Molecular Analysis of the Regulation of csiD,a Carbon Starvation-inducible Gene in Escherichia coli that is Exclusively Dependent on s S and Requires Activation by cAMP-CRP Christoph Marschall 1 , Vale  rie Labrousse 2 , Margit Kreimer 1 Dieter Weichart 1 , Annie Kolb 2 and Regine Hengge-Aronis 1 * 1 Department of Biology University of Konstanz P.O. Box 5560, 78434 Konstanz Germany 2 Unite  de Physicochimie des Macromole Âcules Biologiques UMR 321, Institut Pasteur 25 rue du Dr. Roux 75724 Paris, Cedex, France The general stress-induced sigma subunit s S of Escherichia coli RNA poly- merase is closely related to the vegetative sigma factor s 70 . In view of their very similar promoter speci®city in vitro, it is unclear how sigma factor selectivity in the expression of s S -dependent genes is generated in vivo. The csiD gene is such a strongly s S -dependent gene. In contrast to s S , which is induced in response to many different stresses, csiD, whose expression is driven from a single promoter, is induced by carbon starvation only. To our knowledge, the csiD promoter is the ®rst charac- terized promoter which is not only exclusively dependent on s S -contain- ing RNA polymerase (Es S ), but also requires an activator, cAMP-CRP. In addition, leucine-responsive regulatory protein (Lrp) acts as a positive modulator of csiD expression. Also in vitro,Es S is more ef®cient than Es 70 in csiD promoter binding, open complex formation and run-off transcription, which might be due to the poor match of the csiD 35 region to the s 70 consensus and to transcription by Es S being less dependent on contacts in this region. By DNase I protection experiments, a cAMP-CRP binding site centered at 68.5 nucleotides upstream of the csiD transcriptional start site was identi®ed. While cAMP-CRP stimulates Es 70 binding, it does not promote open complex formation by Es 70 , but does so in conjunction with Es S . With linear templates, cAMP-CRP signi®cantly stimulates Es S -mediated in vitro transcription, whereas transcription by Es 70 is negligible and hardly stimulated by cAMP-CRP. These ®ndings may re¯ect different or less stringent positional requirements for an activator site for Es S than for Es 70 , and indicate that cAMP-CRP contributes to sigma factor selec- tivity at the csiD promoter. In vitro transcription experiments with super- coiled templates, however, revealed signi®cant cAMP-CRP-stimulated transcription also by Es 70 . Yet, under these conditions, H-NS was found to restore Es S speci®city by strongly interfering with cAMP-CRP/Es 70 - dependent transcription. Lrp strongly and cooperatively binds to multiple sites located between positions 14 and 102 (in a way that suggests DNA wrapping around multiple Lrp molecules) and moderately stimu- lates in vitro transcription, especially with Es S . In summary, we conclude that the csiD promoter has an intrinsic prefer- ence for Es S , but that also protein factors such as cAMP-CRP, Lrp and probably H-NS as well as DNA conformation contribute to its strong Es S selectivity. Furthermore, this strong Es S preference in combination with a requirement for high concentrations of the essential activator cAMP-CRP *Corresponding author Present addresses: C. Marschall, Department of Dermatology, Ludwigs-Maximilians-University, 80337 Mu È nchen, Germany; D. Weichart, Institute of Biological Sciences, University of Wales, Aberystwyth, Dyfed SY23 3DA, United Kingdom. Abbreviations used: cAMP, 3 0 ,5 0 -cyclic adenosine monophosphate; CRP, cAMP receptor protein; Lrp, leucine- responsive regulatory protein; E, RNA polymerase core enzyme; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis. J. Mol. Biol. (1998) 276, 339±353 0022±2836/98/070339±15 $25.00/0/mb971533 # 1998 Academic Press Limited