IRIS, Chiron S.r.l., Via Fiorentina 1, 53100 Siena, Italy
Correspondence
Cesira L. Galeotti
cesira_galeotti{at}chiron.it
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ABSTRACT |
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INTRODUCTION |
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In E. coli there are several lytic transglycosylases (MltA, MltB, MltC, MltD and Slt70) that bear little sequence conservation, which are however at least partly functionally redundant. Indeed, E. coli mutants lacking MltA, MltB or Slt70 show a similar reduction in the rate of murein turnover and formation of low-molecular-mass murein degradation products is dramatically reduced in the triple mutant (Lommatzsch et al., 1997; Kraft et al., 1999
). An important consequence of the diminished rate of murein breakdown to muropeptides shown by this mutant is a concomitant block of inducible
-lactamase expression, since murein turnover products seem to function as signalling compounds that interact with AmpR, the regulator of ampC (
-lactamase) gene expression (Jacobs et al., 1997
; Kraft et al., 1999
).
Lytic transglycosylases catalyse cleavage of the -1,4-glycosidic bond between N-acetylmuramic acid and N-acetylglucosamine and belong to the family of murein hydrolases that are involved in the maintenance of bacterial cell-wall integrity during cell elongation and division. Enlargement and reshaping of the cell wall require the action of both synthetic and hydrolytic enzymes, since cleavage of bonds in the murein sacculus is a pre-requisite for insertion of new murein subunits in the pre-existing structure (reviewed by Höltje, 1998
). In fact, coordination of the broad variety of activities required for cell-wall synthesis seems to be achieved through the formation of a multienzyme complex that includes both murein synthases and hydrolases (Romeis & Höltje, 1994
; von Rechenberg et al., 1996
; Vollmer et al., 1999
). Sequestering of the hydrolytic enzymes in a multienzyme complex would also be a means to control their activity which, if unrestricted, would lead to cell death through autolysis (Höltje, 1998
). Indeed, overexpression of MltA results in spheroplast formation and lysis at 30 °C (Lommatzsch et al., 1997
) while overproduction of MltB leads to rapid cell lysis at 37 °C (Ehlert et al., 1995
).
Redundancy of such a potentially suicidal function possibly implies that several strategies are needed by the cell to keep all the paralogues under control in a coordinated fashion. Here it is shown that the sequence encoding the signal peptide of the MltA homologue GNA33 contains elements of dyad symmetry that exert a strong negative regulatory effect on expression of its gene. We propose that formation of a stemloop structure within the signal peptide sequence may represent a mechanism of post-transcriptional regulation of GNA33 expression.
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METHODS |
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The 5'PhoC and 3'PhoC primers were used to amplify by PCR the Morganella morganii phoC coding sequence from the pBlueScript-phoC plasmid (kindly given by G. M. Rossolini, University of Siena, Italy). The PCR product was digested with NdeI and XhoI and inserted into the NdeI/XhoI sites of the pET-21b(+) and pET-lac vectors to obtain the T7-phoCwt and Lac-phoCwt constructs, respectively.
To construct the 33-phoC fusion, the 60 bp GNA33 signal peptide sequence was fused in-frame to the region of phoC encoding the mature PhoC using two PCR steps. In the first step the 33Pho1 and 3'PhoC primers were used. The sequence immediately downstream from the signal peptide cleavage site, 5'-ATCCCGGCAGG-3', was changed to 5'-ATCCCGGCCGG-3' to create an EagI site (underlined), and an XhoI site was inserted at the 3' end of the PCR fragment. The second PCR step was performed using the 33Pho2 and 3'PhoC primers and an NdeI site was created at the 5' end of the resulting PCR fragment. The PCR product was digested with NdeI and XhoI and inserted into the NdeI/XhoI sites of the pET-21b(+) and pET-lac vectors to obtain the T7-33phoC and Lac-33phoC constructs, respectively.
Construction of GNA33 signal peptide mutations.
To generate mutations in the GNA33 signal-peptide-encoding sequence, this was substituted with synthetic DNA linkers containing degenerate codons in the different portions targeted for mutagenesis. Linkers were designed for insertion between the NdeI/EagI sites of the pET-33phoC vector. The linker used to create mutations in the Loop1 region was obtained by annealing the 33Loop1 and 33Loop1c oligonucleotides, while the one used to create mutations in the Stem1 region was prepared by annealing 33Stem1 and 33Stem1c. Similarly, mutation 33-S1b' was obtained using a synthetic NdeI/EagI linker (Table 2).
Protein analysis.
At different time intervals a number of cells corresponding to an OD600 of 1 were centrifuged and the pellet was resuspended by boiling in 1 % SDS, 50 mM Tris/HCl (pH 6·8), 1 mM DTT for 510 min. Total protein concentrations were determined using the Bio-Rad DC protein assay system. BSA (Sigma) was used as a standard. Separation of protein samples was performed by SDS-PAGE through 420 % Tris-Glycine separating gels (Novex) using standard protocols (Laemmli, 1970). After electrophoresis, the gels were stained with Gelcode Blue (Pierce) or transferred to nitrocellulose membranes (MSI). Mouse polyclonal anti-GNA33 antiserum or rabbit polyclonal anti-PhoC antiserum were used as primary antibodies. Bands were visualized with Super Signal Chemiluminescent Substrate (Pierce).
RNA analysis.
Total RNA was extracted with NucleoSpin RNA II Kit (MacheryNagel) from E. coli liquid cultures at 0, 30 and 90 min after induction of expression. RNA samples (5 µg) were separated on 1·5 % agarose/formaldehyde gels and then dry-blotted and UV-cross-linked onto nylon membranes (MSI). Hybridization was carried out at 65 °C for 16 h in 7 % SDS, 1 mM EDTA, 0·5 M Na2HPO4 (pH 7·2). The DNA probe used for detecting GNA33 transcripts was obtained by amplifying the full-length GNA33 coding sequence and labelling the PCR product with [-32P]dATP and [
-32P]dCTP (800 Ci mmol1) using a random priming method. The DNA probe specific for the phoC transcripts was amplified by PCR and labelled in the same way. After hybridization, membranes were washed at 65 °C in 1 % SDS, 1 mM EDTA, 40 mM Na2HPO4 (pH 7·2) and exposed to X-OMAT AR films (Kodak). The hybridization signals were also quantified using a Phosphor imager and IMAGEQUANT software (Molecular Dynamics).
PhoC reporter assays.
The M. morganii PhoC phosphatase activity using p-nitrophenyl phosphate (pNPP) as a substrate was assayed by measuring the released p-nitrophenol at 414 nm at pH 12. After growth in liquid medium, a number of cells corresponding to an OD600 of 1 were centrifuged and the pellet was resuspended in 50 µl sterile H2O with the addition of 10 µl 0·5 M EDTA to inhibit E. coli phosphatases. After 15 min at room temperature, 50 µl 2 M sodium acetate, 50 µl 100 mM pNPP and 840 µl H2O were added to the cell suspension and incubated at 37 °C for 30 min. The reaction was stopped by adding 2 ml 2 M NaOH and absorbance of the samples was measured at 414 nm. The PhoC enzymic activity was assayed also on plates of TPMG (Tryptose phosphate/phenolphthalein/methyl green) indicator medium containing Tryptose-phosphate agar (Difco) pH 7·2, supplemented with 1 mg phenolphthalein diphosphate ml1 (tetrasodium salt; Sigma) and 50 µg methyl green ml1 (Sigma). Colonies producing PhoC develop a green colour after incubation at 37 °C overnight.
Computer analysis.
Nucleic acid secondary structure predictions were obtained by the MFold program which uses the method of Zuker (1989) to determine optimal and suboptimal secondary structures for an RNA or DNA molecule. The output files can be represented graphically using the PlotFold program. MFold and PlotFold are included in the University of Wisconsin Genetics Computer Group (GCG) sequence analysis package (v10.3).
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RESULTS |
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The Western blot analysis presented in Fig. 1(b) shows that the amount of T7 lysozyme produced from either pLysS or pLysE is sufficient to reduce considerably the basal T7-driven expression of GNA33 (samples at 0 min in Fig. 1b
). In the induced condition, both plasmids have a positive effect on levels of GNA33, with pLysS showing a greater influence than pLysE. Interestingly, the presence of several additional copies of the T7 promoter (or of lac-repressor binding sites) on the pACYC-T7 control plasmid seems to result in an increase in GNA33 comparable with that produced by the presence of pLysE, although no reduction of basal expression is observed with the pACYC-T7 plasmid.
A higher level of GNA33 production by cells carrying the pLysS plasmid is correlated with cell lysis 60 min after induction of expression, while the strain expressing T7 lysozyme at a higher level (pLysE) but producing a lower amount of GNA33 shows only a slight reduction of growth (Fig. 1c). This correlation between the level of protein expression and the degree of cell lysis strongly suggests that GNA33 and MltA are functionally related, since overexpression of MltA has been reported to cause cell lysis (Lommatzsch et al., 1997
). However, these results suggest also that toxicity of the GNA33 protein cannot account for the low expression level observed in the absence of the pLys plasmids.
In a different approach, also aimed at increasing expression of GNA33 in a secreted form, the sequence encoding the mature portion of GNA33 was fused in-frame with the signal peptide sequence from another N. meningitidis lipoprotein, NMB1946 (Jennings et al., 2002). Substitution of the GNA33 signal peptide sequence with that of NMB1946 was found to give a level of GNA33 comparable to that obtained for the native clone expressed in the presence of pLysS. However, in contrast with what was observed for the native construct, expression of T7 lysozyme from pLysS in cells containing the GNA33 clone with the substituted signal peptide sequence did not increase the amount of protein produced. On the contrary, a reduction of GNA33 level was observed in cells carrying either of the pLys plasmids (Fig. 1b
, lower panel).
Regulation of expression is exerted at the transcriptional/post-transcriptional level
Taken together, the data obtained from the analysis of GNA33-m and GNA33 protein expression suggest that the 60 bases encoding its signal peptide are involved in down-regulating GNA33 synthesis and, at least partly, regulation of its expression is exerted at the transcriptional level.
Northern blot analysis of GNA33 transcription at 30 min after induction of expression in cells from the same cultures used for protein analysis (Fig. 1b) showed significant differences in steady-state mRNA levels (Fig. 2
a). A quantitative evaluation of these differences is presented graphically in Fig. 2(b)
. The greatest difference was observed between the GNA33-m and the GNA33 samples, with an approximately 35-fold decrease in the clone that contains the construct with the signal peptide sequence. The presence in the same clone of the additional T7-based expression cassettes (pACYC-T7) or of the pLysS plasmid is sufficient to up-regulate GNA33 expression by a factor of 10-fold, thus bringing the level of GNA33 transcript to values only threefold lower than that found for GNA33-m (Fig. 2b
). A greater inhibition of T7 RNAP in cells bearing the pLysE plasmid, however, gave a reduction of GNA33 mRNA comparable with that observed in cells carrying only the GNA33 construct. The reproducibility of these results was confirmed in three independent experiments.
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Expression of the reporter was analysed in cells transformed with the two GNA33 signal peptide fusion constructs and, as a control, in cells containing the same vectors carrying the full-length phoC gene (phoCwt). As shown in Fig. 4, the influence of the GNA33 signal peptide sequence is not dependent on the promoter used for expression and is confined to the 60 bp fused to the reporter gene. The phoC signal peptide sequence does not exert the same influence (see phoCwt samples) and allows high expression from both vectors as demonstrated by the Northern blot analysis shown in Fig. 4(a)
. Moreover, as observed for the GNA33 transcript, the T7-based 33-phoC clone displays an increased level of steady-state 33-phoC mRNA in the presence of the pLysS plasmid (data not shown).
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Design and screening of mutations that increase expression
In a search for motifs or features that could help to explain the down-regulation effect exerted by the GNA33 signal peptide sequence, the putative secondary structure drawn in Fig. 5(a) was obtained using the MFold program (Zuker, 1989
). Based on this hypothetical stemloop structure, two sets of partially degenerate linkers were designed that would introduce mutations in two different regions of the signal peptide sequence: in the first 19 bp (L1) and between bases 2036 (S1). The screening of GNA33 signal peptide mutants was performed by the PhoC plate assay using the TPMG indicator medium. On this medium PhoC-producing E. coli strains grow as green-stained colonies, while non-producing cells give white colonies (Thaller et al., 1994
). In a first screening for L1 mutants, over 200 colonies were obtained that showed different shades of green on TPMG medium. These were tested again on the same medium and the eight clones that showed a darker green colour were selected for further analysis. Following the same screening procedure, 12 mutants in the S1 region were isolated. An example of the PhoC plate assay used for the screening of mutants is given in Fig. 5(b)
. Sequence analysis of the signal peptide region in the selected clones showed that most contained multiple point mutations and several contained in-frame deletions (Fig. 5c
). Interestingly, although the approach used for obtaining mutations was designed to give only third-base point substitutions, a significant number of deletions were found among the small number of mutants selected on the basis of high expression. Moreover, of all the mutants analysed those containing deletions displayed the darkest green colony colour on TPMG plates.
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Western blot analysis using PhoC-specific antibodies confirmed that, in different degrees, all the selected mutations up-regulate synthesis of the reporter protein. Examples are given in Fig. 6 where it is shown that the amount of PhoC produced from the 33-phoC construct is increased at least 5- to 10-fold in the 33-L1 and 33-S1e deletion mutants and approximately threefold in the 33-S1G single substitution mutant. Preliminary analysis of phoC localization in these mutants indicates that, while the 33-L1 deletion causes loss of the capability of translocating the reporter, phoC is exported to the periplasm in the 33-S1e deletion as well as in all the substitution mutants analysed (data not shown).
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Northern blot analysis of the same samples at 30 min from induction is presented in Fig. 7(a) and quantification of the relative abundance of phoC-specific transcripts is reported in the graph shown in Fig. 7(b)
. The level of phoC-specific mRNA is highest in the 33-S1e mutant, which shows an increase of approximately 300-fold with respect to the 33-phoC sample. A lower, but significant increment was found in the other deletion mutants, with values ranging from 10- to 23-times the level of the 33-phoC transcript. Point mutations with more than 3 bases substituted (L1f, S1c, S1i) showed a 3·6- to 5·8-fold increase in the level of steady-state mRNA, while single (S1G), double (S1GG) and triple (S1b) substitutions in the signal peptide sequence resulted in no significant increase of phoC-specific transcript.
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Furthermore, a compensatory mutant of the 33-S1b clone (33-S1b') containing three additional point mutations designed to re-establish the formation of the stemloop structure (Fig. 8) shows a significant decrease in expression of the reporter. In fact, both a quantitative evaluation of Northern blot analysis and determination of the PhoC specific activity in cell extracts of this mutant indicated a greater than threefold reduction of expression (data not shown).
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DISCUSSION |
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The results presented in this report show that the 60 bases encoding the signal peptide of the MltA homologue GNA33 of N. meningitidis exert a negative effect on GNA33 expression in E. coli. The cause of the considerable reduction in the GNA33 protein level, however, could not be attributed to toxicity of GNA33 when translocated to the periplasm in non-physiological amounts, since overexpression could be achieved through either modulation of the rate of transcription or substitution of the signal peptide sequence. Indeed, overexpression of the secreted form was toxic in that it resulted in cell lysis, but high-level production of GNA33 could be obtained in spite of its toxicity. Interestingly, analysis of the steady-state GNA33 transcripts in the different clones showed an even greater difference in mRNA levels, thus suggesting that the presence of the GNA33 signal peptide sequence down-regulates expression at the transcriptional/post-transcriptional level.
It is apparent from the data presented herein that a fine-tuning of transcription rate is required for an increase in GNA33 production. Titration of the T7 RNAP due to the presence of additional copies of the T7 promoter in trans, in the pACYC-T7 clone, is sufficient to give high-level accumulation of GNA33 mRNA. Also, a moderate inhibition of the T7 RNAP by T7 lysozyme results in a considerable increase in steady-state mRNA and in higher protein production (Figs 1b and 2). The finding that the presence of T7 lysozyme leads to a significant accumulation of GNA33 mRNA is consistent with the model presented by Zhang & Studier (1997)
for the mechanism of T7 RNAP inhibition by T7 lysozyme. The authors proposed that lysozyme binds RNAP in the initiation conformation and prevents its conversion to an elongating complex, but it does not interfere with the polymerase during elongation. It has been suggested also that lysozyme would increase pausing of the RNAP at specific sites that favour a transition in the RNAP, from the elongation to the initiation conformation (Zhang & Studier, 1997
; Lyakhov et al., 1998
).
The data presented in this work indicate that slowing down transcription may result in a higher level of GNA33 mRNA possibly through a better coupling of translation to transcription, as it is well known that the T7 RNAP travels far ahead of ribosomes' (Iost et al., 1992; Iost & Dreyfus, 1995
). However, substitution of the T7 promoter with Plac gives the same pattern of expression, thus demonstrating that the inhibitory effect exerted by the GNA33 signal peptide sequence is not influenced by the RNAP used but rather by the activity of the RNAP or, more specifically, by its rate of transcription. A fast RNAP would leave behind an unprotected segment of the newly synthesized transcript that would be exposed to degradation. Alternatively, in the presence of elements of symmetry in the sequence of the transcript, a long stretch of unbound mRNA could fold into a stable secondary structure and inhibit the proceeding of translation. This would ultimately lead to degradation of the transcript.
Recently, computational analyses of mRNA secondary structures in coding regions of 10 bacterial genomes have found a highly significant and widespread bias toward local secondary structure potential in many ORFs. These results have been interpreted as indicative of widespread regulation of translation and/or mRNA decay in prokaryotes by mechanisms involving coding-region hairpins (Katz & Burge, 2003).
Indeed, we propose that 40 out of the 60 nucleotides comprising the GNA33 signal peptide sequence could fold into a GC-rich stemloop structure (Fig. 5a). The finding that point mutations or deletions that would disrupt or abolish this hypothetical secondary structure result in a significant increase of reporter mRNA levels suggests that the formation of the putative stemloop regulates expression by affecting mRNA accumulation. In particular, it is interesting to note that a single third-base substitution (33-S1G) is sufficient to augment several-fold the amount of reporter protein produced, even if the accumulation of mRNA seems unchanged with respect to the wild-type sample. This result is particularly surprising since substitution of a frequent codon with a synonymous infrequent one, as occurs in this mutant, has been reported to have inhibitory effects on translation (Deana et al., 1998
). Therefore, since an increased efficiency of translation due to codon bias cannot account for this finding, we propose that even a small difference in the folding of the putative secondary structure might provide a more fruitful coupling of translation to transcription. Furthermore, additional support for the formation of such a secondary structure and its role in regulation of expression can be found in the spontaneous deletion mutants. These mutants, which lack most of the sequence participating in the stemloop structure, have been isolated at surprisingly high frequency by analysing clones with the highest expression levels of the reporter.
In conclusion, it is postulated that the activity of RNAP, or more specifically the elongation rate of the mRNA and/or its translatability, controls the folding of the GNA33 signal peptide sequence into a secondary structure which, when formed, negatively regulates expression. It is tempting to speculate that, since RNAP activity presumably is in turn regulated by growth rate and cell division, the formation of the stemloop structure described herein may represent a control point for GNA33 synthesis during cell division.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 23 September 2003;
revised 8 January 2004;
accepted 26 January 2004.
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