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

Abstract

Protein turnover is essential in all living organisms for the maintenance of normal cell physiology. In eukaryotes, most cellular protein turnover involves the ubiquitin-proteasome pathway, in which proteins tagged with ubiquitin are targeted to the proteasome for degradation. In contrast, most bacteria lack a proteasome but harbor proteases for protein turnover. However, some actinobacteria, such as mycobacteria, possess a proteasome in addition to these proteases. A prokaryotic ubiquitination-like tagging process in mycobacteria was described and was named pupylation: proteins are tagged with Pup (prokaryotic ubiquitin-like protein) and directed to the proteasome for degradation. We report pupylation in another actinobacterium, Streptomyces coelicolor. Both the morphology and life cycle of Streptomyces species are complex (formation of a substrate and aerial mycelium followed by sporulation), and these bacteria are prolific producers of secondary metabolites with important medicinal and agricultural applications. The genes encoding the pupylation system in S. coelicolor are expressed at various stages of development. We demonstrated that pupylation targets numerous proteins and identified 20 of them. Furthermore, we established that abolition of pupylation has substantial effects on morphological and metabolic differentiation and on resistance to oxidative stress. In contrast, in most cases, a proteasome-deficient mutant showed only modest perturbations under the same conditions. Thus, the phenotype of the pup mutant does not appear to be due solely to defective proteasomal degradation. Presumably, pupylation has roles in addition to directing proteins to the proteasome.

Importance: Streptomyces spp. are filamentous and sporulating actinobacteria, remarkable for their morphological and metabolic differentiation. They produce numerous bioactive compounds, including antifungal, antibiotic, and antitumor compounds. There is therefore considerable interest in understanding the mechanisms by which Streptomyces species regulate their complex physiology and production of bioactive compounds. We studied the role in Streptomyces of pupylation, a posttranslational modification that tags proteins that are then directed to the proteasome for degradation. We demonstrated that the absence of pupylation had large effects on morphological differentiation, antibiotic production, and resistance to oxidative stress in S. coelicolor. The phenotypes of pupylation and proteasome-defective mutants differed and suggest that pupylation acts in a proteasome-independent manner in addition to its role in proteasomal degradation.

FIG 1

Pupylation and proteasome genes from mycobacteria and streptomycetes. (A) Comparison of the regions containing the pupylation and proteasome genes in Mycobacterium tuberculosis (Mt), Mycobacterium smegmatis (Ms), Streptomyces coelicolor (Sc), Streptomyces avermitilis (Sa), and Streptomyces griseus (Sg). Genes represented by arrows of the same color are orthologues, except those represented by white arrows, which are not syntenic in actinobacteria. The only difference between Streptomyces strains is the presence, in some strains, of a gene encoding a putative endonuclease VII (SCO1645 in S. coelicolor and SAV_6680 in S. avermitilis). The proteins SCO1641 (a putative transporter of the MFS family), SCO1642 (a putative LacI-like repressor), and SCO1645, and their orthologues in other Streptomyces species, are not predicted to be involved in functions related to pupylation or proteasomal degradation. (B) Alignment of Pup proteins from these five species.

FIG 2

Immunoblots for the detection of pupylated proteins with anti-His₆ antibodies. (A) Proteins extracted from mycelium grown in liquid R2YE medium for the wild-type strain (lane 1), the wild-type strain harboring the empty vector pSET152 (lane 2), and the wild-type strain harboring pSET-E*-His-pup (lane 3). (B) Proteins extracted from mycelium grown in liquid YEME medium for the wild-type strain (lane 1), the wild-type strain harboring the empty vector pSET152 (lane 2), and the wild-type strain harboring pSET-E*-His-pup with the same exposure time as that for lanes 1 and 2 (lane 3) and a shorter exposure time (lane 4).

FIG 3

Morphological differentiation on solid SFM (A and B) and R2YE (C and D) media. The wild-type strain (M145), the pup mutant (Δpup), the pup mutant with the empty vector pSET152 (Δpup+pSET152), the pup mutant with pSET-E*-His-pup (Δpup+pSET-E*His-pup), and the proteasome mutant (prcB::pOJ260) are shown after 5 days of growth.

FIG 4

Scanning electron microscopy of mycelium grown on R2YE medium. Scanning electron micrographs of surface cultures grown on R2YE medium for 5 days are shown for the wild-type strain (M145), the pup mutant (Δpup), the pup mutant with the empty vector pSET152 (Δpup+pSET152), the pup mutant with pSET-E*-His-pup (Δpup+pSET-E*His-pup), and the proteasome mutant (prcB::pOJ260). The same magnification is used for all images. Bar, 10 μm.

FIG 5

Time courses of actinorhodin (ACT) and prodiginine (RED) production in liquid R2YE medium. The production of ACT and RED is shown for the wild-type strain (M145), the pup mutant (Δpup), the pup mutant with the empty vector pSET152 (Δpup + pSET152), the pup mutant with pSET-E*-His-pup (Δpup + pSET-E*His-pup), and the proteasome mutant (prcB::pOJ260).

FIG 6

Response to oxidative stress. The wild-type strain (M145), the pup mutant (Δpup), the pup mutant with the empty vector pSET152 (Δpup + pSET152), the pup mutant with pSET-E*-His-pup (Δpup + pSET-E*His-pup), and the proteasome mutant (prcB::pOJ260) were exposed to oxidative stress caused by H₂O₂ on R2YE medium. (A) Diffusion assay showing the inhibition zone caused by H₂O₂. (B) CFU counts on medium with or without H₂O₂.