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

Abstract

A transcriptome comparison of a wild-typeBacillus subtilisstrain growing under glycolytic or gluconeogenic conditions was performed. In particular, it revealed that theywkAgene, one of the four paralogues putatively encoding a malic enzyme, was more transcribed during gluconeogenesis. Using alacZreporter fusion to theywkApromoter, it was shown thatywkAwas specifically induced by external malate and not subject to glucose catabolite repression. Northern analysis confirmed this expression pattern and demonstrated thatywkAis cotranscribed with the downstreamywkBgene. TheywkAgene product was purified and biochemical studies demonstrated its malic enzyme activity, which was 10-fold higher with NAD than with NADP (kcat/Km102 and 10 s−1 mM−1, respectively). However, physiological tests with single and multiple mutant strains affected inywkAand/or inywkAparalogues showed thatywkAdoes 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 ofywkAexpression. Genetic analysis andin vitrostudies with purified YufM protein showed that YufM binds just upstream ofywkApromoter and activatesywkAtranscription in response to the presence of malate in the extracellular medium, transmitted by YufL.ywkAandyufL/yufMcould thus be renamedmaeAformalicenzyme andmalK/malRformalatekinase sensor/malate responseregulator, respectively.