Transposon mutagenesis in Mycoplasma hyopneumoniae using a novel mariner-based system for generating random mutations
© Maglennon et al.; licensee BioMed Central Ltd. 2013
Received: 30 October 2013
Accepted: 2 December 2013
Published: 21 December 2013
Mycoplasma hyopneumoniae is the cause of enzootic pneumonia in pigs, a chronic respiratory disease associated with significant economic losses to swine producers worldwide. The molecular pathogenesis of infection is poorly understood due to the lack of genetic tools to allow manipulation of the organism and more generally for the Mycoplasma genus. The objective of this study was to develop a system for generating random transposon insertion mutants in M. hyopneumoniae that could prove a powerful tool in enabling the pathogenesis of infection to be unraveled. A novel delivery vector was constructed containing a hyperactive C9 mutant of the Himar1 transposase along with a mini transposon containing the tetracycline resistance cassette, tetM. M. hyopneumoniae strain 232 was electroporated with the construct and tetM-expressing transformants selected on agar containing tetracycline. Individual transformants contained single transposon insertions that were stable upon serial passages in broth medium. The insertion sites of 44 individual transformants were determined and confirmed disruption of several M. hyopneumoniae genes. A large pool of over 10 000 mutants was generated that should allow saturation of the M. hyopneumoniae strain 232 genome. This is the first time that transposon mutagenesis has been demonstrated in this important pathogen and could be generally applied for other Mycoplasma species that are intractable to genetic manipulation. The ability to generate random mutant libraries is a powerful tool in the further study of the pathogenesis of this important swine pathogen.
Belonging to the class Mollicutes, mycoplasmas are characterised by their lack of a cell wall and small genome size, and are considered to be the smallest free-living self-replicating organisms and as such are of considerable interest in synthetic biology . Respiratory disease is a major problem facing swine producers and Mycoplasma hyopneumoniae, the cause of enzootic pneumonia (EP), is a swine-specific mycoplasma of global prevalence that is one of the leading causes of disease [2, 3]. EP is characterised by a chronic non-productive cough that is most evident during the growing and fattening stages of production, although all ages of animal may be affected . Mortality rates are usually low, but morbidity may be high with associated economic losses due to increased medication costs, lower growth rates and lower feed conversion efficiencies . The pathogenesis of EP involves entry of M. hyopneumoniae into the respiratory tract by inhalation, largely from nose-to-nose contact with other pigs , and colonisation of the ciliated epithelial cells of the trachea, bronchi and bronchioles [5, 6]. Adherence of the organism to the epithelium causes ciliostasis and loss of cilia, thereby preventing effective clearance of debris, pathogens and mucus from the airways . Additionally, M. hyopneumoniae may cause direct cell damage by production of cytotoxic metabolites such as hydrogen peroxide . The chronic nature of infection may result from modulation of the host immune response by M. hyopneumoniae[2, 9] and possibly by variable expression of bacterial surface antigens, enabling the organism to evade effective clearance . M. hyopneumoniae infection can be associated with and exacerbated by co-infection with other viral pathogens such as Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) and Porcine Circovirus type 2 , and with upper respiratory tract bacteria such as Actinobacillus pleuropneumoniae, Streptococcus suis, Haemophilus parasuis and Pasteurella multocida. Commercial M. hyopneumoniae vaccines are in widespread use in the pig industry where they are reported to lessen the economic effects of the disease by reducing clinical signs and lung lesions and by improving performance parameters [3, 11, 12]. However, current vaccines are not completely effective and do not prevent colonisation of the respiratory tract with M. hyopneumoniae or eliminate infection from the herd .
Central to the design of more effective vaccines is an understanding of the pathogenesis of EP. Despite the availability of whole genome sequences of several M. hyopneumoniae strains and the small genome size, the functions of most of the protein coding sequences are still unclear. The chronic nature of M. hyopneumoniae infection implies a complicated relationship between the pathogen and the host that is poorly understood. The small genome size of M. hyopneumoniae offers an appealing opportunity to understand the molecular basis of disease formation, and to unravel the interplay between the host and the pathogen. Traditionally the exploration of gene function in a bacterium is investigated through the generation of random insertional mutants by transposon mutagenesis. Typically for other Mycoplasmas, the Tn4001 transposon derived from Staphylococcus aureus has been utilised [15–19]. As reviewed by Halbedel and Stülke , a mini Tn4001 transposon is used containing an antimicrobial resistance cassette flanked by inverted repeat sequences. The mini transposon and transposase enzyme are delivered into the organism by transformation and expression of the resistance cassette enables selection of mutated organisms. Insertions in essential genes result in lethality but many transformants have mutations in genes that are not necessary for growth in vitro, but may encode for particular pathogenicity determinants that are necessary for growth, survival, invasion or disease in the animal. Such mutants could be exploited in a number of different assays, including in vivo screening of mutants using the powerful technique of signature tagged mutagenesis . Through the screening of large numbers of random mutants in vivo, mutants that are attenuated in vivo can be identified. Such mutants may encode for virulence factors and may be useful in the identification of live attenuated vaccine candidates.
Recently we described the first successful transformation of M. hyopneumoniae strain 232 using an artificial self-replicating oriC plasmid system . This system allowed us to optimise a set of transformation conditions, antimicrobial selection cassettes and promoter sequences for M. hyopneumoniae. In this study, the results of that work have been used further to develop a transposon mutagenesis system for M. hyopneumoniae. A key feature of this system is the use of a Himar1 transposon, belonging to the Mariner family of transposons. Insertion occurs at any TA dinucleotide  offering an excellent potential for complete coverage of the AT-rich genome of M. hyopneumoniae. We describe the development of this system and its use in generating a large pool of several thousand M. hyopneumoniae mutants. This is a major advancement in the study of this important swine pathogen and potentially other Mycoplasmas.
Materials and methods
Sequences of oligonucleotides.
Oligonucleotide sequence (5’-3’)
M. hyopneumoniae strain 232  was grown in Friis broth medium at 37 °C in a static incubator . For growth on solid medium, Friis medium was solidified by addition of 0.8% w/v purified agar (Oxoid Ltd, Basingstoke, UK) and 0.01% w/v DEAE-dextran (Sigma-Aldrich Ltd, Gillingham, UK) and incubated at 37 °C with 5% CO2. For the selection of transformants, tetracycline hydrochloride (Sigma-Aldrich Ltd, Gillingham, UK) was added to Friis broth medium and Friis agar medium at final concentrations of 0.5 μg/mL and 0.2 μg/mL respectively. Molecular cloning was performed using Escherichia coli strain DH5α, grown in Luria-Bertani medium according to standard methods . Transformants were selected by the addition of 100 μg/mL ampicillin (Sigma-Aldrich Ltd, Gillingham, UK) to the medium. Additionally, for the selection of transformants containing the tetM gene, tetracycline hydrochloride was added to a final concentration of 5 μg/mL.
Transformation of mycoplasmas
M. hyopneumoniae strain 232 was grown to mid-late logarithmic phase in Friis broth as determined by an acid colour change in the phenol red pH indicator. Mycoplasmas were harvested by centrifugation of culture at 9000 × g for 10 min at 4 °C and washed three times in electroporation buffer (272 mM sucrose, 8 mM HEPES, pH 7.4). One hundred microliters of cells (corresponding to 3 mL culture) were incubated on ice with approximately 10 μg plasmid DNA for 30 min. Electroporation was performed in a 0.2 cm cuvette (Bio-Rad Ltd, Hemel Hempstead, UK) at 2.5 kV, 100 Ω, 25 μF and 900 μL ice-cold Friis medium were immediately added. After 15 min incubation on ice, cells were transferred to a 1.5 mL tube and incubated at 37 °C for 3 h. Culture was then plated onto Friis agar containing tetracycline and incubated at 37 °C in 5% CO2 for up to 18 days. Individual tetracycline-resistant transformants were picked using a sterile pipette tip into Friis broth medium containing tetracycline and grown for 5–7 days until evidence of growth as determined by the pH indicator.
Determination of transposon insertion sites
Transposon insertion site determination was based on the method described by Chaudhuri et al. . Genomic DNA was extracted from M. hyopneumoniae strain 232 culture using a phenol-chloroform method . Presence of tetM and plasmid backbone was determined by PCR using primer pairs TetMF/TetMR and Tpn1/Tpn2 respectively. Two and a half micrograms of DNA were digested overnight at 37 °C with 10 units of Alu I, which cuts the 892 kbp genome of M. hyopneumoniae strain 232 a total of 2239 times and generates blunt ends. Alu I also cuts within the tetM transposon sequence six times. The digested DNA was purified using a MinElute PCR Purification kit (Qiagen Ltd, Manchester, UK). Oligonucleotide linkers were attached to the digested DNA fragments. To generate the linker, 10 μM oligonucleotides Linker-A and Linker-B (Table 1) were heated together in a boiling water bath for 3 min in annealing buffer (10 mM Tris–HCl, 50 mM NaCl, 1 mM EDTA, pH 8.0) and then allowed to cool for 1 h at room temperature. Fifty nanograms of Alu I digested DNA were blunt-ligated to 4 μL annealed oligonucleotides Linker-A and Linker-B in a 10 μL reaction using a Quick Ligation kit (NEB Ltd, Hitchin, UK) for 1 h at room temperature. The ligated DNA was purified using a MinElute PCR Purification kit (Qiagen Ltd, Manchester, UK). PCR was performed using HotStarTaq (Qiagen Ltd, Manchester, UK) according to the manufacturer’s instructions. Thermocycler conditions were as follows: 95 °C for 15 min; 33 cycles of 94 °C for 45 s, 55 °C for 60 s, 72 °C for 120 s; 72 °C for 10 min. PCR products were visualised by electrophoresis in 1.6% agarose. Transposon insertion sites were determined by the direct sequencing of PCR products.
Southern blotting was performed based on previously described methods . Total DNA was extracted from 20 mL mycoplasma broth culture using a phenol-chloroform method [30, 32] and 2.5 μg were digested to completion with Hin dIII. DNA was separated by electrophoresis on 0.9% agarose, blotted onto Hybond-N + membrane (GE Healthcare Ltd, Little Chalfont, UK) and then fixed to the membrane by exposure to UV light. A digoxigenin (DIG)-labeled probe specific for the tetM gene was generated from PCR-amplified DNA (primers TetMF and TetMR, Table 1) using a DIG-High Prime DNA Labelling and Detection Starter Kit II (Roche Applied Science Ltd, Burgess Hill, UK). This kit was also used to perform pre-hybridisation and hybridisation in accordance with the manufacturer’s instructions. The membrane was autoradiographed at room temperature using CL-XPosure Film (Fisher Scientific Ltd, Loughborough, UK).
Construction of transposon delivery vectors
Plasmid pTn4001-RVC1 (Figure 1A) was constructed containing the Tn4001 transposase and a mini Tn4001 transposon consisting of the tetM gene under control of the spiralin gene promoter sequence of Spiroplasma citri bounded by inverted repeats. We previously optimised a set of transformation conditions for M. hyopneumoniae using an oriC-based self-replicating plasmid system, and showed that tetM under control of the spiralin gene promoter region was successfully expressed in M. hyopneumoniae strain 232 allowing the selection of transformants on Friis agar plates containing 0.2 μg/mL tetracycline . However, despite three separate attempts at transforming M. hyopneumoniae strain 232 with pTn4001-RVC using our optimised conditions and our oriC plasmid pMHO-2  as a positive control for transformation, we obtained no transformants. This suggested that the Tn4001 transposase was not functional in M. hyopneumoniae.
Transformation frequencies using Himar1 constructs.
Mean transformation frequency (transformants/CFU) [SE]
2.5 × 10-6 [1.3 × 10-6]
9.0 × 10-8 [2.4 × 10-8]
9.3 × 10-6 [4.5 × 10-6]
3.0 × 10-7 [4.6 × 10-8]
Analysis of transformants
Analysis of transposon insertion sites
Insertion sites of transposon mutants
Transposon insertion sites of M. hyopneumoniae strain 232 mutants.
Transposon insertion location
P97 ciliary adhesion
Outer membrane protein
Outer membrane protein
PTS system ascorbate-specific transporter subunit IIC
PTS system fructose-specific transporter subunit IIABC
PTS system ascorbate-specific transporter subunit IIC
Regulatory region of glpD
Generation of a large pool of M. hyopneumoniae mutants
We sought to determine whether our pMHC9-1 transposon delivery vector could be used to produce a large pool of random mutants, sufficient to provide an adequate number of “hits” in all of the non-essential genes of M. hyopneumoniae. A pool of at least 10 000 individual mutants would be expected to give a very high probability of an insertion into every one of the 692 protein coding sequences in the M. hyopneumoniae 232 genome determined by Minion et al. . It was calculated that a pool of 10 000 individuals would, on average, contain a mycoplasma cell with an insertion every 89 bp, and that there would be 14.45 insertions per coding sequence. From the Poisson distribution, 10 000 independent insertions would give a 99.99995% probability of an insertion into each one of the 692 coding sequences. 20 individual transformations were performed using plasmid pMHC9-1 with approximately 106 CFU per transformation. After 14 days growth at 37 °C on agar plates containing tetracycline, colonies were counted and harvested together in one large pool. A total of 11 759 colonies were counted across all 20 plates, representing an average transformation frequency of 3.4 × 10-4 transformants/CFU (standard error 5.3 × 10-5).
The inability to efficiently genetically manipulate M. hyopneumoniae has stood as a hurdle to advancements in the understanding of the pathogenesis of enzootic pneumonia in pigs. We recently reported the successful transformation of M. hyopneumoniae using an artificial plasmid system containing the origin of replication of M. hyopneumoniae and an antimicrobial resistance cassette, that was capable of self-replicating and maintenance in transformed bacteria . We have used this system to optimise a number of conditions required for the successful transformation of M. hyopneumoniae by electroporation with plasmid DNA. This knowledge has now enabled the design and construction of a transposon-based system for generating random mutants in M. hyopneumoniae.
The Tn4001 transposon is effective in generating insertional mutations in a number of different Mycoplasma species [16, 18, 19, 34] but even using our optimised transformation conditions, we were unable to demonstrate transposition in M. hyopneumoniae. A Himar1 transposon delivery vector that was shown to be active in M. gallisepticum generated very low numbers of transformants in M. hyopneumoniae with a very restricted range of unique insertion sites . By using an M. hyopneumoniae strain 232-specific promoter sequence to control the Himar1 expression and switching the wild-type Himar1 transposase for a hyperactive mutant C9 form, the frequency of transposition increased significantly. Analysis of transformants confirmed that resistance to tetracycline was due to a transposition event into the mycoplasma genome and it appeared that transposition occurred at a single site in each individual transformant. The transposon insertions appeared to be stable over serial passages and were maintained even in the absence of antimicrobial selection. These features are desirable when the phenotypes of mutants are studied in vitro, and particularly for in vivo studies of pools of mutants by TraDIS or signature-tagged mutagenesis. Additionally, we did not encounter any “pseudoresistant” colonies in our “no DNA” control transformations, which have been documented in transposon mutagenesis systems described for other mycoplasmas [16, 26]. “Pseudoresistant” colonies can complicate the isolation of true transformants.
In the course of this study, it was evident that there were significant differences in the transformation frequencies obtained using plasmid pMHC9-1. We suspect that differences in transformation frequency can be partially accounted for by variations in the number of cells electroporated and/or the growth phase of the mycoplasma culture used. It appeared that an increase in the number of cells transformed did not necessarily result in an increase in transformation frequency. However, even where similar numbers of cells were electroporated, there could be significant differences in transformation frequencies, implying that other factors could be important. Growth of M. hyopneumoniae is assessed and monitored by a simple change in the phenol red pH indicator incorporated into the medium, owing to the fact that turbidity is not generated. Thus pH can serve as a rough guide for growth phase, but the actual number of cells can only be estimated after plating the culture out and allowing growth on agar medium for 5–7 days. Additionally, complex and undefined medium is used to grow M. hyopneumoniae, with 20% equine/porcine serum and freshly prepared yeast extract contributing to variations in batches of medium.
Transposon insertion sites were determined for a limited number of individual transformants using a linker PCR technique and direct sequencing of PCR products by Sanger sequencing. From our limited analysis, we found transposon insertions in a number of M. hyopneumoniae genes of both known and unknown function, and in non-coding regions. We observed a clustering of transposon insertions in mhp447, a large open-reading frame of unknown function. The Himar1 transposon inserts at 5’-TA-3’ dinucleotides and we considered that the large number of insertions may be due to an increase in AT content. However, analysis of this ORF showed that the AT content was not unusual at 0.681 compared to 0.714 for the entire M. hyopneumoniae strain 232 genome. Alternatively, it is possible that there are hotspots for Himar1 insertions in the M. hyopneumoniae strain 232 genome, that may be affected by factors such as DNA topology, as is the case with other transposons, including Mariner elements . It is difficult to draw any conclusions from analysis of such a small pool of mutants, but determination of the insertion sites of a large pool of mutants by next generation sequencing would answer the question of whether favoured insertion sites do occur. Using plasmid pMHC9-1, we were able to produce a large pool of mutants. Over 10 000 colonies were generated through a number of transformations that, given an even distribution should be expected to have a high probability of insertions into all of the genes of M. hyopneumoniae. This pool of mutants may prove a valuable tool in future studies of M. hyopneumoniae in allowing a list of minimal essential genes to be determined. Such a pool of mutants could also be subjected to an in vivo screen for attenuated mutants using powerful functional genomic techniques such as TraDIS.
A novel transposon delivery system has been generated for M. hyopneumoniae, allowing for the first time the production of large numbers of random transposon insertion mutants. This system has been used to generate mutants with random transposon insertions in individual M. hyopneumoniae protein coding and non-coding sequences. It has also been used to generate a large pool of over 10 000 transposon insertions. These mutants are powerful tools in elucidating the functions of M. hyopneumoniae genes, and in turn unraveling the pathogenesis of this important swine pathogen.
This work was supported by a Longer and Larger (LoLa) grant from the Biotechnology and Biological Sciences Research Council (grant numbers BB/G020744/1, BB/G019177/1, BB/G019274/1 and BB/G018553/1), the UK Department for Environment, Food and Rural Affairs and Zoetis awarded to the Bacterial Respiratory Diseases of Pigs-1 Technology (BRaDP1T) consortium Additional file 1. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. The BRaDP1T Consortium comprises: Duncan J. Maskell, Alexander W. (Dan) Tucker, Sarah E. Peters, Lucy A. Weinert, Jinhong (Tracy) Wang, Shi-Lu Luan, Roy R. Chaudhuri (University of Cambridge); Andrew N. Rycroft, Gareth A. Maglennon, Dominic Matthews (Royal Veterinary College); Brendan W. Wren, Jon Cuccui, Vanessa Terra (London School of Hygiene and Tropical Medicine); and Paul R. Langford, Janine T. Bossé, Yanwen Li (Imperial College London). We thank David Lampe for the gift of the pET29b + C9 plasmid from which the hyperactive C9 mutant Himar1 transposase was derived. We thank Joel Renaudin for the gift of the pSRT2 plasmid.
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