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Transcriptional analysis of Ralstonia eutropha genes related to poly-(R)-3-hydroxybutyrate homeostasis during batch fermentation

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Abstract

Poly-(R)-3-hydroxybutyrate (PHB) homeostasis in Ralstonia eutropha takes place at the interface of the cytosol and the hydrophobic PHB granule. PHB synthesis and degradation are therefore intimately linked to the process of granule assembly and breakdown. Unraveling this time-dependent three-dimensional process requires an understanding of the kinetics of synthesis of relevant proteins. Reverse transcriptase quantitative PCR and quantitative Western blotting were carried out on batch cultures of R. eutropha H16 in order to gain insight into how expression of the PHB-related genes phaA, phaB, phaC, phaP, phaR, phaZ1a, phaZ1b, and phaZ1c changed during a cell growth phase, a PHB production phase, and a PHB utilization phase. phaA, phaB, phaC, phaR, and phaZ1a were transcribed throughout cell growth, PHB production, and PHB degradation. PHB-mediated induction of PhaP expression was shown to occur at the transcriptional level, with transcript levels increasing during PHB production and decreasing during PHB utilization. Levels of PhaP correlated strongly with levels of PHB. Levels of phaZ1b transcript and protein increased sharply during production and decreased during degradation, but transcript accumulation did not depend on PHB production as in the case of phaP. No evidence of phaZ1c expression was found under the experimental conditions used in this study.

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Acknowledgements

This work was supported by NIH Grant 49171 to JoAnne Stubbe and Anthony J. Sinskey and by NIH grant 5T32GM08334 to Jiamin Tian.

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Authors and Affiliations

  1. Department of Biology, Massachusetts Institute of Technology, Cambridge, MA, 02139, USA

    Adam G. Lawrence, Joerg Schoenheit, JoAnne Stubbe & Anthony J. Sinskey

  2. Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA, 02139, USA

    Aimin He, Jiamin Tian, Pinghua Liu & JoAnne Stubbe

  3. Department of Health Sciences and Technology, Massachusetts Institute of Technology, Cambridge, MA, 02139, USA

    Anthony J. Sinskey

  4. Departments of Biology and Health Sciences and Technology, Massachusetts Institute of Technology, Bldg. 68-370, 77 Massachusetts Ave, Cambridge, MA, 02139, USA

    Anthony J. Sinskey

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  1. Adam G. Lawrence
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  2. Joerg Schoenheit
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  3. Aimin He
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  4. Jiamin Tian
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  5. Pinghua Liu
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  6. JoAnne Stubbe
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  7. Anthony J. Sinskey
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Correspondence to Anthony J. Sinskey.

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Lawrence and Schoenheit made equal contributions to this work

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Lawrence, A.G., Schoenheit, J., He, A. et al. Transcriptional analysis of Ralstonia eutropha genes related to poly-(R)-3-hydroxybutyrate homeostasis during batch fermentation. Appl Microbiol Biotechnol 68, 663–672 (2005). https://doi.org/10.1007/s00253-005-1969-3

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  • DOI: https://doi.org/10.1007/s00253-005-1969-3

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