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Applied and Environmental Microbiology, July 2005, p. 3442-3452, Vol. 71, No. 7
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.7.3442-3452.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Functional Identification of Novel Genes Involved in the Glutathione-Independent Gentisate Pathway in Corynebacterium glutamicum

Xi-Hui Shen, Cheng-Ying Jiang, Yan Huang, Zhi-Pei Liu, and Shuang-Jiang Liu*

State Key Laboratory of Microbial Resources at Institute of Microbiology, Chinese Academy of Sciences, Beijing 100080, People's Republic of China

Received 4 November 2004/ Accepted 11 January 2005

Corynebacterium glutamicum used gentisate and 3-hydroxybenzoate as its sole carbon and energy source for growth. By genome-wide data mining, a gene cluster designated ncg12918-ncg12923 was proposed to encode putative proteins involved in gentisate/3-hydroxybenzoate pathway. Genes encoding gentisate 1,2-dioxygenase (ncg12920) and fumarylpyruvate hydrolase (ncg12919) were identified by cloning and expression of each gene in Escherichia coli. The gene of ncg12918 encoding a hypothetical protein (Ncg12918) was proved to be essential for gentisate-3-hydroxybenzoate assimilation. Mutant strain RES167{Delta}ncg12918 lost the ability to grow on gentisate or 3-hydroxybenzoate, but this ability could be restored in C. glutamicum upon the complementation with pXMJ19-ncg12918. Cloning and expression of this ncg12918 gene in E. coli showed that Ncg12918 is a glutathione-independent maleylpyruvate isomerase. Upstream of ncg12920, the genes ncg12921-ncg12923 were located, which were essential for gentisate and/or 3-hydroxybenzoate catabolism. The Ncg12921 was able to up-regulate gentisate 1,2-dioxygenase, maleylpyruvate isomerase, and fumarylpyruvate hydrolase activities. The genes ncg12922 and ncg12923 were deduced to encode a gentisate transporter protein and a 3-hydroxybenzoate hydroxylase, respectively, and were essential for gentisate or 3-hydroxybenzoate assimilation. Based on the results obtained in this study, a GSH-independent gentisate pathway was proposed, and genes involved in this pathway were identified.

* Corresponding author. Mailing address: Institute of Microbiology, Chinese Academy of Sciences, Beijing 100080, People's Republic of China. Phone: 86-10-62527118. Fax: 86-10-62652317. E-mail: shuangjiang{at}hotmail.com.

Applied and Environmental Microbiology, July 2005, p. 3442-3452, Vol. 71, No. 7
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.7.3442-3452.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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