The ISME Journal

Abstract Marine and estuary sediments contain a variety of uncultured archaea whose metabolic and ecological roles are unknown. De novo assembly and binning of high-throughput metagenomic sequences from the sulfate–methane transition zone in estuary sediments resulted in the reconstruction of three partial to near-complete (2.4–3.9 Mb) genomes belonging to a previously unrecognized archaeal group. Phylogenetic analyses of ribosomal RNA genes and ribosomal proteins revealed that this group is distinct from any previously characterized archaea. For this group, found in the White Oak River estuary, and previously registered in sedimentary samples, we propose the name ‘Thorarchaeota’. The Thorarchaeota appear to be capable of acetate production from the degradation of proteins. Interestingly, they also have elemental sulfur and thiosulfate reduction genes suggesting they have an important role in intermediate sulfur cycling. The reconstruction of these genomes from a deeply branched, widespread group expands our understanding of sediment biogeochemistry and the evolutionary history of Archaea.

Abstract New and emerging environmental pathogens pose some of the greatest threats to modern aquaculture, a critical source of food protein globally. As with other intensive farming practices, increasing our understanding of the biology of infections is important to improve animal welfare and husbandry. The gill infection epitheliocystis is increasingly problematic in gilthead seabream (Sparus aurata), a major Mediterranean aquaculture species. Epitheliocystis is generally associated with chlamydial bacteria, yet we were not able to localise chlamydial targets within the major gilthead seabream lesions. Two previously unidentified species within a novel β-proteobacterial genus were instead identified. These co-infecting intracellular bacteria have been characterised using high-resolution imaging and genomics, presenting the most comprehensive study on epitheliocystis agents to date. Draft genomes of the two uncultured species, Ca. Ichthyocystis hellenicum and Ca. Ichthyocystis sparus, have been de novo sequenced and annotated from preserved material. Analysis of the genomes shows a compact core indicating a metabolic dependency on the host, and an accessory genome with an unprecedented number of tandemly arrayed gene families. This study represents a critical insight into novel, emerging fish pathogens and will be used to underpin future investigations into the bacterial origins, and to develop diagnostic and treatment strategies.

Abstract Microorganisms in biological wastewater treatment plants require adaptive strategies to deal with rapidly fluctuating environmental conditions. At the population level, the filamentous bacterium Candidatus Microthrix parvicella (Ca. M. parvicella) has been found to fine-tune its gene expression for optimized substrate assimilation. Here we investigated in situ substrate assimilation by single cells of Ca. M. parvicella using nano-scale secondary-ion mass spectrometry (nanoSIMS). NanoSIMS imaging highlighted phenotypic heterogeneity among Ca. M. parvicella cells of the same filament, whereby 13C-oleic acid and 13C-glycerol-3-phosphate assimilation occurred in ≈21–55% of cells, despite non-assimilating cells being intact and alive. In response to alternating aerobic–anoxic regimes, 13C-oleic acid assimilation occurred among subpopulations of Ca. M. parvicella cells (≈3–28% of cells). Furthermore, Ca. M. parvicella cells exhibited two temperature optima for 13C-oleic acid assimilation and associated growth rates. These results suggest that phenotypic heterogeneity among Ca. M. parvicella cells allows the population to adapt rapidly to fluctuating environmental conditions facilitating its widespread occurrence in biological wastewater treatment plants.

Abstract Previous studies on enhanced biological phosphorus removal (EBPR) have focused on reconstructing genomic blueprints for the model polyphosphate-accumulating organism Candidatus Accumulibacter phosphatis. Here, a time series metatranscriptome generated from enrichment cultures of Accumulibacter was used to gain insight into anerobic/aerobic metabolism and regulatory mechanisms within an EBPR cycle. Co-expressed gene clusters were identified displaying ecologically relevant trends consistent with batch cycle phases. Transcripts displaying increased abundance during anerobic acetate contact were functionally enriched in energy production and conversion, including upregulation of both cytoplasmic and membrane-bound hydrogenases demonstrating the importance of transcriptional regulation to manage energy and electron flux during anerobic acetate contact. We hypothesized and demonstrated hydrogen production after anerobic acetate contact, a previously unknown strategy for Accumulibacter to maintain redox balance. Genes involved in anerobic glycine utilization were identified and phosphorus release after anerobic glycine contact demonstrated, suggesting that Accumulibacter routes diverse carbon sources to acetyl-CoA formation via previously unrecognized pathways. A comparative genomics analysis of sequences upstream of co-expressed genes identified two statistically significant putative regulatory motifs. One palindromic motif was identified upstream of genes involved in PHA synthesis and acetate activation and is hypothesized to be a phaR binding site, hence representing a hypothetical PHA modulon. A second motif was identified ~35 base pairs (bp) upstream of a large and diverse array of genes and hence may represent a sigma factor binding site. This analysis provides a basis and framework for further investigations into Accumulibacter metabolism and the reconstruction of regulatory networks in uncultured organisms.

Abstract The candidate archaeal phylum ‘Aigarchaeota’ contains microorganisms from terrestrial and subsurface geothermal ecosystems. The phylogeny and metabolic potential of Aigarchaeota has been deduced from several recent single-cell amplified genomes; however, a detailed description of their metabolic potential and in situ transcriptional activity is absent. Here, we report a comprehensive metatranscriptome-based reconstruction of the in situ metabolism of Aigarchaeota in an oxic, hot spring filamentous ‘streamer’ community. Fluorescence in situ hybridization showed that these newly discovered Aigarchaeota are filamentous, which is consistent with the presence and transcription of an actin-encoding gene. Aigarchaeota filaments are intricately associated with other community members, which include both bacteria (for example, filamentous Thermocrinis spp.) and archaea. Metabolic reconstruction of genomic and metatranscriptomic data suggests that this aigarchaeon is an aerobic, chemoorganoheterotroph with autotrophic potential. A heme copper oxidase complex was identified in the environmental genome assembly and highly transcribed in situ. Potential electron donors include acetate, fatty acids, amino acids, sugars and aromatic compounds, which may originate from extracellular polymeric substances produced by other microorganisms shown to exist in close proximity and/or autochthonous dissolved organic carbon (OC). Transcripts related to genes specific to each of these potential electron donors were identified, indicating that this aigarchaeon likely utilizes several OC substrates. Characterized members of this lineage cannot synthesize heme, and other cofactors and vitamins de novo, which suggests auxotrophy. We propose the name Candidatus ‘Calditenuis aerorheumensis’ for this aigarchaeon, which describes its filamentous morphology and its primary electron acceptor, oxygen.

Abstract Methylotrophic planktonic bacteria fulfill a particular role in the carbon cycle of lakes via the turnover of single-carbon compounds. We studied two planktonic freshwater lineages (LD28 and PRD01a001B) affiliated with Methylophilaceae (Betaproteobacteria) in Lake Zurich, Switzerland, by a combination of molecular and cultivation-based approaches. Their spatio-temporal distribution was monitored at high resolution (n=992 samples) for 4 consecutive years. LD28 methylotrophs constituted up to 11 × 107 cells l−1 with pronounced peaks in spring and autumn–winter, concomitant with blooms of primary producers. They were rare in the warm water layers during summer but abundant in the cold hypolimnion, hinting at psychrophilic growth. Members of the PRD01a001B lineage were generally less abundant but also had maxima in spring. More than 120 axenic strains from these so far uncultivated lineages were isolated from the pelagic zone by dilution to extinction. Phylogenetic analysis separated isolates into two distinct genotypes. Isolates grew slowly (μmax=0.4 d−1), were of conspicuously small size, and were indeed psychrophilic, with higher growth yield at low temperatures. Growth was enhanced upon addition of methanol and methylamine to sterile lake water. Genomic analyses of two strains confirmed a methylotrophic lifestyle with a reduced set of genes involved in C1 metabolism. The very small and streamlined genomes (1.36 and 1.75 Mb) shared several pathways with the marine OM43 lineage. As the closest described taxa (Methylotenera sp.) are only distantly related to either set of isolates, we propose a new genus with two species, that is, ‘Candidatus Methylopumilus planktonicus’ (LD28) and ‘Candidatus Methylopumilus turicensis’ (PRD01a001B).

Abstract Magnetotactic bacteria (MTB) of the genus ‘Candidatus Magnetobacterium’ in phylum Nitrospirae are of great interest because of the formation of hundreds of bullet-shaped magnetite magnetosomes in multiple bundles of chains per cell. These bacteria are worldwide distributed in aquatic environments and have important roles in the biogeochemical cycles of iron and sulfur. However, except for a few short genomic fragments, no genome data are available for this ecologically important genus, and little is known about their metabolic capacity owing to the lack of pure cultures. Here we report the first draft genome sequence of 3.42 Mb from an uncultivated strain tentatively named ‘Ca. Magnetobacterium casensis’ isolated from Lake Miyun, China. The genome sequence indicates an autotrophic lifestyle using the Wood–Ljungdahl pathway for CO2 fixation, which has not been described in any previously known MTB or Nitrospirae organisms. Pathways involved in the denitrification, sulfur oxidation and sulfate reduction have been predicted, indicating its considerable capacity for adaptation to variable geochemical conditions and roles in local biogeochemical cycles. Moreover, we have identified a complete magnetosome gene island containing mam, mad and a set of novel genes (named as man genes) putatively responsible for the formation of bullet-shaped magnetite magnetosomes and the arrangement of multiple magnetosome chains. This first comprehensive genomic analysis sheds light on the physiology, ecology and biomineralization of the poorly understood ‘Ca. Magnetobacterium’ genus.

Abstract Amoebae serve as hosts for various intracellular bacteria, including human pathogens. These microbes are able to overcome amoebal defense mechanisms and successfully establish a niche for replication, which is usually the cytoplasm. Here, we report on the discovery of a bacterial symbiont that is located inside the nucleus of its Hartmannella sp. host. This symbiont, tentatively named ‘Candidatus Nucleicultrix amoebiphila’, is only moderately related to known bacteria (∼90% 16S and 23S rRNA sequence similarity) and member of a novel clade of protist symbionts affiliated with the Rickettsiales and Rhodospirillales. Screening of 16S rRNA amplicon data sets revealed a broad distribution of these bacteria in freshwater and soil habitats. ‘Candidatus Nucleicultrix amoebiphila’ traffics within 6 h post infection to the host nucleus. Maximum infection levels are reached after 96–120 h, at which time point the nucleus is pronouncedly enlarged and filled with bacteria. Transmission of the symbionts occurs vertically upon host cell division but may also occur horizontally through host cell lysis. Although we observed no impact on the fitness of the original Hartmannella sp. host, the bacteria are rather lytic for Acanthamoeba castellanii. Intranuclear symbiosis is an exceptional phenomenon, and amoebae represent an ideal model system to further investigate evolution and underlying molecular mechanisms of these unique microbial associations.

Abstract The Miscellaneous Crenarchaeota group (MCG) Archaea is one of the predominant archaeal groups in anoxic environments and may have significant roles in the global biogeochemical cycles. However, no isolate of MCG has been cultivated or characterized to date. In this study, we investigated the genetic organization, ecophysiological properties and evolutionary relationships of MCG archaea with other archaeal members using metagenome information and the result of gene expression experiments. A comparison of the gene organizations and similarities around the 16S rRNA genes from all available MCG fosmid and cosmid clones revealed no significant synteny among genomic fragments, demonstrating that there are large genetic variations within members of the MCG. Phylogenetic analyses of large-subunit+small-subunit rRNA, concatenated ribosomal protein genes and topoisomerases IB gene (TopoIB) all demonstrate that MCG constituted a sister lineage to the newly proposed archaeal phylum Aigarchaeota and Thaumarchaeota. Genes involved in protocatechuate degradation and chemotaxis were found in a MCG fosmid 75G8 genome fragment, suggesting that this MCG member may have a role in the degradation of aromatic compounds. Moreover, the expression of a putative 4-carboxymuconolactone decarboxylase was observed when the sediment was supplemented with protocatechuate, further supporting the hypothesis that this MCG member degrades aromatic compounds.

Abstract The glycogen-accumulating organism (GAO) ‘Candidatus Competibacter’ (Competibacter) uses aerobically stored glycogen to enable anaerobic carbon uptake, which is subsequently stored as polyhydroxyalkanoates (PHAs). This biphasic metabolism is key for the Competibacter to survive under the cyclic anaerobic-‘feast’: aerobic-‘famine’ regime of enhanced biological phosphorus removal (EBPR) wastewater treatment systems. As they do not contribute to phosphorus (P) removal, but compete for resources with the polyphosphate-accumulating organisms (PAO), thought responsible for P removal, their proliferation theoretically reduces the EBPR capacity. In this study, two complete genomes from Competibacter were obtained from laboratory-scale enrichment reactors through metagenomics. Phylogenetic analysis identified the two genomes, ‘Candidatus Competibacter denitrificans’ and ‘Candidatus Contendobacter odensis’, as being affiliated with Competibacter-lineage subgroups 1 and 5, respectively. Both have genes for glycogen and PHA cycling and for the metabolism of volatile fatty acids. Marked differences were found in their potential for the Embden–Meyerhof–Parnas and Entner–Doudoroff glycolytic pathways, as well as for denitrification, nitrogen fixation, fermentation, trehalose synthesis and utilisation of glucose and lactate. Genetic comparison of P metabolism pathways with sequenced PAOs revealed the absence of the Pit phosphate transporter in the Competibacter-lineage genomes—identifying a key metabolic difference with the PAO physiology. These genomes are the first from any GAO organism and provide new insights into the complex interaction and niche competition between PAOs and GAOs in EBPR systems.