Biology subjects

Haenelt, I.

Publications and source records attributed to Haenelt, I..

6 recordsLinked to original sources

bioRxiv2026

Cell-envelope conduits enable transfer of megabase-sized double-stranded DNA between cells of the nosocomial pathogen Acinetobacter baumannii

Horizontal gene transfer in Acinetobacter baumannii has been attributed to conjugative pili, transduction, and natural transformation. Here, we describe a previously unrecognized type of chromosomal DNA exchange mediated by cell-envelope conduits that establish direct cytoplasmic continuity between neighboring A. baumannii cells. Cryo-electron tomography reveals that these conduits, which were found in multiple clinical isolates, comprise an outer membrane, a peptidoglycan layer, and an inner membrane. The ~65 nm-thick conduits contain two ~2 nm-thick filaments, which super-resolution microscopy identifies as DNA. Interstrain horizontal gene transfer assays coupled with whole-genome sequencing demonstrate the transfer and homologous recombination of chromosomal segments up to 1.1 Mbp, corresponding to as much as 27% of the A. baumannii genome. Together, these findings provide direct structural and functional evidence for a conduit-mediated large-scale chromosomal DNA exchange, thus expanding the known repertoire of horizontal gene transfer strategies that may contribute to the remarkable genomic plasticity of A. baumannii.

microbiology↗

bioRxiv2023

Sensory kinase KdpD is a tandem serine histidine kinase controlling K+ pump KdpFABC on the translational and post-transcriptional level

Two-component systems (TCSs), consisting of a histidine kinase (HK) and a response regulator, serve signal transduction in bacteria, often regulating transcription in response to environmental stimuli. Here, we identify a tandem serine histidine kinase function for KdpD, previously described as a HK of the TCS KdpDE, which controls production of the K+ pump KdpFABC. We show that KdpD additionally mediates an inhibitory serine phosphorylation of KdpFABC at high K+ levels, using not its C-terminal HK domain but an N-terminal atypical serine kinase (ASK) domain. Sequence analysis of KdpDs from different species highlights that some KdpDs comprise solely ASK and Usp domains. We show that, while Escherichia coli KdpDs ASK responds directly to K+ levels, a shorter version from Deinococcus geothermalis is controlled by second messenger cyclic di-AMP. Our findings add to the growing functional diversity of sensor kinases while simultaneously expanding the framework for regulatory mechanisms in bacterial K+ homeostasis.

microbiology↗

bioRxiv2023

Discovery of a new activator of Slack potassium channels with robust efficacy in models of histamine-independent and chronic itch

Various disorders are accompanied by histamine-independent itching, which is often resistant to the currently available therapies. In this study, we hypothesized that pharmacological activation of Slack (Kcnt1, KNa1.1), a potassium channel highly expressed in itch-sensitive sensory neurons, has therapeutic potential for the treatment of itching. Based on the Slack-activating antipsychotic drug, loxapine, we designed a series of new derivatives with improved pharmacodynamic and pharmacokinetic profiles that enabled us to validate Slack as a pharmacological target in vivo. One of these new Slack activators, compound 6, exhibited negligible dopamine D2 and D3 receptor binding, unlike loxapine. We found that compound 6 displayed potent on-target antipruritic activity in multiple mouse models of acute histamine-independent and chronic itch without motor side effects. These properties make compound 6 a lead molecule for the development of new antipruritic therapies targeting Slack.

pharmacology and toxicology↗

bioRxiv2022

Inhibited KdpFABC resides in an E1 off-cycle state

KdpFABC is a high-affinity prokaryotic K+ uptake system that forms a functional chimera between a channel-like subunit (KdpA) and a P-type ATPase (KdpB). At high K+ levels, KdpFABC needs to be inhibited to prevent excessive K+ accumulation to the point of toxicity. This is achieved by a phosphorylation of the serine residue in the TGES162 motif in the A domain of the pump subunit KdpB (KdpBS162-P). Here, we explore the structural basis of inhibition by KdpBS162 phosphorylation by determining the conformational landscape of KdpFABC under inhibiting and non-inhibiting conditions. Under turnover conditions, we identified a new inhibited KdpFABC conformation that we termed E1-P tight, which is not part of the canonical Post-Albers transport cycle of P-type ATPases. It likely represents the biochemically described stalled E1-P state adopted by KdpFABC upon KdpBS162 phosphorylation. The E1-P tight state exhibits a compact fold of the three cytoplasmic domains and is likely adopted when the transition from high-energy E1-P states to E2-P states is unsuccessful. This study represents a structural characterization of a biologically relevant off-cycle state in the P-type ATPase family and supports the emerging discussion of P-type ATPase regulation by such conformations.

molecular biology↗

bioRxiv2021

Deciphering ion transport and ATPase coupling in the intersubunit tunnel of KdpFABC

KdpFABC, a high-affinity K+ pump, combines the ion channel KdpA and the P-type ATPase KdpB to secure survival at K+ limitation. Here, we apply a combination of cryo-EM, biochemical assays, and MD simulations to illuminate the mechanisms underlying transport and the coupling to ATP hydrolysis. We unambiguously show that ions are transported via an intersubunit tunnel through KdpA and KdpB. At the subunit interface, the tunnel is constricted by a phenylalanine, which, by polarized cation-{pi} stacking, controls K+ entry into the canonical substrate binding site (CBS) of KdpB. Within the CBS, ATPase coupling is mediated by the charge distribution between an aspartate and a lysine. Interestingly, individual elements of the ion translocation mechanism of KdpFABC identified here are conserved among a wide variety of P-type ATPases from different families. This leads us to the hypothesis that KdpB might represent an early descendant of a common ancestor of cation pumps.

biophysics↗

bioRxiv2019

Regulation of lipid saturation without sensing membrane fluidity

Abstract/SummaryCells maintain membrane fluidity by regulating lipid saturation, but the molecular mechanisms of this homeoviscous adaptation remain poorly understood. Here, we have reconstituted the core machinery for sensing and regulating lipid saturation in bakers yeast to directly characterize its response to defined membrane environments. Using spectroscopic techniques and in vitro ubiquitylation, we uncover a unique sensitivity of the transcriptional regulator Mga2 to the abundance, position, and configuration of double bonds in lipid acyl chains and provide unprecedented insight into the molecular rules of membrane adaptivity. Our data challenge the prevailing hypothesis that membrane viscosity serves as the measured variable for regulating lipid saturation. Rather, we show that the signaling output of Mga2 correlates with the size of a single sensor residue in the transmembrane helix, which senses the lateral pressure and/or compressibility profile in a defined region of the membrane. Our findings suggest that membrane property sensors have evolved remarkable sensitivities to highly specific aspects of membrane structure and dynamics, thus paving the way toward the development of genetically encoded reporters for such membrane properties in the future.

biophysics↗