Low-molecular-weight post-translationally modified microcins

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.     

肠道菌分泌的抑菌肽——小菌素

小菌素是由肠道菌分泌的一类小分子抑菌肽,分子量小于10kDa,由细菌质粒或基因组上相关基因簇编码,小菌素的抑菌谱较窄,仅对肠道菌中部分亲缘较近的菌种发挥有效的抑菌效应。编码小菌素的基因簇一般包括几个部分:前体基因,自身免疫基因,分泌基因,转录后修饰基因。与很多微生物通过非核糖体途径分泌抑菌物质不同,小菌素前体通过核糖体途径分泌。目前已发现的小菌素有15种,它们的结构和抑菌机制具有多样性。     

New developments in non-post translationally modified microcins

Microcins are a family of low molecular weight antibiotic peptides produced by Enterobacteriaceae strains and active against related bacteria. According to some features we propose to classify these antibiotic substances into two distinct groups. The class I microcins contain Mcc B17, C7, J25 and D93 that are small molecules (molecular mass inferior to 5 kDa), largely post-translationally modified and with specific intracellular targets. The class II microcins, MccV, E492, H47, L and 24, share several common properties with class IIa Gram-positive bacteriocins: molecular mass ranging from 7 to 10 kDa, absence of modified amino acids, double-glycine type leader peptides, secretion mediated by an ABC transporter and antibacterial activity due to interaction with bacterial membrane. This review discusses common features of the class II microcins and provides new insights into these peptides.     

Characterization of enterobacteria producing the low-molecular-weight antibiotics microcins

A comparative study of the morphological, cultural, physiological, and biochemical properties of the microcinogenic strains EcS 5/98, EcS 6/98, and EcB 214/99 with the known microcin C51 producer Escherichia coli M17(p74) showed that these strains belong to the species E. coli. The strains produced microcins with molecular masses lower than 10 kDa. Microcin biosynthesis was stimulated by a deficiency of nutrients in the cultivation media. Microcins were found to be resistant to thermolysin, but were degraded by pronase, protolichetrem, and the Bacillus mesentericus metalloproteinase. This indicated that microcins are peptides or contain peptides in their molecules. The study of cross immunity to microcins and the sequence of their genetic determinants showed that the microcins of strains EcS 5/98 and EcS 6/98 are of B type, whereas the microcin of strain EcB 214/99 presumably belongs to another type, since it suppresses the growth of the producers of C-type and B-type microcins. The new microcin producers possess antibacterial activity against natural isolates belonging to the genera Escherichia and Salmonella, against a wide range of colicinogenic Escherichia strains, and against the collection Salmonella cultures.     

The microcins

Abstract Microcins are antibiotics of low M _r, constitutively (non-lethally) produced by non-sporulating bacteria, such as Enterobacteriaceae. Their production depends on plasmids and is not inducible by DNA-damaging agents. Hitherto, five types of microcins have been identified by cross-immunity, biochemical and genetic criteria. Microcins have an amino acid or oligopeptide structure and show different mechanisms of action: inhibition of metabolic enzymes (type A) or of DNA replication (type B), or impairment of the cell's energy-generating system (type D). In some cases (microcin B17), a complex genetic system involving up to seven genes may be required for the synthesis. Finally, microcins may play a role in bacterial interactions in natural microbial ecosystems.     

Characterization of Enterobacteria Producing the Low-Molecular-Weight Antibiotics Microcins

A comparative study of the morphological, cultural, physiological, and biochemical properties of the microcinogenic strains EcS 5/98, EcS 6/98, and EcB 214/99 and the known microcin C51 producer Escherichia coli M17(p74) showed that these strains belong to the species E. coli. The strains produced microcins with molecular masses lower than 10 kDa. Microcin biosynthesis was stimulated by a deficiency of nutrients in the cultivation media. The microcins were found to be resistant to thermolysin but were degraded by pronase, protolichetrem, and the Bacillus mesentericus metalloproteinase. This indicated that the microcins are peptides or contain peptides in their molecules. The study of cross immunity to the microcins and the sequencing of their genetic determinants showed that the microcins of strains EcS 5/98 and EcS 6/98 are of B type, whereas the microcin of strain EcB 214/99 presumably belongs to another type, since it suppresses the growth of the producers of C and B-type microcins. The new microcin producers possess antibacterial activity against natural isolates belonging to the genera Escherichia and Salmonella, against a wide range of colicinogenic Escherichia strains, and against collection Salmonella cultures.     

Structure and Mode-of-Action of the Two-Peptide (Class-IIb) Bacteriocins

This review focuses on the structure and mode-of-action of the two-peptide (class-IIb) bacteriocins that consist of two different peptides whose genes are next to each other in the same operon. Optimal antibacterial activity requires the presence of both peptides in about equal amounts. The two peptides are synthesized as preforms that contain a 15–30 residue double-glycine-type N-terminal leader sequence that is cleaved off at the C-terminal side of two glycine residues by a dedicated ABC-transporter that concomitantly transfers the bacteriocin peptides across cell membranes. Two-peptide bacteriocins render the membrane of sensitive bacteria permeable to a selected group of ions, indicating that the bacteriocins form or induce the formation of pores that display specificity with respect to the transport of molecules. Based on structure–function studies, it has been proposed that the two peptides of two-peptide bacteriocins form a membrane-penetrating helix–helix structure involving helix–helix-interacting GxxxG-motifs that are present in all characterized two-peptide bacteriocins. It has also been suggested that the membrane-penetrating helix–helix structure interacts with an integrated membrane protein, thereby triggering a conformational alteration in the protein, which in turn causes membrane-leakage. This proposed mode-of-action is similar to the mode-of-action of the pediocin-like (class-IIa) bacteriocins and lactococcin A (a class-IId bacteriocin), which bind to a membrane-embedded part of the mannose phosphotransferase permease in a manner that causes membrane-leakage and cell death.     

Escherichia coli K12 strains for use in the identification and characterization of colicins

A collection of strains derived from Escherichia coli K12 W3110 and harbouring various colicin or microcin plasmids (18 and 2 representatives, respectively), or carrying well-characterized mutations conferring reduced colicin/microcin sensitivity is described. The strains can be used in typing schemes based on the identification of colicins, in the detection of new types of colicins/microcins, and in the further characterization of previously identified colicins/microcins and their plasmids.     

Isolation and characterization of Escherichia coli strains producing microcins of B and C types

Abstract A screening of 11956 enterobacteria isolates resulted in selection of seven active microcin-producing strains. The microcins were shown to be peptides or their derivatives with a rather broad spectrum of activity, mainly against Gram-negative bacteria. According to cross-immunity criteria, the microcins studied belonged to two of the previously suggested types, B (five strains) and C (two strains). Those of type B could be further classified into two subtypes on the account of differences in the spectrum of antibacterial activity. In five cases out of seven the microcin-producing ability has been attributed to plasmids that the strains harboured. The effect of microcins on sensitive cells was shown to depend on ompR and ompF gene products.     

Posttranslationally modified microcins

Microcins are antibacterial compounds that are encoded in the bacterial genome and synthesized via ribosomal translation. Microcins play an important role in microbial ecology and are promising as antibiotics. To exert their effect, most microcins are incorporated in the membrane of sensitive cells to increase its permeability. The review considers the known classes of posttranslationally modified microcins. These microcins are unusual in structure and inhibit the grown of sensitive cells by entering their cytoplasm and affecting intracellular targets, such as DNA gyrase, DNA-dependent RNA polymerase, and aspartyl-tRNA synthase.     

Posttranslationally modified microcins

Microcins are antibacterial compounds that are encoded in the bacterial genome and synthesized via ribosomal translation. Microcins play an important role in microbial ecology and are promising as antibiotics. To exert their effect, most microcins are incorporated in the membrane of sensitive cells to increase its permeability. The review considers the known classes of posttranslationally modified microcins. These microcins are unusual in structure and inhibit the grown of sensitive cells by entering their cytoplasm and affecting intracellular targets, such as DNA gyrase, DNA-dependent RNA polymerase, and aspartyl-tRNA synthase.     

LlpB represents a second subclass of lectin-like bacteriocins

Bacteriocins are secreted bacterial proteins that selectively kill related strains. Lectin-like bacteriocins are atypical bacteriocins not requiring a cognate immunity factor and have been primarily studied in Pseudomonas. These so-called LlpAs are composed of a tandem of B-lectin domains. One domain interacts with d -rhamnose residues in the common polysaccharide antigen of Pseudomonas lipopolysaccharide (LPS). The other lectin domain is crucial for interference with the outer membrane protein assembly machinery by interacting with surface-exposed loops of its central component BamA. Via genome mining, we identified a second subclass of Pseudomonas lectin-like proteins, termed LlpB, consisting of a single B-lectin domain. We show that these proteins also display bactericidal activity. Among LlpB-resistant transposon mutants of an LlpB-susceptible Pseudomonas strain, a major subset was hit in an acyltransferase gene, predicted to be involved in LPS core modification, hereby suggesting that LlpBs equally attach to LPS for surface anchoring. This indicates that LPS binding and target strain specificity are condensed in a single B-lectin domain. The identification of this second subclass of lectin-like bacteriocins further expands the toolbox of antibacterial warfare deployed by bacteria and holds potential for their integration in biotechnological applications.     

Bacteriocins: mechanism of membrane insertion and pore formation

Lactic acid bacteria produce several types of pore forming peptides. Class I bacteriocins are lantibiotics that contain (methyl)lanthionine residues that may form intramolecular thioether rings. These peptides generally have a broad spectrum of activity and form unstable pores. Class II bacteriocins are small, heat stable peptides mostly with a narrow spectrum of activity. Most bacteriocins interact with anionic lipids that are abundantly present in the membranes of Gram-positive bacteria.'Docking molecules' may enhance the conductivity and stability of lantibiotic pores, while'receptors' in the target membrane may determine specificity of class II bacteriocins. Insertion into the membrane of many bacteriocins is proton motive force driven. Lantibiotics may form pores according to a'wedge-like' model, while class II bacteriocins may enhance membrane permeability either by the formation of a'barrel stave' pore or by a'carpet' mechanism.     

Microbial production of bacteriocins: Latest research development and applications

Bacteriocins are low molecular weight peptides secreted by the predator bacterial cells to kill sensitive cells present in the same ecosystem competing for food and other nutrients. Exceptionally few bacteriocins along with their native antibacterial property also exhibit additional anti-viral and anti-fungal properties. Bacteriocins are generally produced by Gm+, Gm– and archaea bacteria. Bacteriocins from Gm?+?bacteria especially from lactic acid bacteria (LAB) have been thoroughly investigated considering their great biosafety and broad industrial applications. LAB expressing bacteriocins were isolated from fermented milk and milk products, rumen of animals and soil using deferred antagonism assay. Nisin is the only bacteriocin that has got FDA approval for application as a food preservative, which is produced by Lactococcus lactis subsp. Lactis. Its crystal structure explains that its antimicrobial properties are due to the binding of NH₂ terminal to lipid II molecule inhibiting the peptidoglycan synthesis and carboxy terminal forming pores in bacterial cell membrane leading to cell lysis. The hinge region connecting NH₂ and carboxy terminus has been mutated to generate mutant variants with higher antimicrobial activity. In a 50 ton fermentation of the mutant strain 3807 derived from L. lactis subsp. lactis ATCC 11454, 9,960?IU/mL of nisin was produced. Currently, high purity of nisin (>99%) is very expensive and hardly commercially available. Development of more advanced tools for cost-effective separation and purification of nisin would be commercially attractive. Chemical synthesis and heterologous expression of bacteriocins ended in low yields of pure proteins. At present, bacteriocins are almost solely applied in food industries, but they have a great potential to be used in other fields such as feeds, organic fertilizers, environmental protection and personal care products. The future of bacteriocins is largely dependent on getting FDA approval for use of other bacteriocins in addition to nisin to promote the research and applications.     

Ton-dependent colicins and microcins: modular design and evolution

Ton-dependent colicins and microcins are actively taken up into sensitive cells at the expense of energy which is provided by the proton motive force of the cytoplasmic membrane. The Ton system consisting of the proteins TonB, ExbB and ExbD is required for colicin and microcin import. Colicins as well as the outer membrane transport proteins contain proximal to the N-terminus a short sequence, called TonB box, which interacts with TonB and in which point mutants impair uptake. No TonB box is found in microcins. Colicins are composed of functional modules which during evolution have been interchanged resulting in new colicins. The modules define sites of interaction with the outer membrane transport genes, TonB, the immunity proteins, and the activity regions. Six TonB-dependent microcins with different primary structures are processed and exported by highly homologous proteins. Three of these microcins are modified in an unknown way and they have in common specificity for catecholate siderophore receptors.     

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.     

Microcin E492 Amyloid Formation Is Retarded by Posttranslational Modification

Microcin E492, a channel-forming bacteriocin with the ability to form amyloid fibers, is exported as a mixture of two forms: unmodified (inactive) and posttranslationally modified at the C terminus with a salmochelin-like molecule, which is an essential modification for conferring antibacterial activity. During the stationary phase, the unmodified form accumulates because expression of the maturation genes mceIJ is turned off, and microcin E492 is rapidly inactivated. The aim of this work was to demonstrate that the increase in the proportion of unmodified microcin E492 augments the ability of this bacteriocin to form amyloid fibers, which in turn decreases antibacterial activity. To this end, strains with altered proportions of the two forms were constructed. The increase in the expression of the maturation genes augmented the antibacterial activity during all growth phases and delayed the loss of activity in the stationary phase, while the ability to form amyloid fibers was markedly reduced. Conversely, a higher expression of microcin E492 protein produced concomitant decreases in the levels of the modified form and in antibacterial activity and a substantial increase in the ability to form amyloid fibers. The same morphology for these fibers, including those formed by only the unmodified version, was observed. Moreover, seeds formed using exclusively the nonmodified form were remarkably more efficient in amyloid formation with a shorter lag phase, indicating that the nucleation process is probably improved. Unmodified microcin E492 incorporation into amyloid fibers was kinetically more efficient than the modified form, probably due to the existence of a conformation that favors this process.     

环状细菌素研究进展

细菌素是一类由细菌核糖体合成的抗菌肽,是产生菌获得生存优势的重要手段。与大多数线性细菌素不同,环状细菌素具有N端和C端共价连接的特殊结构。这种环状结构赋予环状细菌素良好的耐热性、广泛的pH适应性和抗蛋白酶降解能力,在食品防腐和对治耐药性细菌领域表现出巨大的应用潜能。通过对已发现的环状细菌素结构分析发现,相对于一级结构,其三级结构的相似性更高,可以作为环状细菌素归类的依据。环状细菌素的生物合成机制尚不清楚,但其环化机制是最具价值的研究热点,可为其他一些肽类物质的合成提供支架,从而提高应用潜能。环状细菌素抑菌机制主要是在目标菌株的细胞膜上穿孔,使胞内物质外流,进而导致目标细菌死亡。其有类似于抗生素的抑菌活性和有别于抗生素的抑菌机制,为治疗日益严重的耐药性病原菌提供了可靠备选资源。本文综述了环状细菌素的构效关系、生物合成和抑菌机制方面的研究进展,希望能够对环状细菌素的深入研究和应用提供有价值的参考。     

Bacteriocins of gram-positive bacteria.

In recent years, a group of antibacterial proteins produced by gram-positive bacteria have attracted great interest in their potential use as food preservatives and as antibacterial agents to combat certain infections due to gram-positive pathogenic bacteria. They are ribosomally synthesized peptides of 30 to less than 60 amino acids, with a narrow to wide antibacterial spectrum against gram-positive bacteria; the antibacterial property is heat stable, and a producer strain displays a degree of specific self-protection against its own antibacterial peptide. In many respects, these proteins are quite different from the colicins and other bacteriocins produced by gram-negative bacteria, yet customarily they also are grouped as bacteriocins. Although a large number of these bacteriocins (or bacteriocin-like inhibitory substances) have been reported, only a few have been studied in detail for their mode of action, amino acid sequence, genetic characteristics, and biosynthesis mechanisms. Nevertheless, in general, they appear to be translated as inactive prepeptides containing an N-terminal leader sequence and a C-terminal propeptide component. During posttranslational modifications, the leader peptide is removed. In addition, depending on the particular type, some amino acids in the propeptide components may undergo either dehydration and thioether ring formation to produce lanthionine and beta-methyl lanthionine (as in lantibiotics) or thio ester ring formation to form cystine (as in thiolbiotics). Some of these steps, as well as the translocation of the molecules through the cytoplasmic membrane and producer self-protection against the homologous bacteriocin, are mediated through specific proteins (enzymes). Limited genetic studies have shown that the structural gene for such a bacteriocin and the genes encoding proteins associated with immunity, translocation, and processing are present in a cluster in either a plasmid, the chromosome, or a transposon. Following posttranslational modification and depending on the pH, the molecules may either be released into the environment or remain bound to the cell wall. The antibacterial action against a sensitive cell of a gram-positive strain is produced principally by destabilization of membrane functions. Under certain conditions, gram-negative bacterial cells can also be sensitive to some of these molecules. By application of site-specific mutagenesis, bacteriocin variants which may differ in their antimicrobial spectrum and physicochemical characteristics can be produced. Research activity in this field has grown remarkably but sometimes with an undisciplined regard for conformity in the definition, naming, and categorization of these molecules and their genetic effectors. Some suggestions for improved standardization of nomenclature are offered.     

Antibiotic resistance in Salmonella enterica serovar Typhimurium exposed to microcin-producing Escherichia coli

Microcin 24 is an antimicrobial peptide secreted by uropathogenic Escherichia coli. Secretion of microcin 24 provides an antibacterial defense mechanism for E. coli. In a plasmid-based system using transformed Salmonella enterica, we found that resistance to microcin 24 could be seen in concert with a multiple-antibiotic resistance phenotype. This multidrug-resistant phenotype appeared when Salmonella was exposed to an E. coli strain expressing microcin 24. Therefore, it appears that multidrug-resistant Salmonella can arise as a result of an insult from other pathogenic bacteria.