Department of Microbiology and Immunology, University at Buffalo, State University of New York, Buffalo, New York 14214, USA.
J Bacteriol. 2011 Jul;193(14):3512-24. doi: 10.1128/JB.01410-10. Epub 2011 May 20.
Streptococcus pneumoniae strains lacking the enzyme dihydrolipoamide dehydrogenase (DLDH) show markedly reduced ability to grow on raffinose and stachyose as sole carbon sources. Import of these sugars occurs through the previously characterized raffinose ATP-binding cassette (ABC) transport system, encoded by the raf operon, that lacks the necessary ATP-binding protein. In this study, we identified the raffinose ATP-binding protein RafK and showed that it was directly involved in raffinose and stachyose import. RafK carries a C-terminal regulatory domain present in a subset of ATP-binding proteins that has been involved in both direct regulation of transporter activity (inducer exclusion) and transcription of transporter genes. Pneumococci lacking RafK showed a 50- to 80-fold reduction in expression of the raf operon genes aga (alpha-galactosidase) and rafEFG (raffinose substrate binding and permease genes), and both glucose and sucrose inhibited raffinose uptake through inducer exclusion. Like RafK, the presence of DLDH also activated the expression of raf operon genes, as DLDH-negative pneumococci showed a significantly decreased expression of aga and rafEFG, but DLDH did not regulate rafK or the putative regulatory genes rafR and rafS. DLDH also bound directly to RafK both in vitro and in vivo, indicating the possibility that DLDH regulates raffinose transport by a direct interaction with the regulatory domain of the transporter. Finally, although not as attenuated as DLDH-negative bacteria, pneumococci lacking RafK were significantly outcompeted by wild-type bacteria in colonization experiments of murine lung and nasopharynx, indicating a role for raffinose and stachyose transport in vivo.
缺乏酶二氢乳清酸脱氢酶（DLDH）的肺炎链球菌菌株在以棉子糖和密二糖为唯一碳源的情况下生长能力明显降低。这些糖的摄取是通过先前表征的棉子糖 ATP 结合盒（ABC）转运系统进行的，该系统由 raf 操纵子编码，但缺乏必需的 ATP 结合蛋白。在这项研究中，我们鉴定了棉子糖 ATP 结合蛋白 RafK，并表明它直接参与棉子糖和密二糖的摄取。RafK 携带一个 C 末端调节域，存在于一组 ATP 结合蛋白中，这些蛋白参与了转运体活性的直接调节（诱导物排除）和转运体基因的转录。缺乏 RafK 的肺炎球菌对 raf 操纵子基因 aga（α-半乳糖苷酶）和 rafEFG（棉子糖底物结合和渗透酶基因）的表达降低了 50-80 倍，葡萄糖和蔗糖通过诱导物排除抑制棉子糖摄取。与 RafK 一样，DLDH 的存在也激活了 raf 操纵子基因的表达，因为 DLDH 阴性肺炎球菌 aga 和 rafEFG 的表达显著降低，但 DLDH 不调节 rafK 或假定的调节基因 rafR 和 rafS。DLDH 还在体外和体内直接与 RafK 结合，表明 DLDH 通过与转运体的调节域直接相互作用来调节棉子糖转运的可能性。最后，尽管 RafK 缺失的肺炎球菌没有 DLDH 阴性细菌那么弱，但在小鼠肺和鼻咽定植实验中，RafK 缺失的肺炎球菌明显被野生型细菌竞争淘汰，表明棉子糖和密二糖转运在体内的作用。
