Structure, organization and characterization of the gene cluster involved in the production of microcin E492, a channel‐forming bacteriocin

Abstract

SummaryMicrocin E492 is a low-molecular-weight, channelforming bacteriocin produced and excreted by Klebsiella pneumoniae RYC492. A 13 kb chromosomal DNA fragment from K. pneumoniae RYC492 was sequenced, and it was demonstrated by random Tn5 mutagenesis that most of this segment, which has at least 10 cistrons, is needed for the production of active microcin and its immunity protein. Genes mceG and mceH correspond to an ABC exporter and its accessory protein, respectively, and they are closely related to the colicin V ABC export system. The microcin E492 system also requires the product of gene mceF as an additional factor for export. Despite the fact that this bacteriocin lacks post-translational modifications, genes mceC, mceI and mceJ are needed for the production of active microcin. Genes mceC and mceI are homologous to a glycosyl transferase and acyltransferase, respectively, whereas mceJ has no known homologue. Mutants in these three genes secrete an inactive form of microcin, able to form ion channels in a phospholipidic bilayer, indicating that the mutation of these microcin genes does not alter the process of membrane insertion. On the other hand, microcin isolated from mutants in genes mceC and mceJ has a lethal effect when incubated with spheroplasts of sensitive cells, indicating that the microcin defects in these mutants are likely to alter receptor recognition at the outer membrane. A model for synthesis and export is proposed as well as a novel maturation pathway that would involve conformational changes to explain the production of active microcin E492.

Microcin E492 Amyloid Formation Is Retarded by Posttranslational Modification

“…Consistently, a marked decrease in antibacterial activity occurred when the host carrying pJEM15 was used, which correlated with a decrease of the modified form (1:0. 4 …”

“…Bacterial growth was performed as previously described (3,4,25) with shaking (220 rpm) at 37°C. The overnight culture was diluted 10,000 to 100,000 in M9 minimal medium supplemented with citrate and glucose (26) with the following antibiotics and concentrations: ampicillin (AMP), 100 g/ml; kanamycin (KAN), 50 g/ml; tetracycline (TET), 50 g/ml; and chloramphenicol (CHL), 50 g/ml.…”

“…The group of genes necessary for the production of active MccE492 has been cloned and expressed in Escherichia coli (3,4). This bacteriocin is found in two forms, unmodified (7,887 Da) and posttranslationally modified by the covalent linkage of salmochelin-like molecules of different molecular masses (5).…”

“…MccE492 modified with the diglycosylated DHBS trimer is also found (8,880 Da). Posttranslational modification is carried out by the maturation gene products MceC and MceIJ encoded in the MccE492 gene cluster (4). MceC glycosylates enterochelin and its derivatives generating salmochelin-like molecules, while the covalent linkage of the salmochelin-like molecules to the C-terminus of MccE492 is accomplished by MceIJ (8).…”

Evaluation of E492 Microcin Gene Presence in Klebsiella pneumoniae Collected from Patients Registered to Educational Hospitals of Isfahan

“…Class I comprises three plasmid-encoded peptides with a molecular weight under 5 kDa that have widespread post-translational modifications: microcins J25, C (called C7, C51 or C7/C51), and B17. Class II microcins comprise higher molecular weight peptides in a range of 5 -10 kDa [8,9]. Microcin E492, produced by a strain of Klebsiella pneumoniae, has low-molecular-weight (7.887 Da) with channelforming features.…”