Nienke Buddelmeijer

The molecular mechanism of bacterial lipoprotein modification -how, when and why? 1 2

Keywords: lipoprotein, fatty acid acylation, bacteria, acyltransferase, signal peptidase

and include an important class of virulence factors expressed by many pathogenic species 26 [START_REF] Kovacs-Simon | Lipoproteins of bacterial pathogens[END_REF]. Prolipoprotein is synthesized in the cytoplasm and translocated 27 via Sec or Tat secretion machineries [START_REF] Inouye | Amino acid sequence for 1045 the peptide extension on the prolipoprotein of the Escherichia coli outer membrane[END_REF], Hayashi & Wu, 1985, Gibbons et 28 al., 2007[START_REF] Siboo | Characterization of the accessory 1351 Sec system of Staphylococcus aureus[END_REF][START_REF] Shruthi | Twin arginine translocase pathway 1344 and fast-folding lipoprotein biosynthesis in E. coli: interesting implications and 1345 applications[END_REF][START_REF] Thompson | Investigating lipoprotein biogenesis and function in the model Gram-positive 1385 bacterium Streptomyces coelicolor[END_REF]. Lipoprotein 29 modification occurs in the cytoplasmic (inner) membrane by the sequential action of three 30 membrane-bound enzymes (Fig. 1) [START_REF] Wu | Biogenesis of membrane lipoproteins in 1493 Escherichia coli[END_REF]. Lipoproteins possess a conserved 31 sequence, so-called lipobox, which allows specific recognition by the modification 32 machinery. The invariant cysteine residue in the lipobox becomes lipidated and the first 33 amino acid after complete maturation of lipoprotein. Phosphatidylglycerol:prolipoprotein 34 diacylglyceryl transferase (Lgt) adds an sn-1,2-diacylglyceryl group from 35 phosphatidylglycerol (PG) to the sulfhydryl group of cysteine via a thioether bond. 36

Prolipoprotein signal peptidase (Lsp) cleaves the signal peptide thereby liberating the α-37 amino group of diacylglyceryl-cysteine. The third and last step is N-acylation of 38 diacylglyceride-cysteine by Apolipoprotein N-acyltransferase (Lnt), resulting in mature N-39 acyl-S-diacylglyceryl-cysteine linked proteins. Lgt and Lsp are conserved in all bacterial 40 species, while Lnt has only been identified in proteobacteria and actinomycetes. Recent 41 findings suggest that a non-canonical lipidation pathway exist by the demonstration that 42 lipoproteins of firmicutes and terenicutes (that include mollicutes), whose genome lack a gene 43 encoding Lnt, exist in a triacylated form (Fig. 1) [START_REF] Jan | Acylation and immunological 1077 properties of Mycoplasma gallisepticum membrane proteins[END_REF][START_REF] Kurokawa | The 1120 triacylated ATP binding cluster transporter substrate-binding lipoprotein of 1121 Staphylococcus aureus functions as a native ligand for Toll-like receptor 2[END_REF], Asanuma 44 et al., 2010[START_REF] Serebryakova | The acylation state of surface lipoproteins of mollicute Acholeplasma laidlawii[END_REF]. Various N-terminal modifications were observed: N- phospholipids phosphatidylserine (PS) and phosphatidic acid (PA) [START_REF] Ames | Lipids of Salmonella typhimurium and Escherichia coli: structure and 722 metabolism[END_REF]. The fatty 76 acids of phospholipids of a typical E. coli strain contain about 45% palmitic acid (C16:0), 2% 77 myristic acid (C14:0), 35% palmitoleic acid (C16:1) and 18% cis-vaccenic acid (C18:1cis-11) 78 [START_REF] Cronan | Biosynthesis of membrane lipids. Escherichia coli and 851 Salmonella: molecular and cellular biology[END_REF]. The main fatty acids of major lipoprotein Lpp (murein-lipoprotein or 79

Braun's lipoprotein) of E. coli are palmitic acid (53%), cis-vaccenic acid (20.7%), 9,10-80 methylene-hexadecanoic acid (C17:0) (10.6%) and palmitoleic acid (9.4%) (Hantke & Braun, 81 1973), corresponding to the fatty acid moieties of phospholipids. Phospholipids that compose 82 membranes of Staphylococcus aureus include phosphatidylglycerol, cardiolipin, lysyl-83 phosphatidylglycerol, and mono-and diglucosyl-diglyceride but not 84 phosphatidylethanolamine [START_REF] White | Fatty acid composition of the complex lipids of 1464 Staphylococcus aureus during the formation of the membrane-bound electron transport 1465 system[END_REF], Fischer, 1994). Mass spectrometry data 85 has shown that lipoproteins in S. aureus are modified with C17:0 to C20:0 at the sn-1 position 86 of the diacylglyceryl group and with C15:0 at the sn-2 position. The α-amino group of 87 lipidated diacylglyceryl-cysteine have fatty acids of varying lengths, from C15:0 to C20:0 88 (Kurokawa et al., 2012). The lipid moiety of lipoproteins thus reflects the fatty acid 89 composition of phospholipids and varies among bacterial species. 90 91 Phosphatidylglycerol:prolipoprotein diacylglyceryl transferase (Lgt) 92

Modification of prolipoprotein by Lgt is the first step in lipoprotein modification (Tokunaga 93 et al., 1982). Upon membrane insertion of prolipoprotein by Sec or Tat, Lgt transfers the 94 diacylglyceryl group from phosphatidylglycerol onto the sulfhydryl group of the invariant 95 cysteine in the lipobox resulting in a thioether-linked diacylglyceryl-prolipoprotein and a sn-96 glycerol-1-phosphate by-product [START_REF] Lai | Acyl moieties in phospholipids are tha precursors 1125 for the fatty acids in murein lipoprotein in Escherichia coli[END_REF][START_REF] Sankaran | Lipid modification of bacterial prolipoprotein. Transfer of 1323 diacylglyceryl moiety from phosphatidylglycerol[END_REF]. Signal sequences 97 of lipoproteins share common features including one or several positively charged residues at 98 the amino terminus followed by a hydrophobic stretch of about 20 amino acids that includes 99 becomes the first amino acid in the mature protein [START_REF] Wu | Posttranslational 1496 modification and processing of membrane lipoproteins in bacteria[END_REF][START_REF] Wu | Biosynthesis of lipoproteins. Escherichia coli and Salmonella Cellular and 1484[END_REF], Madan Babu 102 & Sankaran, 2002[START_REF] Babu | A 736 database of bacterial lipoproteins (DOLOP) with functional assignments to predicted 737 lipoproteins[END_REF]. This consensus sequence has been documented in the 103 PROSITE database under pattern entry PS00013/PDOC00013 104 (http://www.expasy.ch/prosite/). Lipobox motifs are similar in firmicutes but more variable in 105 spirochetes [START_REF] Haake | Spirochaetal lipoproteins and pathogenesis[END_REF][START_REF] Sutcliffe | Pattern searches for the identification of putative 1368 lipoprotein genes in Gram-positive bacterial genomes[END_REF], the -3 position in particular is more 106 loosely defined in the latter, where a leucine residue is often replaced by another hydrophobic 107 amino acid. A high percentage of ORFs in spirochetes encode lipoproteins, some of which are 108 highly abundant proteins [START_REF] Haake | Spirochaetal lipoproteins and pathogenesis[END_REF]. For Lpp it has also been demonstrated that residue 109 G14 in the signal peptide sequence is important for lipidation by Lgt, and that a signal 110 sequence shortened by several residues still leads to modified protein [START_REF] Wu | Biochemical characterization of a mutant 1489 lipoprotein of Escherichia coli[END_REF], Yem 111 & Wu, 1977[START_REF] Lin | An Escherichia coli mutant with an amino 1135 acid alteration within the signal sequence of outer membrane prolipoprotein[END_REF][START_REF] Lin | Assembly of the outer membrane lipoprotein in 1139 Escherichia coli[END_REF][START_REF] Inouye | Effects of mutations at glycine 1040 residues in the hydrophobic region of the Escherichia coli prolipoprotein signal peptide 1041 on the secretion across the membrane[END_REF][START_REF] Pollitt | A functional prolipoprotein signal peptide with a 1264 deletion of four amino acid residues from the hydrophobic region[END_REF]. The 112 first biochemical characterization of Lgt was reported in the 1980-s using crude membrane 113 extracts. The optimal pH and reaction temperature for Lgt are pH 7.8 and 37°C, respectively 114 [START_REF] Tokunaga | Prolipoprotein modification and processing 1409 enzymes in Escherichia coli[END_REF]. Lgt is stable in membrane vesicles up to 65°C, the enzyme is active 115 in N-octyl-β-D-glucoside but many other detergents are not compatible with enzyme activity 116 [START_REF] Tokunaga | Prolipoprotein modification and processing 1409 enzymes in Escherichia coli[END_REF][START_REF] Sankaran | Lipid modification of bacterial prolipoprotein. Transfer of 1323 diacylglyceryl moiety from phosphatidylglycerol[END_REF]. PG is specific as acyl donor since PE and CL 117 could not be used as substrate in the in vitro assay using a synthetic peptide, suggesting that 118 the polar headgroup of the phospholipid plays an important role in the Lgt reaction (Sankaran 119 & Wu, 1994). The gene encoding Lgt was identified by screening a collection of temperature 120 sensitive mutants of S. enterica for the accumulation of unmodified Lpp and was shown to be 121 located at the 5' end of thyA [START_REF] Williams | Identification and 1480 genetic mapping of the structural gene for an essential Escherichia coli membrane 1481 protein[END_REF][START_REF] Bell-Pedersen | A transcription terminator in the 761 thymidylate synthase (thyA) structural gene of Escherichia coli and construction of a 762 viable thyA::Kmr deletion[END_REF], Gan et al., 122 1993). The enzyme is localized in the cytoplasmic membrane and spans the membrane by 123 seven transmembrane segments (Fig. 2.) [START_REF] Pailler | Phosphatidylglycerol::prolipoprotein diacylglyceryltransferase (Lgt) of E. coli has seven 1242 transmembrane segments and its essential residues are embedded in the membrane[END_REF]. The majority of essential 124 with its substrates PG and prolipoprotein [START_REF] Qi | Structure-function relationship of bacterial 1274 prolipoprotein diacylglyceryl transferase: functionally significant conserved regions[END_REF][START_REF] Sankaran | Roles of histidine-103 and 1326 tyrosine-235 in the function of the prelipoprotein diacylglyceryl transferase of 1327 Escherichia coli[END_REF], Pailler et al., 126 2012). Residues H103 and Y235 were identified as conserved by comparing four Lgt 127 sequences and were shown to be essential for Lgt activity [START_REF] Sankaran | Roles of histidine-103 and 1326 tyrosine-235 in the function of the prelipoprotein diacylglyceryl transferase of 1327 Escherichia coli[END_REF]. These 128 residues are among twelve residues highly conserved in proteobacteria, firmicutes and 129 actinobacteria [START_REF] Pailler | Phosphatidylglycerol::prolipoprotein diacylglyceryltransferase (Lgt) of E. coli has seven 1242 transmembrane segments and its essential residues are embedded in the membrane[END_REF]. A more recent analysis on conserved residues highlighted 130 the existence of a so-called Lgt signature motif; two amino acids in this sequence (N146 and 131 G154) were shown to be absolutely required for function [START_REF] Pailler | Phosphatidylglycerol::prolipoprotein diacylglyceryltransferase (Lgt) of E. coli has seven 1242 transmembrane segments and its essential residues are embedded in the membrane[END_REF]. Together, these 132 data show that regions H103-G108 and G142-G154 and residues Y26 and Y235 are critical 133 for Lgt function. Initial kinetics studies were performed with crude membrane extracts but 134 further biochemical characterization of the E. coli enzyme turned out difficult due to 135 overproduction defects and instability of the purified enzyme [START_REF] Sankaran | Roles of histidine-103 and 1326 tyrosine-235 in the function of the prelipoprotein diacylglyceryl transferase of 1327 Escherichia coli[END_REF], Selvan 136 & Sankaran, 2008[START_REF] Pailler | Phosphatidylglycerol::prolipoprotein diacylglyceryltransferase (Lgt) of E. coli has seven 1242 transmembrane segments and its essential residues are embedded in the membrane[END_REF]. Lgt has been purified recently from Lactococcus 137 lactis with which kinetic studies were performed (Banerjee & Sankaran, 2012), unfortunately, 138 assay conditions were not comparable with those described previously [START_REF] Sankaran | Roles of histidine-103 and 1326 tyrosine-235 in the function of the prelipoprotein diacylglyceryl transferase of 1327 Escherichia coli[END_REF], 139 Sundaram et al., 2012), making it difficult to compare enzyme activities from different 140 bacterial species. 141 Lgt of S. aureus can complement a temperature sensitive mutant of E. coli, suggesting that 142

Lgt is functionally conserved among bacterial species [START_REF] Qi | Structure-function relationship of bacterial 1274 prolipoprotein diacylglyceryl transferase: functionally significant conserved regions[END_REF]. Deletion mutants of 143 lgt in firmicutes often lead to shedding of lipoproteins into the culture supernatant in non-144 lipidated precursor form with uncleaved signal peptide [START_REF] Leskelä | Lipid modification of prelipoproteins 1131 is dispensable for growth but essential for efficient protein secretion in Bacillus subtilis: 1132 characterization of the Igt gene[END_REF] or processed by 145 an unidentified signal peptidase [START_REF] Stoll | Staphylococcus aureus deficient in lipidation of 1354 prelipoproteins is attenuated in growth and immune activation[END_REF][START_REF] Denham | In the absence of Lgt, lipoproteins are shed 867 from Streptococcus uberis independently of Lsp[END_REF] or Lsp (Baumgärtner 146 et al., 2007[START_REF] Henneke | Lipoproteins are critical TLR2 activating 989 toxins in group B streptococcal sepsis[END_REF]. Two Lgt homologues were identified in Streptomyces 147 coelicolor, Lgt1 and Lgt2, which can both complement the lgt mutant of Streptomyces 148 scabies, suggesting that both enzymes are functional enzymes [START_REF] Widdick | Dissecting the complete lipoprotein 1473 biogenesis pathway in Streptomyces scabies[END_REF] [START_REF] Thompson | Investigating lipoprotein biogenesis and function in the model Gram-positive 1385 bacterium Streptomyces coelicolor[END_REF]. Lipoproteins were purified from the complementing strains and the 151 composition of their N-terminal fatty acid was analyzed by mass spectrometry (Widdick et 152 al., 2011). Several fatty acid acylated peptides were missing in these strains compared to wild 153 type S. scabies, suggesting that subtle differences in diacylglyceryl transferase activity might 154 exist. The gene encoding Lgt in Mycobacterium tuberculosis and two genes in 155 Mycobacterium smegmatis have recently been identified [START_REF] Tschumi | Functional 1420 analyses of mycobacterial lipoprotein diacylglyceryl transferase and comparative 1421 secretome analysis of a mycobacterial lgt mutant[END_REF]. The 156 MSMEG_3222 gene was shown to be responsible for the transfer of diacylglyceryl onto 157 lipoproteins, and although less conserved than MSMEG_3222 the role of the second lgt gene 158 (MSMEG_5408) in lipoprotein modification has not yet been addressed (Tschumi et al., 159 2012). An lgt deletion mutant could not be obtained in M. tuberculosis suggesting that this 160 gene is essential [START_REF] Sassetti | Genes required for mycobacterial growth 1333 defined by high density mutagenesis[END_REF][START_REF] Tschumi | Functional 1420 analyses of mycobacterial lipoprotein diacylglyceryl transferase and comparative 1421 secretome analysis of a mycobacterial lgt mutant[END_REF]. Strikingly, MSMEG_3222 is not 161 essential in M. smegmatis and deletion did not completely abolish incorporation of radioactive 162 palmitate suggesting that either MSMEG_5408 or another, as yet unknown, enzyme possesses 163 Lgt activity but is perhaps less efficient than MSMEG_3222 [START_REF] Tschumi | Functional 1420 analyses of mycobacterial lipoprotein diacylglyceryl transferase and comparative 1421 secretome analysis of a mycobacterial lgt mutant[END_REF]. Absence 164 of Lgt resulted in massive release of lipoproteins in the culture media but none of these 165 proteins contained a signal peptide, suggesting that they are processed by Lsp or other signal 166 peptidases [START_REF] Tschumi | Functional 1420 analyses of mycobacterial lipoprotein diacylglyceryl transferase and comparative 1421 secretome analysis of a mycobacterial lgt mutant[END_REF]. No clear phenotype on cell wall biogenesis was observed. 167

Detailed biochemical analyses and comparison of kinetic parameters of various Lgt enzymes 168 will contribute to a better understanding of the mechanism of the reaction and are predicted to 169 highlight differences and similarities in enzyme activity. The thiol group of prolipoprotein 170 probably performs a nucleophilic attack on the carbonyl group of PG where Lgt provides the 171 catalytic residues to support transfer of diacylglyceryl onto prolipoprotein, and positions the 172 two substrates close together. PG is the sole acyl donor in the Lgt reaction, suggesting that the 173 glycerol-phosphate headgroup plays an important role in substrate recognition by Lgt. 174 Lsp (SPase II) is localized in the cytoplasmic membrane [START_REF] Tokunaga | Prolipoprotein modification and processing 1409 enzymes in Escherichia coli[END_REF] and spans 177 this membrane by four transmembrane segments with both the amino and carboxyl termini 178 facing the cytoplasm [START_REF] Munoa | Membrane topology of 1196 Escherichia coli prolipoprotein signal peptidase (signal peptidase II)[END_REF] (Fig. 2). Five conserved regions were identified, 179

i.e. region I (amino acids residues 23-27 in E. coli sequence), region II (residues 53-58), 180 region III (residues 104-115) and region IV (residues 121-123) and region V (residues 137-181 141) [START_REF] Munoa | Membrane topology of 1196 Escherichia coli prolipoprotein signal peptidase (signal peptidase II)[END_REF][START_REF] Tjalsma | The potential active site of 1388 the lipoprotein-specific (type II) signal peptidase of Bacillus subtilis[END_REF]. The role of these conserved regions in the 182 peptidase reaction has not yet been addressed but probably plays a role in substrate 183 recognition and/or binding. The active site of Lsp is composed of at least two aspartate 184 residues (D114 and D141 in E. coli) [START_REF] Tjalsma | The potential active site of 1388 the lipoprotein-specific (type II) signal peptidase of Bacillus subtilis[END_REF]. Modification of lipoproteins by 185 addition of diacylglyceryl catalyzed by Lgt is a prerequisite for processing by Lsp in 186 proteobacteria [START_REF] Tokunaga | Post-translational modification and 1401 processing of Escherichia coli prolipoprotein in vitro[END_REF]. The Lsp enzymes of actinomycetes have an N-187 terminal extension, truncations of which led to loss of Lsp function, either due to inactivation 188 or instability of the enzyme [START_REF] Thompson | Investigating lipoprotein biogenesis and function in the model Gram-positive 1385 bacterium Streptomyces coelicolor[END_REF]. Biochemical studies with Lsp were 189 performed in the early 1980-s and showed that Lsp is temperature resistant in isolated 190 membranes, but unlike Lnt still active at 80°C [START_REF] Hussain | Mechanism of signal peptide cleavage in the 1029 biosynthesis of the major lipoprotein of the Escherichia coli outer membrane[END_REF]. In detergent-solubilized 191 membranes Lsp is active at a reaction temperature between 37°C-45°C with a pH optimum of 192 7.9 [START_REF] Tokunaga | Prolipoprotein modification and processing 1409 enzymes in Escherichia coli[END_REF]. The enzyme does not require phospholipids and is active in non-193 ionic detergents. 194 Initially two enzymatic activities were shown to be involved in the proteolytic cleavage of 195 lipoproteins, Lsp that cleaves off the signal peptide of diacylglyceryl-proLpp and a signal 196 peptide peptidase responsible for the degradation of signal peptide (SppA or Protease IV) 197 [START_REF] Suzuki | 1372 Murein-lipoprotein of Escherichia coli: a protein involved in the stabilization of bacterial 1373 cell envelope[END_REF][START_REF] Novak | Degradation of a signal peptide by protease IV and 1210 oligopeptidase A[END_REF]. SppA is an intramembrane protease (Pacaud, 198 1982, Pacaud, 1982) utilizing a Ser/Lys dyad for catalysis [START_REF] Wang | Escherichia coli signal peptide peptidase A is a serine-lysine protease with a 1447 lysine recruited to the nonconserved amino-terminal domain in the S49 protease family[END_REF]. It adopts a 199 [START_REF] Xiao | Genetic redundancy, proximity, and functionality of lspA, the 1503 target of antibiotic TA, in the Myxococcus xanthus producer strain[END_REF] [START_REF] An | Identification and characterization of a 725 determinant (eep) on the Enterococcus faecalis chromosome that is involved in 726 production of the peptide sex pheromone cAD1[END_REF], in the absence of Lsp in late-229 stationary-phase cells [START_REF] Denham | In the absence of Lgt, lipoproteins are shed 867 from Streptococcus uberis independently of Lsp[END_REF]. 230

Structural information is not available for any of the lipoprotein modification enzymes, 231 including Lsp. Other bacterial signal peptidases, such as signal peptidase I (leader peptidase) 232 and type IV pilin signal peptidase (prepilin peptidase), share their membrane-embedded 233 characteristics and the hydrophobic nature of their substrates. The catalytic mechanism 234 [START_REF] Paetzel | Structure and mechanism of Escherichia coli type I signal peptidase[END_REF] and the Ser/Lys active site [START_REF] Wang | Escherichia coli signal peptide peptidase A is a serine-lysine protease with a 1447 lysine recruited to the nonconserved amino-terminal domain in the S49 protease family[END_REF] of leader peptidase have been 235

reported. A soluble version of the enzyme, lacking its two N-terminal transmembrane 236 segments, is active in vitro [START_REF] Kuo | Escherichia coli 1106 leader peptidase: production of an active form lacking a requirement for detergent and 1107 development of peptide substrates[END_REF], although slightly less compared to full-length 237 protein and it requires detergents or phospholipids for its activity [START_REF] Tschantz | Characterization 1416 of a soluble, catalytically active form of Escherichia coli leader peptidase: requirement of 1417 detergent or phospholipid for optimal activity[END_REF]. 238

Several crystal structures of the soluble form have been solved [START_REF] Kim | Crystal structure of a bacterial signal Peptide 1100 peptidase[END_REF] and a recent 239 NMR structure suggests that the signal peptide remains in the bilayer in an unstructured 240 conformation and that the active site of leader peptidase functions at the membrane surface 241 (De [START_REF] Bona | Structural 859 studies of a signal peptide in complex with signal peptidase I cytoplasmic domain: the 860 stabilizing effect of membrane-mimetics on the acquired fold[END_REF] or well within the lipid bilayer [START_REF] Paetzel | Structure and mechanism of Escherichia coli type I signal peptidase[END_REF]. The membrane is 242 probably important in stabilizing the signal peptide while under reconstituted conditions the 243 detergents or phospholipids would serve an analogous role. Several inhibitors have been 244

identified including penem-type inhibitors [START_REF] Allsop | Inhibitors of bacterial 718 signal peptidase: a series of 6-(substituted oxyethyl)penems[END_REF] and lipopeptides (Paetzel et 245 al., 2002). Differences in specificity between leader peptidases from different species may be 246 explained by structural variations in the active site [START_REF] Auclair | Signal peptidase I: cleaving the way to 733 mature proteins[END_REF]. Prepilin peptidase is, 247 like Lsp, an aspartic protease that belongs to the class of aspartyl protease I-CLiPs (Erez et 248 al., 2009) and is involved in processing of pre-pilin proteins [START_REF] Lapointe | The type 4 prepilin peptidases comprise a novel family 1128 of aspartic acid proteases[END_REF]. I-249 membrane proteins that can function as regulatory molecules, and are found in many different 251 organisms [START_REF] Erez | How intramembrane proteases bury hydrolytic reactions 894 in the membrane[END_REF]. Prepilin peptidase is an integral membrane protein and 252 processing of prepilin has been postulated to occur at the cytoplasmic side of the membrane, 253 allowing the subsequent addition of methyl groups onto phenylalanine [START_REF] Strom | Mapping of export signals of Pseudomonas aeruginosa pilin 1361 with alkaline phosphatase fusions[END_REF]. 254

A topology model was proposed based on data obtained with reporter fusion proteins and 255 topology prediction programs; prepilin peptidases have eight transmembrane segments with 256 their N-and C-termini are located in the periplasm, and the catalytic Asp dyad is facing the 257 cytoplasm [START_REF] Lapointe | The type 4 prepilin peptidases comprise a novel family 1128 of aspartic acid proteases[END_REF]. The crystal structure of aspartyl protease FlaK, has 258 recently been solved [START_REF] Hu | The crystal structure of GXGD membrane protease FlaK[END_REF]. The two active site Asp residues are located on top of 259 two α-helices, housed in a hydrophilic cavity in the membrane and facing the cytoplasm, 260 similar to the structure of presenilin [START_REF] Hu | The crystal structure of GXGD membrane protease FlaK[END_REF][START_REF] Lu | Three-dimensional structure of human gamma-1142 secretase[END_REF]. Lsp probably shares 261 structural features with both leader peptidase and aspartyl proteases. It interacts with 262 diacylglyceryl-prolipoprotein as leader peptidase with signal peptides of secreted proteins in 263 such a way that the diacylglyceryl-cysteine is accessible to the Asp dyad active site as in 264 aspartyl proteases, with the only difference that the active site is facing the periplasm. 265 266

Globomycin -a specific inhibitor of Lsp 267

Globomycin was first identified in 1978, as an antibiotic produced by various Streptomyces 268 strains with spheroplast-forming (global-shape) activity on E. coli (Inukai et al., 1978, Inukai 269 et al., 1978). It specifically inhibits proteobacteria such as E. coli (MIC 0.4 µg/ml for E. coli 270 B) but not Pseudomonas aeruginosa. It is also less active against firmicutes (for example, 271 MIC >100 µg/ml for S. aureus and B. subtilis) (Inukai et al., 1978). Globomycin is a 272 lipophilic cyclic peptide with molecular formula C 32 H 57 N 5 O 2 and a molecular weight of 655 273 [START_REF] Nakajima | Globomycin, a new 1200 peptide antibiotic with spheroplast-forming activity. III. Structural determination of 1201 globomycin[END_REF] (Fig. 3). Indications that the target of globomycin is Lsp were 274 12 reported in the last paper in a series of articles on globomycin discovery and its 275 characterization by Mamoru Arai's group (Inukai et al., 1978). It was later shown that 276 diacylglyceryl-proLpp that accumulated in the cytoplasmic membrane in cells grown in the 277 presence of globomycin [START_REF] Hussain | Accumulation of glyceride-containing 1025 precursor of the outer membrane lipoprotein in the cytoplasmic membrane of 1026 Escherichia coli treated with globomycin[END_REF] could be converted into mature protein in vitro 278 when cell envelope fractions were incubated under optimal conditions. The gene encoding 279

Lsp was identified in E. coli by selecting clones from the Carbon-Clarke collection for 280 globomycin resistance, or by screening for complementation of a strain that is temperature 281 sensitive in the processing of diacylglyceryl-proLpp [START_REF] Tokunaga | Isolation and characterization of an 1405 Escherichia coli overproducing prolipoprotein signal peptidase[END_REF], Yamagata et al., 282 1983). Kinetics studies showed that globomycin inhibits Lsp in a non-competitive manner 283 with a K i of 36 nM, and a K m value of 6 µM was determined for diacylglyceryl-prolipoprotein 284 substrate [START_REF] Dev | Inhibition of prolipoprotein signal peptidase by 871 globomycin[END_REF]. Globomycin is mycobactericidal at elevated concentrations but 285 killing is independent of Lsp in M. tuberculosis, suggesting an alternative pathway of 286 inhibition in this bacterium [START_REF] Banaiee | LspA-independent action of globomycin on 740 Mycobacterium tuberculosis[END_REF]. Globomycin analogues have been 287

described that are more potent against proteobacteria and exhibit antibacterial activity against 288 firmicutes [START_REF] Kiho | Synthesis and 1092 antimicrobial activity of novel globomycin analogues[END_REF][START_REF] Kiho | Structure-1096 activity relationships of globomycin analogues as antibiotics[END_REF]. A long alkyl side chain, the lipophilic part of 289 the molecule, increases activity and also shows moderate activity against S. aureus. The 290 hydroxyl group of L-Ser is essential but not that of L-allo-Thr [START_REF] Kiho | Structure-1096 activity relationships of globomycin analogues as antibiotics[END_REF]. The lipid 291 moiety of globomycin is probably required to anchor the antibiotic into the membrane to 292 allow correct positioning of the peptidic part for interaction with the enzyme, but also seems 293 to play a direct role in globomycin-enzyme interaction. Differences in membrane composition 294 and/or membrane topology of Lsp might explain differences in antibacterial activities of 295 globomycin and its analogues on Lsp of different bacterial species. Another antibiotic, TA or 296 myxovirescin isolated from Myxococcus xantus, has been shown to target Lsp (Xiao et al., 297 2012). Antibiotic TA is a cyclic macrolactam lactone that is active against proteobacteria and 298 some firmicutes [START_REF] Rosenberg | Bactericidal action of an antibiotic 1306 produced by Myxococcus xanthus[END_REF]. 299 The membrane localization of Lnt of E. coli was demonstrated by Gupta andWu in 1991 302 (Gupta &Wu, 1991), in the same study it was demonstrated that Lnt has a broad pH optimum 303 (pH 6.5-7.5) and that it requires the presence of detergents for its activity in vitro. In 2005 the 304 membrane topology of Lnt and the phenotype of an lnt depletion mutant was reported 305 [START_REF] Robichon | Depletion of apolipoprotein N-1298 acyltransferase causes mislocalization of outer membrane lipoproteins in Escherichia 1299 coli[END_REF]. Several essential residues in Lnt were identified that are mainly 306 located in the catalytic periplasmic domain and include the catalytic triad E-K-C (Vidal-307 Ingigliardi et al., 2007). It was shown that growth of the lnt depletion mutant could be 308 restored by Lnt homologues of proteobacteria, but not actinobacteria (Vidal-Ingigliardi et al., 309 2007). The reaction catalyzed by Lnt involves two steps. In the first step, the sn-1 of PE reacts 310 with the active site cysteine of Lnt resulting in the formation of a thioester-acyl enzyme 311 intermediate and a lysophospholipid by-product [START_REF] Jackowski | Transfer of fatty acids from the 1-position of 1070 phosphatidylethanolamine to the major outer membrane lipoprotein of Escherichia coli[END_REF], Buddelmeijer & 312 Young, 2010[START_REF] Hillmann | Kinetics and phospholipid specificity 1004 of apolipoprotein N-acyltransferase[END_REF]. In the second step, the acyl group is transferred to 313 apolipoprotein resulting in triacylated, mature lipoprotein [START_REF] Buddelmeijer | The essential Escherichia coli Apolipoprotein N-793 acyltransferase (Lnt) exists as an extracytoplasmic thioester acyl-enzyme intermediate[END_REF]. 314

Kinetics studies showed that Lnt follows a ping-pong mechanism whereby lyso-phospholipid 315 produced in the first reaction is released before the second substrate, apolipoprotein, is bound 316 and modified. With PE and a synthetic diacylated peptide (FSL-1) a K m value of 8.0 µM and 317 a V max value of 350 µmol FSL-1 min -1 µmol -1

Lnt were determined, corresponding to a specific 318 activity of 5.6 µmol FSL-1 min -1 mg -1 Lnt . Interestingly, similar kinetics were obtained with PagP 319 that catalyzes the transfer of an acyl group from PE to lipid A [START_REF] Bishop | Transfer of 765 palmitate from phospholipids to lipid A in outer membranes of gram-negative bacteria[END_REF]. Although 320 acyltransferases Lnt and PagP are involved in completely different pathways, their substrates 321 are highly abundant; 5 x 10 5 molecules of Lpp and 2 x 10 6 molecules of lipid A per cell 322 [START_REF] Braun | Chemical characterization, spatial distribution and function of 774 a lipoprotein (murein-lipoprotein) of the E. coli cell wall[END_REF][START_REF] Raetz | Molecular genetics of membrane phospholipid synthesis[END_REF]. Furthermore, similar K m values have been reported for 323 Lsp and Lnt of E. coli for diacylglyceryl-proLpp and apo-lipopeptide, respectively (Dev et al., 324 1985[START_REF] Hillmann | Kinetics and phospholipid specificity 1004 of apolipoprotein N-acyltransferase[END_REF]. 325

Lnt is highly efficient in the second step of the reaction but less in the first step: k cat /K m of 4.8 326

x 10 3 for PE versus 7.3 x 10 5 for FSL-1. Based on the kinetic data, the number of Lnt 327 molecules per cell was estimated between 100 and 200, indicating that the N-acyltransferase 328 reaction is fast and efficient since lipoproteins are very abundant. PE is the preferred substrate 329 with an optimal fatty acid chain length of C16-18, more specifically palmitate or oleate with a 330 saturated acyl chain in position sn-1 and a non-saturated acyl chain at sn-2 (Jackowski & 331 Rock, 1986[START_REF] Hillmann | Kinetics and phospholipid specificity 1004 of apolipoprotein N-acyltransferase[END_REF]. Phospholipids with small polar headgroups can also serve 332 as acyl donor (Gupta et al., 1991[START_REF] Hillmann | Kinetics and phospholipid specificity 1004 of apolipoprotein N-acyltransferase[END_REF]. PagP and LpxA, which is involved 333 in the first step of lipopolysaccharide biosynthesis, possess a carbon-ruler that makes these 334 enzymes highly specific for the chain length of the acyl donor substrate [START_REF] Wyckoff | Hydrocarbon rulers in UDP-N-1500 acetylglucosamine acyltransferases[END_REF], 335 Ahn et al., 2004[START_REF] Williams | Structural basis for the acyl chain selectivity and 1476 mechanism of UDP-N-acetylglucosamine acyltransferase[END_REF]. It is unlikely that Lnt has carbon ruler capacity 336 since the acyl chain length and composition of the phospholipid substrate are variable. On the 337 other hand, the S-diacylglyceryl moiety of the apolipoprotein substrate probably determines 338 substrate specificity due to variations in lipid composition depending on the bacterial species. 339

The gene encoding Lnt was first identified in S. enterica by screening the same library as 340 described for lgt for the accumulation of apol-Lpp [START_REF] Gupta | Characterization of a temperature-954 sensitive mutant of Salmonella typhimurium defective in apolipoprotein N-955 acyltransferase[END_REF]. The mutation causing 341 the temperature sensitive defect in Lnt was a substitution of glutamate 435 to lysine; a similar 342 mutation in E. coli did not result in a temperature sensitive phenotype [START_REF] Robichon | Depletion of apolipoprotein N-1298 acyltransferase causes mislocalization of outer membrane lipoproteins in Escherichia 1299 coli[END_REF]. 343

Lnt activity in Actinomycetes was first reported in M. smegmatis [START_REF] Tschumi | 1425 Identification of apolipoprotein N-acyltransferase (Lnt) in Mycobacteria[END_REF]. A 344 strain of M. smegmatis deleted for lnt was viable and resulted in the accumulation of the 345 apolipoprotein form of two model lipoproteins LppX and LprF, directly demonstrating its N-346 acyltransferase activity [START_REF] Tschumi | 1425 Identification of apolipoprotein N-acyltransferase (Lnt) in Mycobacteria[END_REF][START_REF] Brülle | Cloning, expression and charaterization of Mycobacterium tuberculosis 786 lipoprotein LprF[END_REF] [START_REF] Brülle | Lipoproteins of slow-growing Mycobacteria 781 carry three fatty acids and are N-acylated by apolipoprotein N-acyltransferase 782 BCG_2070c[END_REF]. Phospholipids of mycobacteria possess mainly 349 octadecanoic acid and tuberculostearic acid at the sn-1 position and palmitate at the sn-2 350 position [START_REF] Okuyama | Positional distribution of fatty acids in 1225 phospholipids from Mycobacteria[END_REF], suggesting that Lnt has different substrate specificity in 351 these species. Lnt of M. smegmatis was unable to complement a conditional lnt null mutant of 352 E. coli, suggesting that indeed these enzymes have different substrate specificity. 353 Furthermore, the enzyme was correctly inserted into the cytoplasmic membrane and shown to 354 have a similar membrane topology as Lnt of E. coli [START_REF] Baulard | In vivo 752 interaction between the polyprenol phosphate mannose synthase Ppm1 and the integral 753 31 membrane protein Ppm2 from Mycobacterium smegmatis revealed by a bacterial two-754 hybrid system[END_REF]. Among the 355 essential residues in Lnt of E. coli, W237 and Y388 are not conserved in Lnt of M. smegmatis 356 [START_REF] Vidal-Ingigliardi | Identification of essential 1439 residues in apolipoprotein N-acyltransferase, a member of the CN hydrolase family[END_REF][START_REF] Tschumi | 1425 Identification of apolipoprotein N-acyltransferase (Lnt) in Mycobacteria[END_REF]. These residues were shown to be 357 involved in the second step of the reaction, N-acylation of apolipoprotein, suggesting they 358 might play a role in substrate specificity [START_REF] Buddelmeijer | The essential Escherichia coli Apolipoprotein N-793 acyltransferase (Lnt) exists as an extracytoplasmic thioester acyl-enzyme intermediate[END_REF]. The lnt gene in M. 359 smegmatis (MSMEG_3860 or ppm2) is located adjacent to ppm1 encoding polyprenol 360 monophosphomannose (Ppm) synthase that is involved in the transfer of mannose from GDP-361 mannose to polyprenol phosphate, an important step in the biosynthesis of mannose-362 containing lipoglycans [START_REF] Gurcha | Ppm1, a novel polyprenol monophosphomannose 959 synthase from Mycobacterium tuberculosis[END_REF]. Ppm synthase is essential for viability in M. 363 smegmatis [START_REF] Rana | Ppm1-encoded 1285 polyprenyl monophosphomannose synthase activity is essential for lipoglycan synthesis 1286 and survival in mycobacteria[END_REF]. 364

In M. tuberculosis ppm1 (Rv2051c) encodes a two-domain protein, in which the N-terminal 365 domain is Lnt and the C-terminal domain Ppm synthase. The Lnt domain of Ppm1 enhances 366 Ppm synthase activity [START_REF] Gurcha | Ppm1, a novel polyprenol monophosphomannose 959 synthase from Mycobacterium tuberculosis[END_REF]. In mycobacteria where Lnt and Ppm synthase are 367 encoded by two genes, the proteins were shown to interact [START_REF] Baulard | In vivo 752 interaction between the polyprenol phosphate mannose synthase Ppm1 and the integral 753 31 membrane protein Ppm2 from Mycobacterium smegmatis revealed by a bacterial two-754 hybrid system[END_REF]. In both 368 cases Lnt is thought to stabilize the Ppm synthase [START_REF] Gurcha | Ppm1, a novel polyprenol monophosphomannose 959 synthase from Mycobacterium tuberculosis[END_REF][START_REF] Baulard | In vivo 752 interaction between the polyprenol phosphate mannose synthase Ppm1 and the integral 753 31 membrane protein Ppm2 from Mycobacterium smegmatis revealed by a bacterial two-754 hybrid system[END_REF]. 369

A second lnt-like gene could be identified in M. tuberculosis (Rv2261c and Rv2262c) and M. 370 bovis (BCG_2279c), but the active site cysteine residue is replaced by serine (Brülle et al., 371 2013). The role of these enzymes in lipoprotein modification is currently unknown. glycosylated [START_REF] Espitia | Identification, isolation and partial characterization of 897 Mycobacterium tuberculosis glycoprotein antigens[END_REF][START_REF] Herrmann | Bacterial 992 glycoproteins: a link between glycosylation and proteolytic cleavage of a 19 kDa antigen 993 from Mycobacterium tuberculosis[END_REF], Gonzalez-Zamorano et al., 375 2009[START_REF] Sartain | N-Terminal clustering of the O-glycosylation sites in the 1330 Mycobacterium tuberculosis lipoprotein SodC[END_REF][START_REF] Wehmeier | Glycosylation of the phosphate binding protein, PstS, in 1460 Streptomyces coelicolor by a pathway that resembles protein O-mannosylation in 1461 eukaryotes[END_REF][START_REF] Brülle | Cloning, expression and charaterization of Mycobacterium tuberculosis 786 lipoprotein LprF[END_REF]. Recent findings 376 demonstrated a direct correlation between N-acylation and glycosylation of lipoproteins in 377

Corynebacterium glutamicum [START_REF] Mohiman | The ppm operon is essential for acylation and glycosylation of lipoproteins in 1193 Corynebacterium glutamicum[END_REF]. Lnt (Cg-Ppm2) of C. glutamicum 378 catalyzes N-acylation of apolipoproteins by addition of a palmitoyl moiety similar to M. 379 smegmatis [START_REF] Tschumi | 1425 Identification of apolipoprotein N-acyltransferase (Lnt) in Mycobacteria[END_REF][START_REF] Mohiman | The ppm operon is essential for acylation and glycosylation of lipoproteins in 1193 Corynebacterium glutamicum[END_REF]. Lipid modifications in C. 380 glutamicum included a diacylglyceryl moiety (C16:0, C18:1) and a N-palmitoyl group 381 (C16:0) similar to lipoproteins of E. coli [START_REF] Mohiman | The ppm operon is essential for acylation and glycosylation of lipoproteins in 1193 Corynebacterium glutamicum[END_REF]. N-acylation of lipoproteins 382

was not affected in a Cg-ppm1 mutant but proteins were no longer O-glycosylated with 383 hexose units, indicating that Ppm synthase is required for glycosylation of lipoproteins in C. 384 glutamicum. In a Cg-ppm2 mutant lipoproteins are only diacylated and not glycosylated 385 [START_REF] Mohiman | The ppm operon is essential for acylation and glycosylation of lipoproteins in 1193 Corynebacterium glutamicum[END_REF]. A Cg-ppm1 deletion mutant does not synthesize lipoglycans, 386 furthermore, their biosynthesis is also affected in the absence of Cg-ppm2 (Lnt), probably 387 because Ppm synthase is less stable under these conditions [START_REF] Gibson | Disruption of Cg-Ppm1, a polyprenyl monophosphomannose synthase, 927 and the generation of lipoglycan-less mutants in Corynebacterium glutamicum[END_REF]. 388

Interestingly, Ppm1 of M. tuberculosis is able to complement both N-acylation and 389 glycosylation defects in C. glutamicum [START_REF] Mohiman | The ppm operon is essential for acylation and glycosylation of lipoproteins in 1193 Corynebacterium glutamicum[END_REF] and to restore N-acylation in 390 M. smegmatis [START_REF] Tschumi | 1425 Identification of apolipoprotein N-acyltransferase (Lnt) in Mycobacteria[END_REF]. The Lnt domain of Ppm1 alone does not restore N-391 acylation but does restore glycosylation in C. glutamicum. This strengthens the interpretation 392 that the activities of Lnt and Ppm synthase are tightly linked and illustrate that these enzymes 393 operate in a common pathway implicated in envelope biosynthesis. 394 395 All Streptomyces species have two lnt genes [START_REF] Widdick | Dissecting the complete lipoprotein 1473 biogenesis pathway in Streptomyces scabies[END_REF]. In S. scabies, Lnt1 is 396 responsible for N-acylation of lipoproteins since lipoproteins are diacylated in lnt1 and 397 lnt1lnt2 mutants. In the absence of Lnt2, however, both di-and triacylated proteins were 398 (Widdick et al., 399 2011). Since these bacteria undergo different developmental stages, gene expression and 400 activity of lipoprotein modifying enzymes might be tightly regulated depending on 401 environmental conditions. In this respect it is interesting to note that the degree of fatty acid 402 acylation varies greatly in firmicutes depending on growth conditions. For example, 403 diacylated lipoproteins accumulated in S. aureus during late-exponential growth at low pH 404 (Kurokawa et al., 2012). New protein synthesis is required for both accumulation of 405 diacylated proteins and N-acylation of these proteins during pH upshift. These data suggest 406 that regulation of lnt expression or pH-mediated enzyme activity control N-acylation of 407 lipoproteins. Assuming that phospholipids are also substrate for N-acylation in firmicutes, 408

Lnt-like enzymes of these species probably have different specificities for the acyl-donor 409 substrate than those of proteobacteria. 410 411 After completion of the Lnt reaction, the lysophosphatidylethanolamine, or 2-acyl-412 glycerophosphoethanolamine (2-acyl-GPE) by-product can either be degraded by 413 lysophospholipase PldB [START_REF] Hsu | Isolation and characterization of Escherichia coli K-1018 12 mutants lacking both 2-acylglycerophosphoethanolamine acyltransferase and acyl-1019 acyl carrier protein synthetase activity[END_REF] or recycled in the synthesis of phospholipid via the 414 acyl-acyl carrier protein synthetase/2-acylglycerophosphoethanolamine acyltransferase 415 pathway [START_REF] Cooper | 2-Acylglycerolphosphoethanolamine 822 acyltransferase/acyl-acyl carrier protein synthetase is a membrane-associated acyl 823 carrier protein binding protein[END_REF][START_REF] Jackowski | Sequence and function of the aas gene in 1074 Escherichia coli[END_REF]. 2-acyl-GPE first needs to be 416 translocated to the inner leaflet of the cytoplasmic membrane, a process catalyzed by LplT, 417 before it can be used as a substrate for PL synthesis [START_REF] Harvat | 974 Lysophospholipid flipping across the Escherichia coli inner membrane catalyzed by a 975 transporter (LplT) belonging to the major facilitator superfamily[END_REF]. The Aas enzyme is 418 a cytoplasmic membrane protein [START_REF] Cooper | 2-Acylglycerolphosphoethanolamine 822 acyltransferase/acyl-acyl carrier protein synthetase is a membrane-associated acyl 823 carrier protein binding protein[END_REF]) that contains both enzymatic activities 419 with acyltransferase located at the N-terminal end and acyl ACP synthetase located at the C-420 terminal end [START_REF] Jackowski | Sequence and function of the aas gene in 1074 Escherichia coli[END_REF]. In the first step of the reaction acyl-ACP synthetase 421 adds saturated fatty acids onto ACP and in the second step fatty acid is transferred to the sn-1 422 position of 2-acyl-GPE by the acyltransferase resulting in the formation of new PE. 423 Alternatives for removal of 2-acyl-GPE are the production of PE and GPE from two 424 molecules of GPE [START_REF] Homma | Synthesis of various phospholipids from 2-acyl 1015 lysophospholipids by Escherichia coli extract[END_REF], and another phospholipase that degrades 2-425 acyl-GPE [START_REF] Doi | Lysophospholipase of Escherichia coli[END_REF]. 426 427

Sorting of lipoproteins to the outer membrane 428

The majority of lipoproteins reside in the inner leaflet of the outer membrane (Tokuda et al., 429 2007), although recent examples of surface-exposed lipoproteins have been reported (see 430 below). They reach this cellular location by a specific lipoprotein outer membrane 431 localization pathway (Lol) that is composed of five proteins: a cytoplasmic ATP-ase LolD, 432 two cytoplasmic membrane proteins LolC and LolE, a periplasmic chaperone LolA and an 433 outer membrane receptor LolB (Fig. 4). Sorting and translocation of lipoproteins by Lol is 434 dependent on the lipoprotein-sorting signal determined by specific residues located C-435 terminally to the diacylglyceryl-cysteine. The Lol pathway has been described in great detail 436 in several excellent reviews [START_REF] Tokuda | Bacterial lipoproteins: biogenesis, virulence/pathogenicity and 1397 trafficking. Bacterial membranes: structural and molecular biology[END_REF][START_REF] Zuckert | Secretion of Bacterial Lipoproteins: Through the Cytoplasmic 1532 Membrane, the Periplasm and Beyond[END_REF] and will only be briefly 437 discussed in light of functionality with enzymes involved in lipoprotein modification. N-438 acylation of lipoproteins by Lnt is essential for the Lol-dependent release of lipoproteins from 439 the cytoplasmic membrane (Fukuda et al., 2002), however, overexpression of lolCDE can 440 compensate for lack of N-acylation, in the absence of Lpp, and results in the release of 441 diacylated lipoproteins from the membrane and correct outer membrane localization (Narita 442 & Tokuda, 2011). However, the efficiency of outer membrane targeting is dependent on the 443 nature of the lipoprotein [START_REF] Narita | Overexpression of LolCDE allows deletion of the Escherichia 1207 coli gene encoding apolipoprotein N-acyltransferase[END_REF]. Model lipoproteins containing an outer 444 membrane-sorting signal are not localized in the outer membrane, neither released from the 445 cytoplasmic membrane in absence of Lnt. This suggests that the N-acyl moiety of lipoproteins 446 plays an important role in lipoprotein translocation through the periplasm. Mature lipoprotein 447 first interacts with LolE, is then transferred to LolC, and upon ATP hydrolysis by LolD, the 448 (Okuda & 449 Tokuda, 2009). The interaction between LolA and lipoprotein is weaker than the interaction 450 between LolB and lipoprotein, driving transfer from LolA to LolB and preventing back-451 transfer to LolCE [START_REF] Taniguchi | Mechanisms underlying energy-1380 independent transfer of lipoproteins from LolA to LolB, which have similar unclosed 1381 {beta}-barrel structures[END_REF]. It is unknown how lipoproteins interact with the 452 ABC transporter LolCDE and by which mechanism lipoproteins are released from the 453 cytoplasmic membrane. Crystal structures of LolA and soluble LolB, lacking its lipid-anchor, 454 have been solved [START_REF] Takeda | Crystal 1376 structures of bacterial lipoprotein localization factors, LolA and LolB[END_REF]. The calculated size of the cavity is predicted to 455 accommodate one but is not large enough for three fatty acid chains. Structure-function 456 analyses of LolA of P. aeruginosa showed that one large hydrophobic surface patch is likely 457 to be involved in binding of acyl chains [START_REF] Remans | Hydrophobic surface patches on LolA of 1294 Pseudomonas aeruginosa are essential for lipoprotein binding[END_REF]. Decreasing its hydrophobic 458 nature abolished LolA-dependent release of lipoproteins from the cytoplasmic membrane 459 [START_REF] Remans | Hydrophobic surface patches on LolA of 1294 Pseudomonas aeruginosa are essential for lipoprotein binding[END_REF]. The fatty acid acyl moiety of lipoprotein, together with its sorting 460 signal, clearly plays a role in substrate recognition by the Lol machinery. LolC and LolE 461 share a similar membrane topology and their periplasmic domains share sequence identity 462 with each other as well as with LolB [START_REF] Okuda | Model of mouth-to-mouth transfer of bacterial lipoproteins 1213 through inner membrane LolC, periplasmic LolA, and outer membrane LolB[END_REF]. LolA interacts specifically 463 with LolC and not with LolE. Differences in binding affinity between LolE and LolC for 464 lipoprotein, similar as described for LolA and LolB, may explain the molecular mechanism of 465 transfer. Characterization of the lipid-protein interaction by structural-and biophysical studies 466 is likely to provide more insight into the mechanism of Lol-dependent targeting of 467 lipoproteins to the outer membrane. 468

Inhibitors of the Lol machinery were identified in a chemical genomic approach (Pathania et 469 al., 2009) which utilized a combination of three in vitro assays [START_REF] Ito | A new screening method to identify inhibitors of the Lol (localization of 1062 lipoproteins) system, a novel antibacterial target[END_REF]. Treatment 470 of cells with MAC13243 led to the accumulation of Lpp in the cytoplasmic membrane. It 471 directly interacts with LolA [START_REF] Pathania | Chemical genomics in Escherichia coli identifies an 1257 inhibitor of bacterial lipoprotein targeting[END_REF]. Further characterization of this compound 472 led to the finding that a degradation product is the active species [START_REF] Barker | Degradation of MAC13243 and studies of the interaction of resulting thiourea 748 compounds with the lipoprotein targeting chaperone LolA[END_REF]. This 473 (Barker et al., 474 2013) and seems to inhibit bacterial growth in a non-specific fashion. 475

The Lol machinery is not fully conserved in proteobacteria, LolB is only found in βand γ-476 proteobacteria and also LolCE are often missing [START_REF] Okuda | Lipoprotein sorting in bacteria[END_REF]. Proteomics 477 analyses showed that lipoproteins reside in the outer membrane of diderm actinomycetes but 478 the sorting mechanism is completely unknown. 479 480

Lipoproteins involved in key processes of envelope biosynthesis 481

The most abundant lipoprotein in γ-proteobacteria is Lpp that is covalently linked to murein 482 (peptidoglycan) and the first protein shown to be lipidated [START_REF] Braun | Chemical characterization, spatial distribution and function of 774 a lipoprotein (murein-lipoprotein) of the E. coli cell wall[END_REF]. Lpp is also 483 the most abundant protein in the cell and plays an important role in homeostasis of the cell 484 envelope. A recent study identified a σ E -regulated small RNA, MicL, which specifically 485 targets lpp [START_REF] Guo | MicL, a new 944 sigmaE-dependent sRNA, combats envelope stress by repressing synthesis of Lpp, the 945 major outer membrane lipoprotein[END_REF]. The mechanism of regulation allows decrease of Lpp 486 translation and acceleration of degradation of the stable lpp mRNA. σ E up-regulates proteins 487 that facilitate assembly of outer membrane proteins and transport of LPS under stress 488 conditions. At the same time it down-regulates OMP synthesis by up-regulation of sRNAs 489 such as MicL [START_REF] Guo | MicL, a new 944 sigmaE-dependent sRNA, combats envelope stress by repressing synthesis of Lpp, the 945 major outer membrane lipoprotein[END_REF]. A strain deleted for lpp is very sensitive to EDTA and 490 cationic dyes and somewhat sensitive to detergents and it also leaks periplasmic proteins into 491 the extracellular space [START_REF] Hirota | On the process of cellular division in 1007 Escherichia coli: A mutant of E. coli lacking a murein-lipoprotein[END_REF][START_REF] Yem | Physiological characterization of an Escherichia coli mutant 1521 altered in the structure of murein lipoprotein[END_REF]. One-third of Lpp is covalently 492 linked to peptidoglycan, while two-third is in the cell envelope but not anchored to the cell 493 wall, the so-called free form [START_REF] Inouye | The assembly of a structural lipoprotein in the 1037 envelope of Escherichia coli[END_REF]. There is a dynamic equilibrium between 494 the two forms and the protein is homogeneously distributed over the cell (Hiemstra et al., 495 1986[START_REF] Hiemstra | Distribution of 1000 newly synthesized lipoprotein over the outer membrane and the peptidoglycan sacculus 1001 of an Escherichia coli lac-lpp strain[END_REF]. The enzymes responsible for cross-linking Lpp to peptidoglycan 496 were identified in 2008 [START_REF] Magnet | Identification of the L,D-1152 transpeptidases for peptidoglycan cross-linking in Escherichia coli[END_REF]. YbiS was shown to catalyze the covalent 497 anchoring of Lpp to peptidoglycan, but two other enzymes ErfK and YcfS could also perform 498 this reaction. These enzymes belong to the family of L,D-transpeptidases found in firmicutes 499 [START_REF] Mainardi | A novel peptidoglycan cross-linking enzyme for a beta-1161 lactam-resistant transpeptidation pathway[END_REF][START_REF] Magnet | Specificity of L,D-transpeptidases from 1156 gram-positive bacteria producing different peptidoglycan chemotypes[END_REF]. YbiS is expected to cleave the peptide bond 500 between meso-DAP and D-Ala and to link meso-DAP to the L-Lys residue in Lpp (Magnet et 501 al., 2008). Mis-localization of Lpp in the cytoplasmic membrane is lethal when cross-linked 502 to the peptidoglycan [START_REF] Yakushi | Lethality of the covalent linkage 1511 between mislocalized major outer membrane lipoprotein and the peptidoglycan of 1512 Escherichia coli[END_REF]. The highly abundant Lpp homologue in P. 503 aeruginosa, OprI exist in a free form and is not covalently cross-linked to peptidoglycan 504 [START_REF] Mizuno | Isolation and characterization of major outer 1188 membrane proteins of Pseudomonas aeruginosa strain PAO with special reference to 1189 peptidoglycan-associated protein[END_REF][START_REF] Cornelis | Cloning and analysis of the gene for the major outer membrane lipoprotein from 832 Pseudomonas aeruginosa[END_REF]. When produced in E. coli it is exposed on 505 the surface of cells [START_REF] Cornelis | Development of new cloning vectors for 835 the production of immunogenic outer membrane fusion proteins in Escherichia coli[END_REF]. Interestingly, the free form of E. coli Lpp is also 506 exposed on the cell surface [START_REF] Cowles | The free and bound 847 forms of Lpp occupy distinct subcellular locations in Escherichia coli[END_REF], although detection of Lpp by immunogold 507 labeling was only successful upon permeabilization of the outer membrane with TRIS-EDTA 508 [START_REF] Hiemstra | Induction kinetics and cell surface 996 distribution of Escherichia coli lipoprotein under lac promoter control[END_REF]. Lpp is an elongated trimer composed of long α-helical domains that 509 adopt a coiled coil, which reflects its repetitive characteristics [START_REF] Braun | Sequence of the murein-lipoprotein and the attachment site 778 of the lipid[END_REF], Choi et 510 al., 1986). The last amino acids of the C-terminus form a hydrophobic capping motif that is 511 important for binding to PG and possibly involved in the recognition of lipoprotein by ligase 512

YbiS [START_REF] Zhang | Alterations of the carboxyl-terminal amino acid residues of 1524 Escherichia coli lipoprotein affect the formation of murein-bound lipoprotein[END_REF][START_REF] Shu | Core structure of the outer membrane lipoprotein from 1348 Escherichia coli at 1.9 A resolution[END_REF]. It is thus likely that Lpp has a dual orientation, 513 similar as described for peptidoglycan-associated Lipoprotein P6 of Haemophilus influenza 514 [START_REF] Michel | Dual orientation of the outer membrane lipoprotein 1175 P6 of nontypeable haemophilus influenzae[END_REF], although the correlation between surface-exposed orientation and 515 biological function is not yet known. 516

Surface exposed lipoproteins are abundant in Borrelia, which lacks LPS in the outer 517 membrane (for a recent review see [START_REF] Zuckert | Secretion of Bacterial Lipoproteins: Through the Cytoplasmic 1532 Membrane, the Periplasm and Beyond[END_REF]). The N-terminal membrane-tethering 518 peptide keeps lipoproteins of Borrelia burgdorferi in a translocation compatible state that 519 allow them to be secreted to the cell surface probably via a flippase complex. Several other 520 examples of surface-exposed lipoproteins have been reported in proteobacteria, including 521 proteins that are translocated via type II secretion machinery (PulA) [START_REF] Pugsley | Extracellular pullulanase of Klebsiella 1271 pneumoniae is a lipoprotein[END_REF] or 522 via an autotransporter mechanism (NalP and SphB1) (Coutte et al., 2003, van ). In case of NalP of Neisseria meningitidis, it was demonstrated that lipidation is 524 required for efficient release of surface-exposed proteins by delaying its own release from the 525 cell surface through autocatalytic processing [START_REF] Roussel-Jazede | Lipidation 1309 of the autotransporter NalP of Neisseria meningitidis is required for its function in the 1310 release of cell-surface-exposed proteins[END_REF]. Similarly, non-526 lipidated SphB1 is efficiently secreted but is unable to cleave its substrate filamentous 527 haemagglutinin FHA (Coutte et al., 2003), which is important for virulence of Bordetella 528 pertussis (Coutte et al., 2003). 529

The Lpt pathway transports LPS from the cytoplasmic membrane to the cell surface (Ruiz et 530 al., 2009). LptE is an essential OM lipoprotein that stabilizes the LptD OM channel 531 responsible for correct translocation of LPS [START_REF] Bos | Identification of an outer 769 membrane protein required for the transport of lipopolysaccharide to the bacterial cell 770 surface[END_REF], Chng et al., 2010). Recent 532 studies showed that LptE forms a plug inside the β-barrel structure of LptD (Freinkman et al., 533 2011) and that it changes the biophysical state of LPS to allow transport and OM insertion 534 [START_REF] Malojcic | LptE 1169 binds to and alters the physical state of LPS to catalyze its assembly at the cell surface[END_REF]. The N-terminal lipid anchor is not required for function since a variant 535 of LptE lacking the signal sequence including the cysteine complements a lptE deletion strain 536 and is able to form a complex with LptD (Chng et al., 2010). LptD is inserted into the OM by 537 the β-barrel assembly machine (Bam complex) (Chimalakonda et al., 2011). Four lipoproteins 538 (BamB-E) are part of the Bam complex, of which BamD is essential for the assembly and/or 539 insertion of proteins in the OM [START_REF] Malinverni | YfiO 1165 stabilizes the YaeT complex and is essential for outer membrane protein assembly in 1166 Escherichia coli[END_REF]. BamC is surface exposed (Webb et 540 al., 2012). How the lipid moiety is orientated in the outer membrane is still an open question. 541 Biosynthesis of peptidoglycan is catalyzed by several membrane-bound enzymes, among 542 which the bifunctional penicillin-binding proteins PBP1A and PBP1B. These enzymes each 543 require an outer membrane lipoprotein, LpoA and LpoB, respectively, for their function 544 [START_REF] Paradis-Bleau | Lipoprotein cofactors located in the outer membrane activate bacterial cell wall 1247 polymerases[END_REF][START_REF] Typas | Regulation of 1429 peptidoglycan synthesis by outer-membrane proteins[END_REF]. The Lpo proteins bind specifically to their 545 cognate PBP and stimulate transpeptidase activity. LpoB with an inner membrane retention 546 signal is functional as long as the flexible N-terminal domain is present [START_REF] Typas | Regulation of 1429 peptidoglycan synthesis by outer-membrane proteins[END_REF]. It 547 is currently unkown whether non-lipidated LpoA is functional. 548

The role of lipoprotein modification in virulence 550

The role of individual lipoproteins on virulence of pathogenic bacteria has been studied (for a 551 review see [START_REF] Kovacs-Simon | Lipoproteins of bacterial pathogens[END_REF]). Signaling of the host immune system by 552 lipoproteins and lipopeptides occurs via Toll-like receptor 2, either via TLR2-1 in case of 553 diacylated proteins or via TLR2-6 heterodimers in case of triacylated proteins. The lipid 554 moiety plays an important role in the interaction with these receptors [START_REF] Jin | Crystal structure 1081 of the TLR1-TLR2 heterodimer induced by binding of a tri-acylated lipopeptide[END_REF], Kang et 555 al., 2009). In recent years the effect of lipoprotein modification on virulence has been 556 addressed using lgt and lsp deletion mutants of pathogenic bacteria and depending on the 557 pathogen and the infection model used, various effects on virulence were obtained. 558

For example, impaired colonization of the nasopharynx and a decrease in virulence in 559 pneumonia and septicaemia mouse models was reported for Streptococcus pneumoniae 560 deleted for lgt [START_REF] Petit | Lipid modification of 1260 prelipoproteins is dispensable for growth in vitro but essential for virulence in 1261 Streptococcus pneumoniae[END_REF], Chimalapati et al., 2012). TLR-2 signaling is dependent on 561 pneumococcal lipoproteins and NF-κB and cytokine release were reduced in a ∆lgt stain 562 [START_REF] Tomlinson | TLR-Mediated Inflammatory 1412 Responses to Streptococcus pneumoniae Are Highly Dependent on Surface Expression 1413 of Bacterial Lipoproteins[END_REF]. Intracellular survival of Listeria monocytogenes was affected 563 [START_REF] Baumgärtner | 757 Inactivation of lgt allows systematic characterization of lipoproteins from Listeria 758 monocytogenes[END_REF] and the ∆lgt strain was attenuated in a mouse model (Machata et 564 al., 2008). Lipoproteins are released into the culture supernatant of this stain (Baumgärtner et 565 al., 2007). Inactivation of lsp in L. monocytogenes led to a severe growth defect in 566 macrophages and a reduced capacity of phagosomal escape leading to attenuated virulence 567 [START_REF] Reglier-Poupet | 1289 Maturation of lipoproteins by type II signal peptidase is required for phagosomal escape 1290 of Listeria monocytogenes[END_REF]. Expression of the lsp gene was strongly induced inside 568 phagosomes probably caused by environmental conditions similar to pH-regulation of N-569 acylation in S. aureus [START_REF] Reglier-Poupet | 1289 Maturation of lipoproteins by type II signal peptidase is required for phagosomal escape 1290 of Listeria monocytogenes[END_REF], Kurokawa et al., 2012). Inefficient 570 germination of spores of a lgt deletion strain of Bacillus anthracis led to attenuation of 571 virulence [START_REF] Okugawa | Lipoprotein biosynthesis by prolipoprotein diacylglyceryl transferase is required 1221 for efficient spore germination and full virulence of Bacillus anthracis[END_REF]. Lipoproteins of an lsp deletion mutant of M. tuberculosis 572 are not processed and the strain was attenuated in an animal model of tuberculosis (Sander et 573 , 2004[START_REF] Rampini | LspA 1281 inactivation in Mycobacterium tuberculosis results in attenuation without affecting 1282 phagosome maturation arrest[END_REF]. Reduced innate immune activation was observed with a ∆lgt 574 strain of Streptococcus suis (Wichgers [START_REF] Schreur | Lgt 1468 processing is an essential step in Streptococcus suis lipoprotein mediated innate 1469 immune activation[END_REF], but no difference in virulence 575 was observed between a wild type strain and a lsp deletion mutant in an infection model in 576 piglets [START_REF] De Greeff | 863 Lipoprotein signal peptidase of Streptococcus suis serotype 2[END_REF]. Deletion of either lgt or lsp in Streptococcus sanguinis has 577 only a slight effect on endocarditis virulence [START_REF] Das | Contribution of 855 lipoproteins and lipoprotein processing to endocarditis virulence in Streptococcus 856 sanguinis[END_REF]. No significant effect on 578 virulence was observed in Streptococcus equi [START_REF] Hamilton | Mutation of the maturase lipoprotein attenuates the 966 virulence of Streptococcus equi to a greater extent than does loss of general lipoprotein 967 lipidation[END_REF], Das et al., 2009). 579

Escape of immune recognition caused lethal infections by a ∆lgt strain of S. aureus but a ∆lsp 580 strain was attenuated [START_REF] Bubeck-Wardenburg | Host defenses against 789 Staphylococcus aureus infection require recognition of bacterial lipoproteins[END_REF]. Enhanced lethality was shown in a 581 mouse model for neonatal sepsis by ∆lgt of Streptococcus agalactiae [START_REF] Henneke | Lipoproteins are critical TLR2 activating 989 toxins in group B streptococcal sepsis[END_REF]. 582

Stimulation of the immune system via TLR2 during early stages of infection is important for 583 elimination of pathogens. The examples described here clearly show that both the lipid 584 moiety and the functional characteristics of lipoproteins determine virulence capacity. 585 586

Protein lipidation in other microbes 587

Lipoproteins are predicted in Archaea, but orthologous genes for lipoprotein modifying 588 enzymes could not been identified in their genomes. Recent findings in Haloferax volcanii 589

show that the Tat-dependent secretion of predicted lipoproteins depends on the conserved 590 cysteine in the putative lipobox [START_REF] Gimenez | Haloferax volcanii twin-arginine 931 translocation substates include secreted soluble, C-terminally anchored and 932 lipoproteins[END_REF][START_REF] Storf | Mutational and 1358 bioinformatic analysis of haloarchaeal lipobox-containing proteins[END_REF]. Future studies will 591 provide direct evidence for lipidation of these proteins and will demonstrate whether 592 phospho-, glyco-or phospho-glyco-lipids that constitute archaeal membranes, and are 593 substantially different from their bacterial counterparts, are the source of modification 594 [START_REF] Villanueva | A re-evaluation of the archaeal membrane 1443 lipid biosynthetic pathway[END_REF]. 595

In eukaryotes, including yeast, various types of protein lipidation exist including S-596 palmitoylation, N-myristoylation and S-prenylation [START_REF] Aicart-Ramos | Protein palmitoylation and 711 subcellular trafficking[END_REF]. In the 597 process of S-palmitoylation, palmitate is transferred onto a cysteine residue catalyzed by 598 domains that results in the formation of a thioester-linked palmitoylated protein (Dietrich & 600 Ungermann, 2004). The PAT enzymes are polytopic integral membrane proteins located in 601 the ER or Golgi. The active site is facing the cytoplasm where acyl-CoA functions as the acyl 602 donor. Multiple DHHC enzymes have been identified in Saccharomyces cerevisiae but their 603 substrate specificity is still poorly understood. Recent work on the Erf2-Erf4 PAT complex in 604 S. cerevisiae showed that it catalyzes a two-step reaction in which Erf2 undergoes auto-605 acylation [START_REF] Mitchell | Mutational analysis of 1178 Saccharomyces cerevisiae Erf2 reveals a two-step reaction mechanism for protein 1179 palmitoylation by DHHC enzymes[END_REF]. Erf2 transfers the palmitate group onto the substrate and Erf4 606 is required for stabilization of Erf2 and for the formation of the Erf2-palmitate intermediate 607 [START_REF] Mitchell | The 1183 Erf4 subunit of the yeast Ras palmitoyl acyltransferase is required for stability of the 1184 Acyl-Erf2 intermediate and palmitoyl transfer to a Ras2 substrate[END_REF]. S-palmitoylation is a reversible process due to the action of acyl-608 protein thioesterases. Together these two processes allow for regulated control of membrane-609 associated proteins, such as small GTPase Ras, the function of which depends on their lipid 610

anchor. 611

Several examples have been described of bacterial effector proteins that contain eukaryotic 612 lipidation motifs and that are modified by lipids upon entry into host cells. A farnesyl or 613 geranylgeranyl isopreny lipid moiety is added to cysteine residue of the conserved sequence 614 leading to enhanced membrane affinity and localization of effector proteins to membranes of 615 host cells [START_REF] Ivanov | Lipidation by the host 1066 prenyltransferase machinery facilitates membrane localization of Legionella 1067 pneumophila effector proteins[END_REF]. 616 617

Concluding remarks 618

Lipoproteins are highly abundant in bacteria, and require therefore efficient maturation. The 619 acyltransferases and signal peptidase involved in this pathway are not abundant, estimated to 620 only a few hundred molecules per cell. It is very likely that these enzymes exist in a complex 621 to guarantee complete and efficient lipoprotein modification. The existence of a lipoprotein 622 modification complex was already suggested in 1982 [START_REF] Tokunaga | Post-translational modification and 1401 processing of Escherichia coli prolipoprotein in vitro[END_REF]. This presumed 623 including the Sec and Tat secretion machineries. In this respect it is intriguing to understand 625 how the lipoprotein modification machinery recognizes non-folded-and fully folded 626 substrates that are translocated via Sec and Tat, respectively. The signal peptide of both types 627 of lipoprotein substrates including the early mature region of the precursor protein is thought 628 to exist in a flexible conformation inside the translocon. Lateral opening of the Sec channel 629 can be envisaged for insertion of lipoproteins into the lipid bilayer [START_REF] Chatzi | SecA-mediated targeting and 800 translocation of secretory proteins[END_REF], 630 whether lateral gating of the Tat complex occurs is currently unknown [START_REF] Patel | Protein transport by the bacterial Tat pathway[END_REF]. 631

Once inserted into the membrane, Lgt binds the signal peptide and the first few amino acids 632 of the mature protein and catalyzes the first step in the lipidation pathway. 633Several examples clearly demonstrate that some essential lipoproteins are perfectly functional 634 without their lipid anchor what raises the following questions: what is the role of the lipid 635 moiety, and how does the degree of lipidation influence protein function? Unlike membrane 636 proteins, lipoproteins are expected to be more mobile within lipid bilayers giving them more 637 flexibility, although this has not been experimentally tested. For lipoproteins in monoderm 638 bacteria it has been postulated that the N-terminal lipid anchor keeps the protein close to the 639 membrane while the extended functional domain participates in various biological processes. 640 Many lipoproteins in these organisms fulfill equivalent functions as periplasmic proteins in 641 diderm bacteria. Accessory proteins such as LptE and LpoA/B interact strongly with their 642 partner proteins, for which the lipid part is not essential, although the process in which they 643 are involved might be more efficient when these proteins are present in lipidated form. In the 644 case of lipoproteins that themselves fulfill specific functions and not in complex with other 645 proteins, such as surface-exposed proteases or targeting of proteins to the OM, the lipid 646 anchor becomes essential. The lipoprotein modification pathway seems to be tightly regulated 647 since a lipoprotein can exist in both di-or triacylated form in one bacterial species, depending 648 27 on growth conditions. The fact that multiple copies of genes encoding modification enzymes 649 exist in bacteria that undergo various developmental stages also suggest that regulation of 650 their expression is environmentally controlled. 651

The lipoprotein modification pathway represents an attractive target for antibiotic 652 development. It is essential for the viability of all proteobacteria examined to date, which 653 include many important human pathogens. Furthermore, Lsp and Lnt are membrane-bound 654 with catalytic domains facing the bacterial periplasm. This topology makes at least two steps 655 of lipoprotein biogenesis accessible to small molecules that will be capable of inhibiting 656 enzymes without a need to traverse the relatively impermeable cytoplasmic membrane or risk 657 elimination by the action of efflux pumps. For future studies on lipoprotein modification and 658 function it is important to include proteomic analysis of membrane-bound and secreted 659 lipoproteins and to determine the lipidation state and lipid composition by mass spectrometry 660 [START_REF] Cain | Beyond gene expression: the impact of protein 797 post-translational modifications in bacteria[END_REF]. These approaches not only allow the identification and characterization of 661 specific lipoproteins that act as virulence factors but also demonstrate a correlation between 662 lipidation and the stage of infection. Structural and mechanistic properties of enzymes 663 involved in lipidation of proteins in various organisms will highlight similarities and 664 differences and will provide ways of finding new species specific antibacterial agents. [START_REF] Gan | The 917 umpA gene of Escherichia coli encodes phosphatidylglycerol: prolipoprotein 918 diacylglyceryl transferase (lgt) and regulates thymidylate synthase levels through 919 translational coupling[END_REF] lsp [START_REF] Tokunaga | Isolation and characterization of an 1405 Escherichia coli overproducing prolipoprotein signal peptidase[END_REF][START_REF] Yamagata | Cloning and expression of a gene coding 1515 for the prolipoprotein signal peptidase of Escherichia coli[END_REF] lnt (cutE) (Gupta & Wu, 1991[START_REF] Rogers | Cloning and 1302 characterization of cutE a gene involved in copper transport in Escherichia coli[END_REF], Gupta et al., 1993[START_REF] Robichon | Depletion of apolipoprotein N-1298 acyltransferase causes mislocalization of outer membrane lipoproteins in Escherichia 1299 coli[END_REF]) S. enterica lgt [START_REF] Gan | Isolation and characterization 913 of a temperature-sensitive mutant of Salmonella typhimurium defective in 914 prolipoprotein modification[END_REF] lnt [START_REF] Gupta | Characterization of a temperature-954 sensitive mutant of Salmonella typhimurium defective in apolipoprotein N-955 acyltransferase[END_REF]) M. xantus lsp1, lsp2, lsp3, lsp4 (Xiao & Wall, 2014) M. smegmatis lgt (MSMEG_3222) [START_REF] Tschumi | Functional 1420 analyses of mycobacterial lipoprotein diacylglyceryl transferase and comparative 1421 secretome analysis of a mycobacterial lgt mutant[END_REF] lnt (MSMEG_3860) (ppm2) [START_REF] Tschumi | 1425 Identification of apolipoprotein N-acyltransferase (Lnt) in Mycobacteria[END_REF] M. tuberculosis lgt (Rv1614) [START_REF] Sassetti | Genes required for mycobacterial growth 1333 defined by high density mutagenesis[END_REF][START_REF] Tschumi | Functional 1420 analyses of mycobacterial lipoprotein diacylglyceryl transferase and comparative 1421 secretome analysis of a mycobacterial lgt mutant[END_REF] lsp (Rv1539) [START_REF] Sander | Lipoprotein processing is required for 1320 virulence of Mycobacterium tuberculosis[END_REF][START_REF] Rampini | LspA 1281 inactivation in Mycobacterium tuberculosis results in attenuation without affecting 1282 phagosome maturation arrest[END_REF] ppm1 (Rv2051c) [START_REF] Tschumi | 1425 Identification of apolipoprotein N-acyltransferase (Lnt) in Mycobacteria[END_REF] M. bovis lnt (BCG_2070c) [START_REF] Brülle | Lipoproteins of slow-growing Mycobacteria 781 carry three fatty acids and are N-acylated by apolipoprotein N-acyltransferase 782 BCG_2070c[END_REF]) C. glutamicum lnt (NCgl1424) [START_REF] Mohiman | The ppm operon is essential for acylation and glycosylation of lipoproteins in 1193 Corynebacterium glutamicum[END_REF] S. coelicolor lgt1 (SCO2034), lgt2 (SCO7822) [START_REF] Thompson | Investigating lipoprotein biogenesis and function in the model Gram-positive 1385 bacterium Streptomyces coelicolor[END_REF] lsp (SCO2074) [START_REF] Thompson | Investigating lipoprotein biogenesis and function in the model Gram-positive 1385 bacterium Streptomyces coelicolor[END_REF] lnt1 (SCO1014), lnt2 (SCO1336) [START_REF] Widdick | Dissecting the complete lipoprotein 1473 biogenesis pathway in Streptomyces scabies[END_REF][START_REF] Cordova-Davalos | Lipoprotein N-acyl transferase (Lnt1) is dispensable for 827 protein O-mannosylation by Streptomyces coelicolor[END_REF]) S. scabies lgt (SCAB68531) [START_REF] Widdick | Dissecting the complete lipoprotein 1473 biogenesis pathway in Streptomyces scabies[END_REF] lsp (SCAB68121) [START_REF] Widdick | Dissecting the complete lipoprotein 1473 biogenesis pathway in Streptomyces scabies[END_REF] lnt1 (SCAB83111), lnt2 (SCAB76621) [START_REF] Widdick | Dissecting the complete lipoprotein 1473 biogenesis pathway in Streptomyces scabies[END_REF]) S. lividans lsp [START_REF] Gullon | Transcriptional characterisation of the negative 939 effect exerted by a deficiency in type II signal peptidase on extracellular protein 940 secretion in Streptomyces lividans[END_REF]) Essential genes are shown in red, non-essential genes are shown in blue. Table includes only

2

  Bacterial lipoproteins are exported proteins and often exposed on the bacterial cell surface 25

  ), phosphatidylglycerol (PG), cardiolipin (CL) and minor 75

  Based on studies with Lpp, the consensus sequence of the lipobox was 100 determined as: (L) -3 -(A/S) -2 -(G/A) -1 -C +1 , in which the lipid-modified cysteine at position +1 101

  in the membrane, however, it is currently unknown how Lgt interacts 125

  of the antibiotic TA biosynthetic cluster

  in intramembrane proteolysis liberating membrane-tethered domains of 250

  and Streptomyces are often, in addition to being acylated, 374

  a role for Lnt2 in efficient N-acylation of lipoproteins

  and LolA leads to charging of LolA with lipoprotein

  an analog of A22, an inhibitor of bacterial actin MreB

  (PAT) containing DHHC (Asp-His-His-Cys) cysteine rich 599

  -localize and function together with other essential protein complexes 624

Figure 1 .Figure 2 .Figure 3 .Figure 4 .Page

 1234 Figure legends 671

  published findings. See text for further details.

  double mutant could not be obtained suggesting that Lgt activity is essential in S. coelicolor 150

	F o
	r
	P
	e e r
	R e v i e w
	149	. A
	7

  . The Lsp 225 enzymes can complement an lsp depletion strain of E. coli only in the absence of Lpp 226 suggesting that Lsp-s of M. xanthus are less efficient than Lsp of E. coli. Lipoproteins of 227

	228	Streptococcus uberis are processed by Eep, a homologue of enhanced expression of
		pheromone from Enterococcus faecalis

  . Lnt of mycobacteria 347 transfers either palmitate or tuberculostearic acid (in M. bovis) from phospholipids onto 348

	apolipoprotein
	F o
	r
	P
	e e r
	R e v i e w
	15

  Ulsen et al., 523

	2003
	F o
	r
	P
	e e r
	R e v i e w
	22

Table 1 .

 1 Proceedings of the National Academy of Sciences of 805 the United States of America 108: 2492-2497. 806 807 Chimalapati S, Cohen JM, Camberlein E, MacDonald N, Durmort C, Vernet T, Hermans 808 PW, Mitchell T & Brown JS (2012) Effects of deletion of the Streptococcus pneumoniae 809 lipoprotein diacylglyceryl transferase gene lgt on ABC transporter function and on 810 growth in vivo. PLoS One 7: e41393. 811 812 Chng SS, Ruiz N, Chimalakonda G, Silhavy TJ & Kahne D (2010) Characterization of the 813 two-protein complex in Escherichia coli responsible for lipopolysaccharide assembly at 814 the outer membrane. Proceedings of the National Academy of Sciences of the United States Lipoteichoic acid and lipids in the membrane of Staphylococcus 901 aureus. Medical microbiology and immunology 183: 61-76. 902 903 Freinkman E, Chng SS & Kahne D (2011) The complex that inserts lipopolysaccharide 904 into the bacterial outer membrane forms a two-protein plug-and-barrel. Proceedings of 905 the National Academy of Sciences of the United States of America 108: 2486-2491. 906 907 Fukuda A, Matsuyama S-I, Hara T, Nakayama J, Nagasawa H & Tokuda H (2002) 908 Aminoacylation of the N-terminal cysteine is essential for Lol-dependent release of 909 lipoproteins from membranes but does not depend on lipoprotein sorting signals. J Biol Essentiality of genes encoding lipoprotein modifying enzymes in diderm bacteria.

	F o
	r
	P
	e e r
	R e v i e w

ScholarOne Support 1-434/964-4100FEMS Microbiology Reviews

ScholarOne Support 1-434/964-4100

Acknowledgements 667 I thank Joel Berry, Richard Wheeler and Nicolas Bayan for constructive comments and all 668 members of the Biology and Genetics of the Bacterial Cell Wall Unit at the Institut Pasteur 669 for their support. 670