Department of Microbiology, Lund University, Sölvegatan 12, SE-223 62 Lund, Sweden1
Author for correspondence: Gustav Hambraeus. Tel: +46 46 222 49 80. Fax: +46 46 15 78 39. e-mail: gustav.hambraeus{at}mikrbiol.lu.se
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ABSTRACT |
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Keywords: mRNA secondary structure, subtilisin, amyE, stationary phase
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INTRODUCTION |
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Most of our ideas about bacterial mRNA degradation are based on experiments performed with E. coli. The 5' untranslated leader of an mRNA often contains the major stability determinant(s) (Bechhofer, 1993 ). The rate-limiting step in mRNA degradation is thought to be an endonucleolytic cut in the 5' part of the molecule, which opens it for further endonucleolytic attacks towards the 3' end. The resulting fragments are rapidly degraded by 3' to 5' exoribonucleases (Spickler & Mackie, 2000
). RNase E is the major endoribonuclease executing the rate-limiting attack (Cohen & McDowall, 1997
). The general model for mRNA degradation probably applies to other bacteria but with important modifications in details. For instance, no RNase E homologue has been found in Bacillus subtilis (Kunst et al., 1997
) and different patterns of degradation have been found for the same mRNA species in B. subtilis and E. coli (Persson et al., 2000
).
When B. subtilis enters stationary phase, several new genetic programmes are switched on for sporulation, competence development (Lazazzera et al., 1999 ) or production of extracellular enzymes (Ferrari et al., 1993
). The aprE gene encodes subtilisin, an extracellular proteolytic enzyme produced by stationary-phase cells (Ferrari et al., 1988
). Resnekov et al. (1990)
measured the decay of aprE mRNA and found it to be extremely stable in stationary-phase cells, with a half-life of at least 25 min. This is not a general property of stationary-phase mRNA (Melin et al., 1989
; Resnekov et al., 1992
). Interestingly, when the bacteria were diluted into fresh medium and allowed to resume growth, the stability of aprE mRNA seemed to decrease four- to fivefold. These findings indicated the presence of a specific control mechanism for decay of aprE mRNA related to growth phase and perhaps operating also on mRNA for other extracellular enzymes whose synthesis is induced in stationary-phase cells.
In the present work we re-examined the results of Resnekov et al. (1990) with the primary aim of identifying a possible control mechanism for growth-stage-dependent differential rate of decay of aprE mRNA. However, we found that aprE mRNA has the same extreme stability in both stationary-phase and growing cells. The apparent growth-stage-dependent stability of aprE mRNA can be fully explained by the techniques used by Resnekov et al. (1990)
. Our experiments also demonstrate that the determinant(s) for the extreme stability of aprE mRNA is contained within the leader.
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METHODS |
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Genetic techniques.
B. subtilis was grown to competence as described by Arwert & Venema (1973) . E. coli was made competent as described by Mandel & Higa (1970)
.
Enzyme activities.
ß-Galactosidase activity was detected on TBAB plates containing 40 mg X-Gal l-1. ß-Galactosidase activity in liquid cultures was assayed according to Miller (1972) as described by Glatz et al. (1998)
. Amylase activity was detected on TBAB plates containing 1·5% starch. After incubation for 2 d, the plates were sprayed with an iodine solution (1 g iodine and 2 g potassium iodide in 300 ml distilled water). A clear halo is observed around amylase-positive colonies.
Construction of strains.
A sequence containing the aprE promoter, leader sequence and the first 8 codons of the aprE gene, from -104 to +82 (Ferrari et al., 1988 ) was amplified from chromosomal DNA of B. subtilis BR95 with PCR using primers aprEBam1 and aprEBam2. The fragment was cleaved at both ends by restriction enzyme BamHI and ligated into the BamHI site of the B. subtilis integration plasmid pMD432. Competent E. coli XL-1 Blue was transformed with the ligate with selection for ampicillin resistance. Transformants producing ß-galactosidase were identified on X-Gal plates, and from one of these, pLUS1 was purified and the inserted fragment was sequenced. pLUS1 was then used to transform B. subtilis BR95, with selection for chloramphenicol resistance. Transformants producing ß-galactosidase and lacking amylase activity were isolated. One of these was kept and the strain was named LUS1 (Fig. 1
).
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Plasmid pLUS2 was used to construct two additional strains with modifications in the aprE leader sequence by running PCRs with modified primers. A first PCR was run with primers aprEconstS and aprEdel25inv in one mix and aprEdel25 and aprEBam2 in another. aprEdel25inv and aprEdel25 are complementary to each other and introduce a deletion of 25 nt. The two fragments obtained were mixed and a second PCR was run with primers aprEconstS and aprEBam2. The fragment obtained was ligated to BamHI-cleaved pMD432 and E. coli XL-1 Blue was transformed with the ligate. Ampicillin-resistant transformants were selected and pLUS3 was isolated from one of these. pLUS4 was obtained analogously but with the primers aprEdel25inv and aprEdel25 replaced with the primers aprEsubTTinv and aprEsubTT. These primers introduce T substitutions at +31 and +32. pLUS3 and pLUS4 were used to transform B. subtilis BR95, with selection for chloramphenicol resistance. Transformants were isolated and the resultant strains were named LUS3 and LUS4.
A sequence containing the amyE promoter, leader and part of the coding region, from -100 to +134 (Nicholson & Chambliss, 1986 ), was amplified by PCR with primers amyEBam1 and amyEBam2. The fragment was cleaved with BamHI and ligated to BamHI-cleaved pMD433. E. coli XL-1 Blue was transformed with the ligate, selecting for ampicillin resistance. Plasmid pLUS5a was isolated from one of the transformants. From pLUS5a, the region containing the transcribed amyE part (+1 to +134) was amplified with PCR using primers amyEconst and amyEBam2. amyEconst substitutes the amyE promoter with the glpD promoter (-40 to -1) (Holmberg & Rutberg, 1992
). After cleavage with BamHI, the amplified fragment was ligated to BamHI-cleaved pMD433. Competent E. coli XL-1 Blue was transformed with the ligate, with selection for ampicillin resistance. Transformants producing ß-galactosidase were identified on X-Gal plates and from one of these, pLUS6a was purified.
pLUS5a and pLUS6a were used as templates for PCRs with primer pairs amyEHindIII/lacZHindIII and amyEconstS/lacZHindIII, respectively. After cleavage with HindIII, the fragments were ligated to HindIII-cleaved pDG1664. E. coli XL-1 Blue was transformed with the ligates, selecting for ampicillin resistance. pLUS5b and pLUS6b were isolated and used to transform B. subtilis BR95. Transformants resistant to erythromycin and lincomycin (MLSR) were selected and from each transformation a transformant that produced ß-galactosidase and required threonine for growth was kept. The resultant strains were named LUS5 and LUS6, respectively.
RNA techniques.
Total RNA was extracted from B. subtilis as described by Resnekov et al. (1990) . For measuring mRNA half-lives, rifampicin (100 mg l-1) was added and samples were then removed at intervals for extraction of total RNA. Electrophoresis of RNA for Northern blots was done as described by Thomas (1980)
, and the RNA was then blotted onto Hybond-N filters (Amersham). Twenty micrograms of RNA was added to each well unless otherwise indicated. Single-stranded (ss) DNA probes were generated as described before (Persson et al., 2000
) with the following primers: lacZ60 for lacZ constructs, aprEBam2 for aprE, and amyESeq for amyE. To generate templates for the ssPCR, specific fragments were amplified with PCR using the following primers and templates: glpDBam1 and lacZ60 with pLUM1041, aprESubTT and aprEBam2 with pLUS1, and amyE1 and amyESeq with chromosomal B. subtilis DNA. The glpDBam1lacZ60 fragment was cleaved with BamHI and the lacZ part was isolated prior to the ssPCR. After hybridization, the radioactivity of the bands was quantified with a PhosphorImager (Molecular Dynamics). 23S rRNA and 16S rRNA were used as size markers. Primer extension analysis was performed according to the method of Ayer & Dynan (1988)
. The primer used was lacZseq.
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RESULTS |
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LUS1 and LUS2 were grown as described in Methods. At different times, samples were taken for measurement of OD600 and ß-galactosidase activity. In LUS1, ß-galactosidase activity was detected only after the cells had entered stationary phase, whereas in LUS2, ß-galactosidase was produced throughout exponential growth (Fig. 2). These results are in good agreement with the fact that a major control of aprE expression occurs at initiation of transcription through the AbrB/SpoOA switch (Olmos et al., 1996
).
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A truncated transcript of about 700 nt was seen in the Northern blots for strains LUS1 and LUS2 (Fig. 3b, c
). This represents the 5' part of the full-length transcript and has been found also in other types of transcriptional-translational fusions with lacZ in B. subtilis (Persson et al., 2000
). The truncated transcript decayed at the same rate as the full-length transcript, which further supports the notion that the aprE leader is the major stability determinant also in the fusion transcript.
To examine how changes in the aprE leader transcript would affect mRNA stability, two modifications were introduced into the aprE leader of the lacZ fusion in LUS2. These were chosen so as to significantly alter the secondary structure of the leader (Fig. 4). Prediction of secondary structures is very uncertain. Alternative structures are often suggested with similar free energies. One modification was a deletion of nucleotides +1 to +25, resulting in strain LUS3. In the second modification, a G and an A residue, at positions +31 and +32, respectively, were exchanged for two T residues, resulting in strain LUS4. According to computer-predicted folding (Zuker, 1989
), the RBS of both LUS3 and LUS4 leader is contained within a strong stemloop structure (Fig. 4b
, c
). In LUS3, the stemloop is located at the very 5' end and in LUS4, 22 unpaired bases precede the stemloop.
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DISCUSSION |
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In the present work we have shown that the determinant(s) for the extreme stability of aprE mRNA is located in the 5' leader. A transcript of a transcriptional-translational fusion between the aprE leader and E. coli lacZ was found to have the same stability as the native aprE transcript. Furthermore, substituting the aprE promoter with the constitutive B. subtilis glpD promoter had no effect on the rate of decay of the fusion transcript. By using the glpD promoter one can directly measure the rate of decay of the aprElacZ fusion transcript (or the aprE transcript) in growing and stationary-phase cells. The results of such measurements show that the half-life of the aprElacZ fusion transcript is at least 25 min under all conditions tested. Thus, contrary to previous suggestions (Resnekov et al., 1990 ) there is no growth-stage-related control of aprE mRNA stability. The increased rate of degradation of aprE mRNA on shift-up observed by Resnekov et al. (1990)
is spurious. The shift-up is immediately followed by a selective increase in stable RNA whereas the aprE promoter is turned off. In experiments of Resnekov et al. (1990)
this leads to an apparent increase in degradation rate of aprE mRNA which is simply due to a dilution effect.
There are few data available which compare the stability of a specific B. subtilis mRNA at different growth stages, but increased stability is not a general property of stationary-phase mRNA (Melin et al., 1989 ). In the present work we have also determined the half-life of amyE mRNA, which encodes another stationary-phase-specific B. subtilis exoenzyme, to be 5 min. This shows that extreme stability is not a general property of exoenzyme transcripts. Whatever the physiological reason for the exceptionally high stability of the aprE mRNA, it contributes to a high level of production of subtilisin in the stationary-phase window where the aprE promoter is active.
Very few bacterial transcripts of extreme stability have been characterized, ermC and gsiB being two of the best-known examples in B. subtilis. The half-life of the ermC transcript increases about 20-fold upon exposure of the bacteria to erythromycin (Bechhofer & Zen, 1989 ). Binding of the drug to ribosomes translating a short ORF preceding the coding region for the ErmC protein stalls the ribosomes at this ORF. The stalling protects the whole ermC transcript from degradation, possibly by shielding a nuclease-sensitive site and/or affecting the secondary structure of the transcript. The gsiB transcript, which is produced from a SigB-dependent promoter and encodes a stress-related protein, has a half-life of at least 20 min (Jurgen et al., 1998
). The gsiB gene has a very strong RBS with an optimal spacing to the AUG start codon. Mutations that weaken the gsiB RBS lead to a decrease in the half-life of the transcript, suggesting that a major factor in determining the gsiB mRNA half-life is the interaction between the RBS and 16S rRNA. However, RBS mutations also lead to some changes in the predicted secondary structure of the gsiB mRNA leader sequence, which complicates the interpretation of these experiments.
The free energy of binding of the RBS and the 3' end of 16S rRNA is very similar for gsiB, aprE and amyE (Tinoco et al., 1973 ) and yet the amyE transcript decays at least five times faster than those of the other two genes. It should be noted that the start codon is AUG for gsiB and amyE but GUG for aprE. Thus, a model suggesting the RBS16S rRNA interaction to be a major determinant for mRNA stability is hardly of general validity. However, the interaction between 16S rRNA and the mRNA leader in forming the initiation complex is a function not only of base complementarity but also of the secondary structure of the leader (Yamanaka et al., 1999
). The favoured predicted secondary structure of the wild-type aprE leader is a stemloop at the very 5' end with the RBS contained in a region with weak interactions (Fig. 4a
). The computer prediction also suggests an alternative structure with similar free energy. However, the formation of this structure involves opening the already folded stemloops shown in Fig. 4(a)
. That makes this alternative structure less likely. Changing two bases in the leader (G31 and A32 both changed to T) leads to a large structural change with a perfect stemloop containing the RBS and preceded by 22 unpaired bases (Fig. 4b
). This transcript has a half-life of about 5 min. The same stemloop was obtained by deleting 25 nt in the leader (Fig. 4c
). Also this modification led to a major destabilization of the transcript. Deleting a single-stranded 5' end is known to stabilize some E. coli transcripts (Emory et al., 1992
).
Clearly, the secondary structure of the aprE transcript is important for the extreme stability of aprE mRNA. This may simply depend on occlusion of RNase-sensitive sites, but other factors may also be involved, e.g. specific (temporary or permanent) binding of proteins to the transcript. This is a known mechanism for stabilizing or destabilizing transcripts. For example, B. subtilis glpD mRNA can be stabilized by the GlpP antiterminator protein (Glatz et al., 1996 ) and in E. coli, the ompA and glgC transcripts are destabilized by the Hfq and CsrA proteins, respectively (Vytvytska et al., 1998
, Liu & Romeo, 1997
). Finally, we wish to point out that there are striking sequence similarities between the sequence around the aprE RBS and that of a polypurine-rich sequence in bacteriophage SP82 which functions as a 5' stabilizer in B. subtilis (Hue et al., 1995
).
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ACKNOWLEDGEMENTS |
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Received 30 May 2000;
revised 29 August 2000;
accepted 4 September 2000.