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(R)-Cysteate-nitrogen assimilation by Cupriavidus necator H16 with excretion of 3-sulfolactate: a patchwork pathway

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Abstract

Cupriavidus necator H16 grew exponentially with (R)-cysteate, a structural analogue of aspartate, as sole source of nitrogen in succinate-salts medium. Utilization of cysteate was quantitative and concomitant with growth and with the excretion of the deaminated product (R)-sulfolactate, which was identified thoroughly. The deaminative pathway started with transport of (R)-cysteate into the cell, which we attributed to an aspartate transporter. Transamination to sulfopyruvate involved an aspartate/(R)-cysteate:2-oxoglutarate aminotransferase (Aoa/Coa) and regeneration of the amino group acceptor by NADP+-coupled glutamate dehydrogenase. Reduction of sulfopyruvate to (R)-sulfolactate was catalyzed by a (S)-malate/(R)-sulfolactate dehydrogenase (Mdh/Sdh). Excretion of the sulfolactate could be attributed to the sulfite/organosulfonate exporter TauE, which was co-encoded and co-expressed, with sulfoacetaldehyde acetyltransferase (Xsc), though Xsc was irrelevant to the current pathway. The metabolic enzymes could be assayed biochemically. Aoa/Coa and Mdh/Sdh were highly enriched by protein separation, partly characterized, and the relevant locus-tags identified by peptide-mass fingerprinting. Finally, RT-PCR was used to confirm the transcription of all appropriate genes. We thus demonstrated that Cupriavidus necator H16 uses a patchwork pathway by recruitment of ‘housekeeping’ genes and sulfoacetaldehyde-degradative genes to scavenge for (R)-cysteate-nitrogen.

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Abbreviations

RT-PCR:

Reverse-transcription PCR

SDS-PAGE:

Sodium dodecylsulfate polyacrylamide gel electrophoresis

MALDI-TOF:

Matrix-assisted laser desorption time of flight (mass spectrometry)

Mdh/Sdh:

(S)-malate/(R)-sulfolactate dehydrogenase

Aoa/Coa:

Aspartate/(R)-cysteate:2-oxoglutarate aminotransferase

Gdh:

Glutamate dehydrogenase

TauE:

Sulfite/organosulfonate exporter

Xsc:

Sulfoacetaldehyde acetyltransferase

Pta:

Phosphotransacetylase

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Acknowledgments

We are grateful to Alexandra Roth, Janina von Watzdorf, Onur Yücel and Susanne Wörner who generated data during advanced practical courses, and to the late Botho Bowien for kindly providing C. necator H16. The work was funded by the German Research Foundation (DFG: CO 206/7-1 to AMC and Theo H. M. Smits, and SCHL 1936/1-1 to DS) and by the University of Konstanz.

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Authors and Affiliations

  1. Department of Biology, University of Konstanz, 78457, Konstanz, Germany

    Jutta Mayer, Karin Denger, David Schleheck & Alasdair M. Cook

  2. The Konstanz Research School, Chemical Biology, 78457, Konstanz, Germany

    Jutta Mayer

  3. Institute of Biochemical Engineering, University of the Saarland, 66041, Saarbrücken, Germany

    Klaus Hollemeyer

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  1. Jutta Mayer
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Correspondence to Alasdair M. Cook.

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Communicated by Ursula Priefer.

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Mayer, J., Denger, K., Hollemeyer, K. et al. (R)-Cysteate-nitrogen assimilation by Cupriavidus necator H16 with excretion of 3-sulfolactate: a patchwork pathway. Arch Microbiol 194, 949–957 (2012). https://doi.org/10.1007/s00203-012-0825-y

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