Assembly of outer membrane lipoprotein in an Escherichia coli mutant with a single amino acid replacement within the signal sequence of prolipoprotein.

We have compared the rate of assembly of outer membrane proteins including the lipoprotein in a pair of isogenic mlpA+ (lpp+) and mlpA (lpp) strains by pulse-chase experiments. The rate of assembly of the mutant prolipoprotein into the outer membrane was slightly slower than that of the wild-type lipoprotein. The rate of assembly of protein I and protein H-2 was similar in the wild type and the mutant, whereas the rate of assembly of protein II into the outer membrane was slightly reduced in the mutant strain. The organization of outer membrane was slightly reduced in the mutant strain. The organization of outer membrane proteins in the mutant cells appeared not to be grossly altered, based on the apparent resistance (or susceptibility) of these proteins toward trypsin treatment and their resistance to solubilization by Sarkosyl. Like the wild-type lipoprotein, the mutant prolipoprotein in the outer membrane was resistant to trypsin. On the other hand, the prolipoprotein in the cytoplasmic membrane fraction of the mutant cell envelope was susceptible to trypsin digestion. We conclude from these data that proteolytic cleavage of prolipoprotein is not essential for the translocation and proper assembly of lipoprotein into outer membrane.     

Genetic characterization of an Escherichia coli mutant altered in the structure of murein lipoprotein.

Mutants defective in the structure, biosynthesis, and assembly of murein lipoprotein have been isolated. One of these mutants has been shown to synthesize a structurally altered lipoprotein. The biochemical features of the mutant lipoprotein (lipid deficiency, dimer formation, and a reduced, bound form of lipoprotein) could be attributed to a single mutation (or closely linked mutations) located at 36.4 min of the Escherichia coli map. We propose that this mutant is altered in the structural gene for murein lipoprotein (mlpA). Biochemical studies carried out with a heterogenote, mlpA/F'mlpA+, revealed the biochemical codominance of the wild-type and mutant genes.     

A novel outer membrane lipoprotein, LolB (HemM), involved in the LolA (p20)-dependent localization of lipoproteins to the outer membrane of Escherichia coli.

The Escherichia coli major outer membrane lipoprotein (Lpp) is released from the inner membrane into the periplasm as a complex with a carrier protein, LolA (p20), and is then specifically incorporated into the outer membrane. An outer membrane protein playing a critical role in Lpp incorporation was identified, and partial amino acid sequences of the protein, named LolB, were identical to those of HemM, which has been suggested to play a role in 5-aminolevulinic acid synthesis in the cytosol. In contrast to this suggested role, the deduced amino acid sequence of HemM implied that the gene encodes a novel outer membrane lipoprotein. Indeed, an antibody raised against highly purified LolB revealed its outer membrane localization, and inhibited in vitro Lpp incorporation into the outer membrane. Furthermore, LolB was found to be synthesized as a precursor with a signal sequence and then processed to a lipid-modified mature form. An E.coli strain possessing chromosomal hemM under the control of the lac promoter-operator required IPTG for growth, indicating that hemM (lolB) is an essential gene. Outer membrane prepared from LolB-depleted cells did not incorporate Lpp. When the Lpp-LolA complex was incubated with a water-soluble LolB derivative, Lpp was transferred from LolA to LolB. Based on these results, the outer membrane localization pathway for E.coli lipoprotein is discussed with respect to the functions of LolA and LolB.     

Restriction enzyme cleavage sites surrounding the structural gene for the lipoprotein of the Escherichia coli outer membrane.

The purified messenger ribonucleic acid (mRNA) for the lipoprotein of the Escherichia coli outer membrane was hybridized with fragments obtained by digestion of E. coli chromosomal deoxyribonucleic acid (DNA) with eight different restriction enzymes. For each restriction enzyme digestion, one specific fragment separated by agarose gel electrophoresis was found to hybridize with the lipoprotein mRNA. From the analysis of restriction fragments generated by double digestions with various combinations of restriction enzymes, cleavage sites for the restriction enzymes near the locus of the lipoprotein structural gene (lpp) were mapped. No restriction fragments of DNA from the E. coli lpp-2 mutant hybridized with the lipoprotein mRNA, confirming that the mutant has a deletion mutation in the vicinity of the lpp gene.     

Gene dosage effects of the structural gene for a lipoprotein of the Escherichia coli outer membrane.

The gene dosage effects of the structural gene (lpp) for the lipoprotein of the Escherichia coli outer membrane were examined. A novel F-prime factor containing the lpp gene was constructed. The amount of the free-form lipoprotein in the merodiploid strain carrying the F-prime factor was found to be about two times as great as that in the corresponding haploid strain. On the other hand, the amount of the bound-form lipoprotein, which is vovalently linked to the peptidoglycan, was not significantly different in the merodiploid strain as compared with the corresponding haploid strain. The present results suggest that the lpp gene is expressed constitutively in contrast to another major protein of the E. coli outer membrane, tolG protein (protein II, D. B. Datta et al., J. Bacteriol. 128:834-841, 1976). The F-prime factor isolated may include a portion of the E. coli chromosome (located between 33 and 36 min on the genetic map) that is not covered by any other F-prime factor.     

Alpha-glucan phosphorylase from Escherichia coli. Cloning of the gene, and purification and characterization of the protein

By using a synthetic oligonucleotide probe identical to a part of the gene for the Escherichia coli major outer membrane lipoprotein, we have cloned a gene from E. coli chromosomal DNA. However, the cloned gene was not one of the lipoprotein genes. The amino acid sequence deduced from its nucleotide sequence shows extensive similarities instead to alpha-glucan phosphorylase (EC 2.4.1.1). The gene, glgP, is located immediately downstream from glgA, the gene for glycogen synthase. The glgP gene was inserted into pUC9 vector and expressed in the presence of the lac inducer. The gene product was purified to apparent homogeneity as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In all chromatographies, the protein was eluted accompanied by a low phosphorylase activity. The final preparation showed phosphorolytic activity to various alpha-glucans, although the specific activity was extremely low compared to other alpha-glucan phosphorylases under the standard assay conditions. Its enzymatic activity, however, increased almost linearly as the concentration of glucan increased, reaching a value comparable with those of other phosphorylases. The amino acid sequence deduced was compared with those of alpha-glucan phosphorylases from other sources.     

Isolation and characterization of extragenic suppressor mutants of the tolA-876 periplasmic-leaky allele in Escherichia coli K-12

tolA mutants of Escherichia coli K-12 release periplasmic proteins into the extracellular medium; they are sensitive to growth inhibitors such as cholic acid and tolerant to group A colicins and filamentous bacteriophage. Suppressor mutants of the tolA-876 allele were isolated by selecting for cholic acid resistant clones that did not release periplasmic ribonuclease I. One class of tolA suppressor strains carried mutations in the staA gene (for suppressor of tolA) located a 41 min. tolA-876 staA strains partially recovered a wild-type phenotype: they exported alkaline phosphatase and beta-lactamase into the periplasm and only released very low amounts of periplasmic proteins; moreover, they were sensitive to E1 and A colicins and more resistant than tolA-876 staA+ strains to various growth inhibitors. Furthermore, tolA-876 staA-2 and tolA+staA-2 mutants were 10- to 2700-times more resistant than staA+ strains to bacteriophages TuIa, TuIb and T4, and TuII whose receptors are major outer membrane proteins OmpF, OmpC and OmpA, respectively. SDS-PAGE analysis suggested that cell envelopes of staA or staA+ strains contained similar amounts of these proteins but characterization of strains carrying ompF (or C or A)-phoA gene fusions showed that mutation stA-2 reduced ompF gene expression by a factor of two. Analysis of double mutants strains carrying mutation staA-2 and a tolA, tolB, excC or excD periplasmic-leaky mutation showed that staA suppression was allele specific which suggested that proteins TolA and StaA might directly interact.     

Cloning and analysis of the gene for the major outer membrane lipoprotein from Pseudomonas aeruginosa

The gene for the Pseudomonas aeruginosa outer membrane lipoprotein I was isolated from a genomic library in the phage lambda EMBL3 vector and subsequently subcloned in the low copy-number, wide host-range plasmid vector, pKT240. The cloned gene was highly expressed, resulting in the production of a low molecular-weight protein (8 kD) that was found to be associated with the outer membrane. Sequence analysis showed an open reading frame of 83 amino acids with a putative N-terminal hydrophobic signal peptide of 19 residues immediately followed by the lipoprotein consensus sequence, GLY-CYS-SER-SER (residues 19-22). The predicted amino acid composition of the mature polypeptide and that of the purified lipoprotein I of P. aeruginosa (Mizuno and Kageyama, 1979) were identical. In contrast with other Gram-negative outer membrane lipoproteins, conformation predictions suggested that the mature protein was a single alpha helix.     

MlpA, a lipoprotein required for normal development of Myxococcus xanthus.

The mlpA gene encoding a 236-residue polypeptide has been identified immediately downstream of the oar gene of Myxococcus xanthus (M. Martinez-Canamero, J. Munoz-Dorado, E. Farez-Vidal, M. Inouye, and S. Inouye, J. Bacteriol. 175:4756-4763, 1993). The amino-terminal 21 residues of MlpA encode a typical prokaryotic signal sequence with a putative lipoprotein cleavage site. When expressed in Escherichia coli in the presence of [2-3H]glycerol, 3H-labeled MlpA had a molecular mass of 33 kDa and was found to be associated with the membrane fraction. Globomycin, an inhibitor of signal peptidase II, caused a shift in the mobility of E. coli-expressed MlpA to 35 kDa. Subsequently, a mlpA disruption strain (oar+) was constructed and found to have delayed fruiting body formation (by approximately 36 h), with significantly larger fruiting bodies being produced compared with those of the wild-type strain. Nevertheless, spore yields for the two strains were identical after 120 h of development. These data indicate that MlpA, the lipoprotein identified in M. xanthus, is required for normal fruiting body formation.     

Expression of the Proteus mirabilis lipoprotein gene in Escherichia coli. Existence of tandem promoters

The Ipp gene from Proteus mirabilis was cloned onto pBR322 and expressed in Escherichia coli. The P. mirabilis lpp gene is unique in that it has two tandem promoters transcribing two mRNAs that differ in length by approximately 70 nucleotides at their 5'-ends. The two mRNAs thus encode the identical lipoprotein. The P. mirabilis prolipoprotein has a 19-amino acid signal peptide and a 59-amino acid lipoprotein sequence. In spite of the substantial differences in the amino acid sequence from the E. coli prolipoprotein, the P. mirabilis prolipoprotein is normally modified and processed in E. coli, and the resultant lipoprotein is assembled in the E. coli outer membrane as is the E. coli lipoprotein.     

Cell envelope and shape of Escherichia coli: multiple mutants missing the outer membrane lipoprotein and other major outer membrane proteins.

Starting with an Escherichia coli strain missing the outer membrane lipoprotein, multiple mutants were constructed than in addition to this defect miss the outer membrane proteins II, Ia and Ib, or Ia, Ib, and II. In contrast to all single mutants or strains missing the lipoprotein and polypeptides Ia and Ib, drastic influences on the integrity of the outer membrane and cell morphology were observed in mutants without lipoprotein and protein II. Such strains exhibited spherical morphology. They required increased concentrations of electrolytes for optimal growth, and Mg2+ or Ca2+ were the most efficient. These mutants were sensitive to hydrophobic antibiotics and detergents. Electron microscopy revealed abundant blebbing of the outer membrane, and it could clearly be seen that the murein layer was no longer associated with the outer membrane.     

Effects of inserting eight amino acid residues into the major lipoprotein on its assembly in the outer membrane of Escherichia coli.

A DNA sequence consisting of 24 base pairs was inserted into the structural gene (lpp) coding for the major lipoprotein of the Escherichia coli outer membrane which was carried on a high-copy-number plasmid in which expression was regulated through a lac promoter-operator region. This modification resulted in the insertion of eight amino acid residues, Glu-Glu-Phe-Leu-Glu-Glu-Phe-Leu, between the glutamine residue at position 9 and the leucine residue at position 10 of the wild-type lipoprotein sequence. When production of the mutant lipoprotein was induced by a lac inducer, the cells became swollen, showed unusual morphology, and eventually lysed. When the membrane fraction was analyzed after the induction, the mutant lipoprotein was found to have been normally secreted across the cytoplasmic membrane and assembled in the outer membrane. This lipoprotein was modified with glycerol and palmitic acid and even formed the bound form, which was linked covalently to peptidoglycan. The major difference between the membrane-associated mutant lipoprotein and the wild-type lipoprotein was that the mutant lipoprotein became sensitive to trypsin treatment. These results indicate that the substantial alteration in mutant lipoprotein structure near the amino-terminal end does not interfere with modification of the amino-terminal cysteine residue or cleavage of the signal peptide by the prolipoprotein-specific signal peptidase. However, this mutant lipoprotein assembled in the outer membrane appears to have deleterious effects with respect to envelope structure and cellular morphology and viability.     

Recombinant Porphyromonas gingivalis FimA preproprotein expressed in Escherichia coli is lipidated and the mature or processed recombinant FimA protein forms a short filament in vitro

The gram-negative anaerobic bacterium Porphyromonas gingivalis is an etiologically important pathogen for chronic periodontal diseases in adults. Our previous study suggested that the major structural components of both Fim and Mfa fimbriae in this organism are secreted through their lipidated precursors. In this study, we constructed Escherichia coli strains expressing various fimA genes with or without the 5'-terminal DNA region encoding the signal peptide, and we determined whether lipidation of recombinant FimA proteins occurred in E. coli. Lipidation occurred for a recombinant protein from the fimA gene with the 5'-terminal DNA region encoding the signal peptide but not for a recombinant protein from the fimA gene without the signal-peptide-encoding region, as revealed by [3H]palmitic acid labeling experiments. A TLR2-dependent signaling response was induced by the recombinant protein from the fimA gene with the signal-peptide-encoding region but not by a recombinant protein from the fimA gene with the signal-peptide-encoding region that had a base substitution causing an amino acid substitution (C19A). Electron microscopic analysis revealed that recombinant FimA (A-47?- W-383) protein was autopolymerized to form filamentous structures of about 80?nm in length in vitro. The results suggest that FimA protein, a major subunit of Fim fimbriae, is transported to the outer membrane by the lipoprotein sorting system, and a mature or processed FimA protein on the outer membrane is autopolymerized to form Fim fimbriae.     

Testing the '+2 rule' for lipoprotein sorting in the Escherichia coli cell envelope with a new genetic selection

We report a novel strategy for selecting mutations that mislocalize lipoproteins within the Escherichia coli cell envelope and describe the mutants obtained. A strain carrying a deletion of the chromosomal malE gene, coding for the periplasmic maltose-binding protein (MalE), cannot use maltose unless a wild-type copy of malE is present in trans. Replacement of the natural signal peptide of preMalE by the signal peptide and the first four amino acids of a cytoplasmic membrane-anchored lipoprotein resulted in N-terminal fatty acylation of MalE (lipoMalE) and anchoring to the periplasmic face of the cytoplasmic membrane, where it could still function. When the aspartate at position +2 of this protein was replaced by a serine, lipoMalE was sorted to the outer membrane, where it could not function. Chemical mutagenesis followed by selection for maltose-using mutants resulted in the identification of two classes of mutations. The single class I mutant carried a plasmid-borne mutation that replaced the serine at position +2 by phenylalanine. Systematic substitutions of the amino acid at position +2 revealed that, besides phenylalanine, tryptophan, tyrosine, glycine and proline could all replace classical cytoplasmic membrane lipoprotein sorting signal (aspartate +2). Analysis of known and putative lipoproteins encoded by the E. coli K-12 genome indicated that these amino acids are rarely found at position +2. In the class II mutants, a chromosomal mutation caused small and variable amounts of lipoMalE to remain associated with the cytoplasmic membrane. Similar amounts of another, endogenous outer membrane lipoprotein, NlpD, were also present in the cytoplasmic membrane in these mutants, indicating a minor, general defect in the sorting of outer membrane lipoproteins. Four representative class II mutants analysed were shown not to carry mutations in the lolA or lolB genes, known to be involved in the sorting of lipoproteins to the outer membrane.     

Cloning of genes encoding a 15,000-dalton peptidoglycan-associated outer membrane lipoprotein and an antigenically related 15,000-dalton protein from Haemophilus influenzae.

We have cloned and expressed in Escherichia coli a gene encoding a 15,000-apparent-molecular-weight peptidoglycan-associated outer membrane lipoprotein (PAL) of Haemophilus influenzae. The nucleotide sequence of this gene encodes an open reading frame of 153 codons with a predicted mature protein of 134 amino acids. The amino acid composition and sequence of the predicted mature protein agree with the chemically determined composition and partial amino acid sequence of PAL purified from H. influenzae outer membranes. We have also identified a second gene from H. influenzae that encodes a second 15,000-apparent-molecular-weight protein which is recognized by antiserum against PAL. This protein has been shown to be a lipoprotein. The nucleotide sequence of this gene encodes an open reading frame of 154 codons with a predicted mature protein of 136 amino acids and has limited sequence homology with that of the gene encoding PAL. Southern hybridization analysis indicates that both genes exist as single copies in H. influenzae chromosomal DNA. Both genes encode polypeptides which have amino-terminal sequences similar to those of reported membrane signal peptides and are associated primarily with the outer membrane when expressed in E. coli.     

Isolation of an ompC-like outer membrane protein gene from Salmonella typhi

We have isolated the structural gene for an outer membrane protein of Salmonella typhi, from a genomic library constructed in bacteriophage lambda 1059, using the Escherichia coli ompC gene as a heterologous probe. E. coli ompC codes for an outer membrane pore protein (porin) that is induced preferentially at high osmolarity and high temperature. The S. typhi ompC-like gene was subcloned in pBR322 and introduced into E. coli HB101 and into P678-54, a minicell-producing strain. In both strains it expressed a 38.5-kDa protein, which was incorporated into the outer membrane envelope and comigrated with an S. typhi outer membrane protein which was expressed both at low and high osmolarity in vivo.     

Construction and evaluation of a plasmid vector for the expression of recombinant lipoproteins in Escherichia coli

Outer membrane lipoproteins are emerging as key targets for protective immunity to many bacterial pathogens. Heterologous expression of lipoproteins in Escherichia coli does not always result in high level expression of acylated recombinant protein. Thus, these proteins do not take up their correct membrane topology and are lacking the immunostimulatory properties endowed by the lipid. To this end, we have designed a lipoprotein expression vector (pDUMP) that results in the production of fusion proteins containing the E. coli major outer membrane lipoprotein (Lpp) signal sequence, lipoprotein signal peptidase recognition site, and the +2 outer membrane sorting signal at their N termini. To test the ability of pDUMP to express lipoproteins from heterologous hosts, the surface lipoprotein PsaA from the Gram-positive organism Streptococcus pneumoniae and the outer membrane lipoproteins MlpA from the Gram-negative Pasteurella multocida and BlpA from the spirochete Brachyspira hyodysenteriae were cloned into both hexahistidine fusion vectors and pDUMP. High level expression of antigenically active protein from both the hexahistidine fusion vectors and pDUMP resulted in abundant bands of the predicted molecular masses when analyzed by SDS-PAGE. When grown in the presence of 3[H]palmitic acid, proteins encoded by pDUMP were observed to incorporate palmitic acid whilst the hexahistidine fusion proteins did not. Using mass spectrometry and image analysis we determined the efficiency of lipidation between the three clones to vary from 31.7 to 100%. In addition, lipidated, but not hexahistidine, forms of the proteins were presented on the E. coli surface.     

Diverse effects of phospholipids on lipoprotein sorting and ATP hydrolysis by the ABC transporter LolCDE complex

The LolCDE complex of Escherichia coli releases outer membrane-specific lipoproteins from the inner membrane. Lipoproteins with Asp at +2 remain in the inner membrane since this residue functions as a LolCDE avoidance signal depending on phosphatidylethanolamine. We examined the effects of other phospholipids on lipoprotein sorting in proteoliposomes reconstituted with LolCDE and various synthetic phospholipids. The lipoprotein release and ATP hydrolysis were both low at 2 mM Mg(2+) but very high at 10 mM Mg(2+) in proteoliposomes containing cardiolipin alone. However, the Lol avoidance function was abolished at 10 mM Mg(2+), and the release of lipoproteins with Asp at +2 was as efficient as that of outer membrane-specific lipoproteins. The addition of phosphatidylethanolamine to cardiolipin stimulated the ATP hydrolysis and increased the Lol avoidance function of Asp at +2 at 2 mM Mg(2+). The addition of phosphatidylglycerol to cardiolipin nearly completely inhibited the release of lipoproteins with Asp at +2 even at 10 mM Mg(2+), while that of outer membrane-specific lipoproteins was not. Taken together, these results indicate that three major phospholipids of E. coli differently affect lipoprotein sorting and the activity of LolCDE.