Krom B P, Aardema R, Lolkema J S
Department of Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Haren, The Netherlands.
J Bacteriol. 2001 Oct;183(20):5862-9. doi: 10.1128/JB.183.20.5862-5869.2001.
The genome of Bacillus subtilis contains two genes that code for membrane proteins that belong to the 2-hydroxycarboxylate transporter family. Here we report the functional characterization of one of the two, yxkJ, which codes for a transporter protein named CimHbs. The gene was cloned and expressed in Escherichia coli and complemented the citrate-negative phenotype of wild-type E. coli and the malate-negative phenotype of the E. coli strain JRG4008, which is defective in malate uptake. Subsequent uptake studies in whole cells expressing CimHbs clearly demonstrated the citrate and malate transport activity of the protein. Immunoblot analysis showed that CimHbs is a 48-kDa protein that is well expressed in E. coli. Studies with right-side-out membrane vesicles demonstrated that CimHbs is an electroneutral proton-solute symporter. No indications were found for the involvement of Na(+) ions in the transport process. Inhibition of the uptake catalyzed by CimHbs by divalent metal ions, together with the lack of effect on transport by the chelator EDTA, showed that CimHbs translocates the free citrate and malate anions. Among a large set of substrates tested, only malate, citramalate, and citrate competitively inhibited citrate transport catalyzed by CimHbs. The transporter is strictly stereoselective, recognizing only the S enantiomers of malate and citramalate. Remarkably, though citramalate binds to the transporter, it is not translocated.
枯草芽孢杆菌的基因组包含两个编码膜蛋白的基因，这些膜蛋白属于2-羟基羧酸盐转运蛋白家族。在此，我们报告了其中一个基因yxkJ的功能特性，该基因编码一种名为CimHbs的转运蛋白。该基因被克隆并在大肠杆菌中表达，它弥补了野生型大肠杆菌的柠檬酸盐阴性表型以及大肠杆菌菌株JRG4008的苹果酸盐阴性表型，后者在苹果酸盐摄取方面存在缺陷。随后在表达CimHbs的全细胞中进行的摄取研究清楚地证明了该蛋白的柠檬酸盐和苹果酸盐转运活性。免疫印迹分析表明，CimHbs是一种48 kDa的蛋白，在大肠杆菌中表达良好。对外翻膜囊泡的研究表明，CimHbs是一种电中性质子-溶质同向转运体。未发现Na(+)离子参与转运过程的迹象。二价金属离子对CimHbs催化的摄取有抑制作用，同时螯合剂EDTA对转运无影响，这表明CimHbs转运游离的柠檬酸盐和苹果酸盐阴离子。在测试的大量底物中，只有苹果酸盐、柠苹酸和柠檬酸盐竞争性抑制CimHbs催化的柠檬酸盐转运。该转运体具有严格的立体选择性，只识别苹果酸盐和柠苹酸的S对映体。值得注意的是，尽管柠苹酸与转运体结合，但它不会被转运。
