PAGE 9 OF 10 nucleotide positions 80 and 30. Repressor-binding sites are generally located downstream from nucleotide posi- tion 30. We have also shown that our ATFs can function as both an activator and a repressor as described above (Figure 1B and C). We induced various phenotypic changes in E. coli with our ATFs and the genes associated with the heat-resis- tance phenotype were identiﬁed. We found that the expression of over two hundred genes was signiﬁcantly aﬀected by the T2 ATF that induces a thermotolerance phenotype. RNA for the transcriptomic analysis was iso- lated from exponential-phase cultures to see the biggest diﬀerence in gene expression between the wild-type and the thermotolerant cells. Therefore, the result of the tran- scriptomic analysis presented does not address the diﬀer- ence in the general expression proﬁle, but the gene expression diﬀerence under a speciﬁc condition (4 h after inoculation at 508C) because the wild-type and the thermotolerant cells have very diﬀerent growth rates at 508C. The genes that show the largest increase in gene expression under our experimental condition, including cpxP and ompW, are highly involved in the protection of bacteria against various forms of environmental stres- ses. CpxP is a chaperone involved in resistance to extra- cytoplasmic stress (34,35). This result is consistent with the previous report that many chaperones are heat shock pro- teins, which are expressed in response to elevated tempera- tures or other cellular stresses. It is well known that proper protein folding is disrupted when cells are subjected to heat treatment, and our ﬁndings suggest that CpxP may be involved in protein folding under heat shock condi- tions. OmpW is an outer membrane protein that forms an eight-stranded beta-barrel with a hydrophobic channel (33). The function of OmpW is unknown, but recent data suggest that it may be involved in the protection of bac- teria against various forms of environmental stresses, such as temperature extremes, salinity and availability of nutri- ents or oxygen. We also reason that the overexpression of OmpW might provide structural stability to the cell. It is known that high temperatures lead to an increase in the ﬂuidity of the cellular membranes (37,38). Excessive mem- brane ﬂuidity gives rise to leaky membranes, and, thus a corresponding loss of function of membrane proteins. Therefore, the changes in the E. coli’s plasma membrane through the overexpression of OmpW may be associated with the mutated cell’s ability to withstand elevated temperatures. Overexpression of a single or double gene(s) among the top-candidate genes identiﬁed in the microarray analysis (e.g. cpxP and/or ompW) did not produce a thermotoler- ance phenotype as robust as the phenotype induced by the T2 ATF. It seems that the ability of the ATF to elicit simultaneous modiﬁcation to the expression levels of many genes is critically important for obtaining highly improved thermotolerance phenotypes. For instance, transcriptional proﬁling revealed that T2 ATF represses the marRAB operon, which in turn leads to increased expression of genes such as hdeA and slp that are repressed by products of marRAB (39). HdeA is a chaperon and Slp is an outer membrane lipoprotein and both are involved in resistance to stress conditions (i.e. at low pH) (40) and so Nucleic Acids Research, 2008, Vol. 36, No. 16 e102 are likely to be among the many proteins contributing to the thermotolerance phenotype. The ATF approach that we describe can cause nuanced changes in gene expression. In cases where the expression level of a particular gene is critical to obtaining the desired phenotypes, subtle changes in the gene expression might be necessary to allow cells to achieve them. For instance, the knockout of the marRAB operon failed to impart an improved thermotolerance phenotype, which indicates that the altered level of marRAB expression induced by T2 ATF rather than the complete elimination of these genes is necessary to achieve this phenotype. Additional levels of control over cellular phenotype are also possible by regulating the expression of the ATF. We have observed that our ATF can generate various levels of phe- notypic improvements, such as the diﬀerent degree of thermotolerance, when they were expressed at various levels with diﬀerent expression vectors (Figure 3B). In addition, a conditional induction of a desired phenotype is possible with ATFs under an inducible promoter. We have observed that ATF expressed under an inducible promoter (a tac promoter) can conditionally generate desired phenotypes (data not shown). Moreover, a pheno- typic transfer from one strain to another strain of the same species is straightforward with our approach. The same phenotypic changes were observed when the selected ATFs were expressed in other strains such as E. coli BL21 and XL1-Blue (data not shown). Finally, we have observed that our ATF can generate desired phenotypes more eﬃciently than random mutation. Random muta- genesis approach could generate osmotolerant and cold- tolerant cells, but not to the extent that we have obtained with our ATFs. Indeed, 50/106 thermotolerant cells were obtained under the heat treatment condition (at 558C for 2 h), but cells that are tolerant to heat at 558C could not be obtained by random mutation under our experimental condition. Also, ATFs could generate osmotolerance and cold-tolerance phenotypes (150/106 and 13/106 cells, respectively) more eﬃciently than random mutation (approximately 1/107 cells for each phenotype) under our experimental conditions. Our results and the general applicability of this platform demonstrate that ATFs can be used as a powerful tool for phenotypic engineering and functional genomic analysis. SUPPLEMENTARY DATA Supplementary Data are available at NAR Online. ACKNOWLEDGEMENTS This work was supported in part by grants from 21C Frontier Program of Microbial Genomics and Applica- tions (MG05-0204-1-0), the Molecular & Cellular BioDis- covery Research Program (M1-0106-00-0200) from the Ministry of Science and Technology of Korea, the Korea Science & Engineering Foundation Grant (R01-2005-000- 11010-0), and the Basic Research Program of the Korea Research Foundation Grant (KRF-2004-042-D00072).