Isolation of temperature-sensitive aminoacyl-tRNA synthetase mutants from an Escherichia coli strain harboring the pemK plasmid.
The pem locus, which is responsible for the stable maintenance of the low copy number plasmid R100, contains the pemK gene, whose product has been shown to be a growth inhibitor. Here, we attempted to isolate mutants which became tolerant to transient induction of the PemK protein. We obtained 20 mutants (here called pkt for PemK tolerance), of which 9 were temperature sensitive for growth. We analyzed the nine mutants genetically and found that they could be classified into three complementation groups, pktA, pktB and pktC, which corresponded to three genes, ileS, gltX and asnS, encoding isoleucyl-, glutamyl- and asparaginyl-tRNA synthetases, respectively. Since these amino-acyl-tRNA synthetase mutants did not produce the PemK protein upon induction at the restrictive temperature, these mutants could be isolated because they behaved as if they were tolerant to the PemK protein. The procedure is therefore useful for isolating temperature-sensitive mutants of aminoacyl-tRNA synthetases.
Masuda
Y
Y
Institute of Applied Microbiology, University of Tokyo, Japan.
Tsuchimoto
S
S
Nishimura
A
A
Ohtsubo
E
E
D000604:Amino Acyl-tRNA Synthetases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D001226:Aspartate-tRNA Ligase; D001426:Bacterial Proteins / Q000235:genetics*; D002874:Chromosome Mapping; D002876:Chromosomes, Bacterial; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics / Q000302:isolation & purification; D004268:DNA-Binding Proteins / Q000235:genetics*; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics*; D029968:Escherichia coli Proteins; D005798:Genes, Bacterial; D005816:Genetic Complementation Test; D005838:Genotype; D005975:Glutamate-tRNA Ligase / Q000235:genetics; D007533:Isoleucine-tRNA Ligase / Q000235:genetics; D009154:Mutation; D010957:Plasmids; D012346:RNA, Transfer, Amino Acyl; D013696:Temperature; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; C080273:PemK protein, E coli; D012346:RNA, Transfer, Amino Acyl; D001616:beta-Galactosidase; D000604:Amino Acyl-tRNA Synthetases; D001226:Aspartate-tRNA Ligase; D005975:Glutamate-tRNA Ligase; C029194:asparaginyl-tRNA synthetase; D007533:Isoleucine-tRNA Ligase