Biology subjects

Seifert, R.

Publications and source records attributed to Seifert, R..

10 recordsLinked to original sources

bioRxiv2026

A family of small proteins links c-di-GMP riboswitch signaling with sporulation in Clostridioides difficile

Cyclic diguanosine monophosphate (c-di-GMP) is a ubiquitous bacterial second messenger that coordinates lifestyle transitions, virulence, and developmental processes. In Clostridioides difficile, elevated c-di-GMP levels inhibit sporulation, but the underlying mechanism remained unclear. Here, we identified a conserved family of small, membrane-associated proteins encoded by c-di-GMP riboswitch-regulated genes. Transcriptomic analyses revealed that c-di-GMP represses these genes, and reporter assays demonstrated riboswitch-dependent transcriptional regulation via premature termination mechanism. Overexpression of a single member, CD1980.2, was sufficient to trigger the transcriptional activation of sporulation genes, including sigma factors and their regulons, and to increase spore formation. Conversely, sporulation efficiency decreased proportionally with the number of deleted small protein genes, and the strain lacking all seven genes displayed a severe sporulation defect, underscoring their cumulative and functionally redundant roles. Elevating c-di-GMP levels in the deletion mutant did not further reduce sporulation, supporting a model in which c-di-GMP inhibits spore formation by repressing the expression of this small-protein family. Our work establishes these small proteins, encoded by c-di-GMP riboswitch-regulated genes, as important mediators of developmental output in C. difficile. Their redundancy, conservation, and integration into riboswitch regulatory pathways highlight their central role in spore formation, a process essential for pathogen persistence and transmission. These findings expand the repertoire of components regulated by second-messenger signaling in bacterial physiology.

microbiology↗

bioRxiv2026

Genome degradation in the c-di-GMP signaling pathways and serovar-specific YhjH-YciG interactions rewired motility regulation in Salmonella Paratyphi A

Salmonella enterica serovars Typhimurium (STM) and Paratyphi A (SPA) cause clinically distinct diseases, yet the molecular bases for their different lifestyles remain incompletely understood. Genome degradation, a hallmark of typhoidal Salmonella, results in extensive pseudogenization across multiple functional pathways. Here, we investigate how such gene inactivation rewires cyclic-di-GMP (c-di-GMP) signaling and flagellar motility regulation in SPA vs. STM. We show that YhjH, a conserved phosphodiesterase (PDE), is required for motility in STM but not in SPA, despite retaining PDE activity in both serovars. We demonstrate that this functional divergence is caused by pseudogenization of ycgR in SPA, which truncates the flagellar brake protein YcgR to a nonfunctional peptide, severing the link between c-di-GMP levels and flagellar motor control. Site directed mutagenesis in the YhjH active site and ectopic expression of intact YcgR from STM that restored YhjH-dependent motility regulation in SPA, confirmed this molecular mechanism. Additionally, using a bacterial two-hybrid (BACTH) genetic screen, we identified a serovar-specific interaction between YhjH and the general stress protein YciG in SPA, but not in STM. Computational RNA folding analysis predicted substantial differences in mRNA secondary structure and stability between the SPA and STM yhjH alleles, suggesting a potential role for synonymous mutations in this serovar-specific interaction. Together, these findings reveal how genome degradation can rewire regulatory networks, uncovering a fundamental difference in motility control between typhoidal and non-typhoidal Salmonella and suggest that these differences allow SPA motility under conditions that suppress motility in STM. IMPORTANCESalmonella enterica serovars Typhimurium (STM) and Paratyphi A (SPA) cause fundamentally different diseases in humans, yet the molecular basis for their distinct lifestyles and pathogenicity remains poorly understood. Genome degradation is a hallmark of typhoidal Salmonella, but its functional consequences for regulatory networks are largely unexplored. Here, we demonstrate that pseudogenization of ycgR in SPA dismantles c-di-GMP-mediated flagellar motor control, liberating SPA from an inhibitory brake that suppresses motility in STM. Additionally, we uncover a serovar-specific interaction between the phosphodiesterase YhjH and the general stress protein YciG in SPA, demonstrating that genome degradation can drive regulatory network rewiring beyond simple gene loss. These differences in motility regulation may facilitate the systemic pathogenesis and unique lifestyle of SPA.

microbiology↗

bioRxiv2024

Traction Force Microscopy with DNA FluoroCubes

Mechanical forces at the cell-substrate interface govern processes from migration to differentiation, yet mapping these forces at high spatial resolution remains challenging. Traction force microscopy (TFM) addresses this by quantifying substrate deformations using fiducial markers, which are conventionally fluorescent beads. Here, we introduce fluorescently labeled DNA nanostructures (FluoroCubes) as alternative fiducials grafted onto polydimethylsiloxane (PDMS) substrates. Co-anchored with RGD peptides, FluoroCubes remain stably tethered, resist internalization, and enable dense, minimally perturbing labeling. This surface-functionalized platform is compatible with TIRF microscopy and leverages tunable biotin-NeutrAvidin chemistry for precise control of fiducial density. Using a modified multi-channel optical flow algorithm, we achieve improved displacement sensitivity and force reconstruction resolution compared to conventional algorithms. FluoroCube-functionalized substrates provide a reproducible, high-resolution method for traction force mapping and offer a versatile foundation for future integration with DNA-based molecular sensors to probe interfacial forces at biointerfaces.

biophysics↗

bioRxiv2024

Deciphering rapid cell signaling and control of cell motility by reverse opto-chemical engineering

Cells transform complex environmental stimuli into physiological responses. For time-varying stimuli or motile cells, the perception of the environment depends on the temporal stimulus pattern and cell motion, respectively. Here we report a concept, "reverse optochemical engineering" (ROCE), that uses temporal light patterns and photo-triggers to expose cells to virtual sensory landscapes while recording in real time their physiological responses and motor behavior. We studied cyclic-nucleotide signaling in cell lines, sperm, olfactory neurons, and cardiomyocytes. The technique can be employed for remote control of motility by light. We reprogrammed sperm from a chemotactic to a phototactic cell that is attracted towards light. The method provides new opportunities to decipher the mechanisms and signaling molecules underlying rapid cellular computations, and thus reveal the wire diagram of cellular networks.

biophysics↗

bioRxiv2021

SAVED by a toxin: Structure and function of the CRISPR Lon protease.

CRISPR antiviral defense systems such as the well-known DNA-targeting Cas9- and the more complex RNA-targeting type III systems are widespread in bacteria and archea 1, 2. The type III systems can orchestrate a complex antiviral response that is initiated by the synthesis of cyclic oligoadenylates (cOAs) upon foreign RNA recognition 3-5. These second messenger molecules bind to the CARF (CRISPR associated Rossmann-fold) domains of dedicated effector proteins that are often DNAses, RNAses, or putative transcription factors 6. The activated effectors interfere with cellular pathways of the host, inducing cell death or a dormant state of the cell that is better suited to avoid propagation of the viral attack 7, 8. Among a large set of proteins that were predicted to be linked to the type III systems 9, 10, the CRISPR-Lon protein caught our attention. The protein was predicted to be an integral membrane protein containing a SAVED-instead of a CARF-domain as well as a Lon protease effector domain. Here, we report the crystal structure of CRISPR-Lon. The protein is a soluble monomer and indeed contains a SAVED domain that accommodates cA4. Further, we show that CRISPR-Lon forms a stable complex with the 34 kDa CRISPR-T protein. Upon activation by cA4, CRISPR-Lon specifically cleaves CRISRP-T, releasing CRISPR-T23, a 23 kDa fragment that is structurally very similar to MazF toxins and is likely a sequence specific nuclease. Our results describe the first cOA activated proteolytic enzyme and provide the first example of a SAVED domain connected to a type III CRISPR defense system. The use of a protease as a means to unleash a fast response against a threat has intriguing parallels to eukaryotic innate immunity.

biochemistry↗

bioRxiv2021

Octopamine drives honeybee thermogenesis

In times of environmental change species have two options to survive: they either relocate to a new habitat or they adapt to the altered environment. Adaptation requires physiological plasticity and provides a selection benefit. In this regard, the Western honeybee (Apis mellifera) protrudes with its thermoregulatory capabilities, which enables a nearly worldwide distribution. Especially in the cold, shivering thermogenesis enables foraging as well as proper brood development and thus survival. In this study, we present octopamine signaling as a neurochemical prerequisite for honeybee thermogenesis: we were able to induce hypothermia by depleting octopamine in the flight muscles. Additionally, we could restore the ability to increase body temperature by administering octopamine. Thus we conclude, that octopamine is necessary and sufficient for thermogenesis. Moreover, we show that these effects are mediated by {beta} octopamine receptors. The significance of our results is highlighted by the fact the respective receptor genes underlie enormous selective pressure due to adaptation to cold climates. Finally, octopamine signaling in the service of thermogenesis might be a key strategy to survive in a changing environment.

neuroscience↗

bioRxiv2021

c-di-AMP signaling is required for bile salts resistance and long-term colonization by Clostridioides difficile

To cause disease, the important human enteropathogen Clostridioides difficile must colonize the gastro-intestinal tract but little is known on how this organism senses and responds to the harsh host environment to adapt and multiply. Nucleotide second messengers are signaling molecules used by bacteria to respond to changing environmental conditions. In this study, we showed for the first time that c-di-AMP is produced by C. difficile and controls the uptake of potassium, making it essential for growth. We found that c-di-AMP is involved in biofilm formation, cell wall homeostasis, osmotolerance as well as detergent and bile salt resistance in C. difficile. In a colonization mouse model, a strain lacking GdpP, a c-di-AMP degrading enzyme, failed to persist in the gut in contrast to the parental strain. We identified OpuR as a new regulator that binds c-di-AMP and represses the expression of the compatible solute transporter OpuC. Interestingly, an opuR mutant is highly resistant to a hyperosmotic or bile salt stress compared to the parental strain while an opuCA mutant is more susceptible A short exposure of C. difficile cells to bile salts resulted in a decrease of the c-di-AMP concentrations reinforcing the hypothesis that changes in membrane characteristics due to variations of the cellular turgor or membrane damages constitute a signal for the adjustment of the intracellular c-di-AMP concentration. Thus, c-di-AMP is a signaling molecule with pleiotropic effects that controls osmolyte uptake to confer osmotolerance and bile salt resistance in C. difficile and that is important for colonization of the host. One Sentence Summaryc-di-AMP is an essential regulatory molecule conferring resistance to osmotic and bile salt stresses by controlling osmolyte uptake and contributing to gut persistence in the human enteropathogen Clostridioides difficile.

microbiology↗

bioRxiv2021

A GGDEF domain serves as a spatial on-switch for a phosphodiesterase by direct interaction with a polar landmark protein

In bacteria, the monopolar localization of enzymes and protein complexes can result in a bi-modal distribution of enzyme activity between the dividing cells and heterogeneity of cellular behaviors. In Shewanella putrefaciens, the multidomain hybrid diguanylate cyclase/phosphodiesterase PdeB, which degrades the secondary messenger c-di-GMP, is located at the flagellated cell pole. Here we show how PdeB polar recruitment is mediated by direct interaction between the inactive diguanylate cyclase (GGDEF) domain of PdeB and the C-terminal FimV domain of the polar landmark protein HubP. We demonstrate that this interaction is crucial for full function of PdeB as a phosphodiesterase. Thus, the GGDEF domain serves as a spatially controlled on-switch that effectively restricts PdeBs activity to the flagellated cell pole. We further show that PdeB regulates abundance and activity of at least two crucial surface-interaction factors, the BpfA surface adhesion protein and the MSHA type IV pilus. The heterogeneity in c-di-GMP concentrations that is generated by differences in abundance and temporal polar appearance of PdeB as well as by bi-modal distribution after cell fission orchestrates the population behavior with respect to cell-surface interaction and environmental spreading. SignificancePhenotypic heterogeneity benefits the proliferation of microbial populations in changing environments. Such heterogeneity can be created by recruitment of enzymatic activity to specific cellular compartments, e.g., the cell pole. Here we show how a GGDEF domain of a multidomain phosphodiesterase has adopted the function as a spatial on-switch that is specifically activated upon direct interaction with a polar landmark protein.

microbiology↗

bioRxiv2021

External fertilization is orchestrated by a pH-regulated soluble adenylyl cyclase controlling sperm motility and chemotaxis

The reaction of CO2 with H2O to form HCO3- and H+ is one of the most important chemical equilibria in cells. In mammalian sperm, a soluble adenylyl cyclase (sAC) serves as cellular HCO3- sensor that conveys the equilibrium state via cAMP synthesis to cAMP-signaling molecules. The function of sAC and cAMP in non-mammalian sperm is largely unknown. Here, we identify sAC orthologs in sea urchin and salmon sperm that, surprisingly, are activated by alkaline pH rather than HCO3-. Two amino-acid residues required for HCO3- binding of mammalian sAC are lacking in pH-regulated sAC. Orthologs identified in ten other phyla are also lacking either one of these key residues, suggesting that pH control is widespread among non-mammalian metazoan. The pH-sensitive sAC controls several functions of sperm from external fertilizers. Upon spawning, alkalization triggers cAMP synthesis and, thereby, activates motility of quiescent sperm. Egg-derived chemoattractants also alkalize sperm and elevate cAMP, which then-modulates pacemaker HCN channels to trigger a chemotactic Ca2+ response. Finally, the sAC and the voltage- and cAMP-activated Na+/H+ exchanger sNHE mutually control each other. A picture of evolutionary significance is emerging: motility and sensory signaling of sperm from both internal and external fertilizers rely on cAMP, yet, their sAC is regulated by HCO3- or pHi, respectively. Acidification of aquatic habitats due to climate change may adversely affect pH-sensing by sAC and thereby sexual reproduction in the sea. Statement of significanceAdenylyl cyclases synthesize cAMP, a prominent cellular messenger. A bicarbonate-sensitive AC family member, soluble AC (sAC), is tied to the chemical equilibrium: H2O + CO2 {leftrightarrow} HCO3- (bicarbonate) + H+. The sAC is required for fertilization: Mammals lacking sAC are infertile and sperm immotile. We now identify a new sAC form in sperm of non-mammalian animals that reproduce in the sea. This novel sAC is activated at alkaline pH rather than bicarbonate. It controls sperm motility and chemotaxis. The switch from HCO3- to pH rests on substitution of two amino-acids, which represents an adaptation to aquatic environments low in bicarbonate. Acidification of aquatic habitats due to climate change may adversely affect sAC activity and, thereby, fertilization.

biophysics↗

bioRxiv2018

Accurate action potential inference from a calcium sensor protein through biophysical modeling

Multiphoton imaging of genetically encoded calcium indicators is routinely used to report activity from populations of spatially resolved neurons in vivo. However, since the relationship between fluorescence and action potentials (APs) is nonlinear and varies over neurons, quantitatively inferring AP discharge is problematic. To address this we developed a biophysical model of calcium binding kinetics for the indicator GCaMP6s that accurately describes AP-evoked fluorescence changes in vivo. The models physical interpretation allowed the same parameters to describe GCaMP6s binding kinetics for both in vitro binding assays and in vivo imaging. Using this model, we developed an algorithm to infer APs from fluorescence and measured its accuracy with cell-attached electrical recordings. This approach consistently inferred more accurate AP counts and times than alternative methods for firing rates from 0 to >20 Hz, while requiring less training data. These results demonstrate the utility of quantitative, biophysically grounded models for complex biological data.

neuroscience↗