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Reduced heme levels underlie the exponential growth defect of the Shewanella oneidensis hfq mutant.

Brennan CM, Mazzucca NQ, Mezoian T, Hunt TM, Keane ML, Leonard JN, Scola SE, Beer EN, Perdue S, Pellock BJ - PLoS ONE (2014)

Bottom Line: We have found that the exponential phase growth defect of the hfq mutant in LB is the result of reduced heme levels.Increasing expression of gtrA, which encodes the enzyme that catalyzes the first step in heme biosynthesis, also restores heme levels and exponential phase growth of the hfq mutant.Taken together, our data indicate that reduced heme levels are responsible for the exponential growth defect of the S. oneidensis hfq mutant in LB medium and suggest that the S. oneidensis hfq mutant is deficient in heme production at the 5-ALA synthesis step.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Providence College, Providence, Rhode Island, United States of America.

ABSTRACT
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 Shewanella oneidensis 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 hfq mutant in LB is the result of reduced heme levels. Both heme levels and exponential phase growth of the hfq 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 gtrA, which encodes the enzyme that catalyzes the first step in heme biosynthesis, also restores heme levels and exponential phase growth of the hfq mutant. Taken together, our data indicate that reduced heme levels are responsible for the exponential growth defect of the S. oneidensis hfq mutant in LB medium and suggest that the S. oneidensis hfq mutant is deficient in heme production at the 5-ALA synthesis step.

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Restoring heme biosynthesis rescues the exponential phase growth defect of the hfq mutant.(A) Growth curves for MR-1/pBBAD-SP (MR-1) and hfqΔ/pBBAD-SP (hfqΔ) 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 hfqΔ/pBBAD-SP (hfqΔ) 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 hfqΔ mutant strains containing either pBBAD-SP (vector/ev) or pgtrA grown in either LB Km or in LB Km containing 0.005% arabinose. (D) Heme content analysis of MR-1 and hfqΔ mutant cells containing either pBBAD-SP (vector/ev) or pgtrA 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 hfqΔ mutant are statistically significant (P<0.0025 in an unpaired two-tailed Student's t-test).
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pone-0109879-g004: Restoring heme biosynthesis rescues the exponential phase growth defect of the hfq mutant.(A) Growth curves for MR-1/pBBAD-SP (MR-1) and hfqΔ/pBBAD-SP (hfqΔ) 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 hfqΔ/pBBAD-SP (hfqΔ) 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 hfqΔ mutant strains containing either pBBAD-SP (vector/ev) or pgtrA grown in either LB Km or in LB Km containing 0.005% arabinose. (D) Heme content analysis of MR-1 and hfqΔ mutant cells containing either pBBAD-SP (vector/ev) or pgtrA 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 hfqΔ mutant are statistically significant (P<0.0025 in an unpaired two-tailed Student's t-test).

Mentions: Addition of 50 µM 5-ALA to LB liquid medium completely rescued the exponential phase growth defect of the hfq mutant (Figure 4A) and restored heme levels in the hfq mutant to wild type levels (Figure 4B). However, by 7–8 hours of culture growth, when heme levels in the unsupplemented hfq mutant were indistinguishable from those in wild type cells (Figure 4B and 4D), absorbance values for the hfq mutant plus 5-ALA stopped increasing and were no longer coincident with the MR-1 cultures (Figure 4A). The terminal density of stationary phase hfq mutant cultures was significantly increased by addition of 5-ALA, but these cultures never achieved the terminal density of wild type cultures (Figure 4A). This suggests that factors independent of heme levels are at least partially responsible for the reduced terminal density of the hfq mutant.


Reduced heme levels underlie the exponential growth defect of the Shewanella oneidensis hfq mutant.

Brennan CM, Mazzucca NQ, Mezoian T, Hunt TM, Keane ML, Leonard JN, Scola SE, Beer EN, Perdue S, Pellock BJ - PLoS ONE (2014)

Restoring heme biosynthesis rescues the exponential phase growth defect of the hfq mutant.(A) Growth curves for MR-1/pBBAD-SP (MR-1) and hfqΔ/pBBAD-SP (hfqΔ) 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 hfqΔ/pBBAD-SP (hfqΔ) 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 hfqΔ mutant strains containing either pBBAD-SP (vector/ev) or pgtrA grown in either LB Km or in LB Km containing 0.005% arabinose. (D) Heme content analysis of MR-1 and hfqΔ mutant cells containing either pBBAD-SP (vector/ev) or pgtrA 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 hfqΔ mutant are statistically significant (P<0.0025 in an unpaired two-tailed Student's t-test).
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4214671&req=5

pone-0109879-g004: Restoring heme biosynthesis rescues the exponential phase growth defect of the hfq mutant.(A) Growth curves for MR-1/pBBAD-SP (MR-1) and hfqΔ/pBBAD-SP (hfqΔ) 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 hfqΔ/pBBAD-SP (hfqΔ) 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 hfqΔ mutant strains containing either pBBAD-SP (vector/ev) or pgtrA grown in either LB Km or in LB Km containing 0.005% arabinose. (D) Heme content analysis of MR-1 and hfqΔ mutant cells containing either pBBAD-SP (vector/ev) or pgtrA 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 hfqΔ mutant are statistically significant (P<0.0025 in an unpaired two-tailed Student's t-test).
Mentions: Addition of 50 µM 5-ALA to LB liquid medium completely rescued the exponential phase growth defect of the hfq mutant (Figure 4A) and restored heme levels in the hfq mutant to wild type levels (Figure 4B). However, by 7–8 hours of culture growth, when heme levels in the unsupplemented hfq mutant were indistinguishable from those in wild type cells (Figure 4B and 4D), absorbance values for the hfq mutant plus 5-ALA stopped increasing and were no longer coincident with the MR-1 cultures (Figure 4A). The terminal density of stationary phase hfq mutant cultures was significantly increased by addition of 5-ALA, but these cultures never achieved the terminal density of wild type cultures (Figure 4A). This suggests that factors independent of heme levels are at least partially responsible for the reduced terminal density of the hfq mutant.

Bottom Line: We have found that the exponential phase growth defect of the hfq mutant in LB is the result of reduced heme levels.Increasing expression of gtrA, which encodes the enzyme that catalyzes the first step in heme biosynthesis, also restores heme levels and exponential phase growth of the hfq mutant.Taken together, our data indicate that reduced heme levels are responsible for the exponential growth defect of the S. oneidensis hfq mutant in LB medium and suggest that the S. oneidensis hfq mutant is deficient in heme production at the 5-ALA synthesis step.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Providence College, Providence, Rhode Island, United States of America.

ABSTRACT
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 Shewanella oneidensis 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 hfq mutant in LB is the result of reduced heme levels. Both heme levels and exponential phase growth of the hfq 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 gtrA, which encodes the enzyme that catalyzes the first step in heme biosynthesis, also restores heme levels and exponential phase growth of the hfq mutant. Taken together, our data indicate that reduced heme levels are responsible for the exponential growth defect of the S. oneidensis hfq mutant in LB medium and suggest that the S. oneidensis hfq mutant is deficient in heme production at the 5-ALA synthesis step.

Show MeSH
Related in: MedlinePlus