An insertion sequence (IS901), found in pathogenic strains of Mycobacterium avium, but absent in M. avium complex isolates from patients with acquired immune deficiency syndrome (AIDS), has been isolated and sequenced. This insertion element has a nucleotide sequence of 1472 bp, with one open reading frame (ORF1), which codes for a protein of 401 amino acids. The amino acid sequence, terminal ends and target site of IS901 are similar to those of IS900, present in Mycobacterium paratuberculosis. However, the DNA sequences of these two IS elements exhibit only 60% homology, compared to a DNA homology of 98% between their respective hosts. IS901, like IS900, appears to belong to a family of related insertion elements present in actinomycetes and other bacteria. M. avium strains containing IS901 were found to be more virulent in mice than closely related strains lacking IS901. IS901 may be a useful tool for the study of the genetics of virulence in the M. avium complex and for obtaining stable integration of foreign genes into mycobacteria.

The nucleotide sequence of the unusual plasmid-mediated OXA /I-lactamase is presented, and compared with other fi-lactamases. The OXA enzyme has similar features at the presumed active site, but no other significant regions of homology with other penicillin-reactive enzymes. The active site homology may therefore represent convergent evolution of otherwise dissimilar genes.

The rational use of IS6110 fingerprinting for studies of the molecular epidemiology and evolution of Mycobacterium tuberculosis requires understanding of the dynamics of transposition. In laboratory model systems, it has been shown that transposition is context-sensitive, i.e. it is influenced by the nature of the site in which the insertion sequence is presented. Stimulation of transposition by activation of an adjacent promoter supports the hypothesis that transposition occurs more readily from transcriptionally active locations. In addition, it has been shown that transposition can be enhanced by the expression of the transposase in trans. These findings imply that the frequency of transposition will vary substantially between different strains of M. tuberculosis, and furthermore that a hitherto stable strain may develop more rapid variation due to transposition into an active site. The use of IS6110 fingerprinting for the analysis of longer-range relationships between M. tuberculosis isolates therefore needs to be interpreted with care.

We have constructed a mycobacterial integrative vector by placing two copies of the insertion sequence IS900 flanking a kanamycin-resistance gene into a 'suicide' vector unable to replicate in mycobacteria. The Mycobacterium leprae gene encoding the M. leprae 18 kDa protein was cloned between the two copies of IS900 to provide expression signals. Constructs were introduced into Mycobacterium species smegmatis, vaccae and bovis BCG by electroporation and selection for kanamycin resistance. The expression of the 18 kDa gene was analysed by Western blotting. Integration of the vector into the M. smegmatis chromosome was analysed by Southern blotting. One to five copies of the vector were detected in each transformant. The SIV gag p27 gene and the foot-and-mouth disease virus VP1 140-160 epitope were successfully cloned into the 18 kDa gene and expression in M. smegmatis was obtained.

Mycobacterium paratuberculosis has been isolated from tissue taken from patients with Crohn's disease and has been implicated in the etiology of this disease. On culture, the organisms appear initially as cell wall-deficient, spheroplast-like forms that are difficult to identify by conventional techniques. Here we examine 30 unidentified cultures by the polymerase chain reaction using primers specific for M. paratuberculosis, Mycobacterium tuberculosis, and Mycobacterium avium restriction fragment length polymorphism type A/I and also by a non-species-specific mycobacterial polymerase chain reaction. Six of these cultures, all from Crohn's disease, were shown to contain DNA from M. paratuberculosis. Cultures from both Crohn's disease and controls were found to contain mycobacterial DNA of unknown specific origin.