G, immunodetection of SPA-tagged proteins UbiE, UbiK, and UbiJ after growth in LB medium under aerobic (+O2) or anaerobic (−O2) conditions. Equal loading was verified with immunodetection of the protein LamB and staining of the SDS-PAGE with Coomassie Blue dye. Mw, molecular mass.
Figure 2.
Figure 2.
A, SEC-MALS analysis of UbiK from E. coli (injection of 100 μl of UbiK at 2 mg/ml on a Superdex 200 10/300 GL increase column, 50 mm Tris-HCl, 150 mm NaCl buffer, pH 7.5. Solid and blue lines correspond to refraction index signal and molar mass, respectively. B, far-UV circular dichroism spectrum (185–260 nm) of E. coli UbiK (50 mm Tris-HCl, 150 mm NaCl buffer, pH 7.5).
Figure 3.
Figure 3.
A, crystal structure of the α-helical coiled-coil Brick1 homotrimer from D. discoideum (PDB code 3PP5); B, predicted structural model of E. coli UbiK (Swiss model); C, hypothesized model of the α-helical coiled-coil UbiK homotrimer, based on sequence identity, CD, and SEC-MALS. N and C termini are predicted as disordered regions.
Figure 4.
Figure 4.
Interactions between UbiK and the other Ubi proteins. A bacterial two-hybrid system was used to detect in vivo protein-protein interactions. Values of β-galactosidase activities, calculated in Miller units, with standard errors are shown: pT25-ubiK (A) or pT18-ubiK (B) were analyzed with ubiJ, -E, -I, -K, -B, -D, -G, -A, -X, -H, or -F genes, respectively.
Figure 5.
Figure 5.
A, UbiJ-His and UbiK-S tag were co-expressed from the pETDuet-6his-ubiJ/ubiK-S tag plasmid. Cell extracts were passed over a nickel-nitrilotriacetic acid column. Imidazole gradient was applied and the two proteins were co-eluted. Lane 1, soluble cell extract; lane 2, flow through; lane 3, wash; lanes 4–9, imidazole eluted peak fractions. B, pulldown assays were carried out to investigate the interactions between the UbiK and UbiJ proteins. ubiK, ubiJ, and sufC (negative control) genes were fused at their 5′ end with the malE gene and the chimeric proteins were purified in His6–ubiJ (lanes 1 and 2) or ubiK–SPA (lanes 3 and 4) backgrounds. Soluble extracts of each strain were loaded onto an amylose column, the column was washed and a solution of maltose was applied. MBP–UbiK and His6–UbiJ were co-eluted (lane 2) as MBP–UbiJ and UbiK–SPA (lane 4). C, interactions between UbiK and UbiJ (top), UbiK and the 50 C-terminal amino acids of UbiJ (middle), and UbiK and an empty plasmid (bottom) were analyzed by a yeast two-hybrid reporter system. Diploid strains producing pairs of Ubi proteins were tested on solid medium for activation of the lacZ gene and the intensity of interaction was monitored by a gradient from dark blue (strong interaction) to white (no interaction) (left). The ability to grow on selective medium lacking leucine was also monitored during 16 h at 30 °C (right).
Figure 6.
Figure 6.
SEC-MALS analysis of the two complexes P1 (A) and P2 (B) of UbiK–UbiJ. Samples were run on a Superdex 200 Increase 10/300 GL. Blue, red, and green curves correspond to the molar masses of the UbiK–UbiJ complex, UbiK and UbiJ, respectively (protein-conjugate method). C, crystal structure of the sterol carrier protein SCP2 from Yarrowia lipolytica, with palmitic acid ligand. D, hypothesized model of the E. coli UbiK–UbiJ 2:1 heterotrimer complex, made by Phyre 2. UbiJ monomer is colored orange, UbiK dimer is colored blue and red. The template for the N-terminal domain of UbiJ(1–120) was PDB 4JGX (SCP2 from Y. lipolytica); the template for the C-terminal domain (151–195) was PDB 3QH9 (human coiled-coil liprin) and the template for the middle domain (132–150) was PDB 1M1J (fibrinogen α subunit).
Figure 7.
Figure 7.
ESI-TOF-MS of UbiK–UbiJ P2 supernatant. The m/z 263.22 seen in the UbiK–UbiJ sample corresponds to the exact mass of palmitoleic acid, m/z 263.23 (commercial standard inset).
Figure 8.
Figure 8.
A, UQ8 content of S. enterica WT and ΔubiK cells after growth at 37 °C in LB medium in aerobic (O2) or anaerobic conditions. Mean ± S.E.; n = 4; ns, nonsignificant; ****, p < 0.0001, unpaired two-tailed t test. B and C, Salmonella strains (WT and ΔubiK) were grown overnight under aerobic (gray bars, B) or anaerobic conditions (black bars, B) in LB at 37 °C. Opsonized bacteria were phagocytosed by RAW 264.7 cells. The experiments were carried out at 37 °C (gray bars, C) or 30 °C (black bars, C). Two and 16 h post-infection, mouse macrophages were lysed for enumeration of intracellular bacteria (gentamicin-protected) determined by colony-forming unit (cfu) counts. The values shown represent the proliferation index calculated as a ratio of the intracellular bacteria between 16 and 2 h postinfection. Results are the mean ± S.D. of at least three independent experiments each in triplicate. Asterisks indicate a statistically significant difference between the ubiK mutant and the WT. *, p ≤ 0.05 (Mann-Whitney U test). D, BALB/c mice were inoculated intraperitoneally with a 1:1 mixture of ubiK mutant and wild-type Salmonella strains. Forty-eight hours post-inoculation, spleens were harvested for bacterial counts. Competitive indexes of wild-type versus ubiK mutant strains in mice were determined. Each circle represents one mouse and the horizontal bar corresponds to the mean. A one-sample t test was used to determine whether the CI was significantly different from 1. ***, p ≤ 0.001.
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