A Rainbow Coalition of Lipid Transcriptional Regulators

Summary

Lipids are essential structural constituents of bacterial cell membranes and walls, and their biosynthetic pathways are stringently regulated at both biochemical and genetic levels. The recent surge of new information about transcriptional regulation of bacterial lipid metabolism is highlighted by two studies in this issue of Molecular Microbiology by Hugo Gramajo's research group, who add two transcription factors to the diverse repertoire of lipid biosynthesis regulators. FasR is a Streptomyces coelicolor transcriptional activator of genes in fatty acid synthesis, which supplies substrates for membrane phospholipid and triglyceride storage droplets. MabR is a regulator in Mycobacterium tuberculosis that functions as a repressor of essential genes in the cell wall mycolic acid biosynthetic pathway. MabR also affects the expression of fas, which encodes the multifunctional fatty acid synthase that supports phospholipid and triglyceride synthesis. Despite belonging to the same protein family, the distinct ligand binding domains of FasR and MabR suggest different ligands may regulate their DNA binding. The characterization of FasR/MabR exemplifies the structural and functional diversity of the rainbow coalition of lipid transcriptional regulators that reflects the diverse life styles of bacteria.

Membrane lipid biogenesis is a vital facet of bacterial physiology that is tightly regulated at both biochemical and genetic level. Bacterial survival depends on membrane lipid homeostasis and on the ability to adjust lipid composition to acclimatize the bacterial cell to optimize growth in diverse environments (Zhang and Rock, 2008). The most energetically expensive membrane lipid components to produce are the fatty acids. These phospholipid acyl chains also determine the viscosity of the membrane, which in turn influences many crucial membrane-associated functions. Thus, bacteria have evolved sophisticated mechanisms to finely control the expression of the genes responsible for the metabolism of fatty acids. These regulatory mechanisms adjust the level and activity of biosynthetic enzymes to match the demand for new membrane. In this issue of Molecular Microbiology, two papers from Gramajo laboratory describe structurally-related, but functionally diverse, transcriptional regulators that control the expression of lipid metabolic genes in the actinomycetes (Arabolaza et al., 2010; Salzman et al., 2010). The transcriptional regulator in Streptomyces coelicolor, called FasR, functions as an activator that controls genes involved in fatty acid synthesis destined for phospholipids and triglycerides. The second factor, called MabR, functions as a repressor in Mycobacterium tuberculosis to control the expression of genes involved in cell wall mycolic acid biosynthesis. These manuscripts herald the arrival of a new family of transcription factors to the growing diversity of regulators that control essential genes responsible for the biosynthesis of fatty acids, and their relatives, the mycolic acids.

S. coelicolor FasR functions as an activator of the fabD-fabH-acpP-fabF gene cluster. Genetic disruption of the fasR gene, located just upstream of fadD, results in a significant reduction in the relative mRNA abundance of all genes in the cluster and an impaired growth rate. Chromatin immunoprecipitation showed FasR binds to the fabD promoter and the FasR DNA binding site was located and analyzed by gel shift experiments. Notable phenotypes of the fasR knockout strain are large reductions in the rate of lipid synthesis from acetate, and the amount of total lipid per cell. Like other transcriptional regulators, FasR senses the inhibition of the pathway by antibiotics and compensates by upregulating gene expression. Thus, FasR is an activator of several genes encoding core components of fatty acid synthesis.

The second paper describes a mycobacterial transcription factor, MabR, which belongs to the same protein family as FasR. MabR is a repressor of the fabD-acpM-kasA-kasB gene cluster, which encodes essential components for mycolic acid synthesis. Overexpression of MabR represses transcription and inhibits mycolic acid synthesis. MabR is essential for mycobacterial survival, and knock-down of mabR expression using antisense RNA increases transcription of the fabD gene cluster. The reason for this curious phenomenon is not readily apparent because increased synthesis of KasA and KasB are compatible with growth (Larsen et al., 2002). Increased expression of acpM may be deleterious to growth similar to the growth inhibition caused by elevated expression of acpP in Esherichia coli (Keating et al., 1995), or perhaps there are other essential MabR targets that are responsible. Unlike other bacterial acpP genes that have dedicated, strong promoters, acpM is located in the middle of an operon. Change of MabR expression also alters the expression of the multifunctional fatty acid synthase (fas) indicating a link between the two types of fatty acid synthases in these organisms. The activities of key enzymes in mycolic acid synthesis, such as FabD, FabH, MabA, KasA, and KasB, are modulated by phosphorylation by serine-threonine protein kinases (Molle and Kremer, 2010). Therefore, mycolic acid synthesis is regulated at both transcriptional and post-translational levels to maintain cell wall homeostasis in mycobacteria.

FasR and MabR share high degree of sequence similarity in the carboxy-terminal DNA binding domain, especially within the helix-turn-helix DNA-binding motif. This is consistent with the conserved inverted repeat sequences to which both regulators bind. The operator sequences of FasR and MabR are located at the same distance upstream of the proposed overlapping transcription/translation start sites of the respective fabD genes. Despite the similar location of the FasR and MabR binding sites, FabR is an activator whereas MabR is a repressor. It will be intriguing to determine how they perform their distinct functions by binding at the same location. FasR and MabR homologs are restricted to the actinomycetes. Phylogenetic analysis shows that they segregate into two groups, mycolic acid producers (MabR) and non-producers (FasR). The amino-terminal ligand binding domains of these regulators are more dissimilar than their DNA binding domains. This property separates them into distinct evolutionary subgroups, suggesting that they interact with different ligands.

FasR and MabR add a new protein family to the expanding coalition of bacterial lipid transcriptional activators and repressors (Table 1). FadR was first recognized as a repressor of the fatty acid degradation regulon in Escherichia coli (Cronan, Jr. and Subrahmanyam, 1998), but activates the fabA and fabB genes (Henry and Cronan, Jr., 1992; Campbell and Cronan, Jr., 2001). Detailed structural studies show that FadR constitutively binds DNA, but the FadR-acyl-CoA compolex does not (Zhang and Rock, 2009). This mechanism coordinates fatty acid degradation and synthesis in response to the presence of extracellular fatty acids. FapR is a regulator of the type II fatty acid biosynthesis (fab) and acyltransferase (pls) genes in Bacillus subtilis (Schujman et al., 2003). FapR is released from its DNA binding sites by malonyl-CoA (Schujman et al., 2006). FapR represents an interesting feed-forward control mechanism that places acetyl-CoA carboxylase as a key step that determines the ligand concentration. FabT is a repressor of the fab/acc genes of Streptococcus pneumoniae that belongs to the MarR family of transcription factors (Lu and Rock, 2006). The affinity of FabT for its DNA binding site is enhanced by long-chain acyl-ACPs (Jerga and Rock, 2009), and represents a feedback regulatory mechanism. FabT binds all acyl-ACPs of different chain lengths with approximately equal affinity, but only long-chain acyl-ACPs (>14 carbons) induce the conformational change that allows DNA binding. DesT and FabR are members of the TetR superfamily that respond to fatty acid structure. DesT is a repressor that primarily controls the expression of the desCB operon, which encodes the components of an oxidative Δ9 acyl-CoA desaturase system (Zhu et al., 2006), and secondarily controls the expression of the fabAB operon in Pseudomonas aeruginosa (Subramanian et al., 2010). This factor is a compositional sensor that binds the entire pool of acyl-CoAs with approximately equal affinity (Zhang et al., 2007). Saturated acyl-CoAs stabilize a conformation that cannot bind DNA, while unsaturated acyl-CoAs stabilize the conformation that binds DNA. The structures of two acyl-CoA•DesT complexes illustrate how transcriptional regulators can mechanistically sense the fatty acid unsaturation based on the distortion of the molten hydrophobic core of the ligand binding domain (Miller et al., 2010). FabR is a repressor that controls the expression of the fabB and fabA genes in E. coli (Zhang et al., 2002). Similar to DesT, FabR distinguishes saturated from unsaturated ligands, including both acyl-ACPs and acyl-CoAs, to adjust the expression of fabA and fabB, which govern the composition of acyl chains produced by the de novo pathway (Zhu et al., 2009).

Table 1.

Transcription factors for bacterial fatty acid metabolism.

Transcription Factor	Representative Organisms	Activation1	Repression1	Ligand
FadR	Escherichia coli	fabA, fabB, iclR	fadL, fadD, fadB-A, fadE, fadF, fadI-J	Acyl-CoA
FabR	Escherichia coli	None	fabA, fabB	Acyl-ACP Acyl-CoA
FapR	Bacillus subtilis	None	fabH1-F, fabH2, yhdO, fapR, fabI, fabD, fabG, plsX	Malonyl-CoA
FabT	Streptococcus pneumoniae	None	fabT-H-acpP, fabK-D-G-F-accB-fabZ-accC-D-A	Acyl-ACP
DesT	Pseudomonas aeruginosa	None	desC-B, fabA-B	Acyl-CoA
FasR	Streptomyces coelicolor	fabD-fabH-acpP-fabF	Unknown	Unknown
MabR	Mycobacterium tuberculosis	Unknown	fabD-acpM-kasA-kasB	Unknown

¹Only established direct transcriptional targets are listed. Operons are indicated by connecting the individual genes with dashes.

A key aspect of FasR regulation is the significant decrease in the total amount of cellular lipid in the fasR knockout strain. Previous studies indicated that biochemical regulation of the pathway is the primary mechanism for matching the amount of fatty acids produced by the de novo biosynthetic pathway to the demand for new membrane. E. coli strains lacking FabR (Zhang et al., 2002), B. subtilis strains lacking FapR (Schujman et al., 2003) and S. pneumoniae strains lacking FabT (Lu and Rock, 2006) do not have increased amounts of lipid even though the fab genes are significantly upregulated. This may not be the case in S. coelicolor. Fatty acids produced in S. coelicolor are destined for not only membrane phospholipid, but also triglycerides, a storage lipid that can be mobilized for β-oxidation when carbon becomes depleted in the environment (Arabolaza et al., 2008). Thus, the fatty acids produced in excess of the requirement for membrane phospholipid formation are diverted to a storage droplet. These findings suggest that FasR may be nutritionally regulated to shunt excess carbon into triglycerides when nutrients are abundant. M. tuberculosis also creates a triglyceride energy storage reservoir (Waltermann et al., 2005). MabR does not directly regulate fas expression, but the reduced fas mRNA levels in a mabR overexpressing strain illustrate the coordination of phospholipid, triglyceride and mycolic acid synthesis in mycobacteria.

The discovery of FasR and MabR raises several important questions concerning how these regulators are integrated into bacterial physiology. A key advance will be to identify the ligand(s) that control the DNA binding properties of these factors. The differences in the ligand binding domains, coupled with the different metabolic pathways that are regulated, suggest that distinct regulatory ligands may be involved. It also seems likely that these transcriptional regulators may control a broader set of genes than identified in these first publications. The genes specifically investigated by Gramajo and coworkers are located adjacent to the transcriptional regulators in the chromosomes. However, the coordinate regulation of fas with fabD in M. tuberculosis indicates that the expression of additional lipid and mycolic acid biosynthetic genes is also controlled by these factors either directly or indirectly. In the case of FasR, other candidate genes are those involved in type II fatty acid synthesis along with the acyltransferases involved in phospholipid and triglyceride synthesis. Likewise, it would be interesting to know which of these genes are also impacted by the activity of MabR. In addition, a global analysis of the MabR regulatory activity may provide the answer to why this transcriptional regulator is essential for mycobacterial survival.

So, we are left without a consensus mechanism that bacteria use to transcriptionally regulate lipid biosynthetic genes. Rather, the diverse life styles of bacteria are reflected by the rainbow coalition of transcriptional regulators and ligands used to control this important and energy intensive aspect of intermediary metabolism (Table 1). Proteobacteria produce saturated and unsaturated fatty acids and genetically regulate the pathway using compositional sensors (FabR and DesT). The ligand for the S. pneumoniae FabT repressor of the fab/acc genes is long-chain acyl-ACP, whereas the FapR repressor of B. subtilis controls the fab/pls genes based on the concentration of malonyl-CoA. Two new colors can be added to the “rainbow” with the characterization of FasR and MabR from the actinomycetes.