Transcriptome analysis and anaerobic C-4-dicarboxylate transport in Actinobacillus succinogenes

Abstract

A global transcriptome analysis of the natural succinate producer Actinobacillus succinogenes revealed that 353 genes were differentially expressed when grown on various carbon and energy sources, which were categorized into six functional groups. We then analyzed the expression pattern of 37 potential C-4-dicarboxylate transporters in detail. A total of six transporters were considered potential fumarate transporters: three transporters, Asuc_1999 (Dcu), Asuc_0304 (DASS), and Asuc_0270-0273 (TRAP), were constitutively expressed, whereas three others, Asuc_1568 (DASS), Asuc_1482 (DASS), and Asuc_0142 (Dcu), were differentially expressed during growth on fumarate. Transport assays under anaerobic conditions with [C-14]fumarate and [C-14]succinate were performed to experimentally verify that A.succinogenes possesses multiple C-4-dicarboxlayte transport systems with different substrate affinities. Upon uptake of 5mmol/L fumarate, the systems had substrate specificity for fumarate, oxaloacetate, and malate, but not for succinate. Uptake was optimal at pH 7, and was dependent on both proton and sodium gradients. Asuc_1999 was suspected to be a major C-4-dicarboxylate transporter because of its noticeably high and constitutive expression. An Asuc_1999 deletion (1999) decreased fumarate uptake significantly at approximately 5mmol/L fumarate, which was complemented by the introduction of Asuc_1999. Asuc_1999 expressed in Escherichia coli catalyzed fumarate uptake at a level of 21.6 mu mol.gDW(-1).min(-1). These results suggest that C-4-dicarboxylate transport in A.succinogenes is mediated by multiple transporters, which transport various types and concentrations of C-4-dicarboxylates.