Transposition of Tn5367 in Mycobacterium marinum, Using a ...

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Sep 27, 2002 - oped as a transposon mutagenesis tool for M. tuberculosis, to meet the specific requirements of M. marinum. ... Mailing address: Institut für Medizinische ... Mass). Second-round products were purified using a PCR pu-.
JOURNAL OF BACTERIOLOGY, Mar. 2003, p. 1745–1748 0021-9193/03/$08.00⫹0 DOI: 10.1128/JB.185.5.1745–1748.2003 Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Vol. 185, No. 5

Transposition of Tn5367 in Mycobacterium marinum, Using a Conditionally Recombinant Mycobacteriophage Jan 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

Mycobacterium marinum is a close relative of the obligate human pathogen Mycobacterium tuberculosis. As with M. tuberculosis, M. marinum causes intracellular infection of poikilothermic vertebrates and skin infection in humans. It is considered a valid model organism for the study of intracellular pathogenesis of mycobacteria. Low transformation efficiencies for this species have precluded approaches using mutant libraries in pathogenesis studies. We have adapted the conditionally replicating mycobacteriophage phAE94, originally developed as a transposon mutagenesis tool for M. tuberculosis, to meet the specific requirements of M. marinum. Conditions permissive for phage replication in M. tuberculosis facilitated highly efficient transposon delivery in M. marinum. Using this technique we succeeded in generating a representative mutant library of this species, and we conclude that TM4-derived mycobacteriophages are temperature-independent suicide vectors for M. marinum.

Mycobacterium marinum is a pathogenic, slow-growing member of the genus Mycobacterium (18). It causes fatal infection in salt- and freshwater fish as well as in amphibians. In humans, M. marinum is the causative agent of a disease called swimming pool granuloma (2, 8, 23), a chronic skin infection of the extremities that can convert into a systemic infection in immunocompromised patients such as those with AIDS (10, 17, 21). 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 ␮g of tryptophan/ml, and 40 ␮g of kanamycin/ml. Transposon mutagenesis. M. marinum cultures were grown for 10 days to approximately 2 ⫻ 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

* Corresponding author. Mailing address: Institut fu ¨r Medizinische Mikrobiologie, Immunologie und Hygiene, Klinikum der Universita¨t zu Ko ¨ln, Goldenfelsstr. 21, 50935 Cologne, Germany. Phone: 49-221478-3066. Fax: 49-221-478-3094. E-mail: [email protected]. 1745

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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 ␮g 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⬘-GGCCACGCGTCGACTAGTACNNNNNNNN NN-3⬘) and ARB2 (5⬘GGCCACGCGTCGACTAGTAC-3⬘). Specific primers were RPCRa1 (5⬘-CTTGCTCTTCCGCTTC TTCTC-3⬘) and RPCRa2 (5⬘ CTCTACACCGTCAAGTGCG AAGAG-3⬘) as well as RPCRb1 (5⬘-CAGGCACGTCGAGG TCTTTC-3⬘) and RPCRb2 (5⬘-CTTTCAGATGGATGGCGT AG-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.

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FIG. 1. Southern blot analysis of M. marinum Tn5367 mutants. Lanes 1 to 10 show BamHI digests of total chromosomal DNA hybridized to a labeled IS1096 probe. Lane L, DNA markers (sizes in kilobase pairs are indicated on the left); lane 1, wild type; lanes 2 to 9, Kanr insertion mutants; lane 10, M. smegmatis mc2155.

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 ␮l 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). Using the randomly primed PCR method, sequence analysis of the Tn5367 insertion junctions yielded good-quality sequences for all examined mutants. A BLAST analysis of the sequences revealed different results for the chromosomal DNA adjacent to the ends of the transposon. The sequences obtained using primer RPCRa2 showed the expected GC-rich mycobacterial genomic DNA in all mutants. The majority had a high level of homology to M. marinum and M. tuberculosis. The opposite side of the transposon (sequencing primer RPCRb2) showed an insertion of parts of the cloning vector pYUB552 that flanks Tn5367 in the phAE94 genome. In 2 out of 20 examined mutants, small parts of TM4 genomic DNA adjacent to this cloning vector had additionally integrated into the chromosome. However, further analysis of these sequences revealed that this cointegration of vector and phage DNA stops at different points for all examined mutants and merges

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TABLE 1. Mapping and analysis of the sequences adjacent to Tn5367 insertions Kanr mutant

Duplication

% Sequence similarity to M. marinum strain Ma

Protein or sequence of interrupted gene (% sequence similarity to M. marinum wild type)b

MmuA1 MmuA2 MmuB2 MmuB3 MmuB7 MmuB9

AAAC7TTAC ACCA7ATGG AAGA7GGCT GCTA7CGCG ATGG7ATCG TTGA7TAGC

97 98 64 92 97 92

PE-PGRS protein of M. tuberculosis H37Rv (73) Hypothetical protein Rv2030c of M. tuberculosis H37Rv (72) Putative DNA-binding protein of Streptomyces coelicolor (62) M. tuberculosis sequence from clone y414b (82) Hypothetical protein Rv3903c of M. tuberculosis H37Rv (62) Unknown

a b

Data calculated using the M. marinum BLAST server (Sanger). Data calculated using the National Center for Biotechnology Information database.

into the mycobacterial genome. Southern hybridization and PCR analysis with several primers distributed over the TM4 genome were performed to detect residual phAE94 sequences in the M. marinum mutants. All examined mutants were negative for the presence of the sequences (data not shown). All insertions were flanked by a unique 8-bp target duplication such as was described previously for the transposition of Tn5367 in other mycobacterial strains (3, 11). The sequenced chromosomal DNA produced high-scoring segment pairs when analyzed using the BLASTN program (BLASTN 2.0 MP [Washington University] [http://blast.wustl.edu]) with the almost completed M. marinum genome data (Table 1) at the Sanger Institute website (http://www.sanger.ac.uk/Projects/ M_marinum/blast_server.shtml). An additional BLAST analysis using the National Center for Biotechnology Information database (http://www.ncbi.nlm.nih.gov/blast/) demonstrated that the sequences of some of these mutants showed a high percentage of similarity to those of M. tuberculosis genes (Table 1). These data imply that Tn5367 transposes with no sequence specificity into the genome of M. marinum after the highly efficient vector phAE94 is used for transfection of the cells. Interestingly, using the same method to try to mutagenize Mycobacterium ulcerans, a very closely related slow-growing mycobacterium with an optimal growth temperature of 32°C, also resulted in a high number of mutants. Unfortunately, these mutants showed an extremely low growth rate, with an estimated generation time of 60 h (data not shown). Due to the slow growth, it was not possible to isolate sufficient amounts of DNA to perform a Southern blot analysis. However, by sequencing randomly primed PCR products we were able to identify M. ulcerans sequences adjacent to the transposon. The reason for the inhibition of growth remained unclear, and attempts to colonize the mutants on agar without kanamycin did not accelerate the growth of the bacteria. In contrast to M. marinum, spotting a dilution of phAE94 on top agar containing M. ulcerans and incubating at 32°C leads to turbid plaques, implying that stable lysogens are formed. This shows that phAE94 is a temperate phage of M. ulcerans. Though incubation was at 37°C for several hours during the transposon mutagenesis, an integration of the phAE94 prophage together with phage-derived repressor genes in the Kanr mutants might have occurred, leading to the observed growth inhibition. As additional evidence for successful transposon mutagenesis in M. marinum, a library of approximately 5 ⫻ 104 Kanr M. marinum mutants was pooled and plated on 7H10 agar plates containing 40 ␮g of ethionamide/ml. This compound is a pro-

drug 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. This work was supported by a studentship of Stiftung Maria Pesch. We thank W. R. Jacobs, Jr., for providing phAE77 and phAE94. We also thank F. Portaels for the M. marinum and M. ulcerans strains used in this work and for the use of laboratory facilities. We thank P. L. C. Small for the use of laboratory facilities and Brian Ranger for proofreading. REFERENCES 1. Armstrong, J. A., and P. D. Hart. 1971. Response of cultured macrophages to Mycobacterium tuberculosis, with observations on fusion of lysosomes with phagosomes. J. Exp. Med. 134:713–740. 2. Aubry, A., O. Chosidow, E. Caumes, J. Robert, and E. Cambau. 2002. Sixty-three cases of Mycobacterium marinum infection: clinical features, treatment, and antibiotic susceptibility of causative isolates. Arch. Intern. Med. 162:1746–1752. 3. Bardarov, S., J. Kriakov, C. Carriere, S. Yu, C. Vaamonde, R. A. McAdam, B. R. Bloom, G. F. Hatfull, and W. R. Jacobs, Jr. 1997. Conditionally replicating mycobacteriophages: a system for transposon delivery to Mycobacterium tuberculosis. Proc. Natl. Acad. Sci. USA 94:10961–10966. 4. Barker, L. P., K. M. George, S. Falkow, and P. L. Small. 1997. Differential trafficking of live and dead Mycobacterium marinum organisms in macrophages. Infect. Immun. 65:1497–1504. 5. Chen, T., H. Dong, R. Yong, and M. J. Duncan. 2000. Pleiotropic pigmentation mutants of Porphyromonas gingivalis. Microb. Pathog. 28:235–247. 6. Cirillo, J. D., R. G. Barletta, B. R. Bloom, and W. R. Jacobs, Jr. 1991. A novel transposon trap for mycobacteria: isolation and characterization of IS1096. J. Bacteriol. 173:7772–7780. 7. Clemens, D. L., and M. A. Horwitz. 1995. Characterization of the Mycobacterium tuberculosis phagosome and evidence that phagosomal maturation is inhibited. J. Exp. Med. 181:257–270. 8. Collins, C. H., J. M. Grange, W. C. Noble, and M. D. Yates. 1985. Mycobacterium marinum infections in man. J. Hyg. (London) 94:135–149. 9. DeBarber, A. E., K. Mdluli, M. Bosman, L. G. Bekker, and C. E. Barry III. 2000. Ethionamide activation and sensitivity in multidrug-resistant Mycobacterium tuberculosis. Proc. Natl. Acad. Sci. USA 97:9677–9682. 10. Hanau, L. H., A. Leaf, R. Soeiro, L. M. Weiss, and S. S. Pollack. 1994. Mycobacterium marinum infection in a patient with the acquired immunodeficiency syndrome. Cutis 54:103–105. 11. Harris, N. B., Z. Feng, X. Liu, S. L. Cirillo, J. D. Cirillo, and R. G. Barletta. 1999. Development of a transposon mutagenesis system for Mycobacterium avium subsp. paratuberculosis. FEMS Microbiol. Lett. 175:21–26. 12. Hart, P. D., M. R. Young, A. H. Gordon, and K. H. Sullivan. 1987. Inhibition of phagosome-lysosome fusion in macrophages by certain mycobacteria can be explained by inhibition of lysosomal movements observed after phagocytosis. J. Exp. Med. 166:933–946. 13. Jacobs, W. R., Jr., G. V. Kalpana, J. D. Cirillo, L. Pascopella, S. B. Snapper, R. A. Udani, W. Jones, R. G. Barletta, and B. R. Bloom. 1991. Genetic systems for mycobacteria. Methods Enzymol. 204:537–555. 14. McAdam, R. A., T. R. Weisbrod, J. Martin, J. D. Scuderi, A. M. Brown, J. D.

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