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.     

Lantibiotics and microcins: polypeptides with unusual chemical diversity

Bacterial-derived antimicrobial polypeptides enjoy a large degree of structural and chemical diversity. Two well-studied examples of such polypeptides are the lanthionine-containing lantibiotics produced by a variety of Gram-positive bacteria, and their Gram-negative counterparts, the microcins. Both groups are produced as gene-encoded precursor peptides and undergo post-translational modification to generate the active moieties. Structure elucidation of novel lantibiotics and microcins has recently uncovered further novel structural and chemical features and, combined with the generation of analogue peptides by genetic manipulation, new insights into structure-function relationships have been gained. Furthermore, study of the mode of action of the lantibiotics nisin and mersacidin has revealed their use of a 'docking molecule' in the target cell to facilitate their biological activities. Meanwhile, in vitro studies with microcin B17 have helped to uncover the molecular mechanisms by which post-translational modification results in the formation of heterocyclic oxazole and thiazole rings. From a practical standpoint, both groups of polypeptides represent new lead structures for future development of antimicrobial agents, whilst the identification of the 'docking molecules' represents a step forward in the search for novel targets for future antibiosis.     

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.     

Focus on modified microcins: structural features and mechanisms of action

Microcins are gene-encoded antimicrobial (poly)peptides secreted by Enterobacteriaceae. Produced under conditions of nutrient depletion, they are active against phylogenetically related microbial strains. Therefore, they are considered to play an important role in the microbial competitions within the intestinal flora. Among the limited sample of nine microcins hitherto described, a wide variety of structures and modes of action could be identified. The knowledge on microcins is very uneven, some being extensively studied, and others remaining uncharacterized. In this article, we have focused on a subgroup of highly modified microcins that show very original structures. We present an updated overview on the structures and mechanisms of action of microcins B17, C7 and J25, and on the associated effector proteins, also encoded by the microcin genetic system, which include specific modification enzymes, export proteins, and immunity factors.     

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.     

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。     

Streptolysin S-like virulence factors: the continuing sagA

Streptolysin S (SLS) is a potent cytolytic toxin and virulence factor that is produced by nearly all Streptococcus pyogenes strains. Despite a 100-year history of research on this toxin, it has only recently been established that SLS is just one of an extended family of post-translationally modified virulence factors (the SLS-like peptides) that are produced by some streptococci and other Gram-positive pathogens, such as Listeria monocytogenes and Clostridium botulinum. In this Review, we describe the identification, genetics, biochemistry and various functions of SLS. We also discuss the shared features of the virulence-associated SLS-like peptides, as well as their place within the rapidly expanding family of thiazole/oxazole-modified microcins (TOMMs).     

Microcin B17 blocks DNA replication and induces the SOS system in Escherichia coli

Microcin B17 is a novel peptide antibiotic of low Mr (about 4000) produced by Escherichia coli strains carrying plasmid pMccB17. The action of this microcin in sensitive cells is essentially irreversible, follows single-hit kinetics, and leads to an abrupt arrest of DNA replication and, consequently, to the induction of the SOS response. RecA- and RecBC- strains are hypersensitive to microcin B17. Strains producing a non-cleavable SOS repressor (lexAl mutant) are also more sensitive than wild-type, whereas strains carrying a mutation which causes constitutive expression of the SOS response (spr-55) are less sensitive to microcin. Microcin B17 does not induce the SOS response in cells which do not have an active replication fork. The results suggest that the mode of action of this microcin is different from all other well-characterized microcins and colicins, and from other antibiotics which inhibit DNA replication.     

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.     

The Microbial Toxin Microcin B17: Prospects for the Development of New Antibacterial Agents

Microcin B17 (MccB17) is an antibacterial peptide produced by strains of Escherichia coli harboring the plasmid-borne mccB17 operon. MccB17 possesses many notable features. It is able to stabilize the transient DNA gyrase–DNA cleavage complex, a very efficient mode of action shared with the highly successful fluoroquinolone drugs. MccB17 stabilizes this complex by a distinct mechanism making it potentially valuable in the fight against bacterial antibiotic resistance. MccB17 was the first compound discovered from the thiazole/oxazole-modified microcins family and the linear azole-containing peptides; these ribosomal peptides are post-translationally modified to convert serine and cysteine residues into oxazole and thiazole rings. These chemical moieties are found in many other bioactive compounds like the vitamin thiamine, the anti-cancer drug bleomycin, the antibacterial sulfathiazole and the antiviral nitazoxanide. Therefore, the biosynthetic machinery that produces these azole rings is noteworthy as a general method to create bioactive compounds. Our knowledge of MccB17 now extends to many aspects of antibacterial–bacteria interactions: production, transport, interaction with its target, and resistance mechanisms; this knowledge has wide potential applicability. After a long time with limited progress on MccB17, recent publications have addressed critical aspects of MccB17 biosynthesis as well as an explosion in the discovery of new related compounds in the thiazole/oxazole-modified microcins/linear azole-containing peptides family. It is therefore timely to summarize the evidence gathered over more than 40 years about this still enigmatic molecule and place it in the wider context of antibacterials.     

Microcin H47, a chromosome-encoded microcin antibiotic of Escherichia coli.

Microcin H47 (MccH47) is a novel microcin antibiotic produced by a natural Escherichia coli isolate. In contrast to all the other colicins and microcins examined to date, which are plasmid encoded, the genes for MccH47 synthesis and immunity are located on the chromosome. These genetic determinants were cloned and shown to extend over a continuous DNA region of ca. 10 kb.     

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.     

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.     

Expansion of ribosomally produced natural products: a nitrile hydratase- and Nif11-related precursor family

Background  

A new family of natural products has been described in which cysteine, serine and threonine from ribosomally-produced peptides are converted to thiazoles, oxazoles and methyloxazoles, respectively. These metabolites and their biosynthetic gene clusters are now referred to as thiazole/oxazole-modified microcins (TOMM). As exemplified by microcin B17 and streptolysin S, TOMM precursors contain an N-terminal leader sequence and C-terminal core peptide. The leader sequence contains binding sites for the posttranslational modifying enzymes which subsequently act upon the core peptide. TOMM peptides are small and highly variable, frequently missed by gene-finders and occasionally situated far from the thiazole/oxazole forming genes. Thus, locating a substrate for a particular TOMM pathway can be a challenging endeavor.     

Microcin plasmids: a group of extrachromosomal elements coding for low-molecular-weight antibiotics in Escherichia coli.

Microcins are low-molecular-weight compounds produced and excreted by Enterobacteriaceae. They inhibit the growth of a wide spectrum of microorganisms. Microcin-synthesizing transconjugants were obtained in seven out of eight experiments of conjugational transfer between wild-type microcinogenic strains of Escherichia coli and E. coli strain BM21. The physical analysis of one of the transconjugant strains that has acquired the ability to produce microcin 17 showed the presence of extrachromosomal DNA as a plasmid (pRYC17) of molecular weight 36 X 10(6) (18.3-micron length), which is absent in the "microcincured" derivative strain. pRYC17 was incompatible with plasmids of the IncFII group. Other suspected plasmids containing the information for the synthesis of microcins have not been clearly classified. Strains producing microcins 93, 136, and 140 show a partial incompatibility with IncFIII group of plasmids.     

The antibacterial threaded-lasso peptide capistruin inhibits bacterial RNA polymerase

Capistruin, a ribosomally synthesized, post-translationally modified peptide produced by Burkholderia thailandensis E264, efficiently inhibits growth of Burkholderia and closely related Pseudomonas strains. The functional target of capistruin is not known. Capistruin is a threaded-lasso peptide (lariat peptide) consisting of an N-terminal ring of nine amino acids and a C-terminal tail of 10 amino acids threaded through the ring. The structure of capistruin is similar to that of microcin J25 (MccJ25), a threaded-lasso antibacterial peptide that is produced by some strains of Escherichia coli and targets DNA-dependent RNA polymerase (RNAP). Here, we show that capistruin, like MccJ25, inhibits wild type E. coli RNAP but not mutant, MccJ25-resistant, E. coli RNAP. We show further that an E. coli strain resistant to MccJ25, as a result of a mutation in an RNAP subunit gene, exhibits resistance to capistruin. The results indicate that the structural similarity of capistruin and MccJ25 reflects functional similarity and suggest that the functional target of capistruin, and possibly other threaded-lasso peptides, is bacterial RNAP.     

Cloning of the determinants for microcin D93 production and analysis of three different D-type microcin plasmids

Three different microcin plasmids coding for D-type microcins were analyzed. Two of the plasmids (pMccD93 and pCP101) were small, multicopy plasmids and were closely related. The third plasmid (pCP106) was a conjugative, antibiotic multiresistance plasmid. Although plasmids pCP101 and pCP106 were previously classified as A-type microcin plasmids, we have determined that they are, in fact, D type. Furthermore, the determinants for microcin D93 production were cloned from plasmid pMccD93, and a DNA probe for the region implicated in the synthesis of microcin was obtained. This probe hybridized to plasmid C from Escherichia coli strain V517, indicating that this plasmid might be involved in the synthesis of a D-type microcin. The characteristics of replication of plasmid pCP106 were analyzed and appeared to be similar to those of ColEl plasmids, although pCP106 is a conjugative single-copy plasmid.     

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.