Novel Members of the Cra Regulon Involved in Carbon Metabolism in Escherichia coli

Abstract

Cra (catabolite repressor activator) is a global regulator of the genes for carbon metabolism in Escherichia coli. To gain insights into the regulatory roles of Cra, attempts were made to identify the whole set of regulation targets using an improved genomic SELEX (systematic evolution of ligands by exponential enrichment) system. Surprisingly, a total of 164 binding sites were identified for Cra, 144 (88%) of which were newly identified. The majority of known targets were included in the SELEX chip pattern. The promoters examined by the lacZ reporter assay in vivo were all regulated by Cra. These two lines of evidence indicate that a total of as many as 178 promoters are under the control of Cra. The majority of Cra targets are the genes coding for the enzymes involved in central carbon metabolism, covering all the genes for the enzymes involved in glycolysis and metabolism downstream of glycolysis, including the tricarboxylic acid (TCA) cycle and aerobic respiration. Taken together, we propose that Cra plays a key role in balancing the levels of the enzymes for carbon metabolism.Central carbon metabolism consists of three major pathways, the glycolysis (Embden-Meyerhof-Parnas [EMP]) pathway, the pentose-phosphate (PP) pathway, and the tricarboxylic acid (TCA) cycle, altogether leading to the production of metabolic energy. These three pathways also provide a number of building blocks that are utilized for the construction of cell architecture (for reviews, see references 26, 43, and 50).

Functioning of a metabolic flux sensor in Escherichia coli

“…Interestingly, several enzymes around the presumed flux-signaling metabolite FBP are repressed by Cra (12), raising the question how these enzyme levels could be constant across conditions (Fig. S2).…”

“…FBP is an intermediate of glycolysis and acts as an inhibitor of the transcription factor Cra (9), which is a known regulator of glycolytic and gluconeogenic genes in Escherichia coli (10)(11)(12) (Fig. 1A).…”

Novel regulation targets of the metal-response BasS–BasR two-component system of Escherichia coli

“…The SELEX-chip method was developed for quick mapping of transcription factor-bound DNA segments across the E. coli genome using a DNA tiling array (Teramoto et al, 2010). The Genomic SELEX screening system has been successfully employed for identification of the whole set of regulation targets against a number of E. coli transcription factors, such as RstA (Ogasawara et al, 2007a), PdhR (Ogasawara et al, 2007b), RutR (renamed from YdcC) (Shimada et al, 2007), NemR (renamed from YdhM) (Umezawa et al, 2008), Dan (renamed from YgiP) (Teramoto et al, 2010), LeuO (Shimada et al, 2009, 2011a, Cra (Shimada et al, 2005(Shimada et al, , 2011c and CRP (Shimada et al, 2011b). BasR binding in vitro to the predicted BasSR targets was examined in detail by gel shift and DNase I footprinting assays, while regulation in vivo was examined by Northern blot analysis.…”

Identification of the Set of Genes, Including Nonannotated morA , under the Direct Control of ModE in Escherichia coli

“…The same collection of genomic SELEX fragments as used for SELEX-clos was subjected, after fluorescent labeling, to hybridization with a DNA tilling microarray (Oxford Gene Technology, Oxford, United Kingdom) (11)(12)(13)(14). The fluorescence intensity on each spot was represented as the ratio to sum of the fluorescent intensity of each spot on an array and plotted on the corresponding position on the E. coli genome.…”

“…For this purpose, we employed the genetic systematic evolution of ligands by exponential enrichment (SELEX) screening system, which was developed for identification of the set of regulation targets recognized by DNA-binding transcription factors and was successfully used for the search of regulation targets by AscG (8), AllR (9), CitB (10), Cra (11,12), cyclic AMP receptor protein (CRP) (13), Dan (14), LeuO (15), NemA (16), PdhR (17), RcdA (18), PgrR (19), RstA (20), RutR (21), and TyrR (22). After the genomic SELEX screening, we identified at least 10 binding sites of the ModEmolybdate complex.…”