Development of a system for discovery of genetic interactions for essential genes in Escherichia coli K-12.
Genetic interaction networks are especially useful for functional assignment of genes and gaining new insights into the systems-level organization of the cell. While studying interactions of nonessential genes can be relatively straight-forward via use of deletion mutants, different approaches must be used to reveal interactions of essential genes due to their indispensability. One method shown to be useful for revealing interactions of essential genes requires tagging the query protein. However, this approach can be complicated by mutational effects of potential hypomorphic alleles. Here, we describe a pilot study for a new scheme of systematically studying the interactions of essential genes. Our method uses a low-copy, F-based, complementing plasmid, pFE604T, from which the essential gene is conditionally expressed. The essential gene is expressed at lower levels, producing a moderate growth defect in a query host. Secondary mutations are introduced into the query host by conjugation and the resultant exconjugants are scored for growth by imaging them over time. We report results from studying five essential query genes: dnaN, ftsW, trmD, yrfF and yjgP, showing (on average) interactions with nearly 80 nonessential genes. This system should prove useful for genome-wide analyses of other essential genes in E. coli K-12.
Graduate School of Biological Science, Nara Institute of Science and Technology.
Han Tek
HT
Yong
Natsuko
N
Yamamoto
Rikiya
R
Takeuchi
Yi-Ju
YJ
Hsieh
Tom M
TM
Conrad
Kirill A
KA
Datsenko
Toru
T
Nakayashiki
Barry L
BL
Wanner
Hirotada
H
Mori
D003227:Conjugation, Genetic; D004843:Epistasis, Genetic; D048168:Escherichia coli K12 / Q000235:genetics*; D029968:Escherichia coli Proteins / Q000235:genetics*; D005144:F Factor; D015964:Gene Expression Regulation, Bacterial; D055786:Gene Knockout Techniques; D053263:Gene Regulatory Networks; D005798:Genes, Bacterial; D020043:Genes, Essential; D016680:Genome, Bacterial; D009154:Mutation; D020411:Oligonucleotide Array Sequence Analysis; D015203:Reproducibility of Results
D029968:Escherichia coli Proteins