Fig 1.
Simplistic representation of oxidative phosphorylation pathways in E. coli.
The aerobic (green frame) and micro-aerobic (red frame) respiratory chain are represented together with the tricarboxylic acid cycle and glycolysis. Iron sulfur clusters and known sources of ROS associated with these pathways are also shown.
Fig 2.
Cell cycle profile of iscU and fre mutants.
A) Cells were grown exponentially in AB medium supplemented with 0.2% glucose and 0.5% casamino acids and treated with rifampicin and cephalexin prior to flow cytometric analysis. Each panel represents a minimum of 30000 cells. The average ori/cell (O/C), ori/mass (O/M) relative to wild-type and mass doubling time (τ) are inserted in the histograms. B) ori/ter ratio determined by qPCR analysis. Shown is the mean ± s.d. (n = 3),* = p<0.05. C) IscUC63F and freΔ68 mutations do not alter the sensitivity to hydroxyurea. Sensitivity to hydroxyurea is tested by a diffusion assay on cells plated on LB agar medium. Cells are plated evenly on LB plates, 3μl of 500mM HU was introduced into 3 separated holes and plates incubated at 37°C for 16h.
Fig 3.
The respiro-fermentative metabolism of IscUC63F and freΔ68 mutant cells.
A) Expression profiles of genes involved in the respiratory chain as obtained by whole genome microarray. Expression intensities relative to wild-type are displayed by a two color gradient from blue (low) to yellow (high). Shown, the expression profile of operons encoding succinate dehydrogenase (SDH), 2-oxoglutarate dehydrogenase (OGDHC), NADH dehydrogenase I (NDH-I) and II (NDH-II), formate dehydrogenase (FDH O), cytochrome o oxidase (Cyt bo) and bd-1 (Cyt bd-1). B) Measurement of glucose consumption and acetate production during growth in glucose limited medium (AB minimal medium supplemented with 0.04% glucose). The plot shows the concentration of glucose (blue symbols), and acetate (red symbols) over optical density (no lactate, formate, citrate, ethanol and succinate were detected). Samples for final optical density for wild-type, IscUC63F and freΔ68 were measured at growth arrest (OD450 nm 0.88, 0.75, 0.64 respectively). C) Growth of wild-type, IscUC63F and freΔ68 cultures in AB minimal medium supplemented with 0.04% glucose.
Table 1.
Metabolic parameters of IscUC63F and freΔ68 mutant cells.
Fig 4.
Hda deficiency is suppressed by ArcA overexpression.
A) The hda::cat mutation was introduced into wild-type cells containing plasmid pNDM220 or pNDM-arcA under anaerobic conditions. Cells were restreaked on LB plates with or without the IPTG and incubated aerobically at 32°C for 24 hours. B) Cells were grown exponentially at 32°C in AB medium supplemented with 0.2% glucose and 0.5% casamino acids and treated with rifampicin and cephalexin prior to flow cytometric analysis. When indicated cells were grown with 1mM IPTG. The flow diagram of “hda/pNDM-arcA -IPTG” was obtained from cells grown in presence of 1mM IPTG then washed and regrown in absence of IPTG for 6 hours. Each panel represents a minimum of 30000 cells. The average ori/cell (O/C), ori/mass (O/M) relative to wt/pNDM220 and mass doubling time (τ) are inserted in the histograms. C) The cellular ori/ter ratio determined by qPCR analysis. Shown is the mean ± s.d (n = 3),* = p<0.05.
Fig 5.
Loss of ATP synthase restores growth of Hda deficient cells.
A) The hda::cat mutation was introduced into wild-type,ΔatpA and ΔatpB cells under anaerobic conditions, restreaked on LB plates and incubated under aerobic and anaerobic conditions. B) Wild-type, atpA, atpB, atpA hda and atpB hda mutants cells were grown exponentially in AB medium supplemented with 0.2% glucose and 0.5% casamino acids and treated with rifampicin and cephalexin prior to flow cytometric analysis. Each panel represents a minimum of 30000 cells. The average ori/cell (O/C), ori/mass (O/M) relative to wild-type and mass doubling time (τ) are inserted in the histograms. C) The cellular ori/ter ratio were determined by qPCR analysis. Shown is the mean ± s.d (n = 3),* = p<0.05.
Fig 6.
Cytochrome bd-1 function is required for freΔ68 survival in absence of Hda.
A) The hda::cat mutation was introduced into freΔ68 and freΔ68ΔcydB cells under anaerobic conditions, restreaked on LB plates under aerobic and anaerobic conditions. Plates were incubated at 32°C for 24 hours. B) The hda::cat mutation was introduced into wild-type cells containing plasmid pNDM220 or pNDM220-cyd under anaerobic conditions. Cells were restreaked on LB plates with or without the IPTG and incubated aerobically at 32°C for 24 hours. C) Cells were grown exponentially at 32°C in AB medium supplemented with 0.2% glucose and 0.5% casamino acids and treated with rifampicin and cephalexin prior to flow cytometric analysis. When indicated cells were grown with 1mM IPTG. The flow diagram of “hda/pNDM-cyd -IPTG” was obtained from cells grown in presence of 1mM IPTG that was washed and regrown in absence of IPTG for 16 hours. The average ori/cell (O/C), ori/mass (O/M) relative to wild-type pNDM220 and mass doubling time (τ) are inserted in the histograms. D) The cellular ori/ter ratios were determined by qPCR analysis. Shown is the mean ± s.d (n = 3),* = p<0.05.
Fig 7.
Respiratory pathways in cells carrying hda suppressor mutations.
Representation of the TCA cycle and the respiratory chain components of cells grown aerobically in presence of glucose. For each strain, the predominant respiratory path is framed in green while secondary or repressed paths are framed in red or shaded respectively.