Perfectly Coupled Transcriptional Regulation of Fatty Acid Synthesis in Enterocci, Streptocci, and Lactococci

The fatty acid synthesis operon of Enterococcus faecalis and similar operons in related pathogenic bacteria are unusual in that fabT, the gene encoding the transcriptional repressor, is the first gene of the multigene fatty acid synthesis operon that it regulates (Figure 1). Therefore, the levels of FabT and the operon encoded enzymes are coordinated because they are encoded on the same mRNA. This is the case in E. faecalis (Bi et al. 2014; Hays et al. 2021; Zhu et al. 2019; Zou et al. 2022), the streptococci (e.g., Streptococcus pneumoniae) (Jerga and Rock 2009; Yao and Rock 2015, 2017) (Lambert et al. 2022) and lactococci (Eckhardt et al. 2013). Moreover, the FabT regulatory ligands, acyl-ACPs, which bind FabT to increase operator binding (Jerga and Rock 2009; Zhu et al. 2019; Zou et al. 2022; Lambert et al. 2023, 2022), are produced by genes regulated by FabT. The conservation of this operon arrangement in bacteria that occupy very different environmental niches argues that it plays an important role. This operon arrangement seems highly evolved in that the coding sequences of fabT and fabH, the gene encoding the first pathway enzyme, often minimally overlap or directly abut. Transcription of the FabT repressor gene and the genes responsible for synthesis of the regulatory ligands (acyl-ACPs) are coupled. Each of the regulatory partners controls transcription of the other partner. FabT decreases transcription of the genes required for acyl-ACP synthesis and acyl-ACPs bind FabT and decrease FabT transcription by strengthening autoregulation. The fact that the FabT repressor is encoded on the same mRNA that encodes most of the fatty acid synthesis genes makes these operons examples of the perfectly coupled system of Hlavacek and Savageau (1997). In their extensive mathematical modeling of bacterial regulatory systems, they defined a perfectly coupled system in which the transcription of the regulatory protein is coordinated with the transcription of the regulated enzymes (Hlavacek and Savageau 1997). In complete uncoupling, the other extreme, there is a constant supply of the regulatory protein. Hlavacek and Savageau predict that in negatively regulated systems, such as the FabT operons, perfect coupling provides optimal regulatory performance (Hlavacek and Savageau 1997). Another aspect of fatty acid synthesis conserved in these bacteria is that FabT repression is weak relative to other negatively regulated bacterial operons. Relative to the wild type strain, deletion of the fabT gene results in only a 2- to 5-fold increase in operon transcription without effect on growth (Eckhardt et al. 2013; Jerga and Rock 2009; Lambert et al. 2023; Yao and Rock 2013; Zou et al. 2022; Hays et al. 2021; Faustoferri et al. 2015). A reviewer queried whether weak repression is due to limiting production of FabT or to weak operator binding by the repressor. Electrophoretic gel shift analyses using purified FabT proteins indicate that operator binding is weak. E. faecalis FabT required a 20-fold molar excess of FabT to give complete mobility shift of operator DNA (Zou et al. 2022) whereas in S. pneumoniae and Lactococcus lactis 25-fold and 40-fold molar excesses of FabT were required for complete operator gel shifting (Eckhardt et al. 2013; Lu and Rock 2006). Another gel shift analysis of S. pneumoniae FabT operator binding used 60 nm FabT and 10 pm promoter DNA (Jerga and Rock 2009). Comparison to a known strong repressor, TrpR of the E. coli tryptophan operon, illustrates the weakness of FabT operator binding. In the presence of tryptophan, TrpR repressor quantitatively shifts equimolar TrpO operator DNA fragments, a 1:1 ratio (Beckmann et al. 1993). The above bacterial species and other members of the order Lactobacillales avidly incorporate exogeneous fatty acids into their membrane phospholipids (Eckhardt et al. 2013; Lai and Cronan 2003; Lambert et al. 2023; Lu and Rock 2006; Morgan-Kiss and Cronan 2008; Saito et al. 2018; Hays et al. 2021). Incorporation of de novo synthesized acyl chains into phospholipids is strongly outcompeted by exogeneous fatty acids due to increased FabT repression by acyl-ACPs derived from the acids (see below). In E. faecalis and S. pneumoniae, exogenous oleic acid almost completely displaces the native C18 unsaturated species and becomes the dominant acyl chain (> 85%) of the membrane phospholipids (Zou et al. 2022; Dong et al. 2025) (Parsons et al. 2011). However, the phospholipids of oleate-grown E. faecalis wild type cells contain saturated and C16 unsaturated acyl chains, indicating 10%–15% residual fatty acid synthesis. These low levels are due to FabT repression because deletion of FabT stimulates de novo fatty acid synthesis resulting in a large (9-fold) decrease in oleic acid incorporation into phospholipids (Zou et al. 2022). We propose that the residual fatty acid synthesis in wild type cells grown with exogenous fatty acids provides a buffer to ameliorate exhaustion of the fatty acid supply. The buffer would provide sufficient enzymes and acyl-ACPs to ensure maintenance of a functional membrane phospholipid bilayer until de novo fatty acid synthesis can ramp up. This proposal predicts that strong FabT repression would block synthesis of the buffer and inhibit the transition from exogenous fatty acid utilization to full de novo fatty acid synthesis. Hence, the moderate nature of FabT repression seems a compromise. Weak FabT repression gives partial saving of cellular resources but allows sufficient residual expression to form a buffer. We tested the buffer hypothesis by removing oleate from cultures of a wild type strain overproducing FabT (thereby mimicking a stronger repressor) and the wild type strain carrying the vector plasmid. Resumption of de novo fatty acid synthesis was assayed by [1-14C]acetate incorporation into phospholipid acyl chains. The FabT overproduction strain was unable to restart de novo synthesis whereas the control strain promptly restarted synthesis (Figure 2). A brief digression into fatty acid uptake in these bacteria is required because the pathway that converts exogenous acids into the acyl-ACPs is not a direct activation to acyl-CoA or acyl-ACP species as in proteobacteria. These bacteria utilize the more complex FakAB pathway discovered by Rock and coworkers (Lu et al. 2006; Parsons et al. 2014) (for reviews see Yao and Rock 2015; Yao and Rock 2017; Cronan 2014). FakAB, a two-component fatty acid kinase, activates incoming fatty acids by conversion to acyl-phosphates. A portion of the acyl-phosphate acyl chains is shuttled to ACP to form acyl-ACPs in a freely reversible reaction catalyzed by PlsX (both acyl-phosphates and acyl-ACPs are required for phospholipid synthesis) (Yao and Rock 2015, 2017; Cronan 2014) (Figure 1). The resulting acyl-ACPs bind FabT and decrease expression of both de novo fatty acid synthesis and FabT. Unusual in bacteria is that the enterococci and streptococci have a second ACP gene, acpB, that is not under FabT control (Zou et al. 2025, 2022; Lambert et al. 2023) (Figure 1). Acyl-AcpB species function in exogenous fatty acid uptake but are also FabT regulatory ligands and acyl chain donors in phospholipid synthesis (Zhu et al. 2019; Dong and Cronan 2022). The ACP encoded within the FabT operon, AcpA, is the acyl carrier of fatty acid synthesis (Dong and Cronan 2022) and acyl-AcpA species like acyl-AcpB species function as FabT regulatory ligands and phospholipid acyl chain donors (Figure 1) (Lambert et al. 2023; Zou et al. 2025, 2022; Dong and Cronan 2022). Note that PlsX via acyl-phosphate intermediates allows AcpA-bound acyl chains to be transferred to AcpB and vice versa. Acyl-AcpB species are significantly stronger FabT regulatory ligands than acyl-AcpA species in E. faecalis (Zou et al. 2022) and are essentially the sole FabT repression ligand in S. pyogenes (Lambert et al. 2023). Like E. faecalis AcpB, expression of the other required fatty acid incorporation proteins (PlsX, FakA plus several FakBs) is independent of FabT (Dong et al. 2025; Lambert et al. 2023; Zou et al. 2022, 2025) and thus exogenous fatty acid incorporation is a constitutive pathway in these bacteria. Does the regulation of fatty acid synthesis in E. faecalis and related bacteria differ from well-understood repression pathways such as those of E. coli tryptophan and methionine synthesis? Based on the analysis of Hlavacek and Savageau (Hlavacek and Savageau 1997), this is the case. The FabT fatty acid synthesis operons are perfectly coupled whereas the above E. coli pathways have direct coupling, a form of coupling intermediate between perfect coupling and complete uncoupling (Hlavacek and Savageau 1997). Hlavacek and Savageau cite only one example of perfect coupling, the E. coli bifunctional alanyl-tRNA synthetase, AlaS. AlaS catalyzes alanyl-tRNA synthesis and negatively represses its own transcription dependent on alanine levels (Putney and Schmmel 1981). In the AlaS case perfect coupling comes from the properties of the protein whereas perfect coupling of the above Lactobacillales fatty acid synthesis operons results from cotranscription of the repressor and metabolic genes. There seem few well-studied examples other than the Lactobacillale fabT operons in which the repressor gene is cotranscribed with the genes it regulates. A reviewer suggested the Fe-S cluster biogenesis operon, iscRSUA. However, although the iscR repressor gene is transcribed with the downstream genes, the small regulatory RNA, OxyS, binds the operon mRNA between the iscR and iscS coding sequences to decrease transcription of the downstream genes (Baussier et al. 2024). A second small regulatory RNA, FnrS, also uncouples iscR and iscS transcription by an unknown mechanism (Baussier et al. 2024). Hence, these small RNAs prevent perfect coupling of the iscRSUA operon despite cotranscription of the repressor and catalysis genes. Q. Z. and J. E. C. wrote the original draft and the subsequent and final versions. Q. Z. performed the investigation. All authors have agreed to be responsible for ensuring the accuracy of the presented data. The data that support the findings of this study are available from the corresponding author upon reasonable request.