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. IMPORTANCEStreptomyces 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.T urnover of cellular proteins is required in all living organisms to maintain normal cellular physiology. In eukaryotes, the ubiquitin-proteasome pathway is the major route of protein degradation and turnover (1). Ubiquitin is a 76-residue protein that can be conjugated to target proteins, marking them for degradation by the 26S proteasome complex. In addition to its role in tagging proteins for degradation...

The pupylation machinery is involved in iron homeostasis by targeting the iron storage protein ferritin

“…In mycobacteria and streptomycetes, pupylation was shown to target proteins to proteasomal degradation and to play an important role in the response to several stress conditions, such as nitrosative stress (25), oxidative stress (19), or nitrogen and carbon starvation (20), as well as in the development of cell morphology (21). However, members of other actinobacterial genera such as Corynebacterium do not harbor a prcAB-encoded proteasome; nevertheless, they possess the genes for the pupylation machinery including Pup, Dop, PafA, and Mpa/ARC.…”

“…In this view, protein degradation mediated by pupylation is assumed to recycle amino acids under several stress conditions in Mycobacterium smegmatis (20). Although pupylation was shown to target proteins to proteasome-mediated degradation, not all pupylated proteins are subject to this fate (15,21). Furthermore, the activity of the mycobacterial ATPase Mpa itself was shown to be reversibly regulated by pupylation, which renders Mpa functionally inactive (22).…”

Bacterial Proteasomes: Mechanistic and Functional Insights

“…Additionally, the production of specific antibiotics is abolished in this strain, demonstrating roles for pupylation in distinct cellular processes in this species (83). Notably, in contrast to the increased peroxide resistance of M. tuberculosis Pupproteasome mutants, degradation-defective S. coelicolor is hypersensitive to peroxide (63,83). Thus, defects in Pup-proteasome system components can result in markedly different phenotypes among bacterial genera.…”

“…Although proteasomal degradation appears to be functional in this species, several phenotypes have been identified for S. coelicolor that depend exclusively on pupylation rather than degradation. S. coelicolor pup and pafA mutants, but not 20S CP mutants, cannot properly sporulate, and they undergo changes in the production of several secondary metabolites (63,83). It is unknown how pupylation regulates these processes.…”

Recent advances in understanding Streptomyces

“…This has been reinforced by the cataloguing of pupylation/proteasome targets and the phenotypes of mutants in the system, which show changes in differentiation and antibiotic production levels 62– 64 . Presumably, controlled proteolysis is involved in the death of some hyphal compartments that accompanies stages of growth and development 56 .…”