The cyclic AMP receptor protein is dependent on GcvA for regulation of the gcv operon.

The Escherichia coli gcv operon is transcriptionally regulated by the GcvA, GcvR, Lrp, and PurR proteins. In this study, the cyclic AMP (cAMP) receptor protein (CRP) is shown to be involved in positive regulation of the gcv operon. A crp deletion reduced expression of a gcvT-lacZ fusion almost fourfold in glucose minimal (GM) medium. The phenotype was complemented by both the wild-type crp gene and four crp alleles that encode proteins with amino acid substitutions in known activating regions of CRP. A cyaA deletion also resulted in a fourfold decrease in gcvT-lacZ expression, and wild-type expression was restored by the addition of cAMP to the growth medium. A cyaA crp double deletion resulted in levels of gcvT-lacZ expression identical to those observed with either single mutation, showing that CRP and cAMP regulate through the same mechanism. Growth in GM medium plus cAMP or glycerol minimal medium did not result in a significant increase in gcvT-lacZ expression. Thus, the level of cAMP present in GM medium appears to be sufficient for regulation by CRP. DNase I footprint analysis showed that CRP binds and protects two sites centered at bp 2313 (site 1) and bp 2140 (site 2) relative to the transcription initiation site, but a mutational analysis demonstrated that only site 1 is required for CRP-mediated regulation of gcvT-lacZ expression. Expression of the gcvT-lacZ fusion in a crp gcvA double mutant suggested that CRP’s role is dependent on the GcvA protein. There are two pathways for the production of one-carbon (C1) units in Escherichia coli. Serine hydroxymethyltransferase, the glyA gene product, catalyzes the cleavage of serine to glycine and the transfer of a C1 unit to tetrahydrofolate to form 5,10-methylenetetrahydrofolate and is the primary source of C1 units (24, 26). The glycine cleavage (GCV) enzyme system catalyzes the oxidative cleavage of glycine to form CO2 ,N H 3, and 5,10-methylenetetrahydrofolate, providing a secondary pathway for C1 units (19). The C1 units produced by these pathways are used in cellular biosyntheses of methylated products such as methionine, thymine, and purines (26). It has been proposed that the physiological role of the GCV system may be to balance a cell’s need for glycine and C1 units. The GCV enzyme system is composed of the GcvT, GcvH, and GcvP proteins, encoded by the gcv operon, and lipoamide dehydrogenase, encoded by the unlinked lpd gene. The regulation of the gcv operon is not fully understood, but there are four proteins known to affect gcv expression. The leucineresponsive protein, Lrp, is a global regulator of genes involved in amino acid metabolism (5) and is required for activation of the gcv operon (22, 41). The PurR protein is a negative regulator of nucleotide metabolic genes (14, 20, 31) and mediates a twofold repression of a gcvT-lacZ fusion when cells are grown in the presence of the purine nucleoside inosine (44). The GcvA protein is responsible for controlling gcv operon expression in two distinct ways. GcvA activates gcv expression when cells are grown in the presence of glycine and mediates a PurRindependent repression of gcv when cells are grown in the presence of inosine but without glycine (44, 45). A fourth protein, GcvR, is a GcvA-dependent negative regulator of gcv expression (12). However, GcvR has not been shown to bind to DNA, and its mechanism of regulation is unknown. Here we report a fifth protein that is involved in controlling gcv expression. The cyclic AMP (cAMP) receptor protein (CRP) mediates a fourfold positive effect on gcv expression as measured from a gcvT-lacZ fusion. In vitro binding experiments and a mutational analysis suggest that CRP binds to a site centered at bp 2313 relative to the transcriptional start site for gcv .I n addition, the CRP effect is dependent on a functional gcvA gene and its role may be to antagonize GcvA’s repression of the gcv operon.