Abee T, van der Wal F J, Hellingwerf K J, Konings W N
Department of Microbiology, University of Groningen, Haren, The Netherlands.
J Bacteriol. 1989 Sep;171(9):5148-54. doi: 10.1128/jb.171.9.5148-5154.1989.
The properties of an L-alanine uptake system in Rhodobacter sphaeroides were studied and compared with those of H+/lactose symport in R. sphaeroides 4P1, a strain in which the lactose carrier of Escherichia coli has been cloned and functionally expressed (F. E. Nano, Ph.D. thesis, University of Illinois, Urbana, 1984). Previous studies indicated that both transport systems were active only when electron transfer took place in the respiratory or cyclic electron transfer chain, while uptake of L-alanine also required the presence of K+ (M. G. L. Elferink, Ph.D. thesis, University of Groningen, Groningen, The Netherlands, 1986). The results presented in this paper offer an explanation for these findings. Transport of the nonmetabolizable L-alanine analog 2-alpha-aminoisobutyric acid (AIB) is mediated by a shock-sensitive transport system. The apparently unidirectional uptake of AIB results in accumulation levels which exceed 7 x 10(3). The finding of L-alanine-binding activity in the concentrated crude shock fluid indicates that L-alanine is taken up by a binding-protein-dependent transport system. Transport of the nonmetabolizable lactose analog methyl-beta-D-thiogalactopyranoside (TMG) by the lactose carrier under anaerobic conditions in the dark was observed in cells and membrane vesicles. This indicates that the H+/lactose symport system is active without electron transfer. Uptake of AIB, but not that of TMG, is inhibited by vanadate with a 50% inhibitory concentration of 50 microM, which suggests a role of a phosphorylated intermediate in AIB transport. Uptake of TMG and AIB is regulated by the internal pH. The initial rates of uptake increased with the internal pH, and and pKa values of 7.2 for TMG and 7.8 for AIB. At an internal pH of 7, no AIB uptake occurred, and the rate of TMG uptake was only 30% of the rate at an internal pH of 8. In a previous study, we found that K+ plays an essential role in regulating the internal pH (T. Abee, K. J. Hellingwerf, and W. N. Konings, J. Bacteriol. 170:5647-5653, 1988). The dependence of solute transport in R. sphaeroides on both K+ and activity of an electron transfer chain can be explained by an effect of the internal pH, which subsequently influences the activities of the lactose-and binding-protein-dependent L-alanine transport system.
研究了球形红细菌中L-丙氨酸摄取系统的特性，并将其与球形红细菌4P1中的H⁺/乳糖同向转运系统的特性进行了比较。在球形红细菌4P1中，大肠杆菌的乳糖载体已被克隆并功能性表达（F.E. Nano，博士论文，伊利诺伊大学，厄巴纳，1984年）。先前的研究表明，只有当电子传递在呼吸或循环电子传递链中发生时，这两种转运系统才具有活性，而L-丙氨酸的摄取还需要K⁺的存在（M.G.L. Elferink，博士论文，格罗宁根大学，荷兰格罗宁根，1986年）。本文给出的结果为这些发现提供了解释。不可代谢的L-丙氨酸类似物2-α-氨基异丁酸（AIB）的转运由一种对休克敏感的转运系统介导。AIB明显的单向摄取导致积累水平超过7×10³。在浓缩的粗休克液中发现L-丙氨酸结合活性，这表明L-丙氨酸是通过一种依赖结合蛋白的转运系统摄取的。在细胞和膜泡中观察到，在黑暗厌氧条件下，乳糖载体对不可代谢的乳糖类似物甲基-β-D-硫代吡喃半乳糖苷（TMG）的转运。这表明H⁺/乳糖同向转运系统在没有电子传递的情况下也具有活性。钒酸盐对AIB的摄取有抑制作用，而对TMG的摄取没有抑制作用，其50%抑制浓度为50μM，这表明磷酸化中间体在AIB转运中起作用。TMG和AIB的摄取受内部pH的调节。摄取的初始速率随内部pH升高而增加，TMG的pKa值为7.2，AIB的pKa值为7.8。在内部pH为7时，不发生AIB摄取，TMG的摄取速率仅为内部pH为8时速率的30%。在先前的一项研究中，我们发现K⁺在调节内部pH中起重要作用（T. Abee、K.J. Hellingwerf和W.N. Konings，《细菌学杂志》170:5647 - 5653，1988年）。球形红细菌中溶质转运对K⁺和电子传递链活性的依赖性可以通过内部pH的影响来解释，内部pH随后会影响乳糖和依赖结合蛋白的L-丙氨酸转运系统的活性。
