A simple turbidimetric method for monitoring the inhibition of tRNA-dependent amidotransferase GatCAB.
In eubacteria that lack glutaminyl-tRNA synthetase (GlnRS), a tRNA-dependent amidotransferase (GatCAB) recognizes mischarged Glu-tRNA(Gln) and converts it into Gln-tRNA(Gln). An inhibitor specific for GatCAB could therefore act as an antibiotic with a novel mode of action against multidrug-resistant bacteria such as Staphylococcus strains. However, there is no rapid, simple and efficient screening method for specifically monitoring the inhibition of GatCAB activity. We have focused on developing a simple system for monitoring the inhibition of GatCAB activity using Escherichia coli Top10 co-expressing the ndGluRS and GatCAB genes from Staphylococcus aureus Mu50. First, growth repression was confirmed by introducing ndgluRS from S. aureus Mu50 into E. coli. Then, we verified that co-expression of the gatCAB operon alleviated growth repression in the host E. coli. The screening system consisted of these two transformants and non-expressing E. coli Top10. The transformant harbors both ndGluRS gene and GatCAB operon could be co-expressed in the presence and in the absence of chemical compound of interest. Since there is no inhibitor that inactivates GatCAB activity, we expressed two inactive GatCAB deletion variants, GatCAB(Delta10) and GatCAB(DeltaCHD) together with ndGluRS in E. coli Top10. The expressed E. coli showed repressed growth as well as ndGluRS expressed. These results indicate that if GatCAB activity is inhibited in this co-expressed E. coli, the inhibition can be monitored by the decrease in O.D. of the co-expressed E. coli.
Chatani
Miho
M
Laboratory of Applied Microbiology, Graduate School of Agriculture, Hokkaido University, N9W9, Kita-ku, Sapporo, 060-8589, Japan.
Tanaka
Michiko
M
Nakamura
Akiyoshi
A
Takesue
Nobuchika
N
Tanaka
Isao
I
Asano
Kozo
K
D003001:Cloning, Molecular; D015169:Colony Count, Microbial; D004926:Escherichia coli / Q000187:drug effects* / Q000235:genetics / Q000254:growth & development*; D029968:Escherichia coli Proteins / Q000037:antagonists & inhibitors*; D017353:Gene Deletion; D015870:Gene Expression; D050296:Microbial Viability; D009391:Nephelometry and Turbidimetry / Q000379:methods*; D019884:Nitrogenous Group Transferases / Q000037:antagonists & inhibitors*; D013211:Staphylococcus aureus / Q000201:enzymology* / Q000235:genetics
D029968:Escherichia coli Proteins; D019884:Nitrogenous Group Transferases; C047694:glutamyl-tRNA(Gln) amidotransferase