Growth inhibition of Escherichia coli during heterologous expression of Bacillus subtilis glutamyl-tRNA synthetase that catalyzes the formation of mischarged glutamyl-tRNA1 Gln.
It is known that Bacillus subtilis glutamyl-tRNA synthetase (GluRS) mischarges E. coli tRNA1 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-tRNA1 Ghn 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 tRNA1 Gln misacylation. However, when amounts of up to 10 mM L-glutamine, the cognate amino acid for acylation of tRNA1 Gln, were added to the growth medium, cell growth was unaffected. Overexpression of the B. subtilis gatCAB gene encoding Glu-tRNAGln 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 GlutRNA1 Gln, and converts it to the cognate Gln-tRNA1 Gln species. B. subtilis GluRS-dependent Glu-tRNA1 Gln formation may cause growth inhibition in the transformed E. coli strain, possibly due to abnormal protein synthesis.
Baick
Ji-Won
JW
Department of Food Science and Technology, Dongguk University, Seoul 100-715, Korea.
Yoon
Jang-Ho
JH
Namgoong
Suk
S
Söll
Dieter
D
Kim
Sung-Il
SI
Eom
Soo-Hyun
SH
Hong
Kwang-Won
KW
D001412:Bacillus subtilis / Q000201:enzymology* / Q000235:genetics; D003001:Cloning, Molecular; D015169:Colony Count, Microbial; D003470:Culture Media / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development* / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D005975:Glutamate-tRNA Ligase / Q000235:genetics* / Q000378:metabolism*; D018698:Glutamic Acid / Q000378:metabolism; D005973:Glutamine / Q000378:metabolism; D007544:Isopropyl Thiogalactoside / Q000378:metabolism; D019884:Nitrogenous Group Transferases / Q000235:genetics / Q000378:metabolism; D014176:Protein Biosynthesis; D012351:RNA, Transfer, Gln / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity
D003470:Culture Media; D012351:RNA, Transfer, Gln; D011994:Recombinant Proteins; D005973:Glutamine; D007544:Isopropyl Thiogalactoside; D018698:Glutamic Acid; C412016:Asp-tRNA(Asn) amidotransferase; D019884:Nitrogenous Group Transferases; D005975:Glutamate-tRNA Ligase