Biology subjects

Rozenberg, A.

Publications and source records attributed to Rozenberg, A..

At least 19 recordsLinked to original sources

bioRxiv2026

On rRNA gene cluster variation, phylogeny and local ecological differentiation in the Jaera albifrons complex (Crustacea: Isopoda)

The Jaera albifrons species complex comprises several closely related species of intertidal marine isopods that exhibit limited morphological differentiation and varying degrees of hybridization. In this pilot study, we analyzed regions of the nuclear rRNA genes (D1-D2 and ITS1) from populations of J. albifrons, J. praehirsuta, and J. ischiosetosa to assess their utility as diagnostic markers for species identification and phylogenetic inference. Despite the species morphological distinctness and ecological differences, rRNA variability was remarkably shallow across the complex, with only few variable sites in D1-D2 and ITS1. This contrasted sharply with high interspecific divergence observed in other Jaera species, indicating recent divergence and nascent speciation within the complex. J. ischiosetosa showed the lowest intra-individual variation and was most distinct from the two other species. Overlap between J. albifrons and J. praehirsuta genotypes, consistent with known introgressive hybridization, was considerable and likely stems from introgressive hybridization. Local hybridization patterns identifiable morphologically varied across settlements with of the settlements demonstrating unusually high hybridization rates. Different settlements were found to demonstrate a range of abiotic factors impacting spatial separation of the different species and varying degrees of similarity between the species in rRNA genotypes. Phylogenetic analysis of the genus revealed that the J. albifrons complex originates from among the species of the Mediterranean species group and represents a recent migration from the Mediterranean rather than an early-diverging lineage as previously suggested. These findings highlight the challenges of using rRNA markers for species delimitation in recently diverged and hybridizing taxa and underscore variable hybridization and spatial segregation as key factors shaping coexistence in mixed populations.

genetics↗

bioRxiv2025

The bacterial schizorhodopsins: novel light-driven inward proton pumps from Antarctic Minisyncoccota (Patescibacteria) and cyanobacteria, with implications for the proton-pumping mechanism

Microbial rhodopsins represent a diverse superfamily of light-sensitive seven-transmembrane proteins with expanding phylogenetic diversity driven by advances in metagenomics. Among these, schizorhodopsins constitute a divergent family originally identified as inward proton pumps from Promethearchaeota (Asgard archaea). Here, we report that in addition to archaeal schizorhodopsins, many members of the family originate from bacteria and detail a comprehensive biophysical characterization of novel schizorhodopsins from Antarctic Minisyncoccota (Patescibacteria) and cyanobacteria, designated as paSzR and psSzR, respectively. Both proteins function as light-driven inward proton pumps, as confirmed through pH measurements in Escherichia coli cells. Laser-flash photolysis experiments identified multiple photointermediates (K, L, and M) characteristic of microbial rhodopsin photocycles, though with slower turnover rates compared to archaeal schizorhodopsins. Site-directed mutagenesis of conserved residues in the third and sixth transmembrane helices demonstrates differential structural requirements between paSzR and psSzR. Our phylogenetic reconstruction reveals that most bacterial schizorhodopsins cluster in a single lineage distinct from archaeal variants. These findings significantly expand our understanding of microbial rhodopsin diversity and provide crucial insights into alternative molecular mechanisms for light-driven proton translocation, with implications for microbial ecology in extreme environments. Statement of significanceSchizorhodopsins represent an unusual family of light-driven proteins that pump protons inwardly--opposite to the classical microbial rhodopsin pumps. Originally discovered in archaea, we revealed their unprecedented prevalence in bacteria. We characterized two novel bacterial schizorhodopsins from the Antarctic: paSzR from Minisyncoccota and psSzR from Cyanobacteriota, demonstrating their inward proton-pumping activity. Our phylogenetic reconstruction showed that most bacterial schizorhodopsins form a distinct evolutionary lineage and two exchange events between Minisyncoccota and cyanobacteria. Through targeted mutagenesis, we identified critical residues that govern spectral tuning and transport efficiency, revealing mechanistic differences between members of the family. These findings expand the known diversity of light-energy conversion mechanisms across microbial life and provide insights into alternative proton translocation strategies, with implications for understanding microbial adaptation to extreme environments.

biophysics↗

bioRxiv2025

Ontogenetic sequence of differential gene expression in predator-induced Daphnia pulex

As a reaction to the presence of predators, many Daphnia species develop morphological defenses. These defenses are phenotypically plastic traits as their production is switched off in the absence of the predator. Previous studies identified some neurohumoral factors and candidate genes that are involved in the development of the defenses. However, there is still significant uncertainty regarding the involvement of certain genes and factors, and minimal information is available about the timing of gene expression changes throughout this development. In the current study, we thus performed a candidate-independent gene expression analysis of defense development over juvenile developmental stages in D. pulex. We analyzed transcriptome responses of the microcrustaceans to the natural mix of predator-emitted compounds and also to the pure kairomone which had been identified recently and which we synthesized. With the data obtained we show that the main factor correlating with the global gene expression patterns in Daphnia is molt cycle. Influence of kairomone treatment on the level of global gene expression is evident only in certain stages. The stages generally show unique patterns of kairomone-induced gene expression. However, expression profiles are highly similar for the naturally released and the chemically synthesized kairomone in each one of the stages. A number of genes with regulatory, structural and detoxification roles are differentially expressed as a reaction to the kairomone treatment. The most consistent response was found in the expression levels of ilp-3 coding for an insulin-like peptide. Gene knockdown experiments suggest that this hormone plays a role in the production of the defense. Many of the genes responding to the kairomone treatment have no predicted function stressing the need to investigate gene functions in Daphnia.

genetics↗

bioRxiv2025

Light-harvesting by antenna-containing xanthorhodopsin from an Antarctic cyanobacterium

AbstractMicrobial rhodopsins are light-sensitive proteins vital to various phototrophic and sensory processes in microorganisms. Xanthorhodopsins, with their dual chromophore system involving retinal and carotenoids, have been predominantly studied in the extreme halophilic bacterium Salinibacter ruber and in the primitive thylakoid-less cyanobacterium Gloeobacter violaceus, where they facilitate light- driven outward proton pumping and enhanced light-harvesting. However, their distribution, binding specificity, and ecological significance in cyanobacteria remain poorly understood. Here we report widespread xanthorhodopsin genes in cyanobacterial genomes and characterize a protein from an Antarctic filamentous cyanobacterium that uniquely binds hydroxylated lutein. Through bioinformatics, spectroscopic, functional and structural assays analyses, we determine the properties and ecological role of this cyanobacterial xanthorhodopsin. Our findings highlight xanthorhodopsins role in modulating light-harvesting efficiency in cyanobacteria, particularly in extreme environments. The lutein binding and associated structural changes likely provide selective advantages for adapting to polar light conditions, contributing to cyanobacterial survival in harsh habitats.

microbiology↗

bioRxiv2025

Apusomonad rhodopsins, a new family of ultraviolet to blue light absorbing rhodopsin channels

Apusomonads are a clade of understudied sediment-dwelling bacterivorous protists sister to Opisthokonta. Recently, apusomonads have been found to show a negative phototactic response to blue light. Here, by screening available apusomonad omics data we found genes of a distinct group of microbial rhodopsins, the ApuRs. ApuRs heterologously expressed in mammalian cells absorbed near-UV/violet or blue light, suggesting that ApuRs could be involved in apusomonads photoavoidance response. Electrophysiological measurements indicate that ApuRs are anion-selective rhodopsin channels which evolved independently of the family of channelrhodopsins widespread in other unicellular eukaryotes. Among the known rhodopsin channels, ApuRs collectively demonstrate the most blue-shifted absorption spectra. In ApuRs, the channel opening is triggered by photoisomerization of the retinal from its all-trans form to 13-cis and 11-cis forms. We found that intracellular proton transfer is involved in channel opening and determined the channels open/close kinetics. These findings expand our understanding of the photobiology of heterotrophic flagellates and showcase the significance of these organisms as a source of new rhodopsin families with unanticipated functions.

biophysics↗

bioRxiv2024

Oceanic photosynthesis is directly affected by cyanophage NblA proteins

Marine picocyanobacteria are abundant photosynthetic organisms of global importance. They coexist in the ocean with cyanophages, viruses that infect cyanobacteria. Cyanophages carry many auxiliary metabolic genes acquired from their hosts that are thought to redirect host metabolism for the phages benefit1-5. One such gene is nblA which is present in multiple cyanophage families2,6-8. Under nutrient deprivation the cyanobacterial NblA is responsible for inducing proteolytic degradation of the phycobilisome9-11, the large cyanobacterial photosynthetic light harvesting complex. This increases the pool of amino acids available for essential tasks11, serving as a survival mechanism12. Ectopic expression of different cyanophage nblA genes results in host pigment protein degradation9,6. However, the benefit of the cyanophage-encoded NblA for the cyanophage and the broader impact on the host are unknown. Here, using a recently developed genetic manipulation system for marine cyanophages14, we reveal that cyanophage NblA significantly accelerates the cyanophage infection cycle, directs degradation of the host phycobilisome and other proteins and reduces host photosynthetic light harvesting efficiency. Metagenomic analysis revealed that cyanophages carrying nblA are widespread in the oceans and compose 35% and 65% of oceanic T7-like cyanophages in the surface and deep photic zones, respectively. Our results show a large benefit of the nblA gene to the cyanophage while exerting a negative effect on the host photosynthetic apparatus and host photosynthesis. These findings suggest that cyanophage nblA has an adverse global impact on light harvesting by oceanic picocyanobacteria.

microbiology↗

bioRxiv2024

Light-harvesting by antenna-containing rhodopsins in pelagic Asgard archaea

Aquatic bacterial rhodopsin proton pumps have been recently reported to utilize hydroxylated carotenoids1,2. Here, by combining a marine chromophore extract with purified archaeal rhodopsins identified in marine metagenomes, we report on light energy transfer from diverse hydroxylated carotenoids (lutein, diatoxanthin, and fucoxanthin) to heimdallarchaeial rhodopsins (HeimdallRs)3,4 from uncultured marine planktonic members of the "Ca. Kariarchaeaceae" ("Ca. Asgardarchaeota")5. These light-harvesting antennas absorb in the blue-light range and transfer energy to the green-light absorbing retinal chromophore within HeimdallRs. Furthermore, antenna enhancement of proton pumping by HeimdallRs is also observed under white-light illumination along with a carotenoid-binding-induced structural change in the protein. Our results indicate that the use of light-harvesting antennas in microbial rhodopsins is observed not only in bacteria but also in marine archaea.

microbiology↗

bioRxiv2024

Significance of extended N-terminal region and a first motif residue in a third transmembrane helix of a novel pump-like cation channelrhodopsin HulaCCR

Channelrhodopsins are light-gated ion channels consisting of seven-transmembrane helices and a retinal chromophore, which are used as popular optogenetic tools for modulating neuronal activity. Cation channelrhodopsins (CCRs), first recognized as the photoreceptors in the chlorophyte Chlamydomonas reinhardtii, have since been identified in diverse species of green algae, as well in other unicellular eukaryotes. The CCRs from non-chlorophyte species are commonly referred to as bacteriorhodopsin-like channelrhodopsins, or BCCRs, as most of them feature the three characteristic amino acid residues of the "DTD motif" in the third transmembrane helix (TM3 or helix C) matching the canonical DTD motif of the well-studied archaeal light-driven proton pump bacteriorhodopsin. Here, we report characterization of HulaCCR1, a novel BCCR identified through metatranscriptomic analysis of a unicellular eukaryotic community in Lake Hula, Israel. Interestingly, HulaCCR1 has an ETD motif in which the first residue of the canonical motif is substituted for glutamate. Electrophysiological measurements of the wild-type and a mutant with a DTD motif of HulaCCR1 suggest the critical role of the first glutamate in spectral tuning and channel gating. Additionally, HulaCCR1 exhibits long extensions at the N- and C-termini. Photocurrents recorded from a truncated variant without the signal peptide predicted at the N-terminus were diminished, and membrane localization of the truncated variant significantly decreased, indicating that the signal peptide is important for membrane trafficking of HulaCCR1. These characteristics of HulaCCR1 would be related to a new biological significance in the original unidentified species, distinct from those known for other BCCRs.

biophysics↗

bioRxiv2023

Robust Optogenetic Inhibition with Red-light-sensitive Anion-conducting Channelrhodopsins

Channelrhodopsins (ChRs) are light-gated ion channels widely used to optically activate or silence selected electrogenic cells, such as individual brain neurons. Here, we describe identifying and characterizing a set of anion-conducting ChRs (ACRs) from diverse taxa and representing various branches of the ChR phylogenetic tree. The Mantoniella squamata ACR (MsACR1) showed high sensitivity to yellow-green light ({lambda}max at 555 nm) and was further engineered for optogenetic applications. A single amino-acid substitution that mimicked red-light sensitive rhodopsins like Chrimson shifted the photosensitivity 20 nm towards red light and accelerated photocurrent kinetics. Hence, it was named red and accelerated ACR, raACR. Both wild-type and mutant are capable optical silencers at low light intensities in mouse neurons in vitro and in vivo, while raACR offers a higher temporal resolution.

biophysics↗

bioRxiv2022

In-depth characterization of apoptosis N-terminome reveals a link between caspase-3 cleavage and post-translational N-terminal acetylation

The N-termini of proteins contain information about their biochemical properties and functions. These N-termini can be processed by proteases and can undergo other co- or post-translational modifications. We have developed LATE (LysN Amino Terminal Enrichment), a method that uses selective chemical derivatization of -amines to isolate the N-terminal peptides, in order to improve N-terminome identification in conjunction with other enrichment strategies. We applied LATE alongside another N-terminomic method to study caspase-3 mediated proteolysis both in vitro and during apoptosis in cells. This has enabled us to identify many unreported caspase-3 cleavages, some of which cannot be identified by other methods. Moreover, we have found direct evidence that neo-N-termini generated by caspase-3 cleavage can be further modified by Nt-acetylation. Some of these neo-Nt-acetylation events occur in the early phase of the apoptotic process and may have a role in translation inhibition. This has provided a comprehensive overview of the caspase-3 degradome and has uncovered previously unrecognized crosstalk between post-translational Nt-acetylation and caspase proteolytic pathways.

biochemistry↗

bioRxiv2022

Energy transfer in ubiquitous rhodopsin pumps with xanthophyll antennas

Energy transfer from light-harvesting ketocarotenoids to light-driven proton pumps xanthorhodopsins has been previously demonstrated in two unique cases: an extreme halophilic bacterium1 and a terrestrial cyanobacterium2. Attempts to find carotenoids that bind and transfer energy to rhodopsin proton pumps from the abundant marine and freshwater photoheterotrophs have thus far failed3-5. Here, using functional metagenomics combined with chromophore extraction from the environment, we detected light energy transfer from the widespread hydroxylated carotenoids zeaxanthin and lutein to the retinal moiety of xanthorhodopsins and proteorhodopsins. The light-harvesting carotenoids transfer up to 42% of the harvested energy in the violet/blue-light range to the green-light absorbing retinal chromophore. Our data suggest that these antennas have a significant impact on rhodopsin phototrophy in the worlds lakes, seas and oceans.

microbiology↗

bioRxiv2022

Infection cycle and phylogeny of the Polinton-like virus Phaeocystis globosa virus virophage-14T

Virophages are small dsDNA viruses dependent on a nucleocytoplasmic large-DNA virus infection of a cellular host for replication. Putative virophages infecting algal hosts are classified together with polinton-like viruses, transposable elements widely found in algal genomes, yet the lack of isolated strains raises questions about their existence as independent entities. In this work we isolated and characterized a virophage (PgVV-14T) co-infecting Phaeocystis globosa with the Phaeocystis globosa virus-14T (PgV-14T). PgVV-14T decreases the fitness of its PgV-14T viral host, yet it does not salvage the cellular host population. We found viral-like elements resembling PgVV-14T in Phaeocystis genomes, suggesting that these virophages are capable of integrating to the cellular host genome, bridging the gap between Polinton-like viruses and virophages. This system, with a giant virus, a virophage and endogenous viral elements preying on an algal host, presents an opportunity to gain a better understanding on the evolution of eukaryotes and their viruses.

microbiology↗

bioRxiv2022

WiChR, a highly potassium selective channelrhodopsin for low-light two-photon neuronal inhibition

The electric excitability of muscle, heart and brain tissue relies on the precise interplay of Na+- and K+-selective ion channels. The involved ion fluxes are controlled in optogenetic studies using light-gated channelrhodopsins (ChRs). While non-selective cation-conducting ChRs are well-established for excitation, K+-selective ChRs (KCRs) for efficient inhibition have only recently come into reach. Here, we report the molecular analysis of recently discovered KCRs from the stramenopile Hyphochytrium catenoides and identify a novel type of hydrophobic K+-selectivity filter. Next, we demonstrate that the KCR signature motif is conserved in related stramenopile ChRs. Among them, WiChR from Wobblia lunata features an unmatched 80-fold preference for K+ over Na+, stable photocurrents under continuous illumination and a prolonged open state lifetime. Well expressed in neurons, WiChR allows two-photon inhibition at low irradiance and reduced tissue heating,_recommending WiChR as the long-awaited efficient and versatile optogenetic inhibitor.

neuroscience↗

bioRxiv2022

Proton-transporting heliorhodopsins from marine giant viruses

Rhodopsins convert light into signals and energy in animals and microbes. Heliorhodopsins (HeRs), a recently discovered new rhodopsin family, are widely present in archaea, bacteria, unicellular eukaryotes, and giant viruses, but their function remains unknown. Here we report that a viral HeR from Emiliania huxleyi virus 202 (V2HeR3) is a light-gated proton channel. V2HeR3 absorbs blue-green lights, and the active intermediate contains the deprotonated retinal Schiff base. Site-directed mutagenesis study revealed that E191 in TM6 constitutes the gate together with the retinal Schiff base. E205 and E215 form a proton accepting group of the Schiff base, whose mutations converted the protein into an outward proton pump. Three environmental viral HeRs from the same group, as well as a more distantly related HeR exhibited similar proton-transport activity, indicating that HeR functions might be diverse similarly to type-1 microbial rhodopsins. Some strains of E. huxleyi contain one HeR that is related to the viral HeRs, while its viruses EhV-201 and EhV-202 contain two and three HeRs, respectively. Except for V2HeR3 from EhV-202, none of these proteins exhibit ion-transport activity. Thus, when expressed in the E. huxleyi cell membranes, only V2HeR3 has the potential to depolarize the host cells by light, possibly to overcome the host defense mechanisms or to prevent superinfection. The neuronal activity generated by V2HeR3 suggests that it can potentially be used as an optogenetic tools, like type-1 microbial rhodopsins.

biophysics↗

bioRxiv2021

Characterization of heliorhodopsins detected via functional metagenomics in freshwater Actinobacteria, Chloroflexi and Archaea

Rhodopsins are widespread in microbes residing in diverse aquatic environments across the globe. Recently, a new unusual rhodopsin family, the heliorhodopsins (HeRs), was discovered, distributed among diverse bacteria, archaea, eukarya and even viruses. Here, using functional metagenomics on samples from Lake HaHula and Ein Afek reserve, we found and characterized ten HeRs representing divergent members of the family. The expressed HeRs absorb light in the green and yellow wavelengths and originate from Actinobacteria, Chloroflexi and Archaea. The photocycle of the HeR from Chloroflexi revealed a low accumulation of the M-intermediate that we connect to the lack of two conserved histidine residues in transmembrane helices 1 and 2 in this protein. Another of HeR, from Actinobacteria, exhibited an unusually fast photocycle (166 ms, 5 times faster than HeR-48C12). To further explore the still unresolved question of the HeR function, we performed an analysis of protein families among genes neighboring HeRs, in our clones and thousands of other microbes. This analysis revealed a putative connection between HeRs and genes involved in oxidative stress. At the same time, very few protein families were found to distinguish genes surrounding prokaryotic HeRs from those surrounding rhodopsin pumps. The strongest association was found with the DegV family involved in activation of fatty acids and uncharacterized family DUF2177, which allowed us to hypothesize that HeRs are involved in membrane lipid remodeling. This work further establishes functional metagenomics as a simple and fruitful method of screening for new rhodopsins. SignificanceThe recently discovered divergent rhodopsin family of heliorhodopsins is abundant in freshwater environments. In this study, we sampled a habitat rich in dissolved organic matter to increase our chances of finding spectrally shifted rhodopsins. Using functional metagenomics, diverse heliorhodopsins absorbing green and yellow light were discovered. The metagenomic clones originated from diverse prokaryotic groups: Actinobacteria, Chloroflexi and even Archaea, emphasizing the versatility of the E. coli expression system used. Photocycles of representative heliorhodopsins were measured and exhibited diverse kinetic characteristics. Analysis of genes neighboring heliorhodopsins in diverse prokaryotes revealed their putative connection to membrane lipid re-modeling and oxidative stress. Our findings suggest that functional metagenomics is a productive method for the discovery of new and diverse rhodopsins.

microbiology↗

bioRxiv2020

Lateral gene transfer of anion-conducting channelrhodopsins between green algae and giant viruses

Channelrhodopsins (ChRs) are algal light-gated ion channels widely used as optogenetic tools for manipulating neuronal activity 1,2. Four ChR families are currently known. Green algal 3-5 and cryptophyte 6 cation-conducting ChRs (CCRs), cryptophyte anion-conducting ChRs (ACRs) 7, and the MerMAID ChRs 8. Here we report the discovery of a new family of phylogenetically distinct ChRs encoded by marine giant viruses and acquired from their unicellular green algal prasinophyte hosts. These previously unknown viral and green algal ChRs act as ACRs when expressed in cultured neuroblastoma-derived cells and are likely involved in behavioral responses to light.

microbiology↗

bioRxiv2019

Signature activities of 20S proteasome include degradation of the ubiquitin-tag with the protein under hypoxia

Careful removal of unwanted proteins is necessary for cell survival. The primary constitutive intracellular protease is the 26S proteasome complex, often found in equilibrium with its free catalytic subcomplex- the 20S core particle. Protein degradation by 26S is tightly regulated by prior ubiquitination of substrates, whereas 20S is amenable to substrates with an unstructured segment. Differentiating their contributions to intracellular proteolysis is challenging due to their common catalytic sites. Here, by chemically synthesizing a synoptic set of homogenous ubiquitinated proteins, we ascribe signature features to 20S function and demonstrate a unique property: degrading the ubiquitin-tag along with the target protein. Cryo-EM confirms that a ubiquitinated substrate can induce asymmetric conformational changes to 20S. Mass-spectrometry of intracellular peptidome under hypoxia and in human failing heart identifies the signature properties of 20S in cells. Moreover, the ability of 20S proteasome to clear toxic proteins rapidly, contributes to better survival under these conditions.

biochemistry↗

bioRxiv2019

Evolution of resistance in vitro reveals a novel mechanism of artemisinin activity in Toxoplasma gondii.

Artemisinins are effective against a variety of parasites and provide the first line of treatment for malaria. Laboratory studies have identified several mechanisms for artemisinin resistance in Plasmodium falciparum, including mutations in Kelch13 that are associated with delayed clearance in some clinical isolates, although other mechanisms are likely involved. To explore other potential mechanisms of resistance in parasites, we took advantage of the genetic tractability of T. gondii, a related apicomplexan parasite that shows moderate sensitivity to artemisinin. Resistant populations of T. gondii were selected by culture in increasing drug concentrations and whole genome sequencing identified several non-conservative point mutations that emerged in the population and were fixed over time. Genome editing using CRISPR/Cas9 was used to introduce point mutations conferring amino acids changes in a serine protease homologous to DegP and a serine/threonine protein kinase of unknown function. Single and double mutations conferred a competitive advantage over wild type parasites in the presence of drug, despite not changing EC50 values. Additionally, the evolved resistant lines showed dramatic amplification of the mitochondrial genome, including genes encoding cytochrome b and cytochrome oxidase I. Consistent with prior studies in yeast and mammalian tumor cells that implicate the mitochondrion as a target of artemisinins, treatment of wild type parasites with artemisinin decreased mitochondrial membrane potential, and resistant parasites showed altered morphology and decreased membrane potential. These findings extend the repertoire of mutations associated with artemisinin resistance and suggest that the mitochondrion may be an important target of inhibition in T. gondii.\n\nSignificanceArtemisinins provide important therapeutic agents for treatment of malaria and have potential for use in other infections and in cancer. Their use is threatened by the potential for resistance development, so understanding their mechanism of action and identifying genetic changes that alter sensitivity are important for improving clinical outcomes. Our findings suggest that mutations in novel targets can contribute to the emergence of parasites with increased tolerance to artemisinin treatment and that such mutations can confer a fitness advantage even in the absence of a notable shift in EC50. Our findings also support the idea that inhibition of mitochondrial function may be an important target in T. gondii, as previously suggested by studies in yeast and human cancer cells.

microbiology↗