Inactivation and regulation of the aerobic C4-dicarboxylate transport (dctA) gene of Escherichia coli
Inactivation and regulation of the aerobic C4-dicarboxylate transport (dctA) gene of Escherichia coli
复制标题
DOI：
10.1128/jb.181.18.5624-5635.1999
复制
发表时间：
1999-09-01
影响因子：
3.2
通讯作者：
Andrews, SC
中科院分区：
文献类型：
--
作者：
Davies, SJ;Golby, P;Andrews, SC
编码大肠杆菌需氧C-4-二羧酸转运蛋白（DctA）的基因（dctA）先前被定位到连锁图的79分钟区域。该区域的核苷酸序列揭示了dct 4基因的两个候选者：f428在79.3分钟和o 157 a-o 424-o328（或orfQMP）操纵子在79.9分钟。f428基因编码苜蓿中华根瘤菌和豆科根瘤菌H+/C-4-二羧酸同向转运体，DctA的同源物，而orfQMP操纵子编码的好氧周质结合蛋白依赖的C-4-二羧酸转运系统（DctQ，DctM，和DctP）的红细菌capsulatus的同源物。为了确定这些位点中的哪一个（如果有的话）指定E. coli DctA系统中，分别插入Sp（r）和Ap（r）表达盒使染色体f428和orfM基因失活。所得到的f428突变体不能以富马酸或苹果酸作为唯一碳源进行有氧生长，并且以琥珀酸盐生长不良。此外，在f428突变体中，富马酸盐摄取被消除，琥珀酸盐转运比野生型低10倍。f428突变体的生长和富马酸转运缺陷通过用含f428的质粒转化来补充。未发现orfM突变体的生长缺陷。总之，上述发现证实了f428对应于dctA基因，并表明orfQMP产物在C-4-二羧酸转运中不起作用。用dctA-lacZ（f428-lacZ）转录融合的调节研究表明，dctA受到环AMP受体蛋白（CRP）依赖的分解代谢物抑制和ArcA介导的厌氧抑制，并且由DcuS-DcuR系统响应于C-4-二羧酸盐和柠檬酸盐而弱诱导。有趣的是，在dctA突变体中，dctA的表达是组成型的，相对于C-4-二羧酸诱导，这表明DctA调节其自身的合成。北方印迹分析揭示了一个单一的，单顺反子的dctA转录，并证实，dctA是受调节的分解代谢物阻遏和CRP。逆转录酶介导的引物延伸表明一个单一的转录起始位点集中在一个强烈预测的CRP结合位点下游81 bp。
The gene (dctA) encoding the aerobic C-4-dicarboxylate transporter (DctA) of Escherichia coli was previously mapped to the 79-min region of the linkage map. The nucleotide sequence of this region reveals two candidates for the dct4 gene:f428 at 79.3 min and the o157a-o424-o328 (or orfQMP) operon at 79.9 min. The f428 gene encodes a homologue of the Sinorhizobium meliloti and Rhizobium leguminosarum H+/C-4-dicarboxylate symporter, DctA, whereas the orfQMP operon encodes homologues of the aerobic periplasmic-binding protein-dependent C-4-dicarboxylate transport system (DctQ, DctM, and DctP) of Rhodobacter capsulatus. To determine which, if either, of these loci specify the E. coli DctA system, the chromosomal f428 and orfM genes were inactivated by inserting Sp(r) or Ap(r) cassettes, respectively. The resulting f428 mutant was unable to grow aerobically with fumarate or malate as the sole carbon source and grew poorly with succinate, Furthermore, fumarate uptake was abolished in the f428 mutant and succinate transport was similar to 10-fold lower than that of the wild type. The growth and fumarate transport deficiencies of the f428 mutant were complemented by transformation with an f428-containing plasmid. No growth defect was found for the orfM mutant. In combination, the above findings confirm that f428 corresponds to the dctA gene and indicate that the orfQMP products play no role in C-4-dicarboxylate transport. Regulation studies with a dctA-lacZ (f428-lacZ) transcriptional fusion showed that dctA is subject to cyclic AMP receptor protein (CRP)-dependent catabolite repression and ArcA-mediated anaerobic repression and is weakly induced by the DcuS-DcuR system in response to C-4-dicarboxylates and citrate. Interestingly, in a dctA mutant, expression of dctA is constitutive,vith respect to C-4-dicarboxylate induction, suggesting that DctA regulates its own synthesis. Northern blot analysis revealed a single, monocistronic dctA transcript and confirmed that dctA is subject to regulation by catabolite repression and CRP. Reverse transcriptase-mediated primer extension indicated a single transcriptional start site centered 81 bp downstream of a strongly predicted CRP-binding site.