Novel roles of cAMP receptor protein (CRP) in regulation of transport and metabolism of carbon sources

Abstract

CRP (cAMP receptor protein), the global regulator of genes for carbon source utilization in the absence of glucose, is the best-studied prokaryotic transcription factor. A total of 195 target promoters on the Escherichia coli genome have been proposed to be under the control of cAMP-bound CRP. Using the newly developed Genomic SELEX screening system of transcription factor-binding sequences, however, we have identified a total of at least 254 CRP-binding sites. Based on their location on the E. coli genome, we predict a total of at least 183 novel regulation target operons, altogether with the 195 hitherto known targets, reaching to the minimum of 378 promoters as the regulation targets of cAMP-CRP. All the promoters selected from the newly identified targets and examined by using the lacZ reporter assay were found to be under the control of CRP, indicating that the Genomic SELEX screening allowed to identify the CRP targets with high accuracy. Based on the functions of novel target genes, we conclude that CRP plays a key regulatory role in the whole processes from the selective transport of carbon sources, the glycolysis-gluconeogenesis switching to the metabolisms downstream of glycolysis, including tricarboxylic acid (TCA) cycle, pyruvate dehydrogenase (PDH) pathway and aerobic respiration. One unique regulation mode is that a single and the same CRP molecule bound within intergenic regions often regulates both of divergently transcribed operons.

Figure 1. Identification of cAMP-CRP-binding sites on the E. coli genome.

Genomic SELEX search of cAMP-CRP-binding sequences was performed using the standard procedure in the presence (filled circles) and absence of cAMP (open circles). SELEX fragments were subjected to SELEX-chip analysis using the tilling DNA microarray as described previously (39). Regulation target genes of CRP were predicted from the location of CRP-binding sites (for details see text). The genes associated with the peaks at the cut-off level above 30 are indicated. When the CRP-binding site is located upstream of divergently transcribed genes, two genes are shown, being connected with a flash mark. Genes under light green background represent newly identified CRP targets while those under pale orange background represent the CRP targets listed in Regulon DB . The level of CRP binding (Y-axis) represents the fluorescent intensity ratio between SELEX samples in the presence and absence of CRP.

Figure 2. The consensus sequences of CRP binding.

The binding sequences of cAMP-CRP were subjected to the Logo analysis for determinatin of the consensus sequences for the following samples: (A) the whole set of CRP targets (total 323 sequences) identified by Genomic SELEX screening in this study; (B) the set of CRP targets (165 sequences) that are listed in Regulon DB and identified by Genomic SELEX; (C) the set of CRP targets (129 sequences) that are listed in Regulon DB but not identified by Genomic SELEX; (D) the whole set of CRP targets (294 sequences) listed in Regulon DB.

Figure 3. Analysis of promoter regulation in vivo by CRP using LacZ fusion.

The predicted CRP-target promoters were inserted into the lacZ expression plasmid to construct a collection of promoter-lacZ fusions. The promoter-dependent expression of LacZ was determined in both wild-type E. coli and crp-defective mutants (Y-axis, β-galactosidase activity in Miller unit). Except for the talA and fbaB promoters (shown under gray background), the activity of all other promoters decreased in the absence of activator CRP, indicating the involvement of CRP as an activator. The newly identified promoters are shown under black background. As references, some known CRP target promoters such as ptsG, dgsA, ptsH and glpF (shown under white background) were examined in parallel under the same conditions.

Figure 4. Regulation of the divergently transcribed promoters by CRP.

Some of the divergently aligned promoters are controlled by CRP molecule(s) bound within spacers. The gene organization and the binding sites of CRP within the spacer regions are shown: (A) divergent promoters are regulated by single and the same CRP bound within spacers; (B) divergent promoters are regulated by different CRP molecules bound within spacers; and (C) multiple CRP molecules bind to spacer between divergent promoters, but the role of each CRP remains unidentified. The numbers above DNA indicate the distance between the initiation sites of divergent transcription.

Figure 5. Regulatory roles of CRP.

Upper panel: The genes under the control of CRP are indicated under either purple background (the genes for sugar transport systems) or blue background (the genes for metabolic enzymes). CRP regulates the majority of genes for three pathways of sugar transport (MFS, PTS and ABC). The number in boxes represent in the order from left to right: The number of CRP target genes listed in Regulon DB (white); the number of genes identified to be regulated by CRP in this study (green); the number of genes predicted to be under the control of CRP in this study (yellow); the total number of genes under the control of CRP (orange). The numbers shown in parenthesis represent total number of genes constituting the respective transport systems, including those not regulated by CRP. Lower panel: Most of the genes for the enzymes involved in glycolysis are controlled by Cra while only three genes, fbaA, gapA and pgk, had been identified as the regulation targets of CRP (shown in while under blue background). In this study, we identified a number of novel targets of CRP (shown in green under blue background). Furthermore a number of the genes involved in the metabolism downstream of glycolysis including PDH pathway and aerobic respiration were found to be the targets of CRP regulation. The number of target 0.5 represents such a particular case as fbaB<CRP>yegT (see Table S3), in which one (fbaB) of the divergent promoters is known under the control of CRP but possible regulation of the opposite promoter (yegT) by CRP can not be ruled out.

Figure 6. The hierarchy of regulation network of CRP.

A total of 70 genes for transcription factors are organized under the control of CRP. Transcription factors are classified on the basis of regulation targets: regulators for carbon metabolism (green background), nitrogen metabolism (orange background), and for stress-response (purple background). Some nucleoid proteins play not only architectural roles but also regulatory roles (blue-green background). A number of genes for uncharacterized transcription factors are under the control of CRP (black background). The set of newly identified CRP targets in this study are shown by asterisk.