Identification of Mycobacterium avium DNA sequences that encode exported proteins by using phoA gene fusions

Setting. Mycobacterium avium is the major cause of disseminated infection i n patients with late stage AIDS. Objective: In order to identify M. avium genes that may be involved in bact erial uptake and intracellular survival, a phoA-based reporter system was u sed to identify genes that encoded surface-expressed or exported proteins. Design: PhoA (alkaline phosphatase) is only active if the protein is export ed across the cell membrane into the periplasm. Consequently, detectable Ph oA activity requires the fusion of a promoterless phoA gene with a DNA frag ment containing a functional promoter and export leader sequence. A M. aviu m promoter library was constructed in the phoA reporter plasmid pJEM11 and screened in M. smegmatis for expression of active PhoA. Results: More than 100 independent PhoA(+) recombinants were isolated, of w hich 15 were sequenced. Most of these exhibited varying degrees of homology with published M. avium, M. tuberculosis, M. bovis and M. leprae sequences . Based on sequence homology, one M. avium sequence was identified as a hom ologue of the M. tuberculosis phosphate transport gene phoS2 (Ag88). Anothe r M. avium sequence was homolog with a putative M. tuberculosis cutinase ge ne. Both of these M. avium genes were cloned and sequenced. Several other M . avium sequences were homologous with, as yet, unidentified M. tuberculosi s genes. Conclusion: PhoA fusion technology is applicable to the study of atypical s low growing mycobacteria. Most of the M. avium exported proteins identified in this study are highly homologous with genes from M. tuberculosis and M. leprae. In addition, parallels in gene organization were identified betwee n M. avium and members of the M. tuberculosis complex. (C) 2000 Harcourt Pu blishers Ltd.