Integrating Genome-wide Analyses to Investigate the Regulatory Role of FNR and IscR in Response to Anaerobiosis in Escherichia coli

Escherichia coli contains multiple O 2-sensing transcription factors, including FNR and IscR. FNR is predominantly active under anaerobic conditions, where it ligates an O2–labile [4Fe–4S] cluster for dimerization and DNA binding. Ligation of a [2Fe–2S] cluster by IscR broadens its DNA binding specificity from binding only a Type 2 site to binding both Type 1 and Type 2 sites. Fe–S occupancy of IscR is regulated by O2 availability and a complex pattern of gene expression results under aerobic and anaerobic conditions. We studied the FNR and IscR regulons on a genome–scale and found little overlap between the two regulons. FNR occupancy at many sites was strongly influenced by nucleoid–associated proteins (NAPs). While only a subset of predicted FNR binding sites were bound in vivo, many sites appeared to be masked by the NAPs H–NS, IHF, and Fis. Cells lacking H–NS and its paralog StpA showed increased FNR occupancy at sites normally bound by H–NS, indicating that the genome is not readily accessible for FNR binding. Correlation of FNR ChIP–seq peaks with transcriptomic data showed that less than half of the FNR regulated operons could be attributed to direct FNR binding. FNR bound some promoters without regulating expression, presumably requiring additional condition specific transcription factors.;ChIP–chip analysis of IscR binding revealed that IscR primarily binds Type 2 sites under aerobic and anaerobic conditions. Correlation of IscR binding regions containing a Type 1 or Type 2 site with the transcriptomic data revealed 33 operons directly regulated by IscR under aerobic or anaerobic conditions. The four previously reported promoters containing Type 1 sites and both known and novel promoters containing Type 2 sites were identified. The previous finding that promoters with Type 2 sites are also regulated by IscR primarily under aerobic conditions extended to newly identified operons. Further analyses suggested that multiple mechanisms prevent IscR regulation of Type 2 sites under anaerobic conditions including action of other global transcription factors FNR, ArcA, and Fur. In summary, this work expands our knowledge of the FNR and IscR regulons and the role of combinatorial regulation in the E. coli response to changes in O2 concentration.