The Bacillus subtilis ywkA gene encodes a malic enzyme and its transcription is activated by the YufL/YufM two-component system in response to malate.
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.
Doan
Thierry
T
Génétique Moléculaire et Cellulaire, INRA (UMR216) CNRS (URA1925) and INAP-G, F-78850 Thiverval-Grignon, France.
Servant
Pascale
P
Génétique Moléculaire et Cellulaire, INRA (UMR216) CNRS (URA1925) and INAP-G, F-78850 Thiverval-Grignon, France.
Tojo
Shigeo
S
Department of Biotechnology, Fukuyama University, 985 Sanzo, Higashimura, Fukuyama, Japan.
Yamaguchi
Hirotake
H
Department of Biotechnology, Fukuyama University, 985 Sanzo, Higashimura, Fukuyama, Japan.
Lerondel
Guillaume
G
Génétique Moléculaire et Cellulaire, INRA (UMR216) CNRS (URA1925) and INAP-G, F-78850 Thiverval-Grignon, France.
Yoshida
Ken-Ichi
KI
Department of Biotechnology, Fukuyama University, 985 Sanzo, Higashimura, Fukuyama, Japan.
Fujita
Yasutaro
Y
Department of Biotechnology, Fukuyama University, 985 Sanzo, Higashimura, Fukuyama, Japan.
Aymerich
Stéphane
S
Génétique Moléculaire et Cellulaire, INRA (UMR216) CNRS (URA1925) and INAP-G, F-78850 Thiverval-Grignon, France.
D001412:Bacillus subtilis / Q000201:enzymology / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial / Q000187:drug effects*; D005809:Genes, Regulator; D008293:Malates / Q000494:pharmacology*; D011401:Promoter Regions, Genetic; D014158:Transcription, Genetic / Q000187:drug effects*
D008293:Malates; C030298:malic acid