MceX negatively regulates the expression of the microcin E492 structural gene.

<p>(A) <i>wt</i> and Δ<i>fur E</i>. <i>coli</i> cells transformed with the reporter p<i>mceBA</i>’-‘<i>lacZ</i> were grown in M9 medium supplemented with 100 μM 2,2’-Bipyridyl (low iron availability) or 10 μM FeSO₄ (high iron availability). β-galactosidase activity (expressed in Miller Units) was measured at different growth phases (Early exponential, OD₆₀₀ = 0.4–0.6; Late exponential, OD₆₀₀ = 0.9–1.1; Stationary, OD₆₀₀ = 1.9–2.1). In the construct scheme, the orange circle represents the previously determined transcription start site. (B) Wild type <i>E</i>. <i>coli</i> cells were transformed with pT5-<i>mceX</i> (allowing the IPTG-inducible expression of MceX), or with the backbone plasmid pUC57 as negative control. The activity was expressed as the percentage of β-galactosidase activity after induction respect to the negative control. Error bars represent the standard deviation between 6 independent experiments. ***<i>P</i><0.001, ****<i>P</i><0.0001, ns: not significant.</p

Fur overexpression reduces the antibacterial activity of <i>E</i>. <i>coli</i> cells carrying the gene cluster for microcin E492 production.

<p><i>E</i>. <i>coli</i> cells carrying pMccE492 (allowing the production of active MccE492) were transformed with p15A-<i>fur</i> (driving overexpression of Fur carrying a His-tag) or pACYC184 (control). (A) Two representative clones of p15A-<i>fur</i> containing cells were grown in M9 medium alone or supplemented with IPTG (1 mM), and total protein extracts were prepared after 3 and 24 h of growth. SDS-PAGE analysis of the extracts followed by Coomassie Blue staining revealed a ~17-kDa protein band largely enriched in cells grown in presence of IPTG (black arrow). Immunoblot using an anti-His antibody confirmed the induction of Fur expression in presence of IPTG, although a basal expression was detected after 24 h of growth in absence of inducer. (B) Antibacterial activity of <i>E</i>. <i>coli</i> cells producing MccE492, transformed with p15A-<i>fur</i> or a control plasmid (pACYC184). The antibacterial activity was measured as a function of the growth inhibition halo’s area over a sensitive strain layer, in M9 medium supplemented with IPTG, and then normalized to the value obtained in the control condition (plasmid backbone). Error bars indicate standard deviation from 40 measurements performed for each condition. ***<i>P</i><0.001.</p

Bacterial strains used in this work.

<p>Bacterial strains used in this work.</p

MceX overexpression reduces the antibacterial activity of <i>E</i>. <i>coli</i> cells carrying the gene cluster for microcin E492 production.

<p><i>E</i>. <i>coli</i> cells carrying pMccE492 (wt, in yellow) or pJAM434 (poor producing, in green) were transformed with pT5-<i>mceX</i> (driving overexpression of MceX) or pUC57 (control). Antibacterial activity was measured as a function of the growth inhibition halo’s area over a sensitive strain layer, in M9 medium supplemented with IPTG, and was normalized to the value obtained in the control condition for each case. Error bars indicate standard deviation from 40 measurements performed for each condition. **<i>P</i><0.01, ns: not significant.</p

List and sequence of primers used in this work.

<p>List and sequence of primers used in this work.</p

The expression of <i>mceX</i> and the microcin E492 maturation genes <i>mceJI</i> is regulated by Fur and iron availability.

<p><i>wt</i> and Δ<i>fur E</i>. <i>coli</i> cells transformed with the reporters p<i>mceX</i>’-‘<i>lacZ</i> (A) or p<i>mceJ17</i>’-‘<i>lacZ</i> (B), were grown in M9 medium supplemented with 100 0μM 2,2’-Bipyridyl (low iron availability) or 10 μM FeSO₄ (high iron availability). β-galactosidase activity (expressed in Miller Units) was measured at different phases of growth (Early exponential, OD₆₀₀ = 0.4–0.6; Late exponential, OD₆₀₀ = 0.9–1.1; Stationary, OD₆₀₀ = 1.9–2.1). In the construct schemes, the orange circle represents the experimentally determined transcription start site and the red square represents the functional Fur box. Error bars represent the standard deviation between 6 independent experiments. ***<i>P</i><0.001, ****<i>P</i><0.0001, ns: not significant.</p

<i>mceX</i>, <i>mceJ</i> and <i>mceI</i> genes are transcribed as a single mRNA from a promoter located upstream of the <i>mceX</i> Fur box.

<p>(A) RT-PCR detection of a polycistronic mRNA harboring <i>mceX</i>, <i>mceJ</i> and <i>mceI</i>. The cDNA was prepared from total RNA of <i>E</i>. <i>coli</i> cells transformed with pJAM229. The amplicons originated from each primer pair are represented in the scheme as blue dashed lines, and are marked with white arrowheads in the corresponding gel section. Total RNA non-subjected to reverse transcription was used as negative control (-), while pJAM229 plasmid was used as positive control (+). (B) 5’ RACE assay to determine the transcription start site of the <i>mceX/mceJI</i> unit. Two primers were used (blue arrowheads), one hybridizing to <i>mceX</i> and the other hybridizing to <i>mceJ</i>. A band of 147–190 bp was enriched after TAP treatment when using <i>mceX</i> primer (orange arrowhead). Bands not enriched after TAP treatment were also observed using <i>mceX</i> or <i>mceJ</i> primers (white arrowheads). Total RNA non-subjected to reverse transcription was used as negative control (-). (C) Two reporter constructs were obtained fusing <i>lacZ</i> to the last codon of <i>mceI</i> gene. The <i>mceJI’-‘lacZ</i> construct harbors <i>mceJ</i>, <i>mceX</i>, and its upstream region (including the TSS). The <i>mceJIΔTSS’-‘lacZ</i> construct is identical to <i>mceJI’-‘lacZ</i>, except for lacking a region (shaded in red) comprising the <i>mceX</i> promoter and part of its coding region. (D) β-galactosidase activity was measured in <i>E</i>. <i>coli</i> cells transformed with each of the reporter fusions and grown until different optical density. Error bars correspond to standard deviation of 3 independent experiments.</p

Model of the iron regulatory circuit controlling microcin E492 production, maturation and antibacterial activity.

<p>(A) At low iron availability, no Fur-Fe²⁺ repressor complexes are available to bind the Fur box located in the promoters of genes participating in the synthesis of enterochelin (1) and <i>mceX</i>/<i>mceJI</i> genes (2). Thus, high amounts of enterochelin and salmochelin are produced, and MceJI proteins catalyze the attachment of salmochelin to MccE492 peptide (maturation). Additionally, the MceX regulator partially represses the <i>mceBA</i> genes (3), restricting the production of immature MccE492. In this situation, a high proportion of modified MccE492 is exported, which can enter the target cells through the catechol siderophore receptors, resulting in a high antibacterial activity. The production of a high amount of modified MccE492, likely disfavors its amyloid aggregation, preventing toxin inactivation. (B) At high iron availability, the Fur-Fe²⁺ complexes repress the <i>ent</i> and <i>mceX</i>/<i>mceJI</i> genes, causing a low production of enterochelin and salmochelin, a poor MccE492 maturation process, and the release of the negative regulation exerted by MceX over the <i>mceBA</i> genes, allowing a higher expression of the MccE492 precursor. Hence, unmodified MccE492 is predominantly exported, which is not recognized by the siderophore receptors and thus fails to cause a toxic effect. The high proportion of unmodified MccE492 likely favor its aggregation into amyloid fibers, and thus the loss of antibacterial activity. The scheme includes a simplified representation of the actual <i>ent</i> genes organization.</p

Functional Fur boxes are located upstream of the microcin E492 maturation genes <i>mceJI</i>.

<p>(A) Sequence analysis revealed the presence of putative Fur boxes in the MccE492 gene cluster (red boxes). Solid gray segments represent the regions of the cluster that were cloned in the respective plasmid listed in the left side, and that were used in the FurTA assays showed in (D). (B) Nucleotide sequence of the 5’ upstream regions from genes <i>mceC</i>, <i>mceD</i>, <i>mceE</i> and <i>mceX</i>. Experimentally determined transcription start site for each gene is indicated as “+1”, while -35 and -10 promoter elements are shown in green. The identified putative Fur boxes are shaded in red. (C) Nucleotide sequence aligment of the Fur boxes identified in the promoter regions of <i>mce</i>C, <i>mceD</i>, <i>mceE</i> and <i>mceX</i> genes, as well as the consensus sequence previously defined for <i>E</i>. <i>coli</i>. A different color was assigned to each nucleotide base. The percentage identity of each Fur box with the consensus sequence is showed in parenthesis. The most conserved nucleotide positions of the <i>E</i>. <i>coli</i> consensus are marked with asterisks. (D) FurTA assay results for <i>E</i>. <i>coli</i> H1717 reporter strain transformed with one of the plasmids listed in (A), or with the control plasmids pHC79 and pACYC184. Red coloration after growing in iron-supplemented MacConkey agar plates indicates Fur binding <i>in vivo</i>.</p

Plasmids used in this work.

<p>Plasmids used in this work.</p