Schoberth S M, Chapman B E, Kuchel P W, Wittig R M, Grotendorst J, Jansen P, DeGraff A A
Institut für Biotechnologie-1, Forschungszentrum Jülich, Germany.
J Bacteriol. 1996 Mar;178(6):1756-61. doi: 10.1128/jb.178.6.1756-1761.1996.
For the first time, unidirectional rate constants of ethanol diffusion through the lipid membrane of a microorganism, the bacterium Zymomonas mobilis, were determined, thus replacing indirect inferences with direct kinetic data. The rate constants k1 (in to out) were 6.8 +/- 0.4s(-1) at 29 degrees C and 2.7 +/- 0.3s(-1) at 20 degrees C. They were determined by using 1H selective nuclear magnetic resonance spin magnetization transfer. The measurements were done on l-ml cell suspensions. No addition of radiotracers, withdrawing of aliquots, physical separation methods, or chemical manipulations were required. Until now, the rate constants of ethanol transport in microorganisms have been unknown because ethanol diffuses through the cytoplasmic membrane too quickly for radiolabel approaches. Net velocities of ethanol exchange were calculated from unidirectional rate constants and cytoplasmic volume, which was also determined with the same nuclear magnetic resonance experiments. The results (i) confirmed that ethanol would not be rate limiting during the conversion of glucose by Z. mobilis and (ii) indicated that ethanol can serve as an in vivo marker of cytoplasmic volume changes. This was verified by monitoring for the first time the changes of both cytoplasmic volume and extracytoplasmic and cytoplasmic concentrations of alpha and beta anomers of D-glucose in cell suspensions of a microorganism. These findings may open up new possibilities for kinetic studies of ethanol and sugar transport in Z. mobilis and other organisms.
首次测定了乙醇通过运动发酵单胞菌脂质膜的单向速率常数，从而用直接动力学数据取代了间接推断。在29℃时，从内向外的速率常数k1为6.8±0.4s⁻¹，在20℃时为2.7±0.3s⁻¹。这些速率常数是通过¹H选择性核磁共振自旋磁化转移测定的。测量是在1ml细胞悬液上进行的。无需添加放射性示踪剂、抽取等分试样、物理分离方法或化学操作。到目前为止，由于乙醇通过细胞质膜扩散太快，无法采用放射性标记方法，所以微生物中乙醇运输的速率常数一直未知。乙醇交换的净速度是根据单向速率常数和细胞质体积计算得出的，细胞质体积也是通过相同的核磁共振实验测定的。结果（i）证实了在运动发酵单胞菌将葡萄糖转化的过程中，乙醇不会成为限速因素；（ii）表明乙醇可作为细胞质体积变化的体内标志物。这一点通过首次监测微生物细胞悬液中细胞质体积以及D - 葡萄糖α和β异头物的胞外和胞内浓度变化得到了验证。这些发现可能为运动发酵单胞菌和其他生物体中乙醇和糖运输的动力学研究开辟新的可能性。
