The MoxR ATPase RavA and its cofactor ViaA interact with the NADH:ubiquinone oxidoreductase I in Escherichia coli

Abstract

MoxR ATPases are widespread throughout bacteria and archaea. The experimental evidence to date suggests that these proteins have chaperone-like roles in facilitating the maturation of dedicated protein complexes that are functionally diverse. In Escherichia coli, the MoxR ATPase RavA and its putative cofactor ViaA are found to exist in early stationary-phase cells at 37 °C at low levels of about 350 and 90 molecules per cell, respectively. Both proteins are predominantly localized to the cytoplasm, but ViaA was also unexpectedly found to localize to the cell membrane. Whole genome microarrays and synthetic lethality studies both indicated that RavA-ViaA are genetically linked to Fe-S cluster assembly and specific respiratory pathways. Systematic analysis of mutant strains of ravA and viaA indicated that RavA-ViaA sensitizes cells to sublethal concentrations of aminoglycosides. Furthermore, this effect was dependent on RavA's ATPase activity, and on the presence of specific subunits of NADH:ubiquinone oxidoreductase I (Nuo Complex, or Complex I). Importantly, both RavA and ViaA were found to physically interact with specific Nuo subunits. We propose that RavA-ViaA facilitate the maturation of the Nuo complex.

Figure 1. Expression and localization of RavA and ViaA in E. coli MG1655.

(A) Expression of RavA and ViaA in WT MG1655 grown aerobically in LB at 37°C profiled over 24 hours by quantitative Western blotting. Both ClpP and LepB were used as loading controls. Different amounts of purified RavA, ViaA, and ClpP were used as indicated to provide the necessary quantification standards. Both OD₆₀₀ of the culture and the amount of RavA and ViaA expressed per cell at each time point are shown graphically in the lower panel. Dotted lines trace the expression levels of RavA and ViaA. (B) Total cell lysate and subcellular fractions of WT MG1655 cells grown aerobically to stationary phase in LB at 37°C were Western-blotted for the presence of RavA and ViaA. ClpP and LepB provide localization standards for cytoplasmic and membrane proteins, respectively. The amount of proteins loaded per lane for each blot is as indicated.

Figure 2. Schematic representation of genes showing significant changes in transcript levels as a result of the deletion or overexpression of RavA/ViaA.

Only genes that are functionally relevant to Fe-S clusters assembly and bacterial respiration are shown. Genes that belong to the same functional category are clustered together. In addition, genes that share a common operon are listed, from top to bottom, in the same order as they would appear in the 5′-to-3′ direction within the E. coli genome. Changes in gene transcription are represented as heatmaps generated using Matrix2png expressed as fold-changes with respect to either WT for ΔravA::cat (Set 1), or WT+p11 for WT+pRV (Set 2).

Figure 3. Growth profiles of cells in the presence of sublethal concentrations of aminoglycosides.

Growth profiles for MG1655 WT and the KO mutants ΔravA, ΔviaA and ΔravAviaA grown aerobically in LB at 37°C over 24 hours. Growth of cells was monitored using OD₆₀₀ readings at the designated time points. The cultures were supplemented as follows: (A) no antibiotics; (B) 4 µg/mL kanamycin; (C) 6 µg/mL streptomycin; (D) 0.5 µg/mL tetracycline; and (E) 1.2 µg/mL chloramphenicol. To confirm the phenotypes observed, ΔravA (F), ΔravAviaA (G) and WT cells (H) were complemented with the plasmids p11 (empty vector control), pR, pRV, pR_K52Q, or pR_K52QV. All cultures in the complementation experiments were supplemented with 4 µg/mL kanamycin for stress induction, and 100 µg/mL ampicillin for plasmid maintenance. Error bars were derived from three independent cultures for each strain and for each condition. Details on the E. coli strains and plasmids used are given in Table 1.

Figure 4. Effects of glutathione and 2,2′-dipyridyl on the growth profiles of cells in the presence of sublethal concentrations of kanamycin.

Growth profiles of MG1655 WT and the KO mutants ΔravA, ΔviaA and ΔravAviaA grown aerobically in LB at 37°C without (A, and D) or with kanamycin (B, and E) or streptomycin (C and F). Selected cultures were further supplemented with 750 µM of GSH (A–C) or 250 µM DP (D–F). Kanamycin was added at 4 µg/mL final concentration. Error bars were derived from three independent cultures for each strain and for each condition. In some instances, the error bars are smaller than the symbols used and cannot be seen. (G) DHR fluorescence measurements normalized by OD₆₀₀ for MG1655 WT+p11, ΔravAviaA+p11, ΔravAviaA+pRV and ΔravAviaA+pR_K52QV grown aerobically to late log phase in LB at 37°C supplemented with 4 µg/mL kanamycin in the presence or absence of 8 mM GSH or 250 µM DP. Error bars were derived from three independent cultures for each strain and for each condition. To highlight the statistical significance, the p-values for ΔravAviaA+p11 vs. WT+p11 (indicated with *) and ΔravAviaA+pRV vs. WT+p11 (indicated with **), in the presence of kanamycin, are given in the upper-right corner of the panel.

Figure 5. Growth profiles of selected single-gene knockouts.

Growth profiles of ΔnuoCD (A), ΔnuoF (B), ΔnuoI (C), Δndh (D), ΔcyoB (E) and ΔcyoD (F) transformed with p11, pRV or pR_K52QV plasmids as indicated. To account for the inherent differences in growth rates among the knockouts that are independent of the effects of RavA and ViaA, all growth data collected in the presence of kanamycin were normalized by the corresponding data collected in the absence of the antibiotic.

Figure 6. Physical interactions between RavA and ViaA with specific subunits of the Nuo complex under different growth conditions.

(A–C) WT, ΔravAviaA::cat, ΔviaA::cat DY330 strains having endogenously C-terminally SPA-tagged Nuo subunits were grown to stationary phase in LB under aerobic (A, C) or anaerobic (B, C) conditions. RavA and ViaA that co-purify with the SPA-tagged Nuo subunits were detected by Western blotting using α-RavA and α-ViaA polyclonal antibodies, respectively, whereas the SPA-tagged Nuo subunits were detected using α-FLAG monoclonal antibodies. The ΔravAviaA::cat strain having the SPA-tagged Nuo subunits is shown (A, B) as a control to confirm the identity of the RavA and ViaA bands detected. While the ΔviaA::cat strain having the SPA-tagged Nuo subunits is shown (C) to assess the role of ViaA in facilitating the binding of RavA to SPA-tagged NuoA and NuoF under aerobic conditions, and to SPA-tagged NuoC under anaerobic conditions. (D) X-ray structure of the NADH:ubiquinone oxidoreductase I from Thermus thermophilus (PDB ID: 3M9S), solved at 3.3 Å . The subunits are identified here using the nomenclature for the E. coli NADH:ubiquinone oxidoreductase I (i.e. the Nuo complex) , . The subunits Nqo15 and Nqo16 unique to T. thermophilus are omitted from the structure for clarity. Physical interactions of specific subunits with RavA and ViaA are indicated by the capital letters R and V, respectively. Red letters denote interactions that were identified in aerobically grown E. coli, and blue letters for the interactions in anaerobically grown cells. Asterisks (*) denote the subunits that are necessary for sensitizing the cell to sub-lethal concentrations of kanamycin upon overexpression of RavA and ViaA.