Figure 1.
Identification of Mtb fatty acyl-CoA reductases.
A, Pathway of wax ester biosynthesis. Fatty acyl-CoA reductase (FCR) catalyzes the reduction of fatty acid to fatty alcohol which is condensed with fatty acyl-CoA by wax synthase (WS) to generate wax ester. R, alkyl chain. B, Amino acid sequence alignment of FCR1 (Rv3391) and FCR2 (Rv1543) with the acyl-CoA reductases ACR1 of Acinetobacter, MAQU_2507 and MAQU_2220 of Marinobacter.
Figure 2.
E. coli lysates express enzymatically active FCR1 and FCR2 proteins with negligible background activity.
A, Cell lysates of E. coli expressing the reductases were analyzed by SDS-PAGE followed by coomassie staining. Lane 1: Molecular weight (MW) markers, Lane 2: Untransformed E. coli (BL21) lysate, Lane 3: FCR1-expressing E. coli lysate, Lane 4: FCR2-expressing E. coli lysate. Arrows indicate the approximately 74 kDa FCR1 protein and 40 kDa FCR2 protein. B, Untransformed E. coli BL21 lysate does not show significant acyl-CoA reductase activity. Autoradiogram of TLC analysis of reaction products shown. BL21, lysate of untransformed E. coli BL21 host cells; Radiolabeled palmitoyl-CoA (C16∶0), stearoyl-CoA (C18∶0) or oleoyl-CoA (C18∶1) were provided as substrates. Arrows indicate positions of authentic lipid standards. CHO, fatty aldehydes; FA, fatty acids; FA-OH, fatty alcohols; PL, polar lipids.
Figure 3.
Acyl-CoA reductase activity of FCR1 and FCR2-expressing E. coli lysates.
Acyl-CoA reductase activity was measured with 70 µg protein in a final volume of 250 µl of 0.1 M phosphate buffer for 45 min (FCR1) and 30 min (FCR2). The dependence of enzymatic activity on pH (A, E), NADPH concentration (B, F), oleoyl-CoA concentration (C, G) and inhibition of alcohol and aldehyde formation by thiol-directed reagents (D, H) was determined to confirm the authenticity of the observed acyl-CoA reductase activity in lysates. Values are average ± SD from three independent experiments.
Figure 4.
Southern blot analysis of Mtb Δfcr1 and Δfcr2 mutants.
A, Schematic depiction shows the genomic locations of the primers and probes used in the construction and confirmation of fcr deletion mutants. The sequences of the primers are given in Table S1. B, Genomic DNA from WT Mtb and Δfcr1 mutant was digested with EcoRI and hybridized with the 3′-flank of the Δfcr1 construct as probe (P1). The WT fcr1 contains two EcoRI sites in the deleted part of the gene, the last one being only 48 bp upstream of the 3′ flank region of the construct. When this 3′ flank sequence was used as the probe, it hybridized to a 4930 bp fragment of the EcoRI digested genomic DNA (lane WT). When the hyg cassette replaced the native gene sequence, its EcoRI site was situated 1047 bp upstream of the 3′ flank sequence which resulted in a shift of the WT band to 5929 bp (lane fcr1). C, Genomic DNA from WT Mtb and Δfcr2 mutant was digested with PstI and hybridized with the 5′-flank of the Δfcr2 construct as the probe (P3). Wild-type genomic DNA digested with PstI and probed with the 5′ flank of the disruption construct yielded a hybridization fragment of 3292 bp (lane WT). In contrast PstI digested DNA from the mutant strain showed a smaller band of 1741 bp due to the presence of a PstI site in the 5′ region of the hyg cassette (lane fcr2).
Figure 5.
Mtb Δfcr mutants are impaired in WE biosynthesis under combined MS.
A, Diminished incorporation of 14C-oleate into WE in Mtb Δfcr mutants under combined MS treatment. Mtb cultures exposed to combined MS for 9 days were metabolically labeled with 14C-oleic acid for 4 h. Total lipids (equal proportions across samples) were resolved on silica-TLC and autoradiograms from a typical experiment is shown. WT, wild type Mtb; d-fcr1, fcr1-deletion mutant; C-fcr1, complemented fcr1 mutant; d-fcr2, fcr2-deletion mutant; C-fcr2, complemented fcr2-deletion mutant. Arrows indicate relative positions of authentic lipid standards. WE, wax esters; TG, triacylglycerols; FA, fatty acids; FA-OH, fatty alcohols, PL, polar lipids. B. Loss of fcr1 or fcr2 results in diminished wax ester biosynthesis under combined MS. Complementation restores WE biosynthesis in fcr1-deletion mutant only. Radioactivity from 14C-oleate incorporated into fatty alcohol and WE after 9 days under combined MS was determined and normalized as percent of total radioactivity in the respective lipid extract. Values are average ± SD.
Figure 6.
WE and fatty alcohol synthesis are impaired in Mtb Δfcr mutants under dormancy-inducing nitric oxide treatment.
A, Fatty alcohol and WE formation are diminished in lysates of Mtb fcr1 and fcr2 deletion mutants subjected to NO-stress. Complementation restores lost activity completely in d-fcr1 and partially in d-fcr2 lysates. Mtb cells were exposed to nitric oxide and lysates were assayed for acyl-CoA reductase activity using 14C-oleoyl-CoA as substrate as described in Methods. Autoradiogram of TLC plate from a typical experiment is shown. WT, wild-type; d-fcr1, fcr1-deletion mutant; C-fcr1, complemented fcr1-deletion mutant; d-fcr2, fcr2-deletion mutant; C-fcr2, complemented fcr2-deletion mutant. Arrows indicate relative positions of authentic lipid standards. WE, wax esters; TG, triacylglycerols; FA, fatty acids; FA-OH, fatty alcohols, PL, polar lipids. B, Radioactivity in WE and fatty alcohols was determined by scintillation counting and activities, normalized to total protein content in respective lysates, are shown. Values are average ± SD from duplicate experiments. C, NO-treated Mtb cells displayed severe decrease in non-radiolabeled WE accumulation. The non-radiolabeled total lipids, resolved on silica-TLC, were visualized by charring at 180°C and the TLC plate is shown. WE, wax esters; PL, polar lipids. D, the WE band in each lane of the TLC was quantitated by densitometry using an AlphaInnotech gel documentation system and the WE levels in the mutants, relative to WT (set at 100%), are represented. Values are average ± SD from duplicate experiments.
Figure 7.
Incorporation of 14C-oleate into WE is severely impaired in Mtb Δfcr2 mutant starved in PBS.
Mtb cells were grown in 7H9 medium to mid-log phase and were subjected to starvation in PBS for 72 h. A, Equal proportions of total lipid extracts were analyzed by TLC and a representative autoradiogram is shown. WT, wild-type; d-fcr1, fcr1-deletion mutant; d-fcr2, fcr2-deletion mutant. Arrows indicate relative positions of standard wax esters (WE), triacylglycerols (TAG) and polar lipids (PL). B, Radioactivity incorporated into WE was quantitated by scintillation counting and normalized as percent of radioactivity in the respective total lipid extract. Values are average ± SD from two experiments.
Figure 8.
Mtb Δfcr mutants display higher growth rates under in vitro dormancy.
A, Growth (optical density at 600 nm) of Mtb WT, Δfcr mutants and complemented mutants in liquid culture was measured at 0, 3, 6, 9 and 18 days under the MS treatment. B, Viable bacteria were enumerated as CFUs on 7H10-agar plates at 0, 3, 6, 9 and 18 day time points. WT, wild-type; d-fcr1, fcr1-deletion mutant; C-fcr1, complemented fcr1-deletion mutant; d-fcr2, fcr2-deletion mutant; C-fcr2, complemented fcr2-deletion mutant. Average ± standard deviation from three experiments shown (n = 3); p<0.05.
Figure 9.
Mtb Δfcr mutants show increased nutrient uptake under dormancy-inducing conditions in vitro.
Mtb WT and fcr-deficient mutants and complemented mutants subjected to MS conditions were incubated with [14C]glycerol for different time periods. Washed cell pellets were used to measure the radioactivity inside cells as described in Methods. At 0-day, Mtb cells (in log-phase) displayed very low uptake of radiolabeled glycerol (A). At 9-days (B) and 18-days (C) under MS, in contrast to WT, fcr-deficient mutants and complemented mutants displayed highly elevated levels of [14C]glycerol uptake. WT, wild-type; d-fcr1, fcr1-deletion mutant; C-fcr1, complemented fcr1-deletion mutant; d-fcr2, fcr2-deletion mutant; C-fcr2, complemented fcr2-deletion mutant. DPM, disintegrations per minute.
Table 1.
Mtb Δfcr mutants are severely impaired in developing phenotypic antibiotic tolerance under dormancy-inducing MS conditions.
Figure 10.
Mtb Δfcr mutants show increased metabolic activity and induction of genes involved in energy generation and transcription under dormancy-inducing conditions.
A, Mtb WT and fcr deficient cells subjected to combined MS condition for 0, 9 and 18 days were incubated with Alamar Blue dye for indicated periods of time and fluorescence was measured using a plate reader. B, Relative gene expression values (fold induction of day 0) of each gene for WT and Δfcr mutants at 9 and 18 days under MS. Real-time Taqman RT-PCR measurement was performed to measure relative abundance of transcripts. Relative quantitation method (ddCt) was used with the 7900 HT real-time system and analysis was done using SDS v2.3 software of Applied Biosystems Inc. Samples of starter culture (day 0) were used as calibrator and sigA was used as the endogenous control to normalize expression values.