引言C4-二羧酸酯(C4-DC)在哺乳动物宿主定殖期间作为肠杆菌科的重要生长底物和信号分子出现。本研究探索了主要需氧C4-DC转运体的转录控制,dctA,在不同的碳水化合物条件下。检查了与碳分解代谢物抑制(CCR)和C4-DC调节(DcuS-DcuR)相关的突变体,以更好地了解它们在有氧C4-DC代谢中的调节整合。此外,研究了来自葡萄糖特异性磷酸转移酶系统的需氧C4-DC转运蛋白DctA和EIAGlc之间的相互作用。方法在各种碳水化合物和调节突变体的存在下表征dctA的表达。这是通过将dctA启动子(PdctA)与lacZ报告基因融合来实现的。使用细菌双杂交系统在体内检查了DctA和EIAGlc之间的相互作用。结果dctA启动子区在-81.5位含有I类cAMP-CRP结合位点,在-105.5位含有DcuR结合位点。DcuR,DcuS-DcuR的响应调节器,和cAMP-CRP刺激dctA表达。dctA的表达受多种碳水化合物通过cAMP-CRP的影响,调节cAMP水平。在这里,我们表明EIAGlc与DctA强烈相互作用,当首选碳底物时,可能导致C4-DC的排除,比如糖,是存在的。与乳糖通透酶LacY的经典诱导排斥相反,抑制C4-DC摄取到细胞质中只影响其作为底物的作用,但不能作为诱导剂,因为DcuS检测周质空间中的C4-DC(\'底物排除\')。这项工作显示了cAMP-CRP与DcuS-DcuR调节系统之间的相互作用,以在转录和翻译后水平上调节dctA。结论该研究强调了在转录和翻译后水平上dctA的全局(cAMP-CRP)和特异性(DcuS-DcuR)调节之间的分层相互作用。全球和特异性转录调控的整合,随着EIAGlc对DctA的影响,响应于优选碳源的可用性,微调C4-DC分解代谢。它将DctA归因于控制需氧C4-DC分解代谢的中心作用,并暗示了EIAGlc在转运蛋白上的新作用(通过底物排斥控制底物吸收)。
BACKGROUND: C4-dicarboxylates (C4-DC) have emerged as significant growth substrates and signaling molecules for various Enterobacteriaceae 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, dctA, 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 EIIAGlc from the glucose-specific phosphotransferase system was investigated.
METHODS: The expression of dctA was characterized in the presence of various carbohydrates and regulatory mutants affecting CCR. This was accomplished by fusing the dctA promoter (PdctA) to the lacZ reporter gene. Additionally, the interaction between DctA and EIIAGlc of the glucose-specific phosphotransferase system was examined in vivo using a bacterial two-hybrid system.
RESULTS: The dctA 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 dctA expression. The expression of dctA is subject to the influence of various carbohydrates via cAMP-CRP, which differently modulate cAMP levels. Here we show that EIIAGlc 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 dctA at both transcriptional and posttranslational levels.
CONCLUSIONS: The study highlights a hierarchical interplay between global (cAMP-CRP) and specific (DcuS-DcuR) regulation of dctA at the transcriptional and posttranslational levels. The integration of global and specific transcriptional regulation of dctA, along with the influence of EIIAGlc 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 EIIAGlc on transporters (control of substrate uptake by substrate exclusion).