Cells of Escherichia coli growing on sugars that result in catabolite repression or amino acids that feed into glycolysis undergo a metabolic switch associated with the production and utilization of acetate. As they divide exponentially, these cells excrete acetate via the phosphotransacetylase-acetate kinase pathway. As they begin the transition to stationary phase, they instead resorb acetate, activate it to acetyl coenzyme A (acetyl-CoA) by means of the enzyme acetyl-CoA synthetase (Acs) and utilize it to generate energy and biosynthetic components via the tricarboxylic acid cycle and the glyoxylate shunt, respectively. Here, we present evidence that this switch occurs primarily through the induction of acs and that the timing and magnitude of this induction depend, in part, on the direct action of the carbon regulator cyclic AMP receptor protein (CRP) and the oxygen regulator FNR. It also depends, probably indirectly, upon the glyoxylate shunt repressor IclR, its activator FadR, and many enzymes involved in acetate metabolism. On the basis of these results, we propose that cells induce acs, and thus their ability to assimilate acetate, in response to rising cyclic AMP levels, falling oxygen partial pressure, and the flux of carbon through acetate-associated pathways.

The cyclic AMP receptor protein (CRP) activates transcription of the Escherichia coli acs gene, which encodes an acetate-scavenging enzyme required for fitness during periods of carbon starvation. Two promoters direct transcription of acs, the distal acsP1 and the proximal acsP2. In this study, we demonstrated that acsP2 can function as the major promoter and showed by in vitro studies that CRP facilitates transcription by "focusing" RNA polymerase to acsP2. We proposed that CRP activates transcription from acsP2 by a synergistic class III mechanism. Consistent with this proposal, we showed that CRP binds two sites, CRP I and CRP II. Induction of acs expression absolutely required CRP I, while optimal expression required both CRP I and CRP II. The locations of these DNA sites for CRP (centered at positions ؊69.5 and ؊122.5, respectively) suggest that CRP interacts with RNA polymerase through class I interactions. In support of this hypothesis, we demonstrated that acs transcription requires the surfaces of CRP and the C-terminal domain of the ␣ subunit of RNA polymerase holoenzyme (␣-CTD), which is known to participate in class I interactions: activating region 1 of CRP and the 287, 265, and 261 determinants of the ␣-CTD. Other surface-exposed residues in the ␣-CTD contributed to acs transcription, suggesting that the ␣-CTD may interact with at least one protein other than CRP.The Escherichia coli cyclic AMP (cAMP) receptor protein (CRP; also known as the catabolite activator protein) activates transcription from more than 100 promoters. When bound to its allosteric effector, cAMP, the CRP homodimer binds specific DNA sites near target promoters, enhancing the binding of RNA polymerase holoenzyme (RNAP) and facilitating the initiation of transcription. Simple CRP activation operates through two related mechanisms, designated class I and class II. Both mechanisms depend upon specific interactions between CRP and RNAP. At class I promoters, a CRP dimer binds to DNA at a site centered near position Ϫ61.5, Ϫ71.5, Ϫ82.5, or Ϫ92.5. CRP bound at any of these positions uses a defined surface (activating region 1 [AR1]) in the downstream subunit of CRP to contact a specific surface determinant, 287, of the C-terminal domain of the ␣ subunit of RNA polymerase (␣-CTD). Two additional ␣-CTD determinants contribute to class I activation: the 261 determinant, proposed to interact with the subunit of RNAP, and the 265 determinant, known to interact with DNA, especially AϩT-rich sequences, most notably the UP element.Class I interactions appear to recruit RNAP to promoter DNA by increasing the binding constant for closed-complex formation. At class II promoters, a CRP dimer binds to DNA at a site centered near position Ϫ41.5. When bound at this position, CRP uses AR1 of the upstream subunit of CRP to contact the 287 determinant of the ␣-CTD and a second surface (AR2) to contact the 162 to 165 determinant of the Nterminal domain of ␣. The ␣-CTD 265 determinant also contributes by interacting with DNA. Class II interactions appea...

Independent regulation of the divergent Escherichia coli nrfA and acsP1 promoters by a nucleoprotein assembly at a shared regulatory region FNR-dependent transcription. We conclude that the nrfA-acs intergenic region is folded into an ordered nucleoprotein structure that permits the two divergent promoters to be regulated independently in response to different physiological signals. IntroductionThe expression of many genes required for anaerobic respiration is controlled by the FNR protein, an anaerobically triggered transcription activator (reviewed by Guest et al., 1996). At many target promoters, FNR binds as a dimer to a 22 bp sequence centred near position -41 in relation to the transcription start site. From such sites, FNR activates transcription initiation by interacting directly with RNA polymerase using defined surfaceexposed activating regions (Wing et al., 1995;Williams et al., 1997;Li et al., 1998;Lamberg and Kiley, 2000;Lee et al., 2000). Many FNR-dependent genes are regulated further by the availability of nitrite and nitrate ions in the medium (reviewed by Darwin and Stewart, 1996). This is achieved by the two homologous transcription factors, NarL and NarP. In response to nitrite and nitrate, both proteins are phosphorylated as a result of the action of two sensor kinase proteins, NarX and NarQ. Once phosphorylated, NarL and NarP bind to specific heptamer sequences at target promoters and mediate transcription activation or repression depending on the promoter context.The Escherichia coli nir operon encodes a cytoplasmic NADH-dependent nitrite reductase, the expression of which is co-dependent on both FNR and NarL/NarP (Harborne et al., 1992;Tyson et al., 1994). Recently, it has been shown that FNR-dependent transcription at pnirB is repressed by various nucleoid-associated factors Browning et al., 2000). Two of these factors were identified to be the Fis protein (factor for inversion stimulation) and the IHF protein (integration host factor). Both are versatile, weakly specific, DNA-binding proteins that modulate the expression of a number of promoters (reviewed by McLeod and Johnson, 2001). At the nirB promoter (pnirB), it has been shown that IHF and Fis drive the promoter DNA into an inhibitory architecture, which represses FNR-dependent transcription. NarL and NarP counteract this repression by disrupting this nucleo- protein complex and allowing maximal FNR-dependent activation to occur. Thus, at pnirB, NarL and NarP activate transcription by an indirect mechanism, rather than by interacting directly with the transcription machinery (Browning et al., 2000).The nrf operon also encodes a functional nitrite reductase (Darwin et al., 1993), and there are both striking similarities and important differences between the regulation of the nrfA and nirB promoters. Both are positively controlled by FNR, NarL and NarP but, unlike the nir promoter, pnrfA is also repressed by high levels of phospho-NarL during growth in the presence of high (mM) concentrations of nitrate (Tyson et al., 1994). Anaerobi...

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SummaryExpression from the Escherichia coli nrf operon promoter is activated by the anaerobically triggered transcription factor, FNR, and by the nitrate/nitrite ioncontrolled response regulators, NarL or NarP, but is repressed by the IHF and Fis proteins. Here, we present in vitro studies on the nrf promoter, using permanganate footprinting to measure open complex formation, and DNase I footprinting to monitor binding of the different regulators and the interactions between them. Our results show that open complex formation is completely dependent on FNR and is enhanced by NarL, but is repressed by IHF or Fis. NarL counteracts repression by IHF but is unable to alter repression by Fis. These results suggest mechanisms by which nrf promoter activity is modulated by the different factors. Expression from the nrf promoter is known to be repressed in rich media, especially in the presence of glucose, but the molecular basis of this is not understood. Here, we show that this catabolite repression is relieved by mutations that weaken the DNA site for Fis, improve the DNA site for FNR or improve the promoter ----10 or ----35 elements. Hence, Fis protein is a major factor responsible for catabolite repression at the nrf promoter, and Fis can override activation by FNR and NarL or NarP.