Lactate racemization as a rescue pathway for supplying D-lactate to the cell wall biosynthesis machinery in Lactobacillus plantarum
Lactate racemization as a rescue pathway for supplying D-lactate to the cell wall biosynthesis machinery in Lactobacillus plantarum
复制标题
DOI：
10.1128/jb.187.19.6750-6761.2005
复制
发表时间：
2005-10-01
影响因子：
3.2
通讯作者：
Hols, P
中科院分区：
文献类型：
--
作者：
Goffin, P;Deghorain, M;Hols, P
植物乳杆菌是一种乳酸菌，利用立体特异性 NAD 依赖性乳酸脱氢酶（分别为 LdhD 和 LdhL）产生 D-和 L-乳酸。然而，LdhD 减少糖酵解丙酮酸并不是 D-乳酸产生的唯一途径，因为这种活性缺陷的突变体仍然产生两种乳酸异构体（T. Ferain, J. N. Hobbs, Jr., J. Richardson, N. Bernard, D. Garmyn, P. Hols, N. E. Allen, and J. Delcour, J. Bacteriol. 178:5431-5437， 1996）。该物种中 D-乳酸的产生已被证明与细胞壁生物合成有关，因为它作为胞壁酰五缩酚肽肽聚糖前体的最后一个残基掺入。这一特殊特征导致对高浓度万古霉素的天然耐药性。在本研究中，我们表明植物乳杆菌具有两种 D-乳酸生产途径：LdhD 酶和乳酸消旋酶，其表达需要 L-乳酸。我们报道了一个六基因操纵子的克隆，该操纵子参与乳酸外消旋化活性，并受到 L-乳酸的正向调节。在缺乏LdhD活性的植物乳杆菌菌株中删除该操纵子导致L-乳酸的排他性产生。因此，肽聚糖生物合成受到影响，并且该突变体的生长依赖于 D-乳酸。我们还表明，通过表达乳酸乳球菌的 D-丙氨酰-D-丙氨酸形成 Ddl 连接酶，或通过补充各种 D-2-羟基酸但不补充 D-2-氨基酸，可以部分恢复生长缺陷，从而导致不同的万古霉素耐药水平。这表明植物乳杆菌无法有效合成以 D-丙氨酸结尾的肽聚糖前体，并且该物种的细胞壁生物合成机制专门用于生产以 D-乳酸结尾的肽聚糖前体。在这种情况下，乳酸消旋酶可以为细菌提供在 LdhD 酶失活或抑制后生产 D-乳酸的拯救途径。
Lactobacillus plantarum is a lactic acid bacterium that produces D- and L-lactate using stereospecific NAD-dependent lactate dehydrogenases (LdhD and LdhL, respectively). However, reduction of glycolytic pyruvate by LdhD is not the only pathway for D-lactate production since a mutant defective in this activity still produces both lactate isomers (T. Ferain, J. N. Hobbs, Jr., J. Richardson, N. Bernard, D. Garmyn, P. Hols, N. E. Allen, and J. Delcour, J. Bacteriol. 178:5431-5437, 1996). Production Of D-lactate in this species has been shown to be connected to cell wall biosynthesis through its incorporation as the last residue of the muramoyl-penta-depsipeptide peptidoglycan precursor. This particular feature leads to natural resistance to high concentrations of vancomycin. In the present study, we show that L. plantarum possesses two pathways for D-lactate production: the LdhD enzyme and a lactate racemase, whose expression requires L-lactate. We report the cloning of a six-gene operon, which is involved in lactate racemization activity and is positively regulated by L-lactate. Deletion of this operon in an L. plantarum strain that is devoid of LdhD activity leads to the exclusive production Of L-lactate. As a consequence, peptidoglycan biosynthesis is affected, and growth of this mutant is D-lactate dependent. We also show that the growth defect can be partially restored by expression of the D-alanyl-D-alanine-forming Ddl ligase from Lactococcus lactis, or by supplementation with various D-2-hydroxy acids but not D-2-amino acids, leading to variable vancomycin resistance levels. This suggests that L. plantarum is unable to efficiently synthesize peptidoglycan precursors ending in D-alanine and that the cell wall biosynthesis machinery in this species is specifically dedicated to the production of peptidoglycan precursors ending in D-lactate. In this context, the lactate racemase could thus provide the bacterium with a rescue pathway for D-lactate production upon inactivation or inhibition of the LdhD enzyme.