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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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Heme or 5-ALA supplementation substantially rescues the colony size phenotype of the hfq mutant.Colony size comparisons of MR-1/pBBR1-MCS2 (vector), and hfqΔ/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″) hfqΔ/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 hfqΔ mutant cells using the ferrozine reagent. Concentration of detectable free iron was computed as described in Materials and Methods. Data presented is the mean of three independent cultures. Error bars indicate standard deviations. The difference between iron levels in MR-1 and the hfqΔ mutant is not statistically significant (P = 0.22 in an unpaired two-tailed Student's t-test).
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pone-0109879-g001: Heme or 5-ALA supplementation substantially rescues the colony size phenotype of the hfq mutant.Colony size comparisons of MR-1/pBBR1-MCS2 (vector), and hfqΔ/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″) hfqΔ/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 hfqΔ mutant cells using the ferrozine reagent. Concentration of detectable free iron was computed as described in Materials and Methods. Data presented is the mean of three independent cultures. Error bars indicate standard deviations. The difference between iron levels in MR-1 and the hfqΔ mutant is not statistically significant (P = 0.22 in an unpaired two-tailed Student's t-test).

Mentions: Colonies formed by an S. oneidensis hfq mutant on LB plates are substantially smaller than colonies formed by cells containing a wild type copy of hfq (Figure 1A, [10]). Because the hfq mutant is highly sensitive to peroxide stress, we tested trypticase soy agar (TSA) medium containing 5% sheep blood as a potential qualitative measure to assay whether the hfq mutant produces higher levels of reactive oxygen species. Though the hfq mutant did not produce more heme oxidation (α hemolysis) than wild type cells, we were surprised to observe that growth on sheep blood agar substantially rescued the colony size defect of the hfq mutant (Figure 1B). The hfq mutant small colony defect on TSA without blood (Figure S1A) was comparable to the phenotype observed on LB (Figure 1A).


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)

Heme or 5-ALA supplementation substantially rescues the colony size phenotype of the hfq mutant.Colony size comparisons of MR-1/pBBR1-MCS2 (vector), and hfqΔ/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″) hfqΔ/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 hfqΔ mutant cells using the ferrozine reagent. Concentration of detectable free iron was computed as described in Materials and Methods. Data presented is the mean of three independent cultures. Error bars indicate standard deviations. The difference between iron levels in MR-1 and the hfqΔ mutant is not statistically significant (P = 0.22 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-g001: Heme or 5-ALA supplementation substantially rescues the colony size phenotype of the hfq mutant.Colony size comparisons of MR-1/pBBR1-MCS2 (vector), and hfqΔ/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″) hfqΔ/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 hfqΔ mutant cells using the ferrozine reagent. Concentration of detectable free iron was computed as described in Materials and Methods. Data presented is the mean of three independent cultures. Error bars indicate standard deviations. The difference between iron levels in MR-1 and the hfqΔ mutant is not statistically significant (P = 0.22 in an unpaired two-tailed Student's t-test).
Mentions: Colonies formed by an S. oneidensis hfq mutant on LB plates are substantially smaller than colonies formed by cells containing a wild type copy of hfq (Figure 1A, [10]). Because the hfq mutant is highly sensitive to peroxide stress, we tested trypticase soy agar (TSA) medium containing 5% sheep blood as a potential qualitative measure to assay whether the hfq mutant produces higher levels of reactive oxygen species. Though the hfq mutant did not produce more heme oxidation (α hemolysis) than wild type cells, we were surprised to observe that growth on sheep blood agar substantially rescued the colony size defect of the hfq mutant (Figure 1B). The hfq mutant small colony defect on TSA without blood (Figure S1A) was comparable to the phenotype observed on LB (Figure 1A).

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