The Enterococcus faecalis FabT Transcription Factor Regulates Fatty Acid Biosynthesis in Response to Exogeneous Fatty Acids

Abstract

The phospholipid acyl chains of Enterococcus faecalis can be derived either by de novo synthesis or by incorporation of exogenous fatty acids through the fatty acid kinase complex (Fak)-phosphate acyltransferase (PlsX) pathway. Exogenous fatty acids suppress fatty acid synthesis through the transcriptional repressor FabT, the loss of which eliminated regulation of de novo fatty acid biosynthesis and resulted in decreased incorporation of exogenous unsaturated fatty acids. Purified FabT bound to the promoters of several fatty acid synthesis genes that contain a specific palindromic sequence and binding was enhanced by acylated derivatives of acyl carrier protein B (acyl-AcpB). The loss of the PlsX pathway blocked FabT-dependent transcriptional repression in the presence of oleic acid. Transcriptional repression was partially retained in a E. faecalis ΔacpB strain which showed decreased fatty acid biosynthesis in the presence of exogenous unsaturated fatty acids. The FabT-dependent activity remaining in the ΔacpB strain indicates that acylated derivatives of AcpA were weak enhancers of FabT binding although AcpA is believed to primarily function in de novo fatty acid synthesis.

Keywords: acyl carrier protein; operons; phospholipid; repressor; transcription.

FIGURE 1

Exogenous fatty acids regulate E. faecalis de novo fatty acid biosynthesis by the FabT-transcription factor. (A) The E. faecalis phospholipid synthesis pathway. (B) De novo fatty acid biosynthesis in the wild type E. faecalis in the presence of exogenous fatty acids. (C) De novo fatty acid biosynthesis in the E. faecalis ΔfabT strain in the presence of exogenous fatty acids (100 μM for each fatty acid). (D) De novo fatty acid biosynthesis of E. faecalis wild-type and ΔfabT strains in the presence of 100 or 300 μM oleate. For panels (B,C), At the left of lane 1 a lane of cells grown with 10-hydroxyl stearic acid was deleted since this fatty acid is not found in E. faecalis. In panel (D), three lanes of ΔacpB (ZL318) cells between the WT and ΔfabT groups were deleted because of the missense mutation later detected in fabT gene of this strain (see below).

FIGURE 2

The E. faecalis ΔfabT strain is deficient in incorporation of exogenous fatty acids. (A) Incorporation of [¹⁴C]oleic acid (at left) or [¹⁴C]stearic acid (at right) into the cell membrane phospholipids of E. faecalis wild-type and ΔfabT strains. (B) The acyl chain composition of cell membrane phospholipids of E. faecalis wild-type and ΔfabT strains grown with oleic acid. (C) The acyl chain composition of cell membrane phospholipids of E. faecalis wild-type and ΔfabT strains grown with the cyclopropane fatty acid, methyleneoctadecanoic acid. (D) The acyl chain composition of cell membrane phospholipids of E. faecalis wild-type and ΔfabT strains grown with trans-vaccenic acid. (E) The acyl chain composition of cell membrane phospholipids of E. faecalis wild-type and ΔfabT strains grown with the C17 saturated acid, margaric acid. In panel (A), the lane of ΔacpB (ZL318) cells at the right of the ΔfabT lane was deleted because of the missense mutation detected in its fabT gene (see below).

FIGURE 3

Enterococcus faecalis FabT protein is functional in vitro. (A) Four E. faecalis fab-related DNA gene fragments having putative FabT-binding sites were tested. The sequences tested are indicated by the red stars whereas the fabT gene is referred by the blue arrow. Note that the first fours genes were previously shown to be cotranscribed (Zhu et al., 2019) and a recent report showed that this is the case for the entire eleven gene operon (Hays et al., 2021). (B) The nucleotide sequences of FabT-binding sites of the promoters of fab-related genes above. The underlined sequences indicate the palindromes. (C) EMSA analysis of E. faecalis FabT binding to the fabI/fabO promoter, fabK promoter, acpB promoter, or fabT promoter. (D) EMSA analysis of E. faecalis FabT binding to the E. faecalis fabT promoter (at left) or L. lactis fabT promoter (at right). In panel (D), both fabT promoters with 0.5 μM FabT lanes in the middle of E. faecalis group and L. lactis group were deleted since almost all E. faecalis fabT gene promoter fragments were trapped in the well and did not enter the gel.

FIGURE 4

Exogenous fatty acids enhance transcriptional repression by E. faecalis FabT. (A) The β-galactosidase reporter plasmid constructs. (B) The effects of oleic acid on expression of β-galactosidase driven by the E. faecalis fabT, fabI, or fabO promoter. (C) The effects of exogenous unsaturated fatty acids (at left) or saturated fatty acids (at right) on production of β-galactosidase driven by the E. faecalis fabT promoter. (D) The effects of oleic acid (at left) or palmitic acid (at right) on production of β-galactosidase by the fabT promoter in the E. faecalis ΔfabT strain. (E) The effects of an increased concentration of oleic acid on production of β-galactosidase driven by the in E. faecalis fabT promoter in the ΔfabT strain. (F) The effects of oleic acid on expression of lacZ gene from fabT promoter in E. faecalis ΔfabT-complemented strain. The fatty acid concentration in the cultures was 0.1 mM for panels (B–D,F). In Panel (E) the concentration was 0.3 mM.

FIGURE 5

Enterococcus faecalis acyl-AcpB is formed by the Fak-PlsX system in vitro. (A) Synthesis of palmitoyl-AcpB and oleoyl-AcpB by the E. faecalis Fak-PlsX system. (B) Synthesis of trans-vaccenoyl-AcpB and linoleoyl-AcpB by the E. faecalis Fak-PlsX system. (C) Relative synthesis of palmitoyl-AcpA and palmitoyl-AcpB by the E. faecalis Fak-PlsX system. (D) Relative of synthesis of oleoyl-AcpA and oleoyl-AcpB by the E. faecalis Fak-PlsX system.

FIGURE 6

Enterococcus faecalis acyl-AcpB enhances FabT transcriptional repression. (A) EMSA analysis of E. faecalis FabT binding to the fabT promoter in the presence of increasing concentrations of oleoyl-AcpB. (B) EMSA analysis of E. faecalis FabT binding to fabT promoter in the presence of increasing concentrations of palmitoyl-AcpB. (C) EMSA analysis of E. faecalis FabT binding to the fabT promoter or to a control unrelated DNA fragment. (D) The effects of oleic acid supplementation on expression of β-galactosidase from the fabT promoter in E. faecalis ΔplsX (top panel) and ΔplsX-complemented (lower panel) strains.

FIGURE 7

The regulatory function of E. faecalis AcpB for fatty acid biosynthesis relies on FabT. (A) De novo fatty acid biosynthesis of the E. faecalis wild-type strain overexpressing AcpB and ΔfabT strain overexpressing AcpB. Incorporation of [¹⁴C]oleic acid into the cell membrane phospholipids of the E. faecalis AcpB overexpression strain. (B) GC-MS analysis for proportion of oleic acid incorporation into the cell membrane phospholipids of the E. faecalis AcpB overexpression strain. For panels (A), the lanes of cells with stearic acid at the right of each group were deleted since they were unrelated to this study. (B), Two lanes for the ΔacpB:cat (ZL318) strain and its AcpB overexpression derivative at the right of ΔfabT group were deleted due to the FabT* mutation of this strain.

FIGURE 8

Enterococcus faecalis acyl-AcpA enhances FabT promoter binding in regulation of fatty acid synthesis. (A) E. faecalis ΔacpB strain de novo fatty acid synthesis in the presence of exogenous fatty acids. (B) The effects of oleic acid on expression of lacZ gene from fabT promoter in the E. faecalis ΔacpB strain. (C,D) EMSA analysis for comparison of E. faecalis FabT biding to the fabT promoter region in the presence of oleoyl-AcpA (at left) or oleoyl-AcpB (at right). The triangles denote increasing concentrations of acyl-ACP ligands These are (in μM): 0 for lanes 1, 2, 9, and 10; 0.25 for lanes 3 and 11; 0.5 for lanes 4 and 12; 1 for lanes 5 and 13; 2 for lanes 6 and 14; and 4 for lanes 7, 8, 15, and 16. In panels (C,D), the binding of FabT to the fabT promoters in the presence of either of the two ligands were processed at the same time and the concentration of DNA promoters used for each situation was the same. But more smears for each lane could be detected in the oleoyl-AcpA lanes, which could lead to lower intensity of free DNA at the bottom. This might also be caused by the different buffers used for preparing oleoyl-AcpA and oleoyl-AcpB, respectively. The oleoyl-AcpB was synthesized and eluted in K-MES buffer (pH 6.1) whereas oleoyl-AcpA was synthesized and eluted in Tris-HCl buffer (pH 7.5) since AcpA precipitates in the acidic buffer used in all other EMSA analyses.

FIGURE 9

The FabT*mutation in the acpB:cat strain used previously results in a phenotype that differs from that of the newly constructed ΔacpB strain. (A) Comparison between the two ΔacpB strains of exogenous oleic acid supplementation on β-galactosidase production from the fabT promoter. The pfabT denotes a plasmid encoding wild type FabT. (B) Incorporation of [¹⁴C] acetate into cell membrane phospholipids of the two ΔacpB strains with additions as given. (C) The differing acyl chain compositions of the cell membrane phospholipids of the two ΔacpB strains grown in the presence of oleic acid Comparison of bacterial de novo fatty acid biosynthesis. (D) The differing acyl chain compositions of the cell membrane phospholipids of the two ΔacpB strains grown in the presence of margaric acid.