Acquisition and activity of bacterial symbionts in marine invertebrates

Chemosynthetic symbioses evolved multiple times in a wide diversity of host species and from many different bacterial lineages. The symbionts provide nutrition to the hosts by fixing CO2 into biomass using reduced inorganic compounds as energy sources. This gives the hosts a physiological advantage to colonize and thrive in nutrient poor habitats. Two key questions that have emerged in symbiosis research are 1) how do the hosts acquire their symbionts and 2) what reduced compounds can be used by the symbionts as energy source to fix CO2 into biomass. This PhD thesis consists of two parts that will each deal with one of these two fundamental questions. In the first part of this thesis, two manuscripts describe the symbiont colonization of host tissues in the deep-sea mussel Bathymodiolus from hydrothermal vents. Bathymodiolus harbors its chemosynthetic symbionts intracellularly in gill tissues and, as in all bivalves, the gills grow throughout the mussel's life. This raises the question how the newly developed gill tissues are colonized by symbionts. Symbiont colonization of newly formed gill tissues was investigated using fluorescence in situ hybridization with symbiont-specific probes on semi-thin sections of whole juveniles. In addition, posterior ends of adult gills were also analyzed, as new gill filament formation occurs here. In the smallest juveniles, symbionts had colonized a wide range of epithelial tissues, revealing a widespread distribution of symbionts in many different juvenile organs. In contrast, juveniles larger than 9 mm had symbionts only in their gills. These observations indicate an ontogenetic shift in symbiont colonization from an indiscriminate infection of almost all epithelia in early life stages to spatially restricted colonization of gills in later developmental stages of Bathymodiolus. Analyses of the posterior end of both juvenile and adult gill tissues further showed that all gill filaments except the first most recently formed 7 to 9 filaments harbored symbionts. Newly formed gill tissues of Bathymodiolus are thus initially symbiont free and only later become infected with symbionts as they extend and differentiate, suggesting a life long de novo colonization by the endosymbionts of aposymbiotic host cells. In the second part of this thesis I investigated the physiological capabilities of the symbionts of Olavius algarvensis. This marine worm lacks both a digestive and excretory system. Instead it relies on a symbiotic community of two gammaproteobacterial sulfur oxidizers, two deltaproteobacterial sulfate reducers, and a spirochete for nutrition and waste recycling. External energy sources for the symbiotic association have remained enigmatic because of extremely low concentrations of reduced sulfur compounds and organic substrates in the worms habitat. Using a metaproteomic approach and incubation experiments I showed that hydrogen (H2) and carbon monoxide (CO) are additional energy sources for the symbiosis of O. algarvensis. The finding of elevated CO and H2 concentrations in the worm s habitat further confirmed the ecological importance of both substrates for the worm symbiosis. One of the sulfur-oxidizing symbionts incorporated high amounts of CO2 into its biomass in the presence of CO, which was determined using 13C-labeled bicarbonate in the incubation medium and subsequent nanoSIMS analyses. The metaproteomic study further revealed a high expression of proteins involved in highly efficient pathways and high-affinity uptake transporters for the recycling and conservation of energy, nitrogen, and carbon sources. This indicates that the nutrient-poor nature of the worm s habitat exerted a strong selective pressure in shaping this association

High-Quality Draft Genome Sequences of Two Deltaproteobacterial Endosymbionts, Delta1a and Delta1b, from the Uncultured Sva0081 Clade, Assembled from Metagenomes of the Gutless Marine Worm Olavius algarvensis

Here, we present high-quality metagenome-assembled genome sequences of two closely related deltaproteobacterial endosymbionts from the gutless marine worm Olavius algarvensis (Annelida). The first is an improved draft genome sequence of the previously described sulfate-reducing symbiont Delta1. The second is from a closely related, recently discovered symbiont of O. algarvensis

High-Quality Draft Genome Sequences of the Uncultured Delta3 Endosymbiont (Deltaproteobacteria) Assembled from Metagenomes of the Gutless Marine Worm Olavius algarvensis

Here, we present two high-quality, draft metagenome-assembled genomes of deltaproteobacterial OalgDelta3 endosymbionts from the gutless marine worm Olavius algarvensis. Their 16S rRNA gene sequences share 98% identity with Delta3 endosymbionts of related host species Olavius ilvae (GenBank accession no. AJ620501) and Inanidrilus exumae (GenBank accession no. FM202060), for which no symbiont genomes are available

Fidelity varies in the symbiosis between a gutless marine worm and its microbial consortium

Background: Many animals live in intimate associations with a species-rich microbiome. A key factor in maintaining these beneficial associations is fidelity, defined as the stability of associations between hosts and their microbiota over multiple host generations. Fidelity has been well studied in terrestrial hosts, particularly insects, over longer macroevolutionary time. In contrast, little is known about fidelity in marine animals with species-rich microbiomes at short microevolutionary time scales, that is at the level of a single host population. Given that natural selection acts most directly on local populations, studies of microevolutionary partner fidelity are important for revealing the ecological and evolutionary processes that drive intimate beneficial associations within animal species. Results: In this study on the obligate symbiosis between the gutless marine annelid Olavius algarvensis and its consortium of seven co-occurring bacterial symbionts, we show that partner fidelity varies across symbiont species from strict to absent over short microevolutionary time. Using a low-coverage sequencing approach that has not yet been applied to microbial community analyses, we analysed the metagenomes of 80 O. algarvensis individuals from the Mediterranean and compared host mitochondrial and symbiont phylogenies based on single-nucleotide polymorphisms across genomes. Fidelity was highest for the two chemoautotrophic, sulphur-oxidizing symbionts that dominated the microbial consortium of all O. algarvensis individuals. In contrast, fidelity was only intermediate to absent in the sulphate-reducing and spirochaetal symbionts with lower abundance. These differences in fidelity are likely driven by both selective and stochastic forces acting on the consistency with which symbionts are vertically transmitted. Conclusions: We hypothesize that variable degrees of fidelity are advantageous for O. algarvensis by allowing the faithful transmission of their nutritionally most important symbionts and flexibility in the acquisition of other symbionts that promote ecological plasticity in the acquisition of environmental resources

Aufnahme und Aktivität bakterieller Symbionten in marinen Invertebraten

Chemosynthetic symbioses evolved multiple times in a wide diversity of host species and from many different bacterial lineages. The symbionts provide nutrition to the hosts by fixing CO2 into biomass using reduced inorganic compounds as energy sources. This gives the hosts a physiological advantage to colonize and thrive in nutrient poor habitats. Two key questions that have emerged in symbiosis research are 1) how do the hosts acquire their symbionts and 2) what reduced compounds can be used by the symbionts as energy source to fix CO2 into biomass. This PhD thesis consists of two parts that will each deal with one of these two fundamental questions. In the first part of this thesis, two manuscripts describe the symbiont colonization of host tissues in the deep-sea mussel Bathymodiolus from hydrothermal vents. Bathymodiolus harbors its chemosynthetic symbionts intracellularly in gill tissues and, as in all bivalves, the gills grow throughout the mussel's life. This raises the question how the newly developed gill tissues are colonized by symbionts. Symbiont colonization of newly formed gill tissues was investigated using fluorescence in situ hybridization with symbiont-specific probes on semi-thin sections of whole juveniles. In addition, posterior ends of adult gills were also analyzed, as new gill filament formation occurs here. In the smallest juveniles, symbionts had colonized a wide range of epithelial tissues, revealing a widespread distribution of symbionts in many different juvenile organs. In contrast, juveniles larger than 9 mm had symbionts only in their gills. These observations indicate an ontogenetic shift in symbiont colonization from an indiscriminate infection of almost all epithelia in early life stages to spatially restricted colonization of gills in later developmental stages of Bathymodiolus. Analyses of the posterior end of both juvenile and adult gill tissues further showed that all gill filaments except the first most recently formed 7 to 9 filaments harbored symbionts. Newly formed gill tissues of Bathymodiolus are thus initially symbiont free and only later become infected with symbionts as they extend and differentiate, suggesting a life long de novo colonization by the endosymbionts of aposymbiotic host cells. In the second part of this thesis I investigated the physiological capabilities of the symbionts of Olavius algarvensis. This marine worm lacks both a digestive and excretory system. Instead it relies on a symbiotic community of two gammaproteobacterial sulfur oxidizers, two deltaproteobacterial sulfate reducers, and a spirochete for nutrition and waste recycling. External energy sources for the symbiotic association have remained enigmatic because of extremely low concentrations of reduced sulfur compounds and organic substrates in the worms habitat. Using a metaproteomic approach and incubation experiments I showed that hydrogen (H2) and carbon monoxide (CO) are additional energy sources for the symbiosis of O. algarvensis. The finding of elevated CO and H2 concentrations in the worm s habitat further confirmed the ecological importance of both substrates for the worm symbiosis. One of the sulfur-oxidizing symbionts incorporated high amounts of CO2 into its biomass in the presence of CO, which was determined using 13C-labeled bicarbonate in the incubation medium and subsequent nanoSIMS analyses. The metaproteomic study further revealed a high expression of proteins involved in highly efficient pathways and high-affinity uptake transporters for the recycling and conservation of energy, nitrogen, and carbon sources. This indicates that the nutrient-poor nature of the worm s habitat exerted a strong selective pressure in shaping this association

High-Quality Draft Genome Sequences of the Uncultured Delta3 Endosymbiont (Deltaproteobacteria) Assembled from Metagenomes of the Gutless Marine Worm Olavius algarvensis.

Here, we present two high-quality, draft metagenome-assembled genomes of deltaproteobacterial OalgDelta3 endosymbionts from the gutless marine worm Olavius algarvensis Their 16S rRNA gene sequences share 98% identity with Delta3 endosymbionts of related host species Olavius ilvae (GenBank accession no. AJ620501) and Inanidrilus exumae (GenBank accession no. FM202060), for which no symbiont genomes are available

Symbiont-Mediated Defense against Legionella pneumophila in Amoebae

International audienceLegionella pneumophila is an important opportunistic pathogen for which environmental reservoirs are crucial for the infection of humans. In the environment, free-living amoebae represent key hosts providing nutrients and shelter for highly efficient intracellular proliferation of L. pneumophila, which eventually leads to lysis of the protist. However, the significance of other bacterial players for L. pneumophila ecology is poorly understood. In this study, we used a ubiquitous amoeba and bacterial endosymbiont to investigate the impact of this common association on L. pneumophila infection. We demonstrate that L. pneumophila proliferation was severely suppressed in Acanthamoeba castellanii harboring the chlamydial symbiont Protochlamydia amoebophila The amoebae survived the infection and were able to resume growth. Different environmental amoeba isolates containing the symbiont were equally well protected as different L. pneumophila isolates were diminished, suggesting ecological relevance of this symbiont-mediated defense. Furthermore, protection was not mediated by impaired L. pneumophila uptake. Instead, we observed reduced virulence of L. pneumophila released from symbiont-containing amoebae. Pronounced gene expression changes in the presence of the symbiont indicate that interference with the transition to the transmissive phase impedes the L. pneumophila infection. Finally, our data show that the defensive response of amoebae harboring P. amoebophila leaves the amoebae with superior fitness reminiscent of immunological memory. Given that mutualistic associations between bacteria and amoebae are widely distributed, P. amoebophila and potentially other amoeba endosymbionts could be key in shaping environmental survival, abundance, and virulence of this important pathogen, thereby affecting the frequency of human infection.IMPORTANCE Bacterial pathogens are generally investigated in the context of disease. To prevent outbreaks, it is essential to understand their lifestyle and interactions with other microbes in their natural environment. Legionella pneumophila is an important human respiratory pathogen that survives and multiplies in biofilms or intracellularly within protists, such as amoebae. Importantly, transmission to humans occurs from these environmental sources. Legionella infection generally leads to rapid host cell lysis. It was therefore surprising to observe that amoebae, including fresh environmental isolates, were well protected during Legionella infection when the bacterial symbiont Protochlamydia amoebophila was also present. Legionella was not prevented from invading amoebae but was impeded in its ability to develop fully virulent progeny and were ultimately cleared in the presence of the symbiont. This study highlights how ecology and virulence of an important human pathogen is affected by a defensive amoeba symbiont, with possibly major consequences for public health

Microorganisms with Novel Dissimilatory (Bi)Sulfite Reductase Genes Are Widespread and Part of the Core Microbiota in Low-Sulfate Peatlands ▿ †

Peatlands of the Lehstenbach catchment (Germany) house as-yet-unidentified microorganisms with phylogenetically novel variants of the dissimilatory (bi)sulfite reductase genes dsrAB. These genes are characteristic of microorganisms that reduce sulfate, sulfite, or some organosulfonates for energy conservation but can also be present in anaerobic syntrophs. However, nothing is currently known regarding the abundance, community dynamics, and biogeography of these dsrAB-carrying microorganisms in peatlands. To tackle these issues, soils from a Lehstenbach catchment site (Schlöppnerbrunnen II fen) from different depths were sampled at three time points over a 6-year period to analyze the diversity and distribution of dsrAB-containing microorganisms by a newly developed functional gene microarray and quantitative PCR assays. Members of novel, uncultivated dsrAB lineages (approximately representing species-level groups) (i) dominated a temporally stable but spatially structured dsrAB community and (ii) represented “core” members (up to 1% to 1.7% relative abundance) of the autochthonous microbial community in this fen. In addition, denaturing gradient gel electrophoresis (DGGE)- and clone library-based comparisons of the dsrAB diversity in soils from a wet meadow, three bogs, and five fens of various geographic locations (distance of ∼1 to 400 km) identified that one Syntrophobacter-related and nine novel dsrAB lineages are widespread in low-sulfate peatlands. Signatures of biogeography in dsrB-based DGGE data were not correlated with geographic distance but could be explained largely by soil pH and wetland type, implying that the distribution of dsrAB-carrying microorganisms in wetlands on the scale of a few hundred kilometers is not limited by dispersal but determined by local environmental conditions

Biphasic Metabolism and Host Interaction of a Chlamydial Symbiont

Chlamydiae are obligate intracellular bacteria comprising well-known human pathogens and ubiquitous symbionts of protists, which are characterized by a unique developmental cycle. Here we comprehensively analyzed gene expression dynamics of Protochlamydia amoebophila during infection of its Acanthamoeba host by RNA sequencing. This revealed a highly dynamic transcriptional landscape, where major transcriptional shifts are conserved among chlamydial symbionts and pathogens. Our data served to propose a time-resolved model for type III protein secretion during the developmental cycle, and we provide evidence for a biphasic metabolism of P. amoebophila during infection, which involves energy parasitism and amino acids as the carbon source during initial stages and a postreplicative switch to endogenous glucose-based ATP production. This fits well with major transcriptional changes in the amoeba host, where upregulation of complex sugar breakdown precedes the P. amoebophila metabolic switch. The biphasic chlamydial metabolism represents a unique adaptation to exploit eukaryotic host cells, which likely contributed to the evolutionary success of this group of microbes.This article is published as König L, Siegl A, Penz T, Haider S, Wentrup C, Polzin J, Mann E, Schmitz-Esser S, Domman D, Horn M. 2017. Biphasic metabolism and host interaction of a chlamydial symbiont. mSystems 2:e00202-16. doi: 10.1128/mSystems.00202-16.</p