Identification of the Origin of Replication of the Mycoplasma pulmonis Chromosome and Its Use in...

JOURNAL OF BACTERIOLOGY, Oct. 2002, p. 5426–5435 Vol. 184, No. 190021-9193/02/$04.00�0 DOI: 10.1128/JB.184.19.5426–5435.2002Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Identification of the Origin of Replication of the Mycoplasma pulmonisChromosome and Its Use in oriC Replicative PlasmidsCaio M. M. Cordova,1 Carole Lartigue,2 Pascal Sirand-Pugnet,2 Joel Renaudin,3

Regina A. F. Cunha,1 and A. Blanchard2*University of Sao Paulo, Analises Clinica & Toxicologicas, Faculdade de Ciencias Farmaceuticas, Sao Paulo 05508-900,

Brazil,1 and Laboratoire des Interactions Plantes-Pathogenes2 and Laboratoire de Biologie Cellulaire et Moleculaire,3

INRA-Universite Victor Segalen Bordeaux 2, 33883 Villenave d’Ornon Cedex, France

Received 12 March 2002/Accepted 10 July 2002

Mycoplasma pulmonis is a natural rodent pathogen, considered a privileged model for studying respiratorymycoplasmosis. The complete genome of this bacterium, which belongs to the class Mollicutes, has recently beensequenced, but studying the role of specific genes requires improved genetic tools. In silico comparativeanalysis of sequenced mollicute genomes indicated the lack of conservation of gene order in the regioncontaining the predicted origin of replication (oriC) and the existence, in most of the mollicute genomesexamined, of putative DnaA boxes lying upstream and downstream from the dnaA gene. The predicted M.pulmonis oriC region was shown to be functional after cloning it into an artificial plasmid and after transfor-mation of the mycoplasma, which was obtained with a frequency of 3 � 10�6 transformants/CFU/�g of plasmidDNA. However, after a few in vitro passages, this plasmid integrated into the chromosomal oriC region.Reduction of this oriC region by subcloning experiments to the region either upstream or downstream fromdnaA resulted in plasmids that failed to replicate in M. pulmonis, except when these two intergenic regions werecloned with the tetM determinant as a spacer in between them. An internal fragment of the M. pulmonishemolysin A gene (hlyA) was cloned into this oriC plasmid, and the resulting construct was used to transformM. pulmonis. Targeted integration of this genetic element into the chromosomal hlyA by a single crossing over,which results in the disruption of the gene, could be documented. These mycoplasmal oriC plasmids maytherefore become valuable tools for investigating the roles of specific genes, including those potentiallyimplicated in pathogenesis.

The mollicutes are the smallest free-living microorganismscapable of autoreplication (33). They are related to gram-positive eubacteria from which they evolved by a drastic re-duction of genome size, being thus considered the best repre-sentatives of the concept of a minimal cell. The genomes offour mollicutes have been sequenced to date: Mycoplasmagenitalium (580 kbp, [15]), Mycoplasma pneumoniae (816 kpb[21]), Ureaplasma urealyticum (752 kpb [19]), and Mycoplasmapulmonis (964 kbp [5]). Postgenomic approaches aiming atdeciphering the role of specific genes discovered by in silicoanalysis now require efficient strategies to genetically manipu-late these organisms. The transposon Tn916 or Tn4001 cantranspose in all the mollicute genomes, at least for the speciesamenable to transformation, such as M. genitalium, M. pneu-moniae, and M. pulmonis. In M. genitalium and M. pneumoniae,a global approach of gene inactivation with the transposonTn4001 was used to define the genes that are essential for thelife of these bacteria (22). Derivatives of this transposon, ob-tained by insertion of a chloramphenicol acetyltransferasegene and the tetM tetracycline resistance determinant, werealso shown to transpose in M. pulmonis (12). The use of thesegenetic elements for gene inactivation, which relies on therandom insertion of the transposon in the genome of the or-

ganism, does not allow specific targeting of a gene of interest.In addition, the complementation of mutants would requirethe cloning and expression of genes in these organisms, exper-iments for which there are no genetic tools to date. If we exceptthe cryptic replicating plasmids previously described for Myco-plasma capricolum and Mycoplasma mycoides subsp. mycoides(23, 24), there no other plasmids known to replicate in Myco-plasma species. In contrast, replicative plasmids have beenobtained for the mollicute Spiroplasma citri by cloning theorigin of replication of the chromosome (oriC) into artificialplasmids (34, 41). The resulting oriC plasmids can be used toinactivate targeted genes by homologous recombination (8,41), to support heterologous gene expression in S. citri (35),and to functionally complement mutants obtained by transpo-son insertion (18).

The first aim of this work was to compare the putative oriCregion of mollicutes for which sequence data are available.This analysis served as the basis for the development of oriCplasmids that replicate in M. pulmonis. The minimal sequencesrequired for the replication of these plasmids were found. Toevaluate the use of these vectors for inactivation and/orcomplementation of genes of interest, we have chosen to dis-rupt the M. pulmonis hemolysin A gene.

MATERIALS AND METHODS

Bacterial strains and culture conditions. The M. pulmonis strain used in thiswork was UAB CTIP (5). For subcloning experiments and propagation of plas-mids, the Escherichia coli strain TG1 {supE hsd�5 thi� (lac-proAB)F�[traD36proAB� lacIq lacZ�M15]) was used. Mycoplasmas were grown in modified Hay-

* Corresponding author. Mailing address: INRA, Centre de Recher-che de Bordeaux, Institut de Biologie Vegetale Moleculaire, 71, ave-nue Edouard Bourlaux, BP 81, 33883 Villenave D’Ornon Cedex,France. Phone: (33) 5 57 12 23 93. Fax: (33) 5 57 12 23 69. E-mail:[email protected].

5426

flick medium N (16) without thallium acetate and supplemented with BBLIsoVitalex Enrichment (Becton Dickinson, Sparks, Md.). For growth in solidmedium, mycoplasmas were incubated at 37°C under anaerobic conditions. E.coli cells were grown in Luria-Bertani (LB) broth or in LB agar at 37°C. E. colicells transformed with plasmids were grown in LB medium supplemented with 50�g of ampicillin/ml and 5 �g of tetracycline/ml.

Cloning procedures. The pBOT1 plasmid which contains a 2-kbp oriC regionfrom S. citri was described elsewhere (35). All plasmids constructed in this studywere based on the pSRT2 plasmid (C. Lartigue et al., unpublished data) (Fig. 1),which harbors the tetM gene (25) from the transposon Tn916 inserted into thepBS plasmid (Stratagene, La Jolla, Calif.). In pSRT2, the tetM gene is under thecontrol of the S. citri spiralin gene promoter (40). The 1.9-kbp oriC region of M.pulmonis, containing the dnaA gene with its upstream and downstream intergenicregions (Fig. 1), was PCR amplified with the primers OR1 and OR2 (Table 1).This amplified DNA fragment was cloned into the unique BamHI site of pSRT2,

resulting in the pMPO1 plasmid (Fig. 1). The downstream DnaA box region wasPCR amplified with the primers OR3 and OR2, and the upstream DnaA boxregion was amplified with the primers OR1 and OR4 (Table 1). To clone thesetwo DNA fragments side by side at the BamHI site of the vector pSRT2 forobtaining the pMPO4 plasmid, the upstream and the downstream DnaA boxregions were amplified with the primers OR1 and OR5, and OR2 and OR6,respectively. To clone these two fragments spaced by the tetM determinant, theupstream DnaA box region was first amplified with the primers OR7 and OR8and cloned at the SphI site of pSRT2. The downstream DnaA box region wasthen amplified with the primers OR2 and OR3 and cloned at the BamHI site,resulting in the plasmid pMPO5 (Fig. 1).

Transformation of mycoplasmas by polyethylene glycol (PEG). M. pulmoniscells from 10-ml cultures were transformed by a PEG-mediated method previ-ously described by others (12). Ten micrograms of plasmid DNA was used ineach transformation. After plating, the cultures were incubated at 37°C under

FIG. 1. M. pulmonis oriC region and plasmid constructs developed in this work (pMPO1, pMPO2, pMPO3, pMPO4, and pMPO5), using thevector pSRT2 for cloning. The plasmid pMPO1 was obtained by cloning the 1.9-kpb oriC region into the unique BamHI site of the pSRT2 plasmid.The orientations of the three genes (recD, dnaA, and dnaN) found in the vicinity of the predicted oriC region are indicated by arrows, and thoseof the putative DnaA boxes are indicated by triangles above the intergenic regions. The plasmids pMPO2 and pMPO3 were constructed by cloningthe intergenic regions downstream and upstream from dnaA, respectively, into the unique BamHI site of the pSRT2 plasmid. The plasmid pMPO4was constructed by cloning these two fragments side by side into the BamHI site of the vector pSRT2, and the plasmid pMPO5 was constructedby cloning these two fragments with the tetM determinant in between them into the BamHI site of the vector pSRT2. P, promoter of the S. citrispiralin gene.

VOL. 184, 2002 IDENTIFICATION OF FUNCTIONAL M. PULMONIS oriC 5427

anaerobic conditions and were examined from the third day of incubation forcolony development. Transformants were subcultured in 2 ml of Hayflick liquidmedia, in which the tetracycline concentration was gradually increased from 2 to50 �g/ml. Cloning of transformed mycoplasmas was performed by standardmethodology with three steps of filter cloning procedures using membranes witha pore size of 0.45 �m (39).

DNA isolation and Southern blot hybridization. Mycoplasma genomic DNAwas obtained from a 10-ml mycoplasma culture by modifications of the methoddescribed by Ferris et al. (14). Briefly, cells were collected by centrifugation(20,000 � g, 20 min, 4°C) and resuspended in 1.0 ml of 10 mM Tris-HCl (pH 8.0)containing 50 mM EDTA. To this suspension, 64 �l of a 10% sodium dodecylsulfate solution and 160 �l of proteinase K at a concentration of 10 mg/ml wereadded. The lysate was incubated at 56°C for 60 min and then deproteinized twicewith phenol and once with phenol-chloroform–isoamyl alcohol. The nucleic acidswere ethanol precipitated. The DNA was further purified by RNase A treatmentfollowed by phenol chloroform deproteinization and ethanol precipitation andfinally resuspended in 50 �l of Tris-EDTA buffer.

For Southern blot hybridization, approximately 2 �g of genomic DNA or 20 ngof plasmid DNA were digested by the appropriate restriction enzyme, andsubmitted to electrophoresis in a 0.8% agarose gel. After alkali transfer of theDNA to a positively charged nylon membrane, hybridization was performed inthe presence of 20 ng/ml of digoxigenin-labeled probes. Hybridization was de-tected by incubation of the membranes with anti-digoxigenin antibodies coupledto alkaline-phosphatase and the use of the chemoluminescent substrate CPDStar (disodium-4-chloro-3-[4-methoxyspiro{1,2-dioxietane-3,2�-(5�-chloro)tricy-clo(3.3.1.13,7) decan}-4-yl]phenyl phosphate) (Roche Molecular Biochemicals).

PCR-based detection of disruption events of the hlyA gene. Mycoplasma cellswere collected from 1.0 ml cultures by centrifugation (10,000 � g, 20 min, 4°C)and washed once with PBS. After resuspension in 100 �l of lysis buffer (10 mMTris pH 8.0, 1 mM EDTA, 0.25% Nonidet P-40, 0.25% Tween-20, 100 �g/mlproteinase K), the lysate was first incubated at 56°C for 60 min, then at 95°C for10 min. Nucleic acids were deproteinized once with phenol and twice withphenol-chloroform. After ethanol-precipitation, the DNA was resuspended in 50�l of Tris-EDTA buffer. To detect M. pulmonis hlyA mutants resulting fromdisruption of the chromosomal hlyA by insertion of plasmid sequences, a PCR-based strategy was developed. The wild-type hlyA gene can be PCR amplified byusing the primers HE-L and HE-R located upstream and downstream of thegene, respectively (Table 1; see Fig. 4). When the gene is disrupted by insertionof the plasmid sequence, the sequence is too large to be PCR amplified under thesame conditions and primers. However, the recombination event can be detectedby PCR amplification using the other pair of primers (HE-R and OR2), asindicated in Fig. 4.

Sequence analysis of the oriC regions of mollicutes. DnaA boxes weresearched within the oriC regions of mollicutes using the MEME/MAST software(3). A random sequence containing seven copies of the E. coli DnaA box con-sensus sequence (TTATCCACA) was used as a training sequence. Query se-quences were examined with a window of 150 bp, sliding with an increment of100 bp. The length of the motifs searched was restricted to 9 bp in a first queryand extended to 6 to 11 bp in a second one.

RESULTS

Identification of putative DnaA boxes within oriC regions ofmollicutes. Putative DnaA boxes have been searched by otherswithin the sequenced oriC regions of mollicutes. However,these results were neither compared nor obtained with similarmethods (17, 20, 41). In order to detect these putative DnaAboxes, the MEME/MAST set of softwares for finding con-served motifs in biological sequences was used. The validity ofthe method was first tested with the well-documented oriCregions of S. citri and M. capricolum. By use of a randomsequence containing seven copies of the consensus sequencefor the E. coli DnaA boxes (TTATCCACA) as a trainingsequence, the seven DnaA boxes previously described withinthe functional oriC region of S. citri (42) were detected (Fig. 2).Using the same strategy, the 10 boxes described for the M.capricolum oriC region (17) were found. These findings indi-cated that our method was reliable for detecting putativeDnaA boxes in the genomes of other mollicutes.

In the four sequenced mollicute genomes (M. genitalium, M.pneumoniae, U. urealyticum, and M. pulmonis), the oriC regionshave not been experimentally located, but a number of analy-ses suggest that the oriC regions are situated, as for mostbacteria, in the vicinity of the dnaA gene. Indeed, there is apolarity not only in the orientation of the genes on both sidesof dnaA but also in the base composition asymmetries at thelevel of this gene (5, 26, 31, 37). It should be noted that the

TABLE 1. Sets of primers used in this work for PCR amplification

Primer name Sequencea Annealingtempb (°C) Amplified region Size of

amplified fragment

OR1 5�-ATTAGGGATCCGCACTCTGGTCAGCGCTAGATC-3� 56 Complete oriC region 1.9 kbpOR2 5�-TCGCGGATCCGCCTAACTTGAAAATAAGCTCC-3�

OR2 58 Downstream DnaA boxes 326 bpOR3 5�-GGCTGGATCCGAAAACTTATCCAAGG-3�

OR1 58 Upstream DnaA boxes 262 bpOR4 5�-CGCTAGGATCCCTATTTTGTCAAGGC-3�

OR1 56 Upstream DnaA boxes 262 bpOR5 5�-CGCTAGAATTCCTATTTTGTCAAGGC-3�

OR2 56 Downstream DnaA boxes 326 bpOR6 5�-GGCTGAATTCGAAAACTTATCCAAGG-3�

OR7 5�-AATAGGCATGCGCACTCTGGTCAGGCGTAGATC-3� 56 Upstream DnaA boxes 262 bpOR8 5�-CGCTAGCATGCCTATTTTGTCAAGGC-3�

HE1 5�-CCGCGAATTCGAAAAAGAAGCTGTTGG-3� 56 hlyA gene 688 bpHE2 5�-GGCCCGAATTCGAGAGATGTATTCAATG-3�

HEL 5�-CCCCGAATTCGATAATTGCCTTGCAG-3� 56 hlyA gene and flanking regions 1.05 kbpHER 5�-CAGGTGAATTCGAATTAGCGGCTGAGTC-3�

a Sequences recognized by the restriction enzymes mentioned in the text are underlined.b Annealing temperature used during the PCR.

5428 CORDOVA ET AL. J. BACTERIOL.

FIG. 2. Gene order and putative DnaA boxes within the oriC regions of mollicute genomes. Orientations of the genes are indicated by arrows;intergenic regions flanking the dnaA genes are magnified with their lengths (in nucleotides) indicated. PCR, putative coding region; CHP,conserved hypothetical protein. Putative DnaA boxes are represented by headless arrows. A match with the consensus defined for mollicutes(TT(A/T)TC(C/A)ACA) is symbolized by the following: black, nine of nine; horizontal stripes, eight of nine; white, seven of nine.


asymmetry in the strand base composition which is typicallyfound around dnaA in bacterial genomes could barely be de-tected in the M. pneumoniae genome (31).

Using our method based on motif detection, putative DnaAboxes were found both upstream (five boxes) and downstream(three boxes) from dnaA in the M. pulmonis oriC region (Fig.2). The consensus sequence derived from these eight DnaAboxes is TTATC[C/A]A[C/A]A and resembled the typicalTTATCCACA DnaA box motif of E. coli, with a variability atpositions six and eight (Table 2). Four of the eight M. pulmonisDnaA boxes perfectly matched the consensus, whereas theother four matched at eight positions out of nine. Two AT-richstretches of 31 and 39 bp were found within the intergenicregions upstream and downstream from dnaA, respectively.

Using the same approach, five putative DnaA boxes werefound in the intergenic sequence soJ-dnaA of the M. genitaliumchromosome (Fig. 2). Although these boxes were not identifiedin the initial paper describing the complete sequence of thisbacterium (15), the origin of replication was predicted to belocalized in this untranscribed region by others (26). Withinthe oriC region of U. urealyticum, only one putative DnaA boxmatching 7 of 9 positions with the E. coli consensus was foundin the intergenic region (146 nt) between the genes rpL34 anddnaA (data not shown). No putative DnaA boxes could befound in the vicinity of the dnaA gene of the M. pneumoniaegenome. However, it should be noted that the intergenic re-gion cysA-dnaA is only 68 nt long and that a putative codingregion has been predicted immediately downstream fromdnaA. In addition, as found by others (20), a few putativeDnaA boxes were located within the 747-bp-long intergenicsoj-dnaN intergenic sequence, but only two of them presentedfewer than three mismatches with the E. coli consensus.

From the 31 putative DnaA boxes predicted from the oriCregions of M. pulmonis, M. capricolum, M. genitalium, and S.citri, a mollicutes DnaA box consensus was proposed (TT(A/T)TC(C/A)ACA); the positions three and six presented a vari-ability in comparison with the E. coli consensus.

Functional analysis of M. pulmonis oriC for plasmid repli-cation. Although no functional oriC region was yet isolatedfrom the chromosome of species belonging to the genus My-coplasma, the construction of oriC-based plasmids which rep-

licated in the mollicute S. citri prompted us to investigatewhether similar plasmids could replicate in M. pulmonis.

The first oriC plasmids that were assayed for transformationof M. pulmonis were those derived from the S. citri sequence.The pBOT1 plasmid containing a 2.0-kbp oriC fragment of S.citri (8) was tested. However, transformation of M. pulmonis bythe PEG-mediated methodology could not be achieved withpBOT1. As a control for transformation of M. pulmonis by thePEG-mediated method, the plasmid pAM120 (10), which con-tains the transposon Tn916, was used. It yielded tetracycline-resistant colonies with a frequency of 10�9 transformants/CFU/�g of plasmid DNA. These results suggested that theoriC plasmids are host specific. Therefore, oriC plasmids basedon the M. pulmonis oriC region were constructed.

The plasmid pMPO1 was obtained by combining the 1.9-kbporiC fragment of M. pulmonis with the pBS vector and the tetMgene driven by the S. citri spiralin gene promoter (Fig. 1).Transformation of M. pulmonis with this plasmid succeededwith a frequency of 3 � 10�6 transformants/CFU/�g of plas-mid DNA. With such an efficacy, about 2,000 transformantswere obtained by a single transformation with 10 �g of pMPO1and a starting culture of M. pulmonis titering 6 � 107 CFU/ml.In agar plates, tetracycline-resistant (Tetr) colonies were ob-served from the fifth day after plating. Their growth was slowerthan that of nontransformed cells, for which colonies were seenas early as 2 days after plating. From the third passage in liquidmedium containing tetracycline, the growth rate of trans-formed mycoplasmas was similar to that of nontransformedcells, leading to acidification of the culture medium in less than36 h. Transformation was confirmed by Southern blot hybrid-ization using the tetM gene and the oriC region as probes (Fig.3B; data not shown for the tetM probe). Both sequences couldbe detected in the genetic material extracted from the trans-formants. For at least some of the transformants, there wasevidence of the plasmid autonomously replicating in the bac-teria (Fig. 3B, lanes 3-2P and 3-5P).

Since it was shown previously that the presence of a func-tional dnaA gene is not required for oriC plasmid replication inS. citri (35), subcloning experiments were undertaken to reducethe oriC fragment of pMPO1 to the minimal sequences re-quired for plasmid replication (Fig. 1). In the two plasmids thatwere obtained, pMPO2 and pMPO3,, the oriC fragments cor-respond to the DnaA box region upstream (262 bp) and to theDnaA box region downstream (327 bp) from the dnaA gene,respectively (Fig. 1). None of these two plasmids replicated inM. pulmonis. Similarly, M. pulmonis could not be transformedwith the plasmid pMPO4, which contains these two DnaA boxregions cloned side by side at the BamHI site of the vectorpSRT2 (Fig. 1). In contrast, pMPO5, in which the two DnaAbox regions are spaced by the tetM gene, transformed M. pul-monis with a frequency of 4 � 10�6 transformants/CFU/�g ofplasmid. Subculturing TetR colonies of mycoplasmas trans-formed with pMPO5 revealed that the growth rate of thesetransformants was markedly slower than for those transformedwith the plasmid pMPO1, with up to 1 week being required foracidification of the culture medium. The presence of pMPO5in the transformants was confirmed by Southern blot hybrid-ization with an oriC probe (Fig. 3D).

Stability of oriC-based plasmids in M. pulmonis. To evaluatethe stability of oriC plasmids in M. pulmonis, genomic DNAs

TABLE 2. Putative DnaA boxes within the oriC intergenic regionsof M. pulmonis

DnaA box Sequence StrandMatcha

[cons. M. pulmonis(cons. Mollicutes)]

Positionb

recD/dnaA 1–222Box 1 TTATCCAAA � 9 (8) 59–67Box 2 TTATTAACA � 8 (8) 77–85Box 3 TTATCAACA � 9 (9) 101–109Box 4 TTATCCACA � 9 (9) 143–151Box 5 TTATCAACT � 8 (8) 173–181

dnaA/dnaN 1608–1740Box 6 TTATCCAAG � 8 (7) 1620–1628Box 7 TTATCCAAA � 9 (8) 1673–1681Box 8 TTAACAACA � 8 (8) 1684–1692

a cons., consensus. See the text.b Position in reference to the first nucleotide of the recD/dnaA intergenic

region.


from transformants obtained with either pMPO1 or pMPO5were analyzed by Southern blots after different passages in thepresence of selection pressure (tetracycline). For the plasmidpMPO1 (Fig. 3A), the appearance of the 6.7- and 4.0-kpbHindIII DNA fragments hybridizing with the oriC probe indi-cates that integration of pMPO1 into chromosomal oriC didoccur. In contrast, the presence of the 3.7- and 7.0-kbp hybrid-izing signals should indicate that some of the transformed cellsstill contained wild-type oriC and free plasmid. Three indepen-dent transformants were analyzed after 2, 5, and 20 passages invitro (Fig. 3B). For transformant 2, there was evidence of at

least partial integration of pMPO1 even after only two pas-sages, and integration of the plasmid was complete after 20passages. In contrast, integration of pMPO1 followed a differ-ent time course for transformant 3, with no detectable integra-tion after five passages, indicating that the plasmid was auton-omously replicating in the bacteria, and only partial integrationafter 20 passages (Fig. 3B). An intermediate pattern of inte-gration was obtained with transformant 1.

The scheme of integration of the plasmid pMPO5 is de-picted in Fig. 3C. In contrast to pMPO1, analysis of pMPO5transformants revealed only two bands, corresponding to the

FIG. 3. Stability of oriC plasmids in M. pulmonis. (A) Schematic representation of the integration of the plasmid pMPO1 in the M. pulmonischromosome within the oriC region. The size of HindIII (H) DNA fragments which overlap the oriC probe is indicated. (B) Southern blot analysisof DNA from M. pulmonis transformed with pMPO5 after 2 (2P), 5 (5P), or 20 (20P) passages in liquid medium. The purified DNAs of three M.pulmonis transformants numbered 1 to 3, and those of nontransformed M. pulmonis (NT) or of the plasmid (P) were digested with HindIII beforeelectrophoresis. M. pulmonis oriC was used as a probe. (C) Schematic representation of the integration of the plasmid pMPO5 in the M. pulmonischromosome within the oriC region. The intergenic regions located upstream and downstream of dnaA are shown by boxes with vertical andhorizontal hatchings, respectively. The integration scheme is shown assuming a crossover within the region located upstream of dnaA. The size ofEcoRI (E) DNA fragments which overlap the oriC probe is indicated. If the integration would occur within the other intergenic region, thepredicted sizes of the EcoRI fragments overlapping the oriC probe are 12 and 0.65 kbp (data not shown). An integration with a double crossoverwould be lethal because it would result in the loss of the dnaA sequence from the chromosome. (D) Southern blot analysis of DNA from M.pulmonis transformed with pMPO5 after five passages in liquid medium. The purified DNAs of M. pulmonis transformants (D) (lanes 1 to 5), ofnontransformed M. pulmonis (NT), or of the plasmid (P) were digested with EcoRI before electrophoresis. M. pulmonis oriC was used as a probe.


free plasmid and intact chromosomal oriC, indicating that re-duction of the oriC region led to a stable replicative plasmid. Inall transformants tested, only free plasmid was detected, atleast until the 15th passage (Fig. 3D).

Evidence for specific gene inactivation in M. pulmonis tar-geted by a recombinant oriC-based plasmid. In an attempt toinactivate the hlyA gene through homologous recombination,we first used the classical strategy based on suicide vectors. Aninternal fragment (688 bp) of the M. pulmonis hlyA gene wasPCR amplified with the primers HE1 and HE2 (Table 1) andinserted in the unique EcoRI site of the pSRT2 plasmid.Transformation of M. pulmonis with the recombinant plasmidpSRT2-�hlyA repeatedly yielded no Tetr transformants. Thissuggests that the frequency of recombination events with use ofnonreplicative plasmids is probably very low with M. pulmonis.

Therefore, we used the replicative, oriC plasmids pMPO1and pMPO5 as disruption vectors according to the strategypresented for pMPO5 in Fig. 4. The use of replicative vectorsin these experiments was expected to enhance the opportunityfor recombination between the plasmid and the chromosomeand hence for disruption of the target gene. Similarly to theconstruction of pSRT2-�hlyA, the internal fragment of the M.pulmonis hlyA gene was inserted into the EcoRI site of thepMPO1 and pMPO5 plasmids, yielding the disruption plas-mids pMPO1-�hlyA and pMPO5-�hlyA, respectively. Aftertransformation of M. pulmonis with these constructs, TetR col-onies were obtained with the same frequency as with the pa-rental plasmids. Transformants were subcultured in liquid me-dium in which the tetracycline concentration was graduallyincreased from 2 to 50 �g/ml. Genomic DNA was extractedfrom these cultures after 5, 10, 15, and 20 passages in vitro andanalyzed by Southern blot hybridization with an hlyA probe.Similarly to its parental plasmid, the plasmid pMPO5-�hlyA

replicated as a free genetic element in the transformants, evenafter 20 passages (data not shown). In contrast, the plasmidpMPO1-�hlyA was not stably maintained as a free plasmidbecause it was integrated into chromosomal oriC after a fewpassages. For transformants obtained with both pMPO1-�hlyAand pMPO5-�hlyA, events of integration of the plasmids intothe targeted hlyA gene could not be detected by analysis of thegenetic material by Southern blot hybridization (data notshown). In order to increase the probability of isolating the fewmycoplasma cells in which recombination might have occurred,further experiments were conducted with the pMPO5-�hlyAtransformants.

First, the transformants were grown in the absence of tetra-cycline to determine whether loss of the free plasmid in theabsence of selection could favor the selection of rare recom-bination events, such as plasmid integration at the hlyA gene.Three randomly selected M. pulmonis transformants withpMPO5-�hlyA were cultured for four passages in the presenceof tetracycline. These transformants were then submitted to 1,5, 10, or 15 passages without antibiotic before being plated inthe absence and in the presence of tetracycline. After incuba-tion, colonies were counted on both types of medium, allowingus to determine the percentage of the cells that kept the tetr

genetic determinant, regardless of its chromosomal or plas-midic localization (Fig. 5). When submitted to one passagewithout selective pressure, an average of 42% of colonies weretetracycline resistant. This percentage markedly decreasedwith passage number down to 4.1% after 5 passages, 0.008%after 10 passages, and 0.0005% after 15 passages without tet-racycline. These data indicate that the plasmid pMPO5-�hlyAis not maintained by the mycoplasma cell in the absence ofselective pressure, suggesting that these passages in a mediumwithout antibiotic could be a means for isolating M. pulmonishlyA mutants.

Therefore, M. pulmonis cells transformed with pMPO5-�hlyA were cultured for five passages in selective medium,submitted to five additional passages without tetracycline, andthen plated onto solid medium containing 2 �g of tetracycline/ml. Randomly selected clones (Tetr colonies) were grown in

FIG. 4. Scheme of integration of the plasmid pMPO5-�hlyA intothe M. pulmonis chromosomal hlyA gene by a single crossing-over. Thegenetic organization of pMPO5-�hlyA and the hlyA chromosomal re-gion are schematically shown before and after integration. The size ofthe HindIII (H) fragments which overlap hlyA sequences is indicated.Arrowheads indicate the positions of the primers (HE-L, HE-R, andOR2) used to document this integration. PS, promoter of the S. citrispiralin gene; ori5�, region containing the DnaA boxes upstream fromthe dnaA gene; ori3�, region containing the DnaA boxes downstreamfrom the dnaA gene.

FIG. 5. Stability of the plasmid pMPO5-�hlyA. The percentages ofcolonies that remained Tetr after different passages without selectivepressure (no tetracycline added to the medium) are represented.


liquid medium with antibiotic, and genomic DNA was analyzedby Southern blot hybridization (Fig. 6A). The hybridizationpatterns of all five transformants are almost identical. Accord-ing to Fig. 4, the appearance of the 4.8- and 5.5-kpb HindIIIDNA fragments hybridizing with the hlyA probe clearly indi-cates that integration of the disruption vector into the targetgene did occur. However, the presence of the 6.2- and 4.0-kbphybridizing signals indicates that some of the transformed cellsstill contained the wild-type hlyA gene and free plasmid. Sim-ilar patterns were obtained from all selected transformants,even after rounds of repeated filter cloning. Additional evi-dence of the hlyA gene disruption was obtained by using aPCR-based approach (Fig. 6B). Using the primers OR2 andHER, a 1.05-kbp DNA was amplified, showing that pMPO5-�hlyA had integrated into the M. pulmonis chromosome by onecrossover recombination at the hlyA gene. As expected fromthe Southern blot hybridization patterns (Fig. 6A), PCR am-plification with the primers HE-R and HE-L also yielded pos-itive results, indicating that cells carrying the intact hlyA genewere still present in all five clones tested. In conclusion, al-though integration events could be documented, we were notable to isolate a clone with a disrupted hlyA gene.

DISCUSSION

The complete sequencing of the M. pulmonis genome hasopened the way to functional genomics for this rodent patho-gen, which is considered a model of respiratory mycoplasmosis(4, 38). However, due to the paucity of genetic tools, geneticstudies could not be conducted so far. Transformation of M.pulmonis to antibiotic resistance has been achieved with Tn916(10) and Tn4001 (11), but there is still no suitable vector forcloning genes into this organism. With the aim of developing

plasmid vectors for M. pulmonis, we have constructed oriCplasmids, a strategy which was successfully used with the plantmollicute S. citri (41). As a preliminary step in identifying M.pulmonis oriC, we have compared the putative oriC regions ofmollicutes for which sequence data are available. The resultsindicated a lack of gene order conservation in this region. Forexample, the tandem of the dnaA-dnaN genes is conserved inS. citri, M. capricolum, and M. pulmonis, whereas these genesare divergent and spaced by the soJ gene in the M. pneumoniaeand M. genitalium genomes. The U. urealyticum genome offerseven more diversity because these two genes are 95 kpb apart.

Using software developed for finding conserved motifs, wewere able to locate potential DnaA boxes in the vicinity ofdnaA. The distribution of putative DnaA boxes on both sidesof dnaA was found to be similar for S. citri, M. capricolum, andM. pulmonis but differed for the other three examined molli-cute genomes. In M. pulmonis in particular, the two DnaA boxregions flanking the dnaA gene were found to contain five andthree DnaA boxes, respectively. Additional putative DnaAboxes with a maximum of two mismatches with the mollicuteconsensus were found within the dnaA gene sequences of M.pulmonis (eight boxes), M. capricolum (three boxes), M. geni-talium (three boxes), and S. citri (six boxes).

The functional characterization of M. pulmonis oriC wasachieved by transformation of the mycoplasma with pMPO1.In the mycoplasmal transformants, the plasmid was found toreplicate as a free extrachromosomal element before integrat-ing into the chromosome during passaging, similarly to theplasmid pBOT1 in S. citri (8). In spite of similarities betweenthe oriC regions of M. pulmonis and S. citri, the plasmid pBOT1failed to transform M. pulmonis. This result suggests that host-specific factors regulate the replication of these plasmids. This

FIG. 6. Detection of M. pulmonis hlyA disruption by Southern blot and PCR. (A) Southern blot hybridization using an hlyA probe withHindIII-digested genomic DNA from M. pulmonis clones transformed with the plasmid pMPO5-�hlyA (lanes T1 to T5). NT, nontransformedHindIII-digested M. pulmonis DNA; P, pMPO5-�hlyA plasmid DNA. (B) Agarose gel electrophoresis of PCR products obtained using either theprimers OR2 and HE-R (lanes a) or the primers HE-L and HE-R (lanes b). The PCR with primers OR2 and HE-R will yield a PCR product onlyif there is integration of the vector pMPO5-�hlyA into the M. pulmonis hlyA gene. The PCR with primers HE-L and HE-R will yield a productonly if the chromosomal hlyA is not disrupted. Target DNAs included the purified DNA from 3 M. pulmonis clones transformed with pMPO5-�hlyA (lanes T1 to T3), DNA from nontransformed M. pulmonis culture (NT), DNA from pMPO5-�hlyA plasmid DNA alone (P), a negativecontrol (NC) with no DNA added, and a mixture of 2 ng of the pMPO5-�hlyA plasmid with 50 ng of nontransformed M. pulmonis DNA (NT�P).M, 100-bp DNA ladder (Gibco-Life Technologies).


finding was not totally unexpected because it was described forother bacterial genera. As an example, Mycobacterium tuber-culosis oriC-based plasmids do not replicate in rapidly growingmycobacterial species such as Mycobacterium smegmatis (32).Nevertheless, it remains to be determined if this barrier ofspecificity can be abolished in the case of mollicute species thatare more phylogenetically related, such as S. citri and M. capri-colum.

The search for the minimal oriC sequences required forplasmid replication showed that the two DnaA box regions arenecessary to support replication in M. pulmonis. Although it isuncommon among gram-positive bacteria, a similar situationhas been described for B. subtilis (29). With this organism, itwas suggested that a DNA-loop structure involving protein-mediated interaction between the two DnaA box regionswould be required for the initiation of replication (30). Thishypothesis is supported by our results showing that cloningboth the upstream and the downstream regions from M. pul-monis dnaA did not result in a replicative plasmid (pMPO4)unless these two regions were spaced, either by dnaA (pMPO1)or by tetM (pMPO5). For the mollicute S. citri (35) and forseveral gram-positive bacteria, including Streptomyces spp.(43), M. smegmatis, Mycobacterium bovis, M. tuberculosis, andMycobacterium avium (27), the 3� flanking region of dnaA wassufficient to support plasmid replication. Otherwise, the 5�region can support plasmid replication for Pseudomonas putidaand Pseudomonas aeruginosa (42). It is not possible to drawconclusions about the factors necessary for the function ofchromosomal oriC from the analysis of cloned oriC, because itis known that they have different requirements (2).

The pMPO1 plasmid, which contains an oriC region encom-passing the dnaA gene, was able to replicate in M. pulmonis butintegrated, within a few in vitro passages, chromosomal oriC.Therefore, the use of this plasmid is limited to the targetedintegration of genes into the oriC chromosomal region. Incontrast, the pMPO5 plasmid remained free for at least 15 invitro passages. It therefore represents the first shuttle plasmidvector functioning between E. coli and M. pulmonis.

The use of the oriC plasmid for targeting gene inactivationby homologous recombination was evaluated. The gene hlyAwas chosen because its putative role in virulence suggested thatit would be dispensable, at least in vitro. This gene is also notpresent in the genomes of M. pneumoniae and M. genitalium,which indicates that it is not essential for the life of theseorganisms. Although events of disruption of this gene could bedetected in the cultured transformants with pMPO5-�hlyA, wewere not able to isolate an hlyA mutant. This could be attrib-uted to a polar effect resulting in the disruption of anothergene, located downstream of hlyA and encoding an essentialfunction. However, other attempts in our laboratory with othercandidate genes, such as the mnuA gene (MYPU_6930), whichencodes a putative nuclease, led to similar results, suggestingthat other mechanisms may operate. The length of the homol-ogous sequence could also be a critical factor for the crossing-over to occur between the plasmid-borne and chromosomalsequences. Although we cannot exclude this hypothesis, theconstruct for targeting the mnuA gene carried an 1,134-bpinsert of mnuA homologous sequence, compared to 688 bp forpMPO5-�hlyA, and still did not allow the isolation of mnuAmutants. For these reasons, as the most probable explanation

of our inability to isolate hlyA mutants using oriC-based plas-mids, we favor a mechanism which would result from both theincompatibility of having two functional oriC regions on thechromosome and the resolution of the integrated genetic ele-ment. To solve the problem due to this incompatibility, we arecurrently trying to use a strategy based on a double crossing-over between the target chromosomal gene and the plasmidsequences. Furthermore, to select the supposedly rare eventsof integration, we are currently trying to counter-select thereplication of the oriC plasmid, as proposed for other bacteria(36). Although a case of homologous recombination with M.pulmonis using suicide vectors has been reported (28), furtherstudies indicated that these results were obtained with a strainthat did not belong to the species M. pulmonis but belonged tothe other mollicute species Acholeplasma oculi (1, 9, 28). Ourresults confirmed that homologous recombination for M. pul-monis using suicide vectors is difficult, if not impossible, todetect. This is in contrast with at least one other Mycoplasmaspecies, M. genitalium, for which gene inactivation by this strat-egy has been obtained (6, 7).

If we except the cryptic unstable replicating plasmids previ-ously described for M. capricolum and M. mycoides subsp.mycoides (23, 24), there are no other plasmids known to rep-licate in Mycoplasma species. In addition, M. pulmonis and S.citri are the only mollicutes for which oriC-based plasmids havebeen developed. Therefore, there is the possibility that thisstrategy may be applicable to other members of this bacterialclass for which genetic tools have been scarce (13).

ACKNOWLEDGMENTS

The first two authors, Caio M. M. Cordova and Carole Lartgue,contributed equally to this study.

This work was funded by INRA, the Universite Victor SegalenBordeaux 2, and the Region Aquitaine and by a FASPESP fellowshipgrant, no. 99/07328-2, to C.M.M.C.

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