Genetic Analysis and Complete Primary Structure of Microcin L

Abstract

Escherichia coli LR05, in addition to producing MccB17, J25, and D93, secretes microcin L, a newly discovered microcin that exhibits strong antibacterial activity against related Enterobacteriaceae, including Salmonella enterica serovars Typhimurium and Enteritidis. Microcin L was purified using a two-step procedure including solid-phase extraction and reverse-phase C 18 high-performance liquid chromatography. A 4,901-bp region of the DNA plasmid of E. coli LR05 was sequenced revealing that the microcin L cluster consists of four genes, mclC, mclI, mclA, and mclB. As bacterial resistance to currently used antibiotics is increasing, new pathogenic agents are discovered, and traditional bacterial diseases reappear, increased efforts to search for new antibiotics are needed. Over the past 40 years, research has been restricted largely to improving those well-known compound classes that are active against a standard set of drug targets. As no new classes of antibiotic have been discovered, such insufficient chemical variability exists that there is a potential for serious escalation in clinical microbe resistance. Numerous living organisms are able to produce a variety of ribosomally synthesized antibacterial peptides or proteins involved in their innate defense against microorganisms. During the past 15 years, these compounds have attracted considerable attention, offering many exciting possibilities for the future of antibiotics, in the face of current declining efficacy of conventional treatment (20,22). Of the bacteriocins produced by bacteria, many direct activity against pathogens and, in particular, food-borne microorganisms, such as the grampositive bacterium Listeria monocytogenes (7, 10) and gramnegative bacteria Salmonella enterica and Escherichia coli (34,40).Microcins are secreted by members of the Enterobacteriaceae family, in particular strains of E. coli. They constitute a class of low-molecular-mass peptides (Ͻ10 kDa) that exhibit a narrow antimicrobial spectrum of activity directed against bacterial species phylogenetically related to the producing strains (33). To protect itself, the microcin-producing bacterium exhibits immunity to the action of its own microcin. Recent developments in the biochemical characterization and mode of action allowed us to propose a classification of these peptides into two classes (17, 36). Class I, which includes to date microcins B17, C7, D93, and J25, encompasses peptides with molecular mass below 5 kDa that are highly posttranslationally modified. These microcins display a range of unrelated chemical structures, which in turn results in a variety of action mechanisms (9). Class II includes microcins E492, H47, V, most likely microcin 24, and now microcin L. This second group is more homogeneous and shares several common structural properties with class IIa gram-positive bacteriocins: size ranging from 7 to 10 kDa, absence of modified amino acids, and presence of a consensus motif. Additionally, they are synthesized as precursor peptides with a double-glycine type ...

Characterization of a Novel Microcin That Kills Enterohemorrhagic Escherichia coli O157:H7 and O26

“…2). Two genes, which we have designated mcpB and mcpD, encode homologs of HlyB and HlyD that are known to be the structural components of a microcin transfer system that interact with chromosomally encoded TolC (7,10,18,26). McpA, which contains a CaaX protease domain (PF02517), is thought to be the "activity" protein that processes the microcin, encoded by mcpM, to its mature form prior to transfer from the cell.…”

“…The dedicated transport system involves the products of two plasmid-borne genes, mcpB and mcpD, that share homology with hlyB and hlyD of the E. coli ␣-hemolysin T1SS (9). This multicomponent export system is organized similarly to transport systems for other class II microcins, including MccE492, MccL, and MccV (10,18,26). McpB contains the transmembrane domains and nucleotide-binding domains, including the highly conserved Walker A and B motifs and ABC signature, characteristic of the ABC transporter superfamily of proteins (21).…”

“…To function properly, these secretion systems require the presence of a chromosomally encoded TolC protein on the cell surface (7,10,18,26). Consequently, we constructed a ⌬tolC strain, and this disrupted the ability of E. coli 25 to inhibit E. coli 186 (Fig.…”

Modular Structure of Microcin H47 and Colicin V

“…They comprise four genes: the activity gene, encoding the microcin precursor; the immunity gene, which codes for a peptide that specifically protects the producing cell against its cognate microcin; and two genes encoding proteins that integrate the ABC secretion apparatus. Colicin V (ColV) and microcin L (MccL) are examples of unmodified microcins (10,18). On the other hand, modified microcins of the higher-molecular-mass class are peptides that undergo posttranslational modifications before being secreted.…”

“…By extrapolation, a similar assumption was adopted for the conserved C-terminal sequences of ColV and MccL, which, besides being highly identical, both have a disulfide bond between two cysteines (Fig. 1A) (18). As to the uptake pathways followed by these microcins, it is known that ColV employs the receptor Cir (6), while no data are available for MccL.…”