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Institut de Biotecnologia i Biomedicina and Departament de Bioquímica i Biologia Molecular, Universitat Autònoma de Barcelona, 08193 Bellaterra, Barcelona, Spain
Correspondence
Jaume Piñol
jaume.pinyol{at}uab.es
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Two supplementary figures and a supplementary table are available with the online version of this paper.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Weiner et al., 2000). These data together with the current dearth of identified regulatory genes have compelled some investigators to suggest the presence in these organisms of novel and still unknown mechanisms to regulate gene expression, at both the transcriptional and the translational level.
Mycoplasma genitalium is a human pathogen causing Chlamydia-negative non-gonococcal urethritis and it is phylogenetically related to M. pneumoniae, the causative agent of walking pneumonia and other extra-pulmonary pathologies in humans (Chiner et al., 2003; Jensen, 2004). With only 525 ORFs, M. genitalium has the smallest genome of any organism that can be grown in pure or axenic culture, which makes it an appealing model of a minimal cell. The genome of this micro-organism also shows several regions of repetitive DNA (the MgPa islands) that contain sequences derived from the ORFs related to cytadherence in the MgPa operon (Peterson et al., 1995). Studies concerning transcriptional analysis of M. genitalium ORFs are very scarce and limited to a few ORFs. These works have relied on the use of real-time RT-PCR (Benders et al., 2005) and macroarrays (Musatovova et al., 2006a). In addition, microarrays have been used in the related species M. pneumoniae and M. hyopneumoniae (Madsen et al., 2006a, b; Weiner et al., 2003). Therefore, the availability of promoter-probe vectors could benefit the study of gene expression and regulation in these micro-organisms, since this technique makes possible the detection of transcripts which are not usually considered when using other strategies. Moreover, a promoterless vector based on the lacZ gene is a simple and well-known system for studying gene expression.
Several mycoplasma promoter-probe vectors based on the Tn4001 transposon have been developed (Dybvig et al., 2000; Knudtson & Minion, 1993). Although a reporter vector has been recently described in M. pneumoniae (Halbedel & Stulke, 2006), no specialized promoter-probe vectors are currently available in M. genitalium. We have previously developed the pMTnTetM438 minitransposon vector, which significantly improves transformation efficiency in M. genitalium (Pich et al., 2006b) and has been used to isolate mutants with transposon insertions in ORFs related to gliding motility (Pich et al., 2006a). In the present work, we report the construction of a promoter-probe vector based on the pMTnTetM438 plasmid and the results of a first survey aimed to study in vivo the transcription in M. genitalium. Many transposon insertions were located in ORFs expected to be highly transcribed (i.e. MgPB, P140 adhesin gene). However, we have also detected a considerable amount of transposon insertions in intergenic regions and in the non-coding strand of many ORFs. In the absence of identified regulatory genes, the presence of antisense transcripts may provide the first mechanism to regulate gene expression in M. genitalium.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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) at 37 °C under 5 % CO2 in tissue culture flasks (TPP). To select mycoplasma colonies expressing the lacZ gene, plates were supplemented with 2 µg tetracycline ml–1 and 150 µg X-Gal ml–1.
DNA manipulations.
All primers used in this work are listed in Table 1. Plasmid DNA was obtained by using the Fast Plasmid Mini Eppendorf kit. The purification of PCR products and digested fragments from agarose gels was achieved using the E.Z.N.A. Gel Extraction kit (Omega BIO-TEK). The E. coli lacZ gene from plasmid pMC1871 (Shapira et al., 1983) was amplified by PCR using the primers lac5b and lac3 or alternatively lac5a and lac3 (Table 1). The first set of primers adds the RBS sequence from the Bacillus subtilis citZ gene and a translation start codon in-frame with the 5' end of the lacZ gene. The second set adds only the translation start codon to the lacZ coding sequence. Both PCR products were inserted into the pMTnTetM438 minitransposon (Pich et al., 2006b) between the XhoI and SalI sites to obtain the placRBS+ and placRBS– constructs.
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; Reddy et al., 1996) with some modifications. Briefly, a mid-exponential-phase M. genitalium culture grown in SP-4 medium in 150 cm2 tissue culture flasks was washed three times with electroporation buffer (8 mM HEPES, pH 7.2, 272 mM sucrose). Cells were scraped off and resuspended in electroporation buffer at a concentration of approximately 109 cells ml–1. Then, 90 µl aliquots were placed in 2 mm gapped electroporation cuvettes and mixed with 10 µg plasmid DNA dissolved in electroporation buffer. After 15 min in ice, cells were electroporated at 2.5 kV, 50 µF and 129 
in an electrocell manipulator 600 (BTX) and immediately kept in ice for another 15 min. Then 900 µl SP-4 medium was added and the cells were incubated for 2 h at 37 °C. Aliquots of 200 µl were spread on SP-4 agar plates supplemented with 2 µg tetracycline ml–1 and 150 µg X-Gal ml–1 and incubated at 37 °C in 5 % CO2. Only well-isolated blue colonies were picked, propagated in 96-well plates containing 200 µl SP-4 medium and stored at –80 °C until analysed.
Genomic DNA manipulations.
After growing transformants in 75 cm2 cell culture flasks with 20 ml SP-4 medium, cells were scraped off and genomic DNA was isolated by using the E.Z.N.A. Bacterial DNA kit (Omega BIO-TEK). Southern blot hybridizations were performed by using the 1.13 kb XhoI–EcoRV fragment of the pLacRBS+ plasmid as a probe and the DNA Dig labelling and detection kit (Roche). Sequencing reactions were performed with fluorescent dideoxynucleotides using the primer Ntergal and the BigDye Terminator v3.0 Cycle Sequencing kit (Applied Biosystems) following the recommendations of the manufacturer. Sequence reactions were analysed in an ABI 3100 Genetic Analyzer (Applied Biosystems). The sequences obtained were aligned with the current release of the M. genitalium G-37 genome (version L43967.2, GI:84626123) using the BLAST program (Altschul et al., 1997) to determine the precise insertion sites. The codon usage bias of the highly expressed genes was analysed using the GCUA-WIN program (McInerney, 1998). The transcription terminators in M. genitalium were predicted using the Transterm program (http://nbc11.biologie.uni-kl.de/framed/left/menu/auto/right/transterm/) (Ermolaeva et al., 2000) and those showing a confidence equal or better than 98 % were selected.
Measurement of β-galactosidase (β-Gal) activity.
Cultures were grown in suspension in 10 ml SP-4 medium to mid-exponential phase as determined by medium colour change. Then, 1.5 ml of culture was centrifuged for 15 min at 14 000 g and the cells were resuspended in 75 µl lysis solution. A volume of 10 µl was processed using the ATP Bioluminescence Assay kit HS II (Roche) to determine the ATP content. Measuring ATP instead of turbidity is considered the best method to estimate the mycoplasma cell mass (Robertson & Stemke, 1995). The remainder of the cell lysate was used to determine the β-Gal activity by a modified Miller test (Miller & Hershberger, 1984). Briefly, 65 µl cell lysate was mixed with 585 µl buffer Z (0.06 M Na2HPO4, 0.04 M NaH2PO4, 0.02 M KCl, 1 mM MgSO4, 0.28 % β-mercaptoethanol) and 65 µl chloroform, vortexed vigorously and kept in a bath at 28 °C for 5 min. Then 130 µl ONPG was added to the sample. When the sample became yellow, the reaction was stopped by addition of 325 µl 0.5 M Na2CO3. The time between substrate addition and colour change was measured. Finally, the samples were centrifuged 15 min at 14 000 g and the supernatant absorbance was recorded at 420 nm.
RNA manipulations and primer extension assay.
Total RNA was isolated using the TRI Reagent kit (Invitrogen) following the recommendations of the manufacturer. Before the RT-PCR assays, total RNA was treated with DNase I (New England Biolabs). Retrotranscription reactions in primer extension assays and RT-PCR reactions were performed using a strand-specific primer and the SuperScript First-Strand Synthesis kit (Invitrogen) according to the instructions of the manufacturer. The RT-PCR negative controls were performed using non-reverse-transcribed RNA as a template. In primer extension assays, the retrotranscription reactions were carried out using 10–20 µg total RNA and 2 pmol of a 6-carboxifluorescein phosphoramidite (6-Fam)-labelled primer (Lloyd et al., 2005). The 6-Fam-labelled cDNA was treated with 2 units RNase H for 20 min at 37 °C and precipitated with ethanol. The sample was then redissolved in 10 µl formamide and mixed with 0.5 µl ROX markers (Applied Biosystems). Electrophoresis was performed using an ABI 3130 XL Genetic Analyzer (Applied Biosystems) and the DNA fragments were sized using the GENESCAN analysis software (Applied Biosystems).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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) as well as the TetM438 marker, which effectively confers tetracycline resistance to the M. genitalium transformant cells.
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), as previously reported for colonies of M. gallisepticum and M. pneumoniae transformed with lacZ promoter-probe vectors (Halbedel & Stulke, 2006; Knudtson & Minion, 1993).
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). The Southern blot data were also consistent with the transposon insertion sites derived from sequencing analysis (Table 2).
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; Supplementary Table S1, available with the online version of this paper). Two potentially disruptive insertions were identified in ORFs considered previously as essential in global transposon analyses: mg224 and mg350.1 (Glass et al., 2006; Hutchison et al., 1999). The transposon insertion in clone 49 is located 41 bp downstream of the start codon of mg350.1, which encodes a hypothetical membrane protein. The transposon insertion in clone 39 is located 448 bp downstream of the start codon of the mg224, which is currently annotated as the ftsZ gene, involved in cell division. A comprehensive study of this mutant is in progress; preliminary data do not show an abnormal growth of this mutant in SP-4 medium.
A significant number of insertions (15 out of 49) that led to lacZ expression were found in intergenic regions, suggesting the absence of terminator sequences in many M. genitalium ORFs, in agreement with previous expectations derived from computer analyses which found few (Ermolaeva et al., 2000) or no (Washio et al., 1998) terminator sequences in the M. genitalium genome. Moreover, 50 % of the insertions in intergenic regions were found in MgPa islands. The presence of transcripts derived from MgPa islands was further confirmed by specific RT-PCR amplifications from total RNA of the G-37 (wild-type) strain (Fig. 2b
). This result was unexpected because these regions are relatively long and in some cases are far away from a putative ORF which could drive their transcription. For instance, the distance between the transposon insertion in MgPa II and tRNA-Thr, the closest annotated locus (apart from mg199, which is annotated as a pseudogene and has not been taken into account here, see Supplementary Fig. S1), is 72 kb. In addition, a terminator sequence that could block the upstream transcripts is predicted upstream of the transposon insertion in clone 41 (very close to MgPa VII, Fig. 2d). These results suggest the presence of promoter sequences either inside or in the vicinity of the MgPa islands.
Monitoring gene expression level using the promoterless vector
The background noise of β-Gal activity of the G-37 strain was found to be very low, 0.12±0.07 Miller units per 0.1 nmol ATP. The β-Gal activity values of the different clones with transposon insertions were in the range 8–2500 Miller units per 0.1 nmol ATP. The reliability of the values obtained was assessed by triplicate measures of the β-Gal activity from two independent cultures of one of the clones (Table 3). Once corrected according to the culture biomass by measuring the ATP content, β-Gal activity values were very reproducible and found to be independent of the stage of the culture, at least in a range of 24 h. This demonstrates that promoter fusions with lacZ can be used in a reliable way to quantify the gene expression levels in M. genitalium cell cultures.
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).
There is evidence that highly expressed genes exhibit a bias towards a subset of synonymous codons, which are those most accurately and/or efficiently recognized by the most abundant tRNA species (Sharp et al., 2005). The possible existence of a codon usage bias in the ORFs described above was investigated by comparing the codon usage average in the ORFs of the M. genitalium complete genome with the codon usage average in this subset of highly expressed ORFs. Although most codons did not show any significant bias, the usage for the UAA stop codon was 72 % in the whole genome whereas in highly transcribed ORFs it was 92 %, similar to that described for highly expressed genes of B. subtilis (Rocha et al., 1999). Slight differences were also found for the His CAC codon usage, which was 35 % in the whole genome and 41 % in highly transcribed transposon insertions. However, these differences are very small and support previous computer analyses indicating that highly expressed genes of several species, including M. genitalium, have no discernible differences in codon usage compared to other genes (Henry & Sharp, 2007).
Transposon insertion reveals antisense transcription
Although transposon insertions were found mainly in sense with respect to the putative transcript derived from the interrupted ORF, there was also a considerable number of insertions (14 out of 34) where the lacZ gene was transcribed from the unexpected strand. In addition, transposon insertions in both orientations were identified in mg191, mg269 and mg298 ORFs. The presence of these antisense transcripts was confirmed by RT-PCR from total RNA of the G-37 strain (Figs 2 and 3). This result is in agreement with a recent report by Benders et al. (2005) in which antisense transcription was also detected in the M. genitalium ftsZ operon. It is noteworthy that many of these antisense transcripts give rise to high levels of β-Gal activity. For instance, the β-Gal activity from the mg269 antisense transcript (clone 21) is twofold higher than that from the sense transcript (clone 30) and the β-Gal activity from the mg191 antisense transcript (clone 46) is only threefold lower than that from the sense transcript (clone 51), the most highly expressed lacZ insertion found in our work.
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). In the absence of data corresponding to the size of the transcripts, the start points of some of them were determined by performing consecutive primer extension reactions with RNA from the G-37 strain until a transcriptional start point was found. This procedure was used to locate the transcriptional origin of the mg354 antisense transcript and that corresponding to the transcript from the minus strand of the MgPa VI island. One band of 134 nt revealed the transcriptional start point of the mg354 antisense transcript, which was located upstream of the mg355 ORF (Fig. 4a). The sequence 5'-TATAAT-3' has been identified as the putative –10 box and is probably the promoter region of the mg355 ORF (Fig. 4a). The absence of a terminator sequence between mg354 and mg355 led us to think that the antisense transcript detected in the mg354 ORF is derived from the promoter of the mg355 ORF. The absence of a transcription terminator sequence between the two ORFs was confirmed by RT-PCR (Fig. 3, mg354). On the other hand, two bands of 284 and 285 nt revealed the heterogeneous transcriptional start point of the MgPa VI island transcript, which was located 257 bp upstream of the transposon insertion site in clone 19 (Fig. 4b). The presence of alternative transcriptional start points has been previously reported in several M. genitalium ORFs (Musatovova et al., 2003, 2006a; Pich et al., 2006b). The putative –10 box is 5'-TATACT-3' and shows similarity to the –10 boxes previously described for cytadherence-related operons of M. genitalium (Musatovova et al., 2003). Thus the MgPa island VI transcript is not the result of a run-on transcript including an additional ORF, but it seems to be derived from its own specific promoter located inside this region.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Since pLacRBS+ is promoterless, the lacZ gene is expected to be transcribed only in transposon insertions located in actively transcribed stretches of the M. genitalium genome. When the pLacRBS+ vector was used to transform M. genitalium cells, around 16 % of blue colonies were obtained. This suggests that only a subset of transposon insertions is transcribed strongly enough to give rise to colonies exhibiting a detectable blue colour. The pLacRBS+ vector contains the RBS signal from the B. subtilis citZ gene. Although it could be argued that this sequence could act as a spurious promoter, the low percentage of blue colonies obtained, together with the fact that β-Gal activity values of the different clones are scattered in a range of two orders of magnitude (Table 2), strongly suggests that the β-Gal activity in promoter gene fusions is a measure of the gene expression level at the transposon insertion site. However, the RBS signal seems to be essential for the translation of the lacZ gene because no blue colonies were observed after transformation with a version of the vector without the RBS signal (Halbedel & Stulke, 2006). The presence of one stop codon in each one of the three possible reading frames in the inverted repeats of the minitransposon in pLacRBS+ (Fig. 1b, c) interrupts the translation of any M. genitalium coding sequence at the insertion point, and therefore the lacZ gene will be translated as an additional cistron in the transcripts containing it. This result demonstrates that an RBS box on mRNA efficiently promotes ribosome recruitment in M. genitalium, but does not exclude the possibility that other additional sequences, different from those complementary to the 3' end of 16S RNA, can also fulfil this function.
The pLacRBS– vector contains three stop codons located respectively at positions –17, –25 and –36 bp from the lacZ start codon. As stated above, no blue colonies were obtained using this vector, suggesting that translational coupling can be expected in M. genitalium when a start codon is located less than 17 bp from the previous stop codon. This result is also relevant because the Rho factor has not been identified in Mycoplasma genomes and, in consequence, it is currently considered that polar effects derived from the appearance of a premature stop codon are only possible in genes exhibiting translational coupling (Waldo & Krause, 2006). Thus, our results can be taken as a predictive tool to determine the chance that two cistrons may be translationally coupled.
No transposon insertions were found in several ORFs related to energy metabolism (i.e. pyruvate dehydrogenase and pyruvate kinase) or in the gene coding for the elongation factor Tu. Such ORFs were previously identified as highly transcribed in M. pneumoniae using microarrays (Weiner et al., 2003), but are not detected by our system, probably because they are essential and there is a reduced probability to recover transposon insertions inside these loci without disrupting gene expression.
Transposon insertions were found in the mg350.1 and mg224 ORFs described as essential in previous global transposon analysis (Glass et al., 2006; Hutchison et al., 1999). While mg350.1 is currently annotated as a putative membrane protein, the disruption found in mg224 (annotated as ftsZ) is somewhat puzzling. The mg224 ORF codes for a highly conserved protein involved in cell division which is essential for cell viability in many micro-organisms (Margolin, 2005). Also, it has been reported that truncated forms of FtsZ are not functional (Osawa & Erickson, 2005). Although disruptions in other important genes like those coding for subunits of the DNA polymerase III or tRNA synthetases are not uncommon in global transposon analyses (Glass et al., 2006), the ftsZ gene is not found in the genomes of either Mycoplasma mobile (Jaffe et al., 2004) or the related mollicute Ureaplasma parvum (Glass et al., 2000). Our results also suggest that the ftsZ gene is not essential for cell division in M. genitalium and support the existence of alternative cell division mechanisms in Mollicutes. Further work characterizing the ftsZ mutant obtained may provide valuable information about mycoplasmal cell division mechanisms.
Several transposon insertions were found inside the MgPa islands, suggesting that they are actively transcribed from specific promoters located inside or in the vicinity of these regions. Because no start codons are present inside the coding sequences of the MgPa islands, it is thought that such sequences are untranslated unless they become transferred by recombination to the MgPa operon. In this sense, MgPa islands are currently considered as an antigenic variability reservoir (Iverson-Cabral et al., 2006; Musatovova et al., 2006b) and it is surprising to see that these untranslated regions are actively transcribed. In the same way, there is experimental evidence supporting the presence of a transcriptional terminator sequence in the MgPa island located downstream of the ftsZ operon (Benders et al., 2005). Computer analyses also predict the presence of transcriptional terminator sequences in MgPa VI (Ermolaeva et al., 2000) (Supplementary Fig. S1), and such sequences also appear in most MgPa islands and in the MgPa operon. Moreover, we have identified a transcriptional start point for the minus strand of the MgPa island VI, suggesting the presence of a specific promoter sequence inside it. Since the sequences in the MgPa islands are reminiscent of similar sequences located in the MgPa operon, we propose that the transcriptional regulatory sequences found in the MgPa islands may be implicated in the regulation of the MgPa operon transcription. The presence in a blue colony of a transposon insertion with the lacZ gene in the opposite orientation to the mg191 ORF (clone 46; Table 2), supports the existence of an antisense transcript and suggests the presence of a promoter that could be involved in the regulation of the MgPa operon (Supplementary Fig. S2).
Thirty per cent of the recovered clones show transposon insertions in the opposite strand of the corresponding ORF, suggesting the presence of a large number of antisense transcripts along the M. genitalium genome. Although the presence of one antisense transcript has been recently reported in the ftsZ operon (Benders et al., 2005), our results provide experimental evidence for the generalized presence of antisense transcripts in M. genitalium. The presence of antisense transcripts could be related to the existence of unidentified reading frames in the opposite strand of currently annotated ORFs. However, we failed to detect stretches encoding peptides longer than 100 amino acids inside genes with transposon insertions in the minus strand. In addition, none of the potential peptides display similarity to any other known protein or peptide (data not shown). Besides that, the presence of antisense transcripts has been traditionally linked to the regulation of gene expression in archaea and eubacteria. This control is well documented and occurs at many levels, including premature transcription termination, facilitated mRNA decay, and direct or indirect inhibition of translation (Wagner & Flardh, 2002). Despite most of the reported examples involving negative control, positive control is also possible (Morfeldt et al., 1995). In the absence of known regulatory proteins, antisense RNAs offer a promising and flexible mechanism to regulate gene expression in M. genitalium. In this way, the MgPa operon provides an interesting model to unravel the roles of the antisense RNAs in the control of the Mycoplasma gene expression.
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ACKNOWLEDGEMENTS |
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Edited by: C. Citti
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Received 27 October 2006;
revised 16 April 2007;
accepted 23 April 2007.
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  O. Q. Pich, R. Burgos, M. Ferrer-Navarro, E. Querol, and J. Pinol Role of Mycoplasma genitalium MG218 and MG317 cytoskeletal proteins in terminal organelle organization, gliding motility and cytadherence Microbiology, October 1, 2008; 154(10): 3188 - 3198. [Abstract] [Full Text] [PDF]
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