PAGE 7 OF 10 caused diﬀerential expression of hundreds of genes relative to the wild-type cells containing a control plasmid without the ATF. Speciﬁcally, a total of 202 genes in the transfor- mant with HT2 were diﬀerentially expressed by more than 4-fold compared to the control (133 up-regulated and 69 down-regulated) (Supplementary Tables 4 and 5). Similarly, 107 genes were altered in the transformant with LT2 (61 up-regulated and 46 down-regulated) (Supplemen- tary Tables 6 and 7). Among these genes, 45 genes were altered in a similar fashion in both HT2 and LT2 (25 up-regulated and 20 down-regulated). These results suggest that the T2 ATF is aﬀecting a subset of important genes responsible for thermotolerance, implying that thermotolerance is a phenotype controlled by those genes. Although the transcriptional reprogramming that occurred in the T2 thermotolerant cells was quite broad, there was some clustering of genes that encoded chaperons and proteins involved in membrane functions and protec- tion against environmental stresses (23,31,32) (Supple- mentary Tables 4–7). Genes with no known function were also found to be diﬀerentially expressed in T2 thermotolerant cells relative to the control. We noticed that the genes that showed the largest change in gene expression in T2 thermotolerant cells (both HT2 and LT2), relative to the control, were those involved in the protection of bacteria against various forms of environmental stresses (Table 1). To determine whether these genes are direct targets of the T2 ATF, we analyzed the regulatory sequences to search for sequence elements that matched the putative binding site for the T2 ATF. Indeed, many of the genes aﬀected by the T2 ATF housed binding sites for this transcription factor in their regulatory regions. Among those genes, cpxP, ompW and marRAB exhibited the greatest change in their expression. The T2 ATF was found to bind to the putative T2 ATF- binding sites present in the regulatory regions of cpxP, Table 1. Genes associated with thermotolerance Gene Functiona Increased expression ompW Outer membrane protein cpxP Chaperone involved in resistance to extracytoplasmic stress yliH Conserved protein ybaS Predicted glutaminase ybaT Predicted transporter Decreased expression marR MarR transcriptional repressor marA MarA transcriptional activator marB Multiple antibiotic resistance protein log2 fold-changeb (high/low)c 6.1/5.2 5.3/2.8 4.9/4.1 4.3/1.8 4.2/1.4 3.5/ 1.3 3.2/ 1.2 2.7/ 1.3 aFrom the EcoCyc database (http://ecocyc.org). blog2 fold-change in gene expression between the T2 thermotolerant mutant and the wild-type E. coli containing a control plasmid that lacked an ATF, both grown at 508C (average of duplicate experiments). chigh and low, log2 fold-change of T2 thermotolerant mutant cell expressing the T2 ATF on high- and low-copy plasmids, respectively. Nucleic Acids Research, 2008, Vol. 36, No. 16 e102 ompW and marRAB by a DNA band-shift assay (data not shown), implying that they are direct targets of the T2 ATF. Therefore, cpxP, ompW and marRAB were selected for further study (Table 1). cpxP and ompW, which are known to be involved in resistance to environmental stresses (33–35), were the most highly activated by the T2 ATF. In addition, a number of genes were repressed by the T2 ATF. The largest decrease in gene expression was seen for the marRAB operon that is known to encode proteins that have a role in multiple antibiotic resistance. The MarRAB protein, for example, is a transcription factor that regu- lates the expression of several E. coli genes involved in responses to multiple antibiotics and oxidative stress. Transcription proﬁling revealed that genes regulated by the marRAB operon exhibited the great change in expres- sion levels in the T2 thermotolerant mutant cell, relative to the control. For example, the hdeA and slp genes, which are known to be repressed by MarRAB in wild-type cells, were strongly activated 4- to 16-fold in the T2 cells (Supplementary Table 4). These results imply that the T2 ATF inhibits the expression of MarRAB, which sub- sequently results in either the activation or repression of genes that are diﬀerentially regulated by this versatile tran- scriptional regulatory protein. To examine the functional relevance of these target genes to the thermotolerance phenotype in E. coli, we either overexpressed or knocked out the cpxP and ompW genes. We also knocked out the marRAB operon and examined the response of the mutant strain to heat treatment (Figure 3). When the cpxP or ompW gene was individually overexpressed in wild-type cells, the resulting cells were more resistant to the lethal eﬀects of heat shock than wild-type cells. But they were less heat resistant than the T2 thermotolerant cells (HT2 and LT2) under heat- stress conditions. In addition, co-expression of cpxP and ompW in wild-type cells led to more heat tolerance than the wild-type cells overexpressing cpxP or ompW indi- vidually. The cpxP and ompW co-overexpressing cells were nearly as resistant as the LT2 thermotolerant cell, but much less resistant than the HT2 thermotolerant cell. Despite the increase in heat tolerance of the cpxP- or/ and ompW-overexpressing cells, the basal growth rate of these strains in the absence of heat stress was similar to that of wild-type cells. Furthemore, the cpxP-deleted mutant exhibited a wild-type phenotype, whereas the ompW- deleted mutant was less heat resistant than wild-type cells. Concerning the marRAB, the marRAB knockout mutant did not show any thermotolerance even though the marRAB was highly repressed in the T2 thermotoler- ant cell (HT2). This observation indicates that this operon itself is insuﬃcient to constitute the thermotolerance phe- notype. Thus, MarRAB may not participate directly in the development of thermotolerace of E. coli. Taken together, the individual eﬀect of cpxP, ompW and marRAB on the thermotolerance was not strong enough to confer a HT2-like thermotolerance, even though their expression level was signiﬁcantly inﬂuenced by the T2 ATF in the HT2 thermotolerant cell. Detailed mechanism of these genes involved in the thermotolerance should be further studied.