Understanding life at a systems level is a major aim of biology.The bacterium Escherichia coli offers one of the best opportunities to achieve this goal. It is a metabolically versatile bacterium able to respond to changes in oxygen availability. This ability is a crucial component of its lifestyle, allowing it to thrive in aerobic external environments and under the oxygen-starved conditions of a host gut. The controlled growth conditions of chemostat culture were combined with transcript profiling to investigate transcriptome dynamics during the transition from aerobic to micro-aerobic conditions. In addition to predictable changes in transcripts encoding proteins of central metabolism, the abundances of transcripts involved in homeostasis of redox-reactive metals (Cu and Fe), and cell envelope stress were significantly altered. To gain further insight into the responses of the regulatory networks, the activities of key transcription factors during the transition to micro-aerobic conditions were inferred using a probabilistic modeling approach, which revealed that the response of the direct oxygen sensor FNR was rapid and overshot, whereas the indirect oxygen sensor ArcA reacted more slowly. Similarly, the cell envelope stress sensors RpoE and CpxR reacted rapidly and more slowly, respectively. Thus, it is suggested that combining rapid and slow reacting components in regulatory networks might be a feature of systems in which a signal is perceived by two or more functionally related transcription factors controlling overlapping regulons.Escherichia coli is a metabolically versatile bacterium that is able to grow in the presence and absence of oxygen. To achieve this, it exploits a flexible biochemistry in which aerobic respiration is preferred to anaerobic respiration, which in turn is preferred to fermentation. The preference for aerobic respiration reflects the relative energetic efficiencies of the alternative metabolic modes (1). To exploit the energetic benefits conferred by aerobic respiration, E. coli has two alternative quinol oxidases, cytochrome bo 3 and cytochrome bd (2). Cytochrome bo 3 is a heme-copper protein that is synthesized under aerobic conditions and has a lower affinity for O 2 than the heme protein cytochrome bd, which has a very high affinity for O 2 and is synthesized under micro-aerobic conditions (3-5). In a further adaptation to lower O 2 concentrations, the role of the pyruvate dehydrogenase complex (PDHC), 2 which oxidizes pyruvate to acetyl-CoA and CO 2 , is progressively taken over by pyruvate formate-lyase (PFL), which converts pyruvate to acetyl-CoA and formate (1). These adaptations allow the bacteria to exploit the relatively low levels of O 2 present under micro-aerobic conditions and maintain redox balance.Adaptation to changes in O 2 availability is regulated at the level of transcription by two well characterized systems, FNR and ArcBA (6 -9). FNR is a direct O 2 sensor (10), whereas the ArcBA two-component system senses O 2 availability indirectly by monitoring the redo...

FNR proteins are transcription regulators that sense changes in oxygen availability via assembly-disassembly of [4Fe-4S] clusters. The Escherichia coli FNR protein is present in bacteria grown under aerobic and anaerobic conditions. Under aerobic conditions, FNR is isolated as an inactive monomeric apoprotein, whereas under anaerobic conditions, FNR is present as an active dimeric holoprotein containing one [4Fe-4S] cluster per subunit. It has been suggested that the active and inactive forms of FNR are interconverted in vivo, or that iron-sulphur clusters are mostly incorporated into newly synthesized FNR. Here, experiments using a thermo-inducible fnr expression plasmid showed that a model FNR-dependent promoter is activated under anaerobic conditions by FNR that was synthesized under aerobic conditions. Immunoblots suggested that FNR was more prone to degradation under aerobic compared with anaerobic conditions, and that the ClpXP protease contributes to this degradation. Nevertheless, FNR was sufficiently long lived (half-life under aerobic conditions,~45 min) to allow cycling between active and inactive forms. Measuring the abundance of the FNR-activated dms transcript when chloramphenicol-treated cultures were switched between aerobic and anaerobic conditions showed that it increased when cultures were switched to anaerobic conditions, and decreased when aerobic conditions were restored. In contrast, measurement of the abundance of the FNR-repressed ndh transcript under the same conditions showed that it decreased upon switching to anaerobic conditions, and then increased when aerobic conditions were restored. The abundance of the FNR-and oxygen-independent tatE transcript was unaffected by changes in oxygen availability. Thus, the simplest explanation for the observations reported here is that the FNR protein can be switched between inactive and active forms in vivo in the absence of de novo protein synthesis. INTRODUCTIONThe FNR protein is an oxygen-responsive transcription regulator that controls the expression of more than 100 transcriptional units in Escherichia coli, and coordinates the transition from aerobic to anaerobic growth (Becker et al., 1996;Covert et al., 2004; Gonzalez et al., 2003;Guest et al., 1996;Kang et al., 2005; Salmon et al., 2003;Sawers, 1999;Unden & Bongaerts, 1997). The intracellular concentration of FNR is similar under both aerobic and anaerobic conditions (Sutton et al., 2004a;Unden & Duchene, 1987), but FNR is activated under anaerobic conditions by the acquisition of [4Fe-4S] clusters that promote the formation of homodimers, and enhance DNA binding at sites resembling the consensus sequence TTGAT-N 4 -ATCAA (Bauer et al., 1999;Eiglmeier et al., 1989;Green et al., 1996Green et al., , 2001Green & Paget, 2004;Khoroshilova et al., 1995Khoroshilova et al., , 1997Kiley & Beinert, 1999Lazazzera et al., 1993 Lazazzera et al., , 1996Moore & Kiley, 2001;Patschkowski et al., 2000;Popescu et al., 1998). Four essential cysteine residues (Cys20, 23, 29 and 122) ligate the FNR [4Fe-4S] ...