Biology subjects

Nowack, E. C. M.

Publications and source records attributed to Nowack, E. C. M..

7 recordsLinked to original sources

bioRxiv2026

A step toward synthetic endosymbiosis: introducing Escherichia coli and the trypanosomatid endosymbiont Candidatus Kinetoplastibacterium crithidii into mammalian cells using a polyethylene glycol-based fusion protocol

Synthetic endosymbiosis, the deliberate introduction of bacteria into eukaryotic host cells, provides a tractable model for how endosymbiotic relationships become established, and a route to cells with new metabolic capabilities that never evolved naturally in that lineage. Current methods for introducing bacteria into mammalian cells rely on microinjection, engineered invasion, listeriolysin-mediated phagosomal escape, or naturally permissive hosts. These methods are invasive, low-throughput, or costly, and most require the prospective endosymbiont to be genetically tractable, which excludes endosymbiotic bacteria which are of great interest as potential organelle precursors. Here we describe a protocol based on polyethylene glycol (PEG), long used to fuse mammalian cells, that delivers Escherichia coli into HeLa cells without genetic modification of either partner or the use of specialised equipment. We optimised the governing parameters in two stages: first, bacterial density, gentamicin concentration, and PEG molecular weight and concentration were optimised; then, medium composition and the timing of fusion and recovery. Across four parameters screened, 10% PEG-3350 applied for 4 min to HeLa cells 12 h after seeding, followed by 4 h in gentamicin-containing medium, gave the most consistent delivery. Under these conditions, confocal microscopy and z-stack reconstruction detected mCherry-tagged E. coli inside HeLa cells. Applying the same protocol to Candidatus Kinetoplastibacterium crithidii, a naturally occurring {beta}-proteobacterial endosymbiont of the trypanosomatid Angomonas deanei, we observed the endosymbiont inside the HeLa cells. This protocol offers an accessible entry point for constructing artificial endosymbioses.

synthetic biology↗

bioRxiv2025

A novel host-encoded protein, ETP2, plays an important role in endosymbiont division in the trypanosomatid Angomonas deanei

A single {beta}-proteobacterial endosymbiont, Candidatus Kinetoplastibacterium crithidii, resides in the cytosol of the trypanosomatid Angomonas deanei, and divides at a defined stage of its hosts cell cycle. This endosymbiont has a highly reduced genome of 0.8 Mb and, notably, has lost most essential genes involved in bacterial cell division, resulting in a loss of division autonomy. It has been previously demonstrated that a host-encoded dynamin-like protein, ETP9, plays an indispensable role in the division of the endosymbiont. In this study, we identified a second nucleus-encoded component of the endosymbiont division machinery, termed ETP2, currently annotated as a hypothetical protein. We observed that ETP2 localizes in a cell cycle-dependent manner at the bacterial division site, alongside the bacterium-encoded FtsZ. Furthermore, we demonstrated that ETP2 plays an important role for timely endosymbiont division and segregation, as a large fraction of cells in an etp2 deletion mutant cell line exhibited either long, filamentous endosymbionts accompanied by severely distorted host cells or host daughter cells lacking endosymbionts. In silico analyses revealed that ETP2 is found exclusively in endosymbiont-harboring trypanosomatids and is most likely an intrinsically disordered protein. Collectively, our data suggests that ETP2, alongside the previously described ETP9, is an integral component of the endosymbiont division machinery. This finding highlights the evolution of a complex host-derived molecular mechanism that exerts tight control over its endosymbiont without requiring gene transfers from the bacterium.

evolutionary biology↗

bioRxiv2025

T7 RNA polymerase-based gene expression from a transcriptionally silent rDNA spacer in the endosymbiont-harboring trypanosomatid Angomonas deanei

Eukaryotic life has been shaped fundamentally by the integration of bacterial endosymbionts. The trypanosomatid Angomonas deanei that contains a {beta}-proteobacterial endosymbiont, represents an emerging model to elucidate initial steps in symbiont integration. Although the repertoire of genetic tools for A. deanei is growing, no conditional gene expression system is available yet, which would be key for the functional characterization of essential or expression of toxic proteins. Development of a conditional expression system based on endogenous RNA polymerase II (POLII) is hampered by the absence of information on transcription signals in A. deanei as well as the unusual genetic system used in the Trypanosomatidae that relies on read-through transcription. This mode of transcription can result in polar effects when manipulating expression of genes in their endogenous loci. Finally, only few resistance markers are available for A. deanei yet, restricting the number of genetic modifications that can be introduced into one strain. To increase the range of possible genetic manipulations in A. deanei, and in particular, build the base for a conditional expression system that does not interfere with the endogenous gene expression machinery, here we (i) implemented two new drug resistance markers, (ii) identified the spacer upstream of the rDNA array on chromosome 13 as transcriptionally silent genomic locus, and (iii) used this locus for engineering an ectopic expression system that depends on the T7 RNA polymerase expressed from the {delta}-amastin locus. We show that transgene expression in this system is independent of the activity of endogenous RNA polymerases, reaches expression levels similar to the previously described POLII-dependent expression from the {gamma}-amastin locus, and can be applied for studying endosymbiosis. In sum, the new tools expand the possibilities for genetic manipulations of A. deanei and provide a solid base for the development of an ectopic conditional expression system.

genetics↗

bioRxiv2025

The Paulinella chromatophore transit peptide part2 adopts a structural fold similar to the γ-glutamyl-cyclotransferase fold

The chromatophores of the cercozoan amoeba Paulinella are photosynthetic organelles that evolved from a cyanobacterial endosymbiont. Many nucleus-encoded chromatophore-targeted proteins carry unusual N-terminal targeting signals termed crTPs. The crTPs are bipartite. Whereas crTPpart1 that likely mediates trafficking through the secretory pathway is cleaved off during import, crTPpart2 remains attached to its cargo protein. The function of crTPpart2 is unknown. To contribute to unravel the functional role of crTPpart2, here we elucidated the structures of crTPpart2 from two different chromatophore-targeted proteins by X-ray crystallography at [~]2.3 [A] resolution. Interestingly, the crTPpart2 of both proteins adopts a structural fold. Both structures share a conserved structured core and a flexible N-terminal arm. The structured core resembles proteins of the {gamma}-glutamyl cyclotransferase superfamily within which crTPpart2 structures form a novel protein (sub)-family. The proposed catalytic center typical for proteins with cyclotransferase activity is not conserved in crTPpart2. A Cys pair that is conserved in crTPpart2 of many chromatophore-targeted proteins has been captured as disulfide bridge. Together, our data suggests that chromatophore-targeted proteins are imported in their folded state and that the fold adopted by crTPpart2 plays a functional role during import. The characterization of its structure and flexibility provides important steps towards elucidating this novel protein translocation mechanism.

microbiology↗

bioRxiv2021

A bipartite chromatophore transit peptide and N-terminal processing of protein in the Paulinella chromatophore

The cercozoan amoeba Paulinella chromatophora contains photosynthetic organelles - termed chromatophores - that evolved from a cyanobacterium [~]100 million years ago, independently from plastids in plants and algae. Despite its more recent origin, at least one third of the chromatophore proteome consists of nucleus-encoded proteins that are imported by an unknown mechanism across the chromatophore double envelope membranes. Chromatophore-targeted proteins fall into two classes. Proteins exceeding 250 amino acids carry a conserved N-terminal sequence extension, termed the chromatophore transit peptide (crTP), that is presumably involved in guiding these proteins into the chromatophore. Short imported proteins do not carry discernable targeting signals. To explore whether the import of protein is accompanied by their N-terminal processing, here we used a mass spectrometry-based approach to determine protein N-termini in Paulinella chromatophora and identified N-termini of 208 chromatophore-localized proteins. Our study revealed extensive N-terminal modifications by acetylation and proteolytic processing in both, the nucleus and chromatophore-encoded fraction of the chromatophore proteome. Mature N-termini of 37 crTP-carrying proteins were identified, of which 30 were cleaved in a common processing region. Our results imply that the crTP mediates trafficking through the Golgi, is bipartite and surprisingly only the N-terminal third ( part 1) becomes cleaved upon import, whereas the rest ( part 2) remains at the mature proteins. In contrast, short imported proteins remain largely unprocessed. Finally, this work sheds light on N-terminal processing of proteins encoded in an evolutionary-early-stage photosynthetic organelle and suggests host-derived post-translationally acting factors involved in dynamic regulation of the chromatophore-encoded chromatophore proteome. One sentence summaryProteins targeted to the evolutionary-early-stage photosynthetic organelle of Paulinella carry a bipartite N-terminal targeting sequence that is only partially removed upon protein import.

evolutionary biology↗

bioRxiv2021

Evolution of a putative, host-derived endosymbiont division ring and symbiosis-induced proteome rearrangements in the trypanosomatid Angomonas deanei

The transformation of endosymbiotic bacteria into genetically integrated organelles was central to eukaryote evolution. During organellogenesis, control over endosymbiont division, proteome composition, and physiology largely shifted from the endosymbiont to the host cell nucleus. However, to understand the order and timing of events underpinning organellogenesis novel model systems are required. The trypanosomatid Angomonas deanei contains a {beta}-proteobacterial endosymbiont that divides synchronously with the host1, contributes essential metabolites to host cell metabolism2-5, and transferred one bacterial gene [encoding an ornithine cyclodeaminase (OCD)] to the nucleus2. However, the molecular mechanisms mediating the intricate host/symbiont interactions are largely unexplored. Here we identified seven nucleus-encoded proteins by protein mass spectrometry that are targeted to the endosymbiont. Expression of fluorescent fusion proteins revealed recruitment of these proteins to specific sites within the endosymbiont including its cytoplasm and a ring-shaped structure surrounding its division site. This structure remarkably resembles in shape and predicted functions mitochondrial and plastid division machineries. The endosymbiotic gene transfer-derived OCD localizes to glycosomes instead of being retargeted to the endosymbiont. Hence, scrutiny of protein re-localization patterns that are induced by endosymbiosis, yielded profound insights into how an endosymbiotic relationship can stabilize and deepen over time far beyond the level of metabolite exchange.

microbiology↗

bioRxiv2020

The puzzle of metabolite exchange and identification of putative octotrico peptide repeat expression regulators in the nascent photosynthetic organelles of Paulinella chromatophora

The cercozoan amoeba Paulinella chromatophora contains photosynthetic organelles - termed chromatophores - that evolved from a cyanobacterium, independently from plastids in plants and algae. Despite the more recent origin of the chromatophore, it shows tight integration into the host cell. It imports hundreds of nucleus-encoded proteins, and diverse metabolites are exchanged across the two chromatophore envelope membranes. However, the limited set of chromatophore-encoded transporters appears insufficient for supporting metabolic connectivity or protein import. Furthermore, chromatophore-localized biosynthetic pathways as well as multiprotein complexes include proteins of dual genetic origin, suggesting coordination of gene expression levels between chromatophore and nucleus. These findings imply that similar to the situation in mitochondria and plastids, nuclear factors evolved that control metabolite exchange and gene expression in the chromatophore. Here we show by mass spectrometric analyses of enriched insoluble protein fractions that, unexpectedly, nucleus-encoded transporters are not inserted into the chromatophore inner envelope membrane. Thus, despite the apparent maintenance of its barrier function, canonical metabolite transporters are missing in this membrane. Instead we identified several expanded groups of short chromatophore-targeted orphan proteins. Members of one of these groups are characterized by a single transmembrane helix, and others contain amphipathic helices. We hypothesize that these proteins are involved in modulating membrane permeability. Furthermore, we identified an expanded family of chromatophore-targeted helical repeat proteins. These proteins show similar domain architectures as known organelle-targeted octotrico peptide repeat expression regulators in algae and plants suggesting their convergent evolution as nuclear regulators of gene expression levels in the chromatophore. ImportanceThe endosymbiotic acquisition of mitochondria and plastids >1 billion years ago was central for the evolution of eukaryotic life. However, owing to their ancient origin, these organelles provide only limited insights into the initial stages of organellogenesis. The chromatophore in Paulinella evolved ~100 million years ago and thus, offers the possibility to gain valuable insights into early stages and common rules in organelle evolution. Critical to organellogenesis appears to be the establishment of nuclear control over metabolite exchange and gene expression in the endosymbiont. Here we show that the mechanism generating metabolic connectivity of the chromatophore fundamentally differs from the one for mitochondria and plastids, but likely rather resembles the poorly understood mechanism in various bacterial endosymbionts in plants and insects. Furthermore, we describe a novel class of chromatophore-targeted helical repeat proteins which evolved convergently to plastid-targeted expression regulators and are likely involved in gene expression control in the chromatophore.

evolutionary biology↗