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-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 (k cat/K m 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.

Metabolic Fluxes during Strong Carbon Catabolite Repression by Malate in Bacillus subtilis

“…2H 2 0, 3 g of KH 2 PO 4 , 1 g of NH 4 Cl, 0.5 g of NaCl. The following components were sterilized separately and then added (per liter of final medium): 1 ml of 0.…”

“…The M9 minimal medium consisted of the following components (per liter): 8.5 g of Na 2 HPO 4 . 2H 2 0, 3 g of KH 2 PO 4 , 1 g of NH 4 Cl, 0.5 g of NaCl.…”

“…Apart from repressing the co-utilization of alternative substrates, preferred carbon sources are normally associated with a high specific growth rate and substantial secretion of overflow metabolites such as ethanol, lactate, or acetate. For the Grampositive model bacterium Bacillus subtilis, glucose has long been recognized to be preferred, but recent data indicated that glucose might not be the only preferred carbon source (4,5).…”

Characterization of an oxaloacetate decarboxylase that belongs to the malic enzyme family

“…In Bacillus subtilis, it is possible to identify four paralogous genes encoding MEs (maeA, malS, ytsJ and mleA) but only ytsJ is required for efficient growth on malate as well as on other TCA intermediates [10].…”

Functional Analysis of the YvrGHb Two-Component System ofBacillus subtilis: Identification of the Regulated Genes by DNA Microarray and Northern Blot Analyses

“…1) Twenty-nine adjacent sets of genes encoding the sensor kinase and response regulator are recognized in Bacillus subtilis, which has one of the best-characterized transcriptional control systems among living organisms. Fourteen regulatory systems, CitST, 2) ComPA, 3) CssSR, 4) DctSR, 5) DegSU, 3) DesKR, 6) PhoRP, 7) ResED, 8) YdfHI, 9) BceRS, 10) YufLM, 11,12) LiaRS, 13) YxdJK, 14) and YycFG, 15) have been reported in detail. In 2001, comprehensive DNA microarray analyses were performed to detect candidates of target genes for the B. subtilis two-component systems.…”