Article Figures & Data
Figures
- FIG 1
Genetic regulation of monounsaturated fatty acid biosynthesis genes fabA and fabB in E. coli. In the absence of exogenous fatty acids, FadR binds to a site upstream of the fabA and fabB promoters and acts as an activator of transcription. When present, exogenous fatty acids are transported across the outer membrane by FadL and converted to acyl-CoA by FadD. Acyl-CoA binding to FadR causes a conformational shift that abolishes the DNA binding capabilities of FadR. In both scenarios, FabR binds to a site downstream of FadR and has been shown to bind in the presence or absence of acyl-CoA and/or acyl-ACP. Loss of FadR activation of transcription presumably allows FabR to act as a better repressor of fabA and -B expression.
- FIG 2
Reporter gene expression in the pfaA::lacZY strain in response to a variety of culture parameters known to modulate EPA composition. (A) Various temperature and hydrostatic pressure conditions. (B) Cells cultured at 15°C with different Tween compounds added at 0.05%. (C) Effect of 0.05% oleic (18:1) or lauric (12:0) free fatty acid supplementation on pfaA::lacZY reporter activity. Fatty acid stocks were prepared as potassium salts in 80% ethanol (EtOH). For panels A to C, results from at least six independent experiments are shown as means with error bars representing standard deviation (ns, P > 0.05; ***, P < 0.005; ****, P < 0.0001). (D) Effect of 0.05% Tween 80 supplementation on various fatty acid biosynthetic gene transcript abundances in SS9R as determined by qRT-PCR. Cells grown without supplementation represent the calibrator condition. Error bars represent the standard deviations based on at least three independent biological replicates with duplicate qPCRs (*, P < 0.05; **, P < 0.005).
- FIG 3
Influence of FadR and FabR on expression of the pfa operon. (A) LacZ activities of strains carrying ΔfabR and/or ΔfadR mutations in the presence or absence of 0.05% Tween 80 (18:1). Results of at least six independent experiments are shown as means with error bars representing standard deviation (**, P < 0.05; ***, P < 0.005; ****, P < 0.0001). (B) Relative transcript abundances of pfaA, pfaD, fabA, and fabB in the ΔfabR ΔfadR mutant grown in the presence or absence of Tween 80 (18:1). Cells grown without supplement represent the calibrator condition. Error bars represent the standard deviations based on at least three independent biological replicates with duplicate qPCRs (NS, P > 0.05; ***, P < 0.005).
- FIG 4
The pfaF genetic locus and regulatory phenotypes associated with its disruption. (A) Genetic organization of PfaF locus. The arrowhead indicates the relative position of the mini-Tn5 insertion site in the pfaA::lacZY regulatory mutant. (B) LacZ activity of the pfaF::Tn5 mutant and the complemented strains carrying pMA62 (pFL122 pfaF) or vector (pFL122) only with or without 0.05% Tween 80 (18:1) supplementation (ns, P > 0.05; ****, P < 0.0001). (C) qRT-PCR analysis of pfaA, pfaD, fabA, and fabB transcript abundances in the SS9R pfaF mutant relative to SS9R. Error bars represent the standard deviations based on at least three independent biological replicates with duplicate qPCRs (ns, P > 0.05; **, P < 0.005; ***, P < 0.001).
- FIG 5
Characterization of PfaF binding to the pfaA promoter. (A) Electrophoretic mobility shift assay demonstrating PfaF binding to the pfaA promoter in a concentration-dependent manner. (B) DNase I footprinting analysis of PfaF binding to the pfaA promoter. Purified PfaF was added at the indicated concentrations and subjected to DNase I digestion as described in Materials and Methods. The chromatograms and sequencing traces shown correspond to the coding strand, and the box indicates the region protected from digestion by PfaF. (C) DNA sequence of the pfaA promoter probe used in mobility shift and footprinting assays. The promoter elements (−35 and −10 sites) and transcriptional start site (arrow) were previously determined (25). The region protected by PfaF is indicated by the underlined font. (D) Binding of an FAM-labeled probe by PfaF is reversed by a molar excess of an unlabeled annealed oligonucleotide containing the PfaF binding site (WT). Binding is not affected by a molar excess of a random scrambled oligonucleotide (SCR). (E) Addition of oleoyl-CoA, but not lauroyl-CoA, inhibits the binding of PfaF to the probe. Representative gels at a single concentration (20 μM) of each acyl-CoA with various amounts of PfaF are shown.
- FIG 6
Proposed model of PfaF-mediated regulation of the pfa operon. In the absence of fatty acids, PfaF binds upstream of the pfaA promoter, acting as a positive regulator. In the presence of exogenous unsaturated fatty acids, PfaF binds to acyl-CoA and releases from the promoter region, leading to downregulation of the pfa operon.
Tables
- TABLE 1
Strains and plasmids used in this study
- TABLE 2
Fatty acid compositions of SS9R and various pfaF mutant strains at 15°C
↵a Data represent the averages from at least three independent cultures ± standard deviations.
Supplemental material
- Supplemental file 1 -
Fig. S1 to S4 and Tables S1 and S2
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- Supplemental file 1 -
