The reduction in small ribosomal subunit abundance in ethanol-stressed cells of Bacillus subtilis is mediated by a SigB-dependent antisense RNA

Abstract

One of the best-characterized general stress responses in bacteria is the σB-mediated stress response of the Gram-positive soil bacterium Bacillus subtilis. The σB regulon contains approximately 200 protein-encoding genes and 136 putative regulatory RNAs. One of these σB-dependent RNAs, named S1136-S1134, was recently mapped as being transcribed from the S1136 promoter on the opposite strand of the essential rpsD gene, which encodes the ribosomal primary-binding protein S4. Accordingly, S1136-S1134 transcription results in an rpsD-overlapping antisense RNA (asRNA). Upon exposure of B. subtilis to ethanol, the S1136 promoter was found to be induced, while rpsD transcription was downregulated. By quantitative PCR, we show that the activation of transcription from the S1136 promoter is directly responsible for the downregulation of rpsD upon ethanol exposure. We also show that this downregulation of rpsD leads to a reduced level of the small (30S) ribosomal subunit upon ethanol stress. The activation of the S1136 promoter thus represents the first example of antisense transcription-mediated regulation in the general stress response of B. subtilis and implicates the reduction of ribosomal protein abundance as a new aspect in the σB-dependent stress response. We propose that the observed reduction in the level of the small ribosomal subunit, which contains the ribosome-decoding center, may protect B. subtilis cells against misreading and spurious translation of possibly toxic aberrant peptides under conditions of ethanol stress.

Regulatory RNAs in Bacillus subtilis: a Gram-Positive Perspective on Bacterial RNA-Mediated Regulation of Gene Expression

“…Conceivably, this downregulation could take place via a promoter collision mechanism. In addition, it was found that the S1136 promoter-dependent rpsD downregulation results in a reduced abundance of the small ribosomal subunit upon ethanol stress (70). The observed reduction in the level of the small ribosomal subunit, which contains the ribosome-decoding center, may protect B. subtilis cells against misreading and spurious translation of possibly toxic aberrant peptides in the presence of ethanol.…”

“…The extensive set of putative regulatory RNA segments in B. subtilis, first identified by Nicolas et al (15) and further explored in this review, opens up many possibilities for in-depth follow-up studies (24,50,70,141). This can of course be approached through targeted studies in which the RNA-mediated regulation of a particular gene or regulon is assessed.…”

“…The most obvious function of these regulatory elements is to prevent the presence of large numbers of unbound ribosomal proteins by inhibiting transcription or translation of the downstream genes (32, 68) (see Table S1 in the supplemental material). Two of these switches are present in front of genes for the so-called primary-binding proteins S4 and S15 of the small ribosomal subunit and could thereby directly control ribosome levels (69,70).…”

“…It is also important to search for phenotypes other than downregulation of the opposite gene since this may potentially arise as a simple by-product of antisense transcription. Ideally, a phenotype that could convincingly contribute to fitness should be identified, as was exemplified for rpsD-S1136/S1134 (70).…”

“…The seventh known sense-antisense pair is represented by rpsD-S1136/S1134 (70). rpsD is an essential gene encoding the S4 component of the small ribosomal subunit.…”