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ABSTRACT: We report here the identification of a new lipoprotein, NlpI, in Escherichia coli K-12. The NlpI structural gene (nlpI) is located between the genes pnp (polynucleotide phosphorylase) and deaD (RNA helicase) at 71 min on the E. coli chromosome. The nlpI gene encodes a putative polypeptide of approximately 34 kDa, and multiple lines of evidence clearly demonstrate that NlpI is indeed a lipoprotein. An nlpI::cm mutation rendered growth of the cells osmotically sensitive, and incubation of the insertion mutant at an elevated temperature resulted in the formation of filaments. The altered phenotype of the mutant was a direct consequence of the mutation in nlpI, since it was complemented by the wild-type nlpI gene alone. Overexpression of the unaltered nlpI gene in wild-type cells resulted in the loss of the rod morphology and the formation of single prolate ellipsoids and pairs of prolate ellipsoids joined by partial constrictions. NlpI may be important for an as-yet-undefined step in the overall process of cell division.
Journal of Bacteriology 08/1999; 181(14):4318-25. · 3.83 Impact Factor
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ABSTRACT: We have studied the effect of several structurally related mansonones on the cytotoxicity of plant and bacterial toxins in Vero and BER-40, a brefeldin A-resistant mutant of Vero cells. Mansonone-D (MD), a sesquiterpenoid ortho-naphthoquinone, inhibited the cytotoxicity of ricin, modeccin, Pseudomonas toxin, and diphtheria toxin in Vero cells to different extents. The inhibition of ricin cytotoxicity was dose dependent and reversed upon removal of the drug. Protection of ricin cytotoxicity was also observed in the presence of cycloheximide, indicating that de novo protein synthesis is not required for the protective effect. Although MD inhibited the degradation and excretion of ricin, the binding and internalization of ricin was not affected. In contrast, MD strongly reduced the specific binding of diphtheria toxin in Vero cells. Fluorescence microscopic studies show that MD treatment dramatically alters the morphology of the Golgi apparatus in Vero cells. The kinetic studies reveal that the protection of ricin cytotoxicity is the consequence of decreased toxin translocation to the cytosol in MD-treated cells. The reactive ortho-quinone moiety of MD is important for the protective effect as thespesone, a para-naphthoquinone with a heterocyclic ring structure identical to that of MD, did not inhibit the cytotoxicity of toxins. Thespone, a dehydromansonone-D, lacking two hydrogens from the heterocyclic dihydrofuran ring of MD, inhibited the cytotoxicity of ricin, but was albeit less potent than MD. Neither mansonone-E nor mansonone-H with reactive ortho-quinone moiety, but with a different heterocyclic structure, had any effect on the cytotoxicity of ricin indicating that the protective effect of MD is specifically related to the overall structure of the metabolite.
Journal of Cellular Physiology 08/1998; 176(1):40-9. · 3.87 Impact Factor
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ABSTRACT: Three distinct clones from a Salmonella typhimurium genomic library were identified which suppressed the copper-sensitive (Cu(s)) phenotype of cutF mutants of Escherichia coli. One of these clones, pCUTFS2, also increased the copper tolerance of cutA, -C, and -E mutants, as well as that of a lipoprotein diacylglyceryl transferase (lgt) mutant of E. coli. Characterization of pCUTFS2 revealed that the genes responsible for suppression of copper sensitivity (scs) reside on a 4.36-kb DNA fragment located near 25.4 min on the S. typhimurium genome. Sequence analysis of this fragment revealed four open reading frames (ORF120, ORF627, ORF207, and ORF168) that were organized into two operons. One operon consisted of a single gene, scsA (ORF120), whereas the other operon contained the genes scsB (ORF627), scsC (ORF207), and scsD (ORF168). Comparison of the deduced amino acid sequences of the predicted gene products showed that ScsB, ScsC, and ScsD have significant homology to thiol-disulfide interchange proteins (CutA2, DipZ, CycZ, and DsbD) from E. coli and Haemophilus influenzae, to an outer membrane protein (Com1) from Coxiella burnetii, and to thioredoxin and thioredoxin-like proteins, respectively. The two operons were subcloned on compatible plasmids, and complementation analyses indicated that all four proteins are required for the increased copper tolerance of E. coli mutants. In addition, the scs locus also restored lipoprotein modification in lgt mutants of E. coli. Sequence analyses of the S. typhimurium scs genes and adjacent DNAs revealed that the scs locus is flanked by genes with high homology to the cbpA (predicted curved DNA-binding protein) and agp (acid glucose phosphatase) genes of E. coli located at 22.90 min (1,062.07 kb) and 22.95 min (1,064.8 kb) of the E. coli chromosome, respectively. However, examination of the E. coli chromosome revealed that these genes are absent at this locus and no evidence has thus been obtained for the occurrence of the scs locus elsewhere on the genome.
Journal of Bacteriology 09/1997; 179(16):4977-84. · 3.83 Impact Factor
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ABSTRACT: 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.
Journal of Bacteriology 06/1997; 179(9):2944-8. · 3.83 Impact Factor
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ABSTRACT: The structure-function relationship of bacterial prolipoprotein diacylgyceryl transferase (LGT) Has been investigated by a comparison of the primary structures of this enzyme in phylogenetically distant bacterial species, analysis of the sequences of mutant enzymes, and specific chemical modification of the Escherichia coli enzyme. A clone containing the gene for LGT, lgt, of the gram-positive species Staphylococcus aureus was isolated by complementation of the temperature-sensitive lgt mutant of E. coli (strain SK634) defective in LGT activity. In vivo and in vitro assays for prolipoprotein diacylglyceryl modification activity indicated that the complementing clone restored the prolipoprotein modification activity in the mutant strain. Sequence determination of the insert DNA revealed an open reading frame of 837 bp encoding a protein of 279 amino acids with a calculated molecular mass of 31.6 kDa. S. aureus LGT showed 24% identity and 47% similarity with E. coli, Salmonella typhimurium, and Haemophilus influenzae LGT.S. aureus LGT, while 12 amino acids shorter than the E. coli enzyme, had a hydropathic profile and a predicted pI (10.4) similar to those of the E. coli enzyme. Multiple sequence alignment among E. coli, S. typhimurium, H. influenzae, and S. aureus LGT proteins revealed regions of highly conserved amino acid sequences throughout the molecule. Three independent lgt mutant alleles from E. coli SK634, SK635, and SK636 and one lgt allele from S. typhimurium SE5221, all defective in LGT activity at the nonpermissive temperature, were cloned by PCR and sequenced. The mutant alleles were found to contain a single base alteration resulting in the substitution of a conserved amino acid. The longest set of identical amino acids without any gap was H-103-GGLIG-108 in LGT from these four microorganisms. In E. coli lgt mutant SK634, Gly-104 in this region was mutated to Ser, and the mutant organism was temperature sensitive in growth and exhibited low LGT activity in vitro. Diethylpyrocarbonate inactivated the E. coli LGT with a second-order rate constant of 18.6 M-1S-1, and the inactivation of LGT activity was reversed by hydroxylamine at pH 7. The inactivation kinetics were consistent with the modification of a single residue, His or Tyr, essential for LGT activity.
Journal of Bacteriology 01/1996; 177(23):6820-4. · 3.83 Impact Factor
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ABSTRACT: We investigated the effects of various ceramide (Cer) analogs and related sphingolipids on the cytotoxicities of modeccin, ricin, Pseudomonas toxin, and diphtheria toxin in various cell lines. The most pronounced protective effect by C6Cer, a short-chain cell-permeable Cer analog, was observed in modeccin cytotoxicity in Vero, BER-40, and MDCK cells, whereas the cytotoxicity of diphtheria toxin was not affected by any of the ceramide analogs tested. C6Cer did not affect the binding and internalization of ricin and modeccin in Vero and BER-40 cells. C2Cer and C8Cer also protected against modeccin cytotoxicity, albeit less effectively than C6Cer. However, related sphingolipids including sphingosine, sphingomyelin, lactosylceramide, C18Cer (the naturally occurring ceramide), and dihydro C6Cer had no effect. A correlation was found between the ability of ceramides to inhibit bulk protein secretion and the inhibition of modeccin cytotoxicity by ceramides. Among Cer analogs tested, C6Cer, the most potent inhibitor of modeccin cytotoxicity, strongly inhibited bulk protein secretion in Vero, BER-40, and MDCK cells. PtK1 cells, which were not protected by ceramides against toxins, were resistant to ceramide-induced inhibition of bulk protein secretion. These results confirm that Cer may modulate the intracellular transport of proteins through the Golgi complex. Such Cer-sensitive processes may be involved in the intoxication of cells by plant and bacterial toxins, especially modeccin.
Experimental Cell Research 12/1995; 221(1):1-10. · 3.58 Impact Factor
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ABSTRACT: It has been suggested previously that copper transport in Escherichia coli is mediated by the products of at least six genes, cutA, cutB, cutC, cutD, cutE, and cutF. A mutation in one or more of these genes results in an increased copper sensitivity (D. Rouch, J. Camakaris, and B. T. O. Lee, p. 469-477, in D. H. Hamer and D. R. Winge, ed., Metal Ion Homeostasis: Molecular Biology and Chemistry, 1989). Copper-sensitive cutC and cutF mutants were transformed with a genomic library of E. coli, and copper-tolerant transformants were selected. Two distinct clones were identified, each of which partially restores copper tolerance in both the cutC and cutF mutants of E. coli. Subcloning, physical mapping, and sequence analysis have revealed that the cutC gene is located at 42.15 min on the E. coli genome and encodes a cytoplasmic protein of 146 amino acids and that the cutF gene is located at 4.77 min on the E. coli genome and is allelic to the nlpE gene independently identified by Silhavy and coworkers (W. B. Snyder, L. J. B. Davis, P. N. Danese, C. L. Cosma, and T. J. Silhavy, J. Bacteriol. 177:4216-4223, 1995). Results from the genetic mapping of the copper-sensitive mutations in the cutF mutant and sequencing of the cutC and cutF (nlpE) alleles from both cutC and cutF mutants indicate that both the cutC and cutF mutants are in fact double mutants altered in these two genes, and mutations in both the genes appear to be required for the copper-sensitive phenotype in each mutant.
Journal of Bacteriology 09/1995; 177(15):4207-15. · 3.83 Impact Factor
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ABSTRACT: Ilimaquinone (IQ), a metabolite from sea sponges, has been shown to cause the breakdown of Golgi membranes into small vesicular structure and to inhibit protein transport without eliciting the retrograde transport of the Golgi enzymes to the endoplasmic reticulum [P. A. Takizawa, J. K. Yucel, B. Viet, D. J. Faulkner, T. Deerinck, G. Soto, M. Ellismann, and V. Malhotra, Cell (1993) 73, 1079-1090]. We have found that incubation of Vero cells with IQ inhibited the cytotoxicity of ricin in a dose-dependent manner. The inhibition was reversed upon the removal of IQ. Neither binding and internalization of 125I-ricin nor the translocation of ricin to the cytosol was affected by IQ. However, IQ significantly inhibited the recycling and degradation of internalized 125I-ricin. Preincubation with IQ also prevented the enhancement of ricin cytotoxicity by NH4Cl or nigericin. The inhibition of ricin cytotoxicity by IQ was observed in the presence of cycloheximide, indicating that de novo protein synthesis is not required for IQ-mediated protection of Vero cells from ricin cytotoxicity. In contrast to perinuclear distribution of TRITC-labeled ricin in Vero cells, TRITC-ricin appeared in numerous small vesicles dispersed throughout the cytoplasm in IQ-treated Vero cells. Double labeling with C6-NBD-ceramide and TRITC-labeled ricin showed that these ricin-containing vesicles were distinct from the IQ-induced breakdown product of the Golgi membranes. Like brefeldin A (BFA), IQ inhibited the cytotoxicities of abrin, modeccin, Pseudomonas toxin, and Shiga-like toxin in Vero cells. Unlike BFA, IQ also inhibited the cytotoxicity of diphtheria toxin (DT). Inhibition of DT cytotoxicity was the consequence of a decreased specific binding of the toxin in the IQ-treated cells.
Experimental Cell Research 09/1995; 219(2):671-8. · 3.58 Impact Factor
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ABSTRACT: Using a combination of biochemical, physical, and genetic techniques, we have shown that the umpA gene of Escherichia coli is allelic with the lgt (phosphatidylglycerol:prolipoprotein diacylglyceryl transferase) of Salmonella typhimurium. These genes are essential for the viability of the respective organism and exhibit 92.8% sequence identity at the amino acid level. In E. coli, lgt and thyA (thymidylate synthase) form an operon. Thymidylate synthase levels are regulated by transcription from the lgt promoter and by translational coupling.
Journal of Bacteriology 05/1995; 177(7):1879-82. · 3.83 Impact Factor
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ABSTRACT: We have found that C6 ceramide, a cell-permeable ceramide analog, partially restored the brefeldin A (BFA) sensitivity in a BFA-resistant mutant of Vero cells (BER-40) and in the naturally BFA-resistant Madin-Darby canine kidney (MDCK) cells. Incubation of BER-40 and MDCK cells with low concentrations of C6 ceramide resulted in (i) a pronounced increase in BFA cytotoxicity as measured by the inhibition of [3H]thymidine incorporation and the inhibition of colony formation by BFA, (ii) a significant protection by BFA against ricin cytotoxicity, and (iii) an inhibition of bulk protein secretion by BFA in BER-40 and MDCK cells. Related sphingolipids including sphingosine, sphingomyelin, and lactosylceramide and other unrelated lipid second messengers such as arachidonic acid and 1,2-diacylglycerol did not elicit the protection of BER-40 and MDCK cells against ricin cytotoxicity by BFA. C6 ceramide was the most effective among the ceramides with different acyl chain lengths. Interestingly, dihydro-C6 ceramide, which lacks the trans double bond in the sphingoid base, had no effect. On the other hand, C6 ceramide did not enhance BFA sensitivity in BFA-sensitive Vero cells. The LD50 of C6 ceramide were similar in Vero and BER-40 cells. Fluorescence microscopic studies revealed that C6 ceramide induced the redistribution of beta-COP from the Golgi membranes to a more dispersed localization in both BFA-sensitive and BFA-resistant cell lines, mimicking the effect of BFA. Suboptimal concentration of C6 ceramide also restored the effect of BFA on the beta-COP distribution in BER-40 and MDCK cells. These results indicate that C6 ceramide restores the BFA sensitivity in BFA-resistant BER-40 and MDCK cells.
Journal of Biological Chemistry 03/1995; 270(8):4088-92. · 4.77 Impact Factor
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Methods in Enzymology 02/1995; 248:169-80. · 2.04 Impact Factor
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Methods in Enzymology 02/1995; 250:683-97. · 2.04 Impact Factor
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ABSTRACT: The peptide, MKATKLVLGAVILGSTLLAGCSSN, corresponding to the N-terminal 24 amino acids of Braun's prolipoprotein, was used to study the lipid modification of prolipoprotein in Escherichia coli by measuring the rate of incorporation of either [2-3H]glycerol or [9,10-3H]palmitate from the corresponding labeled phosphatidylglycerol into the peptide. Using E. coli strains containing varying levels of prolipoprotein diacylglyceryl modification activities due to mutations in or overexpression of the gene involved in diacylglyceryl modification (lgt), we have shown that the activities based on the peptide assay correlated well with the prolipoprotein-based assay. Further, we have followed the fate of the lipid substrate, phosphatidylglycerol, during the modification reaction and found that lipid modification of prolipoprotein involves the transfer of diacylglyceryl moiety from phosphatidylglycerol to the sulfhydryl group of the cysteine residue with the concomitant formation of sn-glycerol 1-phosphate. This mechanism is contrary to the previously proposed two-step mechanism of an initial glyceryl transferase followed by O-acyl transfer (Chattopadhyay, P.K., and Wu, H.C. (1977) Proc. Natl. Acad. Sci. U. S. A. 74, 5318-5322). Accordingly, the enzyme that catalyzes this activity has been named phosphatidylglycerol-prolipoprotein diacylglyceryl transferase. The revised pathway for the lipoprotein biogenesis in bacteria consists of three successive reactions catalyzed by prolipoprotein diacylglyceryl transferase, signal peptidase II, and apolipoprotein N-acyltransferase.
Journal of Biological Chemistry 09/1994; 269(31):19701-6. · 4.77 Impact Factor
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ABSTRACT: Lovastatin, a specific inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, inhibits the biosynthesis of cholesterol and the prenylation of proteins. In this study, we have found that lovastatin inhibited the cytotoxicities of modeccin, ricin, Pseudomonas toxin, and diphtheria toxin in Vero and a brefeldin A (BFA)-resistant mutant of Vero cells (BER-40) to different extents. Among these toxins tested, the cytotoxicity of modeccin was most strongly inhibited by lovastatin in a dose-dependent manner. The protective effect of lovastatin was completely reversed by the addition of mevalonic acid, while the addition of cholesterol had no effect on the cytotoxicity of modeccin in lovastatin-treated cells. These results suggest that prenylated proteins are involved in the intoxication process of modeccin. The addition of cycloheximide to the growth medium also reversed the protective effect of lovastatin, suggesting a requirement of de novo protein synthesis for the protection by lovastatin against toxins. In contrast to Vero and BER-40 cells, no significant effect of lovastatin was observed in naturally BFA-resistant cell lines, PtK1 and MDCK cells, even though similar morphological changes and disassembly of the actin microfilaments were induced by lovastatin in these cell lines as observed in Vero and BER-40 cells. Lovastatin did not affect the binding and internalization of ricin and modeccin in Vero and BER-40 cells. Our results suggest that prenylated cellular proteins are involved in intracellular trafficking or processing of protein toxins, especially modeccin. In PtK1 and MDCK cells, such intracellular vesicle trafficking of protein toxins may be regulated by lovastatin-resistant mechanisms.
Experimental Cell Research 07/1994; 212(2):329-37. · 3.58 Impact Factor
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ABSTRACT: Cytotoxic necrotizing factor type 2 (CNF2) produced by Escherichia coli strains isolated from intestinal and extraintestinal infections is a dermonecrotic toxin of 110 kDa. We cloned the CNF2 gene from a large plasmid carried by an Escherichia coli strain isolated from a lamb with septicemia. Hydropathy analysis of the deduced amino acid sequence revealed a largely hydrophilic protein with two potential hydrophobic transmembrane domains. The N-terminal half of CNF2 showed striking homology (27% identity and 80% conserved residues) to the N-terminal portion of Pasteurella multocida toxin. Methylamine protection experiments and immunofluorescence studies suggested that CNF2 enters the cytosol of the target cell through an acidic compartment and induces the reorganization of actin into stress fibers. Since the formation of stress fibers in eukaryotic cells involves Rho proteins, we radiolabeled these small GTP-binding proteins from CNF2-treated and control cells with a Rho-specific ADP-ribosyltransferase. The [32P]ADP-ribosylated Rho proteins from CNF2-treated cells migrated slightly more slowly in SDS/PAGE than did the labeled proteins from the control cells. This shift in mobility of Rho proteins in SDS/PAGE was also observed when CNF2 and the RhoA protein were coexpressed in E. coli. We propose that Rho proteins are the targets of CNF2 in mammalian cells.
Proceedings of the National Academy of Sciences 05/1994; 91(9):3814-8. · 9.68 Impact Factor
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ABSTRACT: 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.
Journal of Bacteriology 11/1993; 175(19):6113-7. · 3.83 Impact Factor
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ABSTRACT: A temperature-sensitive (ts) mutant of Salmonella typhimurium that accumulated unmodified murein prolipoprotein at 42 degrees C but not at 30 degrees C was identified. In vivo and in vitro studies of the biosynthesis of Braun's lipoprotein revealed that this mutant (SE5221) was defective in the glyceryl modification of prolipoprotein. The ts mutation was mapped to 60.6 min of the S. typhimurium chromosome and was linked to argA and cysH. A clone with a 1.4-kilobase S. typhimurium DNA insert that complemented the ts mutation and restored the prolipoprotein modification activity both in vivo and in vitro was isolated. DNA sequencing of the complementing region revealed an open reading frame encoding a protein with 291 amino acids lacking NH2-terminal signal sequence. This open reading frame is immediately 5' to the thyA gene and is allelic to umpA of Escherichia coli. Wild-type strains harboring the cloned gene exhibited elevated levels of prolipoprotein modification activity. At the non-permissive temperature, the mutation affected both growth and viability, and the mutant cells exhibited anomalous cell morphology. The ts phenotype was suppressed by the introduction of a lpp::Tn10 mutation. These results suggest that the cloned gene encodes prolipoprotein glyceryl transferase (lgt), and in the wild-type background, this prolipoprotein modification enzyme is essential for the growth and viability of S. typhimurium.
Journal of Biological Chemistry 09/1993; 268(22):16544-50. · 4.77 Impact Factor
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ABSTRACT: On screening 440 temperature-sensitive (ts) mutants of Salmonella typhimurium, a mutant strain SE5312 which accumulated apolipoprotein (ALP) at 42 degrees C was identified. In vitro assay of apolipoprotein N-acyltransferase activity indicated that the mutant cell envelope contained reduced activity as compared to the wild-type strain. Transduction with a Mud-P22 mapping set placed the ts mutation to 14-17 min region of the S. typhimurium chromosome. P22 transduction using transposon insertions in this region revealed a linkage of the ts mutation to cobD (6%), nag (8%), and corC68 (99%). The ts phenotype was complemented by a 2.3-kilobase EcoRI subclone derived from lambda-phage 170 of Kohara's bank of Escherichia coli. Restriction enzyme analysis of the cloned DNA revealed that this 2.3-kilobase EcoRI fragment included the copper transport (cutE) gene in E. coli. The mutant strain SE5312 was copper-sensitive at 30 degrees C, and the complementing clone conferred copper resistance and restored the ALP N-acyltransferase activity in the mutant cell. Wild-type strain of S. typhimurium harboring this clone exhibited elevated levels of ALP N-acyltransferase activity. These results suggest that the cloned gene encodes the ALP N-acyltransferase. Upon shift to the non-permissive temperature, the viability of the mutant cells decreased, and the mutant cells assumed anomalous morphology. Temperature-resistant revertants could be readily isolated, and a subset of tr revertants contained no detectable lipoprotein. A lpp::Tn10 derivative of the mutant SE5312 was also temperature-resistant. These observations suggest that ALP N-acyltransferase is essential for the growth and viability of S. typhimurium, and this requirement is decreased in the absence of major outer membrane lipoprotein.
Journal of Biological Chemistry 09/1993; 268(22):16551-6. · 4.77 Impact Factor
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ABSTRACT: We have found that cerulenin, an antibiotic that inhibits de novo fatty acid and cholesterol biosynthesis and fatty acylation of proteins, strongly inhibited the cytotoxicity of ricin, modeccin, Pseudomonas toxin, and diphtheria toxin in a brefeldin A (BFA)-resistant mutant of Vero cells (BER-40). The protective effect of cerulenin against ricin was also observed in two other BFA-resistant cell lines, Madin-Darby canine kidney, and PtK1 cells. In contrast to BER-40 cells, no significant effect of cerulenin was observed in Vero cells. Cerulenin did not affect the binding of ricin to the cell-surface receptors, but reduced significantly the internalization of ricin in BER-40 cells; no effect of cerulenin on the binding or internalization of ricin was observed in Vero, PtK1, and Madin-Darby canine kidney cells. Endocytic uptake of fluid-phase markers such as horseradish peroxidase and lucifer yellow was inhibited by cerulenin in BER-40 cells, but the endocytosis of transferrin via the coated pit/coated vesicle pathway was slightly increased. Cerulenin inhibited the degradation and excretion of ricin in BER-40 cells, and this effect of cerulenin was not observed in Vero cells. Furthermore, cerulenin inhibited the bulk protein secretion in a dose-dependent manner, with BER-40 cells being more susceptible than Vero cells. These results suggest that in addition to its effect on endocytosis, cerulenin interferes with the intracellular trafficking or processing of toxin molecules, and the vesicle transport system in BER-40 cells appears to be cerulenin-sensitive. Since addition of fatty acids and cholesterol did not reverse the effects of cerulenin, the protective effect of cerulenin against protein toxins is not due to an inhibition of de novo fatty acids and cholesterol biosynthesis.
Journal of Biological Chemistry 07/1993; 268(17):12596-602. · 4.77 Impact Factor
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ABSTRACT: The intracellular pathway following receptor-mediated endocytosis of cholera toxin was studied using brefeldin A (BFA), which inhibited protein secretion and induced dramatic morphological changes in the Golgi region. In both mouse Y1 adrenal cells and CHO cells, BFA at 1 micrograms/ml caused a 80-90% inhibition of the cholera toxin (CT)-induced elevation of intracellular cAMP. The inhibition of the cytotoxicity of CT by BFA was also observed in a rounding assay of Y1 adrenal cells. The inhibition of CT cytotoxicity by BFA was dose dependent, with the ID50 value similar to the LD50 of BFA in Y1 adrenal cells. Binding and internalization of [125I]-labeled cholera toxin in Y1 adrenal cells was not affected by BFA. Unlike the BFA-sensitive cell lines such as Y1 adrenal and CHO cells, BFA at 1 micrograms/ml did not inhibit the cytotoxicity of CT in PtK1 cells, of which the Golgi structure was BFA-resistant. These results strongly suggest that a BFA-sensitive Golgi is required for the protection of CT cytotoxicity by BFA. In contrast, elevation of the intracellular cAMP by forskolin, which acts directly on the plasma membrane adenylate cyclase, was not affected by BFA. These observations indicate that the intoxication of target cells by CT requires an intact Golgi region for its intracellular trafficking and/or processing. In this respect, CT shares a common intracellular pathway with ricin, Pseudomonas toxin, and modeccin, even though their structures and modes of action are very different.
Journal of Cellular Physiology 03/1993; 154(2):222-8. · 3.87 Impact Factor
