RESUMO
Microcin E492, a channel-forming bacteriocin with the ability to form amyloid fibers, is exported as a mixture of two forms: unmodified (inactive) and posttranslationally modified at the C terminus with a salmochelin-like molecule, which is an essential modification for conferring antibacterial activity. During the stationary phase, the unmodified form accumulates because expression of the maturation genes mceIJ is turned off, and microcin E492 is rapidly inactivated. The aim of this work was to demonstrate that the increase in the proportion of unmodified microcin E492 augments the ability of this bacteriocin to form amyloid fibers, which in turn decreases antibacterial activity. To this end, strains with altered proportions of the two forms were constructed. The increase in the expression of the maturation genes augmented the antibacterial activity during all growth phases and delayed the loss of activity in the stationary phase, while the ability to form amyloid fibers was markedly reduced. Conversely, a higher expression of microcin E492 protein produced concomitant decreases in the levels of the modified form and in antibacterial activity and a substantial increase in the ability to form amyloid fibers. The same morphology for these fibers, including those formed by only the unmodified version, was observed. Moreover, seeds formed using exclusively the nonmodified form were remarkably more efficient in amyloid formation with a shorter lag phase, indicating that the nucleation process is probably improved. Unmodified microcin E492 incorporation into amyloid fibers was kinetically more efficient than the modified form, probably due to the existence of a conformation that favors this process.
Assuntos
Amiloide/metabolismo , Bacteriocinas/metabolismo , Multimerização Proteica , Processamento de Proteína Pós-Traducional , Amiloide/química , Antibacterianos/química , Antibacterianos/metabolismo , Bacteriocinas/química , Dicroísmo Circular , Eletroforese em Gel de Poliacrilamida , Cinética , Klebsiella pneumoniae/metabolismo , Microscopia Eletrônica , Conformação Proteica , Desnaturação Proteica , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
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.