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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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Exogenous gtrA expression rescues the colony size defect of the hfq mutant.(A) Putative two step pathway for the biosynthesis of 5-aminolevulinic acid in Shewanella oneidensis MR-1. (B and C) Colony size comparisons of MR-1/pBBAD-SP (vector), MR-1/pBBAD-gtrA (pgtrA), hfqΔ/pBBAD-SP (vector), and hfqΔ/pBBAD-gtrA (pgtrA), streaked to single colonies on (B) LB Km and (C) LB Km containing 0.005% arabinose. (D and E) Colony size comparisons of MR-1/pBBAD-SP (vector), MR-1/pBBAD-phemL (phemL), hfqΔ/pBBAD-SP (vector), and hfqΔ/pBBAD-phemL (phemL), on (D) LB Km and (E) LB Km containing 0.005% arabinose. Plates were photographed following 24 hours of growth at 30°C.
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pone-0109879-g003: Exogenous gtrA expression rescues the colony size defect of the hfq mutant.(A) Putative two step pathway for the biosynthesis of 5-aminolevulinic acid in Shewanella oneidensis MR-1. (B and C) Colony size comparisons of MR-1/pBBAD-SP (vector), MR-1/pBBAD-gtrA (pgtrA), hfqΔ/pBBAD-SP (vector), and hfqΔ/pBBAD-gtrA (pgtrA), streaked to single colonies on (B) LB Km and (C) LB Km containing 0.005% arabinose. (D and E) Colony size comparisons of MR-1/pBBAD-SP (vector), MR-1/pBBAD-phemL (phemL), hfqΔ/pBBAD-SP (vector), and hfqΔ/pBBAD-phemL (phemL), on (D) LB Km and (E) LB Km containing 0.005% arabinose. Plates were photographed following 24 hours of growth at 30°C.

Mentions: That addition of 5-ALA to the growth medium substantially rescued the small colony phenotype of the heme deficient hfq mutant suggests that a deficiency in heme production might be the result of a defect in 5-ALA synthesis. In S. oneidensis, 5-ALA is synthesized in a putative two-step process involving the genes gtrA and hemL (Figure 3A). GtrA protein converts glutamate donated by glutamyl tRNAGlu into glutamate-1-semialdehyde, which is then converted into 5-ALA by the HemL protein. Since 5-ALA is used exclusively for heme production, we hypothesized that the heme biosynthesis defect of the hfq mutant is due to reduced function of the gtrA gene, the hemL gene, or both.


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)

Exogenous gtrA expression rescues the colony size defect of the hfq mutant.(A) Putative two step pathway for the biosynthesis of 5-aminolevulinic acid in Shewanella oneidensis MR-1. (B and C) Colony size comparisons of MR-1/pBBAD-SP (vector), MR-1/pBBAD-gtrA (pgtrA), hfqΔ/pBBAD-SP (vector), and hfqΔ/pBBAD-gtrA (pgtrA), streaked to single colonies on (B) LB Km and (C) LB Km containing 0.005% arabinose. (D and E) Colony size comparisons of MR-1/pBBAD-SP (vector), MR-1/pBBAD-phemL (phemL), hfqΔ/pBBAD-SP (vector), and hfqΔ/pBBAD-phemL (phemL), on (D) LB Km and (E) LB Km containing 0.005% arabinose. Plates were photographed following 24 hours of growth at 30°C.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0109879-g003: Exogenous gtrA expression rescues the colony size defect of the hfq mutant.(A) Putative two step pathway for the biosynthesis of 5-aminolevulinic acid in Shewanella oneidensis MR-1. (B and C) Colony size comparisons of MR-1/pBBAD-SP (vector), MR-1/pBBAD-gtrA (pgtrA), hfqΔ/pBBAD-SP (vector), and hfqΔ/pBBAD-gtrA (pgtrA), streaked to single colonies on (B) LB Km and (C) LB Km containing 0.005% arabinose. (D and E) Colony size comparisons of MR-1/pBBAD-SP (vector), MR-1/pBBAD-phemL (phemL), hfqΔ/pBBAD-SP (vector), and hfqΔ/pBBAD-phemL (phemL), on (D) LB Km and (E) LB Km containing 0.005% arabinose. Plates were photographed following 24 hours of growth at 30°C.
Mentions: That addition of 5-ALA to the growth medium substantially rescued the small colony phenotype of the heme deficient hfq mutant suggests that a deficiency in heme production might be the result of a defect in 5-ALA synthesis. In S. oneidensis, 5-ALA is synthesized in a putative two-step process involving the genes gtrA and hemL (Figure 3A). GtrA protein converts glutamate donated by glutamyl tRNAGlu into glutamate-1-semialdehyde, which is then converted into 5-ALA by the HemL protein. Since 5-ALA is used exclusively for heme production, we hypothesized that the heme biosynthesis defect of the hfq mutant is due to reduced function of the gtrA gene, the hemL gene, or both.

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