fortuitum may represent an evolutionary intermediate stage between saprophytic mycobacteria like M. smegmatis and the highly pathogenic slow-growing mycobacteria. Conclusion Our study provides detailed information about porin genes of the mspA class in M. fortuitum and their importance for the

Ferrostatin-1 research buy growth rate and susceptibility to antibiotics. Our future studies will concentrate on the elucidation of the role of PorM1 and PorM2 in survival and replication of phagocytosed M. fortuitum. Methods Bacterial strains, cell lines and plasmids Mycobacterial strains (Table 3) were grown in Middlebrook 7H9 medium (BD Biosciences, Heidelberg, Germany), supplemented with 0.05% Tween 80 selleck kinase inhibitor and either ADC (BD Biosciences) or DC (2 g glucose, 0.85 g NaCl, in 100 ml H2O) at 37°C without shaking, or on Mycobacteria 7H11 agar (BD Biosciences), supplemented with ADC (BD Biosciences). For selection of recombinant mycobacteria, media were supplemented when required with 25 to 100 μg ml-1 kanamycin or 100 μg ml-1 hygromycin B.

E. coli DH5α was grown in LB medium at 37°C [35]. Media were supplemented with 100 μg ml-1 kanamycin or 200 μg ml-1 hygromycin B for selection of recombinant E. coli. All plasmids used in this study are described in Table 4. Table 3 Mycobacterial strains used in this work. Strains Characteristics Reference M. smegmatis SMR5 M. smegmatis mc2155 derivative, SMR [42] M. smegmatis ML10 SMR5 derivative, ΔmspA and ΔmspC [4] M. fortuitum DSM 46621 Type strain; HYGR   M. fortuitum 10851/03 Human patient isolate This study M. fortuitum 10860/03 Human patient isolate; HYGR This study M. bovis BCG Copenhagen Vaccine strain   HYG: hygromycin; SM: streptomycin Measurement of growth rates in broth culture To compare the growth rates of M. fortuitum strains, Middlebrook 7H9/DC medium was inoculated with preparatory cultures to obtain an initial OD600 of 0.02. During 16 over days, the optical

densities of the cultures were measured daily. Growth of the strains was monitored by QNZ quantification of the ATP content of the cultures with the luminescence-based kit BacTiter-Glo™ Microbial Cell Viability Assay (Promega). The luminescence was reported as relative light units (RLU) with the microplate luminometer LB96V (EG&G Berthold) [36]. Molecular biology techniques and in silico analysis Common molecular biology techniques were carried out according to standard protocols [35] or according to the recommendations of the manufacturers of kits and enzymes. Transformation of E. coli was performed according to the method of Hanahan [37]. PCR reactions were performed with the following kits: Taq DNA Polymerase (MBI Fermentas, St. Leon-Roth, Germany), BC Advantage GC Polymerase Mix (Takara Bio Europe S.A., Gennevilliers, France), BIO-X-ACT Short Mix and BIOTAQ DNA Polymerase (Bioline GmbH, Luckenwalde, Germany).