A Bacteriolytic Effect of UDP-Sugar Hydrolase on Escherichia coli via Its Overproduction Using a Modified ushA Gene Expression System

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We examined whether UDP-sugar hydrolase (USH) can inhibit the growth of Escherichia coli cells via its overproduction using a modified gene expression system. In the plasmid pUSH20 the ushA gene was placed downstream of the pelB leader sequence of pET20b after deletion of the original leader sequence and the adjacent downstream region encoding the N-terminal 15 amino acid residues of the enzyme. The cytotoxic effects of the modified ushA gene product were demonstrated by the ability of pUSH20 to transform only E. coli BL21(DE3)pLysE, in which T7 promoter-directed ushA gene expression is markedly suppressed. A strain of BL21(DE3)pLysE carrying pUSH20 grew normally in Luria-Bertani broth but was lysed, with accompanying elevation of USH activity, on addition of IPTG. The IPTG-dependent elevation of USH activity was consistent with the functional expression of a modified ushA gene in the BL21(DE3)pLysE cells, being accompanied by inhibition of macromolecular synthesis using N-acetylglucosamine as a precursor in addition to a decrease in the level of UDP-N-acetylglucosamine. Such growth inhibition was not observed in the case of overproduction of the enzyme by increasing the copy number of the original ushA gene. These results suggest that USH can induce cell lysis when it is overproduced, resulting in accelerated hydrolysis of intracellular uncleotide sugars.
公益社団法人日本生物工学会の論文
1996-01-25