The transcription of pafA, encoding the prokaryotic ubiquitin-like protein ligase, is regulated by PafBC.

AIM: Mycobacterium tuberculosis possesses an intracellular tagging and degradation system, which has emerged as a target for development of anti-tuberculosis agents. In this system, PafA is the ligase that marks proteins for degradation by their covalent modification with a protein modifier. Here, we studied pafA transcriptional regulation, which remained elusive despite its importance for M. tuberculosis virulence. MATERIALS & METHODS: Working with Mycobacterium smegmatis, a mycobacterial model organism, we examined the involvement of the global regulators PafB and PafC in pafA regulation. RESULTS: PafBC activated pafA transcription following DNA damage, resulting in efficient cellular recovery. CONCLUSION: The results unraveled the involvement of PafBC in pafA transcription, and revealed the importance of proper PafA regulation in mycobacterial physiology.

How to control an intracellular proteolytic system: Coordinated regulatory switches in the mycobacterial Pup-proteasome system.

Intracellular proteolysis is critical for the proper functioning of all cells, owing to its involvement in a wide range of processes. Because of the destructive nature of protein degradation, intracellular proteolysis is restricted by control mechanisms at almost every step of the proteolytic process. Understanding the coordination of such mechanisms is a challenging task, especially in systems as complex as the eukaryotic ubiquitin-proteasome system (UPS). In comparison, the bacterial analog of the UPS, the Pup-proteasome system (PPS) is much simpler and, therefore, allows for insight into the control of a proteolytic system. This review integrates available information to present a coherent picture of what is known of PPS regulatory switches and describes how these switches act in concert to enforce regulation at the system level. Finally, open questions regarding PPS regulation are discussed, providing readers with a sense of what lies ahead in the field.

Assembling Glycan-Charged Dolichol Phosphates: Chemoenzymatic Synthesis of a Haloferax volcanii N-Glycosylation Pathway Intermediate.

N-glycosylation, the covalent attachment of glycans to select protein target Asn residues, is a post-translational modification performed by all three domains of life. In the halophilic archaea Haloferax volcanii, in which understanding of this universal protein-processing event is relatively well-advanced, genes encoding the components of the archaeal glycosylation (Agl) pathway responsible for the assembly and attachment of an N-linked pentasaccharide have been identified. As elsewhere, the N-linked glycan is assembled on phosphodolichol carriers before transfer to target Asn residues. However, as little is presently known of the Hfx. volcanii Agl pathway at the protein level, the seemingly unique ability of Archaea to use dolichol phosphate (DolP) as the glycan lipid carrier, rather than dolichol pyrophosphate used by eukaryotes, remains poorly understood. With this in mind, a chemoenzymatic approach was taken to biochemically study AglG, one of the five glycosyltransferases of the pathway. Accordingly, a novel regio- and stereoselective reduction of naturally isolated polyprenol gave facile access to S-dolichol via asymmetric transfer hydrogenation under very mild conditions. This compound was used to generate glucose-charged DolP, a precursor of the N-linked pentasaccharide, as well as DolP-glucose-glucuronic acid and DolP-glucuronic acid. AglG, purified from Hfx. volcanii membranes in hypersaline conditions, like those encountered in situ, was subsequently combined with uridine diphosphate (UDP)-glucuronic acid and DolP-glucose to yield DolP-glucose-glucuronic acid. The in vitro system for the study of AglG activity developed here represents the first such tool for studying halophilic glycosyltransferases and will allow for a detailed understanding of archaeal N-glycosylation.

The regulatory significance of tag recycling in the mycobacterial Pup-proteasome system.

Pup, a ubiquitin analog, tags proteins for degradation by the bacterial proteasome. As an intracellular proteolytic system, the Pup-proteasome system (PPS) must be carefully regulated to prevent excessive protein degradation. Currently, those factors underlying PPS regulation remain poorly understood. Here, experimental analysis combined with theoretical modeling of in vivo protein pupylation revealed how the basic PPS design allows stable and controlled protein pupylation. Specifically, the recycling of Pup when targets are degraded allows the PPS to maintain steady-state levels of protein pupylation and degradation at a rate limited by proteasome function, and at a pupylome level limited by Pup concentrations. This design allows the Pup-ligase, a highly promiscuous enzyme, to act in a controlled manner without causing damage, and the PPS to be effectively tuned to control protein degradation. This study thus provides understanding of how the inherent design of an intracellular proteolytic system serves crucial regulatory purposes.

Posttranslational regulation of coordinated enzyme activities in the Pup-proteasome system.

The proper functioning of any biological system depends on the coordinated activity of its components. Regulation at the genetic level is, in many cases, effective in determining the cellular levels of system components. However, in cases where regulation at the genetic level is insufficient for attaining harmonic system function, posttranslational regulatory mechanisms are often used. Here, we uncover posttranslational regulatory mechanisms in the prokaryotic ubiquitin-like protein (Pup)-proteasome system (PPS), the bacterial equivalent of the eukaryotic ubiquitin-proteasome system. Pup, a ubiquitin analog, is conjugated to proteins through the activities of two enzymes, Dop (deamidase of Pup) and PafA (proteasome accessory factor A), the Pup ligase. As Dop also catalyzes depupylation, it was unclear how PPS function could be maintained without Dop and PafA canceling the activity of the other, and how the two activities of Dop are balanced. We report that tight Pup binding and the limited degree of Dop interaction with high-molecular-weight pupylated proteins results in preferred Pup deamidation over protein depupylation by this enzyme. Under starvation conditions, when accelerated protein pupylation is required, this bias is intensified by depletion of free Dop molecules, thereby minimizing the chance of depupylation. We also find that, in contrast to Dop, PafA presents a distinct preference for high-molecular-weight protein substrates. As such, PafA and Dop act in concert, rather than canceling each other's activity, to generate a high-molecular-weight pupylome. This bias in pupylome molecular weight distribution is consistent with the proposed nutritional role of the PPS under starvation conditions.

Survival of mycobacteria depends on proteasome-mediated amino acid recycling under nutrient limitation.

Intracellular protein degradation is an essential process in all life domains. While in all eukaryotes regulated protein degradation involves ubiquitin tagging and the 26S-proteasome, bacterial prokaryotic ubiquitin-like protein (Pup) tagging and proteasomes are conserved only in species belonging to the phyla Actinobacteria and Nitrospira. In Mycobacterium tuberculosis, the Pup-proteasome system (PPS) is important for virulence, yet its physiological role in non-pathogenic species has remained an enigma. We now report, using Mycobacterium smegmatis as a model organism, that the PPS is essential for survival under starvation. Upon nitrogen limitation, PPS activity is induced, leading to accelerated tagging and degradation of many cytoplasmic proteins. We suggest a model in which the PPS functions to recycle amino acids under nitrogen starvation, thereby enabling the cell to maintain basal metabolic activities. We also find that the PPS auto-regulates its own activity via pupylation and degradation of its components in a manner that promotes the oscillatory expression of PPS components. As such, the destructive activity of the PPS is carefully balanced to maintain cellular functions during starvation.

Efficient and simple generation of unmarked gene deletions in Mycobacterium smegmatis.

Genetic research in molecular laboratories relies heavily on directed mutagenesis and gene deletion techniques. In mycobacteria, however, genetic analysis is often hindered by difficulties in the preparation of deletion mutants. Indeed, in comparison to the allelic exchange systems available for the study of other common model organisms, such as Saccharomyces cerevisiae and Escherichia coli, mycobacterial gene disruption systems suffer from low mutant isolation success rates, mostly due to inefficient homologous recombination and a high degree of non-specific recombination. Here, we present a gene deletion system that combines efficient homologous recombination with advanced screening of mutants. This novel methodology allows for gene disruption in three consecutive steps. The first step relies on the use of phage Che9c recombineering proteins for directed insertion into the chromosome of a linear DNA fragment that encodes GFP and confers hygromycin resistance. In the second step, GFP positive and hygromycin resistant colonies are selected, and in the last step, the gfp-hyg cassette is excised from the chromosome, thus resulting in the formation of an unmarked deletion. We provide a detailed gene deletion methodology and demonstrate the use of this genetic system by deleting the prcSBA operon of Mycobacterium smegmatis.

Bacterial proteasome and PafA, the pup ligase, interact to form a modular protein tagging and degradation machine.

Proteasome-containing bacteria possess a tagging system that directs proteins to proteasomal degradation by conjugating them to a prokaryotic ubiquitin-like protein (Pup). A single ligating enzyme, PafA, is responsible for Pup conjugation to lysine side chains of protein substrates. As Pup is recognized by the regulatory subunit of the proteasome, Pup functions as a degradation tag. Pup presents overlapping regions for binding of the proteasome and PafA. It was, therefore, unclear whether Pup binding by the proteasome regulatory subunit, Mpa, and by PafA are mutually exclusive events. The work presented here provides evidence for the simultaneous interaction of Pup with both Mpa and PafA. Surprisingly, we found that PafA and Mpa can form a complex both in vitro and in vivo. Our results thus suggest that PafA and the proteasome can function as a modular machine for the tagging and degradation of cytoplasmic proteins.