Species variation in ATP-dependent protein degradation:: protease profiles differ between mycobacteria and protease functions differ between Mycobacterium smegmatis and Escherichia coli

We report here that the existence of the potentially broad substrate specificity protease Lon (also called La), is evolutionarily discontinuous within the order Actinomycetales. Lon homologues were identified in the fast-growing species Mycobacterium smegmatis, and the slow-growing species Micobacterium avium and Mycobacterium intracellulare. However, Lon homologues were not detected in the slow-growing species Mycobacterium tuberculosis, Mycobacterium bovis, or Mycobacterium leprae; or in the non-mycobacterial Actinomycetale Corynebacterium glutamica. To characterize the function of the Lon protease within the Actinomycetales, a viable M. smegmatis Delta lon strain was constructed, demonstrating that ion is not essential under certain conditions. Surprisingly, ion was also dispensable in M. smegmatis cells already lacking intact 20S proteasome alpha- and beta-subunit genes (called prcA and prcB, respectively). Creation of the later double deletion strain (prcBA::kan Delta lon) necessitated use of a novel gene deletion strategy that does not require an antibiotic resistance marker. The M. smegmatis prcBA::kan Delta lon double mutants displayed wild type (wt) growth rates and wt stress tolerances. In addition, the M, smegmatis prcBA::kan Delta lon double mutants degraded at wt rates the broad spectrum of truncated proteins induced by treating cells with puromycin. This later result was in sharp contrast to those in Escherichia coli, where either Eon or hslUV single mutants are strongly impaired in their degradation of puromycyl peptides (hslV is a prcB homologue). Overall these data suggested that mycobacterial species contain additional ATP-dependent proteases that have broad substrate specificity. Consistent with this suggestion, M. smegmatis and M. tuberculosis each contain at least one homologue of ClpP, the proteolytic subunit common to the ClpAP and ClpXP proteases. (C) 1999 Elsevier Science B.V. All rights reserved.