The alternative role of enterobactin as an oxidative stress protector allows Escherichia coli colony development.

Numerous bacteria have evolved different iron uptake systems with the ability to make use of their own and heterologous siderophores. However, there is growing evidence attributing alternative roles for siderophores that might explain the potential adaptive advantages of microorganisms having multiple siderophore systems. In this work, we show the requirement of the siderophore enterobactin for Escherichia coli colony development in minimal media. We observed that a strain impaired in enterobactin production (entE mutant) was unable to form colonies on M9 agar medium meanwhile its growth was normal on LB agar medium. Given that, neither iron nor citrate supplementation restored colony growth, the role of enterobactin as an iron uptake-facilitator would not explain its requirement for colony development. The absence of colony development was reverted either by addition of enterobactin, the reducing agent ascorbic acid or by incubating in anaerobic culture conditions with no additives. Then, we associated the enterobactin requirement for colony development with its ability to reduce oxidative stress, which we found to be higher in media where the colony development was impaired (M9) compared with media where the strain was able to form colonies (LB). Since oxyR and soxS mutants (two major stress response regulators) formed colonies in M9 agar medium, we hypothesize that enterobactin could be an important piece in the oxidative stress response repertoire, particularly required in the context of colony formation. In addition, we show that enterobactin has to be hydrolyzed after reaching the cell cytoplasm in order to enable colony development. By favoring iron release, hydrolysis of the enterobactin-iron complex, not only would assure covering iron needs, but would also provide the cell with a molecule with exposed hydroxyl groups (hydrolyzed enterobactin). This molecule would be able to scavenge radicals and therefore reduce oxidative stress

A) Type of growth of <i>E. coli fepD</i> and <i>fepG</i> mutants streaked in M9A and incubated overnight in aerobic conditions. B) Levels of reactive oxygen species in <i>E. coli</i> wild-type and <i>fepD</i>, <i>fepG, entS</i> and <i>entE</i> mutants grown in M9 medium. Quantitation of ROS levels was done using the DCFA-DA probe. Fluorescence intensities are relative to that of the control. Control: wt grown in M9 medium. Error bars = SD, n = 3.

<p>A) Type of growth of <i>E. coli fepD</i> and <i>fepG</i> mutants streaked in M9A and incubated overnight in aerobic conditions. B) Levels of reactive oxygen species in <i>E. coli</i> wild-type and <i>fepD</i>, <i>fepG, entS</i> and <i>entE</i> mutants grown in M9 medium. Quantitation of ROS levels was done using the DCFA-DA probe. Fluorescence intensities are relative to that of the control. Control: wt grown in M9 medium. Error bars = SD, n = 3.</p

Reactive oxygen species levels in <i>E. coli entE</i> mutant grown in different culture media.

<p>Quantitation of ROS levels was done using the DCFA-DA probe. Fluorescence intensities are relative to that of the control. Control: wild-type strain grown in M9 medium; <i>entE</i> M9: indicates cells grown in M9 medium, <i>entE</i> LB: indicates cells grown in LB medium, <i>entE</i> M9 1% cas: indicates cells grown in M9 medium supplemented with 1% casamino acids. Error bars = SD, n = 3.</p

Growth of <i>E. coli</i> wild-type (wt) and <i>entE</i> strains in liquid and solid media.

<p>A) Liquid aerated minimal M9 medium cultures of wild-type strain (blue squares), <i>entE</i> strain (green circles) and <i>entE</i> strain in the same media but supplemented with 100 µM FeCl₃ (red triangles). Growth (OD₆₀₀) was determined at the indicated times. B) Lawn growth of wt and <i>entE E. coli</i> strains on M9A. A stationary phase culture of <i>entE E. coli</i> strain was serially diluted (10⁻¹ to 10⁻⁴) and an aliquot of these dilutions was applied on M9A or M9A supplemented with 100 µM FeCl₃. As control, the same dilutions of a wt strain overnight culture were applied on M9A medium. Lawn growth was compared at 8 hours of incubation. C) Colony growth of wt and <i>entE E. coli</i> on LBA. A stationary phase culture of <i>entE E. coli</i> strain was serially diluted and an aliquot of dilutions 10⁻⁶ to 10⁻⁸ were applied on LBA or LBA supplemented with 100 µM FeCl₃. As control, the same dilutions of an overnight culture of the wt strain were applied on LBA medium. After overnight incubation, colonies sizes were compared. D) Activity of the <i>rhyB</i> promoter estimated by β-galactosidase activity as an indirect measure of the intracellular iron content (The higher the promoter expression, the lower the iron content <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084734#pone.0084734-Ma2" target="_blank">[48]</a>). Both wild-type strain and <i>entE</i> mutant respond to iron addition. The plasmid pALM23 carries the <i>ryhB- lacZ</i> fusion.</p

List of strains and plasmids used in this work.

a<p>CGSC, <i>Escherichia coli</i> Genetic Stock Center;</p

Colonies resume growth upon enterobactin, ascorbic acid or casamino acids (cas) addition.

<p>In plates in which no growth was obtained after overnight incubation (10⁻⁵ dilutions), 1 µL of 1 µM enterobactin (A), 5 µL of 1 mM ascorbic acid (B), 5 µL of 2% casamino acids (C), 10 µL of 1 mM FeCl₃ (D) or 10 µL of 1 mM sodium citrate (E) were spotted. After a second overnight incubation, a size gradient of colonies was clearly observed around the spots containing enterobactin (A), ascorbic acid (B) and casamino acids (C). However, no growth was observed with FeCl₃ (D) or citrate (E).</p

Type of growth of <i>entE</i> mutant in solid medium.

<p>One hundred µL of serial dilutions (from 10⁻³ to 10⁻⁸) from a stationary phase culture were plated in the specified solid medium (M9 agar, M9A or LB agar, LBA) and incubated overnight. The type of growth was observed: L, lawn; C, colony, NG, no growth.</p><p>^a+ Fe, indicate medium supplementation with 100 µM FeCl_3.</p>b<p>+ ASC indicate medium supplementation with 1 mM ascorbic acid.</p>c<p>+CAS indicate medium supplementation with 1% casamino acid.</p

Erratum to: Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition) (Autophagy, 12, 1, 1-222, 10.1080/15548627.2015.1100356

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