Differential responses of Bacillus subtilis rRNA promoters to nutritional stress

Abstract

The in vivo expression levels of four rRNA promoter pairs (rrnp(1)p(2)) of Bacillus subtilis were determined by employing single-copy lacZ fusions integrated at the amyE locus. The rrnO, rrnJ, rrnD, and rrnB promoters displayed unique growth rate regulation and stringent responses. Both lacZ activity and mRNA levels were highest for rrnO under all growth conditions tested, while rrnJ, rrnB, and rrnD showed decreasing levels of activity. During amino acid starvation induced by serine hydroxamate (SHX), only the strong rrnO and rrnJ promoters demonstrated stringent responses. Under the growth conditions used, the rrn promoters showed responses similar to the responses to carbon source limitation induced by α-methyl glucoside (α-MG). The ratio of P2 to P1 transcripts, determined by primer extension analysis, was high for the strong rrnO and rrnJ promoters, while only P2 transcripts were detected for the weak rrnD and rrnB promoters. Cloned P1 or P2 promoter fragments of rrnO or rrnJ were differentially regulated. In wild-type (relA(+)) and suppressor [relA(S)] strains under the conditions tested, only P2 responded to carbon source limitation by a decrease in RNA synthesis, correlating with an increase in (p)ppGpp levels and a decrease in the GTP concentration. The weak P1 promoter elements remain relaxed in the three genetic backgrounds [relA(+), relA, relA(S)] in the presence of α-MG. During amino acid starvation, P2 was stringently regulated in relA(+) and relA(S) cells, while only rrnJp(1) was also regulated, but to a lesser extent. Both the relA(+) and relA(S) strains showed (p)ppGpp accumulation after α-MG treatment but not after SHX treatment. These data reveal the complex nature of B. subtilis rrn promoter regulation in response to stress, and they suggest that the P2 promoters may play a more prominent role in the stringent response.

FIG. 1.

Relative promoter strengths of four rrn operons and the veg gene integrated into the amyE locus. (A) β-Galactosidase activity measured in strains with lacZ-rrn promoter fusions with rrnO, rrnJ, rrnB, rrnD, and veg integrated at the amyE locus cultured in the indicated media to produce different growth rates. One-milliliter samples from the growing cultures were removed at a reading of 100 Klett units and were assayed for β-galactosidase activity by the method of Miller (29) as described in Materials and Methods. (B) The steady-state transcript levels driven by the indicated promoters were determined by Northern slot blotting. Five- and 10-μg portions of total RNA prepared from B. subtilis relA⁺ strains grown in MM1-glucose were loaded into individual slots and hybridized with an excess of a ³²P-labeled lacZ probe. Autoradiograms were analyzed by densitometry, and the values shown are expressed relative to the signal from rrnO. (C and D) The stabilities of rrn-lacZ fusion transcripts initiating at the indicated promoters were determined by Northern slot blot hybridization of a lacZ probe to total RNA prepared at the indicated times (minutes) after rifampin treatment. The half-life was calculated by plotting the relative transcript levels obtained from the densitometric measurements of the autoradiograms versus time on semilog graph paper.

FIG. 2.

Relative strengths of individual promoter elements as determined by primer extension analysis. Total RNA was isolated from strains containing the indicated promoter-lacZ fusion constructs and was used for primer extension analysis with a ³²P-labeled primer that hybridizes to the spoVG-lacZ junction present in the constructs. Primer extension products were separated by gel electrophoresis, and arbitrary values were calculated from densitometric scans of autoradiograms. Transcripts initiating at P1 or P2 were quantitated separately. Each experiment was repeated at least 3 times. Error bars represent the standard errors of the means.

FIG. 3.

Effects of nutritional stress on intact rrn promoters. Cultures of B. subtilis containing the indicated promoter-lacZ fusion constructs were grown in MM1-glucose medium and were treated either with serine hydroxamate (A) or with α-methyl glucoside (B) for the indicated times. Total RNA extracted from these cultures was analyzed by Northern slot blotting using a lacZ probe. The resulting filters were subjected to autoradiography and were then scanned. The values shown are average arbitrary densitometric units determined from at least 3 experiments. Error bars represent the standard errors of the means.

FIG. 4.

Effects of nutritional stress on individual rrn promoter elements of rrnO. (A) Autoradiogram of a representative primer extension experiment performed using RNA isolated from B. subtilis relA⁺, relA, and relA(S) strains and an rrnO-specific ³²P-labeled primer. Cells were treated with SHX or α-MG for the indicated times prior to RNA extraction. P1 and P2 primer extension products were separated by gel electrophoresis. A sequence ladder of ³²P-labeled lambda DNA was included as a size marker for the primer extension products. (B and C) Primer extension analysis using RNA from the different relA strains with the integrated rrnO-lacZ fusion and the spoVG-lacZ primer. Cells were grown in MM1-glucose and were treated with either SHX (B) or α-MG (C) for the indicated times. The relative levels of P1 and P2 transcripts were determined as described for Fig. 2.

FIG. 5.

Responses of separated promoter elements to nutritional stress. Wild-type relA⁺ cells containing the indicated lacZ promoter fusion constructs were grown in MM1 and were treated with SHX (A and B) or with α-MG (C and D) for the indicated times. The relative activities of the promoter fragments of rrnO and rrnJ were measured by densitometric scans of autoradiograms from RNA slot blots before and after treatments. The values were calculated as described in the legend for Fig. 3. (E and F) The chemical half-lives of lacZ mRNAs transcribed from the isolated P1 or P2 elements of rrnO and rrnJ were determined and plotted as described in the legend for Fig. 1C and D.

FIG. 6.

Accumulation of (p)ppGpp in B. subtilis relA⁺, relA, and relA(S) strains during nutritional stress. The indicated strains were grown in low-phosphate medium, followed by a 1-h labeling with [³²P]phosphoric acid. Labeled cells were stressed with SHX or α-MG, and samples were removed at the indicated times and were processed for thin-layer chromatography on PEI plates as described in Materials and Methods. The autoradiogram shows results for a representative carbon source starvation experiment.