Horizontal acquisition of a patchwork Calvin cycle by symbiotic and free-living Campylobacterota (formerly Epsilonproteobacteria).

Assie A, Leisch N, Meier DV, et al. Horizontal acquisition of a patchwork Calvin cycle by symbiotic and free-living Campylobacterota (formerly Epsilonproteobacteria). The ISME journal. 2019;14(1):104-122.Most autotrophs use the Calvin-Benson-Bassham (CBB) cycle for carbon fixation. In contrast, all currently described autotrophs from the Campylobacterota (previously Epsilonproteobacteria) use the reductive tricarboxylic acid cycle (rTCA) instead. We discovered campylobacterotal epibionts ("Candidatus Thiobarba") of deep-sea mussels that have acquired a complete CBB cycle and may have lost most key genes of the rTCA cycle. Intriguingly, the phylogenies of campylobacterotal CBB cyclegenes suggest they were acquired in multiple transfers from Gammaproteobacteria closely related to sulfur-oxidizing endosymbionts associated with the mussels, as well as from Betaproteobacteria. We hypothesize that "Ca. Thiobarba" switched from the rTCA cycle to a fully functional CBB cycle during its evolution, by acquiring genes from multiple sources, including co-occurring symbionts. We also found key CBB cycle genes in free-living Campylobacterota, suggesting that the CBB cycle may be more widespread in this phylum than previously known. Metatranscriptomics and metaproteomics confirmed high expression of CBB cycle genes in mussel-associated "Ca. Thiobarba". Direct stable isotope fingerprinting showed that "Ca. Thiobarba" has typical CBB signatures, suggesting that it uses this cycle for carbon fixation. Our discovery calls into question current assumptions about the distribution of carbon fixation pathways in microbial lineages, and the interpretation of stable isotope measurements in the environment

Host-Microbe Interactions in the Chemosynthetic Riftia pachyptila Symbiosis

The deep-sea tubeworm Riftia pachyptila lacks a digestive system but completely relies on bacterial endosymbionts for nutrition. Although the symbiont has been studied in detail on the molecular level, such analyses were unavailable for the animal host, because sequence information was lacking. To identify host-symbiont interaction mechanisms, we therefore sequenced the Riftia transcriptome, which served as a basis for comparative metaproteomic analyses of symbiont-containing versus symbiont-free tissues, both under energy-rich and energy-limited conditions. Our results suggest that metabolic interactions include nutrient allocation from symbiont to host by symbiont digestion and substrate transfer to the symbiont by abundant host proteins. We furthermore propose that Riftia maintains its symbiont by protecting the bacteria from oxidative damage while also exerting symbiont population control. Eukaryote-like symbiont proteins might facilitate intracellular symbiont persistence. Energy limitation apparently leads to reduced symbiont biomass and increased symbiont digestion. Our study provides unprecedented insights into host-microbe interactions that shape this highly efficient symbiosis

Bacterial symbiont subpopulations have different roles in a deep-sea symbiosis

© The Author(s), 2021. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Hinzke, T., Kleiner, M., Meister, M., Schlueter, R., Hentschker, C., Pane-Farre, J., Hildebrandt, P., Felbeck, H., Sievert, S. M., Bonn, F., Voelker, U., Becher, D., Schweder, T., & Markert, S. Bacterial symbiont subpopulations have different roles in a deep-sea symbiosis. Elife, 10, (2021): e58371, https://doi.org/10.7554/eLife.58371.The hydrothermal vent tubeworm Riftia pachyptila hosts a single 16S rRNA phylotype of intracellular sulfur-oxidizing symbionts, which vary considerably in cell morphology and exhibit a remarkable degree of physiological diversity and redundancy, even in the same host. To elucidate whether multiple metabolic routes are employed in the same cells or rather in distinct symbiont subpopulations, we enriched symbionts according to cell size by density gradient centrifugation. Metaproteomic analysis, microscopy, and flow cytometry strongly suggest that Riftia symbiont cells of different sizes represent metabolically dissimilar stages of a physiological differentiation process: While small symbionts actively divide and may establish cellular symbiont-host interaction, large symbionts apparently do not divide, but still replicate DNA, leading to DNA endoreduplication. Moreover, in large symbionts, carbon fixation and biomass production seem to be metabolic priorities. We propose that this division of labor between smaller and larger symbionts benefits the productivity of the symbiosis as a whole.This work was supported by the German Research Foundation DFG (grant MA 6346/2–1 to SM), fellowships of the Institute of Marine Biotechnology Greifswald (TH, MM), a German Academic Exchange Service (DAAD) grant (TH), the NC State Chancellor’s Faculty Excellence Program Cluster on Microbiomes and Complex Microbial Communities (MK), the USDA National Institute of Food and Agriculture, Hatch project 1014212 (MK), the U.S. National Science Foundation (grants OCE-1131095 and OCE-1559198 to SMS), and The WHOI Investment in Science Fund (to SMS). We furthermore acknowledge support for article processing charges from the DFG (Grant 393148499) and the Open Access Publication Fund of the University of Greifswald

Metaproteomics of marine thiotrophic symbioses – analysis of host-microbe interactions and symbiont physiologies with optimized methods

Symbiotic interactions are a key element of biological systems. One powerful strategy to gain insight into these interactions, and into biological systems in general, is the analysis of proteins expressed in situ using metaproteomics. In this thesis, host-microbe interactions in two mutualistic associations between chemosynthetic sulfur-oxidizing endosymbionts and marine invertebrates, the deep-sea tubeworm Riftia pachyptila and the shallow-water clam Codakia orbicularis, were studied by adapted and optimized metaproteomics methods. The Riftia symbiosis, which inhabits hydrothermal vents in the deep sea, and in which the host completely depends on its symbiont for nutrition, has fascinated researchers for about four decades. Yet, the interaction mechanisms between both partners have been understudied so far. Additionally, while different aspects of the host’s biology have been described, a comprehensive analysis has been lacking. Moreover, although only one symbiont 16S rRNA phylotype is present in Riftia, the symbiont population of the same host expresses proteins of various redundant or opposed metabolic pathways at the same time. As the symbionts also exhibit a wide variety in size and shape, symbionts of different size might have dissimilar physiological functions, which remained as of now to be elucidated. In this thesis, we addressed both, the host-symbiont interaction mechanisms, and physiological roles of symbiont subpopulations. A comprehensive Riftia host and symbiont protein database was generated as prerequisite for metaproteomics studies by de novo sequencing the host’s transcriptome and combining it with existing symbiont protein databases. This database was then used for metaproteomics comparisons of symbiont-containing and symbiont-free Riftia tissues, to gain insights into host-symbiont interactions on the protein level. The impact of energy availability on host-symbiont interactions was studied by comparing specimens with stored sulfur (i.e., high energy availability) with specimens in which sulfur storages were depleted. We employed optimized liquid chromatography peptide separation to increase metaproteome coverage. With this analysis, we identified proteins and mechanisms likely involved in maintaining the symbiosis, under varying environmental conditions. We unraveled key interaction mechanisms, i.e.: (i) the host likely digests its symbionts using abundant digestive enzymes, and, at the same time, (ii) a considerable part of the worm’s proteome is involved in creating stable internal conditions, thus maintaining the symbiont population. Furthermore, (iii) the symbionts probably employ eukaryote-like proteins to communicate with the host. (iv) Under conditions of restricted energy availability, the host apparently increases digestion pressure on the symbiotic population to sustain itself. Riftia symbionts of different size apparently have dissimilar metabolic roles, as revealed in this thesis. We enriched symbionts of different sizes using gradient centrifugation. These enrichments were subjected to protein extraction using a protocol optimized for the small sample amount available. Metaproteomics analysis included a gel-based workflow and evaluation of the complex dataset with machine learning techniques. Based on our metaproteomics study, we propose that Riftia symbionts of different cell size correspond to dissimilar physiological differentiation stages. Smaller cells are apparently engaged in cell differentiation and host interactions. Larger cells, on the other hand, seem to be more involved in synthesis of various organic compounds. Supposedly, in large symbionts endoreduplication cycles lead to polyploidy. Our results indicate that the Riftia symbiont employs a large part of its metabolic repertoire at the same time in the stable host environment. The symbiont of the shallow-water clam Codakia orbicularis, which, like the Riftia symbiont, relies on reduced sulfur compounds as energy source and fixes inorganic carbon, is, unexpectedly, also able to fix atmospheric nitrogen, as shown by metaproteomic, genomic and biochemical analysis. Potentially, this benefits the host, as Codakia digests its symbiont and might thus supplement its diet with organic nitrogen fixed by the symbionts in addition to organic carbon in its nitrogen-poor seagrass habitat.Symbiotische Interaktionen sind ein Schlüsselelement biologischer Systeme. Eine leistungsfähige Möglichkeit, Erkenntnisse über diese Interaktionen und auch über biologische Systeme im Allgemeinen zu gewinnen, ist die Analyse von in situ exprimierten Proteinen mittels Metaproteomics. In dieser Arbeit wurden mittels adaptierter und optimierter Metaproteomics-Methoden Wirt-Mikroorganismen-Interaktionen in zwei mutualistischen Assoziationen zwischen chemosynthetischen schwefeloxidierenden Endosymbionten und marinen Invertebraten untersucht, dem Tiefsee-Röhrenwurm Riftia pachyptila und der Flaschwassermuschel Codakia orbicularis. Die Riftia-Symbiose, die Tiefsee-Hydrothermalquellen bewohnt, und in welcher der Wirt komplett abhängig von seinem Symbionten als einziger Nahrungsquelle ist, fasziniert Forscher seit über vier Jahrzehnten. Dennoch wurden Interaktionsmechanismen zwischen beiden Partnern der Symbiose bislang nur wenig untersucht. Zudem wurden einzelne Aspekte der Wirtsbiologie beschrieben, eine umfassende Analyse gab es bislang jedoch nicht. Darüber hinaus besteht die Riftia-Symbiontenpopulation zwar nur aus einem einzelnen 16S rRNA-Phylotypen, aber die Symbionten eines Wirtstieres exprimieren Proteins verschiedener redundanter oder gegenläufiger Stoffwechselwege zur selben Zeit. Da die Symbionten zudem in Größe und Form variieren, ist es möglich, dass Symbionten unterschiedlicher Größe verschiedene physiologische Funktionen haben. Dies wurde bislang nicht untersucht. In dieser Arbeit wurden sowohl Mechanismen der Wirt-Symbionten-Interaktion untersucht, als auch physiologische Rollen unterschiedlicher Symbionten-Subpopulationen. Als Grundlage für Metaproteom-Studien wurde eine kombinierte Wirts- und Symbionten-Proteindatenbank von Riftia erstellt. Dafür wurde das Wirtstranskriptom de novo sequenziert und mit existierenden Symbionten-Proteindatenbanken kombiniert. Die resultierende Datenbank wurde genutzt, um die Metaproteome von symbionten-haltigen Riftia-Wirtsgeweben mit symbiontenfreien Wirtsgeweben zu vergleichen, um Erkenntnisse über Wirt-Symbionten-Interaktionen auf Proteinebene zu gewinnen. Der Einfluss von Energieverfügbarkeit auf diese Interaktionen wurde untersucht, indem Tiere mit gespeichertem Schwefel (entsprechend hoher Energieverfügbarkeit) mit solchen verglichen wurden, in denen die Schwefelspeicher geleert waren. Um die Metaproteom-Abdeckung zu erhöhen, wurde eine optimierte Peptidseparation mittels Flüssigchromatographie genutzt. Auf diese Weise wurden Proteine und Mechanismen identifiziert, die wahrscheinlich am Erhalt der Symbiose unter unterschiedlichen Umweltbedingungen beteiligt sind. Es wurden Schlüsselmechanismen der Wirt-Symbionten-Interaktion identifiziert: (i) Der Wirt verdaut wahrscheinlich seine Symbionten mittels abundanter Verdauungsenzyme, und gleichzeitig (ii) ist anscheinend ein großer Teil der vom Wirt exprimierten Proteine daran beteiligt, stabile interne Bedingungen zu schaffen und damit die Symbiontenpopulation zu erhalten. (iii) Die Symbionten nutzen mutmaßlich eukaryoten-artige Proteine zur Kommunikation mit dem Wirt. (iv) Bei reduzierter Energieverfügbarkeit erhöht der Wirt vermutlich die Verdauung von Symbionten, um sich selbst zu erhalten. Weiters wurde in dieser Arbeit gezeigt, dass Riftia-Symbionten unterschiedlicher Größe sich wahrscheinlich in ihren physiologischen Funktionen unterscheiden. Um die Physiologie von Symbionten unterschiedlicher Größe zu untersuchen, wurden diese mittels Gradientenzentrifugation angereichert. Aus den Anreicherungen wurden Proteine unter Nutzung eines für geringe Probenmengen optimierten Protokolls extrahiert. Die Metaproteom-Analyse wurde mittels eines gelbasierten Verfahrens durchgeführt. Der resultierende komplexe Datensatz wurde mit Techniken des maschinellen Lernens ausgewertet. Nach den Ergebnissen dieser Metaproteom-Studie handelt es sich bei Symbionten unterschiedlicher Größe wahrscheinlich um physiologisch verschiedene Zelldiffenzierungs-Stadien. Kleinere Zellen teilen sich vermutlich aktiv und etablieren Interaktionen mit dem Wirt. Größere Zellen hingegen sind anscheinend primär aktiv in der Synthese verschiedener organischer Verbindungen. In größeren Symbionten scheint zudem Endoreduplikation stattzufinden, die zu Polyploidie führt. Den Ergebnissen dieser Studie zufolge nutzt der Riftia-Symbiont wahrscheinlich simultan einen Großteil seines metabolischen Potentials in der stabilen Wirtsumgebung. Der Symbiont der Flachwasser-Muschel Codakia orbicularis nutzt wie der Riftia-Symbiont reduzierte Schwefelverbindungen als Energiequelle und fixiert anorganischen Kohlenstoff. Darüber hinaus ist der Codakia-Symbiont unerwarteterweise in der Lage, atmosphärischen Stickstoff zu fixieren. Dies wurde mittels metaproteomischer, genomischer und biochemischer Analysen gezeigt. Die Stickstoff-Fixierung durch den Symbionten könnte ein Vorteil für den Wirt der in einer stickstoffarmen Umgebung lebenden Codakia-Symbiose sein, da der C. orbicularis-Wirt seine Symbionten verdaut und somit zusätzlich zu organischem Kohlenstoff auch von den Symbionten fixierten organischen Stickstoff erhalten könnte

Transcriptional response of key metabolic and stress response genes of a nuculanid bivalve, Lembulus bicuspidatus from an oxygen minimum zone exposed to hypoxia-reoxygenation

Highlights: • Transcriptional response to hypoxia-reoxygenation was studied in an OMZ bivalve. • Anaerobic glycolysis and protein quality control pathways were transcriptionally upregulated in hypoxia. • Hypoxia elevated mRNA levels of UCP2 but had no effect on thiol-dependent antioxidants. • No impact of hypoxia-reoxygenation was found on aerobic marker enzymes. • Responses of an OMZ bivalve show parallels to other hypoxia-tolerant bivalves. Abstract: Benthic animals inhabiting the edges of marine oxygen minimum zones (OMZ) are exposed to unpredictable large fluctuations of oxygen levels. Sessile organisms including bivalves must depend on physiological adaptations to withstand these conditions. However, as habitats are rather inaccessible, physiological adaptations of the OMZ margin inhabitants to oxygen fluctuations are not well understood. We therefore investigated the transcriptional responses of selected key genes involved in energy metabolism and stress protection in a dominant benthic species of the northern edge of the Namibian OMZ, the nuculanid clam Lembulus bicuspidatus,. We exposed clams to normoxia (~5.8 ml O2 l−1), severe hypoxia (36 h at ~0.01 ml O2 l−1) and post-hypoxic recovery (24 h of normoxia following 36 h of severe hypoxia). Using newly identified gene sequences, we determined the transcriptional responses to hypoxia and reoxygenation of the mitochondrial aerobic energy metabolism (pyruvate dehydrogenase E1 complex, cytochrome c oxidase, citrate synthase, and adenine nucleotide translocator), anaerobic glycolysis (hexokinase (HK), phosphoenolpyruvate carboxykinase (PEPCK), phosphofructokinase, and aldolase), mitochondrial antioxidants (glutaredoxin, peroxiredoxin, and uncoupling protein UCP2) and stress protection mechanisms (a molecular chaperone HSP70 and a mitochondrial quality control protein MIEAP) in the gills and the labial palps of L. bicuspidatus. Exposure to severe hypoxia transcriptionally stimulated anaerobic glycolysis (including HK and PEPCK), antioxidant protection (UCP2), and quality control mechanisms (HSP70 and MIEAP) in the gills of L. bicuspidatus. Unlike UCP2, mRNA levels of the thiol-dependent mitochondrial antioxidants were not affected by hypoxia-reoxygenation stress. Transcript levels of marker genes for aerobic energy metabolism were not responsive to oxygen fluctuations in L. bicuspidatus. Our findings highlight the probable importance of anaerobic succinate production (via PEPCK) and mitochondrial and proteome quality control mechanisms in responses to oxygen fluctuations of the OMZ bivalve L. bicuspidatus. The reaction of L. bicuspidatus to oxygen fluctuations implies parallels to that of other hypoxia-tolerant bivalves, such as intertidal species

Comparative proteomics of related symbiotic mussel species reveals high variability of host-symbiont interactions

© The Author(s), 2019. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Ponnudurai, R., Heiden, S. E., Sayavedra, L., Hinzke, T., Kleiner, M., Hentschker, C., Felbeck, H., Sievert, S. M., Schlüter, R., Becher, D., Schweder, T., & Markert, S. Comparative proteomics of related symbiotic mussel species reveals high variability of host-symbiont interactions. ISME Journal, 14, (2019): 649–656, doi: 10.1038/s41396-019-0517-6.Deep-sea Bathymodiolus mussels and their chemoautotrophic symbionts are well-studied representatives of mutualistic host–microbe associations. However, how host–symbiont interactions vary on the molecular level between related host and symbiont species remains unclear. Therefore, we compared the host and symbiont metaproteomes of Pacific B. thermophilus, hosting a thiotrophic symbiont, and Atlantic B. azoricus, containing two symbionts, a thiotroph and a methanotroph. We identified common strategies of metabolic support between hosts and symbionts, such as the oxidation of sulfide by the host, which provides a thiosulfate reservoir for the thiotrophic symbionts, and a cycling mechanism that could supply the host with symbiont-derived amino acids. However, expression levels of these processes differed substantially between both symbioses. Backed up by genomic comparisons, our results furthermore revealed an exceptionally large repertoire of attachment-related proteins in the B. thermophilus symbiont. These findings imply that host–microbe interactions can be quite variable, even between closely related systems.Thanks to captain, crew, and pilots of the research vessels Atlantis (ROV Jason cruise AT26–10 in 2014) and Meteor (cruise M82–3 in 2010). We thank Jana Matulla, Sebastian Grund, and Annette Meuche for excellent technical assistance during sample preparation, MS measurements in the Orbitrap Classic, and TEM imaging preparation, respectively. We appreciate Nikolaus Leisch’s help with TEM image interpretation, Inna Sokolova’s advice on bivalve physiology, and Marie Zühlke’s support during manuscript revision. RP was supported by the EU-funded Marie Curie Initial Training Network ‘Symbiomics’ (project no. 264774) and by a fellowship of the Institute of Marine Biotechnology e.V. TH was supported by the German Research Foundation DFG (grant MA 6346/2–1 to SM). The Atlantis cruise was funded by a grant of the US National Science Foundation’s Dimensions of Biodiversity program to SMS (OCE-1136727)