Jean Peduzzi

Microcins, gene-encoded antibacterial peptides from enterobacteria.

Microcins are gene-encoded antibacterial peptides, with molecular masses below 10 kDa, produced by enterobacteria. They are secreted under conditions of nutrient depletion and exert potent antibacterial activity against closely related species. Typical gene clusters encoding the microcin precursor, the self-immunity factor, the secretion proteins and frequently the post-translational modification enzymes are located either on plasmids or on the chromosome. In contrast to most of the antibiotics of microbial origin, which are non-ribosomally synthesized by multimodular enzymes termed peptide synthetases, microcins are ribosomally synthesized as precursors, which are further modified enzymatically. They form a restricted class of potent antibacterial peptides. Fourteen microcins have been reported so far, among which only seven have been isolated and characterized. Despite the low number of known representatives, microcins exhibit a diversity of structures and antibacterial mechanisms. This review provides an updated overview of microcin structures, antibacterial activities, genetic systems and biosyntheses, as well as of their mechanisms of action.

Close amino acid sequence relationship between the new plasmid-mediated extended-spectrum β-lactamase MEN-1 and chromosomally encoded enzymes of Klebsiella oxytoca

Isolated from an Escherichia coli strain MEN-1 is a plasmid-mediated beta-lactamase that confers resistance to methoxy imino third-generation cephalosporins. The protein purified to homogeneity was digested by trypsin, chymotrypsin and endoproteinase Asp-N. Amino acid sequence determinations of the resulting peptides gave rise to the alignment of the 263 residues of the beta-lactamase. From amino acid sequence comparison MEN-1 was found to share more than 72% identity with the chromosomally mediated beta-lactamases of Klebsiella oxytoca. Therefore, MEN-1 is the first transferable extended-spectrum beta-lactamase which is not directly derived from the widespread TEMs or SHV-1 penicillinases with which it presents less than 39% identity.

Isolation and Characterization of Environmental Bacteria Capable of Extracellular Biosorption of Mercury

ABSTRACT Accumulation of toxic metals in the environment represents a public health and wildlife concern. Bacteria resistant to toxic metals constitute an attractive biomass for the development of systems to decontaminate soils, sediments, or waters. In particular, biosorption of metals within the bacterial cell wall or secreted extracellular polymeric substances (EPS) is an emerging process for the bioremediation of contaminated water. Here the isolation of bacteria from soil, effluents, and river sediments contaminated with toxic metals permitted the selection of seven bacterial isolates tolerant to mercury and associated with a mucoid phenotype indicative of the production of EPS. Inductively coupled plasma-optical emission spectroscopy and transmission electron microscopy in conjunction with X-ray energy dispersive spectrometry revealed that bacteria incubated in the presence of HgCl2 sequestered mercury extracellularly as spherical or amorphous deposits. Killed bacterial biomass incubated in the presence of HgCl2 also generated spherical extracellular mercury deposits, with a sequestration capacity (40 to 120 mg mercury per g [dry weight] of biomass) superior to that of live bacteria (1 to 2 mg mercury per g [dry weight] of biomass). The seven strains were shown to produce EPS, which were characterized by Fourier transform-infrared (FT-IR) spectroscopy and chemical analysis of neutral-carbohydrate, uronic acid, and protein contents. The results highlight the high potential of Hg-tolerant bacteria for applications in the bioremediation of mercury through biosorption onto the biomass surface or secreted EPS.

Siderophore peptide, a new type of post-translationally modified antibacterial peptide with potent activity

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 β-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 FeCl3. 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.

The iron–siderophore transporter FhuA is the receptor for the antimicrobial peptide microcin J25: role of the microcin Val11–Pro16 β-hairpin region in the recognition mechanism

The role of the outer-membrane iron transporter FhuA as a potential receptor for the antimicrobial peptide MccJ25 (microcin J25) was studied through a series of in vivo and in vitro experiments. The requirement for both FhuA and the inner-membrane TonB-ExbB-ExbD complex was demonstrated by antibacterial assays using complementation of an fhuA(-) strain and by using isogenic strains mutated in genes encoding the protein complex respectively. In addition, MccJ25 was shown to block phage T5 infection of Escherichia coli, in vivo, by inhibiting phage adhesion, which suggested that MccJ25 prevents the interaction between the phage and its receptor FhuA. This in vivo activity was confirmed in vitro, as MccJ25 inhibited phage T5 DNA ejection triggered by purified FhuA. Direct interaction of MccJ25 with FhuA was demonstrated for the first time by size-exclusion chromatography and isothermal titration calorimetry. MccJ25 bound to FhuA with a 2:1 stoichiometry and a K(d) of 1.2 microM. Taken together, our results demonstrate that FhuA is the receptor for MccJ25 and that the ligand-receptor interaction may occur in the absence of other components of the bacterial membrane. Finally, both differential scanning calorimetry and antimicrobial assays showed that MccJ25 binding involves external loops of FhuA. Unlike native MccJ25, a thermolysin-cleaved MccJ25 variant was unable to bind to FhuA and failed to prevent phage T5 infection of E. coli. Therefore the Val11-Pro16 beta-hairpin region of MccJ25, which is disrupted upon cleavage by thermolysin, is required for microcin recognition.

Purification and Partial Identification of Novel Antimicrobial Protein from Marine Bacterium Pseudoalteromonas Species Strain X153

A marine bacterium, X153, was isolated from a pebble collected at St. Anne du Portzic (France). By 16S ribosomal DNA gene sequence analysis, X153 strain was identified as a Pseudoalteromonas sp. close to P. piscicida. The crude culture of X153 was highly active against human pathogenic strains involved in dermatologic diseases, and marine bacteria including various ichthyopathogenic Vibrio strains. The active substance occurred both in bacterial cells and in culture supernatant. An antimicrobial protein was purified to homogeneity by a 4-step procedure using size-exclusion and ion-exchange chromatography. The highly purified P-153 protein is anionic, and sodium dodecylsulfate polyacrylamide gel electrophoresis gives an apparent molecular mass of 87 kDa. The X153 bacterium protected bivalve larvae against mortality, following experimental challenges with ichthyopathogenic Vibrio. Pseudoalteromonas sp. X153 may be useful in aquaculture as a probiotic bacterium.

Single amino acid substitution between SHV-1 β-lactamase and cefotaxime-hydrolyzing SHV-2 enzyme

SHV‐2 β‐lactamase was purified from an overproducing variant of a clinical isolate of Escherichia coli resistant to cefotaxime. Pure protein was digested by trypsin and Lys‐C endoproteinase. Proteolytic peptides, isolated by reverse‐phase HPLC, were submitted to manual Edman degradation and aligned by homology with the sequence of SHV‐1 β‐lactamase. A putative amino acid sequence was deduced. Structural comparison revealed that SHV‐2 differed from SHV‐1 by only one amino acid, Gly → Ser, at position 213 of the mature protein.

Structural and functional diversity of microcins, gene-encoded antibacterial peptides from enterobacteria.

Microcins are a peculiar class of gene-encoded low-molecular-mass antibacterial peptides secreted by enterobacteria. They contribute to the regulation of microbial competitions within the intestinal microbiota. The genetic systems involved in microcin biosynthesis share a conserved organization. Similar to bacteriocins of Gram-positive bacteria, microcins exert potent antibacterial activity directed against phylogenetically-related bacterial strains, with minimal inhibitory concentrations in the nanomolar range. In contrast to bacteriocins, they display a great structural diversity among the few representatives well characterized until now, that makes difficult the description of microcin subclasses. This review focuses on three microcins, MccE492m that carries a C-terminal posttranslational modification containing a catechol-type siderophore, MccJ25, a cyclic peptide with a unique ‘lasso-type’ structure and MccC7 or C51, with a common N-formylated heptapeptide-nucleotide structure. We show these microcins exhibit ‘Trojan horse’ mechanisms of antibacterial activity: either (i) the microcin structure is a mime of an essential element, permitting its recognition by outer membrane receptors used for vital functions in bacteria and further translocation into the periplasmic space, or (ii) it is secreted as a harmless molecule and further processed in susceptible bacteria to form the toxic entity. When inside target bacteria, microcins bind essential enzymes or interact with the inner membrane to form a bacterial killing structure.

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 µM. 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.

Isolation and Characterization of Two Members of the Siderophore-Microcin Family, Microcins M and H47

ABSTRACT In this paper we provide the first biochemical evidence of the existence of a family of structure-related antimicrobial peptides, the siderophore-microcins, in the Enterobacteriaceae family. We isolated and characterized two novel siderophore-microcins, MccM and MccH47, previously characterized through genetic studies. MccM and MccH47 were expressed from several Escherichia coli strains containing the microcin gene clusters. The spectra of their bactericidal activities were found to be restricted to some species of the Enterobacteriaceae. MccM and MccH47 were unable to inhibit the growth of strains carrying mutations in the fepA, cir, and fiu genes, which showed the requirement of the iron-catecholate receptors for their recognition. The MccM and MccH47 peptide moieties contain 77 and 60 residues, respectively, and are derived from the microcin precursors McmA and MchB, respectively. In addition, both peptides carried a C-terminal posttranslational modification containing a salmochelin-like siderophore moiety also found in MccE492 (X. Thomas et al., J. Biol. Chem., 279:28233-28242, 2004). Interestingly, when MccM was isolated from E. coli Nissle 1917, which lacks the two genes necessary for modification biosynthesis, it was devoid of posttranslational modification. Those two genes could be complemented by their homologues from the MccH47 gene cluster, thereby showing their functional interchangeability between at least two members of the siderophore-microcin family. Finally, from the sequence analysis of the MccE492 gene cluster, we hypothesized the existence of an additional member of the siderophore-microcin family. Therefore, we propose that the siderophore-microcin family contains five representatives.