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 specif icity protease Lon (also called La), is evolutionarily discontinuous within the order Actinomycetales. Lon homologues were identified in the fast-grow ing species Mycobacterium smegmatis, and the slow-growing species Micobacte rium avium and Mycobacterium intracellulare. However, Lon homologues were n ot detected in the slow-growing species Mycobacterium tuberculosis, Mycobac terium bovis, or Mycobacterium leprae; or in the non-mycobacterial Actinomy cetale Corynebacterium glutamica. To characterize the function of the Lon p rotease within the Actinomycetales, a viable M. smegmatis Delta lon strain was constructed, demonstrating that ion is not essential under certain cond itions. Surprisingly, ion was also dispensable in M. smegmatis cells alread y lacking intact 20S proteasome alpha- and beta-subunit genes (called prcA and prcB, respectively). Creation of the later double deletion strain (prcB A::kan Delta lon) necessitated use of a novel gene deletion strategy that d oes not require an antibiotic resistance marker. The M. smegmatis prcBA::ka n Delta lon double mutants displayed wild type (wt) growth rates and wt str ess tolerances. In addition, the M, smegmatis prcBA::kan Delta lon double m utants degraded at wt rates the broad spectrum of truncated proteins induce d 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 pr cB homologue). Overall these data suggested that mycobacterial species cont ain additional ATP-dependent proteases that have broad substrate specificit y. Consistent with this suggestion, M. smegmatis and M. tuberculosis each c ontain at least one homologue of ClpP, the proteolytic subunit common to th e ClpAP and ClpXP proteases. (C) 1999 Elsevier Science B.V. All rights rese rved.