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 ␣-methyl glucoside (␣-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 The major products of all cellular transcription in Escherichia coli and Bacillus subtilis are rRNA and tRNA, which constitute more than 95% of the total RNA (19, 37). There are 7 rrn operons in E. coli and 10 in B. subtilis, each controlled by tandem (P1 and P2) promoters that are tightly regulated in response to changes in nutritional status and other stress conditions (6,7,8,49). A significant number of studies on the regulation of rrn synthesis in E. coli have been published, while only a few reports focusing on the spore-forming Gram-positive bacterium B. subtilis exist.In E. coli, the core (Ϫ10/Ϫ35) region in rrnp 1 promoters is preceded by an UP element that increases promoter activity 20-to 50-fold because it is strongly bound by the C-terminal domains of the two ␣ subunits of RNA polymerase (6, 7).

Dynamic Expression of the Translational Machinery during Bacillus subtilis Life Cycle at a Single Cell Level

“…According to our data (Figures 1, 3 and 5) the rrn operons can be classified into three groups based on expression strength: high - rrnE , medium - rrnB,D,I,J,O and low - rrnA . These results differ slightly from those described previously for exponential phase, whereby the activity of several rrn promoters was assessed using LacZ reporter fusions [42]. Consistent with our data, the strongest rrn promoter activity was associated with the rrnE operon that harbors three binding elements to the housekeeping sigma factor σ A , while the other members contain two such elements (Fig.…”

The ζ Toxin Induces a Set of Protective Responses and Dormancy

“…B. subtilis , like many other Firmicutes, possesses three (p)ppGpp synthetases: a single bifunctional RelA-SpoT enzyme, which modulates the intracellular levels of (p)ppGpp by both synthesis and degradation in response to the cellular nutritional status [37] and two alarmone synthetases, Ssa1 and Ssa2, responsible for the maintenance of basal levels of (p)ppGpp during homeostatic growth [49], [50]. These observations suggest that the pleiotropic effect observed after ζY83C induction could be due to increased levels of RelA and/or the decreased levels of YvdC, resulting in a potential accumulation of (p)ppGpp with subsequent inhibition of DNA replication, metabolic reallocation, induction of the stringent response and expression of the σ B (orthologue of E. coli σ S ) factor [40], [53], [54], [55]. To investigate this hypothesis, we introduced a null relA mutation (Δ relA ) into xylR - P XylA and xylR - P XylA ζY83C strains, creating xylR - P XylA Δ relA and xylR - P XylA ζY83C Δ relA strains, and measured toxin-mediated dormancy and permeability to PI (Table 4).…”

Multiple rRNA operons are essential for efficient cell growth and sporulation as well as outgrowth in Bacillus subtilis

“…The differences among the strains with different combinations of two rrn operons might be a consequence of sequence heterogeneity, the location of the operons on the chromosome, or the transcriptional activity of each rrn operon. It has been reported in E. coli that the promoter activity of an rrn operon depends on its distance from the replication origin (Condon et al, 1992), and different rrn operons are regulated differentially during the stringent response (Samarrai et al, 2011). Thus, the differences in sporulation frequency and growth rate of the strains that carry two copies of different rrn operons might result from differences in individual promoter activities and their regulation.…”