Regulation of L-lactate utilization by the FadR-type regulator L1dR of Corynebacterium glutamicum
Regulation of L-lactate utilization by the FadR-type regulator L1dR of Corynebacterium glutamicum
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DOI：
10.1128/jb.01147-07
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发表时间：
2008-02-01
影响因子：
3.2
通讯作者：
Wendisch, Volker F.
中科院分区：
文献类型：
--
作者：
Georgi, Tobias;Engels, Verena;Wendisch, Volker F.
谷氨酸棒杆菌可以在L-乳酸盐作为唯一碳源和能源上生长。NCg 12816-lldD操纵子编码推定的转运蛋白（NCg 12816）和醌依赖性L-乳酸脱氢酶（LldD）是L-乳酸利用所必需的。DNA亲和层析显示FadR型调节子LldR（由NCg 12814编码）与NCg 12816-lldD的上游区域结合。lldR的过表达导致NCg 12816-lldD mRNA水平的强烈降低和LldD活性的强烈降低，因此，在以L-乳酸盐作为唯一碳源和能源生长的细胞中观察到严重的生长缺陷，但在以葡萄糖、核糖或乙酸盐生长的细胞中未观察到。缺失lldR对这些碳源上的生长没有影响，但在存在和不存在L-乳酸的情况下导致高NCg 12816-lldD mRNA水平和高LldD活性。纯化的His标记的LldR与NCg 12816-lldD启动子的54-bp片段结合，该片段与通过随机扩增cDNA末端-PCR确定的转录起始位点重叠，并含有FadR型调节子典型的推定操纵子基序，其为（-1）TNGTNNACNA（10）。突变分析表明，这个基序与连字符的二分体对称性是必不可少的结合LldD的NCg 12816-lldD启动子。L-乳酸盐，而不是D-乳酸盐，干扰LldR Hi与NCg 12816-lldD启动子的结合。因此，在缺乏L-乳酸的培养基上生长期间，LldR抑制NCg 12816-lldD的表达。在生长培养基中存在L-乳酸或在导致细胞内L-乳酸积累的条件下，诱导L-乳酸利用操纵子。
Corynebacterium glutamicum can grow on L-lactate as a sole carbon and energy source. The NCg12816-lldD operon encoding a putative transporter (NCg12816) and a quinone-dependent L-lactate dehydrogenase (LldD) is required for L-lactate utilization. DNA affinity chromatography revealed that the FadR-type regulator LldR (encoded by NCg12814) binds to the upstream region of NCg12816-lldD. Overexpression of lldR resulted in strongly reduced NCg12816-lldD mRNA levels and strongly reduced LldD activity, and as a consequence, a severe growth defect was observed in cells grown on L-lactate as the sole carbon and energy source, but not in cells grown on glucose, ribose, or acetate. Deletion of lldR had no effect on growth on these carbon sources but resulted in high NCg12816-lldD mRNA levels and high LldD activity in the presence and absence Of L-lactate. Purified His-tagged LldR bound to a 54-bp fragment of the NCg12816-lldD promoter, which overlaps with the transcriptional start site determined by random amplification of cDNA ends-PCR and contains a putative operator motif typical of FadR-type regulators, which is (-1)TNGTNNNACNA(10). Mutational analysis revealed that this motif with hyphenated dyad symmetry is essential for binding of LldD to the NCg12816-lldD promoter. L-Lactate, but not D-lactate, interfered with binding of LldR Hi, to the NCg12816-lldD promoter. Thus, during growth on media lacking L-lactate, LldR represses expression of NCg12816-lldD. In the presence Of L-lactate in the growth medium or under conditions leading to intracellular L-lactate accumulation, the L-lactate utilization operon is induced.