Inactivation of the 20S proteasome in Streptomyces lividans and its influence on the production of heterologous proteins

Abstract

Proteasomes are self-compartmentalizing proteases first discovered in eukaryotes but also occurring in archaea and in bacteria belonging to the order Actinomycetales. In bacteria, proteasomes have so far no known function. In order to evaluate the influence of the 20S proteasome on the production of heterologous proteins by Streptomyces lividans TK24, the production of a number of heterologous proteins, including soluble human tumour necrosis factor receptor II (shuTNFRII) and salmon calcitonin (sCT), was compared with the wild-type TK24, a proteasome-deficient mutant designated PRO41 and a strain complemented for the disrupted proteasome genes (strain PRO41R). S. lividans cells lacking intact proteasome genes are phenotypically indistinguishable from the wild-type or the complemented strain containing functional proteasomes. Using the expression and secretion signals of the subtilisin inhibitor of Streptomyces venezuelae CBS762.70 (Vsi) for shuTNFRII and those of tyrosinase of Streptomyces antibioticus (MelC1) for the production of sCT, both proteins were secreted in significantly higher amounts in the strain PRO41 than in the wild-type S. lividans TK24 or the complemented strain PRO41R. However, the secretion of other heterologous proteins such as shuTNFRI was not enhanced in the proteasome-deficient strain. This suggests that S. lividans TK24 can degrade some heterologous proteins in a proteasome-dependent fashion. The proteasome-deficient strain may therefore be useful for the efficient production of these heterologous proteins.

The Mycobacterium tuberculosis Proteasome Active Site Threonine Is Essential for Persistence Yet Dispensable for Replication and Resistance to Nitric Oxide

“…T. acidophilum proteasome mutants were impaired for survival post heat shock but not under normal growth conditions [38]. Deletion of the proteasome in S. lividans , S. coelicolor and M. smegmatis did not reveal phenotypic defects [39], [40], [41]. The current work proves that the 20S proteasome is not essential for growth of M. tuberculosis .…”

The Absence of Pupylation (Prokaryotic Ubiquitin-Like Protein Modification) Affects Morphological and Physiological Differentiation in Streptomyces coelicolor

“…b Y, YEME medium; R, R2YE medium. c Orthologous proteins detected as confirmed pupylated targets in mycobacterial, Rhodococcus, or Corynebacterium pupylomes (10,11,19,25,50) are underlined. Orthologous proteins identified in these organisms were obtained by a BLAST search through the MAGE genome browser (https://www.genoscope.cns.fr/agc/microscope/home/index.php).…”

“…A bacterial 20S proteasome has also been described for some bacteria, including actinobacteria such as Rhodococcus erythropolis (5), Streptomyces coelicolor (6), Frankia alni (7), Mycobacterium smegmatis (8), and Mycobacterium tuberculosis (9). Unlike its eukaryotic counterpart, the actinobacterial proteasome is not essential (8)(9)(10)(11), although its inactivation can be associated with various deleterious effects. For example, proteasome inactivation in M. tuberculosis impairs growth on agar media and reduces virulence in mice (9,10).…”

“…For example, proteasome inactivation in M. tuberculosis impairs growth on agar media and reduces virulence in mice (9,10). Conversely, proteasome mutants of Streptomyces lividans and M. smegmatis were described to be phenotypically indistinguishable from the wild-type strains under both normal and stress-inducing conditions (8,11). Proteasome inactivation in S. coelicolor has only a modest effect on cell physiology: it was reported to increase resistance to cumene hydroperoxide, an effect associated with increased levels of a haloperoxidase enzyme (12).…”

In vivo gene silencing identifies the Mycobacterium tuberculosis proteasome as essential for the bacteria to persist in mice

“…Bacterial proteasomes, which are unique to actinomycetes 14,15 , are not essential for in vitro growth of Streptomyces coelicolor, S. lividans, or M. smegmatis [16][17][18] . In contrast, prcA and prcB were predicted to be essential for optimal in vitro growth of Mtb 6 .…”