Advance Publication by J-STAGE Genes & Genetic Systems
Received for publication: April 19, 2017 Accepted for publication: July 4, 2017 Published online: October 6, 2017
Matsuoka, 2017
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Genes & Genetic Systems
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Biological functions of glucolipids in Bacillus subtilis
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Satoshi Matsuoka*
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Department of Biochemistry and Molecular Biology, Graduate School of Science
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and Engineering, Saitama University, 255 Shimo-Okubo, Sakura, Saitama,
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Saitama 338-8570, Japan
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*Corresponding author:
Satoshi Matsuoka
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Tel: +81 48-858-9294
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Fax: +81 48-858-3384 E-mail: [email protected]
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Running head: Biological functions of glucolipids in B. subtilis
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Key words: Bacillus subtilis, glucolipid, ugtP, ECF sigma factor, cell wall
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maintenance
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1
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Abstract
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Glyceroglycolipids are very important in Gram-positive bacteria and cyano-
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bacteria. In Bacillus subtilis, a model organism for the Gram-positive bacteria,
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the ugtP mutant, which lacks glyceroglucolipids, shows abnormal morphology.
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Lack of glucolipids has many consequences: abnormal localization of the cyto-
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skeletal protein MreB and activation of some extracytoplasmic function (ECF)
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sigma factors ( M,
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the expression of monoglucosyldiacylglycerol (MGlcDG) by 1,2-diacylglycerol
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3-glucosyltransferase from Acholeplasma laidlawii (alMGS) almost completely
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suppresses the ugtP disruptant phenotype. Activation of ECF sigmas in the ugtP
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mutant is decreased by alMGS expression, and is suppressed to low levels by
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MgSO4
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1,2-diacylglycerol-3-glucose (1-2)-glucosyltransferase producing diglucosyldi-
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acylglycerol (DGlcDG)) are simultaneously expressed,
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pressed to wild type level. These observations suggest that MGlcDG molecules
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are required for maintenance of B. subtilis cell shape and regulation of ECF
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sigmas, and that DGlcDG regulates X activity. The activation of ECF sigmas is
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not accompanied by proteolysis of anti-. Thus, glyceroglucolipids may have the
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specific role of helping membrane proteins function by acting in the manner of
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chaperones.
addition.
V
and X) in the log phase are two examples. Conversely,
When
alMGS
2
and
alDGS
(A.
X
laidlawii
activation is re-
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Introduction
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All living organisms have membranes which compartment cells from the
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outer environment and from each other. A lipid bilayer structure for these mem-
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branes has been proposed by Singer and Nicolson in 1972. In this model,
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membrane proteins are floating in a “sea of lipids” and the proteins can move
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freely in the membrane. Lipid molecules are uniformly distributed in the mem-
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brane. This model is almost correct; however recent studies have demonstrated
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the existence of specific lipid-protein domains, which come about by lipid mole-
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cules and membrane proteins forming domain structures in the membrane, so
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called lipid rafts (Raghunathan et al., 1990). In bacteria, the existence of lipid
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rafts is not certain although some researchers have reported evidence pointing
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to lipid rafts as detergent resistant membrane fractions (Donovan and
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Bramkamp, 2009). On the other hand, localizations of lipid molecules have been
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reported. The presence of CL or anionic phospholipids in specific regions is re-
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quired for many biological functions such as the localization of ClsA (Kusaka et
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al., 2016) and the Min system (Ishikawa et al., in press) in B. subtilis. In cyano-
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bacteria galactolipids play an important role in photosynthesis as galactolipids
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are included in the thylakoid membranes which are the sites of oxygenic photo-
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synthesis (Mizusawa and Wada, 2012); the crystal structures of the photosystem
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I (PSI) and PS II complexes from the thermophilic cyanobacterium Synecho-
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coccus elongatus reveal that these PSI and PSII contain one molecule of mo-
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nogalactosyldiacylglycerol (Jordan et al., 2001), and six monogalactosyldiacyl-
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glycerol and five digalactosyldiacylglycerol molecules, respectively (Umena et al.,
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2011). It is difficult to uncover the biological functions of membrane lipid mole-
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cules because these are products resulting from enzyme reactions, not directly 3
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encoded in genes, and there are only few probes which detect specific lipid
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molecules. To reveal the biological functions of membrane lipids, genes which
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encode lipid synthesis enzymes have been inactivated, and observations of the
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resulting phenotypes have been performed in an attempt to gain clues. Shibuya,
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Dowhan and their colleagues have revealed gene regulation and physiological
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roles of membrane lipids in E. coli using molecular genetics (Shibuya, 1992;
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Mileykovskaya and Dowhan, 2005). However, the knowledge based on E. coli
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studies cannot be naively applied to B. subtilis.
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In this outline, lipid synthesis pathways, cell surface structures, and biolog-
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ical functions of membrane lipids in B. subtilis are reviewed. Then it focusses on
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the biological functions of glucolipids.
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Synthesis of membrane lipids in B. subtilis
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The lipid biosynthesis pathways of phospholipids are shown in Fig. 1. The
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pathway in B. subtilis is almost the same as that in E. coli. The main phospho-
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lipid molecules of E. coli are phosphatidylethanolamine (PE), phosphatidylglyc-
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erol (PG), and cardiolipin (CL). They make up about 70%, 25%, and 5%, re-
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spectively, of phospholipids in the cell membrane. In B. subtilis, PE, PG, and CL
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constitute 30%, 40%, and 5% of the cell membrane lipids, respectively. In addi-
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tion to these phospholipids, diacylglycerol (DG), glucolipids, and lysylphospha-
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tidylglycerol (lysylPG) are contained at a fraction of about 5%, and 10% each in
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B. subtilis cell membranes (Matsuoka et al., 2011b; Hashimoto et al., 2013). The
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B. subtilis cell contains three kinds of glucolipids, monoglucosyldiacylglycerol
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(MGlcDG), diglucosyldiacylglycerol (DGlcDG), and triglucosyldiacylglycerol
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(TGlcDG), which amount to 1.2%, 9.8% and 0.3% of total membrane lipids 4
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(Kawai et al., 2006). These are synthesized processively by UgtP, which trans-
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fers glucose to diacylglycerol from UDP-glucose (Jorasch et al., 1998). In these
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pathways, phosphatidic acid (PA) is synthesized from glycerol-3-phosphate
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(G3P) but the first reactions to lysophosphatidic acid (LPA) synthesis are differ-
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ent between B. subtilis and E. coli (Hara et al., 2008). The gene cdsA, which
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encodes CDP-diglycerol synthase, is an essential gene in both organisms.
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Conditional knockout mutants lacking either PE or acidic phospholipids (PG and
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CL) can be constructed in E. coli. By contrast, pssA, which encodes the key
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enzyme to synthesize PE, can easily be deactivated without obvious defective
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phenotype, and pgsA, which encodes the key enzyme to synthesize PG, is in-
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dispensable in B. subtilis (Salzberg and Helmann, 2008; Hashimoto et al., 2009).
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DgkB, a diacylglycerol kinase of B. subtilis is also essential (Kobayashi et al.,
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2003). In the absence of LtaS homologue genes, dgkB is dispensable because
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accumulation of DG, which is toxic for cells, does not occur (Matsuoka et al.,
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2011b).
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Cell surface and membrane lipids of B. subtilis
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The cell walls of B. subtilis, a model microorganism representative of the
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Gram-positive bacteria, consist of thick multiple layers of peptidoglycan, which is
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the major physical and chemical defense against deadly agents. The envelope
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of Gram-positive bacteria contains further components: polymers of glycerol-
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phosphate, wall teichoic acids (WTA) covalently linked with peptidoglycan and
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lipoteichoic acids (LTA) linked with DGlcDG on the membrane (Schirner et al.,
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2009). In S. aureus, LTA is essential for normal cell growth (Gründling and
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Schneewind, 2007; Oku et al., 2009). B. subtilis has four LTA synthases (the 5
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products of yflE, yfnI, yqgS, and yvgJ), and the products of yflE and yfnI are be-
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lieved to be the major LTA synthases (Wörmann et al., 2011). Therefore, there
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might be a relationship between the phenotype of the ugtP mutant and LTA
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synthases. However, there is no relationship between change in LTA structure
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and the morphology of the ugtP mutant (Hashimoto et al., 2013). The synthesis
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of LTA releases DG moieties of PG. Since LTA is a long polymer, synthesis of a
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single LTA molecule results in the production of dozens of DG molecules. DG is
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converted to PA by DG kinase and reused in the synthesis of phospholipids, one
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of which is PG (Fig. 1). Thus, LTA is synthesized by a cyclic reaction in which DG
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kinase plays a key role. Therefore, the dgkB gene encoding DG kinase is es-
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sential in B. subtilis. DGlcDG is not only important for membrane lipids but also
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as an anchor for LTA.
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Biological function of glucolipids in B. subtilis
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Based on many genetic studies using E. coli cells, it can be taken as proven
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that the physical and chemical properties of membrane lipids are important for
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their functions. In other words, their “acidity” and capacity to form nonbilayers
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are essential for them to function in the membrane. However, recent studies im-
134
ply that lipid molecules have other specific roles that go beyond providing mate-
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rial with certain simple chemical properties. In B. subtilis, PG is essential for
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growth (Hashimoto et al., 2009), whereas in E. coli it is not. This suggests that
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the repertoire of functions a lipid performs is specific in each organism, not a
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universal aspect of the substance itself.
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Although ugtP in B. subtilis, encoding glucosyltransferase, is dispensable, a
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ugtP mutant shows abnormal morphology. The phenotype of cells with ugtP 6
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disruption has been reported to be shorter and rounder for the first time by Price
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et al., 1997. The ugtP mutant cells have also been reported to show strikingly
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abnormal cell morphology (Lazarevic et al., 2005). In addition, the GFP fusion of
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UgtP, which is predicted to be a cytoplasmic protein with no transmembrane
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segment, is localized in the septal region in the form of an open-ring similar to
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the FtsZ-ring (Nishibori et al., 2005). Weart et al. (2007) have proposed that
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UgtP regulates the timing of cell division by adjusting the assembly of FtsZ pro-
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tein based on their finding of dissociation in vitro of FtsZ polymer in the presence
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of UgtP. Disruption of pgcA and gtaB, which are involved in UDP-glucose syn-
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thesis, leads to a cell shape that is known to occur as a consequence of ugtP
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disruption. Since these mutants lack glucolipids, the abnormal morphology of the
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ugtP mutant is presumably caused by the lack of glucolipids (Matsuoka et al.,
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2016).
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Glucolipids are important for the localization of MreB, especially during the
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mid-log phase. MreB had been believed to localize helically along the cell axis
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with MreBH and Mbl in wild type cells (Jones et al., 2001; Graumann, 2007;
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Kawai et al., 2009a; Kawai et al., 2009b). This apparently helical pattern has
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recently been shown to stem from a misinterpretation owing to the increased
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depth of field (Domínguez-Escobar et al., 2011; Garner et al., 2011). In another
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study of cells lacking glucolipids, discrete dots of GFP-MreB failed to appear.
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Instead, faint dots of fluorescence were scattered all over whole cells and the
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MreB amount was labile in absence of glucolipids (Matsuoka et al., 2011a).
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Glucolipids may be required, directly or indirectly, for construction of the MreB
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cytoskeletal structure.
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Glucolipids may also be required for maintenance of cell surface structure, 7
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as it has been observed that the ugtP mutant shows abnormal morphology.
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Therefore, it is believed, the extracytoplasmic function (ECF) sigmas are acti-
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vated in glucolipid-lacking cells. The ECF sigmas, M, V and X, were found to
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be activated in the ugtP mutant cells (Matsuoka et al., 2011a). The analysis of
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the influence of glucolipids produced by UgtP on the activity of ECF sigmas in an
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E. coli heterologous expression system also suggested that glucolipids alone
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may be sufficient to strongly affect the activity of M and V (Seki et al., 2015).
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Furthermore, production of heterologous MGlcDG in the ugtP mutant sup-
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pressed the abnormal morphology and repressed activation of M, V, and X.
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Moreover, when heterologous glucolipid synthases (alMGS and alDGS) from
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Acholeplasma laidlawii were expressed simultaneously, the X activity was spe-
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cifically suppressed to wild type level (Matsuoka et al., 2016). This indicates that
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MGlcDG is required for maintaining normal cell shape and controls the activities
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of a number of ECF sigmas via membrane-anchored anti- factors. Addition of
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magnesium ion allows the cell membrane to attain a condition similar to the
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condition it has when containing glucolipids. We come to the tentative conclusion
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that DGlcDG and/or LTA may be involved in the regulation of X. Thus, each
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glucolipid species plays many different roles in B. subtilis cells.
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Those ECF sigmas that are activated in ugtP mutants are concerned with
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cell envelope maintenance (Cao et al., 2002; Asai et al., 2003). The activation of
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ECF sigmas in the ugtP mutant is not accompanied by regulated intramembrane
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proteolysis. In the case of V activation, no proteolysis of RsiV was observed in
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ugtP mutant cells (Seki et al., 2017). It has been suggested that an appropriate
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length of transmembrane region is required for proper integration of anti-into
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the membrane (Yano et al., 2011). Thus, glucolipids seem to have specific func8
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tions, and aid the function of membrane proteins as “lipid chaperones” (Fig. 2).
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In the absence of glucolipids, membrane proteins do not fold normally and their
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dysfunctionality may lead, in particular, to release of sigma factors. The lack of
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glucolipids affects cell functions in multiple ways.
9
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Acknowledgements
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I thank Professors Emeriti Kouji Matsumoto, Yoshito Sadaie, and Dr. Hiroshi
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Hara for their continuous encouragement. This study was supported by a JSPS
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KAKENHI Grant Number 15K18664.
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Figure Legends
325 326
Fig. 1. Lipid synthesis pathways in B. subtilis and E. coli. (A) Biosynthetic path-
327
ways for phospholipids, glucolipids and LTA in B. subtilis. The gene product cat-
328
alyzing each step is indicated. Abbreviations used are: G3P, glycer-
329
ol-3-phosphate; LPA, lysophosphatidic acid; PA, phosphatidic acid; CDP-DG,
330
(d)CDP-diacylglycerol; PS, phosphatidylserine; PE, phosphatidylethanolamine;
331
PGP, phosphatidylglycerophosphate; PG, phosphatidylglycerol; CL, cardiolipin;
332
lysylPG, lysylphosphatidylglycerol; LTA, lipoteichoic acid; DG, diacylglycerol;
333
MGlcDG,
334
TGlcDG, triglucosyldiacylglycerol. The contents of each lipid are: PE, 30%; PG,
335
40%; CL, 5%; LysylPG, 10%; DG, 5%; MGlcDG, 1.2%; DGlcDG, 9.8%; TGlcDG,
336
0.3%. (B) Phospholipid biosynthetic pathway in E. coli. Abbreviations used are:
337
OPG, osmoregulated periplasmic glucans. For Cls homologues, ClsA, ClsB, and
338
ClsC, refer to Matsumoto et al., 2012. The contents of each lipid are: PE, 75%;
339
PG, 20%; CL, 5%.
monoglucosyldiacylglycerol;
DGlcDG,
diglucosyldiacylglycerol;
340 341
Fig. 2. A model for the roles of glucolipids in the B. subtilis membrane. In wild
342
type cells, ECF sigma is tethered by its cognate membrane anti-and the ac-
343
tivity of ECF sigma is repressed (left). In the absence of glucolipids (in the ugtP
344
mutant), ECF sigma is released because of the dysfunctionality of the misfolded
345
anti-. Then the regulon genes of sigma are expressed (right). Glucolipids con-
346
trol the folding and functionality of anti- by acting like chaperones.
16
Fig. 1 (A) B. subtilis
(B) E. coli
G3P PlsY
LPA
LPA
PlsC
PlsC CdsA
PA
PssA
PgsA
PS
PGP PG
PE MprF
LysylPG
LtaS homologues
Psd
LTA
DgkA
PA
CdsA
UgtP MGlcDG UgtP DGlcDG UgtP
ClsA
CL
DgkB
DG
CDP-DG
G3P
PlsB
TGlcDG
PssA
CDP-DG PgsA PGP
PS Psd
PE
DG
OpgB
PG
OPG Cls
CL
Fig. 2 ugtP cells
WT cells anti-
Unfolded anti- Lack of glucolipids
ECFσ
Repressed ECFσ
Active
Glucolipid
Expression of regulon genes
Received for publication: April 19, 2017 Accepted for publication: July 4, 2017 Published online: October 6, 2017
Matsuoka, 2017
1 2
Genes & Genetic Systems
3 4
Biological functions of glucolipids in Bacillus subtilis
5
Satoshi Matsuoka*
6 7 8
Department of Biochemistry and Molecular Biology, Graduate School of Science
9
and Engineering, Saitama University, 255 Shimo-Okubo, Sakura, Saitama,
10
Saitama 338-8570, Japan
11 12
*Corresponding author:
Satoshi Matsuoka
13
Tel: +81 48-858-9294
14
Fax: +81 48-858-3384 E-mail: [email protected]
15 16 17
Running head: Biological functions of glucolipids in B. subtilis
18
Key words: Bacillus subtilis, glucolipid, ugtP, ECF sigma factor, cell wall
19
maintenance
20
1
21
Abstract
22
Glyceroglycolipids are very important in Gram-positive bacteria and cyano-
23
bacteria. In Bacillus subtilis, a model organism for the Gram-positive bacteria,
24
the ugtP mutant, which lacks glyceroglucolipids, shows abnormal morphology.
25
Lack of glucolipids has many consequences: abnormal localization of the cyto-
26
skeletal protein MreB and activation of some extracytoplasmic function (ECF)
27
sigma factors ( M,
28
the expression of monoglucosyldiacylglycerol (MGlcDG) by 1,2-diacylglycerol
29
3-glucosyltransferase from Acholeplasma laidlawii (alMGS) almost completely
30
suppresses the ugtP disruptant phenotype. Activation of ECF sigmas in the ugtP
31
mutant is decreased by alMGS expression, and is suppressed to low levels by
32
MgSO4
33
1,2-diacylglycerol-3-glucose (1-2)-glucosyltransferase producing diglucosyldi-
34
acylglycerol (DGlcDG)) are simultaneously expressed,
35
pressed to wild type level. These observations suggest that MGlcDG molecules
36
are required for maintenance of B. subtilis cell shape and regulation of ECF
37
sigmas, and that DGlcDG regulates X activity. The activation of ECF sigmas is
38
not accompanied by proteolysis of anti-. Thus, glyceroglucolipids may have the
39
specific role of helping membrane proteins function by acting in the manner of
40
chaperones.
addition.
V
and X) in the log phase are two examples. Conversely,
When
alMGS
2
and
alDGS
(A.
X
laidlawii
activation is re-
41
Introduction
42
All living organisms have membranes which compartment cells from the
43
outer environment and from each other. A lipid bilayer structure for these mem-
44
branes has been proposed by Singer and Nicolson in 1972. In this model,
45
membrane proteins are floating in a “sea of lipids” and the proteins can move
46
freely in the membrane. Lipid molecules are uniformly distributed in the mem-
47
brane. This model is almost correct; however recent studies have demonstrated
48
the existence of specific lipid-protein domains, which come about by lipid mole-
49
cules and membrane proteins forming domain structures in the membrane, so
50
called lipid rafts (Raghunathan et al., 1990). In bacteria, the existence of lipid
51
rafts is not certain although some researchers have reported evidence pointing
52
to lipid rafts as detergent resistant membrane fractions (Donovan and
53
Bramkamp, 2009). On the other hand, localizations of lipid molecules have been
54
reported. The presence of CL or anionic phospholipids in specific regions is re-
55
quired for many biological functions such as the localization of ClsA (Kusaka et
56
al., 2016) and the Min system (Ishikawa et al., in press) in B. subtilis. In cyano-
57
bacteria galactolipids play an important role in photosynthesis as galactolipids
58
are included in the thylakoid membranes which are the sites of oxygenic photo-
59
synthesis (Mizusawa and Wada, 2012); the crystal structures of the photosystem
60
I (PSI) and PS II complexes from the thermophilic cyanobacterium Synecho-
61
coccus elongatus reveal that these PSI and PSII contain one molecule of mo-
62
nogalactosyldiacylglycerol (Jordan et al., 2001), and six monogalactosyldiacyl-
63
glycerol and five digalactosyldiacylglycerol molecules, respectively (Umena et al.,
64
2011). It is difficult to uncover the biological functions of membrane lipid mole-
65
cules because these are products resulting from enzyme reactions, not directly 3
66
encoded in genes, and there are only few probes which detect specific lipid
67
molecules. To reveal the biological functions of membrane lipids, genes which
68
encode lipid synthesis enzymes have been inactivated, and observations of the
69
resulting phenotypes have been performed in an attempt to gain clues. Shibuya,
70
Dowhan and their colleagues have revealed gene regulation and physiological
71
roles of membrane lipids in E. coli using molecular genetics (Shibuya, 1992;
72
Mileykovskaya and Dowhan, 2005). However, the knowledge based on E. coli
73
studies cannot be naively applied to B. subtilis.
74
In this outline, lipid synthesis pathways, cell surface structures, and biolog-
75
ical functions of membrane lipids in B. subtilis are reviewed. Then it focusses on
76
the biological functions of glucolipids.
77 78
Synthesis of membrane lipids in B. subtilis
79
The lipid biosynthesis pathways of phospholipids are shown in Fig. 1. The
80
pathway in B. subtilis is almost the same as that in E. coli. The main phospho-
81
lipid molecules of E. coli are phosphatidylethanolamine (PE), phosphatidylglyc-
82
erol (PG), and cardiolipin (CL). They make up about 70%, 25%, and 5%, re-
83
spectively, of phospholipids in the cell membrane. In B. subtilis, PE, PG, and CL
84
constitute 30%, 40%, and 5% of the cell membrane lipids, respectively. In addi-
85
tion to these phospholipids, diacylglycerol (DG), glucolipids, and lysylphospha-
86
tidylglycerol (lysylPG) are contained at a fraction of about 5%, and 10% each in
87
B. subtilis cell membranes (Matsuoka et al., 2011b; Hashimoto et al., 2013). The
88
B. subtilis cell contains three kinds of glucolipids, monoglucosyldiacylglycerol
89
(MGlcDG), diglucosyldiacylglycerol (DGlcDG), and triglucosyldiacylglycerol
90
(TGlcDG), which amount to 1.2%, 9.8% and 0.3% of total membrane lipids 4
91
(Kawai et al., 2006). These are synthesized processively by UgtP, which trans-
92
fers glucose to diacylglycerol from UDP-glucose (Jorasch et al., 1998). In these
93
pathways, phosphatidic acid (PA) is synthesized from glycerol-3-phosphate
94
(G3P) but the first reactions to lysophosphatidic acid (LPA) synthesis are differ-
95
ent between B. subtilis and E. coli (Hara et al., 2008). The gene cdsA, which
96
encodes CDP-diglycerol synthase, is an essential gene in both organisms.
97
Conditional knockout mutants lacking either PE or acidic phospholipids (PG and
98
CL) can be constructed in E. coli. By contrast, pssA, which encodes the key
99
enzyme to synthesize PE, can easily be deactivated without obvious defective
100
phenotype, and pgsA, which encodes the key enzyme to synthesize PG, is in-
101
dispensable in B. subtilis (Salzberg and Helmann, 2008; Hashimoto et al., 2009).
102
DgkB, a diacylglycerol kinase of B. subtilis is also essential (Kobayashi et al.,
103
2003). In the absence of LtaS homologue genes, dgkB is dispensable because
104
accumulation of DG, which is toxic for cells, does not occur (Matsuoka et al.,
105
2011b).
106 107
Cell surface and membrane lipids of B. subtilis
108
The cell walls of B. subtilis, a model microorganism representative of the
109
Gram-positive bacteria, consist of thick multiple layers of peptidoglycan, which is
110
the major physical and chemical defense against deadly agents. The envelope
111
of Gram-positive bacteria contains further components: polymers of glycerol-
112
phosphate, wall teichoic acids (WTA) covalently linked with peptidoglycan and
113
lipoteichoic acids (LTA) linked with DGlcDG on the membrane (Schirner et al.,
114
2009). In S. aureus, LTA is essential for normal cell growth (Gründling and
115
Schneewind, 2007; Oku et al., 2009). B. subtilis has four LTA synthases (the 5
116
products of yflE, yfnI, yqgS, and yvgJ), and the products of yflE and yfnI are be-
117
lieved to be the major LTA synthases (Wörmann et al., 2011). Therefore, there
118
might be a relationship between the phenotype of the ugtP mutant and LTA
119
synthases. However, there is no relationship between change in LTA structure
120
and the morphology of the ugtP mutant (Hashimoto et al., 2013). The synthesis
121
of LTA releases DG moieties of PG. Since LTA is a long polymer, synthesis of a
122
single LTA molecule results in the production of dozens of DG molecules. DG is
123
converted to PA by DG kinase and reused in the synthesis of phospholipids, one
124
of which is PG (Fig. 1). Thus, LTA is synthesized by a cyclic reaction in which DG
125
kinase plays a key role. Therefore, the dgkB gene encoding DG kinase is es-
126
sential in B. subtilis. DGlcDG is not only important for membrane lipids but also
127
as an anchor for LTA.
128 129
Biological function of glucolipids in B. subtilis
130
Based on many genetic studies using E. coli cells, it can be taken as proven
131
that the physical and chemical properties of membrane lipids are important for
132
their functions. In other words, their “acidity” and capacity to form nonbilayers
133
are essential for them to function in the membrane. However, recent studies im-
134
ply that lipid molecules have other specific roles that go beyond providing mate-
135
rial with certain simple chemical properties. In B. subtilis, PG is essential for
136
growth (Hashimoto et al., 2009), whereas in E. coli it is not. This suggests that
137
the repertoire of functions a lipid performs is specific in each organism, not a
138
universal aspect of the substance itself.
139
Although ugtP in B. subtilis, encoding glucosyltransferase, is dispensable, a
140
ugtP mutant shows abnormal morphology. The phenotype of cells with ugtP 6
141
disruption has been reported to be shorter and rounder for the first time by Price
142
et al., 1997. The ugtP mutant cells have also been reported to show strikingly
143
abnormal cell morphology (Lazarevic et al., 2005). In addition, the GFP fusion of
144
UgtP, which is predicted to be a cytoplasmic protein with no transmembrane
145
segment, is localized in the septal region in the form of an open-ring similar to
146
the FtsZ-ring (Nishibori et al., 2005). Weart et al. (2007) have proposed that
147
UgtP regulates the timing of cell division by adjusting the assembly of FtsZ pro-
148
tein based on their finding of dissociation in vitro of FtsZ polymer in the presence
149
of UgtP. Disruption of pgcA and gtaB, which are involved in UDP-glucose syn-
150
thesis, leads to a cell shape that is known to occur as a consequence of ugtP
151
disruption. Since these mutants lack glucolipids, the abnormal morphology of the
152
ugtP mutant is presumably caused by the lack of glucolipids (Matsuoka et al.,
153
2016).
154
Glucolipids are important for the localization of MreB, especially during the
155
mid-log phase. MreB had been believed to localize helically along the cell axis
156
with MreBH and Mbl in wild type cells (Jones et al., 2001; Graumann, 2007;
157
Kawai et al., 2009a; Kawai et al., 2009b). This apparently helical pattern has
158
recently been shown to stem from a misinterpretation owing to the increased
159
depth of field (Domínguez-Escobar et al., 2011; Garner et al., 2011). In another
160
study of cells lacking glucolipids, discrete dots of GFP-MreB failed to appear.
161
Instead, faint dots of fluorescence were scattered all over whole cells and the
162
MreB amount was labile in absence of glucolipids (Matsuoka et al., 2011a).
163
Glucolipids may be required, directly or indirectly, for construction of the MreB
164
cytoskeletal structure.
165
Glucolipids may also be required for maintenance of cell surface structure, 7
166
as it has been observed that the ugtP mutant shows abnormal morphology.
167
Therefore, it is believed, the extracytoplasmic function (ECF) sigmas are acti-
168
vated in glucolipid-lacking cells. The ECF sigmas, M, V and X, were found to
169
be activated in the ugtP mutant cells (Matsuoka et al., 2011a). The analysis of
170
the influence of glucolipids produced by UgtP on the activity of ECF sigmas in an
171
E. coli heterologous expression system also suggested that glucolipids alone
172
may be sufficient to strongly affect the activity of M and V (Seki et al., 2015).
173
Furthermore, production of heterologous MGlcDG in the ugtP mutant sup-
174
pressed the abnormal morphology and repressed activation of M, V, and X.
175
Moreover, when heterologous glucolipid synthases (alMGS and alDGS) from
176
Acholeplasma laidlawii were expressed simultaneously, the X activity was spe-
177
cifically suppressed to wild type level (Matsuoka et al., 2016). This indicates that
178
MGlcDG is required for maintaining normal cell shape and controls the activities
179
of a number of ECF sigmas via membrane-anchored anti- factors. Addition of
180
magnesium ion allows the cell membrane to attain a condition similar to the
181
condition it has when containing glucolipids. We come to the tentative conclusion
182
that DGlcDG and/or LTA may be involved in the regulation of X. Thus, each
183
glucolipid species plays many different roles in B. subtilis cells.
184
Those ECF sigmas that are activated in ugtP mutants are concerned with
185
cell envelope maintenance (Cao et al., 2002; Asai et al., 2003). The activation of
186
ECF sigmas in the ugtP mutant is not accompanied by regulated intramembrane
187
proteolysis. In the case of V activation, no proteolysis of RsiV was observed in
188
ugtP mutant cells (Seki et al., 2017). It has been suggested that an appropriate
189
length of transmembrane region is required for proper integration of anti-into
190
the membrane (Yano et al., 2011). Thus, glucolipids seem to have specific func8
191
tions, and aid the function of membrane proteins as “lipid chaperones” (Fig. 2).
192
In the absence of glucolipids, membrane proteins do not fold normally and their
193
dysfunctionality may lead, in particular, to release of sigma factors. The lack of
194
glucolipids affects cell functions in multiple ways.
9
195
Acknowledgements
196
I thank Professors Emeriti Kouji Matsumoto, Yoshito Sadaie, and Dr. Hiroshi
197
Hara for their continuous encouragement. This study was supported by a JSPS
198
KAKENHI Grant Number 15K18664.
10
199
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Figure Legends
325 326
Fig. 1. Lipid synthesis pathways in B. subtilis and E. coli. (A) Biosynthetic path-
327
ways for phospholipids, glucolipids and LTA in B. subtilis. The gene product cat-
328
alyzing each step is indicated. Abbreviations used are: G3P, glycer-
329
ol-3-phosphate; LPA, lysophosphatidic acid; PA, phosphatidic acid; CDP-DG,
330
(d)CDP-diacylglycerol; PS, phosphatidylserine; PE, phosphatidylethanolamine;
331
PGP, phosphatidylglycerophosphate; PG, phosphatidylglycerol; CL, cardiolipin;
332
lysylPG, lysylphosphatidylglycerol; LTA, lipoteichoic acid; DG, diacylglycerol;
333
MGlcDG,
334
TGlcDG, triglucosyldiacylglycerol. The contents of each lipid are: PE, 30%; PG,
335
40%; CL, 5%; LysylPG, 10%; DG, 5%; MGlcDG, 1.2%; DGlcDG, 9.8%; TGlcDG,
336
0.3%. (B) Phospholipid biosynthetic pathway in E. coli. Abbreviations used are:
337
OPG, osmoregulated periplasmic glucans. For Cls homologues, ClsA, ClsB, and
338
ClsC, refer to Matsumoto et al., 2012. The contents of each lipid are: PE, 75%;
339
PG, 20%; CL, 5%.
monoglucosyldiacylglycerol;
DGlcDG,
diglucosyldiacylglycerol;
340 341
Fig. 2. A model for the roles of glucolipids in the B. subtilis membrane. In wild
342
type cells, ECF sigma is tethered by its cognate membrane anti-and the ac-
343
tivity of ECF sigma is repressed (left). In the absence of glucolipids (in the ugtP
344
mutant), ECF sigma is released because of the dysfunctionality of the misfolded
345
anti-. Then the regulon genes of sigma are expressed (right). Glucolipids con-
346
trol the folding and functionality of anti- by acting like chaperones.
16
Fig. 1 (A) B. subtilis
(B) E. coli
G3P PlsY
LPA
LPA
PlsC
PlsC CdsA
PA
PssA
PgsA
PS
PGP PG
PE MprF
LysylPG
LtaS homologues
Psd
LTA
DgkA
PA
CdsA
UgtP MGlcDG UgtP DGlcDG UgtP
ClsA
CL
DgkB
DG
CDP-DG
G3P
PlsB
TGlcDG
PssA
CDP-DG PgsA PGP
PS Psd
PE
DG
OpgB
PG
OPG Cls
CL
Fig. 2 ugtP cells
WT cells anti-
Unfolded anti- Lack of glucolipids
ECFσ
Repressed ECFσ
Active
Glucolipid
Expression of regulon genes
