CcpA-dependent regulation of Bacillus subtilis glutamate dehydrogenase gene expression

Abstract

The Bacillus subtilis rocG gene, encoding catabolic glutamate dehydrogenase, was found to be subject to direct CcpA-dependent glucose repression. The effect of CcpA required the presence of both the HPr and Crh proteins. The primary CcpA binding site was identified by mutational analysis and DNase I footprinting. In the absence of inducers of the Roc pathway, rocG was still expressed at a low level due to readthrough transcription. CcpA-dependent repression of rocG readthrough transcription proved to contribute to the slow growth rate of B. subtilis cells in glucose-glutamate medium. Increased readthrough expression of rocG was shown to be partially responsible for the growth defect of ccpA strains in glucose-ammonium medium.

FIG. 1.

(A) Sequence of the rocG regulatory region. The termination codon of yweA, the −12 and −24 promoter regions, the transcription start points, a likely initiation codon, and the core of a putative cre site of the rocG gene are in bold. The directions of transcription and translation are indicated by horizontal arrows. The dyad-symmetry sequence of a putative yweA transcriptional terminator and the region protected by CcpA in DNase I experiments are underlined. The sequence changes in the rocGp1 mutant are indicated. (B) Comparison of rocG cre to the cre consensus sequence (36). Note that the second residue (G) of the core consensus sequence is not conserved in the rocG cre site.

FIG. 2.

Gel mobility shift analysis of the interaction between the rocG promoter and CcpA. Radioactively labeled 259-bp rocGp⁺ (lanes 1 to 9) and rocGp1 (lanes 11 to 19) promoter fragments were incubated with increasing concentrations of purified CcpA. The CcpA concentrations were 0 (lanes 1 and 11), 0.15 nM (lanes 2 and 12), 0.6 nM (lanes 3 and 13), 2.4 nM (lanes 4 and 14), 9.8 nM (lanes 5 and 15), 39 nM (lanes 6 and 16), 156 nM (lanes 7 and 17), 625 nM (lanes 8 and 18), and 2,500 nM (lanes 9 and 19).

FIG. 3.

DNase I footprinting analysis of CcpA binding to the rocG promoter. The 259-bp rocGp⁺ (lanes 1 to 5) and rocGp1 (lanes 6 to 10) promoter fragments, labeled on the template strand, were incubated with purified CcpA and then with DNase I. The sequence of the template strand of pBB907 (7) was determined with oBB56 as a primer. The apparent transcription start sites and the direction of rocG transcription are shown by the bent arrows, and the cre site is indicated by a bracket. The −12 and −24 promoter regions are shown. The protected cre area is indicated by a vertical line to the right of the gel lanes. Lanes 1 and 6, no CcpA; other lanes contained increasing concentrations of purified CcpA: 9.8 nM (lanes 2 and 7), 39 nM (lanes 3 and 8), 156 nM (lanes 4 and 9), and 625 nM (lanes 5 and 10).