Biology subjects

Ulbrich, M. H.

Publications and source records attributed to Ulbrich, M. H..

3 recordsLinked to original sources

bioRxiv2024

Protein aggregation is a consequence of the dormancy-inducing membrane toxin TisB in Escherichia coli

Bacterial dormancy is a valuable strategy to survive stressful conditions. Toxins from chromosomal toxin-antitoxin systems have the potential to halt cell growth, induce dormancy and eventually promote a stress-tolerant persister state. Due to their potential toxicity when overexpressed, sophisticated expression systems are needed when studying toxin genes. Here, we present an optimized plasmid expression system for toxin genes based on an artificial 5 untranslated region. We applied the system to induce expression of the toxin gene tisB from the chromosomal type I toxin- antitoxin system tisB/istR-1 in Escherichia coli. TisB is a small hydrophobic protein that targets the inner membrane, resulting in depolarization and ATP depletion. We analyzed TisB-producing cells by RNA- sequencing and revealed several genes with a role in recovery from TisB-induced dormancy, including the chaperone genes ibpB, spy and cpxP. The importance of chaperone genes suggested that TisB- producing cells are prone to protein aggregation, which was validated by an in vivo fluorescent reporter system. We moved on to show that TisB is an essential factor for protein aggregation upon DNA damage mediated by the fluoroquinolone antibiotic ciprofloxacin in E. coli wild-type cells. The occurrence of protein aggregates correlates with an extended dormancy duration, which underscores their importance for the life cycle of TisB-dependent persister cells. ImportanceProtein aggregates occur in all living cells due to misfolding of proteins. In bacteria, protein aggregation is associated with cellular inactivity, which is related to dormancy and tolerance to stressful conditions, including the exposure to antibiotics. In Escherichia coli, the membrane toxin TisB is an important factor for dormancy and antibiotic tolerance upon DNA damage mediated by the fluoroquinolone antibiotic ciprofloxacin. Here, we show that TisB provokes protein aggregation, which in turn promotes a deeper state of cellular dormancy. Our study suggests that protein aggregation is a consequence of membrane toxins with the potential to affect the duration of dormancy and the outcome of antibiotic therapy.

microbiology↗

bioRxiv2022

mRNA targeting eliminates the need for the signal recognition particle during membrane protein insertion in bacteria

Signal-sequence dependent protein targeting is essential for the spatiotemporal organization of eukaryotic and prokaryotic cells and facilitated by dedicated protein targeting factors, such as the signal recognition particle (SRP). However, targeting signals are not exclusively contained within proteins, but can also be present within mRNAs. By in vivo and in vitro assays, we show that mRNA targeting is controlled by the nucleotide content and by secondary structures within mRNAs. mRNA binding to bacterial membranes occurs independently of soluble targeting factors, but is dependent on the SecYEG-translocon and YidC. Importantly, membrane insertion of proteins translated from membrane-bound mRNAs occurs independently of the SRP pathway, while the latter is strictly required for proteins translated from cytosolic mRNAs. In summary, our data indicate that mRNA targeting acts in parallel to the canonical SRP-dependent protein targeting and serves as an alternative strategy for safeguarding membrane protein insertion when the SRP pathway is compromised.

microbiology↗

bioRxiv2020

Kappa but not delta or mu opioid receptors form homodimers at low membrane densities

Opioid receptors (ORs) have been observed as homo- and heterodimers, but it is unclear if the dimers are stable under physiological conditions, and whether monomers or dimers comprise the predominant fraction in a cell. Here we use three live-cell imaging approaches to assess dimerization of ORs at different expression levels. At high membrane densities, a split GFP assay reveals that {kappa}OR dimerizes, while OR and {delta}OR stay monomeric. In contrast, single-molecule imaging showed no {kappa}OR dimers at low receptor densities. To reconcile our seemingly contradictory results, we used a high-density single-molecule assay to assess membrane protein interactions at densities up to 100x higher than conventional single-molecule imaging. We observe that {kappa}OR is monomeric at low densities and forms dimers at densities that are considered physiological. In contrast, OR and {delta}OR stay monomeric even at the highest densities covered by our approach. The observation of long-lasting {kappa}OR dimers but not higher-order aggregates suggests that ORs dimerize through a single, specific interface.

biochemistry↗