Abstract

It is known that Bacillus subtilis glutamyl-tRNA synthetase (GluRS) mischarges E. coli tRNA_1^Gln with glutamate in vitro. It has also been established that the expression of B. subtilis GluRS in Escherichia coli results in the death of the host cell. To ascertain whether E. coli growth inhibition caused by B. subtilis GluRS synthesis is a consequence of Glu-tRNA_1^ Gln formation, we constructed an in vivo test system, in which B. subtilis GluRS gene expression is controlled by IPTG. Such a system permits the investigation of factors affecting E. coli growth. Expression of E. coli glutaminyl-tRNA synthetase (GlnRS) also ameliorated growth inhibition, presumably by competitively preventing tRNA_1^Gln misacylation. However, when amounts of up to 10 mM L-glutamine, the cognate amino acid for acylation of tRNA_1^ Gln, were added to the growth medium, cell growth was unaffected. Overexpression of the B. subtilis gatCAB gene encoding GlutRNA^Gln amidotransferase (Glu-AdT) rescued cells from toxic effects caused by the formation of the mischarging GluRS. This result indicates that B. subtilis Glu-AdT recognizes the mischarged E. coli GlutRNA_1^Gln, and converts it to the cognate Gln-tRNA_1^Gln species. B. subtilis GluRS-dependent Glu-tRNA_1^Gln formation may cause growth inhibition in the transformed E. coli strain, possibly due to abnormal protein synthesis.

• Keywords: misaminoacylation; tRNA_1^Gln; glutamyl-tRNA synthetase; Glu-tRNA^Gln amidotransferase