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1 Molecular Genetics Laboratory, Instituto de Tecnologia Química e Biológica da Universidade Nova de Lisboa, 2780 Oeiras, Portugal
2 Laboratory of Microbiology, The Rockefeller University, 1230 York Avenue, NY 10021, USA
Correspondence
Alexander Tomasz
tomasz{at}mail.rockefeller.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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-lactam antibiotics (De Jonge & Tomasz, 1993
). Recent experiments have indicated that the resistance protein PBP2A may also function in contexts other than drug resistance, and in cooperation with one of the native PBPs, namely PBP2.
The first evidence for this cooperation was the finding that inhibition of transcription of pbpB, which is the structural gene of PBP2, was lethal in a meticillin-sensitive S. aureus (MSSA), but not in the MRSA strain COL, which expresses PBP2A in a constitutive manner (Pinho et al., 2001b). PBP2 is a bifunctional cell wall synthetic enzyme containing both transglycosylase (TGase) and transpeptidase (TPase) domains (Murakami et al., 1994), and genetic experiments have demonstrated that the essential function of this protein, successfully replaced by PBP2A, is that of the TPase domain (Pinho et al., 2001b). Cooperative functioning of PBP2 and PBP2A has been further documented by the observation that growth of MRSA in the presence of high concentrations of antibiotic requires not only PBP2A, but the functioning of the TGase domain of the native PBP2 as well (Pinho et al., 2001a). Additional evidence for cooperative functioning has come from the recent observation that the presence of PBP2A is able to prevent dislocation of PBP2 from the cell wall growth zone, a phenomenon that occurs in MSSA strains exposed to -lactam antibiotics (Pinho & Errington, 2005).
The purpose of the studies described here was to examine in more detail these intriguing and unusual phenomena, which involve cooperative functioning between two proteins: a cell wall synthetic enzyme native to S. aureus, and an antibiotic-resistance factor acquired by S. aureus from an extra-species source.
In a conditional mutant, we followed the effects of turning off and turning on the transcription of pbpB on the growth, oxacillin resistance, cell structure and cell wall composition and gene expression profiles, and also the production of PBPs 2 (in strain RN4220spac : : pbpB) and 2A in the MRSA strain COLspac : : pbpB. Extended incubation of the bacteria in the absence of pbpB transcription resulted in a gradual decline in the abundance of the mecA transcript, and a drastic reduction in the cellular amounts of PBP2A. The phenomenon appeared to be specific, and reversible by readdition of IPTG to the growth medium.
These surprising findings identify yet another cooperative phenomenon between pbpB and mecA that seems to occur at the level of gene expression. Changes in the transcription of pbpB seem to bring about parallel changes in transcription of the resistance gene mecA – apparently in response to some trans-acting factor that is produced in the cells as a function of the level of transcription of the native pbpB.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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. S. aureus strains were grown in tryptic soy broth (TSB) with aeration at 37 °C, or on tryptic soy agar (TSA) plates at 37 °C. Escherichia coli strains were grown in Luria–Bertani broth (LB) with aeration at 37 °C. For selection and maintenance of S. aureus and E. coli transformants, media were supplemented, as required, with 10 mg erythromycin l–1, 10 mg chloramphenicol l–1 and 100 mg ampicillin l–1, all obtained from Sigma.
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Cultures were centrifuged, bacterial pellets were washed twice with TSB (to remove IPTG), and the bacteria were resuspended in fresh pre-warmed TSB without IPTG to an initial OD620 of 0.05 (time point 1). Both the RN4220spac : : pbpB and COLspac : : pbpB cultures were then incubated in the IPTG-free medium at 37 °C with aeration. Both cultures grew with close-to-normal doubling times for about 2 h, and, by hour 3, the OD620 increase of the MSSA culture (RN4220spac : : pbpB) had come to an abrupt stop (time point 2), while the MRSA culture (COLspac : : pbpB) continued to grow with gradually decreasing generation times for a total of 20 h after the removal of IPTG from the medium. In order to prevent the COLspac : : pbpB culture from entering stationary phase during this extended period, the culture was periodically diluted to an OD620 of 0.05 in fresh TSB without IPTG each time the culture density had reached an OD620 of 0.4. Samples were removed from the COLspac : : pbpB culture after the 11th hour (time point 3) and 20th hour (time point 4) of incubation in the IPTG-free medium. A portion of this culture was diluted to an OD620 of 0.05 in fresh TSB containing a full supplement of IPTG (100 µM), and growth of the culture was monitored. After a lag of about 2 h, the culture of COLspac : : pbpB supplemented with IPTG began to grow at an increasing rate, eventually reaching a virtually normal doubling time. Samples were removed from this culture at time point 5, which corresponded to a total of 26.5 h of incubation from the beginning of the experiment. Determination of OD620 was carried out in a Spectrophotometer Ultraspec III (Pharmacia).
Population analysis profiles (PAPs).
Antibiotic susceptibility of COLspac : : pbpB was determined by PAPs, as described previously (Tomasz et al., 1991), on agar plates containing various concentrations (mg l–1) of oxacillin (Sigma), and supplemented with 0, 50 or 500 µM IPTG. Colonies were counted after incubation of the plates at 37 °C for 48 h.
Luciferin–luciferase assay.
Cultures of pbpB conditional mutants of the sensitive MSSA strain RN4220spac : : pbpB and the MRSA strain COLspac : : pbpB, grown overnight with aeration in TSB supplemented with 100 µM IPTG, were diluted to an OD620 of 0.05 in fresh TSB containing 100 µM IPTG, and incubated with aeration at 37 °C until they reached an OD620 of 0.4. The cultures were centrifuged, and traces of IPTG were removed, as described above. The bacteria were then resuspended in fresh pre-warmed TSB without IPTG to an initial OD620 of 0.05, and the amounts of ATP released by the bacteria were determined indirectly by the luciferin–luciferase assay, which was adapted from O'Neill et al. (2004). The ATP levels (expressed as relative luminescence units) produced by each strain were normalized to the OD620.
DNA methods.
DNA manipulations were performed by standard methods. Restriction enzymes were used as recommended by the manufacturer (New England Biolabs). Routine PCR amplification was performed with Tth DNA polymerase (HT Biotechnology). Wizard Plus Minipreps and Midipreps (Promega) systems were used for plasmid extraction. PCR and digestion products were purified with Wizard PCR Preps and Wizard DNA Clean-up systems (Promega). Ligation reactions were performed with T4 ligase (New England Biolabs). DNA sequencing was done at the Rockefeller University Protein/DNA Technology Center using the BigDye terminator cycle sequencing method, with either a 3700 DNA analyser for capillary electrophoresis, or ABI Prism 377 DNA sequencers for slab gel electrophoresis.
RNA preparation, Northern blotting, and analysis of the relative mRNA abundance.
After extraction of RNA (Sobral et al., 2003), 5 µg of each RNA sample was analysed by electrophoresis in a 1.2 % (w/v) agarose gel containing 0.66 M formaldehyde and MOPS (Sigma). The RNA was blotted onto Hybond-N+ membranes (Amersham) with a turbo blotter alkaline transfer system (Schleicher & Schuell) with 20x SSC. The PCR-amplified DNA probes were labelled with [
-32P]dCTP (Amersham) by using a Ready to Go labelling kit (Amersham), and hybridized under high-stringency conditions. The blots were subsequently washed and autoradiographed. In order to compare the relative abundance of mecA, pbpC and 16S rRNA transcripts produced by COLspac : : pbpB grown in the presence or absence of IPTG, a semi-quantitative method was introduced that allowed normalization of the Northern signals to an internal control (Matsuzaki et al., 2001; Schelert et al., 2004). For this purpose we used the value obtained for the Northern signal of the housekeeping gene pta, which encodes the phosphatase acetyl transferase, and which is constitutively expressed in S. aureus (Enright et al., 2000). The intensities of the mecA, pbpC and 16S rRNA Northern blot signals were divided by that of the pta transcript in the same preparation.
Construction of promoter fusions.
A DNA fragment of 443 bp, encompassing the region upstream of the pta gene from the MRSA strain COL, was amplified by PCR with Pfu Turbo DNA polymerase (Stratagen), and primers PtaBamHI (5'-CGGGATCCGCTTGATCACCAGATTTTG-3') and PtaHindIII (5'CGTAAGCTTCGTTCGTCCTCTCCTTCA-3'). The primers were engineered to carry restriction sites (underlined). The following PCR conditions were used: 94 °C for 4 min, 40 cycles of 94 °C for 45 s, 55 °C for 45 s, 72 °C for 1 min, and a final extension step of 72 °C for 10 min. The purified PCR product was subsequently cloned into plasmid pLC4 to generate the recombinant plasmid pSG3. The recombinant plasmid was then introduced into S. aureus RN4220 electrocompetent cells by electroporation with Gene Pulser apparatus (Bio-Rad), as described by Kraemer & Iandolo (1990), and finally transduced into strain COLspac : : pbpB by using phage 80, as previously described (Oshida & Tomasz, 1992), except that 100 µM IPTG was added to the medium for selecting COLspac : : pbpB+pSG3 transductants.
Enzyme assays.
Catechol 2,3-dioxygenase activity was used to measure the activity of mecA and pta promoters using the assay of Sheehan et al. (1992), except for the lysis of bacteria, which was done by using glass beads and FastPrep 120 (Bio 101 Savant) in 100 mM phosphate buffer (pH 7.5) containing 10 % (v/v) acetone. The reaction mixture, consisting of 100 mM potassium phosphate buffer (pH 7.5), 0.2 mM catechol, and 200 µg crude extract, was incubated at room temperature for 20 min, with A375 readings taken at 2 min intervals in an Ultraspec III spectrophotometer (Pharmacia). Enzymic assays were done in triplicate using crude extracts prepared on three different days. One milliunit corresponds to the formation at room temperature of 1 nmol 2-hydroxymuconic semialdehyde min–1. Specific activity is reported in milliunits per milligram of total protein. Protein concentrations were measured by using the Modified Lowry Protein Assay (Pierce), with bovine serum albumin as a standard. COLspac : : pbpB harbouring the plasmid pLC4 was used as the negative control.
Quantitative real-time PCR (Q-PCR).
The levels of pbpA, pbpB, pbpC, pbpD and mecA mRNA transcripts in strains COL and COLspac : : pbpB grown under different conditions were determined by Q-PCR. Total RNA extracts were used as templates for cDNA synthesis by random priming. To remove genomic DNA contamination, RNA samples were treated with the RNase-free DNase I set (Qiagen), and further purified with the RNeasy Mini Kit (Qiagen), according to the manufacturer's instructions. RT-PCR was performed by using the GeneAmp RNA PCR kit (Applied Biosystems) in a 100 µl reaction mixture under the following conditions: 10 min at 25 °C, 60 min at 48 °C, and 5 min at 94 °C. To quantify cDNA generated by reverse transcription from target RNA, real-time PCR with SYBR Green I was performed by using SYBR Green PCR master mix in the ABI Prism 7900 Sequence Detection System (Applied Biosystems). The 25 µl reaction mixture contained 1x iTaq SYBR Green PCR supermix with ROX (Bio-Rad), forward and reverse primers (each at a concentration of 100 nM), and 5 µl template (reverse transcription product). The primers were designed by Primer Express software (Applied Biosystems) and are listed in Table 2. The thermal conditions were: 2 min at 60 °C and 10 min at 95 °C, followed by 40 cycles at 95 °C for 15 s, 60 °C for 1 min and 72 °C for 30 s. Three different RNA samples of the same culture and the various controls were processed in duplicate. Fluorescence was measured at the end of the annealing-extension phase of each cycle. A threshold value for the fluorescence of all samples was set manually. The reaction cycle at which the PCR product exceeds this fluorescence threshold was identified as the threshold cycle (CT). The CT was then converted to relative quantity of mRNA by using a standard curve. The standard curve was generated, via the Q-PCR program conditions, using serial twofold dilutions (2 ng to 66 pg) of genomic DNA. Relative gene expression was expressed as a ratio of target gene (mecA, pbpA, pbpB, pbpC and pbpD) concentration to housekeeping gene (pta) concentration. To verify the specificity of the PCR amplification products, melting curve analyses were performed using the following thermal cycling profile: 95 °C for 15 s, 60 °C for 15 s, 95 °C for 15 s, with 2 % increment.
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). Protein concentrations were determined using the Modified Lowry Protein Assay (Pierce), with the bovine serum albumin as a standard.
Western blotting analysis.
For detection of PBP2A in the membrane protein fraction, 60 µg of each membrane protein preparation was resolved on an 8 % (w/v) acrylamide/0.06 % (w/v) bisacrylamide gel at a constant current of 20 mA, and transferred to a nitrocellulose membrane by Western blotting, as previously described (Wu et al., 2001). Incubation with a monoclonal antibody against PBP2A of an MRSA strain (Eli Lilly & Co.) was carried out with the ECL Western blot analysis system (Amersham) (Wu et al., 2001).
Penicillin-binding assays.
Membrane proteins (150 µg protein per sample) were labelled with benzyl[14C]penicillin potassium (5.846x1012 Bq mg–1; GE Healthcare), at a final concentration of 20 mg l–1 in a 20 µl volume, for 10 min at 30 °C. Addition of an excess of unlabelled benzylpenicillin [1000 mg l–1 in 10 % (v/v) SDS] was used to stop the reaction. An equal volume of sample buffer [125 mM Tris/HCl, pH 6.8, 4 % (v/v) SDS, 2 % (w/v) glycerol, 100 mg bromophenol blue l–1, and 1.43 M 2-mercaptoethanol] was added to the samples, which were boiled for 5 min at 95 °C. Protein separation on SDS-PAGE was carried out on an 8 % (w/v) acrylamide/0.06 % (w/v) bisacrylamide gel, at a constant current of 20 mA. The gel was exposed to a tritium storage phosphor screen (GE Healthcare) for 2 weeks, and the screen was scanned with a typhoon scanner (GE Healthcare).
Cell wall analysis.
Cell walls were isolated; the peptidoglycan was purified, and hydrolysed with M1 muramidase, and the resulting muropeptides were reduced with borohydride and separated by reverse-phase HPLC, as previously described (De Jonge et al., 1992).
Electron microscopy.
Aliquots (1 ml) of bacterial cultures were harvested by low-speed centrifugation, and fixed with 1 ml 2.5 % (v/v) glutaraldehyde. Electron microscopy was done at the Electron Microscopy Service of The Rockefeller University.
Reproducibility.
All experiments, including growth curves, Northern blots and PAPs, were repeated at least five to seven times, and HPLC profiles were obtained at five different time points in order to assure reproducibility. Details about the reproducibility of Q-PCR and promoter fusion experiments are described below. Determination of protein profiles and the radioactive PBP binding assay (Fig. 2), ATP release and Western blotting were only performed once. Electron micrographs were selected after scanning numerous fields at low-power magnification to make sure that photos presented at high-power magnification represented ‘typical’ structures.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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), and transcription of pbpB and rate of growth (OD620 increase) were followed over time. Transcription of pbpB was turned off within 10 min after the removal of the inducer, as indicated by Northern blotting (data not shown). On the other hand, the culture continued to grow for the first 2 h after the removal of IPTG from the medium, with a doubling time virtually the same as that in the presence of IPTG (55 versus 49 min). After about 3 h growth (time point 2), corresponding to approximately 2.5 generation times, the OD620 increase of strain RN4220spac : : pbpB came to an abrupt halt. Electron microscopic observation showed the accumulation of empty cells, suggesting lysis, and extending the incubation time by a further 4 h resulted in the loss of about 50 % of the viable titre of the culture (data not shown). Testing the growth medium with the luciferin–luciferase assay indicated extensive release of ATP during incubation in the IPTG-free medium (see Fig. 1a–c).
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shows the Coomassie-blue-stained gel, and the PBP pattern obtained by the radioactive-penicillin-binding assay. A diminished amount of the Coomassie-stained material was evident in the position corresponding to that of PBP2 in the stained gel, and PBP2 was no longer detectable in the penicillin-binding assay. Large amounts of PBP4 were observed for the RN4220spac : : pbpB mutant grown in the absence of the inducer, suggesting that PBP2, which has previously been proposed to cooperate functionally with PBP4 in the cross-linking of peptidoglycan (Leski & Tomasz, 2005), may exert some kind of positive control in the translation of this PBP.
Inhibition of pbpB transcription in an MRSA strain: effect on growth, cell structure and cellular amounts of PBP2A
The design of this experiment was exactly the same as that for RN4220spac : : pbpB. MRSA strain COLspac : : pbpB was resuspended in growth medium free of IPTG, and the transcription of pbpB and rate of growth (OD620 increase) were followed over time. Similar to RN4220spac : : pbpB, in COLspac : : pbpB, transcription of pbpB was turned off 10 min after the removal of the inducer (data not shown), but the culture continued to grow for 20 h. During the first 3 h, the doubling time of the culture was virtually the same as that in the presence of IPTG (51 versus 48 min), but from the third hour on, the doubling time began to gradually increase, to reach – between the 11th and the 20th hour of incubation – a value as long as 180 min (see Fig. 1). A portion of this extensively ‘starved’ culture was resuspended in medium containing an optimal concentration of IPTG. Within 30 min after readdition of IPTG, the pbpB transcription signal reappeared (data not shown), and, after a lag of about 2 h, the culture began to grow with a doubling time approaching that of the original culture.
Samples were removed from COLspac : : pbpB grown in the absence of IPTG at several times during the experiment: at the beginning of cultivation (time point 1), after 20 h incubation in the IPTG-free medium (time point 4), and at a time after the readdition of IPTG to the medium when the culture of COLspac : : pbpB had resumed normal growth (time point 5). Electron microscopic thin sections began to show a few cells with extensive morphological abnormalities by the end of the 3-h period of growth in the absence of IPTG (data not shown).
The release of ATP into the growth medium was relatively small compared with the extensive release of ATP observed in samples from RN4220spac : : pbpB starved for the same length of time (see Fig. 1b). Cells with abnormal morphology became predominant in the cultures of COLspac : : pbpB grown for 20 h in the absence of IPTG (see Fig. 3b). The abnormal morphology included a block in cell separation, enlarged and abnormally placed septa, and amorphous cell-wall-like material accumulating at the cell surface. These abnormal structures gradually decreased in number as the bacteria resumed multiplication in the IPTG-containing medium (see Fig. 3b).
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shows the increase in the relative proportions of the monomeric muropeptides 1 and 5, and a parallel decrease in the proportion of the highly cross-linked muropeptides (elution time >90 min) in the cell walls of bacteria grown in the absence of IPTG for 20 h. These structural abnormalities had virtually disappeared from the cell wall of the culture that had resumed growth in the IPTG-containing medium.
Effect of inhibited pbpB transcription on the transcription of mecA, and on the production of PBP2A
Samples were removed from COLspac : : pbpB grown in IPTG-free medium, and also from the same culture in which pbpB transcription was reinitiated (by addition of IPTG to the medium). The samples were used to test the effect on the transcription of a variety of genetic determinants (determined by Northern blotting), and also on the cellular amounts of the mecA gene product PBP2A (determined by Western blotting). All samples were removed from cultures at an OD620 of 0.4.
The extended growth in the absence of pbpB transcription did not alter the transcription of pta, 16S rRNA and pbpC, but removal of IPTG caused an immediate overexpression of mgtB, a mono-functional TGase, which showed normal levels of expression equally promptly upon renewed transcription of pbpB. Most interestingly and unexpectedly, growth in the IPTG-free medium caused a gradual slow decrease in the intensity of the transcriptional signal of mecA, which was then reversed as the culture resumed growth following readdition of IPTG to the medium (Fig. 4a). Testing the transcription of mecA in COL and COL+pMGPII (Pinho et al., 2001b) grown in increasing concentrations of IPTG demonstrated that neither LacI, nor IPTG itself, interfered with expression of mecA.
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Effect of pbpB transcription on the level of oxacillin resistance
An overnight culture of COLspac : : pbpB was plated on three sets of agar media. In the first set, the medium contained no IPTG; in the second and third sets, the medium contained 50 and 500 µM IPTG, respectively. The agar media were then supplemented with increasing concentrations of oxacillin, and the bacterial culture was plated at different cell concentrations for the analysis of PAPs (as described in Methods). As an additional control, strain COL without the Pspac : : pbpB construct was also plated. Fig. 5(a) shows that the IPTG concentration in the agar medium had a profound effect on the shape of the PAP curves: the MIC value for the majority of the cells increased with the concentration of IPTG, which also increased the relative homogeneity of the PAP profile. It is noteworthy that the high-level and homogeneous phenotype of the parental strain COL was not attained, even at the optimal concentration of the inducer. The abundance of pbpB transcript produced by the conditional mutant is never as high as in strain COL due to the disruption of the prfA–pbpB operon, the major transcript of pbpB gene, after integration of the suicide vector with the IPTG-inducible Pspac promoter into the COL chromosome (Pinho et al., 1998). Fig. 5(b) shows the degree of transcription of pbpB at the corresponding concentrations of IPTG, and also demonstrates the existence of only one pbpB transcript in the COL pbpB mutant.
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summarizes the data. Expression of pbpB was suppressed, and transcription of mecA was significantly lower, in COLspac : : pbpB grown in the absence of the inducer. Transcription of mecA increased approximately twofold in parallel with the renewed expression of pbpB following the readdition of IPTG to the growth medium. Transcription of pbpA, pbpC and pbpD remained unaffected.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Presumably, the ‘take-over’ by PBP2A occurred at the time when PBP2 was no longer detectable by the penicillin-binding assay (see Fig. 2). An effective take-over by PBP2A is also supported by the analysis of the peptidoglycan isolated from COLspac : : pbpB after 20 h growth in the IPTG-free medium, when only relatively minor abnormalities were evident (an increase in the proportion of monomeric muropeptides, and some decrease in highly cross-linked oligomers) (see Fig. 3).
Nevertheless, the gradual increase in the doubling time of COLspac : : pbpB, reaching 180 min after 11 h growth in the IPTG-free medium, indicates that the capacity of the mecA gene product PBP2A to fully replace the native PBP2 is not without limits. This was also supported by the accumulation in the culture of structurally abnormal cells, which showed blocked cell separation, widening of the septal areas, and appearance of amorphous cell-wall-like material at the bacterial surface (Fig. 3b). These structural features are very similar to those described in S. aureus with an inhibited autolytic system, which can be fully reversible (Sieradzki & Tomasz, 2006). This interpretation is also consistent with the fact that the morphological abnormalities were reversible in the COLspac : : pbpB culture, since they became extremely rare after resumption of normal growth following the readdition of IPTG to the medium (Fig. 3b).
In order to find the reason for decreasing growth rates of COLspac : : pbpB, we tested the rate of transcription of mecA, and the cellular amounts of PBP2A, after various periods of incubation of the bacteria in IPTG-free medium. Surprisingly, both the abundance of the mecA transcript, and the amounts of PBP2A, showed a significant decline as the time of incubation in the IPTG-free medium progressed, suggesting that the inhibition of transcription of pbpB – directly or indirectly – impacts on the transcription, and also on the translation, of the resistance gene mecA.
Strain COL carries a mecA gene as part of an SCCmec type I cassette, which does not contain functional mecI/mecR1, and the bacterium also lacks the blaI/blaR1 system (Oliveira et al., 2001). In this strain, mecA is constitutively expressed, producing high levels of mecA transcript, and large amounts of PBP2A (Hackbarth & Chambers, 1993).
Our observations suggest that the transcription of this mecA gene lacking the dedicated controlling elements is somehow influenced by the regulatory circuitry that controls transcription of the native pbpB. This apparent ‘regulatory’ connection appears to be specific, since testing the effect of the blocked pbpB transcription on the transcription of a number of other genetic determinants failed to detect similar changes. These determinants included pta, the determinant of 16S rRNA, and pbpC, A and D. Most importantly, the decline in transcription of mecA that was observed in the PBP2-starved bacteria was reversed when the transcription of pbpB was reinitiated by adding the inducer back to the growth medium (Fig. 4). These parallel changes in transcription of pbpB and mecA were confirmed by Q-PCR, and by the results of promoter fusion experiments, which suggests that some factor accumulating in bacteria with an inhibited pbpB transcription can influence the reading of the mecA promoter.
An interesting consequence of the removal of IPTG from the growth medium of COLspac : : pbpB was the overexpression of mgtB, a monofunctional TGase of S. aureus, which rapidly followed the inhibition of pbpB transcription. Readdition of IPTG to the medium caused an equally rapid reduction in the abundance of the mgtB transcript, suggesting the existence of a regulatory system that may provide an alternative TGase for the cells in which the TGase activity of PBP2 is not available. Experiments are in progress to test whether or not the TGase activity of mgtB becomes essential for the bacteria in which PBP2A replaces the transpeptidase activity of PBP2.
A previous study demonstrated that the TGase domain of PBP2 becomes essential for growth and cell wall synthesis when COLspac : : pbpB is exposed to high concentration of oxacillin (Pinho et al., 2001a). In view of the new observation on the overexpression of mgtB under the same conditions, we repeated titration of oxacillin resistance as a function of the concentrations of IPTG in the medium. Fig. 5 shows that both the resistance level of the majority of the bacteria, and the population profile of the culture, were functions of the abundance of the pbpB transcript, indicating that MgtB may not be able to replace efficiently the TGase function of PBP2 in the assembly of peptidoglycan. These data also suggest that the optimal expression of resistance to oxacillin may require a stoichiometric balance between the two proteins PBP2 and PBP2A, which are known to cooperate in cell wall synthesis (Pinho et al., 2001a).
The observation described in this paper, namely that transcription of the resistance determinant mecA is under the ‘control’ of the determinant of an essential native pbp, is not only surprising, but counterintuitive as well, since survival and multiplication of S. aureus with an inhibited pbpB is dependent on the continued expression and function of the resistance gene mecA and its protein product. However, the physiological scenario of a S. aureus pbpB conditional mutant with inhibited PBP2 production is clearly an abnormal one, and the ‘normal’ role of mecA gene in S. aureus is to provide antibiotic resistance, not to replace the native PBP2 in the absence of antimicrobial agents in the medium.
The tuning down of transcription of mecA, and the decline in the cellular amounts of PBP2A, follow the halt in pbpB transcription only after a prolonged lag, suggesting that the link between transcription of these two genes is an indirect one. Possibly, PBP2A somehow ‘senses' the lack of PBP2, the native cell wall synthesis protein with which the resistance protein is clearly able to cooperate in the functional sense (Pinho et al., 2001a). We propose that the new evidence described in this paper, documenting a connection between the transcription of pbpB and mecA, may be an indication of an attempt by the S. aureus cell to integrate the uncontrolled transcription of mecA into a regulatory circuitry that allows harmonious functioning of cell wall synthetic enzymes. The mechanism by which transcription of mecA is downregulated in the absence of pbpB transcription is currently under investigation.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Received 20 April 2006;
revised 23 May 2006;
accepted 24 May 2006.
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), COLspac : : pbpB grown without IPTG (
), COLspac : : pbpB grown with 50 µM IPTG (
) and 500 µpM IPTG (
). (b) Northern blotting analysis of pbpB transcription in COLspac : : pbpB grown in increasing concentrations of IPTG. The parental strain COL was used as a control.