The SsrA-SmpB Ribosome Rescue System Is Important for Growth ofBacillus subtilisat Low and High Temperatures

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Abstract
Bacillus subtilishas multiple stress response systems whose integrated action promotes growth and survival under unfavorable conditions. Here we address the function and transcriptional organization of a five-gene cluster containingssrA, previously known to be important for growth at high temperature because of the role of its tmRNA product in rescuing stalled ribosomes. Reverse transcription-PCR experiments detected a single message for thesecG-yvaK-rnr-smpB-ssrAcluster, suggesting that it constitutes an operon. However, rapid amplification of cDNA ends-PCR andlacZfusion experiments indicated that operon transcription is complex, with at least five promoters controlling different segments of the cluster. One σA-like promoter precededsecG(P1), and internal σA-like promoters were found in both thernr-smpB(P2) andsmpB-ssrAintervals (P3and PHS). Another internal promoter lay in thesecG-yvaKintercistronic region, and this activity (PB) was dependent on the general stress factor σB. Null mutations in the four genes downstream from PBwere tested for their effects on growth. Loss ofyvaK(carboxylesterase E) orrnr(RNase R) caused no obvious phenotype. By contrast,smpBwas required for growth at high temperature (52°C), as anticipated if its product (a small ribosomal binding protein) is essential for tmRNA (ssrA) function. Notably,smpBandssrAwere also required for growth at low temperature (16°C), a phenotype not previously associated with tmRNA activity. These results extend the known high-temperature role ofssrAand indicate that the ribosome rescue system is important at both extremes of theB. subtilistemperature range.