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Journal of Bacteriology, September 2000, p. 4783-4788, Vol. 182, No. 17
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Synthesis and Posttranslational Regulation of Pyruvate Formate-Lyase in Lactococcus lactis

Claus Rix Melchiorsen,1,* Kirsten Væver Jokumsen,1 John Villadsen,1 Mads G. Johnsen,2 Hans Israelsen,2 and José Arnau2

Department of Biotechnology, Center for Process Biotechnology, Technical University of Denmark, DK-2800 Lyngby,1 and Biotechnological Institute, DK-2970 Hørsholm,2 Denmark

Received 20 December 1999/Accepted 15 June 2000

The enzyme pyruvate formate-lyase (PFL) from Lactococcus lactis was produced in Escherichia coli and purified to obtain anti-PFL antibodies that were shown to be specific for L. lactis PFL. It was demonstrated that activated L. lactis PFL was sensitive to oxygen, as in E. coli, resulting in the cleavage of the PFL polypeptide. The PFL protein level and its in vivo activity and regulation were shown by Western blotting, enzyme-linked immunosorbent assay, and metabolite measurement to be dependent on the growth conditions. The PFL level during anaerobic growth on the slowly fermentable sugar galactose was higher than that on glucose. This shows that variation in the PFL protein level may play an important role in the regulation of metabolic shift from homolactic to mixed-acid product formation, observed during growth on glucose and galactose, respectively. During anaerobic growth in defined medium, complete activation of PFL was observed. Strikingly, although no formate was produced during aerobic growth of L. lactis, PFL protein was indeed detected under these conditions, in which the enzyme is dispensable due to the irreversible inactivation of PFL by oxygen. In contrast, no oxygenolytic cleavage was detected during aerobic growth in complex medium. This observation may be the result of either an effective PFL deactivase activity or the lack of PFL activation. In E. coli, the PFL deactivase activity resides in the multifunctional alcohol dehydrogenase ADHE. It was shown that in L. lactis, ADHE does not participate in the protection of PFL against oxygen under the conditions analyzed. Our results provide evidence for major differences in the mechanisms of posttranslational regulation of PFL activity in E. coli and L. lactis.

* Corresponding author. Mailing address: Center for Process Biotechnology, Department of Biotechnology, Technical University of Denmark, Building 223, DK-2800 Lyngby, Denmark. Phone: (45) 45252669. Fax: (45) 45884148. E-mail: CRM{at}ibt.dtu.dk.

Journal of Bacteriology, September 2000, p. 4783-4788, Vol. 182, No. 17
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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