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

University of Cologne, Köln, North Rhine-Westphalia, Germany
Journal of Bacteriology (Impact Factor: 2.81). 04/2003; 185(5):1745-8. DOI: 10.1128/JB.185.5.1745-1748.2003
Source: PubMed


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.

Download full-text


Available from: Jan Rybniker, Jan 03, 2014
  • Source
    • ") carrying the kanamycin resistance transposon Tn5367 ( Shin et al . , 2006 ) was propagated in M . smegmatis mc 2 155 ( Bardarov et al . , 1997 ) and used to infect M . marinum as described previously ( Rybniker et al . , 2003 ) ."
    [Show abstract] [Hide abstract]
    ABSTRACT: Resistance to phagocyte killing is an important virulence factor in mycobacteria. Dictyostelium has been used to study the interaction between phagocytes and bacteria, given its similarity to the mammalian macrophage. Here, we investigated the genes responsible for virulence to Dictyostelium by screening 1728 transposon mutants of the Mycobacterium marinum NTUH-M6094 strain. A total of 30 mutants that permissive for Dictyostelium growth were identified. These mutants revealed interruptions in 20 distinct loci. Of the 20 loci, six genes (losA, mmar_2318, mmar_2319, wecE, mmar_2323 and mmar_2353) were located in the lipooligosaccharide (LOS) synthesis cluster. LOS are antigenic glycolipids and the core LOS structure from LOS-I to LOS-IV have been reported to exist in M. marinum. Two-dimensional thin-layer chromatography (2D-TLC) glycolipid profiles revealed that deletion of mmar_2318 or mmar_2319 resulted in the accumulation of LOS-III and deficiency of LOS-IV. Deletion and complementation of mmar_2318 or mmar_2319 confirmed that these genes both contributed to virulence toward Dictyostelium but not entry and replication inside Dictyostelium. Co-incubation with a murine macrophage cell line J774a.1 or PMA-induced human monocytic cell line THP-1 demonstrated that mmar_2318 or mmar_2319 deletion mutant could grow in macrophages, and their initial entry rate was not affected in J774a.1 but significantly increased in THP-1. In conclusion, although mmar_2319 has been reported to involve LOS biosynthesis in a previous study, we identified a new gene, mmar_2318 that is also involved in the biosynthesis of LOS. Deletion of mmar_2318 or mmar_2319 both exhibits reduction of virulence toward Dictyostelium and increased entry into THP-1 cells.
    Preview · Article · Jan 2016 · Frontiers in Microbiology
  • [Show abstract] [Hide abstract]
    ABSTRACT: Abstract Mycobacteriosis (fish tuberculosis) is a progressive disease of a wide range of wild and captive marine and freshwater fish species. While Mycobacterium marinum, M. fortuitum and M. chelonae are the most frequently reported species to be involved in the disease, several new mycobacteria species have also recently been implicated. Conventional detection / identification of fish mycobacteria is based on histopathology, culture and biochemical characteristics. In this study complementary molecular approaches were developed to assist in Mycobacterium identification. First, a highly specific and sensitive multiplex PCR-based assay, targeting two genes (hsp65 and 16S RNA), was established to simultaneously detect the genus Mycobacterium and identify M. marinum, M. fortuitum or M. chelonae from culture or infected fish tissue, based on presence / absence of specific amplicons. In addition, PCR-restriction enzyme analysis (PRA) and DNA sequence analysis of the 16S-23S internal transcribed spacer (ITS) region and a 441 bp fragment of the hsp65 gene demonstrated the limitations of multiplex PCR (and commercial line probe assays) to differentiate among the species of the M. fortuitum complex. However DNA sequence analysis of the hsp65 gene fragment was found to reliably identify M. fortuitum from closely related species, M. conceptionense and M. senegalense. Reliable identification of novel species (or very similar species) of aquatic mycobacteria requires more extensive DNA sequence comparisons. Thus, multigene (polygenetic) analyses, as used here, provide rapid, accurate and reliable species identification of aquatic mycobacteria. Furthermore, a number of novel species of aquatic mycobacteria, M. stomatepiae, ‘M. angelicum’, ‘M. aemonae’ and M. salmoniphilum were discovered using the polygenetic analysis approach. Correct identification of Mycobacterium species by DNA sequence comparisons relies on accurate database information. Difficulties in this study in assigning M. marine and M. gordonae to their correct taxa suggest errors in the current public sequence repositories. The above methods were successfully applied to detect and identify mycobacteria in field samples including formalin-fixed, paraffin-embedded (FFPE) fish tissue, water and frozen fish tissue.
    No preview · Article · Jan 2008
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Mycobacterium marinum, a close relative of Mycobacterium tuberculosis is being used to study mycobacterial pathogenesis. M. marinum causes a systemic tuberculosis-like infection and disease in ectotherms such as frogs and fish. This review describes the development of M. marinum as a model pathogen and the more recent development of genetically tractable model hosts, namely the zebrafish, Drosophila and Dictyostellium to dissect the complex host-pathogen interactions that lead to tuberculosis.
    Preview · Article · Jan 2004 · Current science
Show more