**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**

120 Lipid Metabolism

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 *in vitro* growth, hampering efforts to characterize the function of individual proteins in their assembly. In contrast, some non-pathogenic species such as *C. glutamicum* can tolerate the loss of major cell wall components, making them useful model systems for delineating

**Figure 1. Mycobacterial plasma membrane and cell wall with flow of key metabolic pathways**. Some of the metabolites are exported to the mycomembrane. SLD, small lipid droplet; LD, lipid droplet; FA-

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. *M. tuberculosis* produces a diversity of lipids unparalleled in bacteria, from simple fatty acids to highly complex long chain structures such as mycolic acids. It has devoted a significant proportion of its coding capacity to lipid metabolism and produces about 250 enzymes dedicated to fatty acid metabolism, which is around five times the number produced by *Escherichia coli* [5]. Lipid biosynthesis places a significant metabolic burden on the organism but is ultimately advantageous, allowing *M. tuberculosis* to survive and replicate in the inhospitable environment of host macrophages. While capable of *de novo* synthesis, these bacteria also scavenge and degrade host cell membrane lipids to acetyl-CoA, via broad families of β-oxidation and other catabolic enzymes, for incorporation into

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

processes involved in the assembly of core cell wall structures.

CoA, fatty acyl-CoA. See text for other abbreviations used in the figure.

their own metabolic pathways and to fuel cellular processes.

Major structural components of the mycobacterial plasma membrane are phospholipids such as cardiolipin (CL), phosphatidylethanolamine (PE), phosphatidylinositol (PI), and glycosylated PIs (*i.e.* phosphatidylinositol mannosides (PIM), lipomannans (LM) and lipoarabinomannans (LAM), see below). The ratio of these phospholipids may vary depending on the species and growth conditions [6-8]. For example, one study indicated that CL, PE, and PI/PIMs represent about 37, 32, and 28%, respectively, of the total phospholipids in the plasma membrane in *M. smegmatis* [9], while another reported the ratio in *Mycobacterium phlei* to be about 50, 10, and 40% [10]. Phosphatidylglycerol (PG), which is abundant in other bacteria, is a relatively minor species in mycobacteria. Deletion of the PI biosynthetic gene has been shown to be lethal in *M. smegmatis* [9], indicating that PI or glycosylated PIs are essential for mycobacterial viability. In *M. tuberculosis*, putative PI synthetase (*Rv2612c*) and PGP synthetase (*Rv2746c*, involved in CL synthesis) genes are predicted to be essential [11], while the PS synthetase gene (*Rv0436c*, involved in PE synthesis) is not [12]. In corynebacteria, major species of phospholipids are PI, PG, CL, and acylphosphatidylglycerol (APG) [13], and PE appears to be absent.

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 [14], allowing microscopic visualization of CL domains. Indeed, using NAO, CLs were found to be enriched in septa and poles of actively dividing *M. tuberculosis* and *M. smegmatis* cells [15, 16]. CL has a nonbilayer structure [17, 18], and carries a small partially immobilized head group that is more exposed to the aqueous environment than those of other glycerophospholipids [19]. Although the physiological function of CL is unclear, its physical properties may indicate that it provides a platform for membrane-protein interactions. Indeed, some mycobacterial enzymes require CL for activity [20-22], although the molecular basis for these observations has not been clarified. Recent fractionation studies in *C. glutamicum* revealed that CL (as well as other phospholipids) is enriched in the plasma membrane [23, 24]. However, a large proportion of CL is also found to be associated with the outer membrane [24], suggesting that some of these phospholipids are exported to the outer membrane in corynebacteria. Similarly, CL is released from *M. bovis* bacillus Calmette-Guerin residing in host phagosomes, and converted to lyso-CL by a host phospholipase A2 [25]. It has been suggested that lyso-CL may influence host immune responses during infection.

Metabolism of Plasma Membrane Lipids in Mycobacteria and Corynebacteria 123

PIM6 species (see below) is viable, but shows severe plasma membrane abnormalities [36], suggesting that higher order PIMs may be involved in the maintenance of plasma

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 *Corynebacterium amycolatum*. Another interesting phospholipid from *C. amycolatum* is acyl-phosphatidylinositol (API), which was identified by electrospray ionization mass spectroscopy [41]. *C. amycolatum* lacks a mycolic acid-based outer membrane, and does not appear to have a fracture plane other than the plasma membrane [42]. Therefore, APG and API are likely to be components of the plasma membrane, and are suggested to play structural roles. Very little is known about their biosynthesis, and acyltransferases responsible for their synthesis remain to be identified for both lipid species.

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

PI 3-phosphate, recently identified in both *M. smegmatis* and *C. glutamicum* [43], may prove to be another interesting example of a functional lipid. It accumulates only transiently upon stimulation by high concentrations of salt, and behaves as if it is involved in a signaling cascade. However, whether PI 3-phosphate represents a mediator of stress responses remains to be addressed. More recently, lysinylated PG was identified as a minor phospholipid species in *M. tuberculosis* [44]. The synthesis of lysinylated PG is mediated by LysX and a *lysX* deletion mutant showed altered phospholipid metabolism and membrane integrity [16, 44], suggesting a regulatory role of lysinylated PG in plasma membrane

Carotenoids are photo-protective pigments and serve to scavenge free radicals or harvest light [45]. Several mycobacterial species are known to produce carotenoids with the notable exception of *M. tuberculosis*, despite the presence of a carotenoid oxidase in the human pathogen [46]. These hydrophobic pigments are thought to be present in the plasma membrane but whether they play structural roles in addition to a photo-protective role

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 [47], Actinobacteria use

membrane integrity.

**2.2. Functional lipids** 

be discussed in a later section.

homeostasis.

remains to be elucidated.

**2.3. Lipid storage for energy and carbon** 

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 [26]. Mycobacteria are no exception [20], but corynebacteria apparently lack the capacity to synthesize PE [27]. Indeed, PE biosynthetic enzymes, such as PS synthetase and PS decarboxylase, appear to be absent in corynebacterial genomes. *Corynebacterium aquaticum* has been reported to possess PE [28], but this species was later reclassified as *Leifsonia aquatica* [29], which belongs to the suborder Micrococcineae of the order Actinomycetales. The functions of PE remain elusive at the molecular level, but it appears to play important roles as a component of the plasma membrane. For example, TBsmr, a small multidrug resistance family protein from *M. tuberculosis*, shows enhanced catalytic activities when PE is supplemented in a reconstituted liposome [30].

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 [35]. In *C. glutamicum*, the outer membrane and plasma membrane were fractionated on sucrose gradients upon cell lysis, and the analysis of these membrane sub-fractions demonstrated that PIMs, LM and LAM are all enriched in the plasma membrane fraction [23]. Another recent study also suggested that PI/PIMs are major components of the plasma membrane of *C. glutamicum* [24]. In the latter study, however, substantial amounts of PI/PIMs were detected in the outer membrane as well. The functional significance of these subcellular localizations, as well as the physiological roles of LM/LAM in each of these locations, remain important questions. The structural importance of PIMs remains unclear as well. For example, a *pimE*-deletion mutant that cannot produce mature PIM6 species (see below) is viable, but shows severe plasma membrane abnormalities [36], suggesting that higher order PIMs may be involved in the maintenance of plasma membrane integrity.

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 *Corynebacterium amycolatum*. Another interesting phospholipid from *C. amycolatum* is acyl-phosphatidylinositol (API), which was identified by electrospray ionization mass spectroscopy [41]. *C. amycolatum* lacks a mycolic acid-based outer membrane, and does not appear to have a fracture plane other than the plasma membrane [42]. Therefore, APG and API are likely to be components of the plasma membrane, and are suggested to play structural roles. Very little is known about their biosynthesis, and acyltransferases responsible for their synthesis remain to be identified for both lipid species.

### **2.2. Functional lipids**

122 Lipid Metabolism

bilayer structure [17, 18], and carries a small partially immobilized head group that is more exposed to the aqueous environment than those of other glycerophospholipids [19]. Although the physiological function of CL is unclear, its physical properties may indicate that it provides a platform for membrane-protein interactions. Indeed, some mycobacterial enzymes require CL for activity [20-22], although the molecular basis for these observations has not been clarified. Recent fractionation studies in *C. glutamicum* revealed that CL (as well as other phospholipids) is enriched in the plasma membrane [23, 24]. However, a large proportion of CL is also found to be associated with the outer membrane [24], suggesting that some of these phospholipids are exported to the outer membrane in corynebacteria. Similarly, CL is released from *M. bovis* bacillus Calmette-Guerin residing in host phagosomes, and converted to lyso-CL by a host phospholipase A2 [25]. It has been

suggested that lyso-CL may influence host immune responses during infection.

is supplemented in a reconstituted liposome [30].

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 [26]. Mycobacteria are no exception [20], but corynebacteria apparently lack the capacity to synthesize PE [27]. Indeed, PE biosynthetic enzymes, such as PS synthetase and PS decarboxylase, appear to be absent in corynebacterial genomes. *Corynebacterium aquaticum* has been reported to possess PE [28], but this species was later reclassified as *Leifsonia aquatica* [29], which belongs to the suborder Micrococcineae of the order Actinomycetales. The functions of PE remain elusive at the molecular level, but it appears to play important roles as a component of the plasma membrane. For example, TBsmr, a small multidrug resistance family protein from *M. tuberculosis*, shows enhanced catalytic activities when PE

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 [35]. In *C. glutamicum*, the outer membrane and plasma membrane were fractionated on sucrose gradients upon cell lysis, and the analysis of these membrane sub-fractions demonstrated that PIMs, LM and LAM are all enriched in the plasma membrane fraction [23]. Another recent study also suggested that PI/PIMs are major components of the plasma membrane of *C. glutamicum* [24]. In the latter study, however, substantial amounts of PI/PIMs were detected in the outer membrane as well. The functional significance of these subcellular localizations, as well as the physiological roles of LM/LAM in each of these locations, remain important questions. The structural importance of PIMs remains unclear as well. For example, a *pimE*-deletion mutant that cannot produce mature 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 *M. smegmatis* and *C. glutamicum* [43], may prove to be another interesting example of a functional lipid. It accumulates only transiently upon stimulation by high concentrations of salt, and behaves as if it is involved in a signaling cascade. However, whether PI 3-phosphate represents a mediator of stress responses remains to be addressed. More recently, lysinylated PG was identified as a minor phospholipid species in *M. tuberculosis* [44]. The synthesis of lysinylated PG is mediated by LysX and a *lysX* deletion mutant showed altered phospholipid metabolism and membrane integrity [16, 44], suggesting a regulatory role of lysinylated PG in plasma membrane homeostasis.

Carotenoids are photo-protective pigments and serve to scavenge free radicals or harvest light [45]. Several mycobacterial species are known to produce carotenoids with the notable exception of *M. tuberculosis*, despite the presence of a carotenoid oxidase in the human pathogen [46]. These hydrophobic pigments are thought to be present in the plasma membrane but whether they play structural roles in addition to a photo-protective role remains to be elucidated.

#### **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 [47], Actinobacteria use

#### 124 Lipid Metabolism

triacylglycerides (TAGs) as a major form of lipid storage, and the presence of TAGs has been reported in *Mycobacterium*, *Streptomyces*, *Rhodococcus* and *Nocardia* [48-52]. Interestingly, corynebacteria seem to lack the capacity to synthesize TAG, indicating that some lineages of Actinobacteria have eliminated this capacity at some point in their evolution. Recent evidence suggests that *M. tuberculosis* accumulates TAG-based lipid droplets while residing in macrophages using fatty acids released from host TAGs, and this process is critical for acquiring a dormancy phenotype [53]. Nevertheless, a mutant defective in accumulating TAG remained viable under *in vitro* dormancy-inducing conditions [54]. These somewhat contradictory observations suggest that our understanding of TAG metabolism in mycobacteria is far from complete. As we illustrate later, there appear to be several redundant genes involved in the final step of TAG synthesis, suggesting that it is an important regulatory step of lipid metabolism in these bacteria.

Metabolism of Plasma Membrane Lipids in Mycobacteria and Corynebacteria 125

metabolism is essential for intracellular survival of the pathogen since it forms the precursors of key membrane components such as plasma membrane phospholipids and outer membrane glycolipids. In particular, mycolic acids, which are very long chain α-alkyl β-hydroxy fatty acids, form the hydrophobic, protective mycomembrane described earlier. *M. tuberculosis* encodes two distinct enzyme systems for biosynthesis of fatty acids, designated FAS (fatty acid synthase) I and II (Fig. 2). Studies on fatty acid synthesis date back to the 1970s when *M. smegmatis* was shown to contain both type I fatty acid synthetase (FAS-I), involving a large multifunctional polypeptide, and type II fatty acid synthetase (FAS-II), consisting of a series of distinct enzymes [64]. The key elongation unit is malonyl-CoA, which is produced by acetyl-CoA carboxylase (ACCase) and the *M. tuberculosis* genome encodes several such enzymes (AccA1-3 and AccD1-6). The resultant malonyl-CoA

**Figure 2. Fatty acid biosynthesis pathways in mycobacteria.** Point of inhibition by the front-line tuberculosis drug isoniazid is indicated. Product profile of FAS-I is bimodal, and C16-C18-CoA and C24- C26-CoA are produced. Dashed lines indicate that some of the fatty acid products are further utilized for

Surprisingly, members of the Corynebacterineae use a eukaryote-like FAS-I system for *de novo* fatty acid synthesis. The single, essential [11], 9.2kb *fas* gene encodes a 326 kDa protein containing all seven domains necessary to perform the iterative series of reactions: acyl transferase, enoyl reductase, β-hydroxyacyl dehydratase, malonyl transferase, acyl carrier protein, β-ketoacyl reductase, and β-ketoacyl synthase [65, 66]. This very large protein elongates acetyl groups by 2-carbon (acetate) units using acetyl-CoA and malonyl-CoA. Early rounds of elongation yield C16 to C18-CoA products that are used for synthesis of membrane phospholipids or to feed into the FAS-II system. More extensive elongation yields C24-C26 products that ultimately form the α-branch of mycolic acids. Unlike *M. tuberculosis*, *C. glutamicum* encodes two *fas* genes (*fasA* and *fasB*) with FasA taking the

is incorporated into fatty acids by the two FAS systems.

mycolic acid production.

*3.1.1. De novo synthesis by FAS-I* 

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 *M. tuberculosis* cells residing in the host, and components of the *mce4* operon have been shown to be involved in cholesterol import [55]. Cholesterol catabolism is critical in the chronic phase of animal infection, and a fully functional catabolic pathway is encoded by the *M. tuberculosis* genome [56]. Furthermore, cholesterol appears to accumulate in the mycobacterial cell envelope, and this might represent a potential form of lipid storage for *M. tuberculosis* during animal infection [57, 58]. Although the authors of this study suggested that cholesterol accumulates in the outer membrane, it remains possible that the plasma membrane is the true site of accumulation. Therefore, in addition to acting as a lipid storage molecule, cholesterol may play roles in plasma membrane structure and function, and these possibilities await further exploration.

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 *M. tuberculosis* has multiple pathways to metabolize propionyl-CoA [62]. Metabolized propionyl-CoA is in part incorporated into TAG [63], and it has been suggested that TAG functions as a sink for reducing equivalents in addition to being a source of carbon and energy.
