Molecular Microbiology (1991) 5(9), 2265-2272 \S901, a new member of a widespread class of atypical insertion sequences, is associated with pathogenicity in Mycobacterium avium Z. M. Kunze,' S. Wall,' R. Appelberg,^ M. T Silva,^ F. Portaels^ and J. J. McFadden^* ' fi/ioiecuiar Microbiology Group, Schooi of Biological Sciences, University of Surrey, Guiidford, Surrey GU2 5XH, UK. ^Centro de Cytoiogia Experimental, Universidade Do Porto, Porto, Portugal. ^institute of Tropicai Medicine, Antwerpen, Beigium. Summary An insertion sequence {\S901), found in pathogenic strains of Mycobacterium avium, but absent in Af. avium complex isolates from patients with acquired immune deficiency syndrome (AIDS), has been iso- lated and sequenced. This insertion element has a nucleotide sequence of 1472bp, with one open read- ing frame (0RF1), which codes for a protein of 401 amino acids. The amino acid sequence, terminal ends and target site of \S901 are similar to those of IS900, present in Mycobacterium paratuberculosis. How- ever, the DNA sequences of these two IS elements exhibit only 60% homoiogy, compared to a DNA homology of 98% between their respective hosts. \S901, like \S900, appears to belong to a family of related insertion eiements present in actinomycetes and other bactena. M. avium strains containing lS90r were found to be more virulent in mice than ciosely related strains lacking \S901. \S901 may be a useful tooi for the study of the genetics of virulence in the Af. avium complex and for obtaining stable integration of foreign genes into mycobacteria. Introduction The Mycobacterium avium complex contains a number of strains that are divided by restriction fragment-length polymorphism (RFLP) analysis {McFadden etai, 1987b) into at least two groups of related strains: M. avium strains comprising serotypes 1-6, 8-10 and Mycobac- terium intraceliulare strains {serotypes 7 and 11-21). M. avium causes disease (mainly tuberculosis) in birds and Received 27 March. 1991; revised 4 June. 1991, "For correspondence, Tel, (0483) 571281, ext, 2671; Fax (0483) 300374. animals and atypical tuberculosis in humans; it is also found in the environment and commonly causes oppor- tunistic infections in immunocompromised patients, par- ticularly patients with acquired immune deficiency syn- drome (AIDS) (Snider et ai, 1987). Two specific pathogens are also very closely related to M. avium, shar- ing greater than 98% DNA homology (MoFadden et ai, 1987a): Mycobacterium paratuberculosis, the causative agent of Johne's disease in ruminants (and implicated in Crohn's disease of humans {McFadden ef ai, 1987a)) and Mycobacterium lepraemurium, the causative agent of rodent leprosy. Both these species differ from M. avium in having complex growth-factor requirements (the iron chelator mycobactin for M. paratubercuiosis) and being very slow growing. However some strains of M. avium iso- lated from animals and birds may also require mycobactin for growth, particularly on initial isolation; in experimental infections these may also cause Johne's disease (Collins et ai., 1983). Previous studies have not detected any genetic differences between M. avium infecting animals and M. avium causing opportunistic infections, particu- larly in AIDS patients. However, M. paratubercuiosis has been shown to differ from M. avium in that it possesses multiple copies of the Insertion sequence IS900 in its genome. This 1.45 kb insertion sequence was isolated as a repetitive DNA element from M. paratuberculosis (McFadden etai, 1987a), and DNA sequence analysis demonstrated it to be an atypical bacterial insertion sequence that lacks terminal inverted repeats and does not generate a target-site duplication on insertion (Green etai, 1989). IS900contains a single open reading frame {ORF) coding for a 399-amino-acid protein that shows homology to proteins encoded by various insertion sequences (ISs) isolated from Streptomyces {Bruton and Chater, 1987; Henderson etai, 1989; 1990; Leskiw etai, 1990). A recently discovered insertion sequence. IS 1000, from Thermus thermophilus (Ashby and Bergquist, 1990), which also lacks terminal inverted repeats and does not produce a target-site duplication, was shown to be related to both IS n o and \S492, a similar element from Pseu- domonas atlantica (Bartiett and Silverman, 1989). Trans- position of iS900in mycobacteria has been demonstrated recently (England etai, 1991) and has been proposed as a method for obtaining stable integration of foreign genes