Deletion of Escherichia coli groEL is complemented by a Rhizobium leguminosarum groEL homologue at 37 degrees C but not at 43 degrees C.
Bacterial Cpn60 proteins (homologues to the Escherichia coli GroEL protein) are often examined for function by testing their ability to complement a temperature sensitive mutation in the E. coli groEL gene. Such tests suffer from two drawbacks: the Cpn600 protein may come from a strain with a lower optimum growth temperature than E. coli, and the requirements for successful complementation in E. coli are likely to be more stringent at 43 degrees C than at lower temperatures. Here we describe the construction of a strain of E. coli where the chromosomal gene for the essential molecular chaperone GroEL has been deleted, with GroEL being expressed from a tightly regulated plasmid borne copy of the gene. The deletion can be transduced into strains expressing heterologous Cpn60 proteins, to test for complementation at any temperature. We show that a Cpn60 protein from the bacterium Rhizobium leguminosarum can function to allow E. coli growth at 37 degrees C but not at 43 degrees C. By switching off the plasmid borne groEL gene, the effects of progressive depletion of GroEL protein from E. coli cells can also be monitored at any temperature.
Ivic
A
A
School of Biological Sciences, University of Birmingham, UK.
Olden
D
D
Wallington
E J
EJ
Lund
P A
PA
D018834:Chaperonin 60 / Q000096:biosynthesis / Q000235:genetics*; D002876:Chromosomes, Bacterial; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics* / Q000254:growth & development / Q000648:ultrastructure; D017353:Gene Deletion; D005816:Genetic Complementation Test; D016296:Mutagenesis; D010957:Plasmids; D016133:Polymerase Chain Reaction; D015183:Restriction Mapping; D016961:Rhizobium leguminosarum / Q000235:genetics*; D013696:Temperature
D018834:Chaperonin 60