Gaines G L, Smith L, Neidle E L
Isogenetics, Inc., Chicago Technology Park, Illinois 60612, USA.
J Bacteriol. 1996 Dec;178(23):6833-41. doi: 10.1128/jb.178.23.6833-6841.1996.
Novel nuclear magnetic resonance spectroscopy techniques, designated metabolic observation, were used to study aromatic compound degradation by the soil bacterium Acinetobacter calcoaceticus. Bacteria which had been rendered spectroscopically invisible by growth with deuterated (2H) medium were used to inoculate cultures in which natural-abundance 1H hydrogen isotopes were provided solely by aromatic carbon sources in an otherwise 2H medium. Samples taken during the incubation of these cultures were analyzed by proton nuclear magnetic resonance spectroscopy, and proton signals were correlated with the corresponding aromatic compounds or their metabolic descendants. This approach allowed the identification and quantitation of metabolites which accumulated during growth. This in vivo metabolic monitoring facilitated studies of catabolism in the presence of multiple carbon sources, a topic about which relatively little is known. A. calcoaceticus initiates aromatic compound dissimilation by forming catechol or protocatechuate from a variety of substrates. Degradation proceeds via the beta-ketoadipate pathway, comprising two discrete branches that convert catechol or protocatechuate to tricarboxylic acid cycle intermediates. As shown below, when provided with several carbon sources simultaneously, all degraded via the beta-ketoadipate pathway, A. calcoaceticus preferentially degraded specific compounds. For example, benzoate, degraded via the catechol branch, was consumed in preference to p-hydroxybenzoate, degraded via the protocatechuate branch, when both compounds were present. To determine if this preference were governed by metabolites unique to catechol degradation, pathway mutants were constructed. Studies of these mutants indicated that the product of catechol ring cleavage, cis,cis-muconate, inhibited the utilization of p-hydroxybenzoate in the presence of benzoate. The accumulation of high levels of cis,cis-muconate also appeared to be toxic to the cells.
一种名为代谢观察的新型核磁共振光谱技术被用于研究土壤细菌醋酸钙不动杆菌对芳香化合物的降解。通过在含氘（2H）培养基中生长而在光谱上不可见的细菌被用于接种培养物，在这种培养物中，天然丰度的1H氢同位素仅由芳香碳源提供，而培养基的其他成分均为2H。对这些培养物培养期间采集的样品进行质子核磁共振光谱分析，质子信号与相应的芳香化合物或其代谢产物相关联。这种方法能够鉴定和定量生长过程中积累的代谢物。这种体内代谢监测有助于研究在多种碳源存在下的分解代谢，而关于这个主题人们了解得相对较少。醋酸钙不动杆菌通过从多种底物形成儿茶酚或原儿茶酸来启动芳香化合物的异化作用。降解通过β-酮己二酸途径进行，该途径包括两个离散的分支，将儿茶酚或原儿茶酸转化为三羧酸循环中间体。如下所示，当同时提供几种碳源且均通过β-酮己二酸途径降解时，醋酸钙不动杆菌优先降解特定化合物。例如，当两种化合物都存在时，通过儿茶酚分支降解的苯甲酸比通过原儿茶酸分支降解的对羟基苯甲酸优先被消耗。为了确定这种偏好是否受儿茶酚降解特有的代谢物控制，构建了途径突变体。对这些突变体的研究表明，儿茶酚环裂解产物顺，顺-粘康酸在苯甲酸存在下抑制对羟基苯甲酸的利用。高水平顺，顺-粘康酸的积累似乎对细胞也有毒性。
