The SsrA-SmpB Ribosome Rescue System Is Important for Growth of Bacillus subtilis at Low and High Temperatures

Abstract

ABSTRACT Bacillus subtilis has 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 containing ssrA , 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 the secG - yvaK - rnr - smpB - ssrA cluster, suggesting that it constitutes an operon. However, rapid amplification of cDNA ends-PCR and lacZ fusion experiments indicated that operon transcription is complex, with at least five promoters controlling different segments of the cluster. One σ A -like promoter preceded secG (P 1 ), and internal σ A -like promoters were found in both the rnr - smpB (P 2 ) and smpB - ssrA intervals (P 3 and P HS ). Another internal promoter lay in the secG - yvaK intercistronic region, and this activity (P B ) was dependent on the general stress factor σ B . Null mutations in the four genes downstream from P B were tested for their effects on growth. Loss of yvaK (carboxylesterase E) or rnr (RNase R) caused no obvious phenotype. By contrast, smpB was 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, smpB and ssrA were 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 of ssrA and indicate that the ribosome rescue system is important at both extremes of the B. subtilis temperature range.