The <i>yonT</i> mRNA is stabilized in strains depleted for RNase III.

<p>(A) Chromosomal context of the <i>yonT/as-yonT</i> toxin/antitoxin cassette present in the SPβ prophage. (B) and (C) Northern blots performed on RNA isolated at times (min) after rifampicin addition in strains depleted for RNase III (CCB288), probed for <i>yonT</i> and as-yonT, respectively. The <i>yonT</i> probe was a uniformly ³²P-labeled riboprobe, synthesized using a PCR fragment containing a T7 promoter as a template (oligos CC1101/1102; <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003181#pgen.1003181.s013" target="_blank">Table S2</a>). The blots were re-probed for 5S rRNA (5S) for normalization. Half-lives are given below each panel.</p

Cleavage of RatA, <i>txpA</i>, and the RatA/<i>txpA</i> hybrid by RNase III and RNase Y.

<p>(A) Cleavage of 5′ labeled <i>txpA</i> (0.5 pmol) alone or hybridized to a two-fold excess of unlabeled RatA by RNase III (3 ng) and RNase Y (2 µg) for the times indicated. A size standard is shown in lane M and an RNase T1 digestion of 5′ labeled <i>txpA</i> is shown in the lane labeled T1. (B) Cleavage of 5′ labeled RatA alone or hybridized to a two-fold excess of unlabeled <i>txpA</i> by RNase III and RNase Y for the times indicated. A size standard is shown in lane M and an RNase T1 digestion of 5′ labeled RatA is shown in the lane labeled T1.</p

The <i>rnc</i> gene can be deleted in strains lacking Skin and SPβ prophages.

<p>(A) Agar plates showing colony growth after transformation of wild-type (WT) strains and strains lacking up to three prophages with 5 µg CCB302 (<i>rnc::spc amyE::Pxyl-rnc</i> Cm) chromosomal DNA, selected for spectinomycin (left panel) or chloramphenicol (right panel) resistance. (B) Histograms showing transformation efficiencies (number of Spc^R colonies/number Cm^R colonies) for the different prophage deficient strains.</p

Doubling time of <i>rnc</i> mutants and parental strains.

<p>Values are the average of 3 independent experiments.</p

Suppressors of the <i>rnc::spc</i> mutation lack the Skin prophage.

<p>(A) Agarose gel showing multiplex PCR analysis of <i>rnc::spc</i> suppressors in wild-type (WT) and ΔSPβ strains. A PCR product corresponding to the reconstituted <i>ypqP</i> and <i>sigK</i> genes is indicative of excision of the SPβ and Skin prophages, respectively. Spontaneous Spc^R colonies show a PCR product for the <i>rnc</i> gene, while successfully deleted <i>rnc</i> strains do not give a corresponding PCR product. A DNA marker (bp) is shown in the lane labeled M. (B) Sequence comparison of the <i>txpA</i> gene in wild-type (wt) and the sup5 mutant shown in panel A.</p

The Essential Function of <em>B. subtilis</em> RNase III Is to Silence Foreign Toxin Genes

<div><p>RNase III–related enzymes play key roles in cleaving double-stranded RNA in many biological systems. Among the best-known are RNase III itself, involved in ribosomal RNA maturation and mRNA turnover in bacteria, and Drosha and Dicer, which play critical roles in the production of micro (mi)–RNAs and small interfering (si)–RNAs in eukaryotes. Although RNase III has important cellular functions in bacteria, its gene is generally not essential, with the remarkable exception of that of <em>Bacillus subtilis</em>. Here we show that the essential role of RNase III in this organism is to protect it from the expression of toxin genes borne by two prophages, Skin and SPβ, through antisense RNA. Thus, while a growing number of organisms that use RNase III or its homologs as part of a viral defense mechanism, <em>B. subtilis</em> requires RNase III for viral accommodation to the point where the presence of the enzyme is essential for cell survival. We identify <em>txpA</em> and <em>yonT</em> as the two toxin-encoding mRNAs of Skin and SPβ that are sensitive to RNase III. We further explore the mechanism of RNase III–mediated decay of the <em>txpA</em> mRNA when paired to its antisense RNA RatA, both <em>in vivo</em> and <em>in vitro</em>.</p> </div

The <i>txpA</i> and RatA RNAs are stabilized in strains depleted for RNase III and RNase Y, respectively.

<p>(A) Chromosomal context of the <i>txpA</i>/RatA toxin/antitoxin cassette present in the Skin prophage. (B) and (C) Northern blots performed on RNAs isolated at times (min) after rifampicin addition (150 µg/ml) in strains depleted for RNase III (CCB288), RNase Y (CCB294) and RNase J1 (CCB034), probed for <i>txpA</i> and RatA, respectively. Northerns were re-probed for 5S rRNA (5S) for normalization. Half-lives are given below each panel. The band labeled D in panel C (RNase J1) is a degradation intermediate of RatA. Note that, in our hands, the <i>txpA</i> mRNA is about 45 nts longer than that proposed in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003181#pgen.1003181-Silvaggi1" target="_blank">[24]</a> and consistent with the presence of a Rho-independent transcription terminator ∼270 nts from the mapped transcription start site. The overlap between RatA and <i>txpA</i> is predicted to be ∼120 nts.</p

Degradation of <i>txpA</i> (AUG→AAG) mRNA by RNase III is RatA-dependent.

<p>Northern blots performed on RNA isolated at times (min) after rifampicin addition in (A) strain CCB467 <i>txpA</i> (AUG→AAG) and (B) strain CCB468 <i>txpA</i> (AUG→AAG) P<i>ratA::ery</i>, depleted or not for RNase III. Northerns were re-probed for 5S rRNA (5S) for normalization. Half-lives are given below each panel.</p

The <i>rnc</i> gene can be deleted in strains lacking TxpA and YonT toxins.

<p>Histogram showing transformation efficiencies (number of Spc^R colonies/number Cm^R colonies) for strains lacking different toxin genes or mRNAs.</p

Degradation of the <i>spoIISAB</i> mRNA depends on both RNase Y and J1.

<p>(A) Structure and predicted degradation pathway(s) of the <i>spoIISAB</i> transcript. ORFs are shown as large white arrows, transcripts as thin black arrows. Scissors indicate cleavage by RNase Y, ‘Pacman’ symbols represent 5′-3′ degradation by RNase J1. A short thick line indicates the position of the probe used. Pσ^A indicates the approximate promoter position and relevant sigma factor mapped in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002520#pgen.1002520-Adler1" target="_blank">[26]</a>. (B) Northern blot of total mRNA isolated at times after addition of rifampicin (rif) from wild-type and RNase Y depleted (<i>Pspac-rny</i>−IPTG) and RNase Y induced (<i>Pspac-rny</i>+IPTG) cells. The blot was probed with 5′-labeled oligo CCB826 (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002520#pgen.1002520.s015" target="_blank">Table S6</a>) and reprobed with oligo HP246 against 5S rRNA. The half-lives of the different RNA species (A, B) from the <i>spoIISAB</i> are given under the Northern blot. Migration positions of RNA markers are shown to the right of the blot. Some cross-hybridization to the marker is visible in the lane between the (−) and (+) IPTG samples. (C) Northern blot of total mRNA isolated at times after addition of rifampicin (rif) from wild-type and RNase J1 depleted (<i>Pspac-rnjA</i>−IPTG) and RNase J1 induced (<i>Pspac-rnjA</i>+IPTG) cells. Description as in panel (B).</p