Accumulation of glyceride-modified pre-penicillinase of Bacillus licheniformis in Escherichia coli treated with globomycin

The membrane penicillinase of Bacillus licheniformis is a glyceride-cysteine lipoprotein whose NH2 terminus is analogous to the major outer membrane lipoprotein of Escherichia coli. When E. coli cells producing B. licheniformis penicillinase were treated with the antibiotic, globomycin, a precursor of the penicillinase, pre-penicillinase, accumulated in the cell. It could be immunoprecipitated with anti-penicillinase antibodies; it contained palmitate; and one of its two cysteine residues was modified by glycerol. The action of globomycin, probably indirectly, also activates protease which acts differently on the pre-penicillinase than does the signal peptidase. The results strongly indicate that the pre-penicillinase is processed by the globomycin-sensitive signal peptidase in E. coli, and the modification of precursor by lipid precedes removal of the signal peptide as it does with the membrane lipoproteins of E. coli.     

Accumulation of prolipoprotein in Escherichia coli mutants defective in protein secretion.

The export of lipoprotein has been found to be affected in both secA and secY mutants of Escherichia coli which are defective in the secretion of a number of outer membrane and periplasmic proteins. The kinetics of accumulation of prolipoprotein upon a temperature shift to 42 degrees C is indistinguishable from that of pre-OmpA protein accumulation in the secA mutant. In both secA and secY mutants, the accumulated prolipoprotein is unmodified with glyceride and localized in the cytoplasmic membrane. We conclude from these results that the early steps in protein export are common to prolipoprotein and non-lipoprotein precursors. The pathways for the export of these two groups of precursor proteins diverge with regard to the modification and processing reactions which are late events in the export process.     

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.     

Existence of abnormal protein aggregates in healthy Escherichia coli cells

Protein aggregation is a phenomenon observed in all organisms and has often been linked with cell disorders. In addition, several groups have reported a virtual absence of protein aggregates in healthy cells. In contrast to previous studies and the expected outcome, we observed aggregated proteins in aerobic exponentially growing and “healthy” Escherichia coli cells. We observed overrepresentation of “aberrant proteins,” as well as substrates of the major conserved chaperone DnaK (Hsp70) and the protease ClpXP (a serine protease), in the aggregates. In addition, the protein aggregates appeared to interact with chaperones known to be involved in the aggregate repair pathway, including ClpB, GroEL, GroES, and DnaK. Finally, we showed that the levels of reactive oxygen species and unfolded or misfolded proteins determine the levels of protein aggregates. Our results led us to speculate that protein aggregates may function as a temporary “trash organelle” for cellular detoxification.     

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.     

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.     

Preferential selection of deletion mutations of the outer membrane lipoprotein gene of Escherichia coli by globomycin.

Globomycin is an antibiotic which inhibits the processing of the prolipoprotein. Eighty globomycin-resistant mutants were independently isolated from Escherichia coli K-12 which had a deletion mutation in chromosomal lipoprotein gene (lpp), but contained a plasmid carrying the wild-type lpp gene. Twenty-six of the mutants did not have the lipoprotein in the membrane fractions. From the analysis of the plasmids of these mutants, all of the lipoprotein-deficient mutations were found to be due to deletion mutations around the lpp gene.     

Roles of histidine-103 and tyrosine-235 in the function of the prolipoprotein diacylglyceryl transferase of Escherichia coli.

Phosphatidylglycerol:prolipoprotein diacylglyceryl transferase (Lgt) is the first enzyme in the posttranslational sequence of reactions resulting in the lipid modification of lipoproteins in bacteria. A previous comparison of the primary sequences of the Lgt enzymes from phylogenetically distant bacterial species revealed several highly conserved amino acid sequences throughout the molecule; the most extensive of these was the region 103HGGLIG108 in the Escherichia coli Lgt (H.-Y. Qi, K. Sankaran, K. Gan, and H. C. Wu, J. Bacteriol. 177:6820-6824, 1995). These studies also revealed that the kinetics of inactivation of E. coli Lgt with diethylpyrocarbonate were consistent with the modification of a single essential histidine or tyrosine residue. The current study was conducted in an attempt to identify this essential amino acid residue in order to further define structure-function relationships in Lgt. Accordingly, all of the histidine residues and seven of the tyrosine residues of E. coli Lgt were altered by site-directed mutagenesis, and the in vitro activities of the altered enzymes, as well the abilities of the respective mutant lgt alleles to complement the temperature-sensitive phenotype of E. coli SK634 defective in Lgt activity, were determined. The data obtained from these studies, in conjunction with additional chemical inactivation studies, support the conclusion that His-103 is essential for Lgt activity. These studies also indicated that Tyr-235 plays an important role in the function of this enzyme. Although other histidine and tyrosine residues were not found to be essential for Lgt activity, alterations of His-196 resulted in a significant reduction of in vitro activity.     

Glucose-sensitive adenylate cyclase in toluene-treated cells of Escherichia coli B.

Toluene treatment of Escherichia coli B makes it possible to measure adenylate cyclase activity directly using [alpha-32-P]-ATP as substrate. In contrast to French press extracts, the activity of adenylate cyclase in toluene-treated cells shows many of the characteristics of the enzyme seen in the intact cell. In both toluene-treated and intact cells the activity of adenylate cyclase is inhibited at least 85% by glucose, while in French press extracts the enzyme activity is much lower and is not sensitive to inhibition by glucose. In toluene-treated cells, glucose inhibits at 10 muM, and the effect is rapid in onset and readily reversible. The activity is not inhibited by glucose 6-phosphate suggesting that glucose is responsible for the inhibition. The measurement of the activity and sensitivity to glucose of adenylate cyclase in toluene-treated cells requires the presence of potassium phosphate in the assay medium. Since it does not increase the activity or sensitivity of the enzyme in the French press extract, it is suggested that potassium phosphate is required for the maintenance of cellular integrity necessary for the activity and sensitivity of adenylate cyclase.     

Sulfate-reducing pathway in Escherichia coli involving bound intermediates.

Although a sulfate-reducing pathway in Escherichia coli involving free sulfite and sulfide has been suggested, it is shown that, as in Chlorella, a pathway involving bound intermediates is also present. E. coli extracts contained a sulfotransferase that transferred the sulfonyl group from a nucleosidephosphosulfate to an acceptor to form an organic thiosulfate. This enzyme was specific for adenosine 3'-phosphate 5'-phosphosulfate, did not utilize adenine 5'-phosphosulfate, and transferred to a carrier molecule that was identical with thioredoxin in molecular weight and amino acid composition. In the absence of thioredoxin, only very low levels of the transfer of the sulfo group to thiols was observed. As in Chlorella, thiosulfonate reductase activity that reduced glutathione-S-SO3- to bound sulfide could be detected. In E. coli, this enzyme used reduced nicotinamide adenine dinucleotide phosphate and Mg2+, but did not require the addition of ferredoxin or ferredoxin nicotinamide adenine dinucleotide phosphate reductase. Although in Chlorella the thiosulfonate reductase appears to be a different enzyme from the sulfite reductase, the E. coli thiosulfonate reductase and sulfite reductase may be activities of the same enzyme.     

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.     

Studies on the modification and processing of prolipoprotein in Escherichia coli. Effects of structural alterations in prolipoprotein on its maturation in wild type and lpp mutants

We have previously shown that an Escherichia coli mutant ( mlpA allele) containing a structurally altered murein prolipoprotein due to substitution of Gly14 by Asp14 , is globomycin resistant. In addition, the mutant prolipoprotein is not modified with glyceride and consequently remains uncleaved. Spontaneous revertants possessing a mature lipoprotein of apparent normal structure can be isolated by EDTA selection. Three revertants were chosen in the present study which included the analysis of kinetics of lipoprotein maturation and the determination of globomycin sensitivity. These pseudorevertants in the lpp gene which could be recognized by the anomalous prolipoprotein mobility in sodium dodecyl sulfate gels, exhibited altered globomycin sensitivity in vivo. Our results indicate that alterations in prolipoprotein structure affect the kinetics of prolipoprotein modification and processing reactions, both in vivo and in vitro. Pulse-chase experiments revealed the transient existence of unmodified prolipoprotein and modified prolipoprotein as biosynthetic intermediates of mature lipoprotein. The rate of prolipoprotein modification appeared to be slightly faster than that of processing in the wild type cell. In contrast, modification of prolipoprotein was rate limiting in a pseudorevertant strain 14R21 , and the processing of 14R21 modified prolipoprotein appeared to proceed more rapidly than that of wild type prolipoprotein, both in vitro and in vivo.     

Effects of prolipoprotein signal peptide mutations on secretion of hybrid prolipo-beta-lactamase in Escherichia coli

Hybrid proteins were constructed by coupling beta-lactamase to the signal sequence (plus nine amino acids) of selected mutant prolipoproteins of Escherichia coli. The mutant prolipoprotein signal peptides contained lesions in two structural domains of the signal peptide, the basic amino-terminal domain and the hydrophobic core domain. We then compared the processing and localization of the mutant prolipo-beta-lactamases to the processing and localization of the comparable mutant prolipoproteins. We show that a mutant signal sequence with an anionic amino terminus exhibits similar limitations in the processing of prolipo-beta-lactamase as previously observed in prolipoprotein. Deletion of four hydrophobic residues from hydrophobic core results in a signal peptide which slowly translocates a fraction of the total mutant hybrid protein synthesized. This signal peptide was previously shown to translocate lipoprotein efficiently. Alteration of this hydrophobic core, which stimulated synthesis of mutant prolipoproteins, does not stimulate synthesis of prolipo-beta-lactamase. Finally mutations that slowed processing of prolipoprotein by affecting the proposed helical structure of the signal peptide had no significant effect on the processing of prolipo-beta-lactamase. These results suggest that the positively charged amino-terminal domain of the signal peptide has a common role in protein secretion regardless of the secretory protein. On the other hand, other domains of the signal peptide exhibit different phenotypes when the secretory protein is changed.     

Neither lipid modification nor processing of prolipoprotein is essential for the formation of murein-bound lipoprotein in Escherichia coli.

The relationship between the modification and processing of prolipoprotein and the formation of murein-bound lipoprotein has been investigated using Escherichia coli mutants altered in the signal sequence of prolipoprotein and an E. coli strain producing OmpF-Lpp hybrid protein. The glyceride-modified prolipoprotein in mutant lppT20 and in globomycin-treated wild-type strain were covalently attached to the peptidoglycan. Likewise, the unmodified prolipoproteins in mutants lppL20, lppV20, and lppG21 were attached to the peptidoglycan. The OmpF-Lpp hybrid protein that is processed but not modified with lipid due to the absence of the cysteine-containing modification site in the hybrid protein was also covalently linked to the peptidoglycan. These results indicate that neither lipid modification nor the processing of prolipoprotein is essential for the formation of murein-bound lipoprotein in E. coli. In contrast, introduction of a charged amino acid residue such as Asp or Arg at the 14th position of prolipoprotein affected not only the lipid modification and processing of the mutant prolipoprotein but also the formation of murein-bound lipoprotein. Replacement of the Gly14 with Glu or Lys partially affected the lipid modification and processing of prolipoprotein; the peptidoglycan of the lppE14 and lppK14 mutants contained a reduced amount of mature lipoprotein but no mutant prolipoprotein. In addition, lpp mutants A20I23I24 and A20I23K24 were found to be defective in both lipid modification/processing of prolipoprotein and the formation of murein-bound lipoprotein. The defective formation of murein-bound lipoprotein in the latter mutants may be related to an alteration in the secondary structure at the modification/processing site of the mutant prolipoproteins.     

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.     

Escherichia coli cyclophilin B binds a highly distorted form of trans-prolyl peptide isomer.

Cyclophilins facilitate the peptidyl-prolyl isomerization of a trans-isomer to a cis-isomer in the refolding process of unfolded proteins to recover the natural folding state with cis-proline conformation. To date, only short peptides with a cis-form proline have been observed in complexes of human and Escherichia coli proteins of cyclophilin A, which is present in cytoplasm. The crystal structures analyzed in this study show two complexes in which peptides having a trans-form proline, i.e. succinyl-Ala-trans-Pro-Ala-p-nitroanilide and acetyl-Ala-Ala-trans-Pro-Ala-amidomethylcoumarin, are bound on a K163T mutant of Escherichia coli cyclophilin B, the preprotein of which has a signal sequence. Comparison with cis-form peptides bound to cyclophilin A reveals that in any case the proline ring is inserted into the hydrophobic pocket and a hydrogen bond between CO of Pro and Neta2 of Arg is formed to fix the peptide. On the other hand, in the cis-isomer, the formation of two hydrogen bonds of NH and CO of Ala preceding Pro with the protein fixes the peptide, whereas in the trans-isomer formation of a hydrogen bond between CO preceding Ala-Pro and His47 Nepsilon2 via a mediating water molecule allows the large distortion in the orientation of Ala of Ala-Pro. Although loss of double bond character of the amide bond of Ala-Pro is essential to the isomerization pathway occurring by rotating around its bond, these peptides have forms impossible to undergo proton transfer from the guanidyl group of Arg to the prolyl N atom, which induces loss of double bond character.     

Cloning and expression of a gene coding for the prolipoprotein signal peptidase of Escherichia coli

An Escherichia coli mutant, Y815, has a temperature-sensitive prolipoprotein signal peptidase. IPTG-induced synthesis of the major outer membrane prolipoprotein (PLP) results in the inhibition of cell growth because of accumulation of PLP in its envelope [J. Bacteriol. (1982) 152, 1163-1168]. The 2000 E. coli strains of Clarke and Carbon's collection were screened for the presence of a plasmid complementing the IPTG-sensitivity of the growth of Y815. One plasmid, pLC3-13, complemented the IPTG-sensitivity. The envelope fraction prepared from Y815 transformed by pLC3-13 showed high activity of the PLP signal peptidase in vitro at high temperature. A 4 kb AccI fragment subcloned onto plasmid pHY001 was shown to carry the gene for the PLP signal peptidase.