Growth and mortality of aerobic anoxygenic phototrophs in the North Pacific Subtropical Gyre.

Aerobic anoxygenic phototrophic (AAP) bacteria harvest light energy using bacteriochlorophyll-containing reaction centers to supplement their mostly heterotrophic metabolism. While their abundance and growth have been intensively studied in coastal environments, much less is known about their activity in oligotrophic open ocean regions. Therefore, we combined in situ sampling in the North Pacific Subtropical Gyre, north of O'ahu island, Hawaii, with two manipulation experiments. Infra-red epifluorescence microscopy documented that AAP bacteria represented approximately 2% of total bacteria in the euphotic zone with the maximum abundance in the upper 50 m. They conducted active photosynthetic electron transport with maximum rates up to 50 electrons per reaction center per second. The in situ decline of bacteriochlorophyll concentration over the daylight period, an estimate of loss rates due to predation, indicated that the AAP bacteria in the upper 50 m of the water column turned over at rates of 0.75-0.90 d-1. This corresponded well with the specific growth rate determined in dilution experiments where AAP bacteria grew at a rate 1.05 ± 0.09 d-1. An amendment of inorganic nitrogen to obtain N:P = 32 resulted in a more than 10 times increase in AAP abundance over 6 days. The presented data document that AAP bacteria are an active part of the bacterioplankton community in the oligotrophic North Pacific Subtropical Gyre and that their growth was mostly controlled by nitrogen availability and grazing pressure.IMPORTANCEMarine bacteria represent a complex assembly of species with different physiology, metabolism, and substrate preferences. We focus on a specific functional group of marine bacteria called aerobic anoxygenic phototrophs. These photoheterotrophic organisms require organic carbon substrates for growth, but they can also supplement their metabolic needs with light energy captured by bacteriochlorophyll. These bacteria have been intensively studied in coastal regions, but rather less is known about their distribution, growth, and mortality in the oligotrophic open ocean. Therefore, we conducted a suite of measurements in the North Pacific Subtropical Gyre to determine the distribution of these organisms in the water column and their growth and mortality rates. A nutrient amendment experiment showed that aerobic anoxygenic phototrophs were limited by inorganic nitrogen. Despite this, they grew more rapidly than average heterotrophic bacteria, but their growth was balanced by intense grazing pressure.

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.

A photoheterotrophic bacterium from Iceland has adapted its photosynthetic machinery to the long days of polar summer.

During their long evolution, anoxygenic phototrophic bacteria have inhabited a wide variety of natural habitats and developed specific strategies to cope with the challenges of any particular environment. Expression, assembly, and safe operation of the photosynthetic apparatus must be regulated to prevent reactive oxygen species generation under illumination in the presence of oxygen. Here, we report on the photoheterotrophic Sediminicoccus sp. strain KRV36, which was isolated from a cold stream in north-western Iceland, 30 km south of the Arctic Circle. In contrast to most aerobic anoxygenic phototrophs, which stop pigment synthesis when illuminated, strain KRV36 maintained its bacteriochlorophyll synthesis even under continuous light. Its cells also contained between 100 and 180 chromatophores, each accommodating photosynthetic complexes that exhibit an unusually large carotenoid absorption spectrum. The expression of photosynthesis genes in dark-adapted cells was transiently downregulated in the first 2 hours exposed to light but recovered to the initial level within 24 hours. An excess of membrane-bound carotenoids as well as high, constitutive expression of oxidative stress response genes provided the required potential for scavenging reactive oxygen species, safeguarding bacteriochlorophyll synthesis and photosystem assembly. The unique cellular architecture and an unusual gene expression pattern represent a specific adaptation that allows the maintenance of anoxygenic phototrophy under arctic conditions characterized by long summer days with relatively low irradiance.IMPORTANCEThe photoheterotrophic bacterium Sediminicoccus sp. KRV36 was isolated from a cold stream in Iceland. It expresses its photosynthesis genes, synthesizes bacteriochlorophyll, and assembles functional photosynthetic complexes under continuous light in the presence of oxygen. Unraveling the molecular basis of this ability, which is exceptional among aerobic anoxygenic phototrophic species, will help to understand the evolution of bacterial photosynthesis in response to changing environmental conditions. It might also open new possibilities for genetic engineering of biotechnologically relevant phototrophs, with the aim of increasing photosynthetic activity and their tolerance to reactive oxygen species.

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.

Intracytoplasmic-membrane development in alphaproteobacteria involves the homolog of the mitochondrial crista-developing protein Mic60.

Mitochondrial cristae expand the surface area of respiratory membranes and ultimately allow for the evolutionary scaling of respiration with cell volume across eukaryotes. The discovery of Mic60 homologs among alphaproteobacteria, the closest extant relatives of mitochondria, suggested that cristae might have evolved from bacterial intracytoplasmic membranes (ICMs). Here, we investigated the predicted structure and function of alphaproteobacterial Mic60, and a protein encoded by an adjacent gene Orf52, in two distantly related purple alphaproteobacteria, Rhodobacter sphaeroides and Rhodopseudomonas palustris. In addition, we assessed the potential physical interactors of Mic60 and Orf52 in R. sphaeroides. We show that the three α helices of mitochondrial Mic60's mitofilin domain, as well as its adjacent membrane-binding amphipathic helix, are present in alphaproteobacterial Mic60. The disruption of Mic60 and Orf52 caused photoheterotrophic growth defects, which are most severe under low light conditions, and both their disruption and overexpression led to enlarged ICMs in both studied alphaproteobacteria. We also found that alphaproteobacterial Mic60 physically interacts with BamA, the homolog of Sam50, one of the main physical interactors of eukaryotic Mic60. This interaction, responsible for making contact sites at mitochondrial envelopes, has been conserved in modern alphaproteobacteria despite more than a billion years of evolutionary divergence. Our results suggest a role for Mic60 in photosynthetic ICM development and contact site formation at alphaproteobacterial envelopes. Overall, we provide support for the hypothesis that mitochondrial cristae evolved from alphaproteobacterial ICMs and have therefore improved our understanding of the nature of the mitochondrial ancestor.

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.

Characterisation of the photosynthetic complexes from the marine gammaproteobacterium Congregibacter litoralis KT71.

Possibly the most abundant group of anoxygenic phototrophs are marine photoheterotrophic Gammaproteobacteria belonging to the NOR5/OM60 clade. As little is known about their photosynthetic apparatus, the photosynthetic complexes from the marine phototrophic bacterium Congregibacter litoralis KT71 were purified and spectroscopically characterised. The intra-cytoplasmic membranes contain a smaller amount of photosynthetic complexes when compared with anaerobic purple bacteria. Moreover, the intra-cytoplasmic membranes contain only a minimum amount of peripheral LH2 complexes. The complexes are populated by bacteriochlorophyll a, spirilloxanthin and two novel ketocarotenoids, with biophysical and biochemical properties similar to previously characterised complexes from purple bacteria. The organization of the RC-LH1 complex has been further characterised using cryo-electron microscopy. The overall organisation is similar to the complex from the gammaproteobacterium Thermochromatium tepidum, with the type-II reaction centre surrounded by a slightly elliptical LH1 antenna ring composed of 16 αß-subunits with no discernible gap or pore. The RC-LH1 and LH2 apoproteins are phylogenetically related to other halophilic species but LH2 also to some alphaproteobacterial species. It seems that the reduction of light-harvesting apparatus and acquisition of novel ketocarotenoids in Congregibacter litoralis KT71 represent specific adaptations for operating the anoxygenic photosynthesis under aerobic conditions at sea.

A bacterium from a mountain lake harvests light using both proton-pumping xanthorhodopsins and bacteriochlorophyll-based photosystems.

Photoheterotrophic bacteria harvest light energy using either proton-pumping rhodopsins or bacteriochlorophyll (BChl)-based photosystems. The bacterium Sphingomonas glacialis AAP5 isolated from the alpine lake Gossenköllesee contains genes for both systems. Here, we show that BChl is expressed between 4°C and 22°C in the dark, whereas xanthorhodopsin is expressed only at temperatures below 16°C and in the presence of light. Thus, cells grown at low temperatures under a natural light-dark cycle contain both BChl-based photosystems and xanthorhodopsins with a nostoxanthin antenna. Flash photolysis measurements proved that both systems are photochemically active. The captured light energy is used for ATP synthesis and stimulates growth. Thus, S. glacialis AAP5 represents a chlorophototrophic and a retinalophototrophic organism. Our analyses suggest that simple xanthorhodopsin may be preferred by the cells under higher light and low temperatures, whereas larger BChl-based photosystems may perform better at lower light intensities. This indicates that the use of two systems for light harvesting may represent an evolutionary adaptation to the specific environmental conditions found in alpine lakes and other analogous ecosystems, allowing bacteria to alternate their light-harvesting machinery in response to large seasonal changes of irradiance and temperature.

Carotenoid responds to excess energy dissipation in the LH2 complex from Rhodoblastus acidophilus.

The functions of both (bacterio) chlorophylls and carotenoids in light-harvesting complexes have been extensively studied during the past decade, yet, the involvement of BChl a high-energy Soret band in the cascade of light-harvesting processes still remains a relatively unexplored topic. Here, we present transient absorption data recorded after excitation of the Soret band in the LH2 complex from Rhodoblastus acidophilus. Comparison of obtained data to those recorded after excitation of rhodopin glucoside and B800 BChl a suggests that no Soret-to-Car energy transfer pathway is active in LH2 complex. Furthermore, a spectrally rich pattern observed in the spectral region of rhodopin glucoside ground state bleaching (420-550 nm) has been assigned to an electrochromic shift. The results of global fitting analysis demonstrate two more features. A 6 ps component obtained exclusively after excitation of the Soret band has been assigned to the response of rhodopin glucoside to excess energy dissipation in LH2. Another time component, ~ 450 ps, appearing independently of the excitation wavelength was assigned to BChl a-to-Car triplet-triplet transfer. Presented data demonstrate several new features of LH2 complex and its behavior following the excitation of the Soret band.

2.4-Å structure of the double-ring Gemmatimonas phototrophica photosystem.

Phototrophic Gemmatimonadetes evolved the ability to use solar energy following horizontal transfer of photosynthesis-related genes from an ancient phototrophic proteobacterium. The electron cryo-microscopy structure of the Gemmatimonas phototrophica photosystem at 2.4 Å reveals a unique, double-ring complex. Two unique membrane-extrinsic polypeptides, RC-S and RC-U, hold the central type 2 reaction center (RC) within an inner 16-subunit light-harvesting 1 (LH1) ring, which is encircled by an outer 24-subunit antenna ring (LHh) that adds light-gathering capacity. Femtosecond kinetics reveal the flow of energy within the RC-dLH complex, from the outer LHh ring to LH1 and then to the RC. This structural and functional study shows that G. phototrophica has independently evolved its own compact, robust, and highly effective architecture for harvesting and trapping solar energy.

Phylum Gemmatimonadota and Its Role in the Environment.

Bacteria are an important part of every ecosystem that they inhabit on Earth. Environmental microbiologists usually focus on a few dominant bacterial groups, neglecting less abundant ones, which collectively make up most of the microbial diversity. One of such less-studied phyla is Gemmatimonadota. Currently, the phylum contains only six cultured species. However, data from culture-independent studies indicate that members of Gemmatimonadota are common in diverse habitats. They are abundant in soils, where they seem to be frequently associated with plants and the rhizosphere. Moreover, Gemmatimonadota were found in aquatic environments, such as freshwaters, wastewater treatment plants, biofilms, and sediments. An important discovery was the identification of purple bacterial reaction centers and anoxygenic photosynthesis in this phylum, genes for which were likely acquired via horizontal gene transfer. So far, the capacity for anoxygenic photosynthesis has been described for two cultured species: Gemmatimonas phototrophica and Gemmatimonas groenlandica. Moreover, analyses of metagenome-assembled genomes indicate that it is also common in uncultured lineages of Gemmatimonadota. This review summarizes the current knowledge about this understudied bacterial phylum with an emphasis on its environmental distribution.

The Influence of Calcium on the Growth, Morphology and Gene Regulation in Gemmatimonas phototrophica.

The bacterium Gemmatimonas phototrophica AP64 isolated from a freshwater lake in the western Gobi Desert represents the first phototrophic member of the bacterial phylum Gemmatimonadota. This strain was originally cultured on agar plates because it did not grow in liquid medium. In contrast, the closely related species G. groenlandica TET16 grows both on solid and in liquid media. Here, we show that the growth of G. phototrophica in liquid medium can be induced by supplementing the medium with 20 mg CaCl2 L-1. When grown at a lower concentration of calcium (2 mg CaCl2 L-1) in the liquid medium, the growth was significantly delayed, cells were elongated and lacked flagella. The elevated requirement for calcium is relatively specific as it can be partially substituted by strontium, but not by magnesium. The transcriptome analysis documented that several groups of genes involved in flagella biosynthesis and transport of transition metals were co-activated after amendment of 20 mg CaCl2 L-1 to the medium. The presented results document that G. phototrophica requires a higher concentration of calcium for its metabolism and growth compared to other Gemmatimonas species.

Study of microbiocenosis of canine dental biofilms.

Dental biofilm is a complex microbial community influenced by many exogenous and endogenous factors. Despite long-term studies, its bacterial composition is still not clearly understood. While most of the research on dental biofilms was conducted in humans, much less information is available from companion animals. In this study, we analyzed the composition of canine dental biofilms using both standard cultivation on solid media and amplicon sequencing, and compared the two approaches. The 16S rRNA gene sequences were used to define the bacterial community of canine dental biofilm with both, culture-dependent and culture-independent methods. After DNA extraction from each sample, the V3-V4 region of the 16S rRNA gene was amplified and sequenced via Illumina MiSeq platform. Isolated bacteria were identified using universal primers and Sanger sequencing. Representatives of 18 bacterial genera belonging to 5 phyla were isolated from solid media. Amplicon sequencing largely expanded this information identifying in total 284 operational taxonomic units belonging to 10 bacterial phyla. Amplicon sequencing revealed much higher diversity of bacteria in the canine dental biofilms, when compared to standard cultivation approach. In contrast, cultured representatives of several bacterial families were not identified by amplicon sequencing.

Distribution of cycle threshold values in RT-qPCR tests during the autumn 2020 peak of the COVID-19 pandemic in the Czech Republic.

Reverse-transcription quantitative PCR (RT-qPCR) is currently the most sensitive method to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which causes coronavirus disease 2019 (COVID-19). We analysed 1927 samples collected in a local public hospital during the autumn 2020 peak of the pandemic in the Czech Republic. The tests were performed using the Seegene Allplex 2019-nCov assay, which simultaneously detects three SARS-CoV-2 genes. In all samples analysed, 44.5 % were negative for all three genes, and 37.6 % were undoubtedly positive, with all three viral genes being amplified. A high degree of correlation between C t values among the genes confirmed the internal consistency of testing. Most of the positive samples were detected between the 15th and 35th cycles. We also registered a small number of samples with only one (13.2 %) or two (4.7 %) amplified genes, which may have originated from either freshly infected or already recovering patients. In addition, we did not detect any potentially false-positive samples from low-prevalence settings. Our results document that PCR testing represents a reliable and robust method for routine diagnostic detection of SARS-CoV-2.

Lineage-Specific Growth Curves Document Large Differences in Response of Individual Groups of Marine Bacteria to the Top-Down and Bottom-Up Controls.

Marine bacterioplankton represent a diverse assembly of species differing largely in their abundance, physiology, metabolic activity, and role in microbial food webs. To analyze their sensitivity to bottom-up and top-down controls, we performed a manipulation experiment where grazers were removed, with or without the addition of phosphate. Using amplicon-reads normalization by internal standard (ARNIS), we reconstructed growth curves for almost 300 individual phylotypes. Grazer removal caused a rapid growth of most bacterial groups, which grew at rates of 0.6 to 3.5 day-1, with the highest rates (>4 day-1) recorded among Rhodobacteraceae, Oceanospirillales, Alteromonadaceae, and Arcobacteraceae. Based on their growth response, the phylotypes were divided into three basic groups. Most of the phylotypes responded positively to both grazer removal as well as phosphate addition. The second group (containing, e.g., Rhodobacterales and Rhizobiales) responded to the grazer removal but not to the phosphate addition. Finally, some clades, such as SAR11 and Flavobacteriaceae, responded only to phosphate amendment but not to grazer removal. Our results show large differences in bacterial responses to experimental manipulations at the phylotype level and document different life strategies of marine bacterioplankton. In addition, growth curves of 130 phylogroups of aerobic anoxygenic phototrophs were reconstructed based on changes of the functional pufM gene. The use of functional genes together with rRNA genes may significantly expand the scientific potential of the ARNIS technique. IMPORTANCE Growth is one of the main manifestations of life. It is assumed generally that bacterial growth is constrained mostly by nutrient availability (bottom-up control) and grazing (top-down control). Since marine bacteria represent a very diverse assembly of species with different metabolic properties, their growth characteristics also largely differ accordingly. Currently, the growth of marine microorganisms is typically evaluated using microscopy in combination with fluorescence in situ hybridization (FISH). However, these laborious techniques are limited in their throughput and taxonomical resolution. Therefore, we combined a classical manipulation experiment with next-generation sequencing to resolve the growth dynamics of almost 300 bacterial phylogroups in the coastal Adriatic Sea. The analysis documented that most of the phylogroups responded positively to both grazer removal and phosphate addition. We observed significant differences in growth kinetics among closely related species, which could not be distinguished by the classical FISH technique.

Structure elucidation of the novel carotenoid gemmatoxanthin from the photosynthetic complex of Gemmatimonas phototrophica AP64.

Gemmatimonas phototrophica AP64 is the first phototrophic representative of the bacterial phylum Gemmatimonadetes. The cells contain photosynthetic complexes with bacteriochlorophyll a as the main light-harvesting pigment and an unknown carotenoid with a single broad absorption band at 490 nm in methanol. The carotenoid was extracted from isolated photosynthetic complexes, and purified by liquid chromatography. A combination of nuclear magnetic resonance (1H NMR, COSY, 1H-13C HSQC, 1H-13C HMBC, J-resolved, and ROESY), high-resolution mass spectroscopy, Fourier-transformed infra-red, and Raman spectroscopy was used to determine its chemical structure. The novel linear carotenoid, that we have named gemmatoxanthin, contains 11 conjugated double bonds and is further substituted by methoxy, carboxyl and aldehyde groups. Its IUPAC-IUBMB semi-systematic name is 1'-Methoxy-19'-oxo-3',4'-didehydro-7,8,1',2'-tetrahydro- Ψ, Ψ carotene-16-oic acid. To our best knowledge, the presence of the carboxyl, methoxy and aldehyde groups on a linear C40 carotenoid backbone is reported here for the first time.

Artificial neural network analysis of microbial diversity in the central and southern Adriatic Sea.

Bacteria are an active and diverse component of pelagic communities. The identification of main factors governing microbial diversity and spatial distribution requires advanced mathematical analyses. Here, the bacterial community composition was analysed, along with a depth profile, in the open Adriatic Sea using amplicon sequencing of bacterial 16S rRNA and the Neural gas algorithm. The performed analysis classified the sample into four best matching units representing heterogenic patterns of the bacterial community composition. The observed parameters were more differentiated by depth than by area, with temperature and identified salinity as important environmental variables. The highest diversity was observed at the deep chlorophyll maximum, while bacterial abundance and production peaked in the upper layers. The most of the identified genera belonged to Proteobacteria, with uncultured AEGEAN-169 and SAR116 lineages being dominant Alphaproteobacteria, and OM60 (NOR5) and SAR86 being dominant Gammaproteobacteria. Marine Synechococcus and Cyanobium-related species were predominant in the shallow layer, while Prochlorococcus MIT 9313 formed a higher portion below 50 m depth. Bacteroidota were represented mostly by uncultured lineages (NS4, NS5 and NS9 marine lineages). In contrast, Actinobacteriota were dominated by a candidatus genus Ca. Actinomarina. A large contribution of Nitrospinae was evident at the deepest investigated layer. Our results document that neural network analysis of environmental data may provide a novel insight into factors affecting picoplankton in the open sea environment.

Characterization of the Aerobic Anoxygenic Phototrophic Bacterium Sphingomonas sp. AAP5.

An aerobic, yellow-pigmented, bacteriochlorophyll a-producing strain, designated AAP5 (=DSM 111157=CCUG 74776), was isolated from the alpine lake Gossenköllesee located in the Tyrolean Alps, Austria. Here, we report its description and polyphasic characterization. Phylogenetic analysis of the 16S rRNA gene showed that strain AAP5 belongs to the bacterial genus Sphingomonas and has the highest pairwise 16S rRNA gene sequence similarity with Sphingomonas glacialis (98.3%), Sphingomonas psychrolutea (96.8%), and Sphingomonas melonis (96.5%). Its genomic DNA G + C content is 65.9%. Further, in silico DNA-DNA hybridization and calculation of the average nucleotide identity speaks for the close phylogenetic relationship of AAP5 and Sphingomonas glacialis. The high percentage (76.2%) of shared orthologous gene clusters between strain AAP5 and Sphingomonas paucimobilis NCTC 11030T, the type species of the genus, supports the classification of the two strains into the same genus. Strain AAP5 was found to contain C18:1ω7c (64.6%) as a predominant fatty acid (>10%) and the polar lipid profile contained phosphatidylglycerol, diphosphatidylglycerol, phosphatidylethanolamine, sphingoglycolipid, six unidentified glycolipids, one unidentified phospholipid, and two unidentified lipids. The main respiratory quinone was ubiquinone-10. Strain AAP5 is a facultative photoheterotroph containing type-2 photosynthetic reaction centers and, in addition, contains a xathorhodopsin gene. No CO2-fixation pathways were found.

Common Presence of Phototrophic Gemmatimonadota in Temperate Freshwater Lakes.

Members of the bacterial phylum Gemmatimonadota are ubiquitous in most natural environments and represent one of the top 10 most abundant bacterial phyla in soil. Sequences affiliated with Gemmatimonadota were also reported from diverse aquatic habitats; however, it remains unknown whether they are native organisms or represent bacteria passively transported from sediment or soil. To address this question, we analyzed metagenomes constructed from five freshwater lakes in central Europe. Based on the 16S rRNA gene frequency, Gemmatimonadota represented from 0.02 to 0.6% of all bacteria in the epilimnion and between 0.1 and 1% in the hypolimnion. These proportions were independently confirmed using catalyzed reporter deposition-fluorescence in situ hybridization (CARD-FISH). Some cells in the epilimnion were attached to diatoms (Fragilaria sp.) or cyanobacteria (Microcystis sp.), which suggests a close association with phytoplankton. In addition, we reconstructed 45 metagenome-assembled genomes (MAGs) related to Gemmatimonadota They represent several novel lineages, which persist in the studied lakes during the seasons. Three lineages contained photosynthesis gene clusters. One of these lineages was related to Gemmatimonas phototrophica and represented the majority of Gemmatimonadota retrieved from the lakes' epilimnion. The other two lineages came from hypolimnion and probably represented novel photoheterotrophic genera. None of these phototrophic MAGs contained genes for carbon fixation. Since most of the identified MAGs were present during the whole year and cells associated with phytoplankton were observed, we conclude that they represent truly limnic Gemmatimonadota distinct from the previously described species isolated from soils or sediments.IMPORTANCE Photoheterotrophic bacterial phyla such as Gemmatimonadota are key components of many natural environments. Its first photoheterotrophic cultured member, Gemmatimonas phototrophica, was isolated in 2014 from a shallow lake in the Gobi Desert. It contains a unique type of photosynthetic complex encoded by a set of genes which were likely received via horizontal transfer from Proteobacteria We were intrigued to discover how widespread this group is in the natural environment. In the presented study, we analyzed 45 metagenome-assembled genomes (MAGs) that were obtained from five freshwater lakes in Switzerland and Czechia. Interestingly, it was found that phototrophic Gemmatimonadota are relatively common in euphotic zones of the studied lakes, whereas heterotrophic Gemmatimonadota prevail in deeper waters. Moreover, our analysis of the MAGs documented that these freshwater species contain almost the same set of photosynthesis genes identified before in Gemmatimonas phototrophica originating from the Gobi Desert.