The SsrA-SmpB ribosome rescue system is important for growth of Bacillus subtilis at low and high temperatures

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 sigma(A)-like promoter preceded secG (P(1)), and internal sigma(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 sigma(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 degrees 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 degrees 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.

FIG. 1.

The five genes in the secG-ssrA cluster can be transcribed as a polycistronic message. (A) Map of the B. subtilis secG-ssrA region showing promoter activities located in this work (P₁, P_B, P₂, and P₃), the heat shock promoter described by Muto et al. (P_HS; reference 41), and the proposed rho-independent terminator sequence following ssrA. A full-length transcript of the region is drawn beneath the kilobase scale, together with the predicted sizes of the RT-PCR products resulting from its amplification with primers FP1, FP2, and RP. (B) RT-PCR full-length transcript detection in total RNA isolated from wild-type B. subtilis. Lanes +RT and −RT indicate with and without reverse transcriptase, lane M contains size markers, and the predicted lengths of the two RT-PCR products are on the right.

FIG. 2.

One σ^A-like and one σ^B-dependent promoter in the secG-yvaK region. (A) Diagram showing promoter location, cDNA from the RACE-PCR experiments, and fragments fused to a lacZ reporter for in vivo assay. (B) RACE-PCR products of RNA extracted from ethanol-stressed cells of wild-type PB2 (lane 1) or sigB null mutant PB153 (lane 2), with negative controls in which the poly(dA) tailing step was omitted for first-strand synthesis products from wild-type (lane 3) or sigB mutant (lane 4) RNA. Size markers are on the left, and the positions of the 596- and 233-bp products (determined by direct DNA sequencing) are indicated on the right. The 5′ ends of these products are located on the DNA sequences in panels D and E. (C) Promoter activity arising from the fragments shown in panel A was measured indirectly by β-galactosidase (β-Gal) accumulation from lacZ transcriptional fusions. Early-exponential-phase cells were stressed by the addition of 5% ethanol at time zero. Longer fragment: filled circles, PB978 (wild type); open circles, PB981 (sigBΔ3). Shorter fragment: filled triangles, PB918 (wild type); open triangles, PB919 (sigBΔ3). (D) yvaM-secG intercistronic region, with the translation start sites in bold (cat for divergently transcribed yvaM and atg for secG). The 5′ end of the 596-bp product from panel B is marked by a filled inverted triangle (P₁), with potential −35 and −10 sequences for σ^A in bold and underlined. (E) secG-yvaK intercistronic region, with the secG termination site (tag) and the yvaK start site (atg) in bold. The 5′ end of the 233-bp product from panel B is marked by a filled inverted triangle (P_B), with potential −35 and −10 sequences for σ^B in bold and underlined.

FIG. 3.

One σ^A-like promoter in the rnr-smpB interval. (A) Diagram of interval labeled as in the Fig. 2 legend. (B) RACE-PCR products of RNAs extracted from ethanol-stressed cells of wild-type PB2 (lane 1) or sigB null mutant PB153 (lane 2) and from unstressed wild-type cells (lane 3). Size markers are on the left, and the 292-bp product is labeled on the right; the 5′ end of this product is located on the sequence in panel D. (C) Promoter activity from the fragments shown in panel A measured by β-galactosidase (β-Gal) accumulation as described in the Fig. 2 legend. Longer fragment: filled circles, PB970. Shorter fragment: filled triangles, PB972. (D) rnr-smpB intercistronic region, with the rnr termination site (tag) and the smpB start site (atg) in bold. The 5′ end of the 292-bp product from panel B (determined by DNA sequencing) is marked by a filled inverted triangle (P₂), with potential −35 and −10 sequences for an extended σ^A promoter in bold and underlined.

FIG. 4.

Two σ^A-like promoter activities in the smpB-ssrA interval. (A) Diagram labeled as in the Fig. 2 legend. (B) RACE-PCR products of RNAs extracted from ethanol-stressed cells of wild-type PB2 (lane 1) or sigB null mutant PB153 (lane 2) and from unstressed wild-type cells (lane 3). Size markers are on the left, and the 241-, 295-, and 370-bp products are labeled on the right. (C) Promoter activity from the fragments shown in panel A, measured as described in the Fig. 2 legend. Longest fragment: filled circles, PB979. Middle fragment: filled triangles, PB982. Shortest fragment: filled squares, PB973. β-Gal, β-galactosidase. (D) smpB-ssrA intercistronic region, with the smpB termination site (taa) and the 5′ end (g) of the mature tmRNA in bold. The estimated 5′ ends of the 370- and 295-bp products from panel B are marked by open inverted triangles (P₃ and P_HS, respectively), with potential −35 and −10 sequences for extended σ^A promoters in bold and underlined. The 5′ end of the 241-bp product from panel B (determined by DNA sequencing) is indicated by the filled inverted triangle (ssrA) and represents the processed (or mature) 10S tmRNA.

FIG. 5.

Growth of an smpB or ssrA mutant is impaired at both high and low temperatures. For high-temperature experiments, cells were grown at 37°C in shake flasks containing either BLB medium or SMM. At A₅₇₈ = 0.5, cells were diluted 1:5 into fresh BLB medium (A) or SMM (B) and transferred to a 52°C water bath shaker. For low-temperature experiments, cells were grown in SMM, diluted 1:5 into fresh SMM (C), and transferred to a 16°C air shaker. All strains were Trp⁺ prototrophs to avoid possible transport effects. Filled circles, PB1017 (wild type); open circles, PB1018 (sigBΔ1), which served as a negative control in the low-temperature experiments (28); filled triangles, PB1019 (smpBΔ1); open triangles, PB1020 (ssrA::cat); filled squares, PB1021 (ssrA::cat with P₃, P_HS, and ssrA in trans).