Microcin 25, a peptide antibiotic excreted by an Escherichia coli strain isolated from human feces, was purified to homogeneity and characterized. Composition analysis and data from gel ifitration indicated that microcin 25 may contain 20 amino acid residues. It has a blocked amino-terminal end. Microcin synthesis and immunity are plasmid determined, and the antibiotic was produced in minimal medium when the cultures entered the stationary phase of growth. The peptide appears to interfere with cell divrision, since susceptible cells filamented when exposed to it. This response does not seem to be mediated by the SOS system.Microcins are a miscellaneous group of low-molecularweight antibiotics produced by a number of enterobacteria, mostly Escherichia coli strains (2). Several features distinguish them from the more widely studied colicins. Microcins are considerably smaller, their synthesis is neither lethal nor controlled by the SOS regulatory circuit, and synthesis takes place during the stationary phase of growth (with the possible exception of microcin E492 from Kiebsiella pneumoniae, the production of which starts in the exponential phase) (3). Moreover, colicins are bactericidal proteins, whereas microcins may exhibit bactericidal or bacteriostatic modes of action. At least for the most extensively characterized microcins, B17 and C7, the genetic systems involved in their production and immunity are considerably more complex than the contiguous arrangement of three genes (synthesis, immunity, and release via lysis) which is a common feature of colicin-producing plasmids (16,31,34). Like those of colicins, the genetic determinants for microcins seem to be invariably plasmid borne. However, Lavinia et al. have recently described a novel member of the family, microcin H47, which is chromosomally encoded (24).Microcins are a promising model system for the study of peptide transport mechanisms and the regulation of gene expression in nonproliferating cells. Studies of them may also contribute to a deeper understanding of the structurefunction relationships of antimicrobial peptides.This article introduces the study of a novel peptide antibiotic which is excreted into the culture medium by a fecal strain of E. coli. We have called it microcin 25 (Mcc25). The purification, initial biochemical characterization, and some properties of Mcc25 are reported. Also, we present results suggesting that it can act as a cell division inhibitor. MATERIALS AND METHODSBacterial strains. The bacteria used in this work are listed in Table 1. All strains are derivatives of E. coli K-12 except for AY25, the natural E. coli producer of Mcc25, and AY29, its nonproducing plasmid-cured derivative. Strain AY25 was isolated in our laboratory from the feces of a newborn infant, as described previously (3).Media. LB rich and M63 minimal media have been de-* Corresponding author.scribed by Miller (29). Minimal medium was supplemented with 0.2% of the specified carbon source, thiamine (1 ,g/ml), and, when appropriate, L-amino acids (20 ,ug/m...

Microcin J25 (MccJ25) is the single representative of the immunity group J of the microcin group of peptide antibiotics produced by Enterobacteriaceae. It induces bacterial filamentation in susceptible cells in a non-SOSdependent pathway [R. A. Salomon and R. Farias (1992) J. Bacteriol. 174, 7428±7435]. MccJ25 was purified to homogeneity from the growth medium of a microcin-overproducing Escherichia coli strain by reverse-phase HPLC. Based on amino acid composition and absolute configuration determination, liquid secondary ion and electrospray mass spectrometry, extensive two-dimensional NMR, enzymatic and chemical degradations studies, the structure of MccJ25 was elucidated as a 21-residue peptide, cyclo(-Val 1 -Gly-Ile-Gly-Thr-Pro-Ile-Ser-Phe-Tyr-Gly-Gly-Gly-AlaGly-His-Val-Pro-Glu-Tyr-Phe 21 -). Although MccJ25 showed high resistance to most of endoproteases, linearization by thermolysin occurred from cleavage at the Phe 21 -Val 1 bond and led to a single peptide, MccJ25-L. While MccJ25 exhibited remarkable antibiotic activity towards Salmonella newport and several E. coli strains (minimal inhibitory concentrations ranging between 0.01 and 0.2 mg´mL ±1 ), the thermolysin-linearized microcin showed a dramatic decrease of the activity, indicating that the cyclic structure is essential for the MccJ25 biological properties. As MccJ25 is ribosomally synthesized as a larger peptide precursor endowed with an N-terminal extremity, the present study shows that removal of this extension and head±tail cyclization of the resulting propeptide are the only posttranslational modifications involved in the maturation of MccJ25, that appears as the first cyclic microcin.Keywords: antibiotic peptide; microcin; NMR.Microcins form a miscellaneous group of low molecular mass peptide antibiotics produced by diverse strains of Enterobacteriaceae, mostly Escherichia coli. They are mainly active against bacterial genera or species phylogenetically related to the producing strains [1,2]. Wild-type bacterial strains producing microcins are resistant to the microcin they produce, i.e. they are self-immune. This feature has been used to classify them into immunity groups [2], the number of which is growing [3,4]. Underlying this classification is the assumption that each immunity group represents a unique structure with an unique mode of action. Microcins, which are supposed to play a part in the stability of intestinal ecosystem [1,2,5,6], could be used as therapeutic agents or for the design of new useful drugs by means of DNA and protein engineering. To develop this research in a rational way, the knowledge of their chemical structures and of the enzymes involved in the biosyntheses appears to be required.Beside ColV, formerly classified as a colicin [7], the other best known microcins are MccB17 and MccC7. Their structures, modes of action and genes involved in their biosynthesis, export and immunity have been described. These microcins are plasmidencoded, ribosomally synthesized small peptides, whose production is induced when cells ...

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