Environmental Microbiology

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 Although metabolic pathways and associated enzymes of anaerobic ammonium oxidation (anammox) of ‘ Ca . Kuenenia stuttgartiensis’ have been studied, those of other anammox bacteria are still poorly understood. reduction to NO is considered to be the first step in the anammox metabolism of ‘ Ca . K. stuttgartiensis’, however, ‘ Ca . Brocadia’ lacks the genes that encode canonical NO‐forming nitrite reductases (NirS or NirK) in its genome, which is different from ‘ Ca . K. stuttgartiensis’. Here, we studied the anammox metabolism of ‘ Ca . Brocadia sinica’. 15 N‐tracer experiments demonstrated that ‘ Ca . B. sinica’ cells could reduce to NH 2 OH, instead of NO, with as yet unidentified nitrite reductase(s). Furthermore, N 2 H 4 synthesis, downstream reaction of reduction, was investigated using a purified ‘ Ca . B. sinica' hydrazine synthase (Hzs) and intact cells. Both the ‘ Ca . B. sinica’ Hzs and cells utilized NH 2 OH and , but not NO and , for N 2 H 4 synthesis and further oxidized N 2 H 4 to N 2 gas. Taken together, the metabolic pathway of ‘ Ca . B. sinica’ is NH 2 OH‐dependent and different from the one of ‘ Ca . K. stuttgartiensis’, indicating metabolic diversity of anammox bacteria.

Summary The C hlamydiae are a phylum of obligate intracellular bacteria comprising important human and animal pathogens, yet their occurrence in the environment, their phylogenetic diversity and their host range has been largely underestimated. We investigated the seasonality of environmental chlamydiae in a Tyrrhenian coastal lake. By catalysed reporter deposition fluorescence in situ hybridization, we quantified the small planktonic cells and detected a peak in the abundance of environmental chlamydiae in early autumn with up to 5.9 × 10 4 cells ml −1 . Super‐resolution microscopy improved the visualization and quantification of these bacteria and enabled the detection of pleomorphic chlamydial cells in their protist host directly in an environmental sample. To isolate environmental chlamydiae together with their host, we applied a high‐throughput limited dilution approach and successfully recovered a V exillifera sp., strain harbouring chlamydiae (93% 16 S r RNA sequence identity to S imkania negevensis ), tentatively named ‘ C andidatus   N eptunochlamydia vexilliferae’. Transmission electron microscopy in combination with fluorescence in situ hybridization was used to prove the intracellular location of these bacteria representing the first strain of marine chlamydiae stably maintained alongside with their host in a laboratory culture. Taken together, this study contributes to a better understanding of the distribution and diversity of environmental chlamydiae in previously neglected marine environments.

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 R ickettsiae comprise intracellular bacterial symbionts and pathogens infecting diverse eukaryotes. Here, we provide a detailed characterization of ‘ C andidatus   J idaibacter acanthamoeba’, a rickettsial symbiont of A canthamoeba . The bacterium establishes the infection in its amoeba host within 2 h where it replicates within vacuoles. Higher bacterial loads and accelerated spread of infection at elevated temperatures were observed. The infection had a negative impact on host growth rate, although no increased levels of host cell lysis were seen. Phylogenomic analysis identified this bacterium as member of the M idichloriaceae . Its 2.4 Mb genome represents the largest among R ickettsiales and is characterized by a moderate degree of pseudogenization and a high coding density. We found an unusually large number of genes encoding proteins with eukaryotic‐like domains such as ankyrins, leucine‐rich repeats and tetratricopeptide repeats, which likely function in host interaction. There are a total of three divergent, independently acquired type IV secretion systems, and 35 flagellar genes representing the most complete set found in an obligate intracellular A lphaproteobacterium . The deeply branching phylogenetic position of ‘ C andidatus   J idaibacter acanthamoeba’ together with its ancient features place it closely to the rickettsial ancestor and helps to better understand the transition from a free‐living to an intracellular lifestyle.

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.

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 The bacterial tree contains many deep‐rooting clades without any cultured representatives. One such clade is ‘ E ndomicrobia’, a class‐level lineage in the phylum E lusimicrobia represented so far only by intracellular symbionts of termite gut flagellates. Here, we report the isolation and characterization of the first free‐living member of this clade from sterile‐filtered gut homogenate of defaunated (starch‐fed) R eticulitermes santonensis . Strain R sa215 is a strictly anaerobic ultramicrobacterium that grows exclusively on glucose, which is fermented to lactate, acetate, hydrogen and CO 2 . Ultrastructural analysis revealed a G ram‐negative cell envelope and a peculiar cell cycle. The genome contains a single set of nif genes that encode homologues of G roup IV nitrogenases, which were so far considered to have functions other than nitrogen fixation. We documented nitrogenase activity and diazotrophic growth by measuring acetylene reduction activity and 15 N 2 incorporation into cell mass, and demonstrated that transcription of nifH and nitrogenase activity occur only in the absence of ammonium. Based on the ancestral relationship to ‘ C andidatus   E ndomicrobium trichonymphae’ and other obligate endosymbionts, we propose the name ‘ E ndomicrobium proavitum ’ gen. nov., sp. nov. for the first isolate of this lineage and the name ‘ E ndomicrobia ’ class. nov. for the entire clade.