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Research Article

Barriers to 3-Hydroxypropionate-Dependent Growth of Rhodobacter sphaeroides by Distinct Disruptions of the Ethylmalonyl Coenzyme A Pathway

Steven J. Carlson, Angela Fleig, M. Kelsey Baron, Ivan A. Berg, Birgit E. Alber
William W. Metcalf, Editor
Steven J. Carlson
Department of Microbiology, Ohio State University, Columbus, Ohio, USA
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Angela Fleig
Department of Microbiology, Ohio State University, Columbus, Ohio, USAInstitut für Biologie II, Mikrobiologie, Albert Ludwigs Universität Freiburg, Freiburg im Breisgau, Germany
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M. Kelsey Baron
Department of Microbiology, Ohio State University, Columbus, Ohio, USA
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Ivan A. Berg
Institut für Biologie II, Mikrobiologie, Albert Ludwigs Universität Freiburg, Freiburg im Breisgau, Germany
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Birgit E. Alber
Department of Microbiology, Ohio State University, Columbus, Ohio, USA
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William W. Metcalf
University of Illinois at Urbana-Champaign
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DOI: 10.1128/JB.00556-18
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ABSTRACT

Rhodobacter sphaeroides is able to use 3-hydroxypropionate as the sole carbon source through the reductive conversion of 3-hydroxypropionate to propionyl coenzyme A (propionyl-CoA). The ethylmalonyl-CoA pathway is not required in this process because a crotonyl-CoA carboxylase/reductase (Ccr)-negative mutant still grew with 3-hydroxypropionate. Much to our surprise, a mutant defective for another specific enzyme of the ethylmalonyl-CoA pathway, mesaconyl-CoA hydratase (Mch), lost its ability for 3-hydroxypropionate-dependent growth. Interestingly, the Mch-deficient mutant was rescued either by introducing an additional ccr in-frame deletion that resulted in the blockage of an earlier step in the pathway or by heterologously expressing a gene encoding a thioesterase (YciA) that can act on several CoA intermediates of the ethylmalonyl-CoA pathway. The mch mutant expressing yciA metabolized only less than half of the 3-hydroxypropionate supplied, and over 50% of that carbon was recovered in the spent medium as free acids of the key intermediates mesaconyl-CoA and methylsuccinyl-CoA. A gradual increase in growth inhibition due to the blockage of consecutive steps of the ethylmalonyl-CoA pathway by gene deletions suggests that the growth defects were due to the titration of free CoA and depletion of the CoA pool in the cell rather than to detrimental effects arising from the accumulation of a specific metabolite. Recovery of carbon in mesaconate for the wild-type strain expressing yciA demonstrated that carbon flux through the ethylmalonyl-CoA pathway occurs during 3-hydroxypropionate-dependent growth. A possible role of the ethylmalonyl-CoA pathway is proposed that functions outside its known role in providing tricarboxylic acid intermediates during acetyl-CoA assimilation.

IMPORTANCE Mutant analysis is an important tool utilized in metabolic studies to understand which role a particular pathway might have under certain growth conditions for a given organism. The importance of the enzyme and of the pathway in which it participates is discretely linked to the resulting phenotype observed after mutation of the corresponding gene. This work highlights the possibility of incorrectly interpreting mutant growth results that are based on studying a single unit (gene and encoded enzyme) of a metabolic pathway rather than the pathway in its entirety. This work also hints at the possibility of using an enzyme as a drug target although the enzyme may participate in a nonessential pathway and still be detrimental to the cell when inhibited.

FOOTNOTES

    • Received 9 September 2018.
    • Accepted 5 November 2018.
    • Accepted manuscript posted online 19 November 2018.
  • Supplemental material for this article may be found at https://doi.org/10.1128/JB.00556-18.

  • Copyright © 2019 American Society for Microbiology.

All Rights Reserved.

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Barriers to 3-Hydroxypropionate-Dependent Growth of Rhodobacter sphaeroides by Distinct Disruptions of the Ethylmalonyl Coenzyme A Pathway
Steven J. Carlson, Angela Fleig, M. Kelsey Baron, Ivan A. Berg, Birgit E. Alber
Journal of Bacteriology Jan 2019, 201 (4) e00556-18; DOI: 10.1128/JB.00556-18

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Barriers to 3-Hydroxypropionate-Dependent Growth of Rhodobacter sphaeroides by Distinct Disruptions of the Ethylmalonyl Coenzyme A Pathway
Steven J. Carlson, Angela Fleig, M. Kelsey Baron, Ivan A. Berg, Birgit E. Alber
Journal of Bacteriology Jan 2019, 201 (4) e00556-18; DOI: 10.1128/JB.00556-18
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KEYWORDS

Rhodobacter sphaeroides
carbon balance
ethylmalonyl-CoA
mesaconate
redox balance

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