Regulation of Aerobic Succinate Transporter <i>dctA</i> of <i>E. coli</i> by cAMP-CRP, DcuS-DcuR, and EIIA^Glc: Succinate as a Carbon Substrate and Signaling Molecule

Abstract

<b><i>Introduction:</i></b> C4-dicarboxylates (C4-DC) have emerged as significant growth substrates and signaling molecules for various <i>Enterobacteriaceae</i> during their colonization of mammalian hosts. Particularly noteworthy is the essential role of fumarate respiration during colonization of pathogenic bacteria. To investigate the regulation of aerobic C4-DC metabolism, the study explored the transcriptional control of the main aerobic C4-DC transporter, <i>dctA</i>, under different carbohydrate conditions. In addition, mutants related to carbon catabolite repression (CCR) and C4-DC regulation (DcuS-DcuR) were examined to better understand the regulatory integration of aerobic C4-DC metabolism into CCR. For initial insight into posttranslational regulation, the interaction between the aerobic C4-DC transporter DctA and EIIA^Glc from the glucose-specific phosphotransferase system was investigated. <b><i>Methods:</i></b> The expression of <i>dctA</i> was characterized in the presence of various carbohydrates and regulatory mutants affecting CCR. This was accomplished by fusing the <i>dctA</i> promoter (P_<i>dctA</i>) to the <i>lacZ</i> reporter gene. Additionally, the interaction between DctA and EIIA^Glc of the glucose-specific phosphotransferase system was examined in vivo using a bacterial two-hybrid system. <b><i>Results:</i></b> The <i>dctA</i> promoter region contains a class I cAMP-CRP-binding site at position −81.5 and a DcuR-binding site at position −105.5. DcuR, the response regulator of the C4-DC-activated DcuS-DcuR two-component system, and cAMP-CRP stimulate <i>dctA</i> expression. The expression of <i>dctA</i> is subject to the influence of various carbohydrates via cAMP-CRP, which differently modulate cAMP levels. Here we show that EIIA^Glc of the glucose-specific phosphotransferase system strongly interacts with DctA, potentially resulting in the exclusion of C4-DCs when preferred carbon substrates, such as sugars, are present. In contrast to the classical inducer exclusion known for lactose permease LacY, inhibition of C4-DC uptake into the cytoplasm affects only its role as a substrate, but not as an inducer since DcuS detects C4-DCs in the periplasmic space (“substrate exclusion”). The work shows an interplay between cAMP-CRP and the DcuS-DcuR regulatory system for the regulation of <i>dctA</i> at both transcriptional and posttranslational levels. <b><i>Conclusion:</i></b> The study highlights a hierarchical interplay between global (cAMP-CRP) and specific (DcuS-DcuR) regulation of <i>dctA</i> at the transcriptional and posttranslational levels. The integration of global and specific transcriptional regulation of <i>dctA</i>, along with the influence of EIIA^Glc on DctA, fine-tunes C4-DC catabolism in response to the availability of other preferred carbon sources. It attributes DctA a central role in the control of aerobic C4-DC catabolism and suggests a new role to EIIA^Glc on transporters (control of substrate uptake by substrate exclusion).