Nucleotide sequence of the Pseudomonas fluorescens signal peptidase II gene (lsp) and flanking genes.

The lsp gene encoding prolipoprotein signal peptidase (signal peptidase II) is organized into an operon consisting of ileS and three open reading frames, designated genes x, orf149, and orf316 in both Escherichia coli and Enterobacter aerogenes. A plasmid, pBROC128, containing a 5.8-kb fragment of Pseudomonas fluorescens DNA was found to confer pseudomonic acid resistance on E. coli host cells and to contain the structural gene of ileS from P. fluorescens. In addition, E. coli strains carrying pBROC128 exhibited increased globomycin resistance. This indicated that the P. fluorescens lsp gene was present on the plasmid. The nucleotide sequences of the P. fluorescens lsp gene and of its flanking regions were determined. Comparison of the nucleotide sequences of the lsp genes in E. coli and P. fluorescens revealed two highly conserved domains in this enzyme. Furthermore, the five genes which constitute an operon in E. coli and Enterobacter aerogenes were found in P. fluorescens in the same order as in the first two species.     

Cloning and nucleotide sequence of the Enterobacter aerogenes signal peptidase II (lsp) gene.

In Escherichia coli, prolipoprotein signal peptidase is encoded by the lsp gene, which is organized into an operon consisting of ileS, lsp, and three open reading frames, designated genes x, orf-149, and orf-316. The Enterobacter aerogenes lsp gene was cloned and expressed in E. coli. The nucleotide sequence of the Enterobacter aerogenes lsp gene and a part of its flanking sequences were determined. A high degree of homology was found between the E. coli ileS-lsp operon and the corresponding genes in Enterobacter aerogenes. Furthermore, the same five genes which constitute an operon in E. coli were found in Enterobacter aerogenes in the same order.     

A Nonessential Signal Peptidase II (Lsp) of Myxococcus xanthus Might Be Involved in Biosynthesis of the Polyketide Antibiotic TA

Myxococcus xanthus is a gram-negative soil bacterium that produces the polyketide antibiotic TA. In this study, we describe the analysis of an M. xanthus gene which encodes a homologue of the prolipoprotein signal peptidase II (SPase II; lsp). Overexpression of the M. xanthus SPase II in Escherichia coli confers high levels of globomycin resistance, confirming its function as an SPase II. The M. xanthus gene encoding the lsp homologue is nonessential for growth, as determined by specific gene disruption. It has been mapped to the antibiotic TA gene cluster, and the disrupted mutants do not produce the antibiotic, indicating a probable involvement in TA production. These results suggest the existence of more than one SPase II protein in M. xanthus, where one is a system-specific SPase II (for TA biosynthesis).     

Mapping of the lipoprotein signal peptidase gene (lsp)

A pBR322 plasmid which contains a fragment of Escherichia coli DNA encoding the lipoprotein signal peptidase gene was used to transform Hfr polA1 strains. Ampr transformants were used as donors in conjugation experiments, and the location of the plasmid amp gene adjacent to the chromosomal lsp gene was determined to be near the thr ara loci of the E. coli chromosome. P1 transduction experiments established that the location of the lsp gene is closely linked to that of dapB , at 0.5 to 0.6 min on the E. coli genetic map. The position of the lsp gene was further determined to be between ileS and dapB by complementation analysis of an E. coli mutant showing temperature-sensitive prolipoprotein signal peptidase activity.     

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.     

Isolation and characterization of an Escherichia coli clone overproducing prolipoprotein signal peptidase

Based on the rationale that Escherichia coli cells containing increased levels of prolipoprotein signal peptidase would be highly resistant to globomycin, a specific inhibitor of the prolipoprotein signal peptidase, we have isolated a clone from the Carbon-Clarke collection, plasmid pLC3-13, which is globomycin-resistant and contains an increased level of prolipoprotein signal peptidase activity. The plasmid pMT521, a subclone of pLC3-13 in pBR322, conferred on its host cells approximately 20 times overproduction of prolipoprotein signal peptidase and an extremely high level of resistance against globomycin. The overproduced prolipoprotein signal peptidase was completely inhibited by the presence of globomycin in the in vitro assay, and the overproduced activity was found in the cell envelope fraction. Several lines of biochemical and genetic evidence suggest that the gene contained in pLC3-13 and its derivative clones is most likely the structure gene (lsp) for prolipoprotein signal peptidase.     

Membrane topology of Escherichia coli prolipoprotein signal peptidase (signal peptidase II)

The lsp gene of Escherichia coli encodes the inner membrane enzyme, signal peptidase II (SPase II). SPase II is comprised of 164 amino acid residues and contains four hydrophobic domains. A series of lsp-phoA and lsp-lacZ gene fusions have been constructed in vitro to determine the topology of SPase II. The fusion junction for each of these gene fusions was determined by DNA sequencing. The lengths of the SPase II fragment in the fusions varied from 12 to 159 amino acid residues. Strains containing SPase II-PhoA fusions to the two predicted periplasmic loops exhibited higher levels of alkaline phosphatase activity than fusions to the predicted cytoplasmic domains. In contrast, SPase II-LacZ fusions at the cytoplasmic and the periplasmic domains of SPase II showed high and low levels of beta-galactosidase activity, respectively, a result opposite to those shown by SPase II-PhoA fusions located at precisely the same amino acid of SPase II. Taken together, these results strongly support the predicted model for SPase II topology, i.e. this enzyme spans the cytoplasmic membrane four times with both the amino and the carboxyl termini facing the cytoplasm.     

Maturation of lipoproteins by type II signal peptidase is required for phagosomal escape of Listeria monocytogenes

Lipoproteins of Gram-positive bacteria are involved in a broad range of functions such as substrate binding and transport, antibiotic resistance, cell signaling, or protein export and folding. Lipoproteins are also known to initiate both innate and adaptative immune responses. However, their role in the pathogenicity of intracellular microorganisms is yet poorly understood. In Listeria monocytogenes, a Gram-positive facultative intracellular human pathogen, surface proteins have important roles in the interactions of the microorganism with the host cells. Among the putative surface proteins of L. monocytogenes, lipoproteins constitute the largest family. Here, we addressed the role of the signal peptidase (SPase II), responsible for the maturation of lipoproteins in listerial pathogenesis. We identified a gene, lsp, encoding a SPase II in the genome of L. monocytogenes and constructed a deltalsp chromosomal deletion mutant. The mutant strain fails to process several lipoproteins demonstrating that lsp encodes a genuine SPase II. This defect is accompanied by a reduced efficiency of phagosomal escape during infection of eucaryotic cells, and leads to an attenuated virulence. We show that lsp gene expression is strongly induced when bacteria are still entrapped inside phagosomes of infected macrophages. The data presented establish, thus, that maturation of lipoproteins is critical for efficient phagosomal escape of L. monocytogenes, a process temporally controlled by the regulation of Lsp production in infected cells.     

Lipoproteins are critical TLR2 activating toxins in group B streptococcal sepsis

Group B streptococcus (GBS) is the most important cause of neonatal sepsis, which is mediated in part by TLR2. However, GBS components that potently induce cytokines via TLR2 are largely unknown. We found that GBS strains of the same serotype differ in released factors that activate TLR2. Several lines of genetic and biochemical evidence indicated that lipoteichoic acid (LTA), the most widely studied TLR2 agonist in Gram-positive bacteria, was not essential for TLR2 activation. We thus examined the role of GBS lipoproteins in this process by inactivating two genes essential for bacterial lipoprotein (BLP) maturation: the prolipoprotein diacylglyceryl transferase gene (lgt) and the lipoprotein signal peptidase gene (lsp). We found that Lgt modification of the N-terminal sequence called lipobox was not critical for Lsp cleavage of BLPs. In the absence of lgt and lsp, lipoprotein signal peptides were processed by the type I signal peptidase. Importantly, both the Deltalgt and the Deltalsp mutant were impaired in TLR2 activation. In contrast to released factors, fixed Deltalgt and Deltalsp GBS cells exhibited normal inflammatory activity indicating that extracellular toxins and cell wall components activate phagocytes through independent pathways. In addition, the Deltalgt mutant exhibited increased lethality in a model of neonatal GBS sepsis. Notably, LTA comprised little, if any, inflammatory potency when extracted from Deltalgt GBS. In conclusion, mature BLPs, and not LTA, are the major TLR2 activating factors from GBS and significantly contribute to GBS sepsis.     

Genetic characterization of a gene for prolipoprotein signal peptidase in Escherichia coli

A mutation (lspA, prolipoprotein signal peptidase) rendering the prolipoprotein signal peptidase temperature-sensitive in Escherichia coli has been analyzed. The mutation was mapped in the dnaJ-rpsT-ileS-dapB region by interrupted mating with various Hfr strains and P1 phage transduction. lambda transducing phage lambda ddapB2 that carries the rpsT-ileS-dapB region was shown to complement the lspA mutation. Plasmid pLC3-13 which had been isolated from Clarke and Carbon's collection as a plasmid carrying the lspA locus was shown to carry the dnaJ and rpsT loci. Complementation analysis with plasmids carrying various DNA fragments derived from pLC3-13 showed that the lspA locus is between the rpsT and ileS loci. The wildtype allele was dominant over the lspA allele.     

Modification and processing of internalized signal sequences of prolipoprotein in Escherichia coli and in Bacillus subtilis

We have cloned the Escherichia coli lipoprotein structural gene (lpp) into a shuttle vector and studied its expression in both E. coli and in Bacillus subtilis. Using in vitro gene fusion techniques, the lpp gene was placed under the control of the promoter for the erythromycin-resistance (ery) gene. This fusion gene directed the synthesis of Braun's prolipoprotein which can be subsequently processed into the mature lipoprotein. In addition to the prolipoprotein, two ery-lpp hybrid proteins containing a 45- and a 22-amino acid extension preceding the NH2 terminus of prolipoprotein, respectively, are also synthesized in E. coli. The synthesis of these three proteins appears to involve the utilization of three distinct translation initiation sites. In B. subtilis, only two proteins are synthesized, the hybrid protein with a 45-amino acid extension and the prolipoprotein. In both E. coli and B. subtilis, the precursor forms of the hybrid proteins are lipid-modified, and they are processed to mature lipoprotein in vivo. These results indicate that internalized signal sequence containing the prolipoprotein modification and processing site (Leu-Ala-Glys-Cys) can function normally and permit the modification of hybrid proteins to lipid-modified precursors which can be subsequently processed by the globomycin-sensitive prolipoprotein signal peptidase.     

Nucleotide sequence of the lspA gene,the structural gene for lipoprotein signal peptidase of Escherichia coli

The nucleotide sequence of the lspA gene coding for lipoprotein signal peptidase of Escherichia coli was determined and the amino acid sequence of the peptidase was deduced from it. The molecular mass and amino acid composition of the predicted lipoprotein signal peptidase were consistent with those of the signal peptidase purified from cells harboring the lspA gene-carrying plasmid. The peptidase most probably has no cleavable signal peptide. The lspA gene was preceded by the ileS gene coding for isoleucyl-tRNA synthetase and the tandem termination codons of the ileS gene overlapped with the initiation codon of the lspA gene.     

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.     

Temperature-sensitive processing of outer membrane lipoprotein in an Escherichia coli mutant.

A mutant of Escherichia coli that accumulated prolipoprotein, a secretory precursor of the outer membrane lipoprotein, was isolated. The prolipoprotein accumulated in this mutant was modified by glyceride, but the in vitro cleavage of the signal peptide of the accumulated prolipoprotein was found to be temperature sensitive. The mutation appears to be located outside the gene for the lipoprotein, thus suggesting that the gene for the signal peptidase for the prolipoprotein was mutated.     

Processing of lipid-modified prolipoprotein requires energy and sec gene products in vivo.

The kinetics of processing of glyceride-modified prolipoprotein that accumulated in globomycin-treated Escherichia coli has been found to be affected by sec mutations, i.e., secA, secE, secY, secD, and secF, and by metabolic poisons which affect proton motive force (PMF). The effect of sec mutations on processing of glyceride-modified prolipoprotein in vivo was not due to a secondary effect on PMF. Neither a secF mutation nor metabolic poisons affected the processing of previously accumulated proOmpA protein in vivo, suggesting that the requirements for functional sec gene products and PMF are specific to the processing of lipoprotein precursors by signal peptidase II.     

Prolipoprotein signal peptidase of Escherichia coli requires a cysteine residue at the cleavage site

A signal peptidase specifically required for the secretion of the lipoprotein of the Escherichia coli outer membrane cleaves off the signal peptide at the bond between a glycine and a cysteine residue. This cysteine residue was altered to a glycine residue by guided site-specific mutagenesis using a synthetic oligonucleotide and a plasmid carrying an inducible lipoprotein gene. The induction of mutant lipoprotein production was lethal to the cells. A large amount of the prolipoprotein was accumulated in the outer membrane fraction. No protein of the size of the mature lipoprotein was detected. These results indicate that the prolipoprotein signal peptidase requires a glyceride modified cysteine residue at the cleavage site.