Substrate-dependent regulation of anaerobic degradation pathways for toluene and ethylbenzene in a denitrifying bacterium, strain EbN1
Substrate-dependent regulation of anaerobic degradation pathways for toluene and ethylbenzene in a denitrifying bacterium, strain EbN1
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DOI：
10.1128/jb.187.4.1493-1503.2005
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发表时间：
2005-02-01
影响因子：
3.2
通讯作者：
Rabus, R
中科院分区：
文献类型：
--
作者：
Kühner, S;Wöhlbrand, L;Rabus, R
甲苯和甲苯的厌氧生物降解分别因其毒性和新颖的反应而受到环境和生物化学的关注。该菌株EbN 1是唯一的厌氧降解烷基苯通过不同的途径，在苯甲酰辅酶A收敛。这两种途径中涉及的基因的组织只是最近确定的菌株EbN 1。在本研究中，全球表达分析（DNA微阵列和蛋白质组学）表明参与了几个迄今未知的蛋白质降解烷基苯。例如，orf 68和orf 57，框架ebd操纵子，涉及在甲苯降解，和ebA 1932和ebA 1936基因，位于7.2 kb上游的bbs操纵子，涉及在甲苯降解。此外，现在可以在完整途径的水平上进行表达研究。生长实验表明，降解能力甲苯和三氯甲烷可以同时诱导，无论基板用于适应。在RNA（实时逆转录-PCR和DNA微阵列）和蛋白质（二维差异凝胶电泳）水平上，通过使用适应于苯甲酸盐、甲苯、甲苯或甲苯和甲苯的混合物厌氧生长的细胞来研究调节。在甲苯适应细胞中特异性地诱导两个甲苯相关操纵子（bbs和toluene-related operons）的表达。与此相反，基因参与厌氧降解的诱导在甲苯和甲苯适应的细胞，表明甲苯可能作为一个无偿的诱导剂。在协议的预测顺序调节的β-内酰胺酶途径，Ebd蛋白（编码亚基β-内酰胺酶脱氢酶）中形成的β-内酰胺酶，但不是在苯乙酮适应细胞，而猿蛋白（亚基预测苯乙酮羧化酶）在这两种条件下形成。
Anaerobic biodegradation of toluene and ethylbenzene is of environmental concern and biochemical interest due to toxicity and novel reactions, respectively. The denitrifying strain EbN1 is unique in anaerobically degrading both alkylbenzenes via different pathways which converge at benzoyl coenzyme A. The organization of genes involved in both pathways was only recently determined for strain EbN1. In the present study, global expression analysis (DNA microarray and proteomics) indicated involvement of several thus-far-unknown proteins in the degradation of both alkylbenzenes. For example, orf68 and orf57, framing the ebd operon, are implicated in ethylbenzene degradation, and the ebA1932 and ebA1936 genes, located 7.2 kb upstream of the bbs operon, are implicated in toluene degradation. In addition, expression studies were now possible on the level of the complete pathways. Growth experiments demonstrated that degradative capacities for toluene and ethylbenzene could be simultaneously induced, regardless of the substrate used for adaptation. Regulation was studied at the RNA (real-time reverse transcription-PCR and DNA microarray) and protein (two-dimensional-difference gel electrophoresis) level by using cells adapted to anaerobic growth with benzoate, toluene, ethylbenzene, or a mixture of toluene and ethylbenzene. Expression of the two toluene-related operons (bss and bbs) was specifically induced in toluene-adapted cells. In contrast, genes involved in anaerobic ethylbenzene degradation were induced in ethylbenzene- and toluene-adapted cells, suggesting that toluene may act as a gratuitous inducer. In agreement with the predicted sequential regulation of the ethylbenzene pathway, Ebd proteins (encoding subunits of ethylbenzene dehydrogenase) were formed in ethylbenzene- but not in acetophenone-adapted cells, while Ape proteins (subunits of predicted acetophenone carboxylase) were formed under both conditions.