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1 Génétique Moléculaire et Cellulaire, INRA (UMR216) CNRS (URA1925) and INAP-G, F-78850 Thiverval-Grignon, France
2 Department of Biotechnology, Fukuyama University, 985 Sanzo, Higashimura, Fukuyama, Japan
Correspondence
Stéphane Aymerich
stef{at}grignon.inra.fr
A transcriptome comparison of a wild-type Bacillus subtilis strain growing under glycolytic or gluconeogenic conditions was performed. In particular, it revealed that the ywkA gene, one of the four paralogues putatively encoding a malic enzyme, was more transcribed during gluconeogenesis. Using a lacZ reporter fusion to the ywkA promoter, it was shown that ywkA was specifically induced by external malate and not subject to glucose catabolite repression. Northern analysis confirmed this expression pattern and demonstrated that ywkA is cotranscribed with the downstream ywkB gene. The ywkA gene product was purified and biochemical studies demonstrated its malic enzyme activity, which was 10-fold higher with NAD than with NADP (kcat/Km 102 and 10 s-1 mM-1, respectively). However, physiological tests with single and multiple mutant strains affected in ywkA and/or in ywkA paralogues showed that ywkA does not contribute to efficient utilization of malate for growth. Transposon mutagenesis allowed the identification of the uncharacterized YufL/YufM two-component system as being responsible for the control of ywkA expression. Genetic analysis and in vitro studies with purified YufM protein showed that YufM binds just upstream of ywkA promoter and activates ywkA transcription in response to the presence of malate in the extracellular medium, transmitted by YufL. ywkA and yufL/yufM could thus be renamed maeA for malic enzyme and malK/malR for malate kinase sensor/malate response regulator, respectively.
The online version of this paper (at http://mic.sgmjournals.org) contains a table listing the oligonucleotides used in this study and a figure showing the overproduction and purification of His6-YufM.
Present address: Institut de Génétique et Microbiologie, Université Paris-Sud, Bât. 409, F-91405 Orsay, France.
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