PAGE 5 OF 10 contained the 3-ZF target sequence downstream of the promoter (+24 to +41) (Figure 1). The ATFs that were constructed by combining the 3-ZF domain with one of various CRP derivatives were shown to eﬃciently regulate transcription of the reporter gene. Among CRP derivatives, CRP D1 showed both strong activation and repression, depending on the position of the 3-ZF target DNA sequence (Figure 1B and C). Therefore, CRP D1 was chosen as an eﬀector domain for the remainder of this study. An ATF that consisted of the 3-ZF domain alone (no eﬀector domain) very poorly either activated or repressed transcription, depending on the position of the 3-ZF target DNA sequences. Construction of ATF libraries ZFs have exquisite sequence speciﬁcity and modularity. Amino acid residues at positions 1, 2, 3 and 6 in the a-helix of a ZF confer its speciﬁcity, recognizing a unique 3-bp of DNA sequence (8,9). We compared the amino acid residues at critical base-contacting positions of ZFs encoded by sequences in the human genome and selected a total of 40 ZFs that specify distinct DNA-bind- ing speciﬁcities (Supplementary Table 2). A library of DNA-binding domains consisting of 3 ZFs each was constructed by combinatorial assembly of the selected 40 ZFs (13,14). This process allowed the prepara- tion of 6.4 104 (= 40 40 40) distinct, 3-ZF DNA- binding domains that targeted selected 9-bp target DNA sequences. These DNA-binding domains were then linked to a potent transcriptional eﬀector domain (CRP D1) to create ATFs (14). Screening for desirable phenotypic changes We introduced the ATF libraries constructed with high- copy plasmid into E. coli and allowed ATFs to be consti- tutively expressed. We then screened the transformed bacteria for speciﬁc phenotypes, such as resistance to heat shock, osmotic pressure and cold shock. The phenotype that we screened for ﬁrst was that of improved thermotolerance (Figure 2A). After heat treat- ment of a pool of 106 ATF transformants at 558C for 2 h, a condition that severely inhibits the growth of wild-type E. coli (17,23), 50 ATF transformants survived. The selected thermotolerant cells not only survived but grew well under the condition of heat treatment (at 558C for 2 h), whereas growth of wild-type cells was completely inhibited under the same condition. In contrast, both wild-type and the selected thermotolerant cells showed similar growth patterns when grown at the optimal growth temperature, 378C, without heat treatment. Second, to screen for cells with improved osmotoler- ance, ATF transformants were grown on a minimal A medium containing 0.6 M NaCl, which almost completely inhibits the growth of wild-type cells (24,25). Of 106 ATF transformants screened, 150 ATF transformants survived on a minimal A medium containing 0.6 M NaCl. The sur- viving cells were then subjected to further screening (Figure 2B). The selected ATF transformants showed an Nucleic Acids Research, 2008, Vol. 36, No. 16 e102 improvement in osmotolerance at the concentration of 0.6 M NaCl. In the absence of osmotic pressure (as encountered in 0.6 M NaCl), both wild-type and the selected osmotolerant cells exhibited similar growth patterns. Finally, we screened for ATFs that induced a cold- tolerance phenotype (26–28) in E. coli (Figure 2C). To screen for cold-tolerant cells, the pool of 106 ATF trans- formants was grown at 158C in LB medium, wherein most wild-type E. coli cells stop their growth. Thirteen of 106 ATF transformants survived at 158C. Only the selected cold-tolerant cells grew well at 158C, even though both the selected cold-tolerant and wild-type cells showed simi- lar growth patterns when grown at the optimal growth temperature, 378C. To conﬁrm that the various phenotypic improvements were indeed caused by the ATFs, the plasmids that encoded the ATFs were rescued from the selected cells and then retransformed into wild-type E. coli. Of 106 wild-type E. coli cells retransformed with each rescued ATF plasmid, 50 transformants were randomly selected and analyzed for their respective phenotypic changes (thermotolerance, osmotolerance and cold-tolerance). The phenotypic changes in all the selected retransformants were the same as those in the primary transformants. The selected ATFs induced only their respective phenotypes and random ATFs did not confer the above phenotypes upon E. coli cells. The identities of the selected ATFs along with their respective phenotypic changes are pre- sented in Supplementary Table 3. Of all the best-growing thermotolerant mutant cells, the T2 thermotolerant cell expressing an ATF on high-copy plasmid (HT2) exhibited the most improved cellular via- bility under the stressful condition (heat treatment at 558C for 2 h). Therefore, the remainder of this study focuses on this particular mutant cell, which expressed the T2 ATF. The T2 ATF was composed of CRP D1 and 3 ZFs (Z13, Z2 and Z25, ordered in the N- to C- terminal direction) (Supplementary Table 3). The putative binding site for the T2 ATF was inferred to be 50-NGA TTC (G/A)CT-30 from the published literature (8,13,29,30). In addition, the T2 ATF generated the diﬀerent degree of thermotolerance, when it was expressed at various levels with diﬀerent expression vectors. The T2 transfor- mants expressing T2 ATF on low-copy plasmid (LT2) were also more heat resistant than wild-type cells. However their heat resistance was less than that of T2 ATF on high-copy plasmid (HT2) under the given heat- stress condition (at 558C for 2 h). This observation sug- gests that various levels of phenotypic changes can be obtained by controlling the level of ATF expression using diﬀerent copy number plasmids. Identification of genes associated with thermotolerance Using cDNA prepared from RNA isolated from the E. coli transformant containing the T2 ATF on low- and high-copy plasmids, we performed genome-scale gene expression proﬁling analyses of the T2 transformant with E. coli DNA microarrays. Transcription proﬁling of the T2 thermotolerant cells revealed that the T2 ATF