Ecology of aerobic anoxygenic phototrophs on a fine-scale taxonomic resolution in Adriatic Sea unravelled by unsupervised neural network.

BACKGROUND: Aerobic anoxygenic phototrophs are metabolically highly active, diverse and widespread polyphyletic members of bacterioplankton whose photoheterotrophic capabilities shifted the paradigm about simplicity of the microbial food chain. Despite their considerable contribution to the transformation of organic matter in marine environments, relatively little is still known about their community structure and ecology at fine-scale taxonomic resolution. Up to date, there is no comprehensive (i.e. qualitative and quantitative) analysis of their community composition in the Adriatic Sea. RESULTS: Analysis was based on pufM gene metabarcoding and quantitative FISH-IR approach with the use of artificial neural network. Significant seasonality was observed with regards to absolute abundances (maximum average abundances in spring 2.136 ± 0.081 × 104 cells mL-1, minimum in summer 0.86 × 104 cells mL-1), FISH-IR groups (Roseobacter clade prevalent in autumn, other Alpha- and Gammaproteobacteria in summer) and pufM sequencing data agglomerated at genus-level. FISH-IR results revealed heterogeneity with the highest average relative contribution of AAPs assigned to Roseobacter clade (37.66%), followed by Gammaproteobacteria (35.25%) and general Alphaproteobacteria (31.15%). Community composition obtained via pufM sequencing was dominated by Gammaproteobacteria clade NOR5/OM60, specifically genus Luminiphilus, with numerous rare genera present in relative abundances below 1%. The use of artificial neural network connected this community to biotic (heterotrophic bacteria, HNA and LNA bacteria, Synechococcus, Prochlorococcus, picoeukaryotes, heterotrophic nanoflagellates, bacterial production) and abiotic environmental factors (temperature, salinity, chlorophyll a and nitrate, nitrite, ammonia, total nitrogen, silicate, and orthophosphate concentration). A type of neural network, neural gas analysis at order-, genus- and ASV-level, resulted in five distinct best matching units (representing particular environments) and revealed that high diversity was generally independent of temperature, salinity, and trophic status of the environment, indicating a potentially dissimilar behaviour of aerobic anoxygenic phototrophs compared to the general bacterioplankton. CONCLUSION: This research represents the first comprehensive analysis of aerobic anoxygenic phototrophs in the Adriatic Sea on a trophic gradient during a year-round period. This study is also one of the first reports of their genus-level ecology linked to biotic and abiotic environmental factors revealed by unsupervised neural network algorithm, paving the way for further research of substantial contribution of this important bacterial functional group to marine ecosystems.

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.

Multi-environment ecogenomics analysis of the cosmopolitan phylum Gemmatimonadota.

Gemmatimonadota is a diverse bacterial phylum commonly found in environments such as soils, rhizospheres, fresh waters, and sediments. So far, the phylum contains just six cultured species (five of them sequenced), which limits our understanding of their diversity and metabolism. Therefore, we analyzed over 400 metagenome-assembled genomes (MAGs) and 5 culture-derived genomes representing Gemmatimonadota from various aquatic environments, hydrothermal vents, sediments, soils, and host-associated (with marine sponges and coral) species. The principal coordinate analysis based on the presence/absence of genes in Gemmatimonadota genomes and phylogenomic analysis documented that marine and host-associated Gemmatimonadota were the most distant from freshwater and wastewater species. A smaller genome size and coding sequences (CDS) number reduction were observed in marine MAGs, pointing to an oligotrophic environmental adaptation. Several metabolic pathways are restricted to specific environments. For example, genes for anoxygenic phototrophy were found only in freshwater, wastewater, and soda lake sediment genomes. There were several genomes from soda lake sediments and wastewater containing type IC/ID ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO). Various genomes from wastewater harbored bacterial type II RuBisCO, whereas RuBisCO-like protein was found in genomes from fresh waters, soil, host-associated, and marine sediments. Gemmatimonadota does not contain nitrogen fixation genes; however, the nosZ gene, involved in the reduction of N2O, was present in genomes from most environments, missing only in marine water and host-associated Gemmatimonadota. The presented data suggest that Gemmatimonadota evolved as an organotrophic species relying on aerobic respiration and then remodeled its genome inventory when adapting to particular environments. IMPORTANCE Gemmatimonadota is a rarely studied bacterial phylum consisting of a handful of cultured species. Recent culture-independent studies documented that these organisms are distributed in many environments, including soil, marine, fresh, and waste waters. However, due to the lack of cultured species, information about their metabolic potential and environmental role is scarce. Therefore, we collected Gemmatimonadota metagenome-assembled genomes (MAGs) from different habitats and performed a systematic analysis of their genomic characteristics and metabolic potential. Our results show how Gemmatimonadota have adapted their genomes to different environments.

Diversity dynamics of aerobic anoxygenic phototrophic bacteria in a freshwater lake.

Aerobic anoxygenic photoheterotrophic (AAP) bacteria represent a functional group of prokaryotic organisms that harvests light energy using bacteriochlorophyll-containing photosynthetic reaction centers. They represent an active and rapidly growing component of freshwater bacterioplankton, with the highest numbers observed usually in summer. Species diversity of freshwater AAP bacteria has been studied before in lakes, but its seasonal dynamics remain unknown. In this report, we analysed temporal changes in the composition of the phototrophic community in an oligo-mesotrophic freshwater lake using amplicon sequencing of the pufM marker gene. The AAP community was dominated by phototrophic Gammaproteobacteria and Alphaproteobacteria, with smaller contribution of phototrophic Chloroflexota and Gemmatimonadota. Phototrophic Eremiobacteriota or members of Myxococcota were not detected. Interestingly, some AAP taxa, such as Limnohabitans, Rhodoferax, Rhodobacterales or Rhizobiales, were permanently present over the sampling period, while others, such as Sphingomonadales, Rhodospirillales or Caulobacterales appeared only transiently. The environmental factors that best explain the seasonal changes in AAP community were temperature, concentrations of oxygen and dissolved organic matter.

Photoheterotrophy by aerobic anoxygenic bacteria modulates carbon fluxes in a freshwater lake.

Lakes are a significant component of the global carbon cycle. Respiration exceeds net primary production in most freshwater lakes, making them a source of CO2 to the atmosphere. Driven by heterotrophic microorganisms, respiration is assumed to be unaffected by light, thus it is measured in the dark. However, photoheterotrophs, such as aerobic anoxygenic photoheterotrophic (AAP) bacteria that produce ATP via photochemical reactions, substantially reduce respiration in the light. They are an abundant and active component of bacterioplankton, but their photoheterotrophic contribution to microbial community metabolism remains unquantified. We showed that the community respiration rate in a freshwater lake was reduced by 15.2% (95% confidence interval (CI): 6.6-23.8%) in infrared light that is usable by AAP bacteria but not by primary producers. Moreover, significantly higher assimilation rates of glucose (18.1%; 7.8-28.4%), pyruvate (9.5%; 4.2-14.8%), and leucine (5.9%; 0.1-11.6%) were measured in infrared light. At the ecosystem scale, the amount of CO2 from respiration unbalanced by net primary production was by 3.69 × 109 g CO2 lower over these two sampling seasons when measured in the infrared light. Our results demonstrate that dark measurements of microbial activity significantly bias the carbon fluxes, providing a new paradigm for their quantification in aquatic environments.