Can assembly factors in Escherichia coli be deleted using DIRex recombineering? : Deconstructing ribosomal assembly in a model organism

Sammanfattning

Bacterial ribosomes are constructed by a diverse group of proteins with varying functions, including assembly factors that attach ribosomal RNA and proteins and fold them into a three-dimensional structure. Most assembly factor genes have yet to be successfully tested through deletion by DIRex, a Lambda-Red based method well suited for deleting genes in the model organism Escherichia coli. To address this, I attempted deletions of a list of 12 assembly factors that previous attempts in this lab had failed to delete using the DIRex method of recombineering by amplifying gene-specific primers and selecting for insertion and excision of an insertable DNA cassette. Successful isolation and genetic sequencing of knockout strains confirmed that deletions of most listed genes were not only possible, the majority succeeded at their first attempt. I was unable to isolate deletions of 3 genes: All mutated colonies of rimJ contained a duplicated wild type rimJ gene, while no mutants of rimP and yhbY grew at all. I concluded that the duplicated rimJ gene lies in a duplication hotspot of the chromosome and that fitness defects may have made isolation of other mutants difficult. To further investigate fitness costs incurred by successful deletions, I also analysed the growth rates of pre-existing and newly created knockout strains at 37 °C and 20 °C. I also transduced deletions onto a strain lacking modifications on its small ribosomal subunit. These strains were also grown at 37 °C and 20 °C. Growth rates of knockout strains and transduced strains varied but were consistently lower than wild type E. coli. Nearly all mutant strains grew more slowly compared to the wild type at 20 °C than at 37 °C. While several transduced strains deviated from their expected combined fitness at both temperatures, I found that ΔSSU + ΔrbfA was the greatest outlier by far with over twice its expected fitness at 20°C. Mutant growth rates at colder temperature may give insight into the importance of assembly factors in protein assembly while the positive epistasis displayed by ΔSSU + ΔrbfA likely occurred due to overlapping defects from both mutant strain, as ΔrbfA targets the small subunit.