The glyoxylate shunt is essential for CO2-requiring oligotrophic growth of Rhodococcus erythropolis N9T-4
The glyoxylate shunt is essential for CO2-requiring oligotrophic growth of Rhodococcus erythropolis N9T-4
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DOI：
10.1007/s00253-015-6500-x
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发表时间：
2015-07-01
影响因子：
5
通讯作者：
Takagi, Hiroshi
中科院分区：
文献类型：
--
作者：
Yano, Takanori;Yoshida, Nobuyuki;Takagi, Hiroshi
红平红球菌N9 T-4显示出需要大气CO2的极端贫营养生长，并且在没有任何额外碳源的无机基础培养基（BM）上形成其菌落。我们建立了一个独特的基因组缺失方法构建的随机突变库的筛选表明，aceA，aceB和pckG基因编码异柠檬酸裂解酶，苹果酸合成酶，磷酸烯醇式丙酮酸羧激酶，分别是必需的生存在BM板。aceA和aceB缺失突变体和pckG缺失突变体分别在含有L-苹果酸和D-葡萄糖的BM平板上生长良好，表明乙醛酸（GO）分流和异源生成对于N9 T-4的贫营养生长是必不可少的。有趣的是，在N9 T-4的无细胞提取物中观察到TCA循环中的大多数酶活性，可能最重要的例外是α-酮戊二酸脱氢酶（KGDH）活性。代替KGDH活性，我们检测到显著水平的α-酮戊二酸脱羧酶（KGD）活性，这是结核分枝杆菌中KGDH复合物的E1组分所表现出的活性。N9 T-4的重组KGD以时间依赖性方式催化α-酮戊二酸脱羧形成琥珀酸半醛（SSA）。由于N9 T-4也显示出可检测的SSA脱氢酶活性，我们得出结论，N9 T-4具有变体TCA循环，其使用SSA而不是琥珀酰辅酶A。这些结果表明，贫营养N9 T-4细胞利用GO分流，以避免损失的碳作为CO2和保存CoA单位在TCA循环。
Rhodococcus erythropolis N9T-4 shows extremely oligotrophic growth requiring atmospheric CO2 and forms its colonies on an inorganic basal medium (BM) without any additional carbon source. Screening of a random mutation library constructed by a unique genome deletion method that we established indicated that the aceA, aceB, and pckG genes encoding isocitrate lyase, malate synthase, and phosphoenolpyruvate carboxykinase, respectively, were requisite for survival on BM plates. The aceA-and aceB deletion mutants and the pckG deletion mutant grew well on BM plates containing L-malate and D-glucose, respectively, suggesting that the glyoxylate (GO) shunt and gluconeogenesis are essential for the oligotrophic growth of N9T-4. Interestingly, most of the enzyme activities in the TCA cycle were observed in the cell-free extract of N9T-4, with perhaps the most important exception being alpha-ketoglutarate dehydrogenase (KGDH) activity. Instead of the KGDH activity, we detected a remarkable level of alpha-ketoglutarate decarboxylase (KGD) activity, which is the activity exhibited by the E1 component of the KGDH complex in Mycobacterium tuberculosis. The recombinant KGD of N9T-4 catalyzed the decarboxylation of alpha-ketoglutarate to form succinic semialdehyde (SSA) in a time-dependent manner. Since N9T-4 also showed a detectable SSA dehydrogenase activity, we concluded that N9T-4 possesses a variant TCA cycle, which uses SSA rather than succinyl-CoA. These results suggest that oligotrophic N9T-4 cells utilize the GO shunt to avoid the loss of carbons as CO2 and to conserve CoA units in the TCA cycle.