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School of Veterinary Science, The University of Melbourne, Parkville, Victoria 3010, Australia
Correspondence
P. F. Markham
pmarkham{at}unimelb.edu.au
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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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). Recently the genome of M. gallisepticum was sequenced completely (Papazisi et al., 2003), allowing prediction of the function of specific genes. However, genetic studies of M. gallisepticum have been limited by the lack of genetic tools for its manipulation, with most studies using transposons Tn916 (Dybvig & Cassell, 1987; Dybvig & Alderete, 1988; Whetzel et al., 2003) and Tn4001 (Bearson et al., 2003; Hedreyda et al., 1993; Hudson et al., 2006; Mahairas & Minion, 1989; Mudahi-Orenstein et al., 2003; Papazisi et al., 2002; Whetzel et al., 2003; Winner et al., 2003) or suicide vectors (Markham et al., 2003) to study gene function. However, the random insertion of the transposon in the genome of the organism does not allow specific targeting of a gene of interest, and random integration can confound analyses of gene expression (Dybvig & Cassell, 1987; Mahairas & Minion, 1989). There has been some success in development of vectors for mollicutes using homologous origins of replication (oriC) and selectable antibiotic resistance markers. These plasmids are able to replicate extrachromosomally and have been used to inactivate target genes by homologous recombination (Cordova et al., 2002; Duret et al., 1999; Janis et al., 2005; Lartigue et al., 2002). In many cases oriC plasmids containing larger oriC regions have been found to integrate into the oriC region in the genomic DNA by homologous recombination following in vitro passage. Plasmids containing a shorter oriC region are more stable and have been used to generate targeted homologous recombination (Cordova et al., 2002; Lartigue et al., 2002; Renaudin et al., 1995). Most of the oriC plasmids of mollicutes show host specificity, but plasmids containing the oriC of Mycoplasma mycoides subsp. mycoides LC and SC replicate in the closely related species Mycoplasma capricolum subsp. capricolum (Lartigue et al., 2003). Mycoplasma imitans, which has been isolated from ducks and geese, is phylogenetically closely related to M. gallisepticum. DNA–DNA hybridization studies show that M. gallisepticum and M. imitans share 40–46 % genetic identity (Bradbury et al., 1993), whilst PFGE and random amplified polymorphic DNA studies suggest a DNA sequence identity of 53–60 % (Bradbury et al., 1993; Marois et al., 2001). Papazisi et al. (2003) reported that the predicted oriC region of M. gallisepticum was located between the dnaN and dnaA genes. The predicted oriC region contains five DnaA box sequences, with the consensus sequence 5'-TTWTMHAMA-3' identical for each DnaA box, and the region between dnaN and soj contains a higher than average content of adenosine and thymidine (80 %). However, the DNA sequence of the M. imitans oriC region has not been determined. The aims of this study were to determine the DNA sequence of the M. imitans oriC region and to produce vectors containing different regions of the oriC of M. gallisepticum and M. imitans to assess their replication and stability during passage. We also attempted to inactivate a target gene in M. gallisepticum using these oriC plasmids. We chose to knockout the expressed VlhA1.1 gene of M. gallisepticum as the genome sequence is available and the target gene is not essential for the survival of the organism (Glew et al., 2000). |
METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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) and M. imitans strain 4229 (Bradbury et al., 1993) were grown in modified Frey's medium to late exponential phase at 37 °C (Whithear, 1993). Mycoplasma pneumoniae strain FH (ATCC No. 15531) (donated by V. Peters, Department of Virology, Royal Children's Hospital, Melbourne) was grown in modified Hayflick's medium in tissue-culture flasks. For the growth and selection of antibiotic-resistant mycoplasma transformants, tetracycline was added to a concentration of 4 µg ml–1 in broth or agar medium (Markham et al., 2003). Escherichia coli strain DH5
was used as the host for gene cloning and was grown in Luria–Bertani (LB) broth at 37 °C and subjected to standard molecular biological techniques (Sambrook & Russell, 2001).
Amplification and DNA sequencing of the oriC region of M. imitans strain 4229.
Part of the oriC region and the dnaA gene of M. imitans was amplified using the primer pairs SW15for and MIsojrev-1, and SW6for and dnaArev, respectively (Table 1), and cloned into pGEM-T for DNA sequencing. The remainder of the oriC region was sequenced directly from genomic DNA. The DNA sequence was aligned with the homologous region from the complete genome of M. gallisepticum strain Rlow (GenBank accession no. NC_004829).
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). The long oriC region, the short oriC region, and the whole oriC region were amplified with the specific primer pairs SW4for and SW5rev, SW6for and SW7rev, and SW4for and SW7rev, respectively (Table 1
). Each PCR contained 5 µl 10x reaction buffer (Invitrogen), 10 µM each deoxynucleoside triphosphate, 2.5 mM MgSO4, 12.5 µM each primer, 1.25 U Platinum Taq High Fidelity DNA polymerase (Invitrogen), 10 ng genomic DNA of M. gallisepticum strain Rlow as template, and water to a final volume of 50 µl. PCRs were performed in a thermocycler (iCycler, Bio-Rad) under the following conditions: 94 °C for 4 min, followed by 35 cycles of 94 °C for 30 s, 45 °C for 2 min, and 68 °C for 4 min, with a final extension at 68 °C for 7 min. PCR products were separated by agarose gel electrophoresis and extracted from gel slices using the QIAEX II kit (Qiagen) according to the manufacturer's instructions. The long and short oriC regions were cloned separately into pGEM-T (Promega), into which the tetracycline-resistance gene had already been inserted (tetM/pGEM-T) (Markham et al., 2003). The long oriC region was inserted between the SphI and NcoI cleavage sites, and the short oriC region was inserted between the PstI and SalI sites (Fig. 1a). The resulting constructs were designated pGTLori and pGTSori. A further construct was produced that contained both the regions upstream and downstream from the soj gene, and was named pGTDori. The whole oriC region was amplified similarly and cloned into pGEM-T, and the tetM gene was then cloned into the SpeI site of the vector, resulting in pGTWori (Fig. 1a). The oriC region of M. imitans was amplified from the genome of M. imitans strain 4229 using the MIdnaNfor and MIsojrev-2 primer pair (Table 1), introduced into the cloning site of pGEM-T, and the tetM gene was then ligated into the SpeI cleavage site of the vector (Fig. 1b). To determine the minimal oriC region for plasmid replication in M. gallisepticum, different regions upstream of the soj gene were amplified by PCR with specific primer pairs (Table 1). To generate the oriC plasmids containing the PCR products shown in Fig. 2, each PCR product was cloned separately into pGEM-T, and the tetM gene was then ligated into the SpeI site of the vector.
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. Briefly, 5 ml cultures of mycoplasmas were grown to late exponential phase and harvested by centrifugation at 12 000 g for 5 min in a bench-top centrifuge at room temperature. The cells were washed twice in 250 µl ice-cold HEPES–sucrose buffer (8 mM HEPES, 272 mM sucrose, pH 7.4). The cell pellet was then resuspended in 100 µl HEPES–sucrose buffer containing 
10 µg plasmid DNA and transferred to a Gene Pulser cuvette with a 0.2 cm electrode gap (Bio-Rad), and the mixture was pulsed using a Gene Pulser (Bio-Rad) set at 2.5 kV, 100 
and 25 µF. The cells were resuspended in 1 ml broth and incubated at 37 °C until the medium showed a colour change, after which 500 µl culture was plated onto agar medium containing 4 µg tetracycline ml–1. The plates were incubated at 37 °C for 7–10 days in the dark in an airtight jar for M. gallisepticum and M. imitans, and incubated for 21 days for M. pneumoniae. Individual tetracycline-resistant colonies were selected and subcultured in 1 ml broth containing 4 µg tetracycline ml–1 and incubated until the medium changed colour. To confirm the presence of the plasmid, tetM was amplified by PCR with the Tetfor and Tetrev primer pair (Table 1). Transformants were passaged by inoculating 1 ml late-exponential-phase culture into 19 ml broth containing 4 µg tetracycline ml–1.
Southern blot analysis.
M. gallisepticum strain S6 and M. imitans strain 4229 genomic DNA, and oriC plasmid DNA were digested to completion with the restriction endonuclease NsiI (New England Biolabs). The fragments were separated in a 0.7 % agarose gel and blotted onto Hybond-N+ membranes (Amersham). The DNA was fixed to the membrane by exposure to UV light for 5 min and the blot was prehybridized in a buffer containing 7 % SDS, 1 % BSA, 1 mM EDTA and 0.25 M Na2HPO4 (pH 7.2) for 2 h at 58 °C (Church & Gilbert, 1984). A probe containing 50 ng of a 1.96 kb PCR product produced with the primer pair SW4for and SW5rev was radiolabelled with [
-32P]ATP using a random-primed DNA labelling kit (Roche). Unincorporated nucleotides were removed by passage through a Bio-Spin 30 chromatography column (Bio-Rad), following the manufacturer's instructions. The radiolabelled probe was denatured by incubation at 100 °C for 10 min and then added to the hybridization buffer and incubated with the membrane at 58 °C overnight. The next day the membrane was washed three times in 2x SSC (0.3 M NaCl, 30 mM sodium citrate, pH 7.0), 0.1 % SDS at 58 °C for 20 min each. The membrane was autoradiographed at room temperature overnight on BioMax film (Kodak). The same method was used to produce probes to detect the ampicillin-resistance gene (ampR) and the oriC region of M. imitans. The ampR gene was amplified from pGEM-T using the AmpRfor and AmpRrev primer pair, and the partial oriC region of M. imitans was amplified using the SW15for and Misojrev-1 primer pair (Table 1), together with the products radiolabelled as described above. The hybridization and washing conditions were the same as those used for the oriC probe.
Targeted gene inactivation by homologous recombination using oriC plasmids.
To construct an oriC plasmid that could integrate into a target gene by homologous recombination, an internal fragment of the vlhA3.03 gene (which also has 96 % DNA identity with vlhA1.1 and vlhA1.2 of M. gallisepticum strain S6), including regions that diverged significantly from most other vlhA genes, was amplified from the genome of M. gallisepticum strain Rlow using the P3.03-F-PstI and P3.03-R-SalI primer pair or the P3.03-F-SphI and P3.03-R-NcoI primer pair (Table 1). The PCR products were digested with the appropriate restriction endonucleases and cloned separately into the PstI and SalI restriction sites in pPLoriC7, which contained 180 bp of the M. gallisepticum oriC region, and into the SphI and NcoI restriction sites in the M. imitans oriC plasmid pMIori (Fig. 3). Approximately 10 µg of these plasmids was introduced into M. gallisepticum strain S6 by electroporation, as described above. Seven days after electroporation, individual tetracycline-resistant colonies were selected from the agar plate and incubated in 500 µl medium containing 4 µg tetracycline ml–1 until a colour change was observed in the medium. The cultures were then screened for the presence of the tetM gene by PCR. To promote homologous recombination, each transformant was passaged 10 times in medium containing tetracycline, and then an additional five times without tetracycline. Transformants were spread on agar containing tetracycline and individual tetracycline-resistant colonies were selected 7 days after plating. Insertion of the construct into vlhA3.03 was assessed by Southern blotting. The genomic DNA from transformants and plasmid DNA were digested with PstI (New England Biolabs) and Southern-transferred. The membrane was hybridized to radiolabelled ampR and the amplified fragment of vlhA3.03, and binding of the probes was detected as described above. The integration of pMIori/
3.03 was confirmed by DNA sequencing of the PCR product that was obtained using the primer pair SW13 for and vlhALeader rev (Table 1).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). All plasmid constructs were introduced into M. gallisepticum strain S6 by electroporation and all, with the exception of pGTSori, could be detected in cultures of tetracycline-resistant transformants; the frequency of transformation was 6x10–7 transformants per c.f.u. These results suggested that the AT-rich regions were important for plasmid replication, and in addition that the dnaA and soj genes were not essential for the replication of the oriC plasmid. In order to determine the replicability of the oriC plasmid of M. gallisepticum in M. imitans strain 4229 and M. pneumoniae strain FH, plasmid pGTLori, harbouring a 1.96 kb region from oriC of M. gallisepticum, was introduced into M. imitans and M. pneumoniae by electroporation. Seven days after transformation, several tetracycline-resistant M. imitans colonies were observed on mycoplasma agar plates containing 4 µg tetracycline ml–1, but no tetracycline-resistant colonies of M. pneumoniae were detected up to 21 days of incubation, in spite of repeated attempts to introduce pGTLori into M. pneumoniae strain FH.
The minimal functional oriC region in M. gallisepticum
Previous studies have reported that oriC plasmids containing larger oriC regions can easily integrate into the oriC region of genomic DNA through homologous recombination after in vitro passage (Cordova et al., 2002; Lartigue et al., 2002; Renaudin et al., 1995). In order to reduce the likelihood of this, a number of plasmids containing different lengths of the oriC region were tested for replication in M. gallisepticum. We amplified the partial oriC regions (oriC1 to oriC9) (Fig. 2) using specific primer pairs (Table 1) and cloned the products into tetM/pGEM-T, and these plasmids were then used to transform M. gallisepticum strain S6. Following transformation, the constructs pPLoriC1, pPLoriC3, pPLoriC5, pPLoriC6 and pPLroiC7, but not pPLoriC2, pPLoriC4, pPLoriC8 or pPLoriC9, were able to replicate in M. gallisepticum (Fig. 2). These results suggested that the minimal oriC region that was functional in M. gallisepticum strain S6 was around 180 bp and included two DnaA boxes and two AT-rich regions.
Determination of the DNA sequence of the oriC region in, and development of an oriC plasmid from, M. imitans
Part of the oriC region and the dnaA gene of M. imitans were amplified using the corresponding M. gallisepticum primer pairs. PCR products and the remainder of the oriC region were sequenced. The 2.17 kb region upstream from the soj gene of M. imitans contained six DnaA boxes, which had a 9 nt consensus sequence (5'-TTWTMHAMA-3'), and was 90 % A+T (Fig. 1b). Alignment of the oriC regions of M. imitans and M. gallisepticum strain R revealed 56 % DNA sequence identity, whilst their dnaA genes had 85 % DNA sequence identity. The pMIori plasmid contained the 2.3 kb region upstream from the soj gene (Fig. 1b). The pMIori plasmid was introduced into M. imitans strain 4229 and M. gallisepticum strain S6 by electroporation and was found to replicate in both species.
Stability of M. gallisepticum oriC plasmids in M. gallisepticum and M. imitans
We investigated the stability of oriC plasmids in transformants by Southern blot analysis after repeated passage. DNA was extracted from cells of transformants after passaging and, together with the genomic DNA of the untransformed organism and the oriC plasmid purified from E. coli, was digested with NsiI; the fragments were separated by gel electrophoresis, Southern-blotted and hybridized to the appropriate radiolabelled probe. Following NsiI digestion, the M. gallisepticum oriC probe is predicted to bind to an extrachromosomal plasmid with a predicted size of 7.0 kb (Fig. 4a). The 1.96 kbp oriC probe bound a 3.9 kb fragment in M. gallisepticum strain S6 (Fig. 4b, lane 1) and to a 7 kb fragment in pGTLori transformants (Fig. 4b, lane 2); a similar-sized fragment was also detected after five passages of the transformants (Fig. 4b, lane 3). The detection of 4.8 and 6 kb fragments by the probe at the 10th passage (Fig. 4b, lane 4) indicated integration of the plasmid into the genome in the oriC region. In transformants obtained with pPLoriC1, which contained a 1.06 kb oriC region, the oriC probe hybridized to the 4.5 kb fragment predicted for an extrachromosomal plasmid (Fig. 4b, lanes 5 and 6). The detection of 3.4 and 4.8 kb fragments at the 10th passage indicated that the plasmid had integrated into the genome (Fig. 4b, lane 7). To determine the stability of pPLoriC7, the replicative plasmid containing the shortest section of the oriC region, we performed Southern blot analysis on two transformants passaged under antibiotic selection pressure (4 µg tetracycline ml–1) after 5, 10 and 15 passages. Due to the low sensitivity of the oriC probe for the 180 nt oriC region, a longer probe that detects the ampR gene, which would indicate the location of the oriC plasmid, was used. It was expected that the probe would not hybridize to DNA from untransformed M. gallisepticum but would hybridize with a 3.5 kb DNA fragment after NsiI digestion of the DNA extracted from transformants containing extrachromosomal plasmid, whilst hybridization to a 4.0 kb fragment would suggest that the plasmid had integrated into the chromosome (Fig. 5a). The probe did not hybridize to untransformed M. gallisepticum (Fig. 5b, lane 1), but bound to a 3.5 kb fragment derived from the oriC plasmid, and to a similar-sized band in both transformants at the fifth passage (Fig. 5b, lanes 2, 3 and 6). At the 10th passage a 4.0 kb fragment was detected in all clones, but extrachromosomal plasmid was also detected (Fig. 5b, lanes 4 and 7). By the 15th passage, extrachromosomal plasmid could not be detected and the plasmid appeared to have integrated into the chromosome of each transformant (Fig. 5b, lanes 5 and 8). One possible reason for the integration of pPLoriC7 was the selection pressure exerted by the antibiotic. To investigate if this was the cause, we passaged both parental M. gallisepticum and a transformant containing pPLoriC7 in broth containing tetracycline at 0.4 or 1 µg ml–1. Parental M. gallisepticum could only grow in broth containing 0.4 µg tetracycline ml–1. Neither extrachromosomal nor integrated forms of the plasmid could be detected in the transformant after five passages in media containing 0.4 µg tetracycline ml–1, suggesting that 0.4 µg tetracycline ml–1 was an inadequate concentration to select for transformants. In the transformant grown in 1 µg tetracycline ml–1, pPLoriC7 had completely integrated into the genome by the 15th passage. There appeared to be no difference in the rate of integration of the plasmid into the chromosome in transformants cultured in low concentrations of tetracycline, suggesting that antimicrobial selection pressure did not influence the rate of chromosomal integration. The stability of pGTLori was tested in two transformants of M. imitans strain 4229, which were selected on solid media containing 4 µg tetracycline ml–1 and then passaged 15 times in broth with tetracycline. Southern blot analysis was performed at the fifth, 10th and 15th passages of each transformant using the radiolabelled M. gallisepticum oriC probe. The oriC probe did not hybridize with the oriC region of untransformed M. imitans (results not shown). At all passage levels examined, a 7 kb DNA band, indicative of the extrachromosomal form of pGTLori, was detectable in both transformants (results not shown).
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, lanes 2–8). At the 15th passage the 7.4 kb extrachromosomal form of plasmid was still detectable in both transformants (Fig. 6a, lanes 5 and 8). In M. gallisepticum, the oriC probe, which contained part of the M. imitans soj gene, hybridized to a 3.9 kb band containing the M. gallisepticum soj gene (Fig. 6b, lane 2). A 7.4 kb band, corresponding to the extrachromosomal plasmid, was detectable until the 15th passage in transformant C1 (Fig. 6b, lane 5). However, the plasmid had integrated into the genome by the 10th passage, although not into the oriC region (Fig. 6b, lanes 4, 5, 7 and 8).
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3.03-transformed M. gallisepticum S6 produced several tetracycline-resistant colonies after 7 days of incubation. To investigate the integration site of the plasmid, Southern blot analysis was performed using DNA extracted from 16 pPLoriC7/3.03 transformants that had been passaged 15 times. Identical membranes were hybridized to the radiolabelled ampR gene or the internal fragment of the vlhA3.03 gene. The ampR probe bound to a 6.5 kb band in the digest of the plasmid pPLoriC7/3.03 and in most passaged transformants to a 10.5 kb band, corresponding to the predicted size of the fragment containing the chromosomal oriC region of M. gallisepticum S6 after the plasmid had integrated into this region. However, no band indicative of targeted homologous recombination was detected (results not shown).
Tetracycline-resistant colonies of the pMIori/3.03 transformants of M. gallisepticum S6 were passaged 15 times and the integration site of the plasmid was investigated by hybridizing Southern blots with radiolabelled ampR gene and vlhA3.03 probes. Several bands were detected in all passaged transformants, indicating that the plasmid had integrated at least twice into the M. gallisepticum genome. The vlhA3.03 probe detected bands of a different size in one of the transformants. This transformant was passaged an additional five times in media that did not contain tetracycline, to allow the transformant to cure itself of the extrachromosomal plasmid, and then inoculated onto mycoplasma agar containing 4 µg tetracycline ml–1. Two single tetracycline-resistant colonies were selected for Southern blot analysis. The ampR probe detected an 8.77 kb band in the digest of pMIori/3.03, while an 15 kb band was detected in these transformants (Fig. 7 lanes P, 2 and 3). The gene that was predicted to have been interrupted was amplified using the primer pair SW13 for and vlhALeader rev, generating a PCR product of 2 kb. DNA sequencing of the PCR product and searches of the databases determined that the amplicon was identical to part of the vlhA1.2 gene (previously pMGA1.2) of M. galliseptiucm S6 and the section of the vlhA3.03 gene that was contained within the plasmid construct. The product also contained part of the pGEM-T vector. This suggested that pMIori/3.03 had integrated into the vlhA1.2 gene rather than the vlhA1.1 gene.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Lartigue et al., 2003). Apart from M. pulmonis, which belongs to the Hominis phylogenic group, all these species belong to the Spiroplasma phylogenic group, and all have a conserved gene order within the oriC region (Fig. 8), which contains the dnaA gene. The functional oriC plasmids for these species require regions upstream and downstream from the dnaA gene, with the exception of the smallest oriC plasmid for S. citri, which contains only a 163 bp region downstream from the dnaA gene (Cordova et al., 2002; Lartigue et al., 2002, 2003). The gene order in the oriC regions of M. gallisepticum and M. imitans is similar to those of M. pneumoniae and Mycoplasma genitalium (Fig. 8). These mycoplasmas belong to the Pneumoniae phylogenetic group and the putative oriC regions surround the soj gene but lie upstream of the dnaA gene (Cordova et al., 2002; Papazisi et al., 2003). We produced four oriC plasmid constructs that contained the soj gene and regions upstream and downstream of the soj gene in M. gallisepticum. Only the plasmids containing the region upstream from the soj gene could be detected in M. gallisepticum, indicating that only the AT-rich sequences found in this region were essential for plasmid replication, even though the region downstream from the soj gene included two DnaA boxes. This suggests that, at least in M. gallisepticum, the soj gene is not required for replication of an oriC plasmid. The M. gallisepticum oriC plasmid, pGTLori, and the M. imitans oriC plasmid pMIori were able to replicate in both species. Alignment of the oriC region of M. imitans with that of M. gallisepticum showed that the sequences were very different, but that the surrounding genes had high levels of DNA sequence identity. Both species contained similar DnaA box consensus sequences. Therefore, M. gallisepticum and M. imitans might be expected to support replication of heterologous oriC plasmids. In contrast to M. imitans, M. pneumoniae did not appear to support replication of the M. gallisepticum oriC plasmid pGTLori. Though both species have a conserved gene order in the oriC region (Fig. 8), the DNA sequence of their oriC regions appears to be poorly conserved, and alignment of the peptide sequence of the DnaA proteins of M. gallisepticum and M. pneumoniae revealed only 23 % peptide sequence identity. Several AT-rich clusters were identified in the putative oriC region of M. pneumoniae, but consensus DnaA box nonamers were not found. This suggests that the DnaA boxes of M. pneumoniae may have a more relaxed consensus sequence. The minimal oriC region of M. gallisepticum that was found to be functional in M. gallisepticum strain S6 was 180 bp in size. This region was upstream from the soj gene and included two DnaA boxes and two AT-rich regions of 31 and 22 bases (Fig. 2). The size of the minimal oriC region in M. gallisepticum was similar to that required in the functional oriC vector for S. citri, which was a 163 bp region downstream from the dnaA gene containing three DnaA boxes and two AT-rich regions (Lartigue et al., 2002). For M. gallisepticum strain S6, we found that only two DnaA boxes were necessary for plasmid replication, and this is the smallest oriC region capable of supporting plasmid replication in mycoplasmas. Both pGTLori and pPLoriC1, which contained larger sections of the oriC region, integrated readily into the chromosome during passage, as has been seen with the oriC plasmids pBOT1 in S. citri and pMPO1 in M. pulmonis (Cordova et al., 2002; Renaudin et al., 1995). We produced a smaller oriC plasmid in an attempt to generate a vector that would not integrate readily into the genome. While pPLoriC7 did not integrate into the chromosome as rapidly as pGTLori and pPLoriC1, it had integrated completely into the chromosome by the 15th passage. In M. pulmonis, the minimal oriC regions necessary for plasmid replication are 262 bp upstream and 327 bp downstream from the dnaA gene. This oriC plasmid (pMPO5) remains extrachromosomal for at least 15 passages (Cordova et al., 2002). The oriC plasmid pGTLori could replicate in M. imitans strain 4229 and appeared to be more stable in M. imitans than in M. gallisepticum. In Southern blot analysis, free plasmid was detectable until the 15th passage, with only limited integration into the chromosome. This suggested that the oriC plasmid of M. imitans might be more stable in M. gallisepticum than the homologous oriC plasmid. However, while some plasmid remained extrachromosomal until the 15th passage, a portion had integrated into the genome at sites outside the oriC region by the 10th passage. Interestingly, an extrachromosomal form of pMIori was found in all passaged transformants of M. imitans. These results suggest that the homologous recombination system in M. gallisepticum has a higher efficiency than that of M. imitans.
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). However, pPLoriC7, containing the 180 bp M. gallisepticum oriC region, integrated readily into the chromosomal oriC region by the 15th passage, and attempts to inactivate vlhA3.03 using pPLoriC7 were unsuccessful, with the plasmid integrating into the chromosomal oriC region in all transformants rather than into the target gene region, even though the vlhA3.03 fragment in the plasmid was 986 bp in size.
Of the 16 M. gallisepticum transformants containing pMIori/3.03, only one showed evidence of an interruption of a vlhA gene in Southern blot analysis. The inactivated gene was identified as vlhA1.2. At the DNA level, vlhA1.2 and vlhA1.1 are 98 % identical, so this result is not unexpected. Targeted gene inactivation has, to our knowledge, only been achieved once before in M. gallisepticum, but use of the M. imitans oriC plasmid could improve the efficiency of recombination. Thus, in this study, we constructed several oriC plasmids that could replicate successfully in M. gallisepticum and M. imitans. This is the first report, to our knowledge, of oriC plasmids for members of the Pneumoniae phylogenetic group. In other mollicutes, oriC plasmids have been used to inactivate genes or to express exogenous genes (Cordova et al., 2002; Duret et al., 1999; Janis et al., 2005; Lartigue et al., 2002), so these oriC plasmids are likely to be useful tools for genetic research in M. gallisepticum and M. imitans.
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ACKNOWLEDGEMENTS |
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Edited by: C. Citti
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Received 9 April 2008;
revised 8 June 2008;
accepted 9 June 2008.
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