Parasitism of Iron-siderophore Receptors of Escherichia Coli by the Siderophore-peptide Microcin E492m and its Unmodified Counterpart

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.     

Investigations of the MceIJ-catalyzed posttranslational modification of the microcin E492 C-terminus: linkage of ribosomal and nonribosomal peptides to form "trojan horse" antibiotics

MceIJ is a two protein complex responsible for attachment of a C-glycosylated and linearized derivative of enterobactin, an iron scavenger (siderophore) and product of nonribosomal peptide synthetase machinery, to the C-terminal serine residue of microcin E492 (MccE492), an 84 aa ribosomal antibiotic peptide produced by Klebsiella pneumoniae RYC492. The MceIJ-catalyzed formation of the glycosyl ester linkage between MccE492 and the siderophore requires ATP and Mg(II) as cofactors. This work addresses the ATP utilization, mechanism of C-terminal carboxylate activation, and substrate scope of MceIJ. Formation of the ribosomal peptide-nonribosomal peptide linkage between the MccE492 C-terminal decapeptide and monoglycosylated enterobactin (MGE) requires cleavage of the alpha,beta bond of ATP and formation of a putative peptidyl-CO-AMP intermediate. Attack of the peptidyl-CO-AMP carbonyl by the deprotonated C4' hydroxyl of the glucose moiety forms a glycosyl ester linkage with release of AMP. Site-directed mutagenesis of the three cysteines and five histidines in MceI to alanines reveals that these residues are not required structurally or catalytically. MceIJ recognizes all glycosylated enterobactin derivatives formed by the MccE492 gene cluster members MceC ( C-glycosyltransferase) and MceD (esterase) in vitro and a MGE derivative lacking the C6' hydroxyl moiety. The protein complex also accepts and modifies the C-terminal decapeptide substrate fragments of the structurally related microcins H47, I47, and M. MccE492 C-terminal decapeptides bearing fluorescein and biotin moieties on the N-terminus are also substrates for MceIJ, which provides a route for the chemoenzymatic synthesis of enterobactin conjugates with peptide linkages.     

Microcin E492 antibacterial activity: evidence for a TonB-dependent inner membrane permeabilization on Escherichia coli

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.     

Bactericidal activity of both secreted and nonsecreted microcin E492 requires the mannose permease

Microcin E492 (MccE492) is a bactericidal protein secreted by Klebsiella pneumoniae that is active against various species of Enterobacteriaceae. Interaction of MccE492 with target cells leads to the depolarization and permeabilization of their inner membranes. Several MccE492-specific proteins are required for the maturation and secretion of active MccE492. Surprisingly, the expression of only MceA, the polypeptide backbone of MccE492, is shown here to be toxic by itself. We refer to this phenomenon as endogenous MceA bactericidal activity to differentiate it from the action of extracellularly secreted MccE492. The toxicity of endogenous MceA is enhanced by an efficient targeting to the inner membrane. However, a periplasmic intermediate state is not required for MceA toxicity. Indeed, endogenous MceA remains fully active when it is fused to thioredoxin-1, a fast-folding protein that promotes retention of the C terminus of MceA in the cytoplasm. The C-terminal domain of MccE492 is required only for delivery from the extracellular environment to the periplasm, and it is not required for inner membrane damage. A common component is absolutely essential for the bactericidal activity of both endogenous MceA and extracellular MccE492. Indeed, toxicity is strictly dependent on the presence of ManYZ, an inner membrane protein complex involved in mannose uptake. Based on these findings, we propose a new model for cell entry, inner membrane insertion, and toxic activity of MccE492.     

Extensive post-translational modification, including serine to D-alanine conversion, in the two-component lantibiotic, lacticin 3147

Lacticin 3147 is a two-component bacteriocin produced by Lactococcus lactis subspecies lactis DPC3147. In order to further characterize the biochemical nature of the bacteriocin, both peptides were isolated which together are responsible for the antimicrobial activity. The first, LtnA1, is a 3,322 Da 30-amino acid peptide and the second component, LtnA2, is a 29-amino acid peptide with a mass of 2,847 Da. Conventional amino acid analysis revealed that both peptides contain the thioether amino acid, lanthionine, as well as an excess of alanine to that predicted from the genetic sequence of the peptides. Chiral phase gas chromatography coupled with mass spectrometry of amino acid composition indicated that both LtnA1 and LtnA2 contain D-alanine residues and amino acid sequence analysis of LtnA1 confirmed that the D-alanine results from post-translational modification of a serine residue in the primary translation product. Taken together, these results demonstrate that lacticin 3147 is a novel, two-component, D-alanine containing lantibiotic that undergoes extensive post-translational modification which may account for its potent antimicrobial activity against a wide range of Gram-positive bacteria.     

The activity of microcin E492 from Klebsiella pneumoniae is regulated by a microcin antagonist

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.     

Characterization of plant-growth-promoting traits of Acinetobacter species isolated from rhizosphere of Pennisetum glaucum

A total of 31 Acinetobacter isolates were obtained from the rhizosphere of Pennisetum glaucum and evaluated for their plant-growth-promoting traits. Two isolates, namely Acinetobacter sp. PUCM1007 and A. baumannii PUCM1029, produced indole acetic acid (10-13 microgram/ml). A total of 26 and 27 isolates solubilized phosphates and zinc oxide, respectively. Among the mineral-solubilizing strains, A. calcoaceticus PUCM1006 solubilized phosphate most efficiently (84 mg/ml), whereas zinc oxide was solubilized by A. calcoaceticus PUCM1025 at the highest solubilization efficiency of 918%. All the Acinetobacter isolates, except PUCM1010, produced siderophores. The highest siderophore production (85.0 siderophore units) was exhibited by A. calcoaceticus PUCM1016. Strains PUCM1001 and PUCM1019 (both A. calcoaceticus) and PUCM1022 (Acinetobacter sp.) produced both hydroxamate- and catechol-type siderophores, whereas all the other strains only produced catechol-type siderophores. In vitro inhibition of Fusarium oxysporum under iron-limited conditions was demonstrated by the siderophore-producing Acinetobacter strains, where PUCM1018 was the most potent inhibitor of the fungal phytopathogen. Acinetobacter sp. PUCM1022 significantly enhanced the shoot height, root length, and root dry weights of pearl millet seedlings in pot experiments when compared with controls, underscoring the plant-growth-promoting potential of these isolates.     

Isolation and purification of siderophores produced by cyanobacteria,Synechococcus sp. PCC 7942 andAnabaena variabilis ATCC 29413

New siderophores were isolated and purified from the spent growth medium of the cyanobacteriaSynechococcus sp. PCC 7942 (Anacystis nidulans R2) andAnabaena variabilis ATCC 29413 by solvent extraction and thin-layer chromatography. For each species the siderophore was released into the medium when the cells were grown at low iron concentrations and was not found in the medium of cells grown in iron-sufficient medium. Through a series of biological and chemical tests, combined with spectral analysis, the dihydroxamate nature of each siderophore was confirmed. The siderophores produced bySynechococcus sp. PCC 7942 andA. variabilis had distinct relative molecular masses of 310–313 Da and 520–525 Da, respectively. Neither of the two strains produced Arnow-positive extracellular organics, which indicate the excretion of extracellular catechol-type siderophores.     

Characterization and structure identification of an antimicrobial peptide, hominicin, produced by Staphylococcus hominis MBBL 2-9

Hominicin, antimicrobial peptide displaying potent activity against Staphylococcus aureus ATCC 25923, methicillin-resistant S. aureus (MRSA) ATCC 11435 and vancomycin-intermediate S. aureus (VISA) CCARM 3501, was purified by chloroform extraction, ion-exchange column chromatography and reverse-phase HPLC from culture supernatant of Staphylococcushominis MBBL 2-9. Hominicin exhibited heat stability up to 121 °C for 15 min and activity under both acidic and basic conditions (from pH 2.0 to 10.0). Hominicin was cleaved into two fragments after treatment with proteinase K, resulting in the loss of its antibacterial activity, while it was resistant to trypsin, α-chymotrypsin, pepsin and lipase. The molecular mass of hominicin determined by mass spectrometry was 2038.4 Da. LC-mass spectrometry and NMR spectroscopy analyses of the two fragments revealed the sequence of hominicin as DmIle-Dhb-Pro-Ala-Dhb-Pro-Phe-Dhb-Pro-Ala-Ile-Thr-Glu-Ile-Dhb-Ala-Ala-Val-Ile-Ala-Dmp, which had no similarity with other antimicrobial peptides previously reported. The present study is the first report of this novel antimicrobial peptide, which has uncommon amino acid residues like the ones in Class I group and shows potent activity against clinically relevant S. aureus, MRSA and VISA.     

DNA binding ligands with in vivo efficacy in murine models of bacterial infection: optimization of internal aromatic amino acids

DNA binding ligands with potent antimicrobial activity against Gram-positive bacteria were further optimized by variation of the internal aromatic amino acids. This modification led to compounds with improved in vivo efficacy in lethal murine models of peritonitis (methicillin-resistant S. aureus, MRSA) and lung infection (S. pneumoniae).     

Hipposin,a histone-derived antimicrobial peptide in Atlantic halibut (Hippoglossus hippoglossus L.)

A novel 51-residue antimicrobial peptide (AMP) from the skin mucus of Atlantic halibut (Hippoglossus hippoglossus L.) was isolated using acid extraction, and cationic exchange and reversed phase chromatography. The complete amino acid sequence of the AMP, termed hipposin, was determined by automated Edman degradation and mass spectrometry to be SGRGKTGGKARAKAKTRSSRAGLQFPVGRVHRLLRKGNYAHRVGAGAPVYL. The N-terminal amino group was acetylated. The theoretical mass of hipposin was calculated to be 5458.4 Da, which was in good agreement with the mass of 5459 Da determined by matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS). Hipposin was shown to be derived from histone H2A by PCR amplifying the encoding sequences from Atlantic halibut genomic DNA. The peptide showed sequence similarity with the 39-mer AMP buforin I of Asian toad and the 19-mer AMP parasin I of catfish. Fifty of the fifty-one residues in hipposin were identical to the N-terminal region of histone H2A from rainbow trout. Hipposin showed strong antimicrobial activity against several Gram-positive and Gram-negative bacteria and activity could be detected down to hipposin concentrations of 0.3 microM (1.6 microg/ml). Hipposin without N-terminal acetylation was prepared by solid-phase peptide synthesis and shown to have the same antimicrobial activity as the natural acetylated peptide. Thus, hipposin is a new broad-spectrum histone-derived AMP found in the skin mucus of Atlantic halibut.     

I-conotoxins in vermivorous species of the West Atlantic: peptide sr11a from Conus spurius

Peptide sr11a was purified from the venom of Conus spurius, a vermivorous cone snail collected in the Yucatan Channel, in the Western Atlantic. Its primary structure was determined by automatic Edman degradation after reduction and alkylation. Its molecular mass, as determined by MALDI-TOF mass spectrometry (average mass 3650.77 Da), confirmed the chemical data (calculated average mass, 3651.13 Da). The sequence of peptide sr11a (CRTEGMSCgamma gamma NQQCCWRSCCRGECEAPCRFGP&; gamma, gamma-carboxy-Glu; &, amidated C-terminus) shows eight Cys residues arranged in the pattern that defines the I-superfamily of conotoxins. Peptide sr11a contains two gamma-carboxy-Glu residues, a post-translational modification that has been found in other I-conotoxins from species that live in the West Pacific: r11e from the piscivorous Conus radiatus, and kappa-BtX from the vermivorous Conus betulinus. Peptide sr11a is the eighth I-conotoxin isolated from a Conus venom and the first I-conotoxin from a species from the Western Atlantic. Peptide sr11a produced stiffening of body, limbs and tail when injected intracranially into mice.     

Characterization of a high-affinity iron transport system in Acinetobacter baumannii.

Analysis of a clinical isolate of Acinetobacter baumannii showed that this bacterium was able to grow under iron-limiting conditions, using chemically defined growth media containing different iron chelators such as human transferrin, ethylenediaminedi-(o-hydroxyphenyl)acetic acid, nitrilotriacetic acid, and 2,2'-bipyridyl. This iron uptake-proficient phenotype was due to the synthesis and secretion of a catechol-type siderophore compound. Utilization bioassays using the Salmonella typhimurium iron uptake mutants enb-1 and enb-7 proved that this siderophore is different from enterobactin. This catechol siderophore was partially purified from culture supernatants by adsorption chromatography using an XAD-7 resin. The purified component exhibited a chromatographic behavior and a UV-visible light absorption spectrum different from those of 2,3-dihydroxybenzoic acid and other bacterial catechol siderophores. Furthermore, the siderophore activity of this extracellular catechol was confirmed by its ability to stimulate energy-dependent uptake of 55Fe(III) as well as to promote the growth of A. baumannii bacterial cells under iron-deficient conditions imposed by 60 microM human transferrin. Polyacrylamide gel electrophoresis analysis showed the presence of iron-regulated proteins in both inner and outer membranes of this clinical isolate of A. baumannii. Some of these membrane proteins may be involved in the recognition and internalization of the iron-siderophore complexes.     

The production in vivo of microcin E492 with antibacterial activity depends on salmochelin and EntF

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.     

Genome-based cryptic gene discovery and functional identification of NRPS siderophore peptide in Streptomyces peucetius

Identification of secondary metabolites produced by cryptic gene in bacteria may be difficult, but in the case of nonribosomal peptide (NRP)-type secondary metabolites, this study can be facilitated by bioinformatic analysis of the biosynthetic gene cluster and tandem mass spectrometry analysis. To illustrate this concept, we used mass spectrometry-guided bioinformatic analysis of genomic sequences to identify an NRP-type secondary metabolite from Streptomyces peucetius ATCC 27952. Five putative NRPS biosynthetic gene clusters were identified in the S. peucetius genome by DNA sequence analysis. Of these, the sp970 gene cluster encoded a complete NRPS domain structure, viz., C-A-T-C-A-T-E-C-A-T-C-A-T-C domains. Tandem mass spectrometry revealed that the functional siderophore peptide produced by this cluster had a molecular weight of 644.4 Da. Further analysis demonstrated that the siderophore peptide has a cyclic structure and an amino acid composition of AchfOrn–Arg–hOrn–hfOrn. The discovery of functional cryptic genes by analysis of the secretome, especially of NRP-type secondary metabolites, using mass spectrometry together with genome mining may contribute significantly to the development of pharmaceuticals such as hybrid antibiotics.     

Isolation and identification of ferrioxamine G and E inHafnia alvei

Summary Under conditions of iron-deprivationHafnia alvei (Enterobacteriaceae) produces ferrioxamine G as the principal siderophore. Maximum hydroxamate siderophore production occurred at medium iron limitation. The ferrioxamines were extracted, purified by gel filtration and chromatography on silica gel yielding a major and a minor siderophore fraction. The minor siderophore fraction contained three siderophores, among which ferrioxamine E could be identified by HPLC and FAB mass spectrometry. Reductive hydrolysis of the ferrioxamine G fraction yielded succinic acid and a mixture of diaminopentane and diaminobutane, as determined by gas-liquid chromatography and GLC/MS. HPLC and FAB mass spectrometry confirmed that the ferrioxamine G fraction consisted of two different species, G₁ and G₂, possessing molecular masses of 671 Da and 658 Da respectively.     

Structural and functional characterization of two novel peptide toxins isolated from the venom of the social wasp Polybia paulista

Two novel inflammatory peptides were isolated from the venom of the social wasp Polybia paulista. They had their molecular masses determined by ESI-MS and their primary sequences were elucidated by Edman degradation chemistry as: Polybia-MPI: I D W K K L L D A A K Q I L-NH2 (1654.09 Da), Polybia-CP: I L G T I L G L L K S L-NH2 (1239.73 Da). Both peptides were functionally characterized by using Wistar rat cells. Polybia-MPI is a mast cell lytic peptide, which causes no hemolysis to rat erythrocytes and presents chemotaxis for polymorphonucleated leukocytes (PMNL) and with potent antimicrobial action both against Gram-positive and Gram-negative bacteria. Polybia-CP was characterized as a chemotactic peptide for PMNL cells, presenting antimicrobial action against Gram-positive bacteria, but causing no hemolysis to rat erythrocytes and no mast cell degranulation activity at physiological concentrations.     

A novel small antifungal peptide from Bacillus strain B-TL2 isolated from tobacco stems

A novel small antifungal peptide produced by a Bacillus strain B-TL2 isolated from tobacco stems was purified. The purification procedure consisted of ammonium sulfate precipitation, cation exchange chromatography on CM-Sepharose Fast Flow column and reverse-phase HPLC on SOURCE 5RPC column. After the final isolation step, one peptide with antifungal activity, designated as BTL, was obtained. The molecular mass of the purified BTL was determined as 2500 Da and 2237.7 Da by SDS-PAGE and matrix-assisted laser desorption/ionization time of flight mass spectrometry, respectively. The N-amino acid sequence of BTL was determined to be NH(2)-KQQLATEAESAGPIL, which shows relatively low identity to other antimicrobial peptides from bacteria. The peptide exhibited strong inhibitory activity against mycelial growth of Bipolaris maydis, Alternaria brassicae, Aspergillus niger, Cercospora personata. The purified BTL displayed thermostability, almost retaining 100% activity at 100 degrees C for 15 min.     

Barley lipid transfer protein, LTP1, contains a new type of lipid-like post-translational modification.

In plants a group of proteins termed nonspecific lipid transfer proteins are found. These proteins bind and catalyze transfer of lipids in vitro, but their in vivo function is unknown. They have been suggested to be involved in different aspects of plant physiology and cell biology, including the formation of cutin and involvement in stress and pathogen responses, but there is yet no direct demonstration of an in vivo function. We have found and characterized a novel post-translational modification of the barley nonspecific lipid transfer protein, LTP1. The protein-modification bond is of a new type in which an aspartic acid in LTP1 is bound to the modification through what most likely is an ester bond. The chemical structure of the modification has been characterized by means of two-dimensional homo- and heteronuclear nuclear magnetic resonance spectroscopy as well as mass spectrometry and is found to be lipid-like in nature. The modification does not resemble any standard lipid post-translational modification but is similar to a compound with known antimicrobial activity.