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Microcin E492 is a channel-forming bacteriocin that is found in two forms, namely, a posttranslationally modified form obtained by the covalent linkage of salmochelin-like molecules to serine 84 and an unmodified form. The production of modified microcin E492 requires the synthesis of enterochelin, which is subsequently glycosylated by MceC and converted into salmochelin. mceC mutants produced inactive microcin E492, and this phenotype was reversed either by complementation with iroB from Salmonella enterica or by the addition of exogenous salmochelin. Cyclic salmochelin uptake by Escherichia coli occurred mainly through the outer membrane catecholate siderophore receptor Fiu. The production of inactive microcin E492 by mutants in entB and entC was reverted by the addition of the end product of the respective mutated pathway (2,3-dihydroxybenzoic acid and enterochelin/salmochelin, respectively), while mutants in entF did not produce active microcin E492 in the presence of enterochelin or salmochelin. The EntF adenylation domain was the only domain required for this microcin E492 maturation step. Inactivation of the enzymatic activity of this domain by site-directed mutagenesis did not prevent the synthesis of active microcin E492 in the presence of salmochelin, indicating that the adenylation activity is not essential for the function of EntF at this stage of microcin E492 maturation. 相似文献
3.
Delphine Destoumieux-Garzón Jean Peduzzi Xavier Thomas Chakib Djediat Sylvie Rebuffat 《Biometals》2006,19(2):181-191
Microcin E492 (MccE492) is an antibacterial peptide naturally secreted by Klebsiella pneumoniae RYC492. Initially described as an 84-residue unmodified peptide, it was also recently isolated in a posttranslationally modified
form, MccE492m. The production of MccE492m is dependent on the synthesis of enterobactin and the mceABCDEFGHIJ gene cluster. The posttranslational modification was characterized as a trimer of N-(2,3-dihydroxybenzoyl)-l-serine (DHBS) linked to the Ser84-carboxylate via a β-d-glucose moiety. MccE492m was shown to bind ferric ions through the trimer of DHBS. This is the first example of a novel type
of antibacterial peptide termed siderophore-peptide. Recognition of MccE492m, but also of the unmodified MccE492, was shown
to be mediated by the catecholate siderophore receptors FepA, Cir and Fiu at the outer membrane of E. coli. The siderophore-type modification was shown to be responsible for a significant enhancement of the microcin antibacterial
activity. Therefore, we propose that MccE492 and MccE492m use iron-siderophore receptors for uptake into the target bacteria
and that improvement of MccE492 antimicrobial activity upon modification results from an increase in the microcin/receptor
affinity. 相似文献
4.
Cloning and expression in Escherichia coli of genetic determinants for production of and immunity to microcin E492 from Klebsiella pneumoniae.
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M Wilkens J E Villanueva J Cofr J Chnaiderman R Lagos 《Journal of bacteriology》1997,179(15):4789-4794
Microcin E492 is a polypeptide antibiotic that is produced and excreted by Klebsiella pneumoniae RYC492. The genetic determinants for microcin synthesis and immunity were cloned in Escherichia coli VCS257 into the cosmid vector pHC79, starting from total DNA of K. pneumoniae RYC492. The microcin E492 expressed in E. coli had the same properties as that of K. pneumoniae, i.e., the same molecular weight, the ability to form ionic channels in planar phospholipid bilayers, and essentially identical biological properties. Microcin E492 expression in E. coli, like that in K. pneumoniae, was mainly in the exponential phase of growth, declining in the stationary phase. The immunity determinant was subcloned into the same vector, and its expression was found to disappear in the stationary phase. This phenomenon is not dependent on rpoS, the stationary-phase sigma factor. 相似文献
5.
Structure, organization and characterization of the gene cluster involved in the production of microcin E492, a channel-forming bacteriocin 总被引:1,自引:0,他引:1
Rosalba Lagos Marcelo Baeza Gino Corsini Claudio Hetz Erwin Strahsburger José Antonio Castillo Cecilia Vergara Octavio Monasterio 《Molecular microbiology》2001,42(1):229-243
Microcin E492 is a low-molecular-weight, channel-forming bacteriocin produced and excreted by Klebsiella pneumoniae RYC492. A 13 kb chromosomal DNA fragment from K. pneumoniae RYC492 was sequenced, and it was demonstrated by random Tn5 mutagenesis that most of this segment, which has at least 10 cistrons, is needed for the production of active microcin and its immunity protein. Genes mceG and mceH correspond to an ABC exporter and its accessory protein, respectively, and they are closely related to the colicin V ABC export system. The microcin E492 system also requires the product of gene mceF as an additional factor for export. Despite the fact that this bacteriocin lacks post-translational modifications, genes mceC, mceI and mceJ are needed for the production of active microcin. Genes mceC and mceI are homologous to a glycosyl transferase and acyltransferase, respectively, whereas mceJ has no known homologue. Mutants in these three genes secrete an inactive form of microcin, able to form ion channels in a phospholipidic bilayer, indicating that the mutation of these microcin genes does not alter the process of membrane insertion. On the other hand, microcin isolated from mutants in genes mceC and mceJ has a lethal effect when incubated with spheroplasts of sensitive cells, indicating that the microcin defects in these mutants are likely to alter receptor recognition at the outer membrane. A model for synthesis and export is proposed as well as a novel maturation pathway that would involve conformational changes to explain the production of active microcin E492. 相似文献
6.
Identification and Properties of the Genes Encoding Microcin E492 and Its Immunity Protein 总被引:2,自引:0,他引:2
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The gene coding for the immunity protein (mceB) and the structural gene of microcin E492 (mceA), a low-molecular-weight channel-forming bacteriocin produced by a strain of Klebsiella pneumoniae, have been characterized. The microcin gene codes for a precursor protein of either 99 or 103 amino acids. Protein sequencing of the N-terminal region of microcin E492 unequivocally identified this gene as the microcin structural gene and indicated that this microcin is synthesized as a precursor protein that is cleaved at either amino acid 15 or 19, at a site resembling the double-glycine motif. The gene encoding the 95-amino-acid immunity protein (mceB) was identified by cloning the DNA segment that encodes only this polypeptide into an expression vector and demonstrating the acquisition of immunity to microcin E492. As expected, the immunity protein was found to be associated with the inner membrane. Analysis of the DNA sequence indicates that these genes belong to the same family as microcin 24, and they do not share structural motifs with any other known channel-forming bacteriocin. The organization of the microcin- and immunity protein-encoding genes suggests that they are coordinately expressed. 相似文献
7.
Arranz R Mercado G Martín-Benito J Giraldo R Monasterio O Lagos R Valpuesta JM 《Journal of structural biology》2012,178(1):54-60
Microcin E492 is a low-molecular weight, channel-forming bacteriotoxin that generates amyloid structures. Using electron microscopy and image processing techniques several structural conformations can be observed. Prior to the conditions that induce amyloid formation and at its initial stage, microcin E492 molecules can be found in two main types of oligomers: a pentameric, pore-like structure consisting of globular monomers of ~25? diameter, and long filaments made up of stacked pentamers. The equilibrium between these structures depends on the properties of the solvent, because samples kept in methanol mainly show the pentameric structure. Amyloid induction in aqueous solvent reveals the presence, together with the above mentioned structures, of several amyloid structures such as flat and helical filaments. In addition, X-ray diffraction analysis demonstrated that the fibrils formed by microcin E492 presented cross-β structure, a distinctive property of amyloid fibrils. Based on the study of the observed structures we propose that microcin E492 has two conformations: a native one that assembles mainly into a pentameric structure, which functions as a pore, and an amyloid conformation which results in the formation of different types of amyloid filaments. 相似文献
8.
Víctor de Lorenzo 《Archives of microbiology》1984,139(1):72-75
The production of a dialysable peptidic antibacterial named microcin E492 by the strain of faecal originKlebsiella pneumoniae RYC492 has previously been reported. In this paper, a procedure to extract this antibiotic from liquid cultures of the producer strain is described. This method was based in the quantitative retention of the microcin on the hydrophobic matrix Bondapak C18 and led to highly active pigment- and salf-free concentrates appropriate for further purification by high pressure liquid chromatography. The characterization of purified preparations indicated that microcin E492 was a basic and hydrophobic peptide with an apparent molecular mass of about 5,000, acid- and heat-resistant and much more active in minimal than in rich medium. These properties are discussed with regard to the likely ecological role of the microcin in the microbial ecosystem of the intestine.Abbreviations AU
Antibiotic Unit
- CFU
Colony-forming units
- HPLC
High Pressure Liquid Chromatography
- Mr
Relative molecular mass
- RP
Reversed phase
- TEAP
Triethylamine-phosphoric acid 相似文献
9.
Destoumieux-Garzón D Thomas X Santamaria M Goulard C Barthélémy M Boscher B Bessin Y Molle G Pons AM Letellier L Peduzzi J Rebuffat S 《Molecular microbiology》2003,49(4):1031-1041
The mechanism of action of microcin E492 (MccE492) was investigated for the first time in live bacteria. MccE492 was expressed and purified to homogeneity through an optimized large-scale procedure. Highly purified MccE492 showed potent antibacterial activity at minimal inhibitory concentrations in the range of 0.02-1.2 microM. The microcin bactericidal spectrum of activity was found to be restricted to Enterobacteriaceae and specifically directed against Escherichia and Salmonella species. Isogenic bacteria that possessed mutations in membrane proteins, particularly of the TonB-ExbB-ExbD complex, were assayed. The microcin bactericidal activity was shown to be TonB- and energy-dependent, supporting the hypothesis that the mechanism of action is receptor mediated. In addition, MccE492 depolarized and permeabilized the E. coli cytoplasmic membrane. The membrane depolarization was TonB dependent. From this study, we propose that MccE492 is recognized by iron-siderophore receptors, including FepA, which promote its import across the outer membrane via a TonB- and energy-dependent pathway. MccE492 then inserts into the inner membrane, whereupon the potential becomes destabilized by pore formation. Because cytoplasmic membrane permeabilization of MccE492 occurs beneath the threshold of the bactericidal concentration and does not result in cell lysis, the cytoplasmic membrane is not hypothesized to be the sole target of MccE492. 相似文献
10.
Low-molecular-weight post-translationally modified microcins 总被引:2,自引:0,他引:2
Microcins are a class of ribosomally synthesized antibacterial peptides produced by Enterobacteriaceae and active against closely related bacterial species. While some microcins are active as unmodified peptides, others are heavily modified by dedicated maturation enzymes. Low-molecular-weight microcins from the post-translationally modified group target essential molecular machines inside the cells. In this review, available structural and functional data about three such microcins--microcin J25, microcin B17 and microcin C7-C51--are discussed. While all three low-molecular-weight post-translationally modified microcins are produced by Escherichia coli, inferences based on sequence and structural similarities with peptides encoded or produced by phylogenetically diverse bacteria are made whenever possible to put these compounds into a larger perspective. 相似文献
11.
Bieler S Estrada L Lagos R Baeza M Castilla J Soto C 《The Journal of biological chemistry》2005,280(29):26880-26885
The aggregation of proteins into amyloid fibrils is the hallmark feature of a group of late-onset degenerative diseases including Alzheimer, Parkinson, and prion diseases. We report here that microcin E492, a peptide naturally produced by Klebsiella pneumoniae that kills bacteria by forming pores in the cytoplasmic membrane, assembles in vitro into amyloid-like fibrils. The fibrils have the same structural, morphological, tinctorial, and biochemical properties as the aggregates observed in the disease conditions. In addition, we found that amyloid formation also occurs in vivo where it is associated with a loss of toxicity of the protein. The finding that microcin E492 naturally exists both as functional toxic pores and as harmless fibrils suggests that protein aggregation into amyloid fibrils is an evolutionarily conserved property of proteins that can be successfully employed by bacteria to fulfill specific physiological needs. 相似文献
12.
Thomas X Destoumieux-Garzón D Peduzzi J Afonso C Blond A Birlirakis N Goulard C Dubost L Thai R Tabet JC Rebuffat S 《The Journal of biological chemistry》2004,279(27):28233-28242
Microcin E492 (MccE492, 7886 Da), the 84-amino acid antimicrobial peptide from Klebsiella pneumoniae, was purified in a post-translationally modified form, MccE492m (8717 Da), from culture supernatants of either the recombinant Escherichia coli VCS257 strain harboring the pJAM229 plasmid or the K. pneumoniae RYC492 strain. Chymotrypsin digestion of MccE492m led to the MccE492m-(74-84) C-terminal fragment that carries the modification and that was analyzed by mass spectrometry and nuclear magnetic resonance at natural abundance. The 831-Da post-translational modification consists of a trimer of N-(2,3-dihydroxybenzoyl)-l-serine linked via a C-glycosidic linkage to a beta-d-glucose moiety, itself linked to the MccE492m Ser-84-carboxyl through an O-glycosidic bond. This modification, which mimics a catechol-type siderophore, was shown to bind ferric ions by analysis of the collision-induced dissociation pattern obtained for MccE492m-(74-84) by electrospray ion trap mass spectrometry experiments in the presence of FeCl(3). By using a series of wild-type and mutant isogenic strains, the three catechol-type siderophore receptors Fiu, Cir, and FepA were shown to be responsible for the recognition of MccE492m at the outer membrane of sensitive bacteria. Because MccE492m shows a broader spectrum of antibacterial activity and is more potent than MccE492, we propose that by increasing the microcin/receptor affinity, the modification leads to a better recognition and subsequently to a higher antimicrobial activity of the microcin. Therefore, MccE492m is the first member of a new class of antimicrobial peptides carrying a siderophore-like post-translational modification and showing potent activity, which we term siderophore-peptides. 相似文献
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14.
The activity of microcin E492 from Klebsiella pneumoniae is regulated by a microcin antagonist 总被引:1,自引:0,他引:1
Abstract Microcin E492 is a polypeptide antibiotic that is produced and excreted by Klebsiella pneumoniae . Different growth conditions of the producer strain affect microcin activity. The production of a microcin antagonist is responsible for the changes in microcin activity. The microcin antagonist is induced when cells are iron-deprived, resulting in a low microcin activity. The microcin antagonist was purified using a procedure developed for the isolation of a catechol-type siderophore, and its activity was titrated using purified microcin. The inhibitory effect of the microcin antagonist is not observed when this compound is forming a complex with iron. The same inhibitory effect on microcin activity was obtained using purified enterochelin from Escherichia coli . The microcin antagonist was identified as enterochelin through thin-layer chromatography. 相似文献
15.
H D Paik S S Bae S H Park J G Pan 《Journal of industrial microbiology & biotechnology》1997,19(4):294-298
Bacillus thuringiensis subsp tochigiensis HD868 was identified as a bacteriocin producer which exhibited a bactericidal effect against closely related species. This
bacteriocin designated as tochicin, was partially purified by 75% ammonium sulfate precipitation followed by subsequent
dialysis. This partially purified tochicin showed a narrow antibacterial spectrum of activity against most of 20 typical
B. thuringiensis strains and a strain of B. cereus, but not against other bacteria and yeasts tested. The antibacterial activity of tochicin on sensitive indicator cells disappeared
completely by proteinase K treatment (1 mg ml−1), which indicates its proteinaceous nature. Tochicin was very stable throughout the range of pH 3.0–9.0 and was relatively
heat-stable at 90°C, but bacteriocin activity was not detected after boiling for 30 min. The relationship between cell growth
and bacteriocin production was studied in a semi-defined medium. Tochicin activity was detected at the mid-log growth phase,
reached the maximum at the early stationary phase, but decreased after the stationary phase. Direct detection of tochicin
activity on sodium dodecyl sulfate-polyacrylamide gel suggested it has an apparent molecular mass of about 10.5 kDa. Tochicin
exhibited a bactericidal activity against B. thuringiensis subsp thompsoni HD522 in phosphate buffer (pH 7.0).
Received 02 December 1996/ Accepted in revised form 25 August 1997 相似文献
16.
Mikhail Metelev Marina Serebryakova Dmitry Ghilarov Youfu Zhao Konstantin Severinov 《Journal of bacteriology》2013,195(18):4129-4137
Escherichia coli microcin B (Ec-McB) is a posttranslationally modified antibacterial peptide containing multiple oxazole and thiazole heterocycles and targeting the DNA gyrase. We have found operons homologous to the Ec-McB biosynthesis-immunity operon mcb in recently sequenced genomes of several pathovars of the plant pathogen Pseudomonas syringae, and we produced two variants of P. syringae microcin B (Ps-McB) in E. coli by heterologous expression. Like Ec-McB, both versions of Ps-McB target the DNA gyrase, but unlike Ec-McB, they are active against various species of the Pseudomonas genus, including human pathogen P. aeruginosa. Through analysis of Ec-McB/Ps-McB chimeras, we demonstrate that three centrally located unmodified amino acids of Ps-McB are sufficient to determine activity against Pseudomonas, likely by allowing specific recognition by a transport system that remains to be identified. The results open the way for construction of McB-based antibacterial molecules with extended spectra of biological activity. 相似文献
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It was earlier shown that expression of the microcin C51 operon in Escherichia coli cells is activated upon decelerated growth of cells during their transition to the stationary growth phase and depends on the sigmaS subunit of RNA polymerase. Using a single-copy construct containing the cloned promoter region of the microcin C51 operon and a promoterless lac operon (P(mcc)-lac), it was shown that the promoter of the microcin operon was also induced by stress caused by the transition of cells at the exponential growth phase into the medium without glucose as a sole carbon source. Activation of P(mcc)-lac expression upon severe glucose starvation occurred in rpoS+ and rpoS- strains. In cells carrying the rpoD800 mutation that renders the sigma70 subunit of RNA polymerase temperature-sensitive, an activation of P(mcc)-lac expression was observed at nonpermissive temperature, in contrast to its complete inhibition in E. coli cells at the phase of delayed growth. Other stressors-nitrogen starvation, high temperatures, osmotic shock, tetracycline and chloramphenicol-did not activate P(mcc)-lac expression in cells at the exponential growth phase. 相似文献
19.
Escherichia coli G3/10 is a component of the probiotic drug Symbioflor 2. In an in vitro assay with human intestinal epithelial cells, E. coli G3/10 is capable of suppressing adherence of enteropathogenic E. coli E2348/69. In this study, we demonstrate that a completely novel class II microcin, produced by probiotic E. coli G3/10, is responsible for this behavior. We named this antibacterial peptide microcin S (MccS). Microcin S is coded on a 50.6 kb megaplasmid of E. coli G3/10, which we have completely sequenced and annotated. The microcin S operon is about 4.7 kb in size and is comprised of four genes. Subcloning of the genes and gene fragments followed by gene expression experiments enabled us to functionally characterize all members of this operon, and to clearly identify the nucleotide sequences encoding the microcin itself (mcsS), its transport apparatus and the gene mcsI conferring self immunity against microcin S. Overexpression of cloned mcsI antagonizes MccS activity, thus protecting indicator strain E. coli E2348/69 in the in vitro adherence assay. Moreover, growth of E. coli transformed with a plasmid containing mcsS under control of an araC PBAD activator-promoter is inhibited upon mcsS induction. Our data provide further mechanistic insight into the probiotic behavior of E. coli G3/10. 相似文献
20.
Microcin J25 is a 2,107-Da, plasmid-encoded, cyclopeptide antibiotic produced by Escherichia coli. We have isolated lacZ fusions to mcjA (encoding the 58-amino-acid microcin precursor) and mcjB and mcjC (which are required for microcin maturation), and the regulation of these fusions was used to identify factors that control the expression of these genes. The mcjA gene was found to be dramatically induced as cells entered the stationary phase. Expression of mcjA could be induced by resuspending uninduced exponential-phase cells in spent supernatant obtained from an early-stationary-phase culture. Induction of mcjA expression was not dependent on high cell density, pH changes, anaerobiosis, or the buildup of some inducer. A starvation for carbon and inorganic phosphate induced mcjA expression, while under nitrogen limitation there was no induction at all. These results taken together suggest that stationary-phase induction of mcjA is triggered by nutrient depletion. The mcjB and mcjC genes were also regulated by the growth phase of the culture, but in contrast to mcjA, they showed substantial expression already during exponential growth. Induction of the microcin genes was demonstrated to be independent of RpoS, the cyclic AMP-Crp complex, OmpR, and H-NS. Instead, we found that the growth-phase-dependent expression of mcjA, mcjB, and mcjC may be explained by the concerted action of the positively acting transition state regulators ppGpp, Lrp, and integration host factor. Measurements of microcin J25 production by strains defective in these global regulators showed a good correlation with the reduced expression of the fusions in such mutant backgrounds. 相似文献