Role of TnrA in nitrogen source-dependent repression of Bacillus subtilis glutamate synthase gene expression

Abstract

Synthesis of glutamate, the cell's major donor of nitrogen groups and principal anion, occupies a significant fraction of bacterial metabolism. In Bacillus subtilis, the gltAB operon, encoding glutamate synthase, requires a specific positive regulator, GltC, for its expression. In addition, the gltAB operon was shown to be repressed by TnrA, a regulator of several other genes of nitrogen metabolism and active under conditions of ammonium (nitrogen) limitation. TnrA was found to bind directly to a site immediately downstream of the gltAB promoter. As is true for other genes, the activity of TnrA at the gltAB promoter was antagonized by glutamine synthetase under certain growth conditions.

FIG. 1

The gltCA intergenic region. Likely transcription start sites of the gltC and gltA genes, their −10 and −35 regions, and the gltA initiation codon are in boldface. Directions of transcription and translation are indicated by horizontal arrows. The mutations in the region are shown above the sequence with their allele numbers. The apparent GltC-binding Box I and Box II sequences (2) are underlined and double underlined, respectively. A TnrA binding sequence is underlined with dashes. Above this site the nucleotides that appear to interact with TnrA in the DNase I protection assay are shown by a horizontal line, and the TnrA consensus sequence (15) is presented. The fusions in pLG200, pLG203, and pLG219 contain fragments with coordinates from −160 to +119. The vertical arrows indicate upstream or downstream junctions for the truncated fusions in plasmids pLG700 (positions −56 to +119), pLG702 (positions −45 to +119), pLG708 (positions −160 to +24), and pLG711 (positions −160 to +3). The numbering of the sequence is with respect to the gltA transcription start point (2).

FIG. 2

Effects of glnA and tnrA mutations on gltA-lacZ expression. Strains LG219 (wild type), BB1194 (glnA73), and BB1195 (glnA73 tnrA) containing a gltAp19-lacZ fusion (lightly shaded bars) and strains LG219/24 (wild type) and BB1199 (glnA73) containing a gltAp19/24-lacZ fusion with the gltAp24 mutation in the TnrA binding site (darkly shaded bars) were grown to mid-exponential phase in glucose-glutamine minimal medium and assayed for β-galactosidase activity. Strains of the LG219 series contain a gltAp19 mutation (Fig. 1) within their fusions. This mutation does not effect expression or regulation from the gltA promoter (Table 2).

FIG. 3

Gel mobility shift assay with crude extracts. Cells of strains SMY (tnrA⁺) and BB278 (tnrA) were grown in glucose-proline medium to mid-exponential phase, harvested, and disrupted. The supernatant fluid from low-speed centrifugation was used as a crude extract for assays of binding to a radioactive 346-bp DNA fragment corresponding to positions −175 to +119 of the gltAB transcription unit. Lane 1, probe only; lanes 2 and 3, 1 and 4 μl of SMY extracts; lanes 4 and 5, 1 and 4 μl of BB278 extracts.

FIG. 4

Gel mobility shift analysis of the interaction between the gltA promoter and TnrA. A radioactively labeled 291-bp fragment containing the gltA promoter region with coordinates from −160 to +119 relative to the gltA transcription start point (lane 1) was incubated with increasing concentrations of purified TnrA (lanes 2 to 10). The TnrA concentrations were 5.5 (lane 2), 11 (lane 3), 22 (lane 4), 44 (lane 5), 88 (lane 6), 175 (lane 7), 350 (lane 8), 700 (lane 9), and 1,400 nm (lane 10).

FIG. 5

DNase I footprinting analysis of TnrA binding to the gltA promoter. The 346-bp gltAp⁺ (left) and gltAp24 (right) promoter fragments, labeled on the nontemplate strand, were incubated with purified TnrA and then with DNase I. The sequence of the nontemplate strand of pIPC119 (2) determined using oBB13 as a primer is shown on the left. The apparent transcription start sites of gltA and gltC and the directions of transcription are shown by the bent arrows. The region of interaction with TnrA is indicated by a vertical line. Lanes 1, no TnrA; lane 2, 1.2 nM TnrA; lanes 3, 4.6 nM TnrA; lanes 4, 18.4 nM TnrA; lanes 5, 73.8 nM TnrA; lanes 6, 295 nM TnrA. The righthand panel has no lane corresponding to lane 2.