Anfora Andrew T, Welch Rodney A
Department of Medical Microbiology & Immunology, University of Wisconsin-Madison, 1300 University Ave., Room 481 MSC, Madison, WI 53706, USA.
J Bacteriol. 2006 Sep;188(18):6622-8. doi: 10.1128/JB.00634-06.
d-Serine is an amino acid present in mammalian urine that is inhibitory to Escherichia coli strains lacking a functional dsdA gene. Counterintuitively, a dsdA strain of E. coli clinical isolate CFT073 hypercolonizes the bladder and kidneys of mice relative to wild type during a coinfection in the murine model of urinary tract infection. We are interested in the mechanisms for uptake of d-serine in CFT073. d-Serine enters E. coli K-12 via CycA, the d-alanine transporter and d-cycloserine sensitivity locus. CFT073 cycA can grow on minimal medium with d-serine as a sole carbon source. The dsdX gene of the dsdCXA locus is a likely candidate for an additional d-serine transporter based on its predicted amino acid sequence similarity to gluconate transporters. In minimal medium, CFT073 dsdX can grow on d-serine as a sole carbon source; however, CFT073 dsdX cycA cannot. Additionally, CFT073 dsdXA cycA is not sensitive to inhibitory concentrations of d-serine during growth on glycerol and d-serine minimal medium. d-[(14)C]serine uptake experiments with CFT073 dsdX cycA harboring dsdX or cycA recombinant plasmids confirm that d-serine is able to enter E. coli cells via CycA or DsdX. In whole-cell d-[(14)C]serine uptake experiments, DsdX has an apparent K(m) of 58.75 microM and a V(max) of 75.96 nmol/min/mg, and CycA has an apparent K(m) of 82.40 microM and a V(max) of 58.90 nmol/min/mg. Only d-threonine marginally inhibits DsdX-mediated d-serine transport, whereas d-alanine, glycine, and d-cycloserine inhibit CycA-mediated d-serine transport. DsdX or CycA is sufficient to transport physiological quantities of d-serine, but DsdX is a d-serine-specific permease.
D-丝氨酸是一种存在于哺乳动物尿液中的氨基酸，对缺乏功能性dsdA基因的大肠杆菌菌株具有抑制作用。与直觉相反，在尿路感染的小鼠模型中，大肠杆菌临床分离株CFT073的dsdA菌株在与野生型共感染期间，相对于野生型，会在小鼠的膀胱和肾脏中过度定殖。我们对CFT073中D-丝氨酸的摄取机制感兴趣。D-丝氨酸通过CycA进入大肠杆菌K-12，CycA是D-丙氨酸转运蛋白和D-环丝氨酸敏感位点。CFT073 cycA可以在以D-丝氨酸作为唯一碳源的基本培养基上生长。基于dsdCXA位点的dsdX基因与葡萄糖酸盐转运蛋白预测的氨基酸序列相似性，它可能是另一种D-丝氨酸转运蛋白的候选基因。在基本培养基中，CFT073 dsdX可以在以D-丝氨酸作为唯一碳源的条件下生长；然而，CFT073 dsdX cycA则不能。此外，CFT073 dsdXA cycA在甘油和D-丝氨酸基本培养基上生长时，对抑制浓度的D-丝氨酸不敏感。用携带dsdX或cycA重组质粒的CFT073 dsdX cycA进行的D-[(14)C]丝氨酸摄取实验证实，D-丝氨酸能够通过CycA或DsdX进入大肠杆菌细胞。在全细胞D-[(14)C]丝氨酸摄取实验中，DsdX的表观K(m)为58.75微摩尔，V(max)为75.96纳摩尔/分钟/毫克，CycA的表观K(m)为82.40微摩尔，V(max)为58.90纳摩尔/分钟/毫克。只有D-苏氨酸对DsdX介导的D-丝氨酸转运有轻微抑制作用，而D-丙氨酸、甘氨酸和D-环丝氨酸抑制CycA介导的D-丝氨酸转运。DsdX或CycA足以转运生理量的D-丝氨酸，但DsdX是一种D-丝氨酸特异性通透酶。
