Abstract
Microcin E492 is a pore-forming bacteriocin with toxic activity against Enterobacteriaceae, which undergoes amyloid aggregation as a mechanism to regulate its toxicity. To be active, it requires the posttranslational attachment to the C-terminus of a glycosylated enterochelin derivative (salmochelin), a process carried out by the proteins MceC, MceI and MceJ encoded in the MccE492 gene cluster. Both microcin E492 and salmochelin have a proposed role in the virulence of the bacterial pathogen Klebsiella pneumoniae. Besides, enterochelin is produced as a response to low iron availability and its synthesis is controlled by the global iron regulator Fur. Since the production of active microcin E492 depends on enterochelin biosynthesis, both processes could be coordinately regulated. In this work, we investigated the role of Fur in the expression of the microcin E492 maturation genes mceCJI. mceC was not regulated by Fur as it occurs with its homolog iroB in Salmonella enterica. We demonstrated that mceJI along with the previously uncharacterized gene mceX are transcribed as a single mRNA, and that Fur binds in vivo to a Fur box located upstream of the mceX-mceJI unit. Also, we established that the expression of these genes decreased in a condition of high iron availability, while this effect is abrogated in a Δfur background. Furthermore, our results indicated that MceX acts as a negative regulator of microcin E492 structural gene expression, coupling its synthesis to the iron-dependent regulatory circuit. Consequently, fur or mceX overexpression led to a significant decrease in the antibacterial activity of cells producing microcin E492. Altogether these results show that both the expression of microcin E492 maturation genes mceJI, and MceX the negative regulator of microcin E492 synthesis, are coordinated with the enterochelin production by Fur, depending on the iron levels in the medium.
Highlights
Microcin E492 (MccE492) is an ~8-kDa pore-forming bacteriocin that is produced and excreted by Klebsiella pneumoniae RYC492 [1,2]
Using lacZ reporter fusions we provided evidence indicating that expression of mceX/mceJI amplification the primers used were JVO5475/JVO5476 (mceX) and mceJI is downregulated by increasing iron concentrations in a Ferric Uptake Regulator (Fur)-dependent mechanism, and we established that MceX acts as a negative regulator of the mceB form a single transcriptional unit (mceBA) unit, coupling the MccE492 protein expression to the iron regulation circuit
The evidence provided in this study indicates that iron metabolism and MccE492 maturation are processes coordinately regulated
Summary
Microcin E492 (MccE492) is an ~8-kDa pore-forming bacteriocin that is produced and excreted by Klebsiella pneumoniae RYC492 [1,2]. MccE492 has antibacterial activity against Enterobacteriaceae, induces apoptosis in some human tumor cell lines, and forms amyloid fibers as a mechanism to regulate its activity [3,4,5]. Once synthesized, it undergoes posttranslational modification (maturation) by the attachment to the C-terminus of glycosylated derivatives of enterochelin (salmochelin), a siderophore widely used by Enterobacteriaceae. It was found that genes involved in the production of these molecules are highly prevalent among hypervirulent K. pneumoniae strains [9,10,11], and likely play a role in the pathogenesis of this species, recently declared as an urgent pathogen trait due its hypervirulence and multi-drug resistance (reviewed in [12,13])
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