Construction of the first shuttle vectors for gene cloning and homologous recombination in Mycoplasma agalactiae

Abstract

Mycoplasma agalactiae is a worldwide ruminant pathogen that causes significant economic losses by inflicting contagious agalactia in sheep and goats. The development of efficient control strategies requires a better understanding of the mycoplasma factors that promote successful infection. However, lack of genetic tools has been a major impediment in studying the pathogenic mechanisms of M. agalactiae. This study describes the identification and cloning of the M. agalactiae origin of replication (oriC) in order to construct the first shuttle vectors for targeted gene disruption, gene complementation and expression studies. Additionally, this report provides the first evidence of the occurrence of homologous recombination and the functionality of heterologous tetM determinant in this pathogen.

1. Introduction

Mycoplasmas are wall-less eubacteria characterized by small genomes with low G + C content that include several species known to cause widespread diseases in humans and animals [1]. Mycoplasma agalactiae is a significant pathogen of small ruminants and the main etiological agent of the syndrome contagious agalactia in sheep and goats. These chronic and difficult-to-eradicate infections, clinically manifested as mastitis, arthritis and keratoconjunctivitis, lead to considerable economic losses [2]. So far, very little is known about the biology of this pathogen as only a few surface proteins have been characterized at the molecular level. These include the P48 [3], the P80 [4], the P30 [5] and the Vpma family [6,7] whose exact role in disease progression is still unknown. Indeed, failure to efficiently control M. agalactiae infections is mainly due to our lack of knowledge concerning the nature of the M. agalactiae factors that are involved in virulence and pathogenesis, which in turn, can be largely attributed to our inability to genetically manipulate this pathogen. To fill this gap, development of adequate gene transfer methods and genetic tools have become a prerequisite.

We have recently demonstrated the amenability of M. agalactiae to genetic transformation and to random transposon-mutagenesis using the transposon Tn4001mod [8]. However, this tool is inappropriate for targeted gene disruption that requires homologous recombination between chromosomal sequences and extrachromosomal copies usually carried by suicide vectors. Considering the poor transformation efficiency of mycoplasmas, it is likely that such a rare event, if occurring in M. agalactiae, might not be detectable. The use of replicating vectors, besides providing a tool for complementation, may improve the likelihood of recombination [9,10]. Due to the paucity of natural plasmids, only a limited number of replicating vectors have been obtained for four mycoplasma species, namely Spiroplasma citri [11], M. pulmonis [12], M. mycoides and M. capricolum [9,10] by cloning their origin of replication (oriC) into standard vectors. Though the oriC plasmids that harbour the chromosomal dnaA gene, flanked by DnaA box sequences, replicate efficiently in their respective hosts, functionality of heterologous oriC plasmids in related mollicute species has also been demonstrated in some cases [9]. Since attempts to transform M. agalactiae with heterologous oriC plasmids of M. pulmonis, a close phylogenetic relative, were unsuccessful, the aim of the current study was to develop replicating vectors for M. agalactiae that would serve as efficient tools for gene manipulation and expression.

2. Materials and methods

2.1. Bacterial strains and culture conditions

Except for the initial cloning of oriC, where the M. agalactiae strain 5632 [13] was used, all subsequent studies were carried out with the type strain PG2 [14] grown at 37 °C in standard SP-4 medium supplemented with 500 U/ml of penicillin [15]. Mycoplasma transformants were selected on SP-4 agar plates containing 2 μg/ml of tetracycline. Escherichia coli DH10B was used for subcloning and for amplifying different oriC plasmids. E. coli transformants were grown in standard Luria–Bertani (LB) medium [16] supplemented with 50 μg/ml of ampicillin and 10 μg/ml of tetracycline.

2.2. Cloning of the M. agalactiae oriC region

Genomic DNA from M. agalactiae strain 5632 was prepared by incubation of the cells in lysis buffer (10 mM Tris pH 8, 10 mM EDTA, 10 mM NaCl, 0.5% SDS, 0.1 mg/ml proteinase K) overnight at 37 °C followed by phenol–chloroform extraction [16]. Approximately 1 μg of this DNA was digested to completion by EcoRI. The digested products were separated by electrophoresis, on 1% agarose gel followed by transfer on nylon membranes for Southern analysis using digoxigenin (DIG)-labeled AGA33 fragment [13], and on 1% low melting point agarose preparative gel. Based on the Southern blot results, fragments ranging from 6.5 to 7.5 kb were recovered from a gel slice using Aga-rACE™ (Promega) followed by ethanol precipitation and resuspension in water. For the construction of genomic sub-library, approximately 25 ng of the gel-purified DNA fragments were ligated to 5 ng of EcoRI digested and Shrimp Alkaline Phosphatase (Roche) treated pBluescriptKS (Stratagene) using the Ligafast kit (Promega). The ligation mix was transformed into E.coli and transformants were selected on LB agar supplemented with ampicillin (50 μg/ml), 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside (X-Gal; 40 μg/ml) and isopropylthio-β-d-galactoside (IPTG; 40 μg/ml) [16]. Ninety-six recombinant clones were randomly picked and their crude DNA content was analysed on Southern Blots using the AGA33 probe. This identified a recombinant plasmid, pMM12-1, carrying a 6.9 kb EcoRI insert. A 5.3 kb BamHI–NotI fragment of the pIVT-1 plasmid [17], bearing the tetM gene, was then cloned into the corresponding sites of pMM12-1. The resulting construct was designated pMM20-1. The 6.9 kb insert of pMM20-1 was further subcloned by partial HindIII digestions to generate smaller constructs pMM21-4 and pMM21-7, carrying 2.7 and 1.3 kb inserts, respectively. Plasmid pMMΔoriC was obtained by EcoRI digestion of pMM20-1 and self-ligation of the 8.2 kb fragment containing the tetM/pBluescriptKS portions.

2.3. Transformation of M. agalactiae

Approximately 3 μg of each plasmid construct was introduced into mycoplasma cells (10⁸–10⁹), at late log phase, by electroporation [8]. The cells were incubated in non-selective SP-4 medium for 2 h at 37 °C before plating them on SP-4 agar containing 2 μg/ml of tetracycline. The plates were examined for colony development from the fifth day of incubation at 37 °C. Transformants were subcultured in 1 ml of SP-4 broth containing 2 μg/ml of tetracycline. For pMM21-7, the transformants were subcultured in SP-4, as well as in Aluotto broth [8,18], and the tetracycline concentration was gradually increased from 2 to 10 μg/ml. A minimum of four independent transformations were made for each of the oriC constructs.

2.4. PCR-based detection of tetM in picked transformants

Crude DNA extracts were prepared from 1 ml cultures [8]. PCR assays were conducted using 2-4 μl of crude DNA as template in 25 μl reaction mixtures, with 1 U of Taq DNA polymerase (Promega) in 1× buffer supplied by the manufacturer, 200 μM dNTPs, 2 mM MgCl₂ and 1.4 μM of each primer, TetF (5′-CATGTG-GAGATAGAAC-3′) and TetR (5′-GATATTCCTGT-GGCGC-3′). The amplification was performed in a Perkin–Elmer GeneAmp thermal cycler over 30 cycles, each consisting of 40 s at 95 °C, 40 s at 54 °C and 40 s at 72 °C besides the initial denaturation step of 5 min at 95 °C and the concluding chain termination at 72 °C for 5 min. In the presence of free or integrated oriC plasmids, this PCR resulted in a 430 bp tetM-product which could be detected by agarose gel electrophoresis.

2.5. DNA isolation, manipulation and Southern hybridization

Plasmid DNA was isolated from E. coli and mycoplasma cells using E.Z.N.A.^® Plasmid Miniprep Kit (Peqlab Biotechnologie GmbH). Total genomic DNA from mycoplasma cells was isolated either by the phenol:chloroform extraction method [16] or by QIAamp^® DNA Mini Kit (Qiagen). Restriction endonucleases (Promega) were used according to the manufacturer's instructions. Southern hybridizations were performed with DIG-labeled probe corresponding to the 430 bp tetM-PCR product (described above) or to the 1.2 kb oriC-PCR product. The latter was obtained using primers Ori21-4F (5′-TGCGTTAACACTGAAGTC-3′) and T3ISLrev (5′-AGAGCAGAATTCAATTAACCCTC-ACTACTAAAG-3′) under standard PCR conditions employing 30 cycles of denaturation (94 °C/1 min), annealing (52 °C/1 min) and extension (72 °C/1 min) besides the starting denaturation step of 5 min at 94 °C and a concluding chain termination for 5 min at 72 °C. Hybridization was detected using anti-DIG antibody coupled to alkaline phosphatase and the chemoluminescent substrate CPD Star as recommended by the manufacturer (Roche Molecular Biochemicals).

2.6. Primers and sequencing

The sequencing, as well as the synthesis of all the primers used in the present study was carried out at VBC-Genomics Bioscience Research GmbH, Vienna. The smallest oriC insert was sequenced at one end using primer Seqori1 (5′-ATGGCGGTTTGTTGGAGGTC-3′) which corresponds to the region of the tetM determinant adjoining the insert. The sequence at the other end of the insert was obtained using the standard T3 primer.

3. Results and discussion

3.1. Cloning of oriC of M. agalactiae and development of replicating shuttle vectors

The recent proof of the amenability of M. agalactiae to genetic transformation [8] triggered a greater interest in developing new cloning and shuttle vectors for this pathogen. Initially, we tested the M. pulmonis oriC plasmids, pMPO1 and pMPO5 [12], for transformation in M. agalactiae. Though the control transformations using modified Tn4001 carrying tetM determinant gave a reasonable number of tetracycline resistant clones, pMPO1 and pMPO5 failed to yield any transformant. These results complied with previous findings, where it was suggested that oriC plasmids of mollicutes might be host specific [9].

In a previous study, a DNA fragment (AGA33) of the M. agalactiae type strain PG2 was cloned and shown by sequence analysis to carry a part of the dnaA gene [13]. This fragment was used to probe a pBluescriptKS EcoRI genomic sub-library of M. agalactiae strain 5632 to identify and isolate the oriC locus of this mycoplasma species. This identified a recombinant plasmid, pMM12-1, carrying a 6.9 kb EcoRI insert. Detailed genetic analysis indicated that the cloned dnaA gene was flanked by sequences of about 1 and 5.5 kb that were expected to harbor the DnaA-boxes usually found in the vicinity of the dnaA gene and considered essential for replication [9,12]. The tetM gene, with its own promoter and terminator, was cloned into pMM12-1 to provide tetracycline resistance. The resulting construct, namely pMM20-1 (Fig. 1) when transformed into PG2, gave tetracycline resistant transformants with a frequency of ~2 × 10⁻⁷ (Table 1). This transformation frequency was much higher than that obtained for Tn4001 [8] and, more importantly, no ‘pseudoresistant’ clones could be detected. This negated the need for overnight growth and selection in selective broth before the final plating of transformants. The latter step was essential for Tn4001 transformations as they often led to a high percentage of pseudoresistant gentamicin clones which were difficult to distinguish from true transformants [8]. PCR analyses confirmed that the tetracycline resistance correlated with the presence of the tetM determinant in all pMM20-1 transformants, as they yielded the expected 430 bp tetM-PCR product (data not shown). Presence of the free and integrated pMM20-1 plasmid, in a randomly selected transformant, was demonstrated by Southern blot hybridization using a tetM- specific probe (Fig. 2(b), lane C3). Although pMM20-1 is predominantly seen as a chromosomal integrant, small amounts of the linearized free plasmid form are clearly visible in the Southern blots (Fig. 2(b), lane C3). Furthermore, plasmid pMM20-1 was recovered and amplified from E. coli clones transformed with cell lysates obtained from pMM20-1 mycoplasma transformants. Hence, pMM20-1 was demonstrated to be the first shuttle vector which could replicate both in E. coli and M. agalactiae. As a control, the same experiment when performed with plasmid pMMΔoriC, which was constructed by deleting the EcoRI insert of pMM20-1 (Fig. 1), failed to yield any transformants.

Fig. 1.

Schematic representation of the plasmids used in this study. The recombinant plasmid pMM20-1 corresponds to the pBluescript KS vector backbone (double line) ligated to the tetM determinant (thick single line) and to a 6.9 kb DNA fragment of M. agalactiae (thin single line) that carries the dnaA gene. Plasmids pMMΔ oriC, pMM21-4 and pMM21-7 were derived from pMM20-1 by total or partial deletion of the insert. Numbers mentioned below the dotted arrows indicate the approximate size of the fragments and bla designates the ampicillin resistance gene. Restriction enzymes: C, ClaI; E, EcoRI; EV, EcoRV; H, HindIII; N: NotI; P, PstI; S, SalI; Sm, SmaI; X, XbaI.

Table 1.

Transformation frequencies^a obtained after electroporation of oriC plasmids into M. agalactiae

Plasmid frequencyd	Total CFUb	TetR CFUc	Transformation
pMM20-1	1.54 × 109	295	1.92 × 10−7
pMM21-4	1.52 × 109	1360	8.95 × 10−7
pMM21-7	4.10 × 108	1720	1.32 × 10−6
pMMΔoriC	1.72 × 109	0	0

^aResults obtained from one representative experiment in which the four constructs were used in parallel.

^bTotal colony forming units (CFU) without tetracycline selection after electroporation.

^cTetracycline-resistant CFU obtained after electroporation and 2 h incubation at 37 °C in SP-4 media.

^dTetracycline-resistant CFU/total viable CFU.

Fig. 2.

Free versus integrated oriC plasmids in M. agalactiae transformants. (a) Schematic representation of the integration of oriC plasmids (P: pMM20-1, pMM21-4 or pMM21-7) into genomic DNA of M. agalactiae PG2. The size of the chromosomal ClaI DNA fragment bearing the oriC is designated as G, whereas X and Y correspond, respectively, to the right and left sequences flanking the oriC within the fragment G. A single putative homologous recombination event between the oriC copy carried by any of the three plasmids and the chromosomal oriC region is represented by crossed lines. This crossing over would lead to the integration of the plasmid into the chromosome and would segregate the two oriC copies onto two ClaI fragments GY and GX. Regardless of the plasmid used, the length of the GY fragment that carries the oriC and the tetM and bla markers is constant, while that of the GX fragment varies accordingly with the size of the plasmid inserts. (b) Replication versus integration of the oriC plasmids as observed in Southern blot hybridization using the tetM specific probe. ClaI-digested DNA from non-transformed M. agalactiae (NT) and from three transformants C1, C2 and C3, respectively, obtained using plasmids pMM21-7 (P1), pMM21-4 (P2) and pMM20-1 (P3), was probed with a DIG-labeled tetM specific probe. (c) Localization of the integrated plasmids at the chromosomal oriC locus of M. agalactiae PG2. Southern blot analysis was performed as in (b) except that the DNA was probed with a 1.2 kb DIG-labeled fragment specific to the oriC of M. agalactiae. DNA size standards are indicated in the left margin.

The 6.9 kb oriC fragment of pMM20-1 was subcloned by HindIII partial digestions to generate plasmids pMM21-4 and pMM21-7, carrying 2.7 and 1.3 kb inserts, respectively (Fig. 1). This was carried out with the aim of promoting replication of the oriC plasmids in opposition to their chromosomal integration. It was already shown in other mycoplasma species that very small sequences, corresponding to the DnaA-boxes, were suffcient for replication [12,19]. Upon transformation, both plasmids were found by Southern blot hybridization to replicate successfully in M. agalactiae PG2 (Fig. 2(b), lanes C1 and C2) and to generate higher transformation frequencies compared to pMM20-1 (Table 1). Moreover, pMM21-7 was found in its free plasmid form (Fig. 2(b), lane C1), while pMM21-4 (Fig. 2(b), lane C2) exhibited an intermediate level of chromosomal integration versus free replication (Fig. 2(b) and (c), lane C2). These data suggest that the frequency of plasmid integration into the M. agalactiae chromosome correlates with the size of the inserts.

3.2. Homologous recombination of the plasmids at the chromosomal oriC site

As anticipated, the integration of plasmids pMM20-1 and pMM21-4 in fact occurred by homologous recombination at the chromosomal oriC locus. This was shown by Southern blot hybridization using a 1.2 kb DIG-labeled probe specific to the oriC region. This probe was generated by PCR using primer T3ISLrev located near the EcoRI insertion site of pMM21-7, and primer Ori21-4F designed after partial sequencing of the pMM21-7 oriC insert. Results are illustrated in Fig. 2(c) (lanes C3 and C2) which depicts hybridization of the oriC-specific probe with ClaI-digested DNA. Since the oriC plasmids contain a unique ClaI site in the multiple-cloning site of the cloning vector, plasmid integration via a single homologous recombination event at the chromosomal oriC locus was expected to result in two oriC copies that would be segregated into two fragments upon ClaI digestion (see Fig. 2(a)). As illustrated in Fig. 2(c), lane NT, the chromosomal oriC is carried in non-transformed M. agalactiae PG2 by a fragment larger than 15 kb (depicted as G in Fig. 2(a) and (c)). In pMM21-7 transformant C1, the same ClaI genomic fragment G was detected (Fig. 2(c)) as well as the linearized free replicating plasmid (P), indicating that in this transformant no chromosomal plasmid integration occurred (Fig. 2(b), lane C1). In contrast, clones transformed with pMM21-4 (lane C2) and pMM20-1 (lane C3) displayed three hybridization signals corresponding: (i) to the linearized free plasmid (P), (ii) to a chromosomal fragment carrying one oriC copy along with the plasmid backbone (GY) whose size is too large to be distinguished from that of the fragment G, and (iii) to a chromosomal fragment carrying a second oriC copy (GX) whose length varies according to the size of the insert originally carried by the plasmid used for transformation. As expected, the GY-fragment hybridized with the tetM probe only in transformants C2 and C3 (Fig. 2(b)), while GX was not detected by the probe. This data provides the first evidence of homologous recombination occurring in M. agalactiae and paves the way for developing improved oriC-based cloning vectors for targeted gene disruption and for introducing new genes either on free replicating plasmids or as integrated copies at the chromosomal oriC locus.

3.3. Stability of pMM21-7 in M. agalactiae

To evaluate the stability of the free replicating form of pMM21-7 plasmid in M. agalactiae, independent transformants were subjected to 21 serial passages in selective SP-4 or Aluotto broth. BamH1-digested total DNA obtained from these clones was analysed by Southern blot hybridization using the tetM-specific probe. Results indicated that chromosomal integration of the plasmid, when occurring, was not observed before the 16th passage. This is illustrated in Fig. 3 for three representative clones in which partial or total chromosomal integration of the plasmid has occurred in two clones after the 16th passage (clones T1 and T3), while the free replicating form was maintained in clone T2 during passaging. Clone T1 and T2 were passaged in Aluotto broth whereas clone T3 was passaged in SP-4. The latter is a very rich medium and, unlike Aluotto medium, supports a high rate of growth for M. agalactiae and related mollicutes. A similar pattern of stability was obtained with other tested clones suggesting that a slower growth rate might be associated with increased stability of the plasmid.

Fig. 3.

Stability of the oriC plasmid pMM21-7 in M. agalactiae. Southern blot hybridizations were performed with BamHI digested DNA obtained from three independent pMM21-7 transformants (T1, T2 and T3) which had undergone 6, 16 and 21 additional passages in Aluotto (T1 and T2) or in SP-4 (T3) broth containing tetracycline. The DNA was probed with a DIG-labeled tetM specific probe. BamHI digested DNA corresponding to nontransformed M. agalactiae (NT) and pMM21-7 (P) served as negative and positive controls, respectively.

Data reported in this paper emphasizes the use of oriC-based vectors as genetic tools in mycoplasmas. The resistance marker, tetM, was shown to be functional in both free and integrated forms of the plasmids, suggesting that the oriC locus is a good target for expression of heterologous genes in M. agalactiae. For instance, stably expressed fluorescent markers would be extremely valuable for labeling dividing mycoplasma cells in studies of mycoplasma host–cell interactions. While the free replicating oriC plasmid pMM21-7 offers a tool for gene complementation studies, integrating oriC plasmids are the first evidence of homologous recombination occurring in M. agalactiae. Although results obtained by passaging pMM21-7 transformants indicate that this event is rare, this data are very encouraging as it implies that targeted gene disruptions and integrations are now technically feasible in this pathogen, and thus, widens the horizons of molecular genetic studies in this ruminant pathogen.