Heterologous expression and localization of gentisate transporter Ncg12922 from Corynebacterium glutamicum ATCC 13032.
Ralstonia sp. strain U2 metabolizes naphthalene via gentisate (2,5-dihydroxybenzoate) to central metabolites, but it was found unable to utilize gentisate as growth substrate. A putative gentisate transporter encoded by ncg12922 from Corynebacterium glutamicum ATCC 13032 was functionally expressed in Ralstonia sp. strain U2, converting strain U2 to a gentisate utilizer. After ncg12922 was inserted into plasmid pGFPe with green fluorescence protein gene gfp, the expressed fusion protein Ncg12922-GFP could be visualized in the periphery of Escherichia coli cells under confocal microscope, consistent with a cytoplasmic membrane location. In contrast, GFP was ubiquitous in the cytoplasm of E. coli cells carrying pGFPe only. Gentisate 1,2-dioxygenase activity was present in the cell extract from strain U2 induced with gentisate but at a much lower level (one-fifth) than that obtained with salicylate. However, it exhibited a similar level in strain U2 containing Ncg12922 induced either by salicylate or gentisate.
Xu
Ying
Y
Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, China.
Yan
Da-Zhong
DZ
Zhou
Ning-Yi
NY
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D048230:Corynebacterium glutamicum / Q000235:genetics / Q000378:metabolism*; D003593:Cytoplasm / Q000378:metabolism; D049308:Dioxygenases / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005841:Gentisates / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000235:genetics; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism*; D043367:Ralstonia / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012459:Salicylates / Q000378:metabolism; D013379:Substrate Specificity
D001426:Bacterial Proteins; D005841:Gentisates; D026901:Membrane Transport Proteins; D011993:Recombinant Fusion Proteins; D012459:Salicylates; D049452:Green Fluorescent Proteins; D049308:Dioxygenases; C026247:gentisate 1,2-dioxygenase