Environmental Microbiology

Summary Enhanced biological phosphorus removal (EBPR) exploits the metabolism of polyphosphate‐accumulating organisms (PAOs) to remove excess phosphorus (P) from wastewater treatment. Candidatus Accumulibacter phosphatis (Accumulibacter) is the most abundant and well‐studied PAO in EBPR systems. In a previous study, we detected polyphosphates throughout peripheral bay sediments, and hypothesized that an estuary is an ideal setting to evaluate PAOs in a natural system, given that estuaries are characterized by dynamic dissolved oxygen fluctuations that potentially favour PAO metabolism. We detected nucleotide sequences attributable to Accumulibacter (16S rRNA, ppk1 ) in sediments within three peripheral bays of the Columbia River estuary at abundances rivalling those observed in conventional wastewater treatment plants (0.01%–2.6%). Most of the sequences attributable to Accumulibacter were Type I rather than Type II, despite the fact that the estuary does not have particularly high nutrient concentrations. The highest diversity of Accumulibacter was observed in oligohaline peripheral bays, while the greatest abundances were observed at the mouth of the estuary in mesohaline sediments in the spring and summer. In addition, an approximately 70% increase in polyphosphate concentrations observed at one of the sites between dawn and dusk suggests that PAOs may play an important role in P cycling in estuary sediments.

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 Scale insects are commonly associated with obligate, intracellular microorganisms which play important roles in complementing their hosts with essential nutrients. Here we characterized the symbiotic system of Greenisca brachypodii , a member of the family Eriococcidae. Histological and ultrastructural analyses have indicated that G. brachypodii is stably associated with coccoid and rod‐shaped bacteria. Phylogenetic analyses have revealed that the coccoid bacteria represent a sister group to the secondary symbiont of the mealybug Melanococcus albizziae , whereas the rod‐shaped symbionts are close relatives of Arsenophonus symbionts in insects – to our knowledge, this is the first report of the presence of Arsenophonus bacterium in scale insects. As a comparison of 16S and 23S rRNA genes sequences of the G. brachypodii coccoid symbiont with other gammaprotebacterial sequences showed only low similarity (∼90%), we propose the name ‘ Candidatus Kotejella greeniscae’ for its tentative classification. Both symbionts are transovarially transmitted from one generation to the next. The infection takes place in the neck region of the ovariole. The bacteria migrate between follicular cells, as well as through the cytoplasm of those cells to the perivitelline space, where they form a characteristic ‘symbiont ball’. Our findings provide evidence for a polyphyletic origin of symbionts of Eriococcidae.

Summary Understanding of global methane sources and sinks is a prerequisite for the design of strategies to counteract global warming. Microbial methane oxidation in soils represents the largest biological sink for atmospheric methane. However, still very little is known about the identity, metabolic properties and distribution of the microbial group proposed to be responsible for most of this uptake, the uncultivated upland soil cluster α (USCα). Here, we reconstructed a draft genome of USCα from a combination of targeted cell sorting and metagenomes from forest soil, providing the first insights into its metabolic potential and environmental adaptation strategies. The 16S rRNA gene sequence recovered was distinctive and suggests this crucial group as a new genus within the Beijerinckiaceae , close to Methylocapsa . Application of a fluorescently labelled suicide substrate for the particulate methane monooxygenase enzyme (pMMO) coupled to 16S rRNA fluorescence in situ hybridisation (FISH) allowed for the first time a direct link of the high‐affinity activity of methane oxidation to USCα cells in situ . Analysis of the global biogeography of this group further revealed its presence in previously unrecognized habitats, such as subterranean and volcanic biofilm environments, indicating a potential role of these environments in the biological sink for atmospheric methane.

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 Obligate acidophilic members of the thaumarchaeotal genus Candidatus Nitrosotalea play an important role in nitrification in acidic soils, but their evolutionary and physiological adaptations to acidic environments are still poorly understood, with only a single member of this genus ( Ca . N. devanaterra) having its genome sequenced. In this study, we sequenced the genomes of two additional cultured Ca . Nitrosotalea strains, extracted an almost complete Ca . Nitrosotalea metagenome‐assembled genome from an acidic fen, and performed comparative genomics of the four Ca . Nitrosotalea genomes with 19 other archaeal ammonia oxidiser genomes. Average nucleotide and amino acid identities revealed that the four Ca . Nitrosotalea strains represent separate species within the genus. The four Ca . Nitrosotalea genomes contained a core set of 103 orthologous gene families absent from all other ammonia‐oxidizing archaea and, for most of these gene families, expression could be demonstrated in laboratory culture or the environment via proteomic or metatranscriptomic analyses respectively. Phylogenetic analyses indicated that four of these core gene families were acquired by the Ca . Nitrosotalea common ancestor via horizontal gene transfer from acidophilic representatives of Euryarchaeota. We hypothesize that gene exchange with these acidophiles contributed to the competitive success of the Ca . Nitrosotalea lineage in acidic environments.

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 Marine Thaumarchaeota are abundant ammonia‐oxidizers but have few representative laboratory‐cultured strains. We report the cultivation of Candidatus Nitrosomarinus catalina SPOT01, a novel strain that is less warm‐temperature tolerant than other cultivated Thaumarchaeota. Using metagenomic recruitment, strain SPOT01 comprises a major portion of Thaumarchaeota (4–54%) in temperate Pacific waters. Its complete 1.36 Mbp genome possesses several distinguishing features: putative phosphorothioation (PT) DNA modification genes; a region containing probable viral genes; and putative urea utilization genes. The PT modification genes and an adjacent putative restriction enzyme (RE) operon likely form a restriction modification (RM) system for defence from foreign DNA. PacBio sequencing showed >98% methylation at two motifs, and inferred PT guanine modification of 19% of possible TGCA sites. Metagenomic recruitment also reveals the putative virus region and PT modification and RE genes are present in 18–26%, 9–14% and <1.5% of natural populations at 150 m with ≥85% identity to strain SPOT01. The presence of multiple probable RM systems in a highly streamlined genome suggests a surprising importance for defence from foreign DNA for dilute populations that infrequently encounter viruses or other cells. This new strain provides new insights into the ecology, including viral interactions, of this important group of marine microbes.

Summary Several Chlamydiales families are associated with epitheliocystis, a common condition of the fish gill epithelium. These families share common ancestors with the Chlamydiaceae and environmental Chlamydiae . Due to the lack of culture systems, little is known about the biology of these chlamydial fish pathogens. We investigated epitheliocystis in cultured Orange‐spotted grouper ( Epinephelus coioides ) from North Queensland, Australia. Basophilic inclusions were present in the gills of 22/31 fish and the presence of the chlamydial pathogen in the cysts was confirmed by in situ hybridization. Giant grouper ( Epinephelus lanceolatus ) cultured in the same systems were epitheliocystis free. 16S rRNA gene sequencing revealed a novel member of the Candidatus Parilichlamydiaceae: Ca . Similichlamydia epinephelii. Using metagenomic approaches, we obtained an estimated 68% of the chlamydial genome, revealing that this novel chlamydial pathogen shares a number of key pathogenic hallmarks with the Chlamydiaceae , including an intact Type III Secretion system and several chlamydial virulence factors. This provides additional evidence that these pathogenic mechanisms were acquired early in the evolution of this unique bacterial phylum. The identification and genomic characterization of Ca . S. epinephelii provides new opportunities to study the biology of distantly‐related chlamydial pathogens while shining a new light on the evolution of pathogenicity of the Chlamydiaceae .