Heliorhodopsin Evolution Is Driven by Photosensory Promiscuity in Monoderms

Rhodopsins are light-activated proteins displaying an enormous versatility of function as cation/anion pumps or sensing environmental stimuli and are widely distributed across all domains of life. Even with wide sequence divergence and uncertain evolutionary linkages between microbial (type 1) and animal (type 2) rhodopsins, the membrane orientation of the core structural scaffold of both was presumed universal. This was recently amended through the discovery of heliorhodopsins (HeRs; type 3), that, in contrast to known rhodopsins, display an inverted membrane topology and yet retain similarities in sequence, structure, and the light-activated response. While no ion-pumping activity has been demonstrated for HeRs and multiple crystal structures are available, fundamental questions regarding their cellular and ecological function or even their taxonomic distribution remain unresolved. Here, we investigated HeR function and distribution using genomic/metagenomic data with protein domain fusions, contextual genomic information, and gene coexpression analysis with strand-specific metatranscriptomics. We bring to resolution the debated monoderm/diderm occurrence patterns and show that HeRs are restricted to monoderms. Moreover, we provide compelling evidence that HeRs are a novel type of sensory rhodopsins linked to histidine kinases and other two-component system genes across phyla. In addition, we also describe two novel putative signal-transducing domains fused to some HeRs. We posit that HeRs likely function as generalized light-dependent switches involved in the mitigation of light-induced oxidative stress and metabolic circuitry regulation. Their role as sensory rhodopsins is corroborated by their photocycle dynamics and their presence/function in monoderms is likely connected to the higher sensitivity of these organisms to light-induced damage. IMPORTANCE Heliorhodopsins are enigmatic, novel rhodopsins with a membrane orientation that is opposite to all known rhodopsins. However, their cellular and ecological functions are unknown, and even their taxonomic distribution remains a subject of debate. We provide evidence that HeRs are a novel type of sensory rhodopsins linked to histidine kinases and other two-component system genes across phyla boundaries. In support of this, we also identify two novel putative signal transducing domains in HeRs that are fused with them. We also observe linkages of HeRs to genes involved in mitigation of light-induced oxidative stress and increased carbon and nitrogen metabolism. Finally, we synthesize these findings into a framework that connects HeRs with the cellular response to light in monoderms, activating light-induced oxidative stress defenses along with carbon/nitrogen metabolic circuitries. These findings are consistent with the evolutionary, taxonomic, structural, and genomic data available so far

Ecogenomics sheds light on diverse lifestyle strategies in freshwater CPR

BACKGROUND: The increased use of metagenomics and single-cell genomics led to the discovery of organisms from phyla with no cultivated representatives and proposed new microbial lineages such as the candidate phyla radiation (CPR or Patescibacteria). These bacteria have peculiar ribosomal structures, reduced metabolic capacities, small genome, and cell sizes, and a general host-associated lifestyle was proposed for the radiation. So far, most CPR genomes were obtained from groundwaters; however, their diversity, abundance, and role in surface freshwaters is largely unexplored. Here, we attempt to close these knowledge gaps by deep metagenomic sequencing of 119 samples of 17 different freshwater lakes located in Europe and Asia. Moreover, we applied Fluorescence in situ Hybridization followed by Catalyzed Reporter Deposition (CARD-FISH) for a first visualization of distinct CPR lineages in freshwater samples. RESULTS: A total of 174 dereplicated metagenome-assembled genomes (MAGs) of diverse CPR lineages were recovered from the investigated lakes, with a higher prevalence from hypolimnion samples (162 MAGs). They have reduced genomes (median size 1 Mbp) and were generally found in low abundances (0.02-14.36 coverage/Gb) and with estimated slow replication rates. The analysis of genomic traits and CARD-FISH results showed that the radiation is an eclectic group in terms of metabolic capabilities and potential lifestyles, ranging from what appear to be free-living lineages to host- or particle-associated groups. Although some complexes of the electron transport chain were present in the CPR MAGs, together with ion-pumping rhodopsins and heliorhodopsins, we believe that they most probably adopt a fermentative metabolism. Terminal oxidases might function in O$_{2}$ scavenging, while heliorhodopsins could be involved in mitigation against oxidative stress. CONCLUSIONS: A high diversity of CPR MAGs was recovered, and distinct CPR lineages did not seem to be limited to lakes with specific trophic states. Their reduced metabolic capacities resemble the ones described for genomes in groundwater and animal-associated samples, apart from Gracilibacteria that possesses more complete metabolic pathways. Even though this radiation is mostly host-associated, we also observed organisms from different clades (ABY1, Paceibacteria, Saccharimonadia) that appear to be unattached to any other organisms or were associated with 'lake snow' particles (ABY1, Gracilibacteria), suggesting a broad range of potential life-strategies in this phylum. Video Abstract

A freshwater radiation of diplonemids

Diplonemids are considered marine protists and have been reported among the most abundant and diverse eukaryotes in the world oceans. Recently we detected the presence of freshwater diplonemids in Lake Biwa, Japan. However, their distribution and abundances in freshwater ecosystems remain unknown. We assessed abundance and diversity of diplonemids from several geographically distant deep freshwater lakes of the world by amplicon-sequencing, shotgun metagenomics and CARD-FISH. We found diplonemids in all the studied lakes, albeit with low abundances and diversity. We assembled long 18S rRNA sequences from freshwater diplonemids and showed that they form a new lineage distinct from the diverse marine clades. Freshwater diplonemids are a sister-group to marine isolates from coastal and bay areas, suggesting a recent habitat transition from marine to freshwater habitats. Images of CARD-FISH targeted freshwater diplonemids suggest they feed on bacteria. Our analyses of 18S rRNA sequences retrieved from single cell genomes of marine diplonemids shows they encode multiple rRNA copies that may be very divergent from each other, suggesting that marine diplonemid abundance and diversity both have been overestimated. These results have wider implications on assessing eukaryotic abundances in natural habitats by using amplicon-sequencing alone

Microbial and Viral Genomic Diversity in Freshwater Environments

Most abundant microbes from natural environments remain uncultured as traditional culture approaches fail to sufficiently simulate native conditions. Moreover, the efforts to obtain axenic cultures are also hampered due to inter-microbial dependencies. The absence of cultured representatives has several downstream effects, e.g., little chance of experimentation and creating genetic models or hypothesis testing, phage isolation, or even producing complete, closed reference genomes. Lack of all these maintains severe gaps in our abilities to understand microbes in appropriate phylogenetic and ecological contexts and also their habitat at large. This thesis tries to circumvent these issues through a meticulous investigation of freshwater microbes and their viruses using a metagenomic approach in the Římov reservoir

Biotic factors drive bacterioplankton community in a tropical coastal site of the equatorial atlantic ocean

The relationship between latitude and microbial diversity in the ocean is controversial. Niche models predict higher richness at high latitudes in winter, while snapshot field-sampling point towards higher richness at intermediate latitudes, with lower values both towards equatorial and Polar Regions. However, given the dynamic nature of ocean’s ecosystem it is difficult to account for temporal variations in empirical assessments of microbial biodiversity. Here, we compared the components of diversity (richness and evenness) and microbial population stability (coefficient of variation) in two coastal ocean observatories with similar trophic state located in contrasting latitudes, one located in the Equatorial Atlantic Ocean, and one temperate located in the Northwestern Mediterranean Sea, to evaluate which factors drive the dynamics of microbial communities in each site. Our observations support the view that, as animals and plants, microbial communities exhibit higher (or at least similar) richness towards the equator, at least in the coastal ocean. We also found evidence of increasing stability with increasing evenness in tropical microbial communities when compared to the temperate ones. Temperature and silicates drove temperate free-living prokaryotic communities, while tropical ones were driven by stochastic factors such as biotic interactions with eukaryotes. We propose a conceptual framework where microbial community composition would be driven by deterministic factors in higher latitudes and once the factor temperature is removed moving towards the equator, more stochastic factors such as biotic interactions would emerge as the main factors shaping microbial communities. This study highlights the importance of comparative studies on Eulerian time-series distributed at different latitudes to fully understand the diversity patterns of microbial communities in the ocean.Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)A relação entre a latitude e diversidade microbiana no oceano é controversa. Modelos de nicho preveem maior riqueza em altas latitudes no inverno, enquanto amostragens pontuais indicam uma maior riqueza em latitudes intermediárias, com valores mais baixos para regiões equatoriais e polares. No entanto, dada a natureza dinâmica do ecossistema oceânico, é difícil explicar variações temporais da biodiversidade microbiana nas avaliações empíricas. Nesse trabalho comparamos os componentes da diversidade (riqueza e equitabilidade) e estabilidade das populações microbianas (coeficiente de variação) em dois observatórios oceânicos costeiros com estados tróficos semelhantes, localizados em latitudes contrastantes: um localizado no Oceano Atlântico Equatorial e um em clima temperado localizado no noroeste do Mar Mediterrâneo, a fim de avaliar quais fatores estruturam a dinâmica das comunidades microbianas em cada local. Observamos que tal como animais e plantas, as comunidades microbianas exibem maior (ou pelo menos similar) riqueza no equador pelo menos em águas costeiras. Também encontramos evidências de aumento da estabilidade com o aumento da uniformidade nas comunidades microbianas tropicais, quando comparadas com as de clima temperado. De modo geral, temperatura e silicatos foram as variáveis que condicionaram as comunidades procariotas de vida livre no observatório da região temperada, enquanto que no observatório tropical, fatores estocásticos tais como interações bióticas com eucariotos, foram os fatores que mais influenciaram as comunidades bacterianas. Assim, propomos um quadro conceitual onde a composição da comunidade microbiana seria impulsionada por fatores determinísticos em latitudes mais elevadas, enquanto que em latitudes menores, seriam determinados por fatores mais estocásticos, como interações bióticas. Nosso estudo destaca a importância de estudos comparativos utilizando series temporais Eulerianas em diferentes latitudes para entender os padrões de diversidade das comunidades microbianas no oceano

Phenology and ecological role of aerobic anoxygenic phototrophs in freshwaters

Background: Aerobic anoxygenic phototrophic (AAP) bacteria are heterotrophic bacteria that supply their metabolism with light energy harvested by bacteriochlorophyll-a-containing reaction centers. Despite their substantial contribution to bacterial biomass, microbial food webs, and carbon cycle, their phenology in freshwater lakes remains unknown. Hence, we investigated seasonal variations of AAP abundance and community composition biweekly across 3 years in a temperate, meso-oligotrophic freshwater lake. Results: AAP bacteria displayed a clear seasonal trend with a spring maximum following the bloom of phytoplankton and a secondary maximum in autumn. As the AAP bacteria represent a highly diverse assemblage of species, we followed their seasonal succession using the amplicon sequencing of the pufM marker gene. To enhance the accuracy of the taxonomic assignment, we developed new pufM primers that generate longer amplicons and compiled the currently largest database of pufM genes, comprising 3633 reference sequences spanning all phyla known to contain AAP species. With this novel resource, we demonstrated that the majority of the species appeared during specific phases of the seasonal cycle, with less than 2% of AAP species detected during the whole year. AAP community presented an indigenous freshwater nature characterized by high resilience and heterogenic adaptations to varying conditions of the freshwater environment. Conclusions: Our findings highlight the substantial contribution of AAP bacteria to the carbon flow and ecological dynamics of lakes and unveil a recurrent and dynamic seasonal succession of the AAP community. By integrating this information with the indicator of primary production (Chlorophyll-a) and existing ecological models, we show that AAP bacteria play a pivotal role in the recycling of dissolved organic matter released during spring phytoplankton bloom. We suggest a potential role of AAP bacteria within the context of the PEG model and their consideration in further ecological models.ISSN:2049-261