Phenotypic alteration and target gene identification using combinatorial libraries of zinc finger proteins in prokaryotic cells

Abstract

We have developed a method with prokaryotic organisms that uses randomized libraries of zinc finger-containing artificial transcription factors to induce phenotypic variations and to identify genes involved in the generation of a specific phenotype of interest. Combining chromatin immunoprecipitation experiments and in silico prediction of target DNA binding sequences for the artificial transcription factors, we identified ubiX, whose down-regulation correlates with the thermotolerance phenotype in Escherichia coli. Our results show that randomized libraries of artificial transcription factors are powerful tools for functional genomic studies.

FIG. 1.

Phenotypic changes in E. coli induced by artificial ZFPs. (A) Thermotolerant phenotype induced by ZFPs. Selected clones (T-1 to T-10) were cultured for 2 h at 37°C (no treatment) or 50°C (heat shock) in LB containing 1 mM IPTG (isopropyl-β-d-thiogalactopyranoside) and then plated on LB plates. C, E. coli cells transformed with control plasmid pZL1; T, E. coli cells transformed with thermotolerance-inducing ZFPs. (B) Site-directed mutagenesis of the T9 ZFP. The arginine residue in the QTHR1 zinc finger was mutated to alanine, and the ability of the resulting mutated ZFP (designated T9-M) to induce thermotolerance in E. coli was tested. The phenotypic changes were confirmed by plasmid rescue, sequence analysis, and retransformation of E. coli. The triangles drawn above each panel indicate 10-fold serial dilutions (1:1 to 1:10,000, left to right) of spotted cells.

FIG. 2.

Identification of the target gene regulated by T9 that gave rise to the thermotolerance phenotype. (a) Effect of T9 expression on the concentration of ubiX RNA. For the analysis of UbiX gene expression, cDNA synthesis was performed on RNA with a UbiX-R primer (5′-CTG GAA AGA ACC GGA AGA GAT GCT G-3′). Real-time reverse transcription-PCR was performed by using a light cycler (Corbett Research) with the UbiX-F (5′-TGA AAC GAC TCA TTG TAG GCA TCA G-3′) and UbiX-R primer set. The concentration of RNA encoding glyceraldehyde-3-phosphate dehydrogenase was used as an internal control. C, E. coli cells transformed with pZL1; T9, T9 ZFP transformants. (b) Interaction of T9 with potential binding sites located in the ubiX promoter. ubiX, ubiX knockout mutant; T9, T9 ZFP transformants; Ab, antibody. (c) Disruption of the ubiX gene in E. coli and analysis of thermotolerance. E. coli strain DY330 [W3110 DlacU169 gal490 lcI857 D (cro-bioA)] was used for gene disruption by homologous recombination. Gene knockout was performed by targeted homologous recombination as described previously (12). The position of a potential T9-binding site relative to the translation start site is indicated. Binding of the T9 ZFP to this site was confirmed by immunoprecipitation. In contrast, the T9-M protein was unable to bind to this site. In panel c, the triangles drawn above each panel indicate 10-fold serial dilutions (1:1 to 1:10,000, left to right) of spotted cells. ubiX, ubiX knockout mutant; T9, T9 ZFP transformants; C, E. coli cells transformed with pZL1.