Two types of glutamyl‐tRNA synthetase exist: the discriminating enzyme (D‐GluRS) forms only Glu‐tRNAGlu, while the non‐discriminating one (ND‐GluRS) also synthesizes Glu‐tRNAGln, a required intermediate in protein synthesis in many organisms (but not in Escherichia coli). Testing the capacity to complement a thermosensitive E. coli gltX mutant and to suppress an E. coli trpA49 missense mutant we examined the properties of heterologous gltX genes. We demonstrate that while Acidithiobacillus ferrooxidans GluRS1 and Bacillus subtilis Q373R GluRS form Glu‐tRNAGlu, A. ferrooxidans and Helicobacter pylori GluRS2 form Glu‐tRNAGln in E. coli in vivo.