Reduced Heme Levels Underlie the Exponential Growth Defect of the <i>Shewanella oneidensis hfq</i> Mutant

<div><p>The RNA chaperone Hfq fulfills important roles in small regulatory RNA (sRNA) function in many bacteria. Loss of Hfq in the dissimilatory metal reducing bacterium <i>Shewanella oneidensis</i> strain MR-1 results in slow exponential phase growth and a reduced terminal cell density at stationary phase. We have found that the exponential phase growth defect of the <i>hfq</i> mutant in LB is the result of reduced heme levels. Both heme levels and exponential phase growth of the <i>hfq</i> mutant can be completely restored by supplementing LB medium with 5-aminolevulinic acid (5-ALA), the first committed intermediate synthesized during heme synthesis. Increasing expression of <i>gtrA</i>, which encodes the enzyme that catalyzes the first step in heme biosynthesis, also restores heme levels and exponential phase growth of the <i>hfq</i> mutant. Taken together, our data indicate that reduced heme levels are responsible for the exponential growth defect of the <i>S. oneidensis hfq</i> mutant in LB medium and suggest that the <i>S. oneidensis hfq</i> mutant is deficient in heme production at the 5-ALA synthesis step.</p></div

The <i>hfq</i> mutant is deficient in heme production.

<p>(A) Comparison of exponentially-growing MR-1 wild type and <i>hfqΔ</i> mutant cell pellet pigmentation. Both cell pellets are comprised of similar numbers of cells. (B) Superimposed heme assay subtraction spectra (reduced – oxidized) from single samples of exponentially-growing MR-1 wild type and <i>hfqΔ</i> mutant cultures. Data presented is typical of the difference observed for the two strains. (C) Quantification of heme concentrations from MR-1 wild type and <i>hfqΔ</i> mutant cultures. Concentration of detectable heme was computed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109879#s4" target="_blank">Materials and Methods</a>. Data presented is the mean of three independent cultures. Error bars indicate standard deviations. *** indicate that the difference in heme levels between MR-1 and the <i>hfqΔ</i> mutant is statistically significant (P<0.001 in an unpaired two-tailed Student's t-test).</p

Restoring heme biosynthesis rescues the exponential phase growth defect of the <i>hfq</i> mutant.

<p>(A) Growth curves for MR-1/pBBAD-SP (MR-1) and <i>hfqΔ</i>/pBBAD-SP (<i>hfqΔ</i>) grown in either LB Km liquid medium or in LB Km liquid medium supplemented with 50 µM 5-ALA. (B) Heme content analysis of cells from MR-1/pBBAD-SP (MR-1) and <i>hfqΔ</i>/pBBAD-SP (<i>hfqΔ</i>) cultures grown for either 4 hours or 8 hours in either LB Km liquid medium or in LB Km liquid medium supplemented with 50 µM 5-ALA. (C) Growth curves for the wild type MR-1 strain and <i>hfqΔ</i> mutant strains containing either pBBAD-SP (vector/ev) or p<i>gtrA</i> grown in either LB Km or in LB Km containing 0.005% arabinose. (D) Heme content analysis of MR-1 and <i>hfqΔ</i> mutant cells containing either pBBAD-SP (vector/ev) or p<i>gtrA</i> grown in either LB Km or in LB Km containing 0.005% arabinose. Results presented for both growth curve and heme assays are the means from three independent cultures. Error bars in panels (A) and (C) indicate a 99% confidence interval (P = 0.01). Error bars in panels (B) and (D) indicate standard deviations. ** indicates that the difference between heme levels at 4 hours between MR1 and the <i>hfqΔ</i> mutant are statistically significant (P<0.0025 in an unpaired two-tailed Student's t-test).</p

Heme or 5-ALA supplementation substantially rescues the colony size phenotype of the <i>hfq</i> mutant.

<p>Colony size comparisons of MR-1/pBBR1-MCS2 (vector), and <i>hfqΔ</i>/pBBR1-MCS2 (vector) streaked to single colonies on (A) LB Km, (B) TSA containing 5% sheep blood [including higher magnification insets for colony size comparison: (B′) MR-1/pBBR1-MCS2 (vector) colonies and (B″) <i>hfqΔ</i>/pBBR1-MCS2 (vector) colonies], (C) LB Km supplemented with 50 µM FeCl₃, (E) LB Km supplemented with 50 µM hemin, or (F) LB Km supplemented with 50 µM 5-aminolevulinic acid (5-ALA). Plates were photographed following 23–25 hours of growth at 30°C. (D) Quantification of total free iron in wild type MR-1 and <i>hfqΔ</i> mutant cells using the ferrozine reagent. Concentration of detectable free iron was computed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109879#s4" target="_blank">Materials and Methods</a>. Data presented is the mean of three independent cultures. Error bars indicate standard deviations. The difference between iron levels in MR-1 and the <i>hfqΔ</i> mutant is not statistically significant (P = 0.22 in an unpaired two-tailed Student's t-test).</p