Identification and Properties of the Genes Encoding Microcin E492 and Its Immunity Protein

Lagos, Rosalba; Villanueva, Jorge; Monasterio, Octavio

doi:10.1128/jb.181.1.212-217.1999

Cited by 46 publications

(21 citation statements)

References 33 publications

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“…The entire MccE492 gene cluster is contained within a 13kb DNA fragment that has been cloned in E. coli (Wilkens et al, 1997). Ten genes (mceABCDEFGHIJ) (Lagos et al, 2001) (Lagos, Villanueva and Monasterio, 1999). Other genes are required for MccE492 post-translational modification.…”

Section: Recognition/uptake and Mechanism Of Actionmentioning

confidence: 99%

Class II Microcins

Vassiliadis¹,

Destoumieux-Garzón²,

Péduzzi

2011

Prokaryotic Antimicrobial Peptides

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Class II microcins are 4.9 to 8.9 kDa polypeptides produced by and active against enterobacteria. They are classified into two subfamilies according to their structure and their gene cluster arrangement. While class IIa microcins undergo no post-translational modification, class IIb microcins show a conserved C-terminal sequence that carries a salmochelin-like siderophore motif as a posttranslational modification. Aside from this C-terminal end, which is the signature of class IIb microcins, some sequence similarities can be observed within and between class II subclasses, suggesting the existence of common ancestors. Their mechanisms of action are still under investigations, but several class II microcins use inner membrane proteins as cellular targets, and some of them are membraneactive. Like group B colicins, many if not all class II microcins are TonBand energy-dependent and use catecholate siderophore receptors for recognition/translocation across the outer membrane. In that context, class IIb microcins are considered to have developed molecular mimicry to increase their affinity for their outer membrane receptors through their salmochelin-like post-translational modification. 4.2.3.2 Class IIa microcins Microcins (Mcc) belonging to this subclass are characterized by the absence of posttranslational modification. MccV and MccL contain disulfide bond(s) whereas Mcc24 would be a linear unmodified peptide lacking such bond. 4.2.3.2.1 Genetics and structure Class IIa microcin gene clusters (Fig. 1) are composed of only four plasmid-borne genes. MccV, formerly colicin V (ColV) (Fredericq, 1949) was the first antibiotic substance reported to be produced by E. coli (Gratia, 1925). MccV is secreted by various E. coli strains harbouring large (> 80 kb) and low copy number pColV plasmids (Waters and Crosa, 1991). MccL is produced by E. coli LR05 isolated from poultry intestine (Gaillard-Gendron et al., 2000), while Mcc24 (formerly colicin 24) is secreted by the uropathogenic E. coli 2424 and its genetic determinants are located on the 43.5-kb conjugative plasmid p24-2 (O'Brien and Mahanty, 1994). MccV and MccL gene clusters are composed of four genes organized in two converging transcription units (Gilson, Mahanty and Kolter, 1987;

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Section: Recognition/uptake and Mechanism Of Actionmentioning

confidence: 99%

Class II Microcins

Vassiliadis¹,

Destoumieux-Garzón²,

Péduzzi

2011

Prokaryotic Antimicrobial Peptides

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“…In recent years, major progress has been made in the characterization of the genetic determinants involved in MccE492 production. A chromosomal DNA fragment from K. pneumoniae RYC492 responsible for MccE492 production and immunity was cloned (Wilkens et al ., 1997), and the gene cluster characterized (Lagos et al ., 1999; Lagos et al ., 2001), thus allowing the identification of genes encoding the MccE492 precursor, the immunity protein, and associated secretion factors. Very recently, by expressing MccE492 in engineered E. coli , small amounts of the pure molecule were obtained, which allowed the characterization of its primary structure (Pons et al ., 2002).…”

Section: Introductionmentioning

confidence: 99%

Microcin E492 antibacterial activity: evidence for a TonB‐dependent inner membrane permeabilization on Escherichia coli

Destoumieux-Garzón

Thomas

Santamaría

et al. 2003

Molecular Microbiology

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SummaryThe 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 m m m m M. 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 TonBand 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 ironsiderophore 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.

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“…Since the expression of MccE492 in the absence of its immunity protein is lethal to the cell ( Bieler et al, 2006 ), both proteins had to be co-expressed in all the experiments, hampering the possibility to directly compare MccE492 amyloid formation in presence or absence of MceB. However, we believe that it is very unlikely that this protein participates in the amyloidogenic process, because MceB is an integral membrane protein with three transmembrane helixes and not detected in the cytoplasm ( Lagos et al, 1999 ), therefore the neutralization of MccE492 by MceB does not occurs in this compartment. Although the exact mechanism by which the immunity protein neutralizes the MccE492 pore-forming activity is unknown, the interaction would occur at the moment that MccE492 is inserted into the inner membrane preventing the correct insertion to form the pore.…”

Section: Discussionmentioning

confidence: 99%

Identification of Key Amino Acid Residues Modulating Intracellular and In vitro Microcin E492 Amyloid Formation

et al. 2016

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Microcin E492 (MccE492) is a pore-forming bacteriocin produced and exported by Klebsiella pneumoniae RYC492. Besides its antibacterial activity, excreted MccE492 can form amyloid fibrils in vivo as well as in vitro. It has been proposed that bacterial amyloids can be functional playing a biological role, and in the particular case of MccE492 it would control the antibacterial activity. MccE492 amyloid fibril’s morphology and formation kinetics in vitro have been well-characterized, however, it is not known which amino acid residues determine its amyloidogenic propensity, nor if it forms intracellular amyloid inclusions as has been reported for other bacterial amyloids. In this work we found the conditions in which MccE492 forms intracellular amyloids in Escherichia coli cells, that were visualized as round-shaped inclusion bodies recognized by two amyloidophilic probes, 2-4′-methylaminophenyl benzothiazole and thioflavin-S. We used this property to perform a flow cytometry-based assay to evaluate the aggregation propensity of MccE492 mutants, that were designed using an in silico prediction of putative aggregation hotspots. We established that the predicted amino acid residues 54–63, effectively act as a pro-amyloidogenic stretch. As in the case of other amyloidogenic proteins, this region presented two gatekeeper residues (P57 and P59), which disfavor both intracellular and in vitro MccE492 amyloid formation, preventing an uncontrolled aggregation. Mutants in each of these gatekeeper residues showed faster in vitro aggregation and bactericidal inactivation kinetics, and the two mutants were accumulated as dense amyloid inclusions in more than 80% of E. coli cells expressing these variants. In contrast, the MccE492 mutant lacking residues 54–63 showed a significantly lower intracellular aggregation propensity and slower in vitro polymerization kinetics. Electron microscopy analysis of the amyloids formed in vitro by these mutants revealed that, although with different efficiency, all formed fibrils morphologically similar to wild-type MccE492. The physiological implication of MccE492 intracellular amyloid formation is probably similar to the inactivation process observed for extracellular amyloids, and could be used as a mean of sequestering potentially toxic species inside the cell when this bacteriocin is produced in large amounts.

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Identification and Properties of the Genes Encoding Microcin E492 and Its Immunity Protein

Cited by 46 publications

References 33 publications

Class II Microcins

Class II Microcins

Microcin E492 antibacterial activity: evidence for a TonB‐dependent inner membrane permeabilization on Escherichia coli

Identification of Key Amino Acid Residues Modulating Intracellular and In vitro Microcin E492 Amyloid Formation

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