Environmental Microbiology

Summary The symbiotic unicellular cyanobacterium Candidatus Atelocyanobacterium thalassa (UCYN‐A) is one of the most abundant and widespread nitrogen (N 2 )‐fixing cyanobacteria in the ocean. Although it remains uncultivated, multiple sublineages have been detected based on partial nitrogenase ( nifH ) gene sequences, including the four most commonly detected sublineages UCYN‐A1, UCYN‐A2, UCYN‐A3 and UCYN‐A4. However, very little is known about UCYN‐A3 beyond the nifH sequences from nifH gene diversity surveys. In this study, single cell sorting, DNA sequencing, qPCR and CARD‐FISH assays revealed discrepancies involving the identification of sublineages, which led to new information on the diversity of the UCYN‐A symbiosis. 16S rRNA and nifH gene sequencing on single sorted cells allowed us to identify the 16S rRNA gene of the uncharacterized UCYN‐A3 sublineage. We designed new CARD‐FISH probes that allowed us to distinguish and observe UCYN‐A2 in a coastal location (SIO Pier; San Diego) and UCYN‐A3 in an open ocean location (Station ALOHA; Hawaii). Moreover, we reconstructed about 13% of the UCYN‐A3 genome from Tara Oceans metagenomic data. Finally, our findings unveil the UCYN‐A3 symbiosis in open ocean waters suggesting that the different UCYN‐A sublineages are distributed along different size fractions of the plankton defined by the cell‐size ranges of their prymnesiophyte hosts.

Summary Methanotrophic bacteria represent an important biological filter regulating methane emissions into the atmosphere. Planktonic methanotrophic communities in freshwater lakes are typically dominated by aerobic gamma‐proteobacteria, with a contribution from alpha‐proteobacterial methanotrophs and the NC10 bacteria. The NC10 clade encompasses methanotrophs related to ‘ Candidatus Methylomirabilis oxyfera’, which oxidize methane using a unique pathway of denitrification that tentatively produces N 2 and O 2 from nitric oxide (NO). Here, we describe a new species of the NC10 clade, ‘ Ca . Methylomirabilis limnetica’, which dominated the planktonic microbial community in the anoxic depths of the deep stratified Lake Zug in two consecutive years, comprising up to 27% of the total bacterial population. Gene transcripts assigned to ‘ Ca . M. limnetica’ constituted up to one third of all metatranscriptomic sequences in situ . The reconstructed genome encoded a complete pathway for methane oxidation, and an incomplete denitrification pathway, including two putative nitric oxide dismutase genes. The genome of ‘ Ca . M. limnetica’ exhibited features possibly related to genome streamlining (i.e. less redundancy of key metabolic genes) and adaptation to its planktonic habitat (i.e. gas vesicle genes). We speculate that ‘ Ca . M. limnetica’ temporarily bloomed in the lake during non‐steady‐state conditions suggesting a niche for NC10 bacteria in the lacustrine methane and nitrogen cycle.

Summary Microorganisms are main drivers of the sulfur, nitrogen and carbon biogeochemical cycles. These elemental cycles are interconnected by the activity of different guilds in sediments or wastewater treatment systems. Here, we investigated a nitrate‐reducing microbial community in a laboratory‐scale bioreactor model that closely mimicked estuary or brackish sediment conditions. The bioreactor simultaneously consumed sulfide, methane and ammonium at the expense of nitrate. Ammonium oxidation occurred solely by the activity of anammox bacteria identified as Candidatus Scalindua brodae and Ca . Kuenenia stuttgartiensis. Fifty‐three percent of methane oxidation was catalyzed by archaea affiliated to Ca . Methanoperedens and 47% by Ca . Methylomirabilis bacteria. Sulfide oxidation was mainly shared between two proteobacterial groups. Interestingly, competition for nitrate did not lead to exclusion of one particular group. Metagenomic analysis showed that the most abundant taxonomic group was distantly related to Thermodesulfovibrio sp. (87–89% 16S rRNA gene identity, 52–54% average amino acid identity), representing a new family within the Nitrospirae phylum. A high quality draft genome of the new species was recovered, and analysis showed high metabolic versatility. Related microbial groups are found in diverse environments with sulfur, nitrogen and methane cycling, indicating that these novel Nitrospirae bacteria might contribute to biogeochemical cycling in natural habitats.

Summary Cotylorhiza tuberculata is an important scyphozoan jellyfish producing population blooms in the Mediterranean probably due to pelagic ecosystem's decay. Its gastric cavity can serve as a simple model of microbial–animal digestive associations, yet poorly characterized. Using state‐of‐the‐art metagenomic population binning and catalyzed reporter deposition fluorescence in situ hybridization (CARD‐FISH), we show that only four novel clonal phylotypes were consistently associated with multiple jellyfish adults. Two affiliated close to Spiroplasma and Mycoplasma genera, one to chlamydial ‘ Candidatus Syngnamydia’, and one to bacteroidetal Tenacibaculum , and were at least one order of magnitude more abundant than any other bacteria detected. Metabolic modelling predicted an aerobic heterotrophic lifestyle for the chlamydia, which were found intracellularly in Onychodromopsis ‐like ciliates. The Spiroplasma ‐like organism was predicted to be an anaerobic fermenter associated to some jellyfish cells, whereas the Tenacibaculum ‐like as free‐living aerobic heterotroph, densely colonizing the mesogleal axis inside the gastric filaments. The association between the jellyfish and its reduced microbiome was close and temporally stable, and possibly related to food digestion and protection from pathogens. Based on the genomic and microscopic data, we propose three candidate taxa: ‘ Candidatus Syngnamydia medusae’, ‘ Candidatus Medusoplasma mediterranei’ and ‘ Candidatus Tenacibaculum medusae’.

Summary The anaerobic, mesophilic and moderately halophilic strain L21‐Spi‐D4 T was recently isolated from the suboxic zone of a hypersaline cyanobacterial mat using protein‐rich extracts of Arthrospira (formerly Spirulina ) platensis as substrate. Phylogenetic analyses based on 16S rRNA genes indicated an affiliation of the novel strain with the Bacteroidetes clade MgMjR‐022, which is widely distributed and abundant in hypersaline microbial mats and heretofore comprised only sequences of uncultured bacteria. Analyses of the complete genome sequence of strain L21‐Spi‐D4 T revealed a possible specialization on the degradation of cyanobacterial biomass. Besides genes for enzymes degrading specific cyanobacterial proteins a conspicuous transport complex for the polypeptide cyanophycin could be identified that is homologous to typical polysaccharide utilization loci of Bacteroidetes . A distinct and reproducible co‐occurrence pattern of environmental 16S rRNA gene sequences of the MgMjR‐022 clade and cyanobacteria in the suboxic zone of hypersaline mats points to a specific dependence of members of this clade on decaying cyanobacteria. Based on a comparative analysis of phenotypic, genomic and ecological characteristics we propose to establish the novel taxa Salinivirga cyanobacteriivorans gen. nov., sp. nov., represented by the type strain L21‐Spi‐D4 T , and Salinivirgaceae fam. nov., comprising sequences of the MgMjR‐022 clade.

Summary As in many deep underground environments, the microbial communities in subsurface high‐CO 2 ecosystems remain relatively unexplored. Recent investigations based on single‐gene assays revealed a remarkable variety of organisms from little studied phyla in Crystal Geyser (Utah, USA), a site where deeply sourced CO 2 ‐saturated fluids are erupted at the surface. To provide genomic resolution of the metabolisms of these organisms, we used a novel metagenomic approach to recover 227 high‐quality genomes from 150 microbial species affiliated with 46 different phylum‐level lineages. Bacteria from two novel phylum‐level lineages have the capacity for CO 2 fixation. Analyses of carbon fixation pathways in all studied organisms revealed that the Wood‐Ljungdahl pathway and the Calvin‐Benson‐Bassham Cycle occurred with the highest frequency, whereas the reverse TCA cycle was little used. We infer that this, and selection for form II RuBisCOs, are adaptions to high CO 2 ‐concentrations. However, many autotrophs can also grow mixotrophically, a strategy that confers metabolic versatility. The assignment of 156 hydrogenases to 90 different organisms suggests that H 2 is an important inter‐species energy currency even under gaseous CO 2 ‐saturation. Overall, metabolic analyses at the organism level provided insight into the biochemical cycles that support subsurface life under the extreme condition of CO 2 saturation.

Summary Virtually all aphids maintain an obligate mutualistic symbiosis with bacteria from the Buchnera genus, which produce essential nutrients for their aphid hosts. Most aphids from the Lachninae subfamily have been consistently found to house additional endosymbionts, mainly Serratia symbiotica . This apparent dependence on secondary endosymbionts was proposed to have been triggered by the loss of the riboflavin biosynthetic capability by Buchnera in the Lachninae last common ancestor. However, an integral large‐scale analysis of secondary endosymbionts in the Lachninae is still missing, hampering the interpretation of the evolutionary and genomic analyses of these endosymbionts. Here, we analysed the endosymbionts of selected representatives from seven different Lachninae genera and nineteen species, spanning four tribes, both by FISH (exploring the symbionts’ morphology and tissue tropism) and 16S rRNA gene sequencing. We demonstrate that all analysed aphids possess dual symbiotic systems, and while most harbour S. symbiotica , some have undergone symbiont replacement by other phylogenetically‐distinct bacterial taxa. We found that these secondary associates display contrasting cell shapes and tissue tropism, and some appear to be lineage‐specific. We propose a scenario for symbiont establishment in the Lachninae, followed by changes in the symbiont's tissue tropism and symbiont replacement events, thereby highlighting the extraordinary versatility of host‐symbiont interactions.

Summary Until now anaerobic oxidation of VFA at high salt‐pH has been demonstrated only at sulfate‐reducing conditions. Here, we present results of a microbiological investigation of anaerobic conversion of organic acids and alcohols at methanogenic conditions by syntrophic associations enriched from hypersaline soda lakes in Central Asia. Sediment incubation experiments showed active, albeit very slow, methane formation from acetate, propionate, butyrate and C 2 C 4 alcohols at pH 10 and various levels of salinity. Enrichments of syntrophic associations using hydrogenotrophic members of the genus Methanocalculus from soda lakes as partners resulted in several highly enriched cultures converting acetate, propionate, butyrate, benzoate and EtOH to methane. Most syntrophs belonged to Firmicutes , while the propionate‐oxidizer formed a novel lineage within the family Syntrophobacteraceae in the Deltaproteobacteria . The acetate‐oxidizing syntroph was identified as ‘ Ca . Syntrophonatronum acetioxidans’ previously found to oxidize acetate at sulfate‐reducing conditions up to salt‐saturating concentrations. Butyrate and a benzoate‐degrading syntrophs represent novel genus‐level lineages in Syntrophomonadales which are proposed as Candidatus taxons ‘Syntrophobaca’, ‘Syntrophocurvum’ and ‘Syntropholuna’. Overall, despite very slow growth, the results indicated the presence of a functionally competent syntrophic community in hypersaline soda lakes, capable of efficient oxidation of fermentation products to methane at extremely haloalkaline conditions.

Summary The anaerobic oxidation of methane (AOM) is mediated by consortia of anaerobic methane‐oxidizing archaea (ANME) and their specific partner bacteria. In thermophilic AOM consortia enriched from Guaymas Basin, members of the ANME‐1 clade are associated with bacteria of the HotSeep‐1 cluster, which likely perform direct electron exchange via nanowires. The partner bacterium was enriched with hydrogen as sole electron donor and sulfate as electron acceptor. Based on phylogenetic, genomic and metabolic characteristics we propose to name this chemolithoautotrophic sulfate reducer Candidatus Desulfofervidus auxilii. Ca . D. auxilii grows on hydrogen at temperatures between 50°C and 70°C with an activity optimum at 60°C and doubling time of 4–6 days. Its genome draft encodes for canonical sulfate reduction, periplasmic and soluble hydrogenases and autotrophic carbon fixation via the reductive tricarboxylic acid cycle. The presence of genes for pili formation and cytochromes, and their similarity to genes of Geobacter spp., indicate a potential for syntrophic growth via direct interspecies electron transfer when the organism grows in consortia with ANME. This first ANME‐free enrichment of an AOM partner bacterium and its characterization opens the perspective for a deeper understanding of syntrophy in anaerobic methane oxidation.

Summary Termite gut flagellates are typically colonized by specific bacterial symbionts. Here we describe the phylogeny, ultrastructure and subcellular location of ‘ Candidatus Adiutrix intracellularis’, an intracellular symbiont of Trichonympha collaris in the termite Zootermopsis nevadensis . It represents a novel, deep‐branching clade of uncultured Deltaproteobacteria widely distributed in intestinal tracts of termites and cockroaches. Fluorescence in situ hybridization and transmission electron microscopy localized the endosymbiont near hydrogenosomes in the posterior part and near the ectosymbiont ‘ Candidatus Desulfovibrio trichonymphae’ in the anterior part of the host cell. The draft genome of ‘ Ca . Adiutrix intracellularis’ obtained from a metagenomic library revealed the presence of a complete gene set encoding the Wood–Ljungdahl pathway, including two homologs of fdhF encoding hydrogenase‐linked formate dehydrogenases (FDH H ) and all other components of the recently described hydrogen‐dependent carbon dioxide reductase (HDCR) complex, which substantiates previous claims that the symbiont is capable of reductive acetogenesis from CO 2 and H 2 . The close phylogenetic relationship between the HDCR components and their homologs in homoacetogenic Firmicutes and Spirochaetes suggests that the deltaproteobacterium acquired the capacity for homoacetogenesis via lateral gene transfer. The presence of genes for nitrogen fixation and the biosynthesis of amino acids and cofactors indicate the nutritional nature of the symbiosis.