Logo of jbacterPermissionsJournals.ASM.orgJournalJB ArticleJournal InfoAuthorsReviewers
J Bacteriol. May 2007; 189(10): 3729–3737.
Published online Mar 16, 2007. doi:  10.1128/JB.00062-07
PMCID: PMC1913333

The SsrA-SmpB Ribosome Rescue System Is Important for Growth of Bacillus subtilis at Low and High Temperatures[down-pointing small open triangle]

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 σA-like promoter preceded secG (P1), and internal σA-like promoters were found in both the rnr-smpB (P2) and smpB-ssrA intervals (P3 and PHS). Another internal promoter lay in the secG-yvaK intercistronic region, and this activity (PB) was dependent on the general stress factor σB. Null mutations in the four genes downstream from PB 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.

Bacteria must coordinate multiple stress response networks to grow and survive under changing environmental conditions. Here we focus on the transcriptional organization and stress-related function of the secG-yvaK-rnr-smpB-ssrA gene cluster in Bacillus subtilis. We were drawn to this cluster by transcriptional profiling experiments designed to identify elements of the general stress regulon controlled by σB, which is activated by diverse stimuli to provide broad resistance against future challenges (reviewed in references 20 and 46). The profiling studies indicate that the adjacent yvaK and rnr genes are under σB control (8, 22, 45, 47), and from sequence inspection, two of the studies suggested that a σB-dependent promoter precedes yvaK (22, 45). However, this promoter was sufficiently divergent from most well-defined σB promoters to be missed by a hidden Markov model analysis (47), and it has not been experimentally characterized.

As we shall describe elsewhere, we pooled and reanalyzed the available profiling data in an effort to identify new elements of the σB regulon that may have escaped detection in the individual studies (P. Fawcett, A. Weigel, and C. W. Price, unpublished data). Of relevance here, the reanalysis suggested that, in addition to yvaK and rnr, the adjacent smpB gene was also partly under σB control. Given that the only rho-independent terminator sequence for the five-gene cluster lies downstream from ssrA, the reanalysis left open the possibility that ssrA expression was also influenced by σB. This regulation would have been overlooked by the profiling studies, which did not include ssrA in their arrays because it does not encode a protein product.

The ssrA gene codes for tmRNA, which functions as both a tRNA and an mRNA to rescue ribosomes that are unproductively stalled on a message (reviewed in references 31 and 59). As part of this trans-translation process, a peptide tag is added to the C-terminal end of the released protein, thus targeting it for proteolysis (17, 35, 58). The presence of ssrA in all of the sequenced prokaryotic genomes indicates the biological importance of the trans-translation system (31). However, ssrA is known to be essential in only a few species, and for enteric bacteria and B. subtilis its loss is largely manifested in stress-related phenotypes. For example, an Escherichia coli ssrA mutant has a decreased ability to grow at 45°C (36) and also exhibits some regulatory anomalies and a variety of phage developmental phenotypes (31, 59). In another example, loss of ssrA function significantly affects pathogenesis in Salmonella enterica and Yersinia pseudotuberculosis, in part because of an inability of the mutants to survive in macrophages (4, 29, 43). And in B. subtilis, ssrA is important for growth at 52°C or under conditions of cadmium or ethanol stress (41). In all of the cases tested, ssrA activity requires the SmpB protein, which mediates the binding of ssrA to the ribosome and performs at least one additional downstream function in the trans-translation process (33, 34, 52, 58).

Given that the ssrA gene is known to have stress-related functions in B. subtilis, control of its expression by the general stress factor σB was an intriguing possibility. However, the work of Muto et al. (41) indicated the presence of a heat shock promoter in the smpB-ssrA intercistronic region, and additional promoters controlling the cluster could not be excluded. We therefore focused on the transcriptional organization of the secG-ssrA region with the aim of locating its principal promoter activities. This analysis found at least five, including a weak σB-dependent promoter preceding yvaK and also strong σA-like promoters preceding smpB and ssrA that would facilitate their differential expression. These data, in turn, allowed us to make nonpolar null mutations to probe the importance of individual gene products for growth at the extremes of the B. subtilis temperature range. On the basis of these results, we suggest that the genes in the secG-ssrA cluster form a functional module beneficial to members of the order Bacillales.

MATERIALS AND METHODS

Bacterial strains and genetic methods.

The B. subtilis strains used in this study are listed in Table Table1.1. Standard recombinant DNA methods were according to reference 49, and transformation of B. subtilis PB2 and derivatives was according to reference 14. Plasmids used for these constructions are shown in Table Table2.2. To locate promoter activity, fragments were PCR amplified and cloned into transcriptional fusion vector pDG268, which integrates in single copy at the amyE locus (2). To make in-frame smpBΔ1, removing triplets 4 to 153, a four-primer fragment (24) was cloned into the pCP115 vector (6) to create pJH13; smpBΔ1 was then substituted for the wild-type allele via a two-step replacement procedure (51). To complement the ssrA::cat insertion in trans, a fragment was PCR amplified and cloned into the pDG1730 vector (19), placing P3, PHS, and ssrA in single copy at amyE. DNA sequences were confirmed for all of the constructs.

TABLE 1.
B. subtilis strains used in this study
TABLE 2.
Plasmid constructions used in this study

Reverse transcription (RT)-PCR and rapid amplification of cDNA ends (RACE)-PCR experiments.

Shake cultures were grown at 37°C to mid-exponential phase in buffered Luria broth (BLB) medium lacking salt (5) and then diluted 1:25 into fresh BLB medium. At mid-exponential phase, some cultures were subjected to a 10-min ethanol stress (5%, vol/vol) while others were allowed to grow untreated before harvesting and RNA extraction. Total RNA was isolated with the RNeasy kit (QIAGEN Inc., Valencia, CA) according to the manufacturer's instructions, except that the lysozyme concentration was increased to 15 mg/ml and a homogenization step (passage through a 20-gauge needle) was added.

For RT-PCR, RNA was extracted from wild-type (PB2) cells and the RT reaction was done according to reference 49. One microgram of total RNA and 25 pmol of gene-specific primer 5′-GATTACTTAAGCGTCTACG-3′ were incubated at 65°C for 10 min, and then Transcriptor reverse transcriptase (Roche Applied Science, Mannheim, Germany) was added and incubation was continued at 50°C for 90 min. The resulting cDNA was amplified in a PCR with Taq polymerase (Promega, Madison, WI). As a negative control, the RT reaction was performed without the addition of reverse transcriptase. To detect a yvaK-to-ssrA complement, the forward primer FP1 (5′-CGGATGTAAGGATGCTGGGA-3′) was used with the reverse primer RP (5′-GGTTTCACTCATCTTCTTGCC-3′); to detect a secG-to-ssrA complement, the FP2 forward primer (5′-GGTTATCGTCAGCATTGC-3′) was used with the same reverse primer. PCR products were analyzed by agarose gel electrophoresis.

For RACE-PCR (16), RNA was extracted from wild-type (PB2) or sigB mutant (PB153) cells. RACE-PCRs were done as previously described (47), with a primer complementary to the poly(A) tail (5′-GACCACGCGTATCGATGTCGACT16V-3′ [where V = A, C, or G]) and two nested, gene-specific primers, (i) YVAK1 (outer, 5′-GTATGTACAAGTTCTTCAGGC-3′) and YVAK2 (inner, 5′-GACGCCATGTCCTTCATATTGAG-3′); (ii) SMPB1 (5′-GTTATAGCGGTTTCCCTG-3′) and SMPB2 (5′-GGGCTGACGTGCATATTGTGG-3′); or (iii) SSRA1 (5′-TCTTCTTACGTTCTCAGA-3′) and SSRA2 (5′-CGCAAGCGTAGCCTACTTGGA-3′). PCR products were separated on agarose gels, purified and sequenced.

β-Galactosidase accumulation assays.

Shake cultures were grown at 37°C in BLB medium and diluted 1:25 into fresh medium as described for the RNA experiments above. At mid-exponential phase, cultures were subjected to ethanol stress (5%, vol/vol). Samples were collected at the indicated times and assayed according to Miller (39), by using sodium dodecyl sulfate and chloroform to permeabilize the cells. Protein levels were determined with the Bio-Rad protein assay reagent (Bio-Rad Laboratories, Hercules, CA). Activity was defined as ΔA420 × 1,000 per minute per milligram of protein.

Growth experiments.

High-temperature growth experiments were done essentially as described by Holtmann et al. (26), and low-temperature experiments were done essentially as described by Brigulla et al. (7). For high-temperature experiments, wild-type and mutant cells were precultured at 37°C in shake flasks containing either BLB medium or Spizizen's minimal medium with 0.5% glucose (SMM; reference 1). At A578 = 0.5, exponentially growing cells were diluted 1:5 into fresh BLB medium or SMM and transferred to a 52°C water bath shaker. For low-temperature experiments, cells were similarly precultured in SMM, diluted 1:5 into fresh SMM, and transferred to a 16°C air shaker. Growth was monitored with a Klett-Summerson colorimeter with a number 66 (red) filter; units were plotted on a linear scale versus time, in accordance with references 7 and 26.

RESULTS

The secG-ssrA gene cluster can be transcribed as a single polycistronic message.

As shown in Fig. Fig.1A,1A, the secG-ssrA region encodes only a single rho-independent terminator sequence that lies downstream from ssrA, leading us to hypothesize that the five-gene cluster could be transcribed as a single message. We used RT-PCR to look for such a message in RNA isolated from unstressed, exponential-phase B. subtilis cells. Two different forward primers were designed, one to anneal to the secG message and the other to anneal to the yvaK message. In conjunction with a reverse primer designed to anneal to the ssrA message, the secG primer was expected to amplify a product of 4,508 bp and the yvaK primer was expected to amplify a product of 4,087 bp.

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, PB, P2, and P3), the heat shock promoter described by Muto et al. ...

As shown in Fig. Fig.1B,1B, products of the predicted size were found in the reaction mixtures with reverse transcriptase. These products were missing from the reaction mixtures lacking transcriptase, ruling out the possibility that they arose from amplification of contaminating DNA. We conclude that at least some full-length message is transcribed from the secG-ssrA cluster and that these genes constitute an operon. However, further analysis revealed the existence of multiple promoters within this region, some stress inducible. These promoters fell into three sets: (i) two in the secG-yvaK region, (ii) one in the rnr-smpB interval, and (iii) two in the smpB-ssrA interval.

The secG-yvaK region.

Expression profiling experiments previously identified yvaK as a candidate element of the σB regulon (22, 45, 47); its product is a carboxylesterase of unknown function (23). While a putative σB-dependent promoter for yvaK could be located by inspection, it has not been characterized experimentally. With RNA isolated from cells that had been ethanol stressed to increase σB activity, RACE-PCR experiments found several potential 5′ ends for the yvaK message. We could obtain sequences from the two most prominent PCR products (Fig. 2A and B), locating one 5′ end to a site 24 nucleotides (nt) upstream from the ATG initiation triplet of yvaK (Fig. (Fig.2E)2E) and the other to a site 31 nt upstream from the ATG of secG (Fig. (Fig.2D).2D). The yvaK 5′ end was preceded by sequences previously suggested to define the σB-dependent promoter, with GtTTTt at −35 and tGGaAa at −10 (22, 45); this 5′ end was not apparent when RNA extracted from ethanol-stressed cells of a sigB null mutant was used (Fig. (Fig.2B).2B). We provisionally call this promoter PB. By contrast, the secG 5′ end was preceded by sequences resembling a σA promoter, with gTGACA at −35 and TAaAAT at −10. This signal was diminished but still detectable in the sigB null mutant, while more rapidly migrating minor bands increased in intensity, suggesting that they were decay products of the secG message. We provisionally call this promoter P1. The 5′ end associated with P1 was found with a primer within yvaK, indicating that the secG message extends into the yvaK reading frame.

FIG. 2.
One σA-like and one σB-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 ...

To test if the secG-yvaK region contained functional promoter activities, we made two transcriptional fusions to a lacZ reporter gene (Fig. (Fig.2A).2A). One carried a fragment extending from the divergently transcribed yvaM gene preceding secG to a site within yvaK; this would be expected to contain both the P1 and PB activities suggested by RACE-PCR. The other carried a shorter fragment that removed 387 bp from the 5′ end of the first; this would be expected to contain only PB activity. These fusions were placed in single copy at the amyE chromosomal locus. As shown in Fig. Fig.2C,2C, in the wild type the longer fusion manifested promoter activity in unstressed cells, and this activity increased following ethanol stress. In the sigB null mutant, this same fusion had slightly less activity in unstressed cells and only a modest increase following stress. By contrast, in the wild type the shorter fusion had very low activity in unstressed cells and a significant increase following ethanol stress, as is the case with well-characterized σB-dependent fusions. Moreover, this stress-induced increase in activity was abolished in the sigB null mutant. The fusion data are consistent with the RACE-PCR assays and led us to conclude that at least two promoters lie in the secG-yvaK region, i.e., a σB-independent P1 activity preceding secG and a σB-dependent PB activity preceding yvaK. On the basis of the β-galactosidase accumulation assays, PB appears to be relatively weak, consistent with its sequence divergence from more typical σB-dependent promoters.

The rnr-smpB interval.

A similar analysis located promoter activity in the rnr-smpB interval. RACE-PCR produced a single abundant product of about 290 bp that did not appear to differ quantitatively among the three sources of RNA—wild-type cells with or without ethanol stress and sigB mutant cells with ethanol stress (Fig. 3A and B). Sequencing of the PCR product located its 5′ end to a site 47 nt upstream from the ATG initiation triplet of smpB (Fig. (Fig.3D).3D). This 5′ end was preceded by sequences resembling an extended σA promoter (21), here provisionally called P2, with TTGtag at −35 and TGgTAaAAT at −10. We made two lacZ fusions to determine whether the region containing these sequences had promoter activity (Fig. (Fig.3A).3A). The first carried a fragment extending from within the 748th triplet of the upstream rnr gene to a site within smpB, and the second removed 198 bp from the 5′ end of this fragment, deleting the proposed −35, −10, and +1 sequences of P2. The longer fragment indeed contained promoter activity, and this increased less than twofold in ethanol-stressed cells (Fig. (Fig.3C).3C). By contrast, the fusion bearing the shorter fragment had no detectable activity. From the RACE-PCR and fusion results, we conclude that the rnr-smpB interval contains at least one promoter, P2, which is modestly induced by ethanol stress.

FIG. 3.
One σA-like promoter in the rnr-smpB interval. (A) Diagram of interval labeled as in the Fig. Fig.22 legend. (B) RACE-PCR products of RNAs extracted from ethanol-stressed cells of wild-type PB2 (lane 1) or sigB null mutant PB153 (lane ...

The smpB-ssrA interval.

Because of the rapid ssrA transcript processing to yield mature 10S tmRNA, fusion analysis was key in locating two potential promoter activities in the smpB-ssrA interval. RACE-PCR produced three products of 241, 295, and 370 bp from RNAs isolated from ethanol-stressed wild-type and sigB mutant cells (Fig. 4A and B, lanes 1 and 2). Of these, the 370-bp product appeared to be reduced or absent when RNA from unstressed wild-type cells was used (Fig. (Fig.4B,4B, lane 3). DNA sequencing was able to locate the 5′ end for the 241-bp principal product, which was the 5′ end of the mature 10S tmRNA (Fig. (Fig.4D).4D). However, no sequence data could be obtained for the 295- and 370-bp minor products, so we estimated the locations of their 5′ ends on the basis of gel mobility. The 295-bp product appeared to correspond to a transcript from the ssrA heat shock promoter mentioned by Muto and colleagues (41); its estimated 5′ end lay downstream from sequences resembling an extended σA promoter, here called PHS, with TTGAaA at −35 and TGtTATAAT at −10 (Fig. (Fig.4D).4D). The 370-bp product had an estimated 5′ end that lay downstream from another set of sequences resembling an extended σA promoter, here provisionally called P3, with TTGAtt at −35 and TGcTATAcT at −10 (Fig. (Fig.4D4D).

FIG. 4.
Two σA-like promoter activities in the smpB-ssrA interval. (A) Diagram labeled as in the Fig. Fig.22 legend. (B) RACE-PCR products of RNAs extracted from ethanol-stressed cells of wild-type PB2 (lane 1) or sigB null mutant PB153 (lane ...

We made three lacZ fusions to determine whether the sequences containing P3 and PHS were important for promoter activity (Fig. (Fig.4A).4A). The first carried a fragment extending from within the 86th triplet of the upstream smpB gene to a site within ssrA; the second removed 269 bp from the 5′ end of this fragment, deleting the proposed −35, −10, and +1 sequences for P3; and the third removed an additional 110 bp, deleting the proposed −35, −10, and +1 sequences for PHS. The longest fragment had strong promoter activity, and this increased about threefold in ethanol-stressed cells (Fig. (Fig.4C).4C). With the second fragment, this activity significantly decreased, consistent with the loss of P3, and ethanol induction was no longer apparent. With the third fragment, there was no detectable activity, consistent with the loss of PHS. From the fusion results shown in Fig. Fig.4C,4C, P3 is ethanol inducible, a conclusion in accord with the signals present in the RACE-PCR experiment shown in Fig. Fig.4B4B (compare the strengths of the 370-bp signals in lanes 1 and 3). From the sum of the RACE-PCR and fusion results, we conclude that the smpB-ssrA interval contains at least two promoter activities, P3 and PHS.

Loss of smpB or ssrA function has a significant effect on growth at both high and low temperatures.

Muto et al. (41) previously showed that ssrA function was required for robust growth of B. subtilis at high temperature and under conditions of ethanol or cadmium stress. Moreover, additional genes in the B. subtilis cluster are known to have stress- or tmRNA-related functions in other bacteria. For example, RNase R is a processive 3′-to-5′ exonuclease that can degrade RNA with significant secondary structure (11, 44); it has been implicated in quality control of rRNA, as well as degradation of certain aberrant mRNAs recognized by the trans-translation system (12, 48). This latter activity is consistent with the copurification from E. coli extracts of a complex containing tmRNA, SmpB, and substoichiometric amounts of RNase R (32). A role for E. coli RNase R in adapting to environmental transitions is suggested by its elevated level following cold stress, starvation, or entry into stationary phase (9, 10).

In order to uncover their possible roles in stress resistance, we constructed null alleles in the four genes downstream from the PB promoter in the B. subtilis cluster and tested their effects on growth at high and low temperatures. It proved difficult to make strains bearing in-frame deletions within yvaK or rnr, so these were built with the pMUTIN4 plasmid, which disrupts the gene of interest while ensuring continued transcription of downstream genes (53). We were able to construct a strain bearing a deletion (smpBΔ1) that removed most of the smpB coding region; this in-frame deletion did not affect the initiation or termination signals for smpB transcription and translation. We also moved the ssrA::cat insertion of Muto et al. (41) into our genetic background so that all of the constructed strains were isogenic (Table (Table11).

In agreement with the results of Muto et al. (41), we found that the ssrA::cat insertion had a major impact on growth when cells were shifted from 37 to 52°C and that this phenotype was complemented in trans by a fragment containing P3, PHS, and ssrA (Fig. 5A and B). We also established that the smpBΔ1 null allele had the same high-temperature phenotype, as expected if SmpB is required for ssrA function (33, 34, 58). Our significant new findings are that (i) ssrA::cat had an equally striking impact on growth when cells were shifted from 37 to 16°C; (ii) this low-temperature phenotype could be fully complemented by a fragment containing P3, PHS, and ssrA; and (iii) the smpBΔ1 allele had the same phenotype (Fig. (Fig.5C).5C). smpBΔ1 is an in-frame deletion that should not affect the expression of downstream genes, and the complementation test indicated that loss of ssrA function alone was sufficient to cause the low-temperature phenotype. Together, these results argue that it was the absence of a functional ribosome rescue system and not the production of a tmRNA fragment from the interrupted ssrA::cat allele that had the harmful effect. We conclude that smpB and ssrA are each required for robust growth at both low and high temperatures and that the promoters on the P3-PHS-ssrA fragment provide sufficient ssrA expression to support growth under both conditions.

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 ...

In contrast, the yvaK or rnr null allele had no substantial effect on growth at either temperature extreme (data not shown). These rnr results are in agreement with the earlier study of Höper et al. (28), who noted that loss of rnr function had no significant effect on growth or survival in heat or cold.

DISCUSSION

It is uncommon for loss of a single cellular system to have only a slight impact under optimal conditions while significantly affecting growth at both temperature extremes, as we have shown here for the B. subtilis trans-translation apparatus encoded by smpB and ssrA. To our knowledge, the only comparable example is the ability of B. subtilis to transport compatible solutes, which help maintain protein functionality at both the 52°C maximum growth temperature and the 15°C standard used for cold stress experiments (7, 25). The striking phenotype caused by loss of smpB or ssrA function reflects the importance of maintaining cellular translational capacity at the extremes of the temperature range.

At optimal temperatures, absence of a trans-translation system reduces the growth rates of E. coli and B. subtilis only marginally, if at all (33, 36, 41). This absence of a strong phenotype is likely due to a low level of stalling under these conditions and also to the presence of alternative routes of spontaneous or factor-dependent recycling that result in peptidyl-tRNA drop off, a process which ultimately requires peptidyl-tRNA hydrolase to restore the affected tRNA (17, 38, 40, 58). However, trans-translation is more efficient in releasing stalled ribosomes and in reducing the peptidyl-tRNA load (50). Its loss would therefore become more telling at maximum growth temperatures, when unbalanced tRNA pools and a sharp increase in truncated messages significantly increase stalling. Absence of trans-translation in B. subtilis has a greater impact on high-temperature growth than is the case for E. coli (33, 36, 41). This difference may reflect a decreased ability of the alternative routes of ribosome recycling or of peptidyl-tRNA hydrolysis to cope with high-temperature demand in B. subtilis.

We have now shown that absence of trans-translation in B. subtilis has a significant impact on low-temperature growth (Fig. (Fig.5),5), another phenotype not strongly manifested in E. coli (33). What is the role of this system at low temperature? Inefficient translational initiation appears to be the rate-limiting step for bacterial growth in the cold (15), and production of helicases or cold shock proteins is a known adaptation by which to cope with the increased RNA secondary structure that contributes to this low rate (57). On the basis of the results presented here, we suggest that recycling of stalled ribosomes by trans-translation also makes a critical contribution to initiation in cold-stressed B. subtilis. This quality control system would both maintain the pool of actively translating ribosomes and release needed amino acids, thereby facilitating synthesis of the proteins required to adapt to cold conditions.

What is the significance of the gene arrangement found in the secG-ssrA region? This particular organization does not appear to be explained by a need for coregulation. Although all five genes can be transcribed as a single message, presumably from the P1 promoter preceding secG, internal promoters allow differential expression of individual genes and sets of genes. Assuming that our β-galactosidase assays roughly correlate with promoter strength, P2 (preceding smpB) and P3/PHS (preceding ssrA) are particularly strong. We speculate that the strength of these promoters and the translation efficiency of the smpB message have coevolved such that SmpB and tmRNA are produced in a physiologically appropriate stoichiometry during unstressed growth at mesophilic temperatures. If this is the case, the increased transcription of ssrA from P3 and PHS following ethanol or heat stress (Fig. (Fig.4;4; reference 41) implies that tmRNA has an SmpB-independent function or that tmRNA is less stable under these conditions. The latter possibility was first advanced in the model of Hong et al. (27), who suggested that E. coli tmRNA is degraded following its unfolding or cleavage under stress conditions. For example, diverse stresses are known to activate the E. coli RelE and MazF toxins to induce cleavage of both mRNA and tmRNA (see reference 18 and references therein). Toxin-induced cleavage of mRNA is thought to assist stress adaptation by recycling and redirecting ribosomes as needed, but the accompanying cleavage of tmRNA seems to be counterproductive for this adaptation process. Thus, if transcription of B. subtilis ssrA is indeed increased relative to smpB, it may serve to compensate for such a stress-induced instability. A detailed investigation of SmpB and tmRNA synthesis and stability addressed the important role of these processes in controlling cell cycle progression in Caulobacter crescentus (27). On the basis of the initial results reported here and by others (41), a similar study of stress-induced changes in the B. subtilis system may be warranted.

There seems to be little physiological need for transcription from the relatively weak PB promoter preceding yvaK to read into ssrA, for the simple reason that the fragment containing P3/PHS had sufficient promoter activity to fully complement in both heat and cold (Fig. (Fig.5).5). Although we cannot rule out the possibility that PB-directed transcription into ssrA plays a role under special conditions, it seems that PB primarily serves to increase yvaK, rnr, and possibly smpB expression under the same circumstances that promote induction of E. coli rnr, i.e., starvation, entry into stationary phase, or cold stress (9, 10). Indeed, an E. coli rnr mutant has a cold stress phenotype (9). However, the absence of a similar phenotype for a B. subtilis rnr mutant suggests that this activity is not essential under cold conditions or that its stress-related function partly overlaps that of another RNase.

Although a need for coregulation does not appear to have strongly shaped the genetic organization of the secG-ssrA region, the clustering itself supports an inference of a functional relationship among seemingly diverse gene products (56). For example, products that associate in a complex are more likely to be encoded by clustered genes (13), and this driving force may be part of the basis for the rnr-smpB-ssrA arrangement (32, 48). However, physical association does not seem to explain the presence of secG or yvaK in the cluster. secG encodes an auxiliary membrane protein that interacts with the SecA-SecE-SecY translocase; its loss causes a growth defect under conditions in which membrane function is compromised, such as at low temperature or under high secretory demand (54). And yvaK encodes carboxylesterase E, which hydrolyzes short- and medium-length fatty acid esters in vitro (23); its in vivo substrate and function are unknown. Nonetheless, analysis of sequenced genomes indicates that a conserved gene order is one of the strongest predictors of a functional module in which dissimilar gene products together affect a common cellular process (56). Horizontal gene transfer could provide the selection for the assembly and maintenance of such a cluster, thereby facilitating the cotransfer of complex capabilities (reviewed in reference 37). On the basis of the cold-sensitive phenotypes of secG and smpB/ssrA mutants, we suggest that the cluster represents a functional module that confers enhanced adaptation to cold stress. In this view, the YvaK carboxylesterase would also contribute to adaptation, a suggestion reinforced by the common regulation of yvaK and rnr by the general stress factor σB. Such an enhanced adaptation might be especially important if the capacity of the B. subtilis trans-translation system is less than that of E. coli, as has been suggested (40).

Evidence for conservation of the secG-ssrA cluster and suggestion of its horizontal transfer can be found in the STRING database, version 6.3 (55), which shows this particular cluster to be largely restricted to the order Bacillales. Here the five-gene cluster is exactly conserved among some representatives, such as Bacillus cereus and Staphylococcus aureus, but not in others, such as Listeria monocytogenes, in which ssrA lies at a different locus. From the absolute conservation of the secG-yvaK-rnr-smpB arrangement, we infer that these four genes form the critical core of the functional module or were the first to assemble. This inference is supported by the occurrence of the same four-gene cluster within two taxa of the order Lactobacillales, Lactobacillus plantarum and Enterococcus faecalis (55). This restricted occurrence in a related but distinct lineage is consistent with the horizontal transfer of the secG-yvaK-rnr-smpB cluster from the order Bacillales (42). If we are correct in our inference that the cluster forms a functional module with an important role in cold adaptation, its presence in pathogens such as B. cereus and L. monocytogenes likely contributes to their ability to grow at refrigerator temperatures and cause significant food-borne illness.

Acknowledgments

We thank Akira Muto, Eugenio Ferarri, Marc Kolkman, and Wolfgang Schumann for kindly providing strains. We also thank Kenneth Keiler, John Roth, Valley Stewart, and Uwe Völker for helpful discussions.

This research was supported by Public Health Service grant GM42077 from the National Institute of General Medical Sciences.

Footnotes

[down-pointing small open triangle]Published ahead of print on 16 March 2007.

REFERENCES

1. Anagnostopoulos, C., and J. Spizizen. 1961. Requirements for transformation in Bacillus subtilis. J. Bacteriol. 81:741-746. [PMC free article] [PubMed]
2. Antoniewski, C., B. Savelli, and P. Stragier. 1990. The spoIIJ gene, which regulates early developmental steps in Bacillus subtilis, belongs to a class of environmentally responsive genes. J. Bacteriol. 172:86-93. [PMC free article] [PubMed]
3. Band, L., H. Shimotsu, and D. J. Henner. 1984. Nucleotide sequence of the Bacillus subtilis trpE and trpD genes. Gene 27:55-65. [PubMed]
4. Bäumler, A. J., J. G. Kusters, I. Stojiljkovic, and F. Heffron. 1994. Salmonella typhimurium loci involved in survival within macrophages. Infect. Immun. 62:1623-1630. [PMC free article] [PubMed]
5. Boylan, S. A., A. R. Redfield, M. S. Brody, and C. W. Price. 1993. Stress-induced activation of the σB transcription factor of Bacillus subtilis. J. Bacteriol. 175:7931-7937. [PMC free article] [PubMed]
6. Boylan, S. A., M. D. Thomas, and C. W. Price. 1991. Genetic method to identify regulons controlled by nonessential elements: isolation of a gene dependent on alternate transcription factor σB of Bacillus subtilis. J. Bacteriol. 173:7856-7866. [PMC free article] [PubMed]
7. Brigulla, M., T. Hoffmann, A. Krisp, A. Völker, E. Bremer, and U. Völker. 2003. Chill induction of the SigB-dependent general stress response in Bacillus subtilis and its contribution to low-temperature adaptation. J. Bacteriol. 185:4305-4314. [PMC free article] [PubMed]
8. Budde, I., L. Steil, C. Scharf, U. Völker, and E. Bremer. 2006. Adaptation of Bacillus subtilis to growth at low temperature: a combined transcriptomic and proteomic appraisal. Microbiology 152:831-853. [PubMed]
9. Cairrão, F., A. Cruz, H. Mori, and C. M. Arraiano. 2003. Cold shock induction of RNase R and its role in the maturation of the quality control mediator SsrA/tmRNA. Mol. Microbiol. 50:1349-1360. [PubMed]
10. Chen, C., and M. P. Deutscher. 2005. Elevation of RNase R in response to multiple stress conditions. J. Biol. Chem. 280:34393-34396. [PubMed]
11. Cheng, Z. F., and M. P. Deutscher. 2005. An important role for RNase R in mRNA decay. Mol. Cell 17:313-318. [PubMed]
12. Cheng, Z. F., and M. P. Deutscher. 2003. Quality control of ribosomal RNA mediated by polynucleotide phosphorylase and RNase R. Proc. Natl. Acad. Sci. USA 100:6388-6393. [PMC free article] [PubMed]
13. Dandekar, T., B. Snel, M. Huynen, and P. Bork. 1998. Conservation of gene order: a fingerprint of proteins that physically interact. Trends Biochem. Sci. 23:324-328. [PubMed]
14. Dubnau, D., and R. Davidoff-Abelson. 1971. Fate of transforming DNA following uptake by competent Bacillus subtilis. I. Formation and properties of the donor-recipient complex. J. Mol. Biol. 56:209-221. [PubMed]
15. Farewell, A., and F. C. Neidhardt. 1998. Effect of temperature on in vivo protein synthetic capacity in Escherichia coli. J. Bacteriol. 180:4704-4710. [PMC free article] [PubMed]
16. Frohman, M. 1994. On beyond classic RACE (rapid amplification of cDNA ends). PCR Methods Appl. 4:540-558. [PubMed]
17. Fujihara, A., H. Tomatsu, S. Inagaki, T. Tadaki, C. Ushida, H. Himeno, and A. Muto. 2002. Detection of tmRNA-mediated trans-translation products in Bacillus subtilis. Genes Cells 7:343-350. [PubMed]
18. Gerdes, K., S. K. Christensen, and A. Løbner-Olesen. 2005. Prokaryotic toxin-antitoxin stress response loci. Nat. Rev. Microbiol. 3:371-382. [PubMed]
19. Guérout-Fleury, A. M., N. Frandsen, and P. Stragier. 1996. Plasmids for ectopic integration in Bacillus subtilis. Gene 180:57-61. [PubMed]
20. Hecker, M., and U. Völker. 2001. General stress response of Bacillus subtilis and other bacteria. Adv. Microb. Physiol. 44:35-91. [PubMed]
21. Helmann, J. D. 1995. Compilation and analysis of Bacillus subtilis σA-dependent promoter sequences: evidence for extended contact between RNA polymerase and upstream promoter DNA. Nucleic Acids Res. 23:2351-2360. [PMC free article] [PubMed]
22. Helmann, J. D., M. F. Wu, P. A. Kobel, F. J. Gamo, M. Wilson, M. M. Morshedi, M. Navre, and C. Paddon. 2001. Global transcriptional response of Bacillus subtilis to heat shock. J. Bacteriol. 183:7318-7328. [PMC free article] [PubMed]
23. Henke, E., and U. T. Bornscheuer. 2002. Esterases from Bacillus subtilis and B. stearothermophilus share high sequence homology but differ substantially in their properties. Appl. Microbiol. Biotechnol. 60:320-326. [PubMed]
24. Ho, S. N., H. D. Hunt, R. M. Horton, J. K. Pullen, and L. R. Pease. 1989. Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene 77:51-59. [PubMed]
25. Holtmann, G., and E. Bremer. 2004. Thermoprotection of Bacillus subtilis by exogenously provided glycine betaine and structurally related compatible solutes: involvement of Opu transporters. J. Bacteriol. 186:1683-1693. [PMC free article] [PubMed]
26. Holtmann, G., M. Brigulla, L. Steil, A. Schütz, K. Barnekow, U. Völker, and E. Bremer. 2004. RsbV-independent induction of the SigB-dependent general stress regulon of Bacillus subtilis during growth at high temperature. J. Bacteriol. 186:6150-6158. [PMC free article] [PubMed]
27. Hong, S. J., Q. A. Tran, and K. C. Keiler. 2005. Cell cycle-regulated degradation of tmRNA is controlled by RNase R and SmpB. Mol. Microbiol. 57:565-575. [PMC free article] [PubMed]
28. Höper, D., U. Völker, and M. Hecker. 2005. Comprehensive characterization of the contribution of individual SigB-dependent general stress genes to stress resistance of Bacillus subtilis. J. Bacteriol. 187:2810-2826. [PMC free article] [PubMed]
29. Julio, S. M., D. M. Heithoff, and M. J. Mahan. 2000. ssrA (tmRNA) plays a role in Salmonella enterica serovar Typhimurium pathogenesis. J. Bacteriol. 182:1558-1563. [PMC free article] [PubMed]
30. Kalman, S., M. L. Duncan, S. M. Thomas, and C. W. Price. 1990. Similar organization of the sigB and spoIIA operons encoding alternate sigma factors of Bacillus subtilis RNA polymerase. J. Bacteriol. 172:5575-5585. [PMC free article] [PubMed]
31. Karzai, A. W., E. D. Roche, and R. T. Sauer. 2000. The SsrA-SmpB system for protein tagging, directed degradation and ribosome rescue. Nat. Struct. Biol. 7:449-455. [PubMed]
32. Karzai, A. W., and R. T. Sauer. 2001. Protein factors associated with the SsrA•SmpB tagging and ribosome rescue complex. Proc. Natl. Acad. Sci. USA 98:3040-3044. [PMC free article] [PubMed]
33. Karzai, A. W., M. M. Susskind, and R. T. Sauer. 1999. SmpB, a unique RNA-binding protein essential for the peptide-tagging activity of SsrA (tmRNA). EMBO J. 18:3793-3799. [PMC free article] [PubMed]
34. Keiler, K. C., and L. Shapiro. 2003. tmRNA is required for correct timing of DNA replication in Caulobacter crescentus. J. Bacteriol. 185:573-580. [PMC free article] [PubMed]
35. Keiler, K. C., P. R. Waller, and R. T. Sauer. 1996. Role of a peptide tagging system in degradation of proteins synthesized from damaged messenger RNA. Science 271:990-993. [PubMed]
36. Komine, Y., M. Kitabatake, T. Yokogawa, K. Nishikawa, and H. Inokuchi. 1994. A tRNA-like structure is present in 10Sa RNA, a small stable RNA from Escherichia coli. Proc. Natl. Acad. Sci. USA 91:9223-9227. [PMC free article] [PubMed]
37. Lawrence, J. G. 2003. Gene organization: selection, selfishness, and serendipity. Annu. Rev. Microbiol. 57:419-440. [PubMed]
38. Menez, J., R. H. Buckingham, M. de Zamaroczy, and C. K. Campelli. 2002. Peptidyl-tRNA hydrolase in Bacillus subtilis, encoded by spoVC, is essential to vegetative growth, whereas the homologous enzyme in Saccharomyces cerevisiae is dispensable. Mol. Microbiol. 45:123-129. [PubMed]
39. Miller, J. M. 1972. Experiments in molecular genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
40. Moore, S. D., and R. T. Sauer. 2005. Ribosome rescue: tmRNA tagging activity and capacity in Escherichia coli. Mol. Microbiol. 58:456-466. [PubMed]
41. Muto, A., A. Fujihara, K. I. Ito, J. Matsuno, C. Ushida, and H. Himeno. 2000. Requirement of transfer-messenger RNA for the growth of Bacillus subtilis under stresses. Genes Cells 5:627-635. [PubMed]
42. Ochman, H., J. G. Lawrence, and E. A. Groisman. 2000. Lateral gene transfer and the nature of bacterial innovation. Nature 405:299-304. [PubMed]
43. Okan, N. A., J. B. Bliska, and A. W. Karzai. 2006. A role for the SmpB-SsrA system in Yersinia pseudotuberculosis pathogenesis. PLoS Pathog. 2:e6. [PMC free article] [PubMed]
44. Oussenko, I. A., T. Abe, H. Ujiie, A. Muto, and D. H. Bechhofer. 2005. Participation of 3′-to-5′ exoribonucleases in the turnover of Bacillus subtilis mRNA. J. Bacteriol. 187:2758-2767. [PMC free article] [PubMed]
45. Petersohn, A., M. Brigulla, S. Haas, J. D. Hoheisel, U. Völker, and M. Hecker. 2001. Global analysis of the general stress response of Bacillus subtilis. J. Bacteriol. 183:5617-5631. [PMC free article] [PubMed]
46. Price, C. W. 2002. General stress response, p. 161-178. In A. L. Sonenshein, R. Losick, and J. A. Hoch (ed.), Bacillus subtilis and its closest relatives: from genes to cells. ASM Press, Washington, DC.
47. Price, C. W., P. Fawcett, H. Cérémonie, N. Su, C. K. Murphy, and P. Youngman. 2001. Genome-wide analysis of the general stress response in Bacillus subtilis. Mol. Microbiol. 41:757-774. [PubMed]
48. Richards, J., P. Mehta, and A. W. Karzai. 2006. RNase R degrades non-stop mRNAs selectively in an SmpB-tmRNA-dependent manner. Mol. Microbiol. 62:1700-1712. [PubMed]
49. Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
50. Singh, N. S., and U. Varshney. 2004. A physiological connection between tmRNA and peptidyl-tRNA hydrolase functions in Escherichia coli. Nucleic Acids Res. 32:6028-6037. [PMC free article] [PubMed]
51. Stahl, M. L., and E. Ferrari. 1984. Replacement of the Bacillus subtilis subtilisin structural gene with an in vitro-derived deletion mutation. J. Bacteriol. 158:411-418. [PMC free article] [PubMed]
52. Sundermeier, T. R., D. P. Dulebohn, H. J. Cho, and A. W. Karzai. 2005. A previously uncharacterized role for small protein B (SmpB) in transfer messenger RNA-mediated trans-translation. Proc. Natl. Acad. Sci. USA 102:2316-2321. [PMC free article] [PubMed]
53. Vagner, V., E. Dervyn, and S. D. Ehrlich. 1998. A vector for systematic gene inactivation in Bacillus subtilis. Microbiology 144:3097-3104. [PubMed]
54. van Wely, K. H., J. Swaving, C. P. Broekhuizen, M. Rose, W. J. Quax, and A. J. Driessen. 1999. Functional identification of the product of the Bacillus subtilis yvaL gene as a SecG homologue. J. Bacteriol. 181:1786-1792. [PMC free article] [PubMed]
55. von Mering, C., L. J. Jensen, B. Snel, S. D. Hooper, M. Krupp, M. Foglierini, N. Jouffre, M. A. Huynen, and P. Bork. 2005. STRING: known and predicted protein-protein associations, integrated and transferred across organisms. Nucleic Acids Res. 33:D433-D437. [PMC free article] [PubMed]
56. von Mering, C., E. M. Zdobnov, S. Tsoka, F. D. Ciccarelli, J. B. Pereira-Leal, C. A. Ouzounis, and P. Bork. 2003. Genome evolution reveals biochemical networks and functional modules. Proc. Natl. Acad. Sci. USA 100:15428-15433. [PMC free article] [PubMed]
57. Weber, M. H., and M. A. Marahiel. 2002. Coping with the cold: cold shock response in the gram-positive soil bacterium Bacillus subtilis. Philos. Trans. R. Soc. Lond. B Biol. Sci. 357:895-907. [PMC free article] [PubMed]
58. Wiegert, T., and W. Schumann. 2001. SsrA-mediated tagging in Bacillus subtilis. J. Bacteriol. 183:3885-3889. [PMC free article] [PubMed]
59. Withey, J. H., and D. I. Friedman. 2003. A salvage pathway for protein structures: tmRNA and trans-translation. Annu. Rev. Microbiol. 57:101-123. [PubMed]

Articles from Journal of Bacteriology are provided here courtesy of American Society for Microbiology (ASM)
PubReader format: click here to try

Formats:

Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...

Links

Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...