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Genetics and Molecular Biology |
Department of Cell Biology, Graduate school of Biological Sciences, Nara Institute of Science and Technology, 8916-5, Takayama, Ikoma, Nara 630-0101, Japan1
Author for correspondence: Naotake Ogasawara. Tel: +743 72 5430. Fax: +743 72 5439. e-mail: nogasawa{at}bs.aist-nara.ac.jp
Transport systems for C4-dicarboxylates, such as malate, fumarate and succinate, are poorly understood in Gram-positive bacteria. The whole genome sequence of Bacillus subtilis revealed two genes, ydbE and ydbH, whose deduced products are highly homologous to binding proteins and transporters for C4-dicarboxylates in Gram-negative bacteria. Between ydbE and ydbH, genes ydbF and ydbG encoding a sensor–regulator pair, were located. Inactivation of each one of the ydbEFGH genes caused a deficiency in utilization of fumarate or succinate but not of malate. Expression of ydbH, encoding a putative transporter, was stimulated in a minimal salt medium containing 0·05% yeast extract but repressed by the addition of malate to the medium. Inactivation of the putative sensor–regulator pair or solute-binding protein, ydbFG or ydbE, caused complete loss of ydbH expression. The utilization of fumarate and stimulation of ydbH expression resumed in a ydbE null mutant in which ydbFGH were overproduced. Based on these observations, together with analysis of the sequence similarities of the deduced product, we conclude that YdbH is a C4-dicarboxylate-transport protein and its expression is regulated by a C4-dicarboxylate sensor kinase–regulator pair, YdbF and YdbG. Furthermore, it is suggested that YdbE does not directly participate in transport of C4-dicarboxylates, but plays a sensory role in the ydbF–ydbG two-component system, giving rise to specificity or increased efficiency to the system. Deletion analysis of the promoter region of ydbH revealed that a direct repeat sequence was required for the activation of ydbH expression. A catabolite-responsive element (CRE) was also found in the -10 region of the promoter, suggesting negative regulation by a CRE-binding protein.
Keywords: C4-dicarboxylate, Bacillus subtilis, two-component regulatory system, transporter, solute-binding protein
Abbreviations: Dct, dicarboxylic acid transport
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