Chapter 66

Biosynthesis of Lipoproteins

HENRY C. WU

INTRODUCTION

Murein lipoprotein of Escherichia coli was the first example of a lipid-modified protein in which the structure of a covalently bound lipid and its linkage to a protein was elucidated (6, 33). In the ensuing years since the discovery of murein lipoprotein, it has become increasingly clear that the unique structure of the covalently linked lipid, N-acyl-diacylglycerylcysteine, is shared by a large number of structurally and functionally diverse lipoproteins in bacteria. These lipoproteins from a large variety of bacterial species presumably share a common biosynthetic pathway to generate the unique N-terminal lipid-modified amino acid, N-acyl-diacylglycerylcysteine. Much of the current knowledge of the structure and biogenesis of bacterial lipoproteins was obtained from biochemical and genetic studies of murein lipoprotein as a prototype. A number of reviews have addressed various aspects of the structure and biogenesis of lipoproteins in bacteria (8, 36, 91). This chapter will summarize recent progress in the studies of lipoprotein biogenesis in E. coli and Salmonella typhimurium (official designation, Salmonella enterica serovar Typhimurium).

LIPOPROTEINS IN E. COLI

Murein lipoprotein was discovered by virtue of its covalent attachment to peptidoglycan (6). It was subsequently discovered that this protein also exists as the so-called free form in the outer membrane of E. coli (45). Because of its abundance in E. coli cells, it is also referred to as the major outer membrane lipoprotein in E. coli. Fatty acid analyses of murein lipoprotein revealed that the amide-linked fatty acid is predominantly palmitate, whereas the fatty acids in the thioether-linked diacylglyceryl moiety are very similar in composition to those of phospholipids (33). As of early 1993, more than 130 lipoproteins had been identified (8). However, apart from the murein lipoprotein, only a few lipoproteins have been studied by chemical and physical methods for the characterization of the lipid-modified amino acid. In the majority of other lipoproteins, the presumptive evidence for the lipoprotein nature of a given protein comes from one or more of the following studies: (i) visual inspection of the deduced amino acid sequence from a cloned gene showing a cysteine residue in the C-terminal portion of a signal sequence as part of a lipoprotein modification and processing consensus sequence (the lipobox), (ii) demonstration of an inhibition of the processing of the precursor protein to the mature form by globomycin (48), a specific inhibitor of prolipoprotein signal peptidase (or signal peptidase II) (102), (iii) incorporation of [2-³H]glycerol or [³H]palmitate into the putative lipoprotein, and the demonstration of both alkali-labile and alkali-resistant linkages of the [³H]palmitate to the lipoprotein (36), and (iv) identification of glycerylcysteine in the acid hydrolysate of the lipoprotein (36).

On the basis of metabolic labeling of E. coli cell envelope proteins with [³H]palmitate or [2-³H]glycerol and the inhibition of the processing by globomycin, Ichihara et al. identified seven "new" lipoproteins (NLP) (41) in addition to murein lipoprotein and a peptidoglycan-associated lipoprotein which is distinct from murein lipoprotein and is bound to peptidoglycan noncovalently (76). They vary greatly in amounts as well as in apparent molecular weights (from 52,000 [NLP1] to 16,000 [NLP7]) (41). Like murein lipoprotein and peptidoglycan-associated lipoprotein, four of these seven NLPs are located in the outer membrane of the E. coli cell envelope, while two of them are located in the cytoplasmic membrane.

As shown in Fig. 1 and summarized in Table 1, 13 genes encoding lipoproteins in E. coli have been cloned, mapped, and sequenced; a few of these correspond to the NLPs identified biochemically by Ichihara et al. (41). The nucleotide sequences of these genes reveal the presence of the lipobox in the signal sequences of their respective gene products. The primary structures of the mature lipoproteins are as unrelated as those of any other group of proteins, thus accounting for the lack of a common function for these proteins. While the subcellular localizations of the NLPs and some of the lipoproteins listed in Table 1 have been experimentally determined, their topology in a given membrane remains unknown. It is generally assumed that the lipid anchor at the N terminus is inserted into one leaflet of the lipid bilayer to place the otherwise hydrophilic protein at the interface of the membrane and an aqueous compartment; the functional importance of such an anchorage remains to be established.

Fig. 1Map positions of genes encoding lipoproteins and prolipoprotein modification and processing enzymes in E. coli. The genes for prolipoprotein modification and processing enzymes are in boldface.

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Table 1E. coli genes encoding membrane lipoproteins

In addition to the chromosomally encoded lipoproteins in E. coli, there are a number of plasmid- or phage-encoded lipoproteins (Table 1). Structurally and functionally related lipoproteins encoded by colicinogenic factors are involved in the release of colicins from cells harboring these plasmids. The TraT protein encoded by F plasmid is involved in the surface exclusion and serum resistance phenotype of cells harboring F plasmids. A lambda phage-encoded outer membrane lipoprotein is structurally and functionally related to a lipoprotein encoded by ColV2-K94 plasmid; both proteins appear to confer increased serum resistance on the microorganisms. Antigenically related proteins are expressed in E. coli lysogens of other lambdoid phages and in clinical isolates of E. coli (4).

While biochemical and molecular genetic studies have suggested the existence of 10 to 20 lipoproteins in E. coli, with the final number to be determined following the completion of the sequencing of the E. coli genome, the number of lipoproteins with the same lipid structure is increasing steadily on the basis of the criteria outlined above. Statistical analysis of the consensus sequence for prolipoprotein modification and processing reactions suggests that the sequence -Leu(Ala,Val)_–4-Leu_–3-Ala(Ser)_–2-Gly(Ala)_–1-Cys₊₁- (subscripts denote positions) in the signal sequence of a preprotein defines a lipobox which is recognized by the enzymes in the biosynthetic pathway that lead to the formation of N-acyl-diacylglycerylcysteine, a hallmark of bacterial lipoproteins. As mentioned above, the presence of the lipobox sequence in the deduced amino acid sequences of cloned genes has successfully predicted the lipoprotein nature of these proteins.

BIOSYNTHESIS OF LIPOPROTEINS IN E. COLI

Several key findings led to the elucidation of the biosynthetic pathway of lipoproteins in E. coli. Using a semi-in vitro system to study the biosynthesis of the E. coli major outer membrane lipoprotein, Inouye and coworkers identified the precursor form of murein lipoprotein, the prolipoprotein, with a 20-amino-acid signal sequence at its N terminus (47). Subsequently an E. coli lpp mutant (lppG14D) was isolated which contained unmodified and unprocessed prolipoprotein in its outer membrane due to a single amino acid substitution of Gly-14 by Asp (69). The discovery of globomycin as a specific inhibitor of the processing of prolipoprotein in E. coli (48) led to two important observations: (i) the diacylglyceryl-modified murein prolipoprotein accumulates in globomycin-treated cells (40), and (ii) in addition to murein lipoprotein, there are other lipoproteins in E. coli whose processing is inhibited by globomycin in a similar manner (41). These in vivo studies also led to the conclusion that diacylglyceryl modification of prolipoprotein may precede or indeed be a prerequisite for the processing of prolipoprotein by signal peptidase II. This conclusion was verified by the demonstration of posttranslational modification and processing of prolipoprotein in vitro and the identification of a prolipoprotein-specific signal peptidase activity which requires lipid-modified prolipoprotein as the substrate (16, 106). This unique requirement not only provides an explanation for the conservation of the lipobox sequence spanning the modification and cleavage site in the signal sequence of lipoprotein precursors but also stipulates that the specificity of the biosynthetic pathway lies mainly in the recognition of lipobox-containing precursor proteins by phosphatidylglycerol:prolipoprotein diacylglyceryltransferase, the first committed step in this pathway (93).

The biosynthetic pathway for the formation of mature lipoprotein stipulates that three enzymes are involved in the modification and processing of prolipoprotein to form the mature or fully modified lipoprotein (reference 93 and Fig. 2): phosphatidylglycerol:prolipoprotein diacylglyceryltransferase, prolipoprotein signal peptidase (or signal peptidase II), and phospholipid:apolipoprotein transacylase (or apolipoprotein N-acyltransferase). They are encoded by the lgt, lsp, and lnt genes, located at 64, 0.5, and 15 min on the E. coli chromosome, respectively (Fig. 1 and Table 2) (22, 28, 88, 114).

Fig. 2Proposed pathway for the biosynthesis of murein lipoprotein in E. coli.

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Table 2E. coli genes encoding prolipoprotein modification and processing enzymes

lgt

The lgt gene was identified in S. typhimurium as an open reading frame complementing a temperature-sensitive mutant defective in diacylglyceryl modification of the major outer membrane lipoprotein (22). It was found to be allelic with the E. coli umpA gene, a gene defined as encoding a 25-kDa essential membrane protein of unknown function (23, 111). The identification of temperature-sensitive alleles of lgt in both E. coli and S. typhimurium strongly suggests that functional Lgt activity is essential for the growth and viability of E. coli and S. typhimurium. Interestingly, both E. coli and S. typhimurium lgt(Ts) mutations are suppressed by lpp::Tn10 mutation (22, 23). This implies that in these temperature-sensitive lgt mutants, the removal of a major competing substrate (murein lipoprotein) for the limited capacity of the prolipoprotein modification enzyme allows the modification and subsequent processing of an essential but minor lipoprotein to proceed at a rate compatible with their growth and viability. A null mutant of this gene has yet to be isolated.

The predicted lgt gene products from both E. coli and S. typhimurium consist of 291 amino acid residues with a calculated molecular mass of 33 kDa, and there is a 92.8% amino acid sequence identity between the E. coli and the S. typhimurium proteins. The UmpA protein of E. coli has been reported to be 25 kDa in size on the basis of sodium dodecyl sulfate-polyacrylamide gel electrophoresis on analysis (111). The deduced amino acid sequence of the Lgt protein shows that it lacks a typical signal sequence at its NH₂ terminus but contains significant stretches of hydrophobic sequences interrupted by charged hydrophilic segments. The high pI (10.58) predicted for this protein and the presence of hydrophobic segments flanked by positively charged residues suggest that this protein can interact with acidic phospholipids by ionic as well as hydrophobic interactions. Phosphatidylglycerol is the major if not the exclusive diacylglyceryl donor in the prolipoprotein modification reaction catalyzed by the Lgt protein (93).

The lgt gene in E. coli and S. typhimurium is immediately 5' to the thyA gene, which encodes thymidylate synthase. The close linkage of these two genes (separated by 6 bp) suggests that they are cotranscribed and translationally coupled. Recent studies have shown that thymidylate synthase levels are regulated by transcription from the lgt promoter and by translation of lgt mRNA through overlap of the lgt open reading frame and the ribosome-binding site of the thyA mRNA (23). The physiological significance of the cotranscription and translational coupling of these two functionally unrelated genes is not obvious. In Staphylococcus aureus, the lgt gene is not immediately adjacent to the thyA gene (86a).

lsp

The lsp gene of E. coli was cloned on the basis of its ability to confer increased globomycin resistance to host cells and to complement a temperature-sensitive lsp mutant of E. coli (103, 112). The deduced amino acid sequence of E. coli Lsp shows that the E. coli signal peptidase II comprises 164 amino acid residues with a calculated molecular weight of 18,144 and lacking a typical signal sequence (44, 119). The enzyme has been purified to apparent homogeneity (16). The K_m of the purified enzyme for diacylglyceryl-modified murein prolipoprotein is 6 μM (16). Globomycin is a potent, reversible, and noncompetitive inhibitor of signal peptidase II with a K_i of 36 nM (15). Other protease inhibitors such as tosylarginyl methyl ester, pepstatin, HgCl₂, phenylmethylsulfonyl fluoride, chymostatin, and N-ethylmaleimide inhibit this enzyme only at much higher concentrations (16, 87).

Signal peptidase II is located in the cytoplasmic membrane of the E. coli cell envelope (104). Analysis of the hydropathic profile of this enzyme reveals the presence of four hydrophobic segments flanked by charged amino acid residues; it suggests that signal peptidase II is an integral membrane protein with four membrane-spanning segments connected by two periplasmic loops and one positively charged cytoplasmic loop and having both N-terminal and C-terminal positively charged segments located in the cytoplasm (44). The predicted topology is in conformity with the positive inside rule of von Heijne (108) and is verified experimentally by using a series of lsp-phoA and lsp-lacZ gene fusions (77).

The lsp homologs from Enterobacter aerogenes, Pseudomonas fluorescens, and Staphylococcus aureus have been cloned and sequenced (50, 51, 123). The hydropathic profiles of signal peptidase IIs from these bacterial species suggest the same membrane topology as that of the E. coli enzyme, even though each of the four corresponding membrane-spanning segments exhibits relatively low amino acid sequence homology. This observation suggests that an overall similar topology of this enzyme in the cytoplasmic membrane is important for its function, and this is achieved with a similar architecture of the polypeptide in terms of alternating hydrophobic segments and charged hydrophilic loops without a high degree of homology in its primary structure. Despite the overall low sequence homology among these four signal peptidase IIs, there are regions of high sequence homology, suggestive of conserved domains involved in the functioning of this enzyme either in the recognition of the substrates (diacylglycerylcysteine and the lipobox sequence in lipoprotein precursors) or in catalysis (92).

The lsp gene in E. coli is essential since inhibition of signal peptidase II by globomycin is lethal. E. coli strains containing a cloned lsp gene from E. coli or other bacterial species exhibit increased resistance to globomycin, and a spontaneous globomycin-resistant mutant of E. coli contained increased levels of signal peptidase II activity due to the transcription of the lsp gene from a new promoter (74, 105). In addition, a globomycin-resistant and temperature-sensitive allele of the E. coli lsp gene (lspD23G) has been shown to render the mutant cell conditionally lethal upon the expression of the lpp gene (113; R.-M. Dai and H. C. Wu, unpublished data). It is worth noting that the globomycin sensitivity of wild-type E. coli cells is greatly reduced upon the removal of murein lipoprotein as a major substrate of this pathway. E. coli lpp mutants with no or greatly reduced levels of murein lipoprotein or containing unmodifiable mutant prolipoprotein are globomycin resistant (61). On the other hand, E. coli lpp mutants including an lpp deletion mutant remain sensitive to globomycin at high concentrations (61). It is unlikely that the accumulation of lipid-modified Braun’s prolipoprotein causes the cell death in globomycin-treated wild-type cells or in an lsp mutant under the nonpermissive condition, since diacylglyceryl-modified murein prolipoprotein is covalently attached to the peptidoglycan (42, 49), suggesting that the bulk of the prolipoprotein molecules have already been translocated across the cytoplasmic membrane. Alternatively, the growth and viability of E. coli cells require modification and processing of essential, albeit minor, lipoprotein(s), which would account for the sensitivity of lpp mutants to high concentrations of globomycin. According to this hypothesis, the greater globomycin sensitivity of the wild-type E. coli strain than that of lpp mutants is due to the competition of the processing of major outer membrane prolipoprotein with that of the minor essential lipoprotein for the limited activity of signal peptidase II in globomycin-treated cells. Null alleles of the lsp gene have yet to be isolated.

The lsp gene is located at 0.5 min on the E. coli map (88, 114). It is part of a five-gene operon, x-ileS-lsp-orf149-lytB, with multiple promoters located upstream or within gene x for transcription of the entire operon and a promoter located within the ileS gene for the transcription of the lsp gene (30, 74, 75). For each promoter, there is a pair of transcripts with a common 5' end but different 3' ends, one terminating after the lsp gene and the other terminating after the lytB gene. This peculiar genomic organization of functionally unrelated genes (ileS for protein synthesis, lsp for prolipoprotein processing, and lytB involved in penicillin tolerance and control of stringent response) is also found in Enterobacter aerogenes and P. fluorescens but not in Staphylococcus aureus (50, 51, 123). The physiological significance of this genetic organization is unclear.

lnt

The lnt gene, which encodes apolipoprotein N-acyltransferase, was identified as an essential gene located at 14.5 min on the S. typhimurium chromosome (28). Comparison of the phenotypes of the lnt mutant with those of a cutE mutant of E. coli and the identification of a complementing clone derived from an E. coli genomic library for both mutations have led to the conclusion that the cutE gene (89), one of six genes involved in copper transport and homeostasis in E. coli (90), is allelic with the lnt gene. The nucleotide sequence of the E. coli cutE gene predicts a protein of 512 amino acid residues. Analysis of the deduced amino acid sequence does not reveal a typical signal sequence at its amino terminus or the presence of multiple membrane-spanning segments. The sequence does, however, contain hydrophobic amino acids at both N-terminal and C-terminal regions which may anchor this protein in the cytoplasmic membrane. Comparison of the deduced amino acid sequence of this enzyme with the data bank has identified a sequence motif similar to sequences in lipases and esterases from various sources (28).

Apolipoprotein N-acyltransferase is located in the cytoplasmic membrane of the E. coli cell envelope (29). Both in vivo and in vitro studies indicate that any of the three major phospholipids in the E. coli cell envelope (phosphatidylethanolamine, phosphatidylglycerol, and cardiolipin) could serve as an acyl donor (29, 62, 64). Furthermore, N-acylation of apolipoprotein proceeds at a normal rate in an E. coli strain which contains only a trace level of phosphatidylethanolamine due to a null mutation in the phosphatidylserine synthase gene (pss::kan) (27). It has been proposed that the 1-acyl moiety of phosphatidylethanolamine is the primary donor for N-acylation of apolipoprotein in vivo (52). Phosphatidylethanolamine, however, is not the obligatory or exclusive acyl donor for apolipoprotein N-acylation (27). Virtually nothing is known regarding the structural determinant in apolipoprotein as an acyl acceptor in this reaction.

The relationship between apolipoprotein N-acyltransferase activity and copper transport and homeostasis in E. coli and S. typhimurium remains to be elucidated. The deduced amino acid sequence of E. coli CutE protein contains the sequence H-F-Q-M-A-R-M, similar to the motif of H-X-X-M-X-X-M found in copper binding protein CopA from Pseudomonas syringae (72). The significance of this sequence in the activity of this enzyme and the possibility that this enzyme is a metalloenzyme remain to be examined. It is clear, however, that lnt (cutE) mutants of S. typhimurium and E. coli are not only temperature sensitive but also copper sensitive. Both phenotypes are complemented by the cloned lnt (cutE) gene and suppressed by lpp::Tn10 mutation or other lpp mutations which affect the structures or expression of the lpp gene (28). The temperature-sensitive and copper-sensitive phenotypes of the lnt (cutE) mutants suggest that certain lipoproteins in E. coli and S. typhimurium require N-acylation for their functions in the growth, division, or viability of bacterial cells as well as in copper transport and homeostasis.

EXPORT OF LIPOPROTEIN IN E. COLI

The biosynthesis of lipoprotein in bacteria is intimately related to its secretion, since the lipobox sequence defining prolipoprotein modification lies at the junction between the signal sequence and the mature portion of prolipoprotein. Like a typical signal sequence targeting precursor proteins to the cytoplasmic membrane in bacteria and to the endoplasmic reticulum in eukaryotic cells, the signal sequence of lipoprotein precursor has a tripartite structure, a positively charged N-terminal region, an uncharged hydrophobic region in the middle, and a C-terminal region which contains the specific determinants for the recognition and cleavage by distinct signal peptidases. The C-terminal region in each of the the signal sequences of prolipoproteins contains the lipobox which is recognized by prolipoprotein diacylglyceryltransferase; presumably, the alanyl (glycyl or seryl) diacylglycerylcysteine sequence in the diacylglyceryl-modified prolipoprotein is recognized by signal peptidase II.

Proteins which are exported through the cytoplasmic membrane utilize a common secretory or export machinery composed of SecA, SecB, SecD, SecE, SecF, and SecY proteins for targeting and translocation across the cytoplasmic membrane (94). The existence of a signal sequence in the lipoprotein precursor and the fact that the specificity of prolipoprotein modification and processing resides in the lipobox located at the C-terminal region of the signal sequence suggest that the Sec machinery is involved in the biogenesis of bacterial lipoproteins prior to the prolipoprotein modification and processing reactions. Such a temporal relationship between the translocation of prolipoprotein by the Sec machinery and the modification and processing reactions catalyzed by the three enzymes located in the cytoplasmic membrane is supported by studies using the conditionally lethal sec mutants. Murein lipoprotein belongs to the SecB-independent group of exported proteins (35, 109), while other lipoprotein precursors may require SecB protein for targeting and chaperone functions in their export (86). A temperature- or cold-sensitive defect in any of the secA, secD, secE, secF, and secY genes results in the accumulation of unmodified prolipoprotein at the nonpermissive temperature (35, 97, 109). These results indicate that the functions of Sec proteins are epistatic to those of the modification and processing enzymes; i.e., the lipoprotein precursors interact with the export machinery before they encounter the modification and processing enzymes located in the cytoplasmic membrane. Mutant prolipoproteins which are not modifiable with the diacylglyceryl moiety because of substitution of the invariant Cys with Ser or Gly undergo alternative processing by signal peptidase I (25, 34). This finding is consistent with the hypothesis that lipoprotein and nonlipoprotein precursors share a common export pathway before they are modified and/or processed by their respective enzymes. There is no evidence for compartmentalization of the export or processing machinery for lipoprotein and nonlipoprotein precursors.

Contrary to the prediction of this model that the modification and processing enzymes encounter their substrates (prolipoproteins) during or after their translocation by the Sec machinery, in vivo studies suggest that processing of diacylglyceryl-modified prolipoprotein by signal peptidase II requires functional SecA, SecD, SecE, SecF, and SecY proteins, but not SecB protein, as well as an intact proton motive force (60). Sodium azide, which is a specific inhibitor of SecA ATPase, does not inhibit the processing of lipid-modified prolipoprotein by signal peptidase II in vivo. The requirement of functional Sec proteins and proton motive force for the processing of lipid-modified prolipoprotein in vivo differs from the results of in vitro studies of signal peptidase II; purified signal peptidase II processes lipid-modified prolipoprotein without requirement of any cofactor. The discrepancy between in vivo and in vitro results remains to be resolved.

Both the Sec machinery and the biosynthetic enzymes are located in the cytoplasmic membrane of bacteria. Fully modified and processed Braun’s lipoprotein can be detected in the cytoplasmic membrane before it is translocated to the outer membrane (68). Mature lipoprotein is also synthesized in spheroplasts (55). At least two of the new lipoproteins identified by Ichihara et al. (41) were found to be located in the cytoplasmic membrane of the E. coli cell envelope. It has been proposed that the +2 residue of the mature lipoproteins is a sorting signal in bacterial lipoproteins. Lipoproteins with Ser at the +2 position are targeted to the outer membrane, whereas those with Asp at the +2 position are retained in the cytoplasmic membranes (116). Recent studies suggest that lipoproteins with Asp at the +2 position are enriched in distinct domains of the cell envelope which contain markers from both the cytoplasmic and the outer membranes (84), and other structural determinants in membrane proteins may contribute to sorting or retention of lipoproteins to a particular membrane localization (24). Most recently, Matsuyama et al. (70a) identified an E. coli periplasmic 20-kDa protein (p20) which is involved in the translocation of lipoproteins from the inner membrane to the outer membrane of E. coli. The gene (lplA) encoding this periplasmic carrier protein has been cloned and sequenced (70a) and is essential for cell growth (H. Tokuda, personal communication). Furthermore, a gene encoding a putative outer membrane receptor for the p20-Lpp complex has been identified (H. Tokuda, personal communication).

ASSEMBLY OF MUREIN LIPOPROTEIN IN E. COLI

Murein lipoprotein exists in two forms: (i) the free form in the outer membrane and (ii) the bound form covalently attached to the peptidoglycan via a peptide bond between the ε-amino group of the C-terminal lysine residue and the carboxyl group at the optically active l-center of meso-diaminopimelate in the pentapeptide side chain of the peptidoglycan (7). On average, every 10 to 12 murein subunits contain murein-bound lipoprotein in place of d-alanine in the side chain (6). Immunoelectron microscopic studies suggest that the distribution of murein lipoprotein is random over the peptidoglycan structure (37).

Not much is known about the biochemistry and genetics of the covalent attachment of murein lipoprotein to the peptidoglycan. Neither the gene encoding the putative murein lipoprotein:peptidoglycan ligase nor its gene product has been identified. The substrate requirement for this reaction has been studied by using site-specific lpp mutations altered in the structure of murein prolipoprotein. Neither the diacylglyceryl modification nor processing of prolipoprotein is required for the formation of murein-bound lipoprotein (121). Apolipoprotein lacking the amide-linked fatty acid is also attached to the peptidoglycan (28). However, the presence of a charged amino acid residue at the 14th position of the signal sequence of the prolipoprotein prevents the formation of bound-form lipoprotein (121). In addition, an alteration in the secondary structure in the vicinity of the modification and processing sites interferes with the formation of the bound-form lipoprotein (46, 121).

The C-terminal region of murein lipoprotein is highly conserved among homologs in members of the family Enterobacteriaceae. Studies using site-specific mutations in the C-terminal region of murein lipoprotein suggest that the positively charged C-terminal amino acid residues Tyr-76, Arg-77, and Lys-78 are important for the formation of the bound-form lipoprotein; introduction of negatively charged residues into this region affects the formation of the bound-form lipoprotein (122). In addition, the presence of a β-turn structure in this region results in a reduced formation of the bound-form lipoprotein. In contrast, internal amino acid residues (Leu-37 to Ala-57) are not essential for the attachment of lipoprotein to the peptidoglycan; a truncated lipoprotein resulting from an internal deletion in the lpp gene is modified, processed, and covalently attached to the peptidoglycan (120).

FUNCTIONS OF LIPOPROTEINS

The NLPs identified biochemically by Ichihara et al. (41) and those identified by cloning and sequencing (Table 1) vary greatly in their levels of expression and apparent molecular weights. Aside from the conserved sequence at the modification and processing site of each lipoprotein precursor (the lipobox), there is no apparent amino acid sequence homology among distinct lipoproteins. They are, therefore, not expected to have a common function. It is assumed that the lipid moiety at the N terminus serves to anchor an otherwise hydrophilic protein to the cytoplasmic or outer membrane at the interface of membrane and aqueous environment. Null mutations in lpp, nlpA, nlpB, nlpC, nlpD, nlpE, pal, and slp have been obtained (1, 4a, 5, 17, 38, 43, 65, 96a, 115); thus, these genes are not essential for the growth of E. coli under normal laboratory conditions. A number of lipoprotein mutants (lpp, excC [=pal], acrA, acrE [=envC]) exhibited phenotypes suggestive of defects in the structural and functional integrity of the bacterial cell envelope (20, 56, 58, 70, 78, 100, 117). It is intriguing that most of [³H]leucine-labeled proteins cross-linked to peptidoglycan with bifunctional cross-linking reagents were also [³H]palmitate labeled, i.e., presumably lipoproteins, suggesting that most cell envelope lipoproteins mediate noncovalent interactions between the murein sacculus and the inner or outer membrane of the cell envelope (67). Lpp (Braun’s lipoprotein), peptidoglycan-associated lipoprotein, and other cell envelope lipoproteins may be important in maintaining the outer membrane structure of gram-negative bacteria. A number of lipoproteins in E. coli are encoded by globally regulated genes, e.g., osmB, nlpD, and slp, and insights into their potential functions may be gained through a consideration of their respective regulatory signals.

While gram-negative bacteria utilize periplasmic binding proteins as essential components of transport systems, gram-positive bacteria devoid of an outer membrane cannot use the same mechanism. Interestingly, some of the binding proteins in gram-positive bacteria are anchored to the outer surface of the cytoplasmic membrane via the N-acyl-diacylglycerylcysteine at their N termini (26, 83, 95, 96, 99). Similarly, exported proteins often exist in two forms, the membrane-bound form containing the lipid anchor at the N terminus and the excreted form released into the medium (57, 63, 71, 80). The presence of exported proteins such as penicillinases from various Bacillus species as membrane-bound lipoproteins allows the concentration of such activities at the cell surface. In addition to the transport of sugar, iron, phosphate, and oligopeptides, other functions of lipoproteins in gram-positive bacteria include adherence to substrata, host tissues, or other bacteria; protein export; sensory mechanism or chemoreception in signal transduction systems; bacterial sporulation and germination; and bacterial conjugation (98). All of these functions require proteins to be located at the cell surface in order to interact with the environment or the host cells. The formation of a generic lipid anchor provides a simple mechanism of achieving such a surface topology of any protein, even a highly charged and hydrophilic protein.

Lipoproteins are also found to be components of secretory apparatus which translocate exported proteins across the outer membrane of gram-negative bacteria. Examples include PulS, XpsD, MxiJ, and YscJ (2, 14, 39, 73). Two other lipoproteins (PrsA and PrtM) are required for the formation of active secreted enzymes (31, 59). Finally, small lipoproteins facilitate the release of bacteriocins (9, 12, 32, 53, 82, 85, 107, 110), and plasmid- and phage-encoded proteins (TraT, Bor, and Iss) confer resistance to complement-mediated serum killing of the host cells (3, 4, 11, 19, 81).

UNANSWERED QUESTIONS

This chapter summarizes our current understanding of the biosynthesis and export of membrane lipoproteins in bacteria. The genes involved in the biogenesis of membrane lipoproteins in bacteria have been identified and partially characterized. Understanding of the biochemistry of the membrane-bound enzymes participating in the posttranslational modification and processing reactions is in its infancy. Two related unanswered questions remain. First, how many lipoproteins are there in E. coli and S. typhimurium, and second, which ones are essential and for what? The answer to the first question will come with the anticipated completion of the sequence determination of the E. coli genome in the near future. The second question is more challenging, and the answer will require genetic manipulation as well as biochemical insights derived from the sequences of the putative essential lipoprotein genes. The combined studies with lgt, lsp, and lnt mutants of E. coli and S. typhimurium and the effect of globomycin in an E. coli lpp deletion mutant suggest that modification and processing of certain, albeit minor, lipoproteins in bacteria are required for the growth and viability of bacterial cells.

The genes encoding prolipoprotein modification and processing enzymes in E. coli and S. typhimurium are not linked to one another. In fact, each of them belongs to transcriptional units with genes of apparently unrelated functions. Regulation of the synthesis or the activities of these enzymes is one of many unanswered questions regarding the biogenesis of bacterial lipoproteins. Except for signal peptidase II, nothing is known about the topology of Lgt and Lnt proteins in the cytoplasmic membrane or about the structure-function relationship of these enzymes. Comparison of homologous sequences from phylogenetically distinct species of bacteria and the use of site-specific mutagenesis to alter conserved amino acid residues in each enzyme may be a productive approach for such studies. In spite of the abundance of murein lipoprotein, the biosynthesis and export of this major outer membrane lipoprotein are extremely efficient. The possibility of the existence of an enzyme complex consisting of Lgt, Lsp, and Lnt plus other accessory proteins should be considered. Furthermore, in view of the apparent requirement of functional Sec proteins for the processing of diacylglyceryl-modified prolipoprotein in vivo (60), the interaction between the secretory machinery, which is shared by lipoprotein and nonlipoprotein precursors, and the enzymes involved in lipoprotein biosynthesis warrants further investigation.

ACKNOWLEDGMENT

The work in this laboratory cited in this chapter was supported by Public Health Service grant GM 28811 from the National Institutes of Health.