Biogenesis of membrane lipoproteins in Escherichia coli.

Globomycin-resistant mutants of Escherichia coli have been isolated and partially characterized. Approximately 2-5% of these mutants synthesize structurally altered Braun's lipoprotein. The majority of these mutants contain unprocessed and unmodified prolipoprotein. One mutant is found to contain modified, processed, but structurally altered lipoprotein. Mutants containing lipid-deficient prolipoprotein or lipoprotein also show increased resistance to globomycin. These results suggest that the inhibition of processing of modified prolipoprotein by globomycin may require fully modified prolipoprotein as the biochemical target of this novel antibiotic. Our failure to isolate mutant containing cleaved but unmodified lipoprotein among globomycin-resistant mutants is consistent with the possibility that modification of prolipoprotein precedes the removal of signal sequence by a unique signal peptidase. Recent evidence indicates that the minor lipoproteins in the cell envelope of E. coli are also synthesized as lipid-containing prolipoproteins and the processing of these prolipoproteins is inhibited by globomycin. These results suggest the existence of modifying enzymes in E. coli which would transfer glyceryl and fatty acyl moieties to cysteine residues located in the proper sequences of the precursor proteins. This speculation is confirmed by our demonstration that Bacillus licheniformis penicillinase synthesized in E. coli as well as in B. licheniformis is a lipoprotein containing glyceride-cysteine at its NH2-terminus.     

The penicillinase of Bacillus licheniformis is an outer membrane protein in Escherichia coli.

The cloned gene coding for Bacillus licheniformis penicillinase (penP) was introduced into Escherichia coli in a heat-inducible lambda Qam vector. After induction, significant amounts of penicillinase were synthesized in the new host. The cellular location of the penicillinase was found to be almost exclusively the outer membrane fraction of E. coli, and virtually no soluble penicillinase was found. According to sodium dodecyl sulfate-gel electrophoresis, the size of the penicillinase from E. coli was identical to that of the membrane-bound form of the B. licheniformis penicillinase. Gel filtration in the presence of Triton X-100 suggested that the penicillinase from E. coli had amphiphilic properties, as does B. licheniformis membrane penicillinase. These results show that the export of the penicillinase to the outer membrane of E. coli involves the cleavage of the signal peptide from the prepenicillinase, giving an outer membrane component indistinguishable from the membrane penicillinase of B. licheniformis.     

Cloning and nucleotide sequence of the signal peptidase II (lsp)-gene from Staphylococcus carnosus

Abstract Staphylococcus carnosus TM300 is able to synthesize at least seven lipoproteins with molecular masses between 15 and 45 kDa; the proteins are located in the membrane fraction. It can be concluded that this strain also posesses the enzymes involved in lipoprotein modification and prolipoprotein signal peptidase (signal peptidase II) processing. The gene encoding the prolipoprotein signal peptidase, lsp , from Staphylococcus carnosus TM300 was cloned in Escherichia coli and sequenced. The deduced amino acid sequence of the Lsp showed amino acid similarities with the Lsp's of S. aureus , Enterobacter aerogenes, E. coli , and Pseudomonas fluorescens . The hydropathy profile reveals four hydrophobic segments which are homologous to the putative transmembrane regions of the E. coli signal peptidase II. E. coli strains carrying lsp of S. carnosus exhibited an increased globomycin resistance.     

Lipoprotein-28, a cytoplasmic membrane lipoprotein from Escherichia coli. Cloning, DNA sequence, and expression of its gene

Escherichia coli contains several lipoproteins in addition to the major outer membrane lipoprotein (Ichihara, S., Hussain, M., and Mizushima, S. (1981) J. Biol. Chem. 256, 3125-3129). We cloned the gene for one of these new lipoproteins by using a synthetic 15-mer oligonucleotide probe identical to the DNA sequence at the signal peptide cleavage site of the major lipoprotein. The DNA sequence of the cloned gene revealed an open reading frame encoding a 272-amino acid protein with a signal peptide of 23 amino acid residues. The amino acid sequence of the putative cleavage site region of the signal peptide, -Leu-Leu-Ala-Gly-Cys-, is identical to that of the major lipoprotein. When the cloned gene was expressed in E. coli, a gene product with an apparent molecular weight of approximately 29,000 was identified which agrees well with the calculated molecular weight (27,800). The product was labeled with [3H]glycerol, and a precursor molecule of increased molecular weight was accumulated when cells were treated with globomycin, a specific inhibitor for prolipoprotein signal peptidase. We thus designed the gene product as lipoprotein-28. Unlike the major lipoprotein, lipoprotein-28 was found to be localized in the cytoplasmic membrane. A possible orientation of lipoprotein-28 in the E. coli envelope is discussed.     

Mechanism of signal peptide cleavage in the biosynthesis of the major lipoprotein of the Escherichia coli outer membrane

On treatment of Escherichia coli cells with globomycin, a glyceride-containing precursor of the major outer membrane lipoprotein accumulates in the cytoplasmic membrane (Hussain, M., Ichihara, S., and Mizushima, S. (1980) J. Biol. Chem. 255, 3707-3712). When the envelope fraction from such cells was incubated in a suitable buffer, this precursor could be processed to the mature lipoprotein. The processing involved removal of the signal peptide and subsequent acylation of the NH2 terminus thus bared. Two types of peptidase and an acylation enzyme(s) were found to be involved in these processes. The enzyme that cleaves the signal peptide, called signal peptidase in this paper, had many unique properties: being highly resistant to high temperature, having a wide optimum pH range, and being highly sensitive to detergents. The other peptidase(s), called signal peptide peptidase in this paper, was assumed to be responsible for the digestion of the signal peptide that had been cleaved from the precursor lipoprotein. This enzyme was rather heat-sensitive. Thus the processing from the precursor to the mature lipoprotein at a high temperature resulted in accumulation of a peptide that was most probably the intact signal peptide. The third enzyme(s) involved in the processing was the one that is responsible for acylation of the newly bared NH2 terminus of the lipoprotein. The enzyme activity was also lost at 80 degrees C. In the light of these findings, the biosynthetic pathway of the lipoprotein is discussed.     

Rapid assay and purification of a unique signal peptidase that processes the prolipoprotein from Escherichia coli B

A simple and accurate assay for prolipoprotein signal peptidase activity has been described that is based on the solubility of the signal peptide in 80% acetone. The unprocessed precursor and the mature form of the lipoprotein are quantitatively recovered in the precipitate. The signal peptide, from the acetone supernatant utilizing the purified signal peptidase, contains labeled methionine at its NH2 terminus and has Mr = 2200 (S.E. = 69). A specific signal peptidase that processes the modified form of Braun's prolipoprotein to its correct mature form has been purified. This enzyme is globomycin sensitive and has been purified 35,000-fold from the membranes of Escherichia coli by extraction at pH 4.0 with 2% Triton X-100 and heating, followed by conventional column chromatography at room temperature. This prolipoprotein signal peptidase has a pH optimum at 6.0, is not inhibited by EDTA, and requires 1 mM dithiothreitol for stability. The monomer molecular weight of this specific signal peptidase is 17,800 (S.E. = 900) as determined by sodium dodecyl sulfate-gel electrophoresis.     

EnvC, a new lipoprotein of the cytoplasmic membrane of Escherichia coli

Abstract A gene product with an apparent molecular mass of approximately 39000 Da can be identified in the cytoplasmic membrane of Escherichia coli upon expression of cloned envC . In this communication we report that the product was labelled with [³H]glycerol and [³H]palmitic acid, and a precursor molecule of increased molecular mass was accumulated when cells were treated with globomycin, a specific inhibitor for the prolipoprotein signal peptidase. The same precursor molecule was encoded by an envC mutant gene, in which the cysteine residue in a pentapeptide sequence, Leu-Ile-Ala-Gly-Cys²⁴ within the amino terminal region of EnvC, was replaced by tryptophane (Trp²⁴). This protein was not labelled with [³H]glycerol. The results demonstrate that the envC gene product represents a new lipoprotein of the cytoplasmic membrane of E. coli .     

Optimization of globomycin analogs as novel gram-negative antibiotics

Discovery of novel classes of Gram-negative antibiotics with activity against multi-drug resistant infections is a critical unmet need. As an essential member of the lipoprotein biosynthetic pathway, lipoprotein signal peptidase II (LspA) is an attractive target for antibacterial drug discovery, with the natural product inhibitor globomycin offering a modestly-active starting point. Informed by structure-based design, the globomycin depsipeptide was optimized to improve activity against E. coli. Backbone modifications, together with adjustment of physicochemical properties, afforded potent compounds with good in vivo pharmacokinetic profiles. Optimized compounds such as 51 (E. coli MIC 3.1 μM) and 61 (E. coli MIC 0.78 μM) demonstrate broad spectrum activity against gram-negative pathogens and may provide opportunities for future antibiotic discovery.     

Accumulation of alkaliphilic Bacillus penicillinase cleaved within the signal sequence in cytoplasm of Escherichia coli.

Alkaliphilic Bacillus penicillinase produced by Escherichia coli is distributed in several subcellular compartments according to cultivation conditions. The penicillinase that accumulated in particular subcellular fractions of E. coli grown under different conditions was purified and characterized. Periplasmic or extracellular penicillinase (24 kDa) was mature protein, indicating that the putative precursor (27 kDa) was processed at the correct amino acid residue, probably by signal peptidase I. Cytoplasmic penicillinase contained two unusual proteins (25 kDa) that are produced by proteolytic cleavage of the precursor within its signal sequence.     

Evidence that extracellular export of the endoglucanase encoded by egl of Pseudomonas solanacearum occurs by a two-step process involving a lipoprotein intermediate

Pseudomonas solanacearum is an important phytopathogen that produces a variety of extracellular enzymes. Previous reports suggested that one of these, a 43-kDa beta-1,4-endoglucanase (EGL), is initially synthesized with a 45-residue leader sequence that is removed during export. Experiments with globomycin presented here also suggest that the primary precursor of EGL (ppEGL) has a 45-residue leader sequence but that only the first 19 residues of the leader sequence are removed by signal peptidase II during initial export across the inner membrane. Further analysis suggested that the resultant 46-kDa intermediate precursor (pEGL) is a transient fatty acylated lipoprotein and is located on the periplasmic side of the inner membrane of P. solanacearum. Although Escherichia coli could synthesize ppEGL, modify it with palmitate, and remove the first 19 residues of the leader sequence during export across the inner membrane, only P. solanacearum could export pEGL across the outer membrane and remove the remaining 26 residues of the leader sequence producing the mature, extracellular EGL. The second step of the export process requires export machinery not present in E. coli. To our knowledge this represents the first example of a leader sequence with two distinct parts, one removed during export across the inner membrane and the other removed during export across the outer membrane.     

Signal peptide digestion in Escherichia coli. Effect of protease inhibitors on hydrolysis of the cleaved signal peptide of the major outer-membrane lipoprotein

Upon incubation of the envelope fraction of Escherichia coli a precursor of the major outer membrane lipoprotein that accumulates in the cytoplasmic membrane of the globomycin-treated cell is processed to the mature form [Hussain, M., Ichihara, S., and Mizushima, S. (1980) J. Biol. Chem. 255, 3707-3712; (1982) J. Biol. Chem. 257, 5177-5182]. When this precursor-containing envelope fraction was incubated in the presence of protease inhibitors such as antipain, leupeptin, chymostatin and elastatinal, a new peptide appeared on a polyacrylamide gel at the position where the signal peptide was expected to appear. This was proved to be the signal peptide of the lipoprotein from the following facts: (a) its appearance is in proportion to the appearance of the lipoprotein and disappearance of the precursor; (b) when the cleavage of the signal peptide from the precursor was inhibited by globomycin, the peptide did not appear on the gel; and (c) the results of labeling of the peptide with [3H]leucine, [35S]methionine and [3H]arginine were consistent with the amino acid composition of the signal peptide. The signal peptide thus accumulated in the envelope fraction was hydrolyzed by an enzyme named 'signal peptide peptidase' when the envelope fraction was washed to remove the inhibitors. The hydrolysis was inhibited by re-addition of these inhibitors. The signal peptide peptidase hydrolyzed the signal peptide only after its cleavage from the lipoprotein precursor.     

Modification, processing, and subcellular localization in Escherichia coli of the pCloDF13-encoded bacteriocin release protein fused to the mature portion of beta-lactamase.

A fusion between the pCloDF13-derived bacteriocin release protein and beta-lactamase was constructed to investigate the subcellular localization and posttranslational modification of the bacteriocin release protein in Escherichia coli. The signal sequence and 25 of the 28 amino acid residues of the mature bacteriocin release protein were fused to the mature portion of beta-lactamase. The hybrid protein (Mr, 31,588) was expressed in minicells and whole cells and possessed full beta-lactamase activity. Immunoblotting of subcellular fractions revealed that the hybrid protein is present in both the cytoplasmic and outer membranes of E. coli. Radioactive labeling experiments in the presence or absence of globomycin showed that the hybrid protein is modified with a diglyceride and fatty acids and is processed by signal peptidase II, as is the murein lipoprotein. The results indicated that the pCloDF13-encoded bacteriocin release protein is a lipoprotein which is associated with both membranes of E. coli cells.     

A distinct signal peptidase for prolipoprotein in escherichia coli

We have previously demonstrated the modification and processing of Escherichia coli prolipoprotein (Braun's) in vitro (Tokunaga M, Tokunaga H. Wu HC: Proc Natl Acad Sci USA 79:2255, 1982). Using this in vitro assay of prolipoprotein signal peptidase and globomycin selection, we have isolated and partially characterized an E coli mutant which contained a higher level of prolipoprotein signal peptidase activity. In contrast, the procoat protein signal peptidase activity was not increased in this mutant as compared to the wild-type strain. Furthermore, E coli strains containing cloned procoat protein signal peptidase gene were found to contain elevated levels of procoat protein signal peptidase, but normal levels of prolipoprotein signal peptidase. These two signal peptidase activities were also found to exhibit different stabilities during storage at 4°C. Thus biochemical, immunological, and genetic evidence clearly indicate that prolipoprotein signal peptidase is distinct from procoat protein signal peptidase in E coli.     

Secretion by Pseudomonas aeruginosa: fate of a cloned gram-positive lipoprotein deletion mutant.

In gram-positive organisms, glyceride-cysteine thioether lipoproteins are frequently associated with secretion. They constitute membrane-bound forms retained by the cell but releasable late in growth phase. Most gram-negative organisms secrete very few proteins to the culture fluid; thioether lipoproteins in such organisms, typified by the enteric bacterium Escherichia coli, are integral outer membrane components for the most part. Unusual among gram-negative organisms, however, are Pseudomonas strains, known for extracellular export of a number of proteins. To examine whether a fundamental difference exists between the processing of lipoproteins in Pseudomonas strains and in nonsecretory gram-negative organisms, we examined the fate in Pseudomonas aeruginosa and E. coli of a cloned gram-positive secretory lipoprotein, Bacillus licheniformis penicillinase. A nonlipoprotein deletion mutant of the same gene was also examined in P. aeruginosa, and its processing was compared with that in E. coli. No important differences were found between P. aeruginosa and E. coli for either the lipoprotein or its deletion mutant. Thus, the contrast in secretory abilities of the two organisms does not appear to result from a difference in their general secretory systems.     

The [NiFeSe] hydrogenase from Desulfovibrio vulgaris Hildenborough is a bacterial lipoprotein lacking a typical lipoprotein signal peptide

Desulfovibrio vulgaris Hildenborough has a membrane-bound [NiFeSe] hydrogenase whose mode of membrane association was unknown since it is constituted by two hydrophilic subunits. This work shows that this hydrogenase is a bacterial lipoprotein bound to the membrane by lipidic groups found at the N-terminus of the large subunit, which is unusual since it is missing the typical lipoprotein signal peptide. Nevertheless, the large subunit has a conserved four residue lipobox and its synthesis is sensitive to the signal peptidase II inhibitor globomycin. The D. vulgaris [NiFeSe] hydrogenase is the first example of a bacterial lipoprotein translocated through the Tat pathway.     

A lipopeptide-encoding sequence upstream from the IysA gene of Pseudomonas aeruginosa

An open reading frame (ORF) of 141 bp was observed upstream from the Pseudomonas aeruginosa lysA gene. The translation product of this ORF contains a signal peptide with a lipoprotein box, Ile-Ala-Ala-Cys, at the predicted signal peptidase cleavage site. The Escherichia coli phoA gene without its signal sequence was fused in frame to this ORF in a broad host-range plasmid. The resulting construct expressed a hybrid protein exhibiting alkaline phosphatase activity in phoA mutants of both E. coli and P. aeruginosa. This indicates that the ORF encodes a peptide, part of which acts as an export signal. The hybrid peptide was identified by immunoblotting with alkaline phosphatase antiserum. The accumulation of a precursor form was observed when P. aeruginosa cells carrying this gene fusion on a plasmid were treated with globomycin. Moreover, the mature form could be labelled with 2-[3H]-glycerol, indicating that lipidic residues may be linked to the hybrid protein. Taken together, these results strongly suggest that the ORF encodes a lipopeptide. We propose that the gene is called IppL.     

An alternate pathway for the processing of the prolipoprotein signal peptide in Escherichia coli

Previous studies showed that when the signal sequence plus 9 amino acid residues from the amino terminus of the major lipoprotein of Escherichia coli was fused to beta-lactamase, the resulting hybrid protein was modified, proteolytically processed, and assembled into the outer membrane as was the wild-type lipoprotein (Ghrayeb, J., and Inouye, M. (1983) J. Biol. Chem. 259, 463-467). We have constructed several hybrid proteins with mutations at the cleavage site of the prolipoprotein signal peptide. These mutations are known to block the lipid modification of the lipoprotein at the cysteine residue, resulting in the accumulation of unprocessed, unmodified prolipoprotein in the outer membrane. The mutations blocked the lipid modification of the hybrid protein. However, in contrast to the mutant lipoproteins, the cleavage of the signal peptides for the mutant hybrid proteins did occur, although less efficiently than the unaltered prolipo-beta-lactamase. The mutant prolipo-beta-lactamase proteins were cleaved at a site 5 amino acid residues downstream of the prolipoprotein signal peptide cleavage site. This new cleavage between alanine and lysine residues was resistant to globomycin, a specific inhibitor for signal peptidase II. This indicates that signal peptidase II, the signal peptidase which cleaves the unaltered prolipo-beta-lactamase, is not responsible for the new cleavage. The results demonstrate that the cleavage of the signal peptide is a flexible process that can occur by an alternative pathway when the normal processing pathway is blocked.     

Synthesis of precursor maltose-binding protein with proline in the +1 position of the cleavage site interferes with the activity of Escherichia coli signal peptidase I in vivo.

The residues occupying the -3 and -1 positions relative to the cleavage site of secretory precursor proteins are usually amino acids with small, neutral side chains that are thought to constitute the recognition site for the processing enzyme, signal peptidase. No restrictions have been established for residues positioned +1 to the cleavage site, although there have been several indications that mutant precursor proteins with a proline at +1 cannot be processed by Escherichia coli signal peptidase I (also called leader peptidase). A maltose-binding protein (MBP) species with proline at +1, designated MBP27-P, was translocated efficiently but not processed when expressed in E. coli cells. Unexpectedly, induced expression of MBP27-P was found to have an adverse effect on the processing kinetics of five different nonlipoprotein precursors analyzed, but not precursor Lpp (the major outer membrane lipoprotein) processed by a different enzyme, signal peptidase II. Cell growth also was inhibited following induction of MBP27-P synthesis. Substitutions in the MBP27-P signal peptide that blocked MBP translocation across the cytoplasmic membrane and, hence, access to the processing enzyme or that altered the signal peptidase I recognition site at position -1 restored both normal growth and processing of other precursors. Since overproduction of signal peptidase I also restored normal growth and processing to cells expressing unaltered MBP27-P, it was concluded that precursor MBP27-P interferes with the activity of the processing enzyme, probably by competing as a noncleavable substrate for the enzyme's active site. Thus, although signal peptidase I, like many other proteases, is unable to cleave an X-Pro bond, a proline at +1 does not prevent the enzyme from recognizing the normal processing site. When the RBP signal peptide was substituted for the MBP signal peptide of MBP27-P, the resultant hybrid protein was processed somewhat inefficiently at an alternate cleavage site and elicited a much reduced effect on cell growth and signal peptidase I activity. Although the MBP signal peptide also has an alternate cleavage site, the different properties of the RBP and MBP signal peptides with regard to the substitution of proline at +1 may be related to their respective secondary structures in the processing site region.     

A gene at 59 minutes on the Escherichia coli chromosome encodes a lipoprotein with unusual amino acid repeat sequences.

We report a 1.432-kb DNA sequence at 59 min on the Escherichia coli chromosome that connects the published sequences of the pcm gene for the isoaspartyl protein methyltransferase and that of the katF or rpoS (katF/rpoS) gene for a sigma factor involved in stationary-phase gene expression. Analysis of the DNA sequence reveals an open reading frame potentially encoding a polypeptide of 379 amino acids. The polypeptide sequence includes a consensus bacterial lipidation sequence present at residues 23 to 26 (Leu-Ala-Gly-Cys), four octapeptide proline- and glutamine-rich repeats of consensus sequence QQPQIQPV, and four heptapeptide threonine- and serine-rich repeats of consensus sequence PTA(S,T)TTE. The deduced amino acid sequence, especially in the C-terminal region, is similar to that of the Haemophilus somnus LppB lipoprotein outer membrane antigen (40% overall sequence identity; 77% identity in last 95 residues). The LppB lipoprotein binds Congo red dye and has been proposed to be a virulence determinant in H. somnus. Utilizing a plasmid construct with the E. coli gene under the control of a phage T7 promoter, we demonstrate the lipidation of this gene product by the incorporation of [3H]palmitic acid into a 42-kDa polypeptide. We also show that treatment of E. coli cells with globomycin, an inhibitor of the lipoprotein signal peptidase, results in the accumulation of a 46-kDa precursor. We thus designate the protein NlpD (new lipoprotein D). E. coli cells overexpressing NlpD bind Congo red dye, suggesting a common function with the H. somnus LppB protein. Disruption of the chromosomal E. coli nlpD gene by insertional mutagenesis results in decreased stationary-phase survival after 7 days.