Bacteria of the Corynebacterineae, a suborder of the Actinobacteria, comprise
All these bacteria share a common feature, a distinctive multilaminate cell wall composed of peptidoglycan, complex polysaccharides, and both covalently linked lipids and free lipids/lipoglycans (Fig. 1). Among them, mycolic acids are the hallmark of these species. These long chain α-branched, β-hydroxylated fatty acids are covalently linked to the arabinogalactan polysaccharide layer. This mycolic acid layer is complemented by a glycolipid layer to form an outer “mycomembrane” analogous to the outer membrane of Gram-negative bacteria. [1, 2]. The outer leaflet of the mycomembrane is composed of a variety of lipids including trehalose dimycolates (TDMs), glycopeptidolipids (GPLs), phthiocerol dimycocerosates (PDIMs), sulfolipids, phenolic glycolipids (PGLs), and lipooligosaccharides. Some of these lipids are widely distributed while others are restricted to particular species. For example, TDMs and their structural equivalents are found in both mycobacteria and corynebacteria, while PDIMs and PGLs are restricted to a subset of mycobacteria. The structure and hydrophobic properties of the cell wall make it a potent permeability barrier that is responsible for intrinsic resistance of mycobacteria to an array of host microbiocidal processes, many antibiotics and sterilization conditions [3, 4]. Many of the cell wall components of pathogenic mycobacterial species are essential for pathogenesis and
Studies on mycobacteria and corynebacteria provide a unique opportunity to illustrate the complexity and diversity of lipid metabolic pathways in bacteria. They have a significantly higher lipid content than other bacteria with cell wall lipids comprising ~40% of the dry cell mass.
The plasma membrane provides the platform for lipid metabolism. While some lipid metabolic reactions take place in the cytoplasm or cell wall, the plasma membrane is the pivotal site for the metabolism of lipids. At the same time, this membrane must perform many other functions associated with energy production, nutrient uptake, protein export, and various sensing/signaling reactions. Studies on how these metabolic and cellular processes might be organized within bacterial plasma membranes are in their infancy. Understanding the homeostasis of the plasma membrane is particularly important in Corynebacterineae organisms because this structure must support the high biosynthetic demands of sustaining such a lipid-rich cell wall. In this chapter, we focus our discussion on processes of lipid metabolism that are critical for the biogenesis and maintenance of the plasma membrane, and illustrate the recent progress on our understanding of plasma membrane biogenesis in mycobacteria and corynebacteria.
2. Functions of plasma membrane lipids in mycobacteria and corynebacteria
In this section we will describe the functions of plasma membrane lipids. First, we will describe the functions of major structural phospholipids. We will then describe quantitatively minor lipids, which have important metabolic/physiological functions. Lastly, we will discuss the functions of neutral lipids because their biosynthesis is closely linked to phospholipid metabolism and neutral lipid storage is a critical part of plasma membrane homeostasis.
2.1. Structural lipids
Major structural components of the mycobacterial plasma membrane are phospholipids such as cardiolipin (CL), phosphatidylethanolamine (PE), phosphatidylinositol (PI), and glycosylated PIs (
CL is widely found in both prokaryotes and eukaryotes. It forms aggregates within the membrane bilayer. Nonyl acridine orange (NAO) is a fluorescent dye which is proposed to bind the hydrophobic surface created by the CL cluster , allowing microscopic visualization of CL domains. Indeed, using NAO, CLs were found to be enriched in septa and poles of actively dividing
PE is another major class of glycerophospholipids in mycobacteria. Although PE is generally found in all organisms, it is particularly abundant in bacterial plasma membranes . Mycobacteria are no exception , but corynebacteria apparently lack the capacity to synthesize PE . Indeed, PE biosynthetic enzymes, such as PS synthetase and PS decarboxylase, appear to be absent in corynebacterial genomes.
PIs are an important class of phospholipids, and are known to be further modified by extensive glycosylation. The resultant lipoglycans, termed PIMs, LM, and LAM, are essential structural components of mycobacterial and corynebacterial cell walls. Furthermore, in pathogenic species, they have been suggested to perform additional roles in the modulation of host immune responses in favor of the pathogen through myriad effects on macrophages including cytokine production, inhibition of phagosome maturation and apoptosis [31-34]. PIMs are oligo-mannosylated PIs carrying up to 6 mannose residues while LM/LAM carry much longer mannose polymers with arabinan modifications. It remains controversial if these glycolipids are embedded in the plasma membrane or exported to the outer membrane. A recent study suggests that LM/LAM appear to be anchored to both the plasma membrane and outer membrane . In
It is notable that some unusual phospholipids have been identified in corynebacteria. APG is an acylated form of PG which is widespread in corynebacteria [37-40], and is a major phospholipid species in
2.2. Functional lipids
There are some examples of lipids that appear to play no structural roles in the plasma membrane. They often exist in low quantities but play important functional roles. Among these, polyprenol-phospho-sugars function as sugar donors. Two well-studied examples are polyprenol phosphomannose (PPM) and decaprenol phosphoarabinose (DPA). These molecules are the donors of mannose and arabinose, respectively, and their biosynthesis will be discussed in a later section.
PI 3-phosphate, recently identified in both
Carotenoids are photo-protective pigments and serve to scavenge free radicals or harvest light . Several mycobacterial species are known to produce carotenoids with the notable exception of
2.3. Lipid storage for energy and carbon
Neutral lipids are an important reservoir of stored energy and carbon, and their metabolism is closely linked to plasma membrane phospholipid metabolism. Unlike many other bacteria which use polyhydroxyalkanoates as a lipid storage material , Actinobacteria use triacylglycerides (TAGs) as a major form of lipid storage, and the presence of TAGs has been reported in
Cholesterol has recently been suggested to be an alternative form of lipid storage in mycobacteria. Neither mycobacteria nor corynebacteria have the capacity to synthesize cholesterol. However, cholesterol is taken up by
Catabolism of cholesterol, amino acids and odd-chain-length/methyl branched fatty acids produces propionyl-coenzyme A (CoA). Propionate accumulation has been shown to be toxic in various organisms [59-61], and
3. Structure and metabolism of plasma membrane lipids in mycobacteria and corynebacteria
In this section, we will describe the structure and metabolism of various lipids found in the plasma membrane of mycobacteria and corynebacteria in more detail. Lipids are categorized into the following four classes based on their key structural features.
3.1. Fatty acids
3.1.1. De novo synthesis by FAS-I
Surprisingly, members of the Corynebacterineae use a eukaryote-like FAS-I system for
3.1.2. Elongation by FAS-II
The FAS-II system is commonly found in bacteria and plants and, unlike FAS-I, is composed of a series of separate enzymes, each performing one step in the pathway. FAS-II elongates medium chain fatty acids derived from FAS-I using malonyl-CoA, producing C18-C30 fatty acids . FAS-II has been extensively studied in
β–ketoacyl-ACP reductase MabA reduces the β–keto group with concomitant oxidation of NADPH
β-hydroxyacyl-ACP dehydratase dehydrates the β-hydroxyl to enoyl-ACP
enoyl-ACP reductase InhA, a target of the first-line anti-tuberculosis drug isoniazid (INH) , reduces enoyl-ACP to acyl-ACP with concomitant oxidation of NADPH
β-ketoacyl-ACP synthase KasA/B elongates acyl-ACP by 2 carbon units, forming β-ketoacyl-ACP, which can feed back into step 1.
In this way, the hydrocarbon chain increases by 2 carbons each cycle. Further elongation and processing of the products of FAS-II produces the precursors of the long meromycolate chains that are condensed with the α-branches derived from FAS-I by the large polyketide synthase Pks13 . Reduction of the β–keto group by CmrA forms the mature C60-C90 mycolic acid .
Glycerolipids include both nonpolar lipids and polar phospholipids. Their biosynthesis is overlapping and 1,2-diacyl-
3.2.1. Biosynthesis of PA
The first step of PA biosynthesis is mediated by glycerol phosphate acyltransferase (GPAT) transferring an acyl chain from acyl-CoA to glycerol-3-phosphate, forming acyl-glycerol 3-phosphate. In general, this reaction produces 1-acyl-
3.2.2. TAG Biosynthesis
3.2.3. Utilization of TAG
Under starvation conditions where stored TAG needs to be mobilized for energy production, TAG is catabolized by lipases. In 1977, TAG lipase was purified from stationary phase
3.2.4. Lipid droplet formation
In eukaryotes, lipid droplets form in between the two leaflets of the endoplasmic reticulum membrane . In bacteria, a distinct mechanism of lipid body formation has been proposed. For example, in rhodococci, TAG is formed in the cytoplasmic surface of the plasma membrane. Small lipid droplets are then fused to each other, coated by a monolayer of phospholipids, and released from the surface of the plasma membrane into the cytoplasm as mature lipid droplets . Although no endogenous proteins have been found to associate with lipid droplets in rhodococci or mycobacteria, heterologous expression of known lipid droplet-associated proteins resulted in correct targeting of these proteins to lipid droplets in both
In both eukaryotic and prokaryotic cells, PA is activated by CTP to form CDP-DAG, and this reaction is mediated by CDP-DAG synthase . The synthesis of CDP-DAG commits the pathway to phospholipid biosynthesis, and CDP-DAG is a common precursor for the biosynthesis of all glycerophospholipids in mycobacteria and corynebacteria. The activity of CDP-DAG synthetase is associated with plasma membrane in
CL is composed of four acyl chains, three glycerols and two phosphates, and is structured in a 1,3-diphosphatidylglycerol configuration . It is a common phospholipid in bacteria, and is one of the abundant phospholipids in mycobacteria and corynebacteria. To initiate CL synthesis, PG phosphate synthase first produces PG phosphate (PGP) using CDP-DAG and glycerol 3-phosphate as substrates. An
The precise structure of PE was recently reported as 1-
PI is a major phospholipid in both mycobacteria and corynebacteria and forms the anchor for the PIMs, which are substrates for heavy mannosylation to form LMs and additional arabinosylation to produce LAMs. PI is formed by the PI synthase PgsA (Rv2612c) from CDP-DAG and
All Corynebacterineae synthesize PIMs that are important components of the cell envelope. Polar PIM species can also serve as membrane anchors for LM and LAM. Many of the steps of PIM/LM/LAM biosynthesis have now been elucidated . Extensive genetic and biochemical studies have demonstrated that the synthesis of PIMs occurs linearly in mycobacteria with PI as the starting substrate (reviewed in ) (Fig. 4A). Early steps of the pathway occur on the cytoplasmic face of the plasma membrane. A PIM biosynthetic membrane, enriched in the early steps, has been purified by sucrose gradient fractionation as a membrane subdomain termed PMf, which is distinct from the bulk plasma membrane . Mannosyltransferases performing the early steps utilize the water-soluble mannose donor, GDP-Man, which can be produced from exogenously acquired mannose or via
The next enzyme in the pathway, PimC, has been identified in
AcPIM4 species can be further mannosylated to form more polar PIMs in reactions thought to take place on the periplasmic side of the cytoplasmic membrane. These reactions are performed by glycosyltransferases that require a lipid sugar donor in the form of PPM, since these reactions are amphomycin-sensitive [36, 125-128]. In mycobacteria, AcPIM4 is proposed to be a branch point for synthesis of polar PIM end products and LM/LAM. PimE (Rv1159) has been shown to elongate AcPIM4 with one or more α1-2 linked mannoses to form AcPIM6 . This polytopic membrane protein has sequence similarities with eukaryotic PIG-M mannosyltransferases and localizes to a cell wall-associated plasma membrane subdomain in
Surprisingly, studies in
A subpopulation of PIMs (AcPIM4 in mycobacteria [128, 129, 132] and AcPIM2 in corynebacteria ) can be extended with chains of α1-6 linked mannose to form LM that is further modified with a number of single α1-2 mannose side chains [134-136]. MptB is a PPM-dependent mannosyltransferase involved in extending AcPIM2 to form the proximal α1-6 mannan backbone of LM in
While the general PI
3.4. Prenol lipids
Polyprenol phosphate (Pol-P) is a key carrier lipid in synthesis of the core structures of the mycobacterial cell wall, including peptidoglycan and arabinogalactan. Unlike most bacteria, mycobacteria contain multiple types of Pol-P. For example,
PPM, a β-d-mannosyl-1-monophosphoryldecaprenol, is utilized by periplasmic mannosyltransferases for synthesis of polar PIM species and LMs . C35/C50-P-Man
DPA is the only known donor of arabinose (Ara) for mycobacterial cell wall synthesis, contributing Ara
Carotenoids are isoprenoid pigments widely distributed in biology and mostly based on C40-polyene. Synthesis of these pigments has been poorly studied in mycobacteria but they have proven useful for taxonomic and identification purposes. Mycobacterial pigments are generally yellow or orange and most have been confirmed as carotenoids . While carotenoid genetics has been best studied in plants, the key enzymes of the pathway have been identified in bacteria, including mycobacteria [172-174]. There are two classes of carotenoids in bacteria, carotenes and xanthophylls, the latter of which contain oxygen. Both classes are composed of eight isoprenoid units with a long central chain of double bonded carbons. A consensus pathway for carotenoid biosynthesis in bacteria has been elucidated with orthologues of key enzymes identified in several species of mycobacteria . As described above for prenol lipids, the carotenoid pathway begins with isopentenyl diphosphate and dimethylallyl diphosphate derived from the MEP pathway . Head-to-tail condensation of these terpenes produces geranylgeranyl pyrophosphate (GGPP) due to the activity of GGPP synthase (CrtE). Condensation of two GGPP molecules  is followed by desaturation to phytoene by phytoene synthase (CrtB). Phytoene desaturase (CrtI) converts phytoene to lycoprene followed by cyclization to β-carotene by lycoprene cyclase (CrtY) .
4. Concluding remarks
Lipid metabolism in mycobacteria and corynebacteria is a highly complex network of catabolic and anabolic reactions. While the metabolic pathways and many of the enzymes involved have been actively elucidated over the past decade, substantial efforts are still needed to draw a comprehensive map of lipid metabolism in these organisms. In particular, our understanding of regulatory mechanisms of lipid metabolism is currently at an early stage. In addition, there are very few studies describing the interactions between multiple metabolic pathways of lipid biosynthesis. One promising approach for the comprehensive understanding of lipid metabolism is lipidomics, which is the study of lipid biosynthetic and catabolic pathways at a global level . In the past, metabolic pathways have generally been examined in isolation without consideration of how different pathways might interact with, and influence, one another. Since the plasma membrane is a shared platform for most lipid biosynthetic pathways, and some donors are shared between different pathways (see above), it seems unlikely that the various pathways are truly independent. Recent advances in mass spectrometry (