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Journal of Bacteriology, March 2003, p . 1745-1748, Vol . 185, No . 5 Transposition of Tn5367 in Mycobacterium marinum, Using a Conditionally Recombinant MycobacteriophageJan Rybniker, Martina Wolke, Christiane Haefs, and Georg Plum* Institute for Medical Microbiology, Immunology and Hygiene, University of Cologne, Cologne, Germany Received 27 September 2002/ Accepted 6 December 2002
Though classified as a slow-growing mycobacterium, M . marinum has a relatively short generation time of 4 to 6 h compared to 20 h for Mycobacterium tuberculosis . The optimal growth temperature range for most M . marinum isolates is 25 to 35°C . Like M . tuberculosis and other virulent mycobacteria, M . marinum survives and replicates in host macrophages, where it prevents phagosome maturation (1, 4, 7, 12) . There are also significant genotypic and phenotypic similarities between these species, which implies that M . marinum is a potent model system for the study of mycobacterial pathogenesis (4) . The major advantages over M . tuberculosis are the faster growth and the safer handling of M . marinum in the laboratory . Working with it requires only common laboratory precautions (biosafety level 2) . The complete genome of M . marinum (strain M, human isolate) is currently being sequenced by the Sanger Institute . The available coverage of the genome was 99.99% complete at the time that this paper was submitted . The mycobacteriophages phAE77 and phAE94 have been used as powerful tools for transposition mutagenesis in several fast- and slow-growing mycobacterial species (3, 11) . Both phages are highly efficient in delivering the mycobacterial transposon Tn5367, while they are incapable of replicating at 37°C due to the presence of temperature-sensitive mutations . Tn5367 is an IS1096-derived insertion element containing a kanamycin resistance gene as a selectable marker (14) . PhAE77 is a derivative of the well-described lytic mycobacteriophage D29, whereas the progenitor of phAE94 is mycobacteriophage TM4, which was isolated as a temperate phage of Mycobacterium avium (22) . Both phages are propagated in Mycobacterium smegmatis at the replication-permissive temperature of 32°C and are used for transposon delivery at 37°C . The transposable element Tn5367 has been shown to insert randomly into the genome of M . marinum isolate ATCC 927 after transformation using the electroporation method . However, the transposition frequency was too low for the generation of a representative mutant library and it was suggested that the low transposition frequency derives from a low transformation frequency (20) . In this study we have overcome this problem by using the mycobacteriophage TM4-derived phAE94 as a delivery vector for Tn5367 . We were able to create a comprehensive bank of kanamycin-resistant M . marinum mutants, showing that a TM4-derived vector is a potent tool for the transduction of M . marinum . Strains. A fish isolate of M . marinum (ATCC 927) was grown at 32°C in Middlebrook 7H9 broth enriched with 10% oleic acid-albumin-dextrose complex (without Tween) in a stirring bottle . Using the double-agar-layer method, phAE77 and phAE94 were propagated in M . smegmatis mc2155 at 32°C (13) . After infection, M . marinum cells were plated on Middlebrook 7H10 agar supplemented with 0.1% Tween 80, 0.4% Casamino Acids, 40 µg of tryptophan/ml, and 40 µg of kanamycin/ml . Transposon mutagenesis. M . marinum cultures were grown for 10 days to approximately 2 x 108 CFU/ml (at an optical density at 600 nm of 0.8) . A total of 10 ml of this culture was concentrated by centrifugation and resuspended in 1 ml of MP buffer (50 mM Tris-HCl [pH 7.6], 150 mM NaCl, 2 mM CaCl2) (13) . Then, 1010 PFU of phAE77 or phAE94 was added and the mixture was incubated at the nonpermissive temperature (37°C) for 5 h in a shaking incubator to inhibit a possible lytic or lysogenic cycle of the phage . Adsorption stop buffer (20 mM sodium citrate and 0.2% Tween 80) was added to prevent further phage infections . One-third of this mixture was plated immediately on 7H10 agar with kanamycin and incubated at 32°C . The remaining cells were kept on 37°C for 6 or 12 h before being plated to prevent a lytic or lysogenic cycle of the phage . Transposition frequency was expressed as the number of kanamycin-resistant (Kanr) colonies per milliliter of input cells . Analysis of the Kanr mutants. Kanamycin-resistant M . marinum colonies were picked and grown in 7H9 broth containing 40 µg of this aminoglycoside/ml . Mycobacterial DNA was isolated as previously described (15) . To reveal random transposition of the marker gene, the restriction enzyme BamHI was chosen, as it cuts in the flanking genomic sequences but not within the transposon itself . Digested genomic DNA was blotted onto nylon membrane and hybridized to a non-radioactively-labeled IS1096 probe (RPN3000; Amersham Pharmacia) . The digested DNA of M . smegmatis mc2155, the natural host of IS1096, was used as a positive control . To determine whether the prophage or parts of the prophage had integrated into the genome of the Kanr M . marinum mutants, DNAs of eight different mutants as well as phAE94 genomic DNA as a positive control were digested with PstI, which cuts more frequently in phAE94 than BamHI . Using the system described above, Southern blotting was performed; two PCR products of essential genes of phAE94 were labeled as a probe . The transposon mutants were further characterized by sequencing randomly primed PCR products (5, 16) . This method uses a specific primer from a region within Tn5367 together with an arbitrary primer in a first round of PCR . The product is used as a template for a second, nested PCR using a specific primer also derived from Tn5367 but closer to the flanking region and a subprimer of the arbitrary primer as a second primer . This second-round PCR product can be sequenced using the second specific primer . The arbitrary primers were ARB1 (5'-GGCCACGCGTCGACTAGTACNNNNNNNNNN-3') and ARB2 (5'GGCCACGCGTCGACTAGTAC-3') . Specific primers were RPCRa1 (5'-CTTGCTCTTCCGCTTCTTCTC-3') and RPCRa2 (5' CTCTACACCGTCAAGTGCGAAGAG-3') as well as RPCRb1 (5'-CAGGCACGTCGAGGTCTTTC-3') and RPCRb2 (5'-CTTTCAGATGGATGGCGTAG-3') for the opposite side of the transposon . First- and second-round conditions were the same as those previously described (5) . Reaction procedures were performed in a Peltier thermal cycler model PTC-200 (MJ Research, Waltham, Mass) . Second-round products were purified using a PCR purification kit (Qiagen) and sequenced using the Big-Dye terminator cycle sequencing ready reaction kit (Applied Biosystems) and an ABI Prism 310 genetic analyzer (Applied Biosystems) . Our goal was to adapt the conditionally replicating mycobacteriophage system as a tool for transposon mutagenesis in M . marinum . Using phAE94, the yield of Kanr M . marinum was very high, with up to 105 colonies per ml of transduced culture in a single experiment . In contrast, phAE77 yielded significantly fewer transposon mutants under the same conditions (103 per ml of transduced culture), so further experiments and the generation of the mutant library were done with phAE94 . Interestingly, keeping the infected cells at 37°C for 6 and 12 h had no influence on the number of transposon mutants . Even when the incubation temperature of the cell-phage mixture was decreased to 32°C throughout the whole experiment, the yield of mutants was the same as for the 37°C trial . Spotting 5 µl of a phAE94 dilution on 7H10 top agar plates containing M . marinum did not produce any clear or turbid plaques that would indicate lytic growth or a lysogenic state of the phage in M . marinum . This implies that TM4-derived mycobacteriophages are temperature-independent suicide vectors for M . marinum, which might be an important feature for future experiments with this model bacterium . To determine whether Tn5367 inserted in a random fashion into the M . marinum genome, eight mutants were picked and analyzed by Southern hybridization (Fig . 1) . Seven of eight screened mutants showed a single hybridizable band in different chromosomal locations, indicating a random insertion of the transposon . One transposon mutant displayed two bands of equal intensity, which might have been the result of two transposition events in the chromosome or of the presence of a mixed colony containing two individual mutants . The positive control showed several bands, as there are multiple copies of IS1096 in M . smegmatis (6) .
As additional evidence for successful transposon mutagenesis in M . marinum, a library of approximately 5 x 104 Kanr M . marinum mutants was pooled and plated on 7H10 agar plates containing 40 µg of ethionamide/ml . This compound is a prodrug that requires activation by bacterial enzymes . Drug resistance results from genetic mutation of the enzymes of the activation pathway (9, 19) . As expected, the number of ethionamide-resistant colonies was significantly larger for the transposon mutant library than for the wild-type strain . We observed 45 ethionamide-resistant cells per 105 plated bacteria for the library compared to 8.4 cells per 105 plated mycobacteria of the M . marinum wild-type strain, i.e., an increase by a factor of 5.3 .
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