ubiJ, a new gene required for aerobic growth and proliferation in macrophage, is involved in coenzyme Q biosynthesis in Escherichia coli and Salmonella enterica serovar Typhimurium

Abstract

Ubiquinone (coenzyme Q or Q8) is a redox active lipid which functions in the respiratory electron transport chain and plays a crucial role in energy-generating processes. In both Escherichia coli and Salmonella enterica serovar Typhimurium, the yigP gene is located between ubiE and ubiB, all three being likely to constitute an operon. In this work, we showed that the uncharacterized yigP gene was involved in Q8 biosynthesis in both strains, and we have renamed it ubiJ. Under aerobic conditions, an ubiJ mutant was found to be impaired for Q8 biosynthesis and for growth in rich medium but did not present any defect anaerobically. Surprisingly, the C-terminal 50 amino acids, predicted to interact with lipids, were sufficient to restore Q8 biosynthesis and growth of the ubiJ mutant. Salmonella ubiE and ubiB mutants were impaired in Q8 biosynthesis and in respiration using different electron acceptors. Moreover, ubiE, ubiJ, and ubiB mutants were all impaired for Salmonella intracellular proliferation in macrophages. Taken together, our data establish an important role for UbiJ in Q8 biosynthesis and reveal an unexpected link between Q8 and virulence. They also emphasize that Salmonella organisms in an intracellular lifestyle rely on aerobic respiration to survive and proliferate within macrophages.

Fig 1

Biosynthetic pathway of coenzyme Q₈ in Escherichia coli. The octaprenyl tail is represented by R on carbon 3 of the different biosynthetic intermediates. The names of the enzymes catalyzing the reactions (each labeled with a lowercase letter) are provided. 4-HB, 4-hydroxybenzoic acid; DDMQ₈, C-1-demethyl-C-6-demethoxy-Q₈; Q₈, coenzyme Q₈.

Fig 2

The ubiJ mutant exhibited a growth defect under aerobic conditions. (A) Genetic organization of the ubiE, ubiJ, and ubiB loci in Salmonella Typhimurium. (B) Wild-type (filled circles), ΔubiE (filled squares), ΔubiJ (open circles), and ΔubiB (open squares) strains were grown overnight and then diluted at an OD₆₀₀ of 0.05 in LB medium at 37°C under aerobic conditions. Growth was monitored at 600 nm. (C) The wild type (filled circles) and the ΔubiJ strain transformed with pBAD24 (filled squares), with pubiJ (open squares), and with pubiB (open circles) were grown overnight and then diluted at an OD₆₀₀ of 0.05 in LB under aerobic conditions. Growth was monitored at 600 nm. (D) The strains previously described were grown overnight anaerobically and then diluted at an OD₆₀₀ of 0.05 in LB under anaerobic conditions. Growth was monitored at 600 nm. (E) Wild-type (black bars), ΔubiE (white bars), ΔubiJ (light gray bars), and ΔubiB (dark gray bars) strains were grown under anaerobiosis in minimal medium supplemented with glucose or glycerol as a carbon source and with nitrate, trimethylamine N-oxide (TMAO), or fumarate as an electron acceptor. OD₆₀₀ values were recorded after 16 h of incubation at 37°C. The experiments whose results are shown in panels B, C, and D were performed at least in triplicate. Identical patterns were obtained, and results of a representative experiment are shown. Error bars (E) show standard deviations.

Fig 3

UbiJ is required for Q₈ biosynthesis under aerobiosis. (A) Quantification of cellular Q₈ content in lipid extracts from WT, ΔubiE, ΔubiJ, and ΔubiB cells grown under aerobiosis (black bars) or anaerobiosis (white bars). Error bars show standard deviations. *, not detected. (B) Demethylmenaquinone (DMK₈, black bars) and menaquinone (MK₈, white bars) content in lipid extracts from WT, ΔubiE, ΔubiJ, and ΔubiB cells grown under aerobiosis (+O₂, left) or anaerobiosis (−O₂, right). Error bars show standard deviations. *, not detected. (C) The wild type (circles) and the ubiJ mutant (squares) were grown overnight under either aerobic (open symbols, +O₂) or anaerobic (filled symbols, −O₂) conditions. The cultures were then diluted at an OD₆₀₀ of 0.05 in LB under aerobic conditions. Growth was monitored at 600 nm, and the doubling time was calculated during the exponential phase. (D) The ubiJ mutant was grown overnight aerobically and diluted at an OD₆₀₀ of 0.05 in LB. Different amounts of coenzyme Q₁ were added at time zero, and growth was monitored at 600 nm. The experiments whose results are presented in panels C and D were performed in triplicate. Similar patterns were obtained, and results of a representative experiment are shown.

Fig 4

The 50 C-terminal amino acids of UbiJ are sufficient to sustain Q₈ biosynthesis and aerobic growth of the ubiJ mutant. (A) Schematic representation of the ubiJ plasmids used in this study. UbiJ N-ter represents the plasmid encoding the 120 amino acids at the N terminus, and UbiJ 63C, 50C, and 35C are the plasmids encoding the 63, 50, and 35 amino acids (respectively) at the C terminus. These constructs were cloned in a pBAD24 vector under the control of the pBAD promoter. (B) The wild type (filled circles), the ΔubiJ mutant (filled squares), and the ΔubiJ mutant transformed with pubiJ N-ter (filled triangles), pubiJ 63C (empty circles), pubiJ 50C (empty squares), and pubiJ 35C (empty triangles) were grown overnight and then diluted at an OD₆₀₀ of 0.05 in LB under aerobic conditions. Growth was monitored at 600 nm. This experiment was performed in triplicate, identical patterns were obtained, and results of a representative experiment are shown. (C) Quantification of cellular Q₈ content in lipid extracts from the strains previously described and grown in LB under aerobic conditions. Error bars show standard deviations. *, not detected.

Fig 5

Salmonella UbiJ activity relies on protein synthesis. (A) Schematic representation of the 5′ ends of the pubiJ 50C derivative plasmids. Frameshift mutations were introduced after the start codon of pubiJ 50C, yielding pubiJ 50C+1 (frameshift of one nucleotide) and pubiJ 50C+2 (frameshift of two nucleotides). (B) The wild type (filled circles), the ΔubiJ mutant (filled squares), and the ΔubiJ mutant transformed with pubiJ 50C (empty circles), pubiJ 50C+1 (empty squares), and pubiJ 50C+2 (empty triangles) were grown overnight and then diluted at an OD₆₀₀ of 0.05 in LB with arabinose under aerobic conditions. Growth was monitored at 600 nm. The experiment was performed at least in triplicate, identical patterns were obtained, and results of a representative experiment are shown. (C) The ΔubiJ strain transformed with pubiJ 50C (left), pubiJ 50C+1 (middle), and pubiJ 50C+2 (right) was grown on LB plates with arabinose for 16 h at 37°C. (D) HPLC separation and electrochemical detection (HPLC-ECD) of lipid extracts from 2 mg of the strains described for panel A and grown in LB with arabinose under aerobic conditions. The peaks corresponding to Q₈, demethylmenaquinone (DMK₈), and menaquinone (MK₈) and to the Q₁₀ standard are marked. (E) Western blot analysis of pubiJ (lane 1), pubiJ 50C (lane 2), pubiJ 50C+1 (lane 3), and pubiJ 50C+2 (lane 4) expressed from a pBAD24-derived plasmid in an MG1655 E. coli strain. UbiJ variants were separated using 15% SDS-PAGE, blotted onto PVDF membranes, and hybridized with a His₆ tag antibody.

Fig 6

UbiJ is necessary for Salmonella intracellular proliferation in macrophages. Opsonized bacteria were phagocytosed by RAW 264.7 cells. At 2 and 16 h postinfection, mouse macrophages were lysed for enumeration of intracellular bacteria (gentamicin protected), as determined by CFU counts. The values are proliferation indexes, calculated as the ratio of the number of intracellular bacteria at 16 h to that at 2 h postinfection. (A) Wild-type, ΔubiJ, ΔubiJ/pBAD24, and ΔubiJ/pubiJ strains were grown overnight in LB under aerobic conditions before opsonization. (B) Wild-type, ΔubiE, ΔubiJ, and ΔubiB strains were grown overnight in LB under aerobic (black bars) or anaerobic (white bars) conditions before opsonization. The inset shows the proliferation index ratios (WT/ΔubiJ mutant) calculated from the aerobic (+O₂) and the anaerobic (−O₂) inocula. Results are the means ± standard deviations from at least three independent experiments, each in triplicate.

Fig 7

The E. coli ubiJ gene is also involved in Q₈ biosynthesis and required for aerobic growth. (A) The MG1655 E. coli wild-type strain (filled circles) and ΔubiJ mutant (open circles) were grown overnight and then diluted at an OD₆₀₀ of 0.05 in LB medium at 37°C under aerobic conditions. Growth was monitored at 600 nm. (B) The E. coli ΔubiJ (top) and S. Typhimurium ΔubiJ (bottom) mutants transformed with pBAD24, E. coli ubiJ (pubiJ_E.c.), and S. Typhimurium ubiJ (pubiJ_S.t.) were grown on LB plates with ampicillin 16 h at 37°C. (C) The E. coli ΔubiJ (top) and S. Typhimurium ΔubiJ (bottom) mutants transformed with pTrc99A or pubiJ 50C_E.c. were grown on LB plates with ampicillin 16 h at 37°C. (D) Quantification of cellular Q₈ content in lipid extracts from WT and ΔubiJ cells grown under aerobic conditions. Error bars show standard deviations. *, not detected.