Pupylierung in https Corynebacterium glutamicum\textit{Corynebacterium glutamicum}

Pupylation represents a post-translational, covalent protein modification by the small protein Pup. Hitherto, this mechanism has been investigated exclusively in actinobacteria, which tag certain proteins with Pup for degradation via the proteasome (Pup proteasome system). The actinobacterium $\textit{Corynebacterium glutamicum}$ possesses the genes coding for the known components of the pupylation machinery ($\textit{pup, pafA, arc}$, and $\textit{dop}$) but not for a proteasome. In the course of the present thesis, it was investigated in $\textit{C. glutamicum}$ whether pupylation is active $\textit{in vivo}$, which proteins are pupylation targets and which functions pupylation exhibits in the absence of a proteasome. Enrichment of poly-histidine-tagged Pup and subsequent MS/MS analyses revealed 55 pupylated proteins in $\textit{C. glutamicum}$, of which 60% belong to the categories „metabolism“ or „translation“. Thus, it could be shown for the first time that $\textit{C. glutamicum}$ possesses a pupylation machinery which is active $\textit{in vivo}$. In addition, an assay for $\textit{in vitro}$ pupylation of proteins was developed using recombinant Pup and the Pup ligase PafA from $\textit{C. glutamicum}$. Hereafter, this assay will provide a tool to confirm pupylation of putative targets and to perform $\textit{in vitro}$ assays with pupylated proteins. The physiological role of pupylation in $\textit{C. glutamicum}$ was investigated by characterization of deletion mutants lacking Pup (Δ$\textit{pup}$), the depupylase Dop (Δ$\textit{dop}$) or the AAA+ ATPase ARC that recognizes pupylated proteins (Δ$\textit{arc}$) under standard growth conditions and iron starvation. All mutants exhibited reduced growth in comparison to the wild type under iron starvation. The Δ$\textit{pup}$ mutant showed differences in the transcriptome and the soluble proteome in comparison the wild type under iron starvation, which can be explained by iron-dependent effects of the transcriptional regulators DtxR and RipA. Investigations of deletion mutants lacking the iron storage proteins ferritin (Δ$\textit{ftn}$) and Dps (Δ$\textit{dps}$) and genes encoding components of the pupylation machinery could show that the growth deficiency of Δ$\textit{pup}$ and Δ$\textit{arc}$ mutants can be complemented by the deletion of the iron storage proteins. Furthermore, the exchange of the pupylated lysine residue in ferritin resulted in a growth defect comparable to the $\textit{pup}$ deletion. Hence, the defects of the mutants can be explained by defective pupylation of iron storage proteins ferritin and Dps. In conclusion, pupylation could represent a novel mechanism to release iron in actinobacteria

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

The balance of sufficient iron supply and avoidance of iron toxicity by iron homeostasis is a prerequisite for cellular metabolism and growth. Here we provide evidence that, in Actinobacteria, pupylation plays a crucial role in this process. Pupylation is a posttranslational modification in which the prokaryotic ubiquitin-like protein Pup is covalently attached to a lysine residue in target proteins, thus resembling ubiquitination in eukaryotes. Pupylated proteins are recognized and unfolded by a dedicated AAA+ ATPase (Mycobacterium proteasomal AAA+ ATPase; ATPase forming ring-shaped complexes). In Mycobacteria, degradation of pupylated proteins by the proteasome serves as a protection mechanism against several stress conditions. Other bacterial genera capable of pupylation such as Corynebacterium lack a proteasome, and the fate of pupylated proteins is unknown. We discovered that Corynebacterium glutamicum mutants lacking components of the pupylation machinery show a strong growth defect under iron limitation, which was caused by the absence of pupylation and unfolding of the iron storage protein ferritin. Genetic and biochemical data support a model in which the pupylation machinery is responsible for iron release from ferritin independent of degradation

Pupylierung - ein bakterielles Pendant zur Ubiquitinylierung

Pupylation is a reversible process in which the prokaryotic ubiquitin-like protein Pup is attached to target proteins by an isopeptide bond. It occurs mainly in Actinobacteria and was discovered in Mycobacterium tuberculosis, where pupylated proteins are targeted for proteasomal degradation, thus resembling ubiquitination in eukaryotes. Interestingly, species without a proteasome like Corynebacterium glutamicum also perform pupylation. Key features of this protein modification are summarized here

The iron deficiency response of Corynebacterium glutamicum and a link to thiamine biosynthesis

The response to iron limitation of the Gram-positive soil bacterium Corynebacterium glutamicum was analyzed with respect to secreted metabolites, the transcriptome, and the proteome. During growth in glucose minimal medium, iron limitation caused a shift from lactate to pyruvate as the major secreted organic acid complemented by l-alanine and 2-oxoglutarate. Transcriptome and proteome analyses revealed that a pronounced iron starvation response governed by the transcriptional regulators DtxR and RipA was detectable in the late, but not in the early, exponential-growth phase. A link between iron starvation and thiamine pyrophosphate (TPP) biosynthesis was uncovered by the strong upregulation of thiC. As phosphomethylpyrimidine synthase (ThiC) contains an iron-sulfur cluster, limiting activities of the TPP-dependent pyruvate–2-oxoglutarate dehydrogenase supercomplex probably cause the excretion of pyruvate and 2-oxoglutarate. In line with this explanation, thiamine supplementation could strongly diminish the secretion of these acids. The upregulation of thiC and other genes involved in thiamine biosynthesis and transport is presumably due to TPP riboswitches present at the 5′ end of the corresponding operons. The results obtained in this study provide new insights into iron homeostasis in C. glutamicum and demonstrate that the metabolic consequences of iron limitation can be due to the iron dependency of coenzyme biosynthesis