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.     

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.     

Regulation of membrane phospholipid syntheesis in Escherichia coli during temperature up-shift.

The synthesis of membrane phospholipids and that of stable ribonucleic acid were inhibited during temperature up-shift of both rel+ and relA strains of Escherichia coli. The kinetics of the inhibition of the synthesis of both molecules were correlated with the kinetics of guanosine 5'-diphosphate-3'-diphosphate synthesis. Metabolic down-shift experiments gave similar results.     

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.     

Orientation of the Escherichia coli outer membrane protein OmpX in phospholipid bilayer membranes determined by solid-State NMR

The solid-state NMR orientation-dependent frequencies measured for membrane proteins in macroscopically oriented lipid bilayers provide precise orientation restraints for structure determination in membranes. Here we show that this information can also be used to supplement crystallographic structural data to establish the orientation of a membrane protein in the membrane. This is achieved by incorporating a few orientation restraints, measured for the Escherichia coli outer membrane protein OmpX in magnetically oriented lipid bilayers (bicelles), in a simulated annealing calculation with the coordinates of the OmpX crystal structure. The (1)H-(15)N dipolar couplings measured for the seven Phe residues of OmpX in oriented bilayers can be assigned by back-calculation of the NMR spectrum from the crystal structure and are sufficient to establish the three-dimensional orientation of the protein in the membrane, while the (15)N chemical shifts provide a measure of cross-validation for the analysis. In C14 lipid bilayers, OmpX adopts a transmembrane orientation with a 7 degrees tilt of its beta-barrel axis relative to the membrane normal, matching the hydrophobic thickness of the barrel with that of the membrane.     

Regulation of phospholipid synthesis in Escherichia coli by guanosine tetraphosphate

Phospholipid synthesis has been reported to be subject to stringent control in Escherichia coli. We present evidence that demonstrates a strict correlation between guanosine tetraphosphate accumulation and inhibition of phospholipid synthesis. In vivo experiments designed to examine the pattern of phospholipid labeling with (32)P-inorganic phosphate and (32)P-sn-glycerol-3-phosphate suggest that regulation must occur at the glycerol-3-phosphate acyltransferase step. Assay of phospholipid synthesis by cell-free extracts and semipurified preparations revealed that guanosine tetraphosphate inhibits at least two enzymes specific for the biosynthetic pathway, sn-glycerol-3-phosphate acyltransferase as well as sn-glycerol-3-phosphate phosphatidyl transferase. These findings provide a biochemical basis for the stringent control of lipid synthesis as well as regulation of steady-state levels of phospholipid in growing cells.     

Minimum substrate sequence for signal peptidase I of Escherichia coli

The minimum substrate sequence recognized by signal peptidase I (SPase I or leader peptidase) was defined by measuring the kinetic parameters for a set of chemically synthesized peptides corresponding to the cleavage site of the precursor maltose binding protein (pro-MBP). The minimum sequence of a substrate hydrolyzed by SPase I at a measurable rate was the pentapeptide Ala-Leu-Ala decreases Lys-Ile. The rates of hydrolysis of this substrate, however, were several hundred-fold lower than those observed for the maturation of MBP in Escherichia coli, suggesting that in addition to these minimal sites involved in recognition, other features of pro-MBP are also needed for the optimal rate of signal peptide cleavage by SPase I. One parameter may be the length of the polypeptide chain. Studies of the synthetic peptides showed that decreasing the length of the polypeptide chain of substrates decreased the substrate efficiency measured as kcat/Km. However, in one case a decrease in the length of a peptide corresponding to -7 to +3 positions of pro-MBP to a nonapeptide (-7 to +2) increased the substrate efficiency by about 900-fold. The nonapeptide is the most efficient substrate for the enzyme in vitro so far reported. It is speculated that better peptide substrates are the ones which are able to adopt folded structures.     

Protease IV, a cytoplasmic membrane protein of Escherichia coli, has signal peptide peptidase activity

During export of the outer membrane lipoprotein across the cytoplasmic membrane, the signal peptide of the lipoprotein undergoes two successive proteolytic attacks, cleavage of the signal peptide by signal peptidase and digestion of the cleaved signal peptide by an enzyme called signal peptide peptidase(s) (Hussain, M., Ichihara, S., and Mizushima, S. (1982) J. Biol. Chem. 257, 5177-5182; Hussain, M., Ozawa, Y., Ichihara, S., and Mizushima, S. (1982) Eur. J. Biochem. 129, 233-239). Here we report that protease IV, a cytoplasmic membrane protease, exhibits the signal peptide peptidase activity. The signal peptide peptidase activity was cofractionated with protease IV throughout the entire process of purification of the latter enzyme. Only the signal peptide was digested by the peptidase among membrane proteins. Both the signal peptide peptidase activity and the protease IV activity were inhibited to similar degrees by antipain, leupeptin, chymostatin, and elastatinal that are known to inhibit the signal peptide peptidase activity in the cell envelope. From these results we conclude that protease IV is the signal peptide peptidase that is responsible for signal peptide digestion in the cytoplasmic membrane. The peptidase attacked the signal peptide only after its release from the precursor protein.     

Paradoxical lipid dependence of pores formed by the Escherichia coli alpha-hemolysin in planar phospholipid bilayer membranes

alpha-Hemolysin (HlyA) is an extracellular protein toxin (117 kDa) secreted by Escherichia coli that targets the plasma membranes of eukaryotic cells. We studied the interaction of this toxin with membranes using planar phospholipid bilayers. For all lipid mixtures tested, addition of nanomolar concentrations of toxin resulted in an increase of membrane conductance and a decrease in membrane stability. HlyA decreased membrane lifetime up to three orders of magnitude in a voltage-dependent manner. Using a theory for lipidic pore formation, we analyzed these data to quantify how HlyA diminished the line tension of the membrane (i.e., the energy required to form the edge of a new pore). However, in contrast to the expectation that adding the positive curvature agent lysophosphatidylcholine would synergistically lower line tension, its addition significantly stabilized HlyA-treated membranes. HlyA also appeared to thicken bilayers to which it was added. We discuss these results in terms of models for proteolipidic pores.     

Interaction of some polyhexamethylene biguanides and membrane phospholipids in Escherichia coli

The interaction between some polyhexamethylene biguanides and the cell envelope of Escherichia coli has been investigated. An amine-ended dimer, (AED, n = 2), a polydisperse mixture (ICI plc) available as the active ingredient of Vantocil IB, (PHMB, n = 5.5), and a high molecular weight fraction, (HMW, n = greater than or equal to 10) of PHMB were used. The sensitivity of batch cultures depleted of magnesium (M-dep), phosphorus (P-dep) or glycerol (C-dep) towards the biocides was assessed by monitoring the rate and extent of potassium ion leakage. P-dep suspensions were particularly resistant to all these agents and possessed less than half the quantity of phospholipid of other cell types. This was compensated for by a proportionate increase in fatty acid and neutral lipid content of the cells. The reduction in phospholipid content was accounted for by decreases in phosphatidylglycerol (PG) and phosphatidylethanolamine (PE). Diphosphatidylglycerol (DPG) and phosphatidylserine (PS) content of the cultures remained unaffected by the depleting nutrient. Fourier-transform n.m.r. spectroscopy was used to study proton nuclei during the interaction of HMW, AED and PHMB with various phospholipid-vesicle preparations. The results strongly suggest that the biocides acted preferentially on the acidic phospholipids PG and DPG, rather than towards PE or PS. Resistance of P-dep cultures therefore reflected reductions in PG content. A molecular basis for the interaction of these compounds and membranes is proposed.     

Interaction of some polyhexamethylene biguanides and membrane phospholipids in Escherichia coli

The interaction between some polyhexamethylene biguanides and the cell envelope of Escherichia coli has been investigated. An amine-ended dimer, (AED, n = 2), a polydisperse mixture (ICI plc) available as the active ingredient of Vantocil IB, (PHMB, n = 5.5), and a high molecular weight fraction, (HMW, n =≧ 10) of PHMB were used. The sensitivity of batch cultures depleted of magnesium (M-dep), phosphorus (P-dep) or glycerol (C-dep) towards the biocides was assessed by monitoring the rate and extent of potassium ion leakage. P-dep suspensions were particularly resistant to all these agents and possessed less than half the quantity of phospholipid of other cell types. This was compensated for by a proportionate increase in fatty acid and neutral lipid content of the cells. The reduction in phospholipid content was accounted for by decreases in phosphatidylglycerol (PG) and phosphatidylethanolamine (PE). Diphosphatidylglycerol (DPG) and phosphatidylserine (PS) content of the cultures remained unaffected by the depleting nutrient. Fourier-transform n.m.r. spectroscopy was used to study proton nuclei during the interaction of HMW, AED and PHMB with various phospholipid-vesicle preparations. The results strongly suggest that the biocides acted preferentially on the acidic phospholipids PG and DPG, rather than towards PE or PS. Resistance of P-dep cultures therefore reflected reductions in PG content. A molecular basis for the interaction of these compounds and membranes is proposed.     

Regulation of the balanced synthesis of membrane phospholipids. Experimental test of models for regulation in Escherichia coli

In Escherichia coli, highly effective regulation controls the balanced synthesis of membrane phospholipids, important for optimal growth. Regulation is such that normally about 70% of a common pool of cytosine liponucleotide precursor is utilized by phosphatidylserine synthase and eventually converted to phosphatidylethanolamine, while about 30% is utilized by the competing enzyme phosphatidylglycerophosphate synthase and converted to phosphatidylglycerol (25%) plus cardiolipin (5%). Although the ratio of phosphatidylglycerol to cardiolipin may vary with conditions of growth, the sum of these two lipids remains relatively constant at about 30% of the total. Alternative models, postulating coordinate regulation of the two competing enzymes, or independent feedback regulation are proposed. These models were tested in experiments in which phosphatidylglycerol was continuously removed from growing cells treated with arbutin (4-hydroxyphenyl-O-beta-D-glucoside), causing its conversion to arbutinphosphoglycerol (Bohin, J.-P., and Kennedy, E.P. (1984) J. Biol. Chem. 259, 8388-8393.) The synthesis of phosphatidylglycerol was increased by a factor of 7 in cells treated with arbutin, with only small changes in phospholipid composition and with no significant change in the level of phosphatidylglycerophosphate synthase. The synthesis of phosphatidylethanolamine was not significantly increased, decisively eliminating the model that requires coordinate regulation of phosphatidylserine synthase and phosphatidylglycerophosphate synthase, and supporting the model of independent feedback inhibition, sensitive to very small changes in composition of cellular phospholipids.     

The phase property of membrane phospholipids is affected by the functionality of signal peptides from the Escherichia coli ribose-binding protein

We examined the effects of synthetic signal peptides from the wild-type, export-defective mutant and its revertant species of ribose-binding protein on the phase properties of lipid bilayers. The lateral segregation of phosphatidylglycerol (PG) in the lipid bilayer was detected through quenching between NBD-PGs upon the reconstitution of signal peptide into the liposome made with the Escherichia coli inner membrane composition. The tendency of lipid segregation was highly dependent on the export competency of signal peptides in vivo, with a decreasing order of wild-type, revertant, and mutant species. The colocalizations of pyrene-PG with BODIPY-PG were also induced by the signal peptides, confirming the phase separation of the acidic phospholipid. The wild-type and revertant signal peptides predominantly formed alpha-helical conformations with the presence of acidic phospholipid as determined by circular dichroism spectroscopy. In addition, they restricted the motion of lipid acyl chains as monitored by fluorescence anisotropy of DPH, suggesting a deep penetration of signal peptide into the lipid bilayer. However, the alpha-helical content of mutant signal peptide was only about half that of the wild-type or revertant peptide with a significantly smaller degree of penetration into the bilayer. An association of the defective signal peptides into the membrane was affected by salt extraction, whereas the functional ones were not. The aforementioned results indicate that the functionality of signal peptide is accomplished through its topologies in the membrane and also by its ability to induce lateral segregation of acidic phospholipid. We propose that the clustering of acidic phospholipid by the functional signal peptide is responsible for the formation of non-bilayer membrane structure, thereby promoting an efficient translocation of secretory proteins.     

Dynamic states of phospholipids in Escherichia coli B membrane. Electron spin resonance studies with biosynthetically generated phospholipid spin labels

Mobility of phospholipid hydrocarbons in the Escherichia coli B membrane fractions was studied by labeling phosphatidylethanolamine or phosphatidylglycerol in situ by biosynthetic incorporation of the spin label. For this purpose, CDP-diacylglycerol spin label was synthesized from phosphatidic acid spin label and cytidine 5'-phosphoromorpholidate and purified by thin-layer chromatography. DCP-diacylglycerol spin label was then incorporated into phospholipids biosynthetically. ESR spectra of these E. coli B membrane fractions showed that phosphatidylglycerol tended to interact with membrane proteins through the mediation of Mg2+, whereas phosphatidylethanolamine had less of this tendency and was more involved in the formation of the bulk of the bilayer continuum of the membrane. These conclusions were also supported by labeling membranes with exogenous spin-labeled phospholipids, although there was some indication that exogenous phospholipids were incorporated into sites different from the sites of incorporation of phospholipids newly synthesized in situ.     

Mutational evidence of transition state stabilization by serine 88 in Escherichia coli type I signal peptidase

Type I signal peptidase (SPase I) catalyzes the hydrolytic cleavage of the N-terminal signal peptide from translocated preproteins. SPase I belongs to a novel class of Ser proteases that utilize a Ser/Lys dyad catalytic mechanism instead of the classical Ser/His/Asp triad found in most Ser proteases. Recent X-ray crystallographic studies indicate that the backbone amide nitrogen of the catalytic Ser 90 and the hydroxyl side chain of Ser 88 might participate as H-bond donors in the transition-state oxyanion hole. In this work, contribution of the side-chain Ser 88 in Escherichia coli SPase I to the stabilization of the transition state was investigated through in vivo and in vitro characterizations of Ala-, Cys-, and Thr-substituted mutants. The S88T mutant maintains near-wild-type activity with the substrate pro-OmpA nuclease A. In contrast, substitution with Cys at position 88 results in more than a 740-fold reduction in activity (k(cat)) whereas S88A retains much less activity (>2440-fold decrease). Measurements of the kinetic constants of the individual mutant enzymes indicate that these decreases in activity are attributed mainly to decreases in k(cat) while effects on K(m) are minimal. Thermal inactivation and CD spectroscopic analyses indicate no global conformational perturbations of the Ser 88 mutants relative to the wild-type E. coli SPase I enzyme. These results provide strong evidence for the stabilization by Ser 88 of the oxyanion intermediate during catalysis by E. coli SPase I.     

Outer membrane lipid homeostasis via retrograde phospholipid transport in Escherichia coli

Biogenesis of the outer membrane (OM) in Gram‐negative bacteria, which is essential for viability, requires the coordinated transport and assembly of proteins and lipids, including lipopolysaccharides (LPS) and phospholipids (PLs), into the membrane. While pathways for LPS and OM protein assembly are well‐studied, how PLs are transported to and from the OM is not clear. Mechanisms that ensure OM stability and homeostasis are also unknown. The trans‐envelope Tol‐Pal complex, whose physiological role has remained elusive, is important for OM stability. Here, we establish that the Tol‐Pal complex is required for PL transport and OM lipid homeostasis in Escherichia coli. Cells lacking the complex exhibit defects in lipid asymmetry and accumulate excess PLs in the OM. This imbalance in OM lipids is due to defective retrograde PL transport in the absence of a functional Tol‐Pal complex. Thus, cells ensure the assembly of a stable OM by maintaining an excess flux of PLs to the OM only to return the surplus to the inner membrane. Our findings also provide insights into the mechanism by which the Tol‐Pal complex may promote OM invagination during cell division.     

In vitro insertion of leader peptidase into Escherichia coli membrane vesicles.

Leader peptidase is an integral protein of the Escherichia coli cytoplasmic membrane whose topology is known. We have taken advantage of this knowledge and available mutants of this enzyme to develop a genetic test for a cell-free protein translocation reaction. We report that leader peptidase inserted into inverted plasma membrane vesicles in its correct transmembrane orientation. We have examined the in vitro membrane assembly characteristics of a variety of leader peptidase mutants and found that domains required for insertion in vivo are also necessary for insertion in vitro. These data demonstrate the physiological validity of the in vitro insertion reaction and strengthen the use of this in vitro protein translocation reaction for the dissection of this complex sorting pathway.     

Oxidation of unsaturated phospholipids in membrane bilayer mixtures is accompanied by membrane fluidity changes

Steady-state and time-resolved fluorescence spectroscopy has been used to obtain information on oxidation processes and associated dynamical and structural changes in model membrane bilayers made from single unilamellar vesicles (SUV's) of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) mixed with increasing amounts of 1-stearoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (SAPC). The highly unsaturated arachidonoyl chain containing four double bonds is prone to oxidation. Lipid oxidation was initiated chemically by a proper oxidant and could be followed on line via the fluorescence changes of an incorporated fluorescent lipophilic fatty acid: 4,4-difluoro-5-(4-phenyl-1,3-butadienyl)-4-bora-3a,4a-diaza-s-indacene-3-undecanoic acid (BP-C11). The oxidation rate increases with an increasing amount of SAPC. Size measurements of different SUV's incorporated with a trace amount of a phosphatidylcholine analogue of BP-C11 using fluorescence correlation spectroscopy have demonstrated that an increase of lipid unsaturation results in smaller sized SUV's and therefore to a larger curvature of the outer bilayer leaflet. This suggests that the lipid-lipid spacing has increased and that the unsaturated fatty acyl chains are better accessible for the oxidant. Oxidation results in some characteristic physical changes in membrane dynamics and structure, as indicated by the use of specific fluorescence probes. Fluorescence measurements of both dipyrenyl- and diphenylhexatriene-labelled PC introduced in non-oxidised and oxidised DOPC-SAPC membranes clearly show that the microfluidity (local fluidity at the very site of the probes) significantly decreases when the oxidised SAPC content increases in the lipid mixture. A similar effect is observed from the lateral diffusion experiments using monopyrenyl PC in the same membrane systems: the lateral diffusion is distinctly slower in oxidised membranes.