Growth inhibition of Escherichia coli during heterologous expression of bacillus sublifis glutamyl-tRNA synthetase that catalyzes the formation of mischarged glutamyl-tRNA (Gln)

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 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. subtitis gatCAB gene encoding Glu-tRNA(1)(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 Glu-tRNA tRNA(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.