The signal peptide sequence of a lytic transglycosylase of Neisseria meningitidis is involved in regulation of gene expression

Davide Serruto and Cesira L. Galeotti

IRIS, Chiron S.r.l., Via Fiorentina 1, 53100 Siena, Italy

Correspondence
Cesira L. Galeotti
cesira_galeotti{at}chiron.it


   ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The 60 nucleotides encoding the signal peptide of the Neisseria meningitidis membrane-bound lytic transglycosylase (MltA) homologue GNA33 were found to exert a negative regulatory effect on expression of GNA33 from either a T7- or a Plac-driven system in Escherichia coli. Down-regulation was observed to occur at the transcriptional/post-transcriptional level and could possibly be ascribed to the formation of a stem–loop secondary structure within the signal peptide sequence. Slowing down the transcription rate through inhibition/titration of the RNA polymerase resulted in a considerable increase in mRNA accumulation, suggesting that a better coupling of translation to transcription would impede the formation of the putative secondary structure. Screening of synonymous mutations in the signal peptide sequence that showed high-level expression of an in-frame fusion to a reporter resulted in the isolation of several deletion mutants lacking most of the sequence participating in the putative secondary structure. Interestingly, the increase in the steady-state mRNA level observed in deletion mutants was higher, reaching a 300-fold increment, than that found in substitution mutants. Our results support the hypothesis that the rate of transcription controls the formation of a secondary structure in the region of the GNA33 transcript corresponding to the signal peptide sequence and this, when formed, negatively regulates expression.


Abbreviations: pNPP, p-nitrophenyl phosphate; RNAP, RNA polymerase; TPMG, Tryptose phosphate/phenolphthalein/methyl green


   INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
In a genome-wide screening for Neisseria meningitidis serogroup B antigens (Pizza et al., 2000) that made use of Neisseria ORFs expressed in Escherichia coli to identify vaccine candidates, it was found that the signal peptide sequence of the ORF named GNA33 exerted a negative regulatory effect on GNA33 expression. GNA33 encodes a protein of 441 aa with an N-terminal 20 aa signal sequence and shares 34·5 % sequence identity with the E. coli membrane-bound lytic transglycosylase A (MltA) (Jennings et al., 2002). Recently, functional homology of GNA33 to E. coli MltA has been confirmed by in vitro characterization of its muramidase and lytic transglycosylase activity (Jennings et al., 2002).

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 {beta}-lactamase expression, since murein turnover products seem to function as signalling compounds that interact with AmpR, the regulator of ampC ({beta}-lactamase) gene expression (Jacobs et al., 1997; Kraft et al., 1999).

Lytic transglycosylases catalyse cleavage of the {beta}-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 stem–loop structure within the signal peptide sequence may represent a mechanism of post-transcriptional regulation of GNA33 expression.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Strains and growth conditions.
The bacterial strains used are listed in Table 1. The liquid medium used for bacterial growth was LB. When required, the antibiotics ampicillin, chloramphenicol and tetracycline were added to culture media at concentrations of 100, 30 and 12·5 µg ml–1, respectively. For protein expression in BL21 and BL21(DE3) strains, a single positive colony was inoculated in 5 ml LB medium (plus antibiotic) and grown overnight at 37 °C. The culture was diluted to give an OD600 of 0·1 in 20 ml freshly prepared LB medium and incubated at 37 °C until an OD600 of 0·5 was reached. Expression was induced by the addition of 1 mM IPTG (Sigma) and growth was continued for another 3 h or longer for growth curve determination.


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Table 1. Bacterial strains and plasmids used in this study

 
Plasmids and DNA manipulations.
DNA cloning and E. coli transformation were performed according to standard protocols (Sambrook et al., 1989). Restriction endonucleases and DNA-modifying enzymes were obtained from New England Biolabs and used according to the manufacturer's instructions. The synthetic oligonucleotides used are listed in Table 2.


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Table 2. Oligonucleotides used in this study

 
To construct the pACYC plasmid containing the T7 promoter (pACYC-T7), the 0·5 kb ScaI–PvuII DNA fragment of pACYC184 (Chang & Cohen, 1978) was replaced by the 0·3 kb ScaI–PvuII fragment containing the T7 cloning/expression region amplified by PCR from the pET-21b(+) vector (Novagen). The pET-lac plasmid was made by substituting the T7 promoter and T7 terminator sequences of the pET-21b(+) vector with Plac (which encompasses the –35 box and Pribnow box of the lac operon promoter) and the {rho}-independent trpA terminator, respectively. The Plac cloning/expression region was obtained by two consecutive PCR steps: the first step was performed using primers Plac1 and Ttrp1 and the second step using primers Plac2 and Ttrp2 which introduce a BglII and a StyI site at the 5' and 3' ends of the resulting PCR fragment, respectively, for cloning into the BglII/StyI sites of the pET-21b(+) vector.

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 5–10 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 4–20 % 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 (Machery–Nagel) 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 [{alpha}-32P]dATP and [{alpha}-32P]dCTP (800 Ci mmol–1) 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 ml–1 (tetrasodium salt; Sigma) and 50 µg methyl green ml–1 (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).


   RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Influence of the GNA33 signal peptide sequence on the level of protein expression
In an extensive screening for N. meningitidis serogroup B antigens (Pizza et al., 2000) it was found that different forms of GNA33, a lipoprotein with homology to the E. coli murein transglycosylase MltA, were expressed at considerably different levels in the heterologous T7 expression system of E. coli. The forms analysed differ due to the presence or absence of a signal peptide sequence. The GNA33 ORF devoid of the N-terminal 20 aa that correspond to the signal peptide sequence (GNA33-m) gave the highest expression level, while the full-length form (GNA33) was poorly expressed, as shown by the SDS-PAGE analysis of cell extracts reported in Fig. 1(a).



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Fig. 1. (a) Expression of different forms of GNA33 in E. coli. The mature (GNA33-m) and full-length (GNA33) forms of ORF GNA33, cloned in pET-21b(+), were expressed in E. coli BL21(DE3). Bacteria were grown to an OD600 of 0·5 and induced with 1 mM IPTG at 37 °C for 3 h. Cell extracts were separated in 4–20 % SDS-PAGE and proteins were stained with Gelcode Blue. E. coli BL21(DE3) carrying the pET-21b(+) vector was used as a control (pET). Lane M represents the protein molecular mass marker. (b) Western blot analysis of expression of full-length GNA33 and of GNA33 with the signal peptide replaced by that of NMB1946 (GNA33-sp1946) in BL21(DE3), BL21(DE3)[pACYC-T7], BL21(DE3)[pLysS] and BL21(DE3)[pLysE]. After induction of expression with IPTG, samples were taken at the timepoints indicated. Bacteria were lysed and cell extracts were analysed by Western blotting with an anti-GNA33 antibody. Total proteins in cell extracts were quantified and 10 µg of each sample was used for the immunoblot. (c) Overexpression of GNA33 leads to cell lysis. Strains were grown in LB broth at 37 °C and growth was monitored by measuring OD600. At the time indicated by the arrow, expression of GNA33 was induced by the addition of 1 mM IPTG to the cultures.

 
On the assumption that overexpression of GNA33 in the ‘native’ form may have adverse effects on growth, as indeed has been observed for overexpression of the homologue MltA (Lommatzsch et al., 1997), E. coli cells bearing the full-length GNA33 construct were transformed with pLys plasmids from which T7 lysozyme is expressed at different levels. In fact, a more stringent regulation of T7 transcription by T7 lysozyme, a specific inhibitor of T7 RNA polymerase (RNAP), has been reported to improve expression levels of genes whose products are toxic for the host cell (Studier, 1991). As a control for the presence of a second plasmid, the same strain was transformed also with a construct of plasmid pACYC184 containing an empty T7 expression cassette (pACYC-T7).

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. 2a). 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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Fig. 2. Inhibition of T7 RNAP up-regulates GNA33 expression. (a) Northern blot analysis of GNA33 steady-state mRNA at 30 min post-induction in cells from the same cultures used for protein analysis in Fig. 1(b). Total RNA ({approx}5 µg) from cells expressing the mature form (GNA33-m) or the full-length GNA33 (GNA33) in the presence of pLys plasmids was separated in a denaturing agarose gel in the presence of ethidium bromide (upper panel), blotted and hybridized to a GNA33-specific probe (lower panel). Quantification of the relative amount of GNA33 mRNA is represented in graphical form in (b). Lane M indicates the 0·24–9·5 kb RNA ladder (Invitrogen) used as a reference.

 
The signal peptide sequence is required for down-regulation, irrespective of which promoter is controlling expression
To verify if down-regulation of GNA33 expression was determined uniquely by the presence of the signal peptide sequence and/or specifically related to the T7-dependent system used, two constructs were made which contained an in-frame fusion of the 60 bp encoding the GNA33 signal peptide to a reporter gene. The fusion was cloned in the pET-21b vector and in a pET-lac plasmid constructed by substituting the pET expression elements with the E. coli lac promoter and trpA terminator, as outlined in Fig. 3.



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Fig. 3. Schematic representation of the two pET-based vectors constructed for expression of the GNA33 signal peptide sequence fused to the portion of the phoC reporter gene encoding the mature peptide (mature PhoC). Upstream of the lac operator, which allows expression to be induced by IPTG from either promoter (P), are reported the sequences of the T7 promoter (PT7) and of the lac promoter (Plac) used in this study. The T7 terminator present in the T7-33phoC construct is substituted with the trpA terminator in the Lac-33phoC plasmid.

 
The Morganella morganii phoC gene (Thaller et al., 1994) was chosen as a reporter in this study for the following reasons. PhoC is a 27 kDa irrepressible acid phosphatase efficiently secreted when expressed in E. coli. Its secretion is mediated by a signal peptide of 20 aa. The enzymic activity pertaining to this phosphatase can be easily assayed with different substrates and under different growth conditions (Thaller et al., 1994, 1998). The relative stability of the substrates used in the PhoC assays allows quantification of the activity synthesized during growth in liquid medium, while accumulation of the reporter can be monitored during growth on plates.

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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Fig. 4. The GNA33 signal peptide sequence down-regulates expression of the phoC reporter irrespective of the promoter used. (a) Northern blot analysis of expression of the phoCwt and 33-phoC constructs in the pET-21b (T7) or pET-lac (Lac) vector. Total RNA ({approx}5 µg) from cells expressing the full-length phoC reporter gene (phoCwt), or the in-frame fusion of the GNA33 signal peptide sequence to the reporter (33-phoC), were separated in denaturing agarose gel, blotted and hybridized to a phoC-specific probe. BL21(DE3) cells transformed with the pET-21b vector were used as a control (pET). (b) Graphical representation of the PhoC specific activity measured in cells of the same cultures as in (a). (c) SDS-PAGE of cell lysates of BL21 carrying the pET-lac vector with the full-length phoC gene (Lac-phoCwt) or the 33-phoC construct (Lac-33phoC). BL21 cells transformed with the pET-lac vector were used as a control (pET).

 
The results shown in Fig. 4 also demonstrate that accumulation of the PhoC reporter protein is easily evaluated by SDS-PAGE analysis of cell extracts (Fig. 4c) and, in parallel, the same samples can be used to assess the PhoC enzymic activity (Fig. 4b). It should be noted that, while there is a remarkable difference between the relative amount of PhoC found at time zero and that seen at other timepoints (Fig. 4c), the level of active acid phosphatase produced remains approximately constant at all the timepoints analysed (Fig. 4b).

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 stem–loop 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 20–36 (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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Fig. 5. Design and screening of mutations that increase expression. (a) Putative mRNA secondary structure predicted for the GNA33 region encoding the signal peptide by the MFold program (Zuker, 1989). The L1 and S1 regions used to design synonymous mutations are indicated. (b) Screening of GNA33 signal peptide sequence mutations by the PhoC plate assay using the TPMG indicator medium (Thaller et al., 1994). BL21(DE3) cells transformed with the pET-21b vector were used as a control (pET). (c) Alignment of mutations with the wild-type GNA33 signal peptide sequence (highlighted in black). Base substitutions are highlighted in grey, while dots represent deletions. (d) Prediction of the mRNA secondary structure in the mutants is represented diagrammatically. The region used for the MFold analysis of each clone begins at the T7 transcription start site and includes the signal peptide coding region. The position of the translation start site is indicated by an arrow in the top diagram (GNA33).

 
Analysis of expression in deletion and point mutants
A more detailed analysis of PhoC levels in several representative mutants was carried out by determining the relative amount of reporter mRNA and protein in cells grown in liquid medium. The same culture of each mutant was used to prepare RNA and cell extracts for phoC-specific Northern and Western blot analyses. It is noteworthy that cells of all the deletion mutants analysed were found to resume growth, after dilution from a stationary-phase culture, with a 3 h delay. Therefore, to enable comparison of these mutants with the other samples, cultures of all deletion mutants were inoculated 3 h earlier. In this way, induction of expression and data collection could be carried out in parallel for all samples.

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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Fig. 6. Influence of mutations in the GNA33 signal peptide sequence on expression of the PhoC reporter. Expression of the reporter protein in BL21(DE3) was analysed by Western blotting with an anti-PhoC antibody. Samples were taken at the timepoints indicated, bacteria were lysed and total proteins in cell extracts were quantified. Ten micrograms of each sample was used for the immunoblot.

 
Another interesting observation that can be drawn from the results of the Western blot analysis presented in Fig. 6 concerns the relative amount of the reporter found at the different timepoints. After induction (time zero), the amount of PhoC found in cell extracts decreases gradually and is lowest at 3 h in all clones except for the 33-S1e mutant, which behaves in the opposite way. Evaluation of PhoC specific activity in cell extracts of all the mutants at the different timepoints also suggests that the S1e mutation confers an opposite trend to the rate of synthesis of PhoC over time (data not shown). One interpretation for this result is that mRNA stability is greatly increased by the S1e deletion.

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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Fig. 7. Expression of the reporter in deletion/substitution mutants. (a) Northern blot analysis of phoC-specific transcripts at 30 min post-induction in deletion (lanes 3–6) or substitution (lanes 8–13) mutants of the GNA33 signal peptide sequence. Total RNA ({approx}5 µg) from cells expressing the full-length phoC reporter gene (lane 1), the GNA33 signal peptide sequence fusion to the reporter (lane 2) or mutants of the latter were separated in a denaturing agarose gel (upper panel), blotted and hybridized to a phoC-specific probe (lower panel). (b) Graphical representation of the relative amount of steady-state phoC mRNA in the same samples as in (a). The amount of phoCwt transcript (black bar) was arbitrarily taken as 100 %. (c) Determination of PhoC specific activity in cells from the same cultures as in (a) and (b) was carried out as described in Methods. The absorbance measured at 414 nm in samples incubated with the PhoC substrate pNPP (5 mM) at 37 °C for 30 min is presented in graphical form. The data represent the mean values of three independent experiments. Error bars indicate standard deviation.

 
PhoC specific activity measured in the cell extracts of the same cultures used for mRNA analysis is reported in Fig. 7(c), where it should be noted that the range of PhoC production correlates with the type of mutation present in the clone. Single/multiple substitution mutants show a moderate increase in reporter activity, while deletion clones produce the highest amount of PhoC.

Furthermore, a compensatory mutant of the 33-S1b clone (33-S1b') containing three additional point mutations designed to re-establish the formation of the stem–loop 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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Fig. 8. A compensatory mutant of 33-S1b which restores inhibition of expression. Compensatory nucleotide substitutions that were introduced in mutant 33-S1b' to re-establish the putative mRNA secondary structure predicted for the GNA33 region encoding the signal peptide (see also Fig. 5a) are indicated in bold on the left.

 
For each of the mutants described above a prediction of secondary structure was determined using the MFold program (Zuker, 1989). The predictions of optimal secondary structures that best fitted the experimental data were obtained by analysing the nucleotide sequence between the T7 transcription start site and the end of the signal peptide coding region. As shown in Fig. 5(d), extending the analysis to include the 5' non-coding region does not alter the prediction of the stem–loop structure within the wild-type GNA33 signal peptide sequence. Moreover, the secondary structure predicted for all mutants, except L1f, is identical in the non-coding portion of the sequence, while significant differences are found within the signal peptide coding region. Only in the compensatory S1b' mutant does the predicted structure coincide with that determined for the wild-type sequence (Fig. 5d).


   DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
It has been proposed that expression of lytic murein transglycosylase activities in the cell must be tightly controlled to ensure integrity of the cell wall during both cell elongation and division (Höltje, 1998; Nanninga, 1998). However, a detailed definition of the role played by the different lytic murein transglycosylase homologues and a description of mechanisms for regulating their activities in the cell are still missing.

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 stem–loop 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 stem–loop 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 stem–loop 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 stem–loop structure described herein may represent a control point for GNA33 synthesis during cell division.


   ACKNOWLEDGEMENTS
 
We are very grateful to G. M. Rossolini for providing the phoC plasmid, anti-PhoC antibodies and for helpful advice. We thank M. M. Giuliani for GNA33-specific antibodies, V. Scarlato for many valuable discussions and critical reading of the manuscript and G. Corsi for artwork.


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Received 23 September 2003; revised 8 January 2004; accepted 26 January 2004.



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