WO2014174257A2 - Procédés - Google Patents

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WO2014174257A2
WO2014174257A2 PCT/GB2014/051220 GB2014051220W WO2014174257A2 WO 2014174257 A2 WO2014174257 A2 WO 2014174257A2 GB 2014051220 W GB2014051220 W GB 2014051220W WO 2014174257 A2 WO2014174257 A2 WO 2014174257A2
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hyopneumoniae
sequence
plasmid
gene
optionally
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WO2014174257A3 (fr
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Andrew Nelson RYCROFT
Gareth Adam MAGLENNON
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The Royal Veterinary College
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The Royal Veterinary College
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Priority claimed from GB201307241A external-priority patent/GB201307241D0/en
Priority claimed from GB201319245A external-priority patent/GB201319245D0/en
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies

Definitions

  • the present invention relates to genetic manipulation of Mycoplasma hyopneumoniae.
  • Mycoplasmas are a genus of bacteria that are characterised by their lack of a cell wall and a small genome size, and are considered to be the smallest free-living self- replicating organisms. As such, they are of considerable interest in synthetic biology (Gibson et al.). They are responsible for a number of important diseases in humans and animals.
  • Mycoplasma hyopneumoniae is the cause of enzootic pneumonia, a chronic swine respiratory disease of global economic importance to pig producers [1]. Partial control of M.
  • hyopneumoniae is achieved by a combination of management practices, antimicrobials and immunisation with commercially available vaccines [1]. Although current vaccines improve performance parameters and reduce the severity of lung lesions, there is a need for more effective vaccines, particularly in response to concerns regarding the current overuse of antimicrobials in production animals. Progress in understanding the molecular basis of M. hyopneumoniae pathogenesis is essential to improved vaccine development, but has been severely hampered by the lack of tools to genetically manipulate the organism [2]. Advances have been made with other mycoplasmas using transposon mutagenesis to generate random insertion mutants that can be screened for the expression of virulence factors using in vivo or in vitro model systems [3-9].
  • a few mycoplasmas have also been transformed with artificial plasmids that are capable of self-replication [10-14]. These plasmids contain the origin of replication (or/ ' C) of the Mycoplasma along with an antimicrobial resistance gene. Following the transformation of mycoplasmas, transformants expressing the resistance gene are selected for by addition of antimicrobial.
  • oriC plasmids have proven valuable in demonstrating the amenability of Mycoplasmas to genetic manipulation and in some cases they have been useful in promoting the generation of mutations in target genes by homologous recombination [1 1 , 12]. The ability to reproducibly transform M. hyopneumoniae has been a long anticipated, but until now, unachieved, development in the study of this important organism.
  • EP enzootic pneumonia
  • hyopneumoniae causes direct damage to epithelial cells by production of cytotoxic metabolites such as hydrogen peroxide (Razin et al., 1998).
  • cytotoxic metabolites such as hydrogen peroxide
  • a chronic inflammatory process ensues that is characterised histologically by hyperplasia of lymphoid follicles surrounding bronchioles and infiltration of mononuclear cells into peribronchiolar tissue and alveolar septae (Livingston et al., 1972; Rodriguez et al., 2004).
  • the chronic nature of disease may result from modulation of the host immune response by M.
  • M. hyopneumoniae infection can be associated with and exacerbated by co-infection with other pathogens involved in the Porcine Respiratory Disease Complex (PRDC) such as Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) and Porcine Circovirus type 2 (Thacker and Minion, 2012).
  • PRDC Porcine Respiratory Disease Complex
  • PRRSV Porcine Reproductive and Respiratory Syndrome Virus
  • Thacker and Minion, 2012 Porcine Circovirus type 2
  • M. hyopneumoniae a respiratory tract bacteria
  • Streptococcus suis a respiratory tract bacteria
  • Haemophilus parasuis a respiratory tract bacteria
  • Pasteurella multocida a respiratory tract bacteria
  • M. hyopneumoniae a respiratory tract bacteria
  • Studies of M. hyopneumoniae on pig herds are currently limited by a combination of management practices, anti-microbial medications and vaccination (Maes et al., 2008).
  • Commercial 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 (Maes et al., 1998; Maes et al., 1999; Maes et al., 2008).
  • current vaccines are not completely effective and do not prevent colonisation of the respiratory tract with M. hyopneumoniae or eliminate infection from the herd (Thacker et al.,
  • M. hyopneumoniae 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.
  • the Tn4001 transposon derived from Staphylococcus aureus has been utilised (Cao et al., 1994; Chopra-Dewasthaly et al., 2005; Dybvig et al., 2000; Hedreyda et al., 1993; Pour-El et al., 2002).
  • 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 (Mazurkiewicz et al., 2006), for example. 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, or in other vaccine approaches.
  • 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.
  • a first aspect of the invention provides a plasmid for transforming Mycoplasma hyopneumoniae comprising an or/ ' C region comprising both AT-rich regions from the predicted or/ ' C region of M. hyopneumoniae strain 232 [2], or comprising a variant thereof that retains the ability to act as an or/ ' C region in M. hyopneumoniae and has at least 90% sequence identity, optionally at least 95% or 98% sequence identity with a sequence comprising both AT-rich regions from the predicted or/ ' C region of M. hyopneumoniae 232.
  • the or/ ' C region of the plasmid may comprise both AT-rich regions as identified in Figure 1A. These AT-rich regions are considered to flank the DnaA gene.
  • the upstream AT-rich sequence is 208 bp in size and is considered to have the sequence TTTTTCTCCTTTT I I I I G CTTAAATTTAAGTTTTA GAA GTTATTTTAAG GTATTTTAAA AAAATATTGTTAAAATTTAAAAC CAAAATCAACTTTATTAAATGTG CTAAATAAAG TTG ATAAAATGTTTGCAAAAACATTTTTGTTGTTTTAAACAAAACAAATTGATTTAAAAATT ATACTACAAAATTAAAG G AAAATTTATAAA .
  • the down stream AT-rich sequence is 144 bp in size and is considered to have the sequence AAATATTAAACAG CTTTTTG GG C AAAAAGTACTATAAATTAAAGTG GTTTTTTATCTTT TCTACATTTCTACACCCTTATTATTTTTATTATTAAAACTAAATAAAAATTAACTAAATA TAATATAATTATTAAAGGTAAAAAT.
  • the or/ ' C region of the plasmid may comprise a sequence having at least 90, 95 or 98% sequence identity with the upstream AT-rich sequence indicated above, and/or having at least 90, 95 or 98% of the length of any such sequence (with any deletions preferably being at the ends of the sequence).
  • the or/ ' C region of the plasmid may comprise a sequence having at least 90, 95 or 98% sequence identity with the downstream AT-rich sequence indicated above, and/or having at least 90, 95 or 98% of the length of any such sequence (with any deletions preferably being at the ends of the sequence).
  • the or/ ' C region of the plasmid may comprise a sequence of about 700bp comprising both AT-rich regions as identified in Figure 1A.
  • the or/ ' C region of the plasmid may comprise the DnaA gene from M. hyopneumoniae or a sequence having at least 70, 80, 90, 95 or 98% sequence identity with the DnaA gene.
  • Percent (%) sequence identity will be well known to those skilled in the art, and is defined as the percentage of nucleic acid residues in a candidate sequence that are identical with the nucleic acid residues of the sequence of interest, after aligning the sequences and introducing gaps, if necessary to achieve maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity.
  • % identity values may be obtained for example, using available computer programs such as WU-BLAST-2 (Altschul ef a/., Methods in Enzvmoloav 266:460-480 (1996).
  • WU-BLAST-2 Altschul ef a/., Methods in Enzvmoloav 266:460-480 (1996).
  • DNAStar (Lasergene) may be used, though the other programs indicated are equally suitable.
  • the oriC region of the plasmid may comprise or consist of the following sequence, considered to be 2121 bp in length and to comprise the DnaA gene and both of the AT rich regions
  • the oriC region of the plasmid may comprise or consist of a variant thereof that retains the ability to act as an oriC region in M. hyopneumoniae and has at least 90, 95 or 98% sequence identity with said sequence. It is considered that other sequences considered (see the examples below) not to act as an oriC region have a lower % sequence identity than indicated above.
  • the ability of a variant sequence to act as an oriC region may be assessed by, for example, replacing the sequence known to act as an oriC region in M. hyopneumoniae with the variant sequence and determining its ability to act as an oriC region, for example by assessing whether transformed colonies are obtained on transforming M. hyopneumoniae (for example M. hyopneumoniae 232) with the plasmid containing the variant oriC region, for example under the same conditions as transformed colonies are obtained with a plasmid containing the sequence known to act as an oriC region.
  • M. hyopneumoniae for example M. hyopneumoniae 232
  • At least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% of the number of transformed colonies obtained with the plasmid containing the known oriC region are obtained with the plasmid containing the variant oriC region.
  • Transformed colonies may be identified using a marker such as tetracycline resistance (for example as described in the Examples), as will be well known to those skilled in the art.
  • the plasmid may further comprise a marker gene.
  • the marker gene may comprise a promoter derived from M. hyopneumoniae or the spiralin gene promoter of Spiroplasma citri[15], or the promoter from the tetM (tetracycline resistance) gene, for example from Staphylococcus aureus.
  • the promoter may be the P97 gene (1 B) or Idh gene (1 C) promoter, for example as shown in Figure 1 or indicated below; or a variant thereof that is capable of acting as a promoter in M. hyopneumoniae, for example M. hyopneumoniae 232 and has at least 90% sequence identity with the relevant promoter sequence, for example as shown in Figure 1 or indicated below.
  • the promoter may be a promoter comprising the motif TATAAT up to 200bp upstream of the native gene start codon.
  • the promoter may be a promoter comprising the motif TAAAAT up to 200bp upstream
  • promoter regions typically (but not always) have the motif TATAAT or TAAAAT within approximately 200 bp upstream of the start codon. This has been described in more detail by Weber Sde et al 2012 in DNA Research, for example. Suitable marker sequences will be well known to those skilled in the art.
  • the tetM resistance gene (conferring resistance to tetracycline) may be used, as may the pac (puromycin-N-acetyl-transferase) sequence (conferring resistance to puromycin).
  • pac puromycin-N-acetyl-transferase sequence
  • the pac gene has, for example, been utilised in the pMiniHimar1 BSC1 transposon mutagenesis system described in Example 6.
  • the teM gene is present in the pMHC9-1 transposon construct described in Example 3.
  • antimicrobial resistance cassettes examples include the cat (chloramphenicol acetyltransferase) sequence (conferring resistance to chloramphenicol) and the aacA-aphD determinant (conferring resistance to aminoglycosides such as gentamicin). Both of these resistance genes have been shown to be functional in conferring resistance to respective antimicrobials in other Mycoplasma spp.
  • the P97 gene promoter sequence is considered to be:
  • lactate dehydrogenase gene promoter sequence is considered to be:
  • lactate dehydrogenase gene and P97 ciliary adhesin gene promoter sequences from M. hyopneumoniae are functional in driving the expression of tetM in M. hyopneumoniae. See, for example, Example 1.
  • spiralin gene (from Spiroplasma citri) promoter sequence is active in driving tetM expression and pac expression in M. hyopneumoniae using the same system.
  • Example 3 we show that the P97 promoter sequence is a suitable promoter for controlling expression of the Himarl transposase enzyme.
  • Other predicted M. hyopneumoniae promoter sequences for example promoter sequences that are constitutively active (or variants thereof), could alternatively be used to drive expression of a marker gene or other gene, for example a transposase gene, for example the Himarl transposase gene, as discussed further below.
  • a marker gene or other gene for example a transposase gene, for example the Himarl transposase gene, as discussed further below.
  • seven further potential promoter sequences from the M. hyopneumoniae strain 232 genome are seven further potential promoter sequences from the M. hyopneumoniae strain 232 genome.
  • the promoter sequence may be, for example, a promoter sequence as shown below or a variant thereof that is capable of acting as a promoter in M. hyopneumoniae, for example M. hyopneumoniae 232 and has at least 90% sequence identity with the relevant promoter sequence, for example as shown in Figure 1 or indicated below.
  • Tuf (elongation factor) (682581-682880)
  • hyopneumoniae may be assessed by, for example, replacing the sequence known to act as a promoter in M. hyopneumoniae with the variant sequence and determining its ability to act as a promoter, for example by assessing whether marked colonies are obtained on transforming M. hyopneumoniae (for example M. hyopneumoniae 232) with the plasmid containing the variant promoter controlling a marker (for example tetracycline resistance), for example under the same conditions as marked colonies are obtained with a plasmid containing the sequence known to act as an promoter controlling the same marker.
  • a marker for example tetracycline resistance
  • the method may similarly be performed using a transposon-based system as described herein, though it may most conveniently be performed using the plasmid based system as indicated above.
  • the plasmid may, for example, comprise an epitope-tagged sequence under the control of a promoter sequence wherein the promoter sequence is active in M. hyopneumoniae, (or putative promoter sequence, as appropriate), where the epitope is optionally GPF, optionally hemagglutinin tag, his6 tag or other epitope which will be well known to those skilled in the art.
  • the plasmid may further (compared with any of the previous embodiments) comprise a sequence that can undergo recombination with the homologous DNA in the bacterial chromosome, thus causing disruption of the homologous gene at a specific site (site- directed mutagenesis).
  • the plasmid may further comprise a further sequence that has at least 80, 90, 95 or 98% identity over a sequence length of at least 100, more typically at least 300, 400 or 500 nucleotides, for example between 300, 400 or 500 and 3000, 2000 or 1500 nucleotides, for example between 500 and 1500 nucleotides, with a sequence of M. hyopneumoniae (for example M. hyopneumoniae 232) chromosomal nucleic acid.
  • M. hyopneumoniae for example M. hyopneumoniae 232
  • a further aspect of the invention provides a method of transforming M. hyopneumoniae, the method comprising the step of exposing a M. hyopneumoniae cell to a plasmid according to the first aspect of the invention.
  • the method may further comprise the step of performing electroporation on the cell in the presence of the plasmid according to the first aspect of the invention.
  • the method may further comprise the step of culturing the exposed (and optionally electroporated) cell under conditions where transformed cells have a selective growth advantage or otherwise can be identified.
  • a further aspect of the invention provides an M. hyopneumoniae cell transformed with a plasmid according to the first aspect of the invention.
  • the plasmid may remain as a separate plasmid within the cell or may be integrated or partially integrated into the cell's genomic DNA.
  • a further aspect of the invention provides a vaccine composition comprising a transformed M. hyopneumoniae cell of the invention.
  • the oriC based plasmid is considered to be able to, for example, introduce DNA into M. hyopneumoniae that could then undergo recombination with the homologous DNA in the bacterial chromosome, thus causing disruption of the homologous gene at a specific site (site-directed mutagenesis). See, for example, Examples 2 and 5.
  • Such a mutant could be useful as, for example, a live, attenuated vaccine for protection of pigs against porcine enzootic pneumonia.
  • the mutated/disrupted gene may be, for example, the gene for P46, P97 or P146 adhesion proteins, involved in cell adhesion.
  • Other suitable genes may be identified making use of the transformation techniques of the present invention, which may be used, for example, in identifying sequences involved in virulence of this pathogen.
  • suitable genes may be identified through formation of and analysis of mutant cells, for example as described further below, for example in Examples 2 to 5.
  • the transformed M. hyopneumoniae of the invention for example in a vaccine composition of the invention, may comprise a gene, for example the gene for P46, P97 or P146 adhesion proteins, or a gene identified as involved in virulence of M.
  • the transformed M. hyopneumoniae of the invention for example in a vaccine composition of the invention may be a live-attenuated cell e.g. a cell that has reduced ability to replicate and is not capable of causing disease in a normal (not immunocompromised) animal.
  • a cell is a M. hyopneumoniae cell, for example a M. hyopneumoniae 232 cell.
  • M. hyopneumoniae cell for example a M. hyopneumoniae 232 cell.
  • M. hyopneumoniae 232 for example M.
  • hyopneumoniae can be recognised making use of PCR-based techniques [16-18].
  • M. hyopneumoniae 232 can be recognised making use of whole genome DNA sequencing techniques [2]. Examples of techniques for identifying M. hyopneumoniae are also indicated in the Examples, for example Example 6.
  • a further aspect of the invention provides a plasmid, transformed M. hyopneumoniae cell or vaccine composition of the invention for use in medicine.
  • a further aspect of the invention provides a plasmid, transformed M. hyopneumoniae cell or vaccine composition of the invention for use in aiding in treating or preventing infection by M. hyopneumoniae or disease caused by infection by M. hyopneumoniae.
  • a further aspect of the invention provides a method for aiding in treating or preventing infection by M. hyopneumoniae or disease caused by infection by M. hyopneumoniae, the method comprising administering to a subject a transformed M.
  • hyopneumoniae cell typically a live, attenuated M. hyopneumoniae organism
  • vaccine composition typically comprising a live, attenuated M. hyopneumoniae organism
  • the vaccine would be used on the farm. It would typically be given by the respiratory route (intranasal or as a spray or aerosol) to piglets or to sows before farrowing. It is also possible that the vaccine could be given by a non-respiratory route such as intramuscular or subcutaneous injection.
  • the vaccine may be formulated or administered alongside other agents intended as vaccines (for example as part of a combination vaccine), as will be well known to those skilled in the art.
  • the vaccine may be formulated or administered with other pharmaceutically acceptable components, which may be considered to be adjuvant components, as will also be well known to those skilled in the art.
  • a further aspect of the invention comprises a method for identifying a sequence capable of acting as an or/ ' C region in M. hyopneumoniae the method comprising the step of comparing the ability of a candidate sequence to act as an or/ ' C region with the ability of a sequence comprising both AT-rich regions from the predicted or/ ' C region of M. hyopneumoniae 232 (for example as set out above) to act as an or/ ' C region in M. hyopneumoniae.
  • a sequence may be identified as capable of acting as an or/ ' C region if transformed colonies are obtained on transforming M.
  • hyopneumoniae for example M hyopneumoniae 232
  • the plasmid containing the candidate oriC region for example under the same conditions as transformed colonies are obtained with a corresponding plasmid containing the sequence known to act as an oriC region.
  • a corresponding plasmid containing the sequence known to act as an oriC region Preferably at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% of the number of transformed colonies obtained with the plasmid containing the known oriC region are obtained with the plasmid containing the candidate or/ ' C region.
  • a further aspect of the invention comprises a method for identifying a sequence capable of acting as a promoter in M. hyopneumoniae the method comprising the step of comparing the ability of a candidate sequence to act as a promoter with the ability of the spiralin gene promoter of Spiroplasma citri, promoter of the M. hyopneumoniae P97 gene or M. hyopneumoniae Idh gene, (or other promoter sequence active as a promoter in M. hyopneumoniae, for example a sequence indicated above as active as a promoter in M. hyopneumoniae) to act as a promoter in M. hyopneumoniae.
  • a sequence may be identified as capable of acting as a promoter if marked colonies are obtained on transforming M. hyopneumoniae (for example M. hyopneumoniae 232) with the plasmid (typically a plasmid comprising an oriC region as set out above) containing the candidate promoter controlling a marker (for example tetracycline resistance), for example under the same conditions as marked colonies are obtained with a corresponding plasmid containing the sequence known to act as an promoter controlling the same marker (for example a promoter and/or marker indicated above).
  • a marker for example tetracycline resistance
  • a further aspect of the invention comprises a method for identifying a sequence capable of acting as a selection marker in M. hyopneumoniae the method comprising the step of comparing the ability of a candidate sequence (for example with at least 90% sequence identity to a sequence known to confer resistance to a compound (challenge compound) in another organism), under the control of a promoter known to work in . hyopneumoniae, to confer resistance to a challenge compound, with the ability of a construct not containing the candidate sequence to confer resistance to the challenge compound.
  • a sequence may be identified as capable of acting as a marker if marked colonies are obtained on titrations of the challenge compound upon transforming M. hyopneumoniae (for example M.
  • the plasmid typically a plasmid comprising an or/ ' C region as set out above
  • the candidate marker under the control of a promoter (for example the spiralin gene promoter of Spiroplasma citri), for example under the same conditions as no marked colonies are obtained with a corresponding plasmid lacking the candidate marker sequence; or under similar conditions (which may be the same conditions, or may be conditions adapted for a different marker, for example using a different antibiotic) in which marked colonies are obtained with a known marker sequence.
  • a promoter for example the spiralin gene promoter of Spiroplasma citri
  • similar conditions which may be the same conditions, or may be conditions adapted for a different marker, for example using a different antibiotic
  • at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% of the number of marked colonies obtained with the plasmid containing a known marker sequence are obtained with the plasmid containing the candidate marker sequence.
  • a further aspect of the invention provides a nucleic acid construct comprising a nucleic acid sequence encoding a transposase enzyme and a promoter sequence, wherein the promoter sequence is active in M. hyopneumoniae;
  • the promoter sequence comprises a promoter sequence from hyopneumoniae, or spiralin gene promoter of Spiroplasma citri or tetM promoter sequence from S. aureus;
  • the promoter sequence from M. hyopneumoniae is a constitutively active promoter sequence, optionally a promoter sequence from the M. hyopneumoniae Idh, P97, secD, Tuf, rpoB, P146, ATP transporter ATP binding protein, Asparagine-tRNA synthetase or Translation elongation factor gene.
  • promoter sequences are indicated above.
  • the promoter sequence may be a variant thereof that is capable of acting as a promoter in M. hyopneumoniae, for example M. hyopneumoniae 232, and has at least 90% sequence identity with the relevant promoter sequence, for example as shown in Figure 1 or indicated above.
  • transposase-encoding sequence is under the control of the promoter sequence, as will be well known to those skilled in the art.
  • transposase and examples of transposases will also be well known to those skilled in the art. See, for example, discussion in Example 6, section 1.5.2 and references cited therein.
  • DNA transposons class II transposons
  • TIRs terminal inverted repeats
  • the families include Superfamily Tc1 /mariner, PIF ⁇ 'Harbinger, hAT, Mutator, Merlin, Transib, P, piggyBac and CACTA.
  • Active transposases of the TC1 /mariner family include Mos and Hima (Lampe et al (1999)). Others indicated in Munoz-Lopez and Garcia_Perez, 2010 include Tc1 , Tc3, Minos, Famarl, Osmar5, Fot1, Impala, ISY100, Mboumar-9, Sleeping Beauty, Frog Prince and Hsmarl. See also, for example, PLoS One. 2013 Sep 11 ;8(9):e73227. doi: 10.1371/journal.pone.0073227.
  • the transposase enzyme is a Mariner family transposase, optionally Himarl transposase or Himarl C9 mutant transposase.
  • a transposase is considered to be particularly useful in relation to Mycoplasma, for example M. hyopneumoniae, in view of its AT-rich genome and the insertion site of TcMmariner transposases, for example Himar 1 transposase or the C9 mutant, being at a TA dinucleotide.
  • the C9 transposase is a hyperactive mutant of Himarl with two amino acid substitutions, Q131 R and E137K, that are considered to result in an increase in activity relative to wild type.
  • variants are also included, for example with at least 90% amino acid sequence identity with the amino acid sequence of Himarl or Himarl C9 mutant and typically at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% of the mutant-forming activity of Himarl or Himarl C9 mutant, for example as judged by comparison of the number of independent transformed colonies (for example of M. hyopneumoniae) recovered with the Himarl or Himar 1 C9 mutant sequence and with the mutated Himarl or Himar 1 C9 sequence, for example as set out in the Examples.
  • the coding sequence of the Himarl gene is;
  • the amino acid sequence of the Himarl transposase is considered to be; MEKKEFRVLIKYCFLKGKNTVEAKTWLDNEFPDSAPGKSTIIDWYAKFKRGEMSTEDGE RSGRPKEWTDENIKKIHKMILNDRKMKLIEIAEALKISKERVGHIIHQYLDMRKLCAKVW PRELTFDQKQQRVDDSERCLQLLTRNTPEFFRRYVTMDETWLHHYTPESNRQSAEWT ATGEPSPKRGKTQKSAGKVMASVFWDAHGIIFIDYLEKGKTINSDYYMALLERLKVEIAA KRPHMKKKKVLFHQDNAPCHKSLRTMAKIHELGFELLPHPPYSPDLAPSDFFLFSDLKR MLAGKKFGCNEEVIAETEAYFEAKPKEYYQNGIKKLEGRYNRCIALEGNYVE
  • the coding sequence of the Himarl C9 mutant gene is considered to be; ATG G AAAAAAAG G AATTTCGTGTTTTG ATAAAATACTGTTTTC TG AAG G G AAAAAATA CAGTGGAAGCAAAAACTTGGCTTGATAATGAGTTTCCGGACTCTGCCCCAGGGAAA TCAAC AATAATTG ATTGGTATG CAAAATTC AAGC GTG GTG AAATG AGC AC G GAG G AC GGTGAACGCAGTGGACGCCCGAAAGAGGTGGTTACCGACGAAAACATCAAAAAAAT C C AC AAAATG ATTTTGAATG AC C GTAAAATG AAGTTG ATCG AG ATAG C AG AG AG GC CTT AAAGATATCAAAGGAACGTGTTGGTCATATCATTCATCAATATTTGGATATGCGGAA GCTCTGTGCGAAATGGGTGCCGCGCGAGCTCACATTTGACCAAAAACAACGACGTG TTGATGATTCTAAGCGGTGTTTGCAGCTGTTAACTCGTAATACACCCGAGTTT
  • the amino acid sequence of the Himarl C9 mutant transposase is considered to be;
  • transposase proteins from different Tc1 /mariner elements are not very similar in sequence, but they have two characteristic domains: an amino-terminal region containing the helix-turn-helix (HTH) motif necessary for recognition and binding of TIRs, and a carboxy-terminal domain harbouring the catalytic motif formed by three amino acids DDD ⁇ mariner family) or DDE (Tc1 -like family).
  • the first and second D residues are typically separated by 92 amino acids, whereas the separation of the second and third residues is variable, between 31 and 39 amino acids in the different families forming the superfamily TcMmariner.
  • Other features include a nuclear localisation signal (NLS) and a WVPHEL linker motif, which may participate in the interaction between transposase monomers. Typically the transposase will have one or more or all such features.
  • the Hsmarl transposase (reported in Claeys et al 2010, Nuc Acids Res and also a Mariner family transposase) may also be suitable. It is considered that the Tn4001 transposase (which is a member of the Mutator family) may not be functional in M. hyopneumoniae. Thus, the transposase may be a transposase other than a Mutator family transposase; or other than the 4001 transposase. The sequence of the Tn4001 transposase may be found at http://www.uniprot.org/uniprot/P19775, for example.
  • the transposase Tn916 is functional in Mycoplasma species.
  • the large size and tendency to insert into hotspots in the genome make Tn916 less preferred in relation to the current invention, as it may be difficult to achieve even coverage or saturation of the genome with mutant.
  • the transposase may be a transposase other than the Tn916 transposase.
  • the nucleic acid construct (which may typically be circular but which may be, for example, linear) may further comprise other transposon/mini transposon components, for example may comprise (two) inverted repeats and optionally a marker gene, optionally comprising a promoter sequence active in M, hyopneumoniae,
  • the promoter sequence comprises a promoter sequence from M. hyopneumoniae, or spiralin gene promoter of Spiroplasma citri or tetM promoter sequence from S. aureus ⁇ as discussed above;
  • the promoter sequence from M. hyopneumoniae is a constitutively active promoter sequence, optionally a promoter sequence from the M. hyopneumoniae Idh, P97, secD, Tuf, rpoB, P146, ATP transporter ATP binding protein, Asparagine-tRNA synthetase or Translation elongation factor gene (as discussed above, including variants).
  • a further nucleic acid construct may be used alongside the nucleic acid construct comprising a sequence encoding a transposase.
  • the further nucleic acid construct may comprise transposon/mini transposon components, for example may comprise (two) inverted repeats and optionally a marker gene, optionally comprising a promoter sequence active in M. hyopneumoniae, optionally wherein the promoter sequence comprises a promoter sequence from M. hyopneumoniae, or spiralin gene promoter of Spiroplasma citri or tetM promoter sequence from S. aureus;
  • the promoter sequence from M. hyopneumoniae is a constitutively active promoter sequence, optionally a promoter sequence from the M. hyopneumoniae Idh, P97, secD, Tuf, rpoB, P1 6, ATP transporter ATP binding protein, Asparagine-tRNA synthetase or Translation elongation factor gene (as discussed above, including variants).
  • Tc1 /mariner terminal inverted repeats may typically be between 17 and 1100bp in length, for example between 19 and 50bp in length. Requirements for TIRs are discussed further in, for example,
  • sequence considered to be the one unit of the repeat sequence may be or comprise; TAACAGGTTGGCTGATAAGTCCCCGGTCT
  • Both repeats of the inverted repeat may have this 29bp sequence, which is based on the sequence described by Lampe et al (1996).
  • the repeat sequence may alternatively be
  • the marker gene may be a selectable marker gene, optionally an antibiotic resistance gene, optionally a gene conferring resistance to puromycin (optionally puromycin-N-acetyl-transferase), tetracycline (optionally tetM), chloramphenicol (optionally chloramphenicol acetyltransferase) or aminoglycosides such as gentamicin (optionally aacA-aphD). Marker sequences are also discussed above.
  • the marker gene is located between the inverted repeats, for example such that the marker gene sequence would be expected to be inserted into the chromosomal nucleic acid by the action of the transposase on the construct comprising the inverted repeats.
  • the inverted repeats typically flank the marker gene in a 5'-3' direction towards the marker gene. The sequence within the construct would therefore read (for example); TAACAGGTTGGCTGATAAGTCCCCGGTCT - promoter - marker gene - AGACCGGGGACTTATCAGCCAACCTGTTA
  • transposon and transposase into the cell on the same or different nucleic acid construct(s), for example DNA (or modified DNA) construct(s).
  • DNA or modified DNA construct(s).
  • the transposon and genome are "mixed” together in vitro along with tranpsosase enzyme and tranposition occurs.
  • the mutated genomes are then transferred back into mycoplasmas and the genome is mutated by allelic exchange.
  • the nucleic acid construct typically is a DNA construct, as will be apparent to those skilled in the art. It may alternatively be a synthetic or modified nucleic acid construct, as also well known to those skilled in the art. It is not considered that there are any special requirements for the form of the nucleic acid constructs that may be used for transforming M. hyopneumoniae or other Mycoplasma.
  • a further aspect of the invention provides a M. hyopneumoniae or other Mycoplasma cell transformed with or comprising a construct or constructs of the invention (for example a nucleic acid construct comprising a transposase-encoding sequence under the control of a promoter active in M. hyopneumoniae; and the same or a different construct comprising inverted repeats and optionally a marker gene comprising a promoter active in M. hyopneumoniae).
  • the cell may comprise the construct or constructs as extrachromosomal constructs, or at least the inverted repeat sequence (and typically marker gene sequence, if present) may be integrated into the chromosomal nucleic acid.
  • the plasmid constructs typically may be propagated in E.coli strain DH5a (although other competent strains would suffice). It is considered that the transposase promoter is active in E. coli but this does not appear to interfere with propagation.
  • Methods for transforming M. hypopneumoniae cells are indicated above and discussed in the Examples. For example, electroporation may be used. Factors such as growth phase and cell density may affect the efficiency of transformation, as noted in the Examples. Known techniques for transforming other Mycoplasma cells may be used with other Mycoplasma cell types; or methods as described herein for M. hyopneumoniae may be used, for example electroporation.
  • a further aspect of the invention provides a method for forming a collection of mutant M. hyopneumoniae or other Mycoplasma cells, the method comprising the steps of exposing a sample or samples of M. hyopneumoniae or other Mycoplasma cells to multiple copies of a construct encoding a transposase, optionally a construct of the invention comprising a sequence encoding a transposase and a promoter sequence active in M. hyopneumoniae; and a construct (separate or otherwise) comprising inverted repeats and optionally a marker gene optionally comprising a promoter sequence active in M. hyopneumoniae; and
  • the construct encoding the transposase is a construct in which transcription of the sequence encoding the transposase is under the control of a promoter sequence active in M. hyopneumoniae.
  • said construct or constructs lead to the insertion of the sequence in the construct (for example transposon vector construct) between the inverted repeats into the chromosomal nucleic acid. It is expected that the insertion occurs at TA dinucleotides (typically when a Tc1 /mariner transposase is used, for example a Himarl or Himarl C9 mutant transposase). Thus, a collection of cells with typically differently mutated chromosomal sequence can be generated.
  • a further aspect of the invention provides a method for forming a collection of mutant M. hyopneumoniae or other Mycoplasma cells, the method comprising the steps of exposing a sample or samples of M. hyopneumoniae or other Mycoplasma nucleic acid (for example genomic nucleic acid, for example genomic nucleic acid intended to be complete) to a transposase, optionally a Mariner family transposase, optionally Himarl or Himarl C9 mutant; and to a construct comprising inverted repeats and optionally a marker gene optionally comprising a promoter sequence active in M. hyopneumoniae; and transforming M. hyopneumoniae or other Mycoplasma cells with the exposed nucleic acid; optionally incubating under conditions where allelic exchange is expected to occur; and optionally culturing the putative transformed cells under conditions such that transformed cells can be detected.
  • a further aspect of the invention provides a collection of M. hyopneumoniae or other Mycoplasma cells transformed with or comprising a construct or constructs according to the invention encoding a transposase, optionally a construct of the invention comprising a sequence encoding a transposase and a promoter sequence active in M. hyopneumoniae; and (in a separate construct or otherwise) comprising inverted repeats and optionally a marker gene optionally comprising a promoter sequence active in M. hyopneumoniae;
  • the collection comprises at least 10, 20, 50, 100, 200, 400, 500, 800 or 1000 different transformants.
  • the transformants comprise the genomic insertion at a TA dinucleotide of the inverted repeats and the intervening DNA, optionally a marker gene, optionally comprising a promoter sequence active in M. hyopneumoniae (for example as discussed above),
  • the promoter sequence comprises a promoter sequence from M. hyopneumoniae, or spiralin gene promoter of Spiroplasma citri or tetM promoter sequence from S. aureus ⁇ as discussed above;
  • the promoter sequence from M. hyopneumoniae is a constitutively active promoter sequence, optionally a promoter sequence from the M. hyopneumoniae Idh, P97, secD, Tuf, rpoB, P146, ATP transporter ATP binding protein, Asparagine-tRNA synthetase or Translation elongation factor gene (as discussed above, including variants);
  • the marker gene is optionally an antibiotic resistance gene, optionally a gene conferring resistance to puromycin (optionally puromycin-N-acetyl-transferase), tetracycline (optionally tetM), chloramphenicol (optionally chloramphenicol acetyltransferase) or aminoglycosides such as gentamicin (optionally aacA-aphD), optionally any gene conferring resistance to a compound as identified from the invention..
  • a further aspect of the invention provides a method for analysing gene function in M. hyopneumoniae or another Mycoplasma, the method comprising the step of providing a collection of M. hyopneumoniae or other Mycoplasma transformants according to the preceding aspect of the invention;
  • the method may further comprise the steps of culturing a sample of the collection of transformants under selective conditions, for example in vitro or in vivo, and identifying transformants, and the location of their insertion, that have a selective advantage or selective disadvantage under those selective conditions, for example using techniques such as Signature Tagged Mutagenesis (STM) or TraDIS, as known to those skilled in the art and discussed further in the Examples.
  • STM Signature Tagged Mutagenesis
  • TraDIS TraDIS
  • a further aspect of the invention provides a vaccine composition of the invention comprising a gene that has undergone site-directed mutagenesis as a consequence of transformation with a plasmid according to any one of claims 1 to 6, wherein the gene is a gene that has been identified by a screening method of the invention.
  • a further aspect of the invention provides a vaccine composition comprising a M. hyopneumoniae cell transformed with a transposon system constructs of the invention.
  • the transformed M. hyopneumoniae cell may have been identified by a screening method of the invention.
  • a further aspect of the invention provides a vaccine composition
  • a vaccine composition comprising a polypeptide encoded at least in part by a coding portion of a gene (for example at least 21 nucleotides, for example at least 30, 39, 45 or 60 nucleotides of the coding portion of the gene) that has been identified by a screening method according to the invention.
  • the polypeptide is not the wild-type M. hyopneumoniae polypeptide.
  • the polypeptide may be mutated or truncated relative to the wild-type M. hyopneumoniae polypeptide.
  • a further aspect of the invention provides a plasmid of the invention typically comprising a marker sequence, wherein the plasmid further comprises a nucleic acid sequence with the ability to undergo homologous recombination with the Mycoplasma hyopneumoniae chromosome wherein the nucleic acid sequence with the ability to undergo homologous recombination may comprise a sequence that has at least 80, 90, 95 or 98% identity over a sequence length of at least 100, more typically at least 300, 400 or 500 nucleotides, for example between 300, 400, 500, 600, 700, 800, 900 and 3000, 2000 or 1500 nucleotides, for example between 500 and 1500 nucleotides, for example between 700 and 1500 nucleotides with a sequence of M.
  • hyopneumoniae for example M. hyopneumoniae 232
  • the said sequence with the ability to undergo homologous recombination with the M. hyopneumoniae chromosome nucleic acid is not a sequence otherwise required in the plasmid, for example the Or/C sequence, nor any promoter sequence otherwise required in the plasmid. It may be desirable to use a promoter sequence, for example, in the plasmid that is not naturally present in wild-type M. hyopneumoniae in order to avoid or minimise the possibility of unwanted homologous recombination through this sequence.
  • the plasmid may comprises two nucleic acid sequences with the ability to undergo homologous recombination with the Mycoplasma hyopneumoniae chromosome as defined in the preceding aspect of the invention, separated by a marker sequence, optionally a selectable marker gene flanked by recombinase recognition sites, optionally FRT flip recombinase recognition sites or, for example, Cre-LoxP system recognition sites.
  • the FRT recognition sequences are discussed in Example 2 below, for example.
  • a LoxP recognition site for Cre recombinase is considered to be, for example, ATAACTTC GTATA - NNNTANNN - TATACGAAGTTAT. This is a 34 bp recognition site that flanks the DNA region to be 'excised'
  • the 34 bp sequence consists of 13 bp regions flanked by an 8 bp spacer with variable nucleotide sequence (N).
  • a further aspect of the invention provides a system comprising a plasmid according to the preceding aspect of the invention and a further plasmid of the invention comprising a marker sequence, wherein the marker sequence in the first said plasmid is different from the marker sequence in the further plasmid, optionally wherein the marker sequence in the first plasmid confers resistance to a different challenge compound to the marker sequence in the further plasmid; and wherein optionally the further plasmid comprises a sequence encoding a recombinase, optionally a flip recombinase, optionally under the control of a promoter active in M. hyopneumoniae.
  • Such a system may be useful in generating transformed cells with mutated genomic nucleic acid that does not include a marker sequence, for example does not include an antibiotic resistance gene.
  • a system may be useful in generating M. hyopneumoniae cells with one or more gene disruptions, mutations or fusions.
  • Such cells may be useful for research purposes or as vaccine candidates. Further information is provided in Example 2 below, for example further information on recombinases and recombination sequences, which will in any case be well known to those skilled in the art.
  • a further aspect of the invention provides a M. hyopneumoniae cell transformed with a plasmid or a system of the preceding two aspects of the invention.
  • the M. hyopneumoniae cell may have genomic nucleic acid which comprises a marker sequence, optionally a selectable marker sequence, as discussed above, flanked by recombinase recognition sites, optionally FRT flip recombinase recognition sites or Cre- LoxP recognition sites; or wherein the genomic nucleic acid of the cell comprises a recombinase recognition site. It is considered that a "scar" is left following recombination, where the scar may be recognised by the presence of the FRT (or other) recognition site - one FRT (or other) site is considered to remain in the chromosome following resolution with flip recombinase (for example).
  • a further aspect of the invention provides a plasmid of the invention wherein the plasmid further comprises a nucleic acid sequence encoding an epitope tag in-frame upstream or downstream of a sequence with the ability to undergo homologous recombination (as discussed above), such that following homologous recombination the epitope tag is expected to be expressed in-frame with the target protein.
  • a plasmid may be useful in generating a transformed Mycoplasma, for example M. hyopneumoniae cell, in which it is expected that the tagged target protein is expressed, which may be useful when investigating gene function, for example.
  • a further aspect of the invention provides a method for expressing an epitope tagged or other fusion protein coding sequences in M. hyopneumoniae the method comprising the steps of introducing a nucleotide sequence downstream of a promoter that functions in M. hyopneumoniae consisting of a epitope coding sequence (or other fusion sequence) inframe with a protein coding sequence in the plasmid according to the invention.
  • a 2.1 kbp or/ ' C region of M. hyopneumoniae 232 was predicted based on the location of putative DnaA boxes (black arrowheads), the location of the dnaA gene and the presence of short AT-rich regions of 144 bp and 208 bp with an AT content of 0.83 and 0.84 respectively (1A).
  • the P97 gene (1 B) and Idh gene (1 C) promoter sequences were predicted based on proximity to the ATG start codon (shown in bold) and putative TATA box location (underlined).
  • the oriC sequence was cloned into the Ncol and Spel restriction sites of pGEM-T to generate plasmid pMHO. The tetM gene with the E.
  • M. hyopneumoniae 232 was transformed with each plasmid and transformants (pMHO-2 in 1 E) grown in Friis medium with tetracycline selection. After 3 passages, plasmid DNA was extracted from two individual clones (C1 and C2) and analysed by agarose gel electrophoresis along with plasmid (P) DNA control (1 F).
  • the predicted or/ ' C region of M. hyopneumoniae 232 is shown with the location of two putative DnaA boxes (black arrowheads) lying within AT-rich intergenic regions (grey boxes).
  • the or/ ' C region from plasmid pMHO (or/C-i) was reduced in a series of steps to produce five further plasmids (por/ ' C-ii, por/ ' C-iii, por/ ' C-iv, por/C-iii/iv and por/C-iv/v).
  • hyopneumoniae 232 was transformed in triplicate with each plasmid and the mean transformation frequencies (TF) are shown, expressed as the number of transformants per colony forming unit of mycoplasma transformed.
  • Figure 3 Individual transformants generated with four plasmids containing various oriC regions were passaged up to 19 times in Friis medium containing tetracycline (3A). Genomic DNA was extracted from cultures and analysed by Southern hybridization using a probe annealing to the bla ampicillin resistance gene. For each plasmid, there was no decline in extrachromosomal DNA over time, and plasmid DNA could still be readily detected after 19 passages.
  • Plasmid pTn4001-RVC1 was constructed by replacing the pac gene of pMiniTn4001 PsPuro with tetM from plasmid pSRT2 (1A).
  • Plasmid pMiniHimar1 BSC1 contains a mini transposon incorporating pac conferring resistance to puromycin and the wild-type Himarl transposase downstream of the Tn4001 native promoter sequence (1 B) (Cook, 2013).
  • the pac in pMiniHimar1 BSC1 has been replaced with tetM from pSRT2 (1 C).
  • the promoter sequence of the M In plasmid pMHWT-1 , the promoter sequence of the M.
  • hyopneumoniae strain 232 P97 gene has been placed upstream of the wild-type Himarl gene (1 D).
  • the wild-type Himarl gene has been replaced with the hyperactive C9 mutant version of the gene (1 E) (Lampe et al., 1999).
  • M. hyopneumoniae strain 232 was transformed with plasmid pMHC9-1 and within 14 days, tetracycline resistant colonies formed on Friis agar. No colonies were observed on 'no DNA' control transformations. Individual colonies exhibited a similar appearance to non-transformed M. hyopneumoniae colonies.
  • Figure 6
  • M. hyopneumoniae strain 232 was transformed with pMHC9-1. PCR was performed on extracted total DNA from 15 individual transformants (lanes 1-15) for identification of the tetM gene (6A) and the pGEM-T plasmid backbone (6B). A further 1 1 transformants underwent a further two rounds of 'colony purification'. Total DNA was extracted and linker PCR performed using two primer pairs at either end of the transposon (left and right) (6C). The same 11 transformants were passaged 15 times in Friis medium without selection, and all subsequently retained their resistance to tetracycline. Linker PCR was repeated using the same two primer pairs (left and right) (62D). Control samples for PCR reactions included: 'no template' control (NT); untransformed M. hyopneumoniae strain 232 (NC); plasmid DNA positive control (PC).
  • NT 'no template' control
  • NC untransformed M. hyopneumonia
  • Transposon insertion sites were determined by linker PCR and sequencing of PCR products for 44 individual mutants. The location of these insertion sites in the M. hyopneumoniae strain 232 genome is depicted by lines emanating from the circular genome (drawn to scale). Thicker lines indicate the presence of more than one insertion at a given location.
  • Plasmid pOriCP146 was generated by cloning a 974 bp fragment of the M. hyopneumoniae 232 ORF into the Mlu ⁇ and Pst ⁇ restriction enzyme cut sites of pOriC- iv/v.
  • the 974 bp fragment was located a short distance from the promoter region (Pr) and start codon (ATG) of the P146 gene (9A).
  • the plasmid was designed such that a single cross over homologous recombination event would result in integration of the entire plasmid into the chromosome of the organism and causing disruption of the P146 open reading frame.
  • Figure 10
  • Primer sets A and B were designed to amplify DNA at either end of the plasmid/chromosome integration site.(C). Following 19 passages of with tetracycline and then a further 6 passages without tetracycline (to promote further loss of extrachromosomal plasmid DNA), PCR was performed using primer sets A (D) and B (E) on total DNA extracts from mycoplasma cultures transformed with pOriCP146.
  • Plasmid pOriC379 was designed to allow targeted gene disruption in the ORF of mhp379 following plasmid integration by homologous recombination (A). Following successful transformation of M. hyopneumoniae 232 with pOriC379, extrachromosomal plasmid DNA could be detected in five clones following 19 serial passages in tetracycline, similar to plasmid DNA derived from E. coli (P) but not in untransformed M. hyopneumoniae 232 (232) (B).
  • Plasmid pOriCpepF was designed to promote targeted integration within the pepF open reading frame following a homologous recombination event (A). Following 19 passages of six randomly selected pOriCpepF transformed clones, extrachromosomal plasmid DNA was readily detected in clone 2 and a feint plasmid band was also detected in clone 1 (B). No extrachromosomal plasmid DNA was detected in clones 3-6 or in untransformed mycoplasma cells (232). Similar findings were apparent following an additional six passages without tetracycline selection pressure (C).
  • the loss of protein band is easier to discern in enhanced image ( Figures 13b and c).
  • Example 1 Development of a self-replicating plasmid system for Mycoplasma hyopneumoniae
  • Mycoplasma hyopneumoniae is a prevalent swine respiratory pathogen that is a major cause of economic loss to pig producers.
  • Current vaccines only offer partial control and there is a need for improved preventative strategies.
  • a major barrier to advances in understanding the pathogenesis of M. hyopneumoniae and in developing new vaccines is the lack of tools to genetically manipulate the organism.
  • the Or/ ' C of M. hyopneumoniae strain 232 [2] was predicted based on homology with other Mycoplasma species for which a functional Or/ ' C has been identified [11 , 12, 14].
  • the Or/ ' C encompasses short AT-rich regions flanking the dnaA gene and containing DnaA boxes.
  • a search of the M. hyopneumoniae 232 genome [2] revealed single DnaA boxes (each with two mismatches with the E. coli consensus sequence 5'- TTATCCACA-3') upstream and downstream of the dnaA gene within AT-rich regions (figure 1A).
  • Plasmid pMHO (figure 1 D) was generated by cloning a ⁇ 2.1 kbp sequence (indicated above and in the claims) into pGEM-T (Promega Ltd, UK), encompassing the dnaA gene and the flanking AT-rich regions.
  • the tetM gene (conferring tetracycline resistance) was chosen as a selection marker given successful use with other Mycoplasma species [10, 12].
  • the tetM gene and promoter from Enterococcus faecalis were amplified from plasmid plVT-1 [5] and cloned into pMHO to produce pMHO-1. M.
  • hyopneumoniae 232 [2] was grown in Friis medium (see Supplementary Files) at 37 °C in a static incubator in airtight culture tubes until late logarithmic phase ( ⁇ 10 8 CFU/ml), as determined by an acid colour change in the phenol red pH indicator.
  • Cells were collected by centrifugation (9000 xg at 4°C for 10 minutes) and washed three times in electroporation buffer (272 mM sucrose, 8 mM HEPES, pH 7.4). After the final wash, 100 ⁇ cells ( ⁇ 2 ml_ of mycoplasma culture) were incubated on ice with 10 ⁇ g plasmid DNA for 30 minutes.
  • Electroporation (2.5 kV, 100 ⁇ and 25 ⁇ ) was performed in a 0.2 cm cuvette using a Gene Pulser system (Bio-Rad Ltd, UK) and 900 ⁇ _ chilled Friis medium immediately added. Cells were incubated for 3 hours at 37 °C and then plated onto Friis agar containing 2 ⁇ g/mL tetracycline hydrochloride (Sigma-Aldrich Ltd, UK). Triplicate repeats were performed for all transformations, and after 14 days incubation at 37 °C in 5% C0 2 , no growth was observed for pMHO-1 or 'no DNA' controls. We considered that the tetM promoter sequence may not be active in M.
  • tetM gene of pMHO-1 was replaced with the tetM gene from plasmid pSRT2 [1 1] to produce pMHO-2 (figure 1 D). This utilises the spiralin gene promoter of Spiroplasma citri [15] that has be shown to be active in several Mycoplasma species [1 1 , 12]. Additionally, the predicted promoter regions of the M.
  • hyopneumoniae 232 P97 ciliary adhesin gene [19] (619bp) and lactate dehydrogenase ⁇ Idh) gene (421 bp) were placed upstream of the tetM gene to produce plasmids pMHO-3 and pMHO-4 respectively (figure 1 D).
  • the MIC for tetracycline was found to be 0.03 ⁇ g/mL in liquid medium and 0.015 ⁇ g/mL in solid medium [20].
  • hyopneumoniae 232 was transformed with each plasmid and plated onto Friis agar containing a low concentration of tetracycline (0.2 ⁇ g/mL). After 14 days incubation, tetracycline resistant colonies were present for all four oriC plasmids but not for the 'no DNA' control, demonstrating activity of the four promoter sequences. Morphologically, colonies were identical to untransformed M. hyopneumoniae colonies, but colony growth lagged approximately 3 days behind (figure 1 E).
  • the highest number of transformants was generated using pMHO-2 (2.5x10 4 /CFU) with fewer for pMHO-3 (5.8x10 5 /CFU) and pMHO-1 (1.7x10 "5 /CFU) and least with pMHO-4 (8.3x10 "6 /CFU).
  • Individual colonies were picked from agar and grown in Friis medium containing 0.5 ⁇ g/mL tetracycline, after which they were sub-cultured every 2-3 days in 1 ⁇ g/mL tetracycline. After three serial passages, plasmid DNA was extracted from 10ml culture using a PureLink Plasmid Mini kit (Invitrogen Ltd, UK) and separated by electrophoresis on 1% agarose.
  • Electroporation voltage was found to have a profound effect on transformation frequency; decreasing voltage to 2kV resulted in a 10-fold drop in transformation frequency, and below 2kV no transformants were obtained (data not shown).
  • M. hyorhinis [21], M. pulmonis [22], M. conjunctivae [23] and M. ovipneumoniae [24] exhibited a similar arrangement of genes in the or/ ' C region to M. hyopneumoniae, with AT-rich regions and DnaA boxes in the intergenic regions flanking the dnaA gene.
  • the PCR-amplified oriC regions were used to replace the M. hyopneumoniae 232 or/ ' C in plasmid pMHO-2.
  • M. hyopneumoniae 232 cells were electroporated in triplicate with each of the plasmids in two separate experiments and plated out onto Friis agar containing 0.2 ⁇ 9/ ⁇ _ tetracycline. As expected, tetracycline resistant colonies were obtained with the pHMO-2 plasmid within 10 days. However, after 14 days, no colonies were visible in any culture transformed with plasmids containing the or/ ' C regions of the other Mycoplasma species.
  • pOr/C-iii and pOr/C-iv containing the upstream and downstream intergenic regions respectively.
  • pOr/C-iii contained the AT-rich region upstream of the dnaA gene, and also extended downstream into the dnaA gene to include three putative DnaA boxes. Transformation of M. hyopneumoniae 232 failed to produce any transformants, showing that each AT-rich region could not function as an oriC independently.
  • hyopneumoniae 232 control (figure 3A). Additionally, we did not observe a reduction in plasmid DNA quantity that may be suggestive of plasmid integration into the M. hyopneumoniae genome by homologous recombination.
  • hyopneumoniae genome would generate a 4.6 kbp Hindlll digestion fragment that would be detected by a DIG-labeled probe annealing to the tetM gene.
  • a DIG-labeled probe annealing to the tetM gene.
  • M. hyopneumoniae 232 Transformation of M. hyopneumoniae 232 with pMHOpuro resulted in a transformation frequency of 1.exlO ⁇ /CFU compared to 4.6x10 5 for plasmid pMHO-2 (containing the tetM gene).
  • puromycin may be a suitable selection marker for M. hyopneumoniae isolates that are resistant to tetracycline.
  • M. hyopneumoniae The inability to genetically manipulate M. hyopneumoniae has been a major obstacle to progress in understanding the pathogenesis of the organism.
  • simple artificial self- replicating plasmids containing the or/ ' C of M. hyopneumoniae and an antimicrobial resistance gene we have shown for the first time that the organism is susceptible to transformation.
  • the transformation conditions optimized by Hedreyda et al and used widely in other Mycoplasmas are suitable for M. hyopneumoniae [7].
  • other important factors include titration of the tetracycline concentration to a level that can readily be overcome by the tetM resistance protein and use of a large excess of plasmid DNA.
  • the choice of promoter affected the expression of the tetM gene, and as is the case with other Mycoplasmas, the spiralin gene promoter of Spiroplasma citri is a suitable choice for M. hyopneumoniae.
  • Our oriC system allowed us to assess the function of putative M. hyopneumoniae promoter sequences, and confirmed the identity of two promoter sequences that were active in controlling tetM expression. The P97 promoter sequence was recently predicted and we have verified that this is functional in our plasmid system [19]. Using the criteria identified by Weber Sde et al [19], we also predicted the location of the Idh promoter, and again demonstrated it's activity in our plasmid system.
  • M. hyopneumoniae specific promoters will be valuable in the generation of further tools for the genetic manipulation of the organism.
  • the origin of replication of M. hyopneumoniae lies in the region of the dnaA gene and includes two short AT-rich regions. It has been suspected that the or/ ' C of M. hyopneumoniae lies in this region, but unusually within the AT-rich regions there are only two DnaA boxes that resemble the £. coli consensus sequence, whereas for other Mycoplasma species, there are typically multiple DnaA boxes [2]. Additional DnaA boxes are present within the dnaA gene, but these are not required for a functional or/ ' C.
  • oriC regions of Mycoplasmas showing a high degree of homology to M. hyopneumoniae would function in M. hyopneumoniae, and enable stable plasmid maintenance without integration by homologous recombination.
  • oriCs are very specific to their host species and none of the or/ ' C regions from four closely related Mycoplasma species functioned in M. hyopneumoniae.
  • the minimum M. hyopneumoniae sequence that functioned in an or/ ' C plasmid was ⁇ 700bp in length and included both AT-rich regions.
  • This plasmid was maintained extrachromosomally over at least 19 passages with selection pressure, equivalent to over 8 weeks post-transformation. However, when selection pressure was removed, tetracycline resistance was rapidly lost within 5 passages. Having demonstrated that plasmids carrying a high degree of homology with M. hyopneumoniae can undergo integration by homologous recombination, it is likely that this plasmid will be useful in generating targeted disruptions in genes of interest. By incorporating sequences encoding mycoplasma genes of interest, it is likely that we will be able to promote targeted homologous recombination and disruption of these genes, as has been shown with other Mycoplasmas [1 1 , 12]. In summary, we have shown that M.
  • hyopneumoniae is indeed susceptible to genetic manipulation by transformation with self-replicating plasmids. In doing so, we have developed a system that we consider is useful in genetic studies of M. hyopneumoniae. Additionally, we consider that our findings will allow development of a transposon- mediated system to generate random mutants of M. hyopneumoniae, which will take us a step closer to unraveling the pathogenic mechanisms of this organism.
  • PCR was performed using the Phusion High Fidelity kit (NEB Ltd, UK) according to the manufacturers instructions. Oligonucleotide sequences used in PCR reactions are shown in table 1. Molecular cloning was performed using standard methods as described by Sambrook and Russell [25]. For cloning, E. Coli strain DH5a was grown in Luria-Bertani medium with 100 ⁇ g/ml ampicillin.
  • Oligonucleotide sequences used in this study are shown along with the size of the PCR product generated and the template DNA used. Restriction enzyme sites (where applicable) that were added to the 5' end of the oligonucleotide are underlined. Forward (F) and reverse (R) oligonucleotides are shown.
  • Dybvig K French CT, Voelker LL: Construction and use of derivatives of transposon Tn4001 that function in Mycoplasma pulmonis and Mycoplasma arthritidis. J Bacteriol 2000, 182:4343-4347. 6. Hahn TW, Mothershed EA, Waldo RH, 3rd, Krause DC: Construction and analysis of a modified Tn4001 conferring chloramphenicol resistance in Mycoplasma pneumoniae. Plasmid 1999, 1:120-124.
  • Example 2 Methods for generating targeted mutants with a small scar site
  • Example 1 describes the generation of an oriC based plasmid system for M. hyopneumoniae.
  • One of the potential applications of this system is utility in producing targeted gene disruptions in M. hyopneumoniae by promoting homologous recombination at target sites.
  • an approximately 1000 bp region of a selected target gene can be incorporated into the oriC plasmid.
  • the plasmid can be used to drive integration of the entire plasmid into the target site by homologous recombination, therefore disrupting an open reading frame. This has been accomplished as detailed in Example 4b.
  • Mutant M. hyopneumoniae carrying the flip recombinase plasmid can be selected for using puromycin (for example). Expression of the recombinase will lead to 'excision' of the tetM marker (for example) at the FRT recognition sites and the resulting M. hyopneumoniae mutant will retain a disrupted ORF but with only a very small scar.
  • the 34bp minimal FRT site sequence has the sequence 5GAAGTTCCTATTCiciagaaaGiATAGGAACTTC 3' for which flippase (Flp) binds to both 13-bp 5'-GAAGTTCCTATTC-3' arms flanking the 8 bp spacer, i.e. the site-specific recombination (region of crossover) in reverse orientation.
  • FRT-mediated cleavage occurs just ahead from the asymmetric 8bp core region ( 5' tctagaaa 3 on the top strand and behind this sequence on the bottom strand.
  • Example 3 A novel Hima transposon mutagenesis system for generating random Mycoplasma (for example hyopneumoniae) gene mutants
  • 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 Himarl transposase along with a mini transposon containing the tetracycline resistance cassette, fefM M.
  • hyopneumoniae strain 232 was electroporated with the construct and tefM-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.
  • PCR was performed using a Phusion High Fidelity kit (NEB Ltd, Hitchin, UK) according to the manufacturer's instructions. Sequences of oligonucleotides used in the study are shown in table 2.
  • bp base pair
  • TetM1 and TetM2 primers
  • Mariner transposon plasmids were constructed from the plasmid pMiniHimar1 BSC1 (figure 4B) (Cook, 2013).
  • pMHWT-2 To construct pMHWT-2, firstly the 2293 bp tetM gene in pSRT2 (Cordova et al., 2002) was amplified by PCR (primers TetM3 and TetM4) and used to replace the puromycin N-acetyl-transferase (pac) gene of pMiniHimar1 BSC1 using the Mlul and Sbfl restriction sites (figure 4C).
  • pac puromycin N-acetyl-transferase
  • pMiniHimar1 BSC1 contains the Himarl transposase gene downstream of the native promoter sequence from the Tn4001 transposase.
  • the 1050 bp Himarl transposase gene minus the Tn4001 promoter sequence was PCR- amplified from pMiniHimar1 BSC1 (primers Tpn1 and Tpn2), introducing an Xhol cut site at the 3' end and Ndel and Ncol restriction sites at the 5' end.
  • This PCR product was cloned into Ndel and Xhol sites of pMHWT-2, replacing the Himarl gene and Tn4001 promoter sequence, and introducing a new Ncol cloning site.
  • the 619 bp P97 ciliary adhesin gene promoter sequence was amplified from M. hyopneumoniae strain 232 (Minion et al., 2004) genomic DNA with Ncol and Ndel restriction sites added (primers P97-1 and P97-2).
  • the P97 promoter sequence was then cloned into the Ncol and Ndel restriction sites upstream of the Himarl transposase gene, placing Himarl under its control, and completing plasmid pMHWT-1 .
  • the 1050 bp hyperactive C9 mutant of Himarl was PCR amplified from plasmid pCAM45 (May et al., 2004), adding Xhol and Ncol restriction sites (primers Tpn1 and Tpn2).
  • This PCR product was used to replace the wild-type Himarl transposase gene of pMHWT-1 , generating plasmid pMHC9-1 such that the C9 mutant Himarl is under control of the P97 promoter sequence.
  • 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.
  • tetracycline hydrochloride (Sigma- Aldrich Ltd, Gillingham, UK) was added to Friis broth medium and Friis agar medium at final concentrations of 0.5 ⁇ g/mL and 0.2 ⁇ g/mL respectively.
  • Molecular cloning was performed using Escherichia coli strain DH5a, grown in Luria-Bertani medium according to standard methods (Sambrook and Russell, 2001 ).
  • Transformants were selected by the addition of 100 ⁇ g/mL ampicillin (Sigma-Aldrich Ltd, Gillingham, UK) to the medium.
  • tetracycline hydrochloride was added to a final concentration of 5 ⁇ g/mL.
  • 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 xg for 10 minutes at 4 °C and washed three times in electroporation buffer (272 mM sucrose, 8 mM HEPES, pH 7.4). 100 ⁇ cells (corresponding to 3 mL culture) were incubated on ice with approximately 10 ⁇ g plasmid DNA for 30 minutes.
  • Electroporation was performed in a 0.2 cm cuvette (Bio- Rad Ltd, Hemel Hempstead, UK) at 2.5 kV, 100 ⁇ , 25 ⁇ and 900 ⁇ ice-cold Friis medium were immediately added. After 15 minutes incubation on ice, cells were transferred to a 1.5 mL tube and incubated at 37 °C for 3 hours. 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.
  • Transposon insertion site determination was based on the method described by Chaudhuri et al (Chaudhuri et al., 2009). Genomic DNA was extracted from M. hyopneumoniae strain 232 culture using a phenol-chloroform method (Bashiruddin, 1998). 2.5 ⁇ g DNA were digested overnight at 37 °C with 10 units of Alul, which cuts the 892 kbp genome of M. hyopneumoniae strain 232 a total of 2239 times and generates blunt ends. Alul also cuts within the ieiM-containing 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.
  • 10 ⁇ oligonucleotides Linker-A and Linker-B (table 1 ) were heated together in a boiling water bath for 3 minutes in annealing buffer (10 mM Tris- HCI, 50 mM NaCI, 1 mM EDTA, pH 8.0) and then allowed to cool for 1 hour at room temperature.
  • 50 ng of Alul digested DNA were blunt-ligated to 4 ⁇ annealed oligonucleotides Linker-A and Linker-B in a 10 ⁇ reaction using a Quick Ligation kit (NEB Ltd, Hitchin, UK) for 1 hour 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 minutes; 33 cycles of 94 °C for 45 s, 55 °C for 60 s, 72 °C for 120 s; 72 °C for 10 minutes. PCR products were visualised by electrophoresis in 1.6% agarose. Transposon insertion sites were determined by the direct sequencing of PCR products. Southern analysis
  • a digoxigenin (DIG)-labeled probe specific for the tetM gene was generated from PCR- amplified DNA (primers TetMF and TetMR, Table 2) 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).
  • Plasmid pTn4001-RVC1 (figure 4A) 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.
  • hyopneumoniae strain 232 allowing the selection of transformants on Friis agar plates containing 0.2 tetracycline (Maglennon et al., 2013; Example 1 ).
  • M. hyopneumoniae strain 232 with pTn4001-RVC using our optimised conditions and our oriC plasmid pMHO-2 (Maglennon et al., 2013; Example 1 ) as a positive control for transformation, we obtained no transformants. This suggested that the Tn4001 transposase was not functional in M. hyopneumoniae.
  • a Himarl transposon delivery vector (plvliniHimar1 BSC1 ) has been prepared that successfully generated transposon insertion mutants in the M. gallisepticum R(i 0W ) strain and very low numbers of transposon insertion mutants in M. hyopneumoniae strain 232 (Cook PhD thesis prepared 2013 but not in the public domain [and example 3]).
  • pMiniHimar1 BSC1 consists of the Himarl transposase under control of the promoter sequence from Tn4001 and a transposon carrying the pac gene conferring resistance to puromycin (figure 4B). Low numbers of mutants (less than 10 per transformation) were generated in M.
  • Plasmid pMiniHimar1 BSC1 failed to generate any puromycin resistant transformants compared to an average of 1.5 x 10 "4 transformants/CFU for the pMHOpuro positive control. To determine whether tetM selection would increase the yield of transformants, pac was replaced with tetM in plasmid pMHWT-2 (figure 4C).
  • An alternative hypothesis for the paucity of transformants was that the Tn4001 promoter sequence controlling Himarl transposase expression was poorly active in M. hyopneumoniae, even although there had been no need to replace the Tn4001 promoter sequence in other systems in which this transposase had been used.
  • pMHWT-1 (figure 4D) was made by replacing the Tn4001 promoter sequence regulating Himarl with the P97 gene promoter of M. hyopneumoniae strain 232 (Maglennon et al., 2013; Example 1 ).
  • Plasmid pMHC9-1 (figure 4E) was constructed by replacing the wild-type Himarl transposase with the C9 mutant form which exhibits increased activity in vitro (Lampe et al., 1999). Additionally, as a positive control for transformation, mycoplasmas were transformed with the or/C-based plasmid pMHO-2 (containing tetM).
  • Transformed cells formed discrete and well-separated colonies allowing the isolation of individual colonies. Additionally, colonies were relatively non-adherent to the agar surface and could be easily removed by gentle washing with Friis medium. Importantly, in none of the experiments described did we observe colonies in our 'no DNA' control transformations, indicating the absence of pseudo-resistance or spontaneous resistance to tetracycline.
  • tetracycline resistance was due to transposition into the host chromosome and not, for example, due to maintenance of extrachromosomal plasmid DNA 15 individual colonies from pMHC9-1 transformed M. hyopneumoniae strain 232 were analysed. Each was transferred into 1 ml Friis broth medium containing 0.5 ⁇ g mL tetracycline.
  • PCR for a region of the pUC19 plasmid backbone failed to amplify DNA for any transformant showing that tetracycline resistance was not due to maintenance of extrachromosomal plasmid DNA, or due to integration of the entire plasmid, for example by homologous recombination at the P97 promoter sequence site (figure 6B).
  • transposon insertion sites were found to match.
  • Southern analysis was performed on total DNA extracted from the same 1 1 transformants to confirm that only single transposon insertions were present. For 10 of the mutants, a single band was present using a DNA probe specific for tetM contained within the transposon (figure 7). For mutant 1 , a small intense band was present along with a larger feint band. Only one insertion site was identified for this transformant by linker PCR and this insertion site was confirmed by DNA sequencing. It is possible that two bands were generated by Southern analysis by a failure to adequately digest the DNA to completion with Hindi 11.
  • Insertion site sequencing of a further 33 individual transformants was performed so that the locations of a total of 44 transformants were mapped to the M. hyopneumoniae strain 232 genome (figure 8). Of these 44 transformants, 41 contained unique insertion sites (table 4). Mutants 49, 68 and 161 all contained an insertion at the nucleotide 549968 in the M. hyopneumoniae strain 232 genome and mutants 56 and 66 contained identical insertion sites at nucleotide 574428. It was not possible to determine whether these mutants arose from a single transposition event or from multiple events at the same location.
  • the Tn4001 transposon is effective in generating insertional mutations in a number of different Mycoplasma species (Chopra-Dewasthaly et al., 2005; French et al., 2008; Hedreyda et al., 1993; Pour-El et al., 2002) but even using our optimised transformation conditions, we were unable to demonstrate transposition in M. hyopneumoniae.
  • a Himarl 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 (Cook, thesis; Example 3). By using an 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 Himarl 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.
  • 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 Himarl 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 (Claeys Bouuaert and Chalmers, 2013). 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.
  • 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 non-essential 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 constructed 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.
  • the system is also considered to be useful for other mycoplasmas.
  • P159 is a proteolytically processed, surface adhesin of Mycoplasma hyopneumoniae: defined domains of P159 bind heparin and promote adherence to eukaryote cells. Mol Microbiol 60, 669-686.
  • TetM2 Xbal GAAATCTAGATTATATAACAACTTAAATTAC
  • TetM3 Sbfl CGATCCTGCAGGCAGAATTAAAAGTTAGTG
  • the locations of transposon insertions within the M. hyopneumoniae strain 232 genome for 44 individual pMHC9-1 transformants are shown.
  • the open reading frame (ORF) or non-coding region (NCR) containing the insertion is shown and, where known, the function of the protein encoded.
  • Example 4 In vitro and in vivo screening of . hyopneumoniae mutant pool
  • genes necessary for growth in vitro it is possible to deduce which genes are not necessary for in vitro growth, and that these genes may therefore have a role in causing disease/survival in the animal. These genes may therefore serve as potential vaccine candidates.
  • a large pool of pMHC9-1 M. hyopneumoniae mutants (for example) is screened in pigs.
  • the pool of mutants is grown up in broth culture and the insertion sites of all of the individual mutants determined by TraDIS.
  • the entire pool of mutants is then used to infect several pigs.
  • individual animals are culled and the lungs harvested.
  • M. hyopneumoniae organisms are re-isolated from the lungs by culturing in broth and the transposon insertion sites of all of the residual mutants are determined by TraDIS.
  • mutants By a 'subtraction' of the output pool of mutants from the input pool, mutants are identified that have lost ability to survive in the pig. Such mutants may have transposon insertion sites in genes that represent potential virulence factors for M. hyopneumoniae, and these may be useful as candidates for vaccine studies.
  • transposon insertion sites of a large pool of transposon mutants by next generation sequencing, and have found that there are approximately 33700 independent transposon insertions into the genome of M. hyopneumoniae 232.
  • a number of the genes of 232 have no, or very few, insertions indicating that they may be essential genes. Some have a number of insertions less than might be expected indicating that the mutation may render the organism less fit to grow and compete with the others.
  • a self-replicating plasmid was produced (pOriC-iv/v) (Example 1 ) that contains the minimum functional or/ ' C of M. hyopneumoniae strain 232.
  • the plasmid is stably maintained in transformed M. hyopneumoniae 232 cells when selection for tetM expression is present using tetracycline, but the cells are rapidly 'cured' of the plasmid when selection is removed.
  • This plasmid was used as part of a strategy to generate targeted gene disruptions in M. hyopneumoniae strain 232 by promoting homologous recombination within selected regions of the chromosome.
  • plasmid pOriCP146 Using the minimum oriC plasmid pOriC-iv/v, we generated plasmid pOriCP146 by inserting a 974 bp homologous region of the P146 gene of M. hyopneumoniae strain 232 (figure 9A). Containing the or/ ' C of M. hyopneumoniae this plasmid is maintained by transformed cells over serial passages in the presence of tetracycline, and was hypothesised to increase the frequency of homologous recombination.
  • homologous recombination may be an infrequent event in M. hyopneumoniae, and that the frequency may be increased by exposing the cells to plasmid DNA containing the homologous sequence for a prolonged period of time.
  • plasmid pOriC-iv/v we generated plasmid pOriCP146 by inserting the same 974 bp homologous region of the P146 gene of M. hyopneumoniae strain 232 (figure 1). Containing the or/ ' C (functional origin of replication) of M.
  • this plasmid should be maintained by transformed cells over serial passages in the presence of tetracycline, affording a wider window for homologous recombination to occur.
  • the particular region of the P146 gene targeted for homologous recombination was chosen such that integration of plasmid DNA would lead to a disruption in the open reading frame and production of a truncated protein (figure 9A). Having demonstrated that plasmid integration could be targeted to a selected region within the P146 gene, two further target regions within the M.
  • hyopneumoniae 232 genome were selected: the pepF gene encoding the protein oligoendopeptidase F and the open reading frame of mhp379, which encodes a nuclease and is part of an operon involved in nucleotide acquistion.
  • a 974 bp region of the M. hyopneumoniae strain 232 P146 ORF was amplified by PCR using primers P146F and P146R. These primers incorporate Mlu ⁇ and Pst ⁇ restriction enzyme recognition sites at their 5' ends respectively to facilitate cloning of the fragment into plasmid pOriC-iv/v, generating plasmid pOriCP146. Plasmid pOriCpepF was generated by cloning a 948 bp region of the pepF ORF, produced by PCR using primers pepF-F and pepF-R, into the same Mlul and Pstl restriction sites of pOriC-iv/v.
  • Plasmid pOriC379 was generated by cloning a 549 bp fragment of the mhp379 ORF, produced by PCR using primers mhp379-F and mhp379-R, into pOriC-iv/v. M. hyopneumoniae strain 232 cells were cultured and prepared for transformation as previously described.
  • Mycoplasmas were electroporated (2.5 kV, 25 ⁇ , 100 ⁇ ) with 7.5 ⁇ g of plasmid DNA, for example pOriCP146, in a 0.2 cm electroporation cuvette and following recovery for 3 hours in Friis broth medium, were plated onto Friis agar containing 0.2 ⁇ g/ml tetracycline. Plates were incubated for approximately 14 days and then examined for the presence of mycoplasma colonies using a stereoscopic microscope. Colonies were picked using a sterile pipette tip into Friis broth medium containing 0.5 ⁇ g/ml tetracycline and cultured for 3-5 days.
  • Plasmid DNA was analysed by agarose gel electrophoresis using a 1 % w/v gel. Total DNA was analysed by Southern analysis using standard methods (Sambrook & Russell 2001 ) and as described in Maglennon et al 2013. Briefly, approximately 2.5 ⁇ g/ml DNA were digested to completion with Hind ⁇ , separated by electrophoresis on 1 % w/v agarose and transferred onto Hybond N+ membrane (GE Healthcare Ltd, UK). Hybridisation was performed using a DIG-labelled DNA probe that anneals to a region of the tetM resistance cassette in plasmid pOriCP146.
  • total DNA was analysed for the presence of plasmid integration by using a PCR strategy to amplify regions of DNA spanning the presumed integration sites.
  • PCR was performed using a Qiagen HotStarTaq kit (Qiagen Ltd, UK) according to the manufacturers instructions.
  • Plasmid DNA was extracted from approximately 10 ml_ broth culture and confirmed the presence of plasmid pOriC-iv/v in six randomly selected clones. ( Figure 10). However, free plasmid DNA was not readily detectable in four randomly selected pOriCP146 clones, despite successful growth in the presence of tetracycline. It was suspected that the plasmid DNA containing the tetM resistance cassette was still present, but that it had integrated into the M. hyopneumoniae 232 chromosomal DNA.
  • hyopneumoniae 232 chromosomal DNA at the P146 gene target site by homologous recombination
  • a PCR approach was adopted. Using two sets of PCR oligonucleotide primers (primer set A (P146-intF / P146-intR) and set B (P146-intF2 / P146-intR2)), we attempted to amplify DNA spanning the suspected integration site at either end of the integrated plasmid (figure 10). In all four colonies, a PCR product of the correct anticipated size was generated, but no PCR product was generated for control reactions consisting of circular plasmid DNA template and genomic DNA from untransformed M. hyopneumoniae 232. Additionally, the PCR products generated were sequenced which confirmed that plasmid DNA had indeed integrated into the chromosomal DNA at the target site.
  • hyopneumoniae 232 with plasmids pOriCpepF and pOriC379 many tetracycline resistant colonies were generated on Friis agar containing 0.2 ⁇ g/mL tetracycline yet as expected no colonies were generated for 'no DNA' controls. Individual colonies were randomly selected and grown in Friis broth medium containing 0.5 ⁇ g mL tetracycline. Plasmid DNA was extracted from approximately 10 mL culture grown from five or six colonies for each plasmid and analysis showed that plasmid DNA could readily be identified for all clones (data no shown).
  • Mycoplasmas were then subcultured a further 19 times in Friis medium containing 0.5 ⁇ g/mL tetracycline and a further plasmid DNA extract performed. Analysis by agarose gel electrophoresis showed that plasmid pOriC379 could be identified in all five clones, demonstrating that the continued tetracycline resistance was likely to be due, at least in part, to the presence of extrachromosomal plasmid DNA (figure 1 1 B). However, using PCR analysis (primers mhp379-intF and mhp379-intR), it was possible to amplify a region of DNA spanning the anticipated target site of integration (figure 1 1 C).
  • PCR was performed using primers pepF- intF/pepF-intR (primer set C) and pepF-intF2/pepF-intR2 (primer set D) to amplify DNA spanning the targeted plasmid integration site within the M. hyopneumoniae 232 genome (figure 12). It was possible to detect PCR product for all six clones demonstrating that plasmid integration had occurred. Sequencing of the PCR products provided additional confirmation that integration had occurred at the anticipated target site.
  • the location of the P146 ORF targeted for homologous recombination is such that we are likely to have achieved disruption of the ORF leading to production of a truncated protein with altered or loss of function. It is possible that this mutant may exhibit an altered phenotype in in vitro assays of cellular attachment and/or in vivo pathogenesis testing. More importantly, this work illustrates that targeted gene disruptions are achievable in M. hyopneumoniae. Additionally, the targeting of exogenous DNA sequences to specific endogenous sequences makes possible the ability to modify coding sequences with epitope tags or other mutations, rearrangements, fusions or deletions. For example incorporating a region of a gene into the plasmid in-frame with the gfp coding sequence allows, following homologous recombination, the expression of the gfp tagged gene under its native promoter from its native locus.
  • P146, mhp379 and pepF Three specific sites (P146, mhp379 and pepF) were chosen within the M, hyopneumoniae strain 232 genome for targeted integration of plasmid DNA by homologous recombination following a single crossover event.
  • the mhp379 ORF was chosen because it encodes a cell surface nuclease and is part of an operon that is involved in nucleotide acquisition (Schmidt et al 2007). The primary reason for choosing this ORF was that if we could disrupt this ORF, then we may be able to detect a phenotypic change in vitro.
  • M. hyopneumoniae expresses potent nuclease activity that can readily be detected using a number of simple assays.
  • Disruption of M. hyopneumoniae 232 genomic DNA using this method may be a useful means of studying the function of genes that are not essential for in vitro growth and survival, but that may be associated with virulence/pathogenesis in the host.
  • gene disruption is possible, currently using this technique, a large amount of foreign DNA is integrated into the chromosome of the host cell. It is considered that by modification of the technique, double crossover recombination events can be generated that will result in integration of the tetM determinant alone.
  • Example 6 Development of genetic tools for functional genomic analysis of Mycoplasma hyopneumoniae.
  • Mycoplasma hyopneumoniae is the cause of enzootic pneumonia: a respiratory disease of pigs worldwide.
  • Growth of M. hyopneumoniae on solid medium was improved following assessment of the purity of the solidifying agar. Incorporation of 2Mg/ml kanamycin into solid medium inhibited the growth of M.hyorhinis allowing the positive selection of M. hyopneumoniae.
  • a transposon delivery system for M. hyopneumoniae was required for the generation of mutant genotypes.
  • M.gallisepticum R(low) infection model was established in chickens with an optimal infectious dose of 107CFU/bird with maximum re-isolation from the trachea and lungs after 7 days post infection. These data will be used to perform an in vivo screen of the large M.gallisepticum transposon library generated. Transformation of M. hyopneumoniae with pMini-H/marl BSC1 by electroporation was successfully achieved following method adaptations. Southern blot and PCR analysis confirmed the generation of six different transposon mutants, suggesting transformation had occurred but was very inefficient. Sequencing of transposon insertion sites identified insertions into 4 different ORF and one intergenic region. This is the first report of the successful introduction of a Mariner- based transposon into Mycoplasma species. With further improvements, this transposon delivery system will allow functional genomic analysis of this poorly understood and economically important swine pathogen.
  • Figure 2.3-1 Schematic diagram of Circular Polymerase Extension Cloning (CPEC).
  • Figure 2.8-1 An example of airsacculitis observed at necropsy following intra-tracheal inoculation with M. gallisepticum strain R (low) 76
  • Figure 3.2-2 Growth of M. hyopneumoniae 277 on different solid media preparations.
  • Figure 3.2-4 Growth of M. hyorhinis on different solid media preparations
  • Figure 3.2-5 Growth of M. hyopneumoniae 277 on different solid media preparations.
  • Figure 3.2-6 Growth of M. hyopneumoniae 325 on different solid media preparations.
  • Figure 3.2-7 PCR detection of M. hyopneumoniae and M. hyorhinis 90
  • Figure 3.2-8 Suppression of M. hyorhinis growth following kanamycin selection on solid medium 93
  • FIG.2-4 PCR analysis of M. hyorhinis and M. gallisepticum transformation with Mini-
  • Figure 5.2-3 PCR amplification of fragments 1 to 4 129
  • Figure 5.2-5 Ligation of Fragment 1 and the pUC18 plasmid backbone 131
  • Figure 5.2-6 Ligation of Fragment 2 and p(JC18+FR1 +FR3 132
  • Figure 5.2-9 Restriction digest of pUC18+FR4a and pUC18+FR1 +FR2+FR3 with Nde ⁇ and Kpn ⁇ 135
  • Figure 5.2-10 Fragment FR4b lx ⁇ 4001 transposase gene promoter region 135
  • Figure 5.2-12 Region of puromycin resistance gene and spiralin gene promoter sequenced from Mini-Tn4007Pspuro and pUC18+FR1 +FR2+FR3 137
  • Figure 5.2-13 Growth of E. coli JM 09 colonies containing plasmid construct pUC18+FR1 +FR2+FR3 on selective agar containing 100 g/ml puromycin 138
  • Figure 5.2-14 Schematic diagram of the construction of pMini-H/mar1 BSC1 139
  • Figure 6.2-1 M. gallisepticum growth selective medium following electroporation. .. 147
  • Figure 6.2-2 PCR analysis of M. gallisepticum following electroporation with Mini-
  • Figure 6.2-4 Southern analysis of M. gallisepticum transformants 150
  • Figure 6.2-5 Southern analysis of re-isolated M. gallisepticum PG31 transformants.
  • Figure 6.2-6 PCR analysis of M. gallisepticum R (low) following electroporation with
  • Figure 6.2-7 PCR analysis to determine the absence of the delivery vector following electroporation of M. gallisepticum R (low) 55
  • Figure 6.2-8 Southern analysis of M. gallisepticum R (low) transformants 156
  • Figure 7.2-1 PCR analysis to confirm the presence of the Mini-H/ma/i transposon and the loss of the plasmid backbone 166
  • Figure 7.2-2 Growth of M. hyopneumoniae 232 puromycin resistant colonies 169
  • Figure 7.2-3 PCR analysis to confirm the presence of the Mini-H/ma/i transposon.
  • Figure 7.2-4 PCR analysis to confirm the loss of the pMini-H/ma/i BSC1 plasmid backbone 171
  • Figure 7.2-5 PCR confirmation of M. hyopneumoniae 232 species 172
  • Figure 7.2-6 Southern analysis of transformed M. hyopenumoniae 232 isolated colonies.
  • Figure 7.2-7 Growth of M. hyopneumoniae 232 colonies following electroporation.
  • Figure 7.2-8 PCR analysis to confirm the presence of the Mini-H/ma/i transposon.
  • Figure 7.2-9 PCR analysis to confirm the loss of the pMini-H/ma/i BSC1 plasmid backbone 178
  • FIG.2-1 1 Southern analysis of genomic DNA extracted from M. hyopneumoniae mini-H/ma/i transposon mutants and digested with EcoRI 180
  • Figure 7.2-12 Analysis of M. hyopneumoniae 232 transposon mutant pools by linker PCR 182
  • FIG. 7.2-16 Western blot analysis of M. hyopneumoniae 232 mini-H/ma/i transposon mutants 187
  • FIG.2-2 Average daily weight gain of males and females following intra-tracheal infection with M. gallisepticum strain R (low) 200
  • Figure 8.2-3 Varying amounts of mucus observed in the trachea of chickens following intra-tracheal infection with M. gallisepticum strain R (low) 202
  • Figure 8.2-4 (A-D): Re-isolation of M. gallisepticum from different sites within the respiratory tract at 3, 7, 10 and 14 days post infection 206
  • Figure 8.2-5 Re-isolation of M. gallisepticum from total lung tissue at 3, 7, 10 and 14 days post infection 207

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Abstract

L'invention concerne un plasmide pour la transformation de Mycoplasma hyopneumoniae comprenant une région OriC comprenant les deux régions riches en AT à partir d'une région OriC prédite de M. hyopneumoniae 232, ou comprenant un variant de celle-ci qui conserve la capacité à agir en tant que région OriC dans M. hyopneumoniae et qui présente au moins 90 % d'identité de séquence avec une séquence comprenant les deux régions riches en AT à partir d'une région OriC prédite de M. hyopneumoniae 232. Une cellule M. hyopneumoniae transformée par un plasmide selon l'invention peut être utile dans une composition de vaccin. L'invention concerne également une construction d'acide nucléique qui comprend une séquence d'acide nucléique codant pour une enzyme transposase et une séquence promotrice, la séquence promotrice étant active dans M. hyopneumoniae, la séquence promotrice comprenant éventuellement une séquence promotrice à partir de M. hyopneumoniae, ou un gène de la spiraline promoteur de Spiroplasma citri ou une séquence promotrice tetM à partir de S. aureus, la séquence promotrice à partir deM. hyopneumoniae étant éventuellement une séquence promotrice active constitutive, éventuellement une séquence promotrice à partir de M. hyopneumoniae ldh, P97, secD, Tuf, rpoB, P146, protéine de liaison ATP transporteur ATP, asparagine-ARNt synthétase ou gène de facteur d'élongation de translation. L'enzyme transposase peut être une transposase de la famille Mariner, éventuellement une transposase Himar1 ou une transposase de mutant C9 Himar1.
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WO2020243085A1 (fr) * 2019-05-24 2020-12-03 The Trustees Of Columbia University In The City Of New York Système de transposon de cas modifié pour des transpositions d'adn programmable et dirigées sur un site

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2873733B1 (fr) 2012-07-10 2019-06-26 Hipra Scientific, S.L.U. Vecteurs pour la transformation de mycoplasma hyopneumoniae, souches transformantes de m.hyopneumoniae, et leur utilisation
EP3546580A1 (fr) * 2012-07-10 2019-10-02 Hipra Scientific, S.L.U. Vecteurs pour la transformation de mycoplasma hyopneumoniae, souches transformantes de m.hyopneumoniae, et leur utilisation
WO2020243085A1 (fr) * 2019-05-24 2020-12-03 The Trustees Of Columbia University In The City Of New York Système de transposon de cas modifié pour des transpositions d'adn programmable et dirigées sur un site

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