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

Abstract

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 -313 (site 1) and bp -140 (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.

FIG. 1

CRP binding sites in the gcv control region. The transcription start site for gcvT is indicated as +1. The nucleotide sequences of the CRP binding sites centered at bp −313 and −140 relative to the transcription initiation site are shown. The inverted repeat sequence known to be important for CRP binding is in capital letters. Nucleotides conserved with respect to the CRP consensus site are underlined. The arrows indicate the nucleotide changes in the mutants gcvT-lacZ_Δ−341−306T−307G−308T and gcvT-lacZ−139A−152T. The consensus CRP binding site is indicated for comparison.

FIG. 2

Gel mobility shift assay for the binding of CRP to gcv DNA. The wild-type 759-bp gcv fragment was used as target for lanes 1 to 5. The 5′-end-truncated 606-bp fragment was used as the target for lanes 6 to 10. Where indicated, 20 mM cAMP was included. The CRP dimer was added at a concentration of either 10 nM (+) or 100 nM (++).

FIG. 3

Gel mobility shift assay for the binding of CRP to gcv DNA. The wild-type 759-bp gcv fragment was used as the target. The CRP dimer was added at the following concentrations: 0, 2.5, 5.0, 10, 25, 50, and 100 nM (lanes 1 to 7, respectively). cAMP was included in all reaction mixtures at a final concentration of 2 mM. The arrow denotes the unbound fragment.

FIG. 4

Protection from DNase I digestion of gcv DNA by CRP plus cAMP. The ³²P-labeled wild-type 759-bp gcv fragment was incubated with dilutions of CRP and digested with DNase I (see Materials and Methods). cAMP (2 mM) was included in all reaction mixtures. The digestion products were electrophoresed on a denaturing 5% polyacrylamide gel adjacent to the Maxam-Gilbert sequencing reaction mixtures of the labeled DNA probe (not shown). (A and B) Long and short runs, respectively, of the digestion products. Lane 1, no protein; lanes 2 to 6, 5, 10, 25, 50, and 100 nM CRP dimer, respectively. The brackets indicate the two sites protected from digestion by DNase I.

FIG. 5

Hypothetical model for repression of gcv. There is evidence that GcvA must bind to its three target sites for repression (47). GcvR is also required for repression (12, 13), but it is unknown if GcvR-GcvA contacts are required or if GcvR performs some other function. Lrp binds gcv DNA in the region depicted and bends DNA (29), possibly to allow the formation of a nucleoprotein complex.