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Methanogenic archaea are a diverse, polyphyletic group of strictly anaerobic prokaryotes capable of producing methane as their primary metabolic product. It has been over three decades since minimal standards for their taxonomic description have been proposed. In light of advancements in technology and amendments in systematic microbiology, revision of the older criteria for taxonomic description is essential. Most of the previously recommended minimum standards regarding phenotypic characterization of pure cultures are maintained. Electron microscopy and chemotaxonomic methods like whole-cell protein and lipid analysis are desirable but not required. Because of advancements in DNA sequencing technologies, obtaining a complete or draft whole genome sequence for type strains and its deposition in a public database are now mandatory. Genomic data should be used for rigorous comparison to close relatives using overall genome related indices such as average nucleotide identity and digital DNA–DNA hybridization. Phylogenetic analysis of the 16S rRNA gene is also required and can be supplemented by phylogenies of the mcrA gene and phylogenomic analysis using multiple conserved, single-copy marker genes. Additionally, it is now established that culture purity is not essential for studying prokaryotes, and description of Candidatus methanogenic taxa using single-cell or metagenomics along with other appropriate criteria is a viable alternative. The revisions to the minimal criteria proposed here by the members of the Subcommittee on the Taxonomy of Methanogenic Archaea of the International Committee on Systematics of Prokaryotes should allow for rigorous yet practical taxonomic description of these important and diverse microbes.

Background Ammonia-oxidizing archaea (AOA) are among the most ubiquitous and abundant microorganisms in the ocean, and play a primary role in the global nitrogen cycle. Antarctic polynyas are ecological hotspots with the highest primary productivity in the Southern Ocean during summer, and their production and formation reflect anthropogenic climate change. Strong seasonal variations in AOA abundance from winter to summer may have affected marine nitrification. We utilized metagenomics and metatranscriptomics to gain insights into the physiology and metabolism of AOA in polar oceans, which are associated with ecosystem functioning. Results A polar-specific ecotype of AOA, from the “Candidatus Nitrosomarinus”-like group, was observed to be dominant in the Amundsen Sea Polynya (ASP), West Antarctica, during a succession of summer phytoplankton blooms. AOA had the highest transcriptional activity among prokaryotes during the bloom decline phase (DC). Expression rank profiles for transcripts of key genes involved in ammonia oxidation (e.g., genes encoding subunits of ammonia monooxygenase, NirK, ferredoxin, and plastocyanin) and carbon fixation (3-hydroxypropionate/4-hydroxybutyrate cycle) indicated that AOA are actively involved in nitrification in the bloom DC. The highly ranked transcripts of flavodoxin and superoxide dismutase genes imply that AOA are exposed to iron limitation and ROS toxicity, respectively. The expression of high-affinity ammonia transporter (Amt2) gene, together with the absence of urea utilization-related genes, indicates that ammonia is a major source of AOA nitrification. The Phn system may aid AOA in acquiring phosphorus-containing compounds. The expression patterns of cdvA and cdvB also support the active cell division and cellular processes of AOA in the bloom DC. Conclusions The polar-specific ecotype AOA of “Candidatus Nitrosomarinus”-like group was found to be the representative AOA in the ASP, West Antarctica. Metatranscriptomic analysis of key genes involved in ammonia oxidation, carbon fixation, transport, and cell division indicated that this polar ecotype AOA was actively involved in nitrification in the bloom DC in the ASP. This study revealed the physiological and metabolic traits of this key polar-type AOA in response to phytoplankton blooms in the ASP and provided insights into AOA functions in polar oceans.

Short-chain alkanes (SCA; C2-C4) emitted from geological sources contribute to photochemical pollution and ozone production in the atmosphere. Microorganisms that oxidize SCA and thereby mitigate their release from geothermal environments have rarely been studied. In this study, propane-oxidizing cultures could not be grown from acidic geothermal samples by enrichment on propane alone, but instead required methane addition, indicating that propane was co-oxidized by methanotrophs. “Methylacidiphilum” isolates from these enrichments did not grow on propane as a sole energy source but unexpectedly did grow on C3 compounds such as 2-propanol, acetone, and acetol. A gene cluster encoding the pathway of 2-propanol oxidation to pyruvate via acetol was upregulated during growth on 2-propanol. Surprisingly, this cluster included one of three genomic operons (pmoCAB3) encoding particulate methane monooxygenase (PMO), and several physiological tests indicated that the encoded PMO3 enzyme mediates the oxidation of acetone to acetol. Acetone-grown resting cells oxidized acetone and butanone but not methane or propane, implicating a strict substrate specificity of PMO3 to ketones instead of alkanes. Another PMO-encoding operon, pmoCAB2, was induced only in methane-grown cells, and the encoded PMO2 could be responsible for co-metabolic oxidation of propane to 2-propanol. In nature, propane probably serves primarily as a supplemental growth substrate for these bacteria when growing on methane.

Natural and anthropogenic wetlands are major sources of the atmospheric greenhouse gas methane. Methane emissions from wetlands are mitigated by methanotrophic bacteria at the oxic–anoxic interface, a zone of intense redox cycling of carbon, sulfur, and nitrogen compounds. Here, we report on the isolation of an aerobic methanotrophic bacterium, ‘ Methylovirgula thiovorans ' strain HY1, which possesses metabolic capabilities never before found in any methanotroph. Most notably, strain HY1 is the first bacterium shown to aerobically oxidize both methane and reduced sulfur compounds for growth. Genomic and proteomic analyses showed that soluble methane monooxygenase and XoxF-type alcohol dehydrogenases are responsible for methane and methanol oxidation, respectively. Various pathways for respiratory sulfur oxidation were present, including the Sox–rDsr pathway and the S 4 I system. Strain HY1 employed the Calvin–Benson–Bassham cycle for CO 2 fixation during chemolithoautotrophic growth on reduced sulfur compounds. Proteomic and microrespirometry analyses showed that the metabolic pathways for methane and thiosulfate oxidation were induced in the presence of the respective substrates. Methane and thiosulfate could therefore be independently or simultaneously oxidized. The discovery of this versatile bacterium demonstrates that methanotrophy and thiotrophy are compatible in a single microorganism and underpins the intimate interactions of methane and sulfur cycles in oxic–anoxic interface environments.

A Gram-stain-negative, aerobic, non-motile and coccoid methanotroph, strain IM1T, was isolated from hot spring soil. Cells of strain IM1T were catalase-negative, oxidase-positive and displayed a characteristic intracytoplasmic membrane arrangement of type I methanotrophs. The strain possessed genes encoding both membrane-bound and soluble methane monooxygenases and grew only on methane or methanol. The strain was capable of growth at temperatures between 15 and 48 °C (optimum, 30–45 °C) and pH values between pH 4.8 and 8.2 (optimum, pH 6.2–7.0). Based on phylogenetic analysis of 16S rRNA gene and PmoA sequences, strain IM1T was demonstrated to be affiliated to the genus Methylococcus . The 16S rRNA gene sequence of this strain was most closely related to the sequences of an uncultured bacterium clone FD09 (100 %) and a partially described cultured Methylococcus sp. GDS2.4 (99.78 %). The most closely related taxonomically described strains were Methylococcus capsulatus TexasT (97.92 %), Methylococcus capsulatus Bath (97.86 %) and Methyloterricola oryzae 73aT (94.21 %). Strain IM1T shared average nucleotide identity values of 85.93 and 85.62 % with Methylococcus capsulatus strains TexasT and Bath, respectively. The digital DNA–DNA hybridization value with the closest type strain was 29.90 %. The DNA G+C content of strain IM1T was 63.3 mol% and the major cellular fatty acids were C16 : 0 (39.0 %), C16 : 1 ω7c (24.0 %), C16 : 1 ω6c (13.6 %) and C16 : 1 ω5c (12.0 %). The major ubiquinone was methylene-ubiquinone-8. On the basis of phenotypic, genetic and phylogenetic data, strain IM1T represents a novel species of the genus Methylococcus for which the name Methylococcus geothermalis sp. nov. is proposed, with strain IM1T (=JCM 33941T=KCTC 72677T) as the type strain.

The excessive input of nutrients into groundwater can accelerate eutrophication in associated surface water systems. This study combined hydrogeochemistry, multi isotope tracers, and microbiological data to estimate nutrient sources and the effects of groundwater-surface water interactions on the spatiotemporal variation of nutrients in groundwater connected to a large weir-regulated river in South Korea. δ

Nitrification, the oxidation of ammonia to nitrate, is an essential process in the biogeochemical nitrogen cycle. The first step of nitrification, ammonia oxidation, is performed by three, often co-occurring guilds of chemolithoautotrophs: ammonia-oxidizing bacteria (AOB), archaea (AOA), and complete ammonia oxidizers (comammox). Substrate kinetics are considered to be a major niche-differentiating factor between these guilds, but few AOA strains have been kinetically characterized. Here, the ammonia oxidation kinetic properties of 12 AOA representing all major cultivated phylogenetic lineages were determined using microrespirometry. Members of the genus Nitrosocosmicus have the lowest affinity for both ammonia and total ammonium of any characterized AOA, and these values are similar to previously determined ammonia and total ammonium affinities of AOB. This contrasts previous assumptions that all AOA possess much higher substrate affinities than their comammox or AOB counterparts. The substrate affinity of ammonia oxidizers correlated with their cell surface area to volume ratios. In addition, kinetic measurements across a range of pH values supports the hypothesis that—like for AOB—ammonia and not ammonium is the substrate for the ammonia monooxygenase enzyme of AOA and comammox. Together, these data will facilitate predictions and interpretation of ammonia oxidizer community structures and provide a robust basis for establishing testable hypotheses on competition between AOB, AOA, and comammox.

A facultatively anaerobic and Gram-stain-negative bacterium, strain GY_GT, was isolated from a river (Daedeock-cheon) in Daejeon, Republic of Korea. The isolate was catalase-positive, oxidase-positive and formed yellow colonies. Strain GY_GT was phylogenetically classified as belonging in the genus Sphingorhabdus. Its closely related strains were Sphingorhabdus wooponensis 03SU3-PT (97.1 % similarity), Sphingorhabdus buctiana T5T (96.9 %), Sphingorhabdus contaminans JC216T (96.5 %), Sphingorhabdus rigui 01SU5-PT (96.5 %) and Sphingorhabdus planktonica G1A_585T (96.3 %) based on 16S rRNA gene sequences. The growth conditions for GY_GT were at 10–45 °C (optimum, 25 °C), pH 6–10 (pH 7) and 0–4% NaCl (0.5–1.5 %). Strain GY_GT could utilize turanose, d-fructose-6-phosphate, glucuronamide, α-keto-glutaric acid and acetoacetic acid. The major fatty acids of strain GY_GT were summed features 8 (C18 : 1 ω7c/C18 : 1 ω6c; 40.6 %) and 3 (C16 : 1 ω6c/C16 : 1 ω7c; 24.7 %). The major quinone required for respiration was Q-10. The polar lipids of strain GY_GT were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine and sphingolipid. The G+C content of the genome was 57.7 mol%. The average nucleotide identity and average amino acid identity values between strains GY_GT and S. wooponensis were 71.0 and 72.7 %, respectively. Based on phylogenetic and phenotypic attributes, we suggest that strain GY_GT is a novel species in the genus Sphingorhabdus and propose the name Sphingorhabdus pulchriflava. The type strain is GY_GT (=KCTC 62791T=JCM 32855T).

Members of the family Thaspiviridae have linear dsDNA genomes of 27 to 29 kbp and are the first viruses known to infect mesophilic ammonia-oxidizing archaea of the phylum Thaumarchaeota. The spindle-shaped virions of Nitrosopumilus spindle-shaped virus 1 possess short tails at one pole and measure 64±3 nm in diameter and 112±6 nm in length. This morphology is similar to that of members of the families Fuselloviridae and Halspiviridae. Virus replication is not lytic but leads to growth inhibition of the host. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family Thaspiviridae, which is available at ictv.global/report/thaspiviridae.

Nitrification, the oxidation of ammonia to nitrate, is an essential process in the biogeochemical nitrogen cycle. The first step of nitrification, ammonia oxidation, is performed by three, often co- occurring guilds of chemolithoautotrophs: ammonia-oxidizing bacteria (AOB), archaea (AOA), and complete ammonia oxidizers (comammox). Substrate kinetics are considered to be a major niche-differentiating factor between these guilds, but few AOA strains have been kinetically characterized. Here, the ammonia oxidation kinetic properties of 12 AOA representing all major phylogenetic lineages were determined using microrespirometry. Members of the genus Nitrosocosmicus have the lowest substrate affinity of any characterized AOA, which are similar to previously determined affinities of AOB. This contrasts previous assumptions that all AOA possess much higher substrate affinities than their comammox or AOB counterparts. The substrate affinity of ammonia oxidizers correlated with their cell surface area to volume ratios. In addition, kinetic measurements across a range of pH values strongly supports the hypothesis that – like for AOB – ammonia and not ammonium is the substrate for the ammonia monooxygenase enzyme of AOA and comammox. Together, these data will facilitate predictions and interpretation of ammonia oxidizer community structures and provide a robust basis for establishing testable hypotheses on competition between AOB, AOA, and comammox.

The third domain Archaea was known to thrive in extreme or anoxic environments based on cultivation studies. Recent metagenomics-based approaches revealed a widespread abundance of archaea, including ammonia-oxidizing archaea (AOA) of Thaumarchaeota in non-extreme and oxic environments. AOA alter nitrogen species availability by mediating the first step of chemolithoautotrophic nitrification, ammonia oxidation to nitrite, and are important primary producers in ecosystems, which affects the distribution and activity of other organisms in ecosystems. Thus, information on the interactions of AOA with other cohabiting organisms is a crucial element in understanding nitrogen and carbon cycles in ecosystems as well as the functioning of whole ecosystems. AOA are self-nourishing, and thus interactions of AOA with other organisms can often be indirect and broad. Besides, there are possibilities of specific and obligate interactions. Mechanisms of interaction are often not clearly identified but only inferred due to limited knowledge on the interaction factors analyzed by current technologies. Here, we overviewed different types of AOA interactions with other cohabiting organisms, which contribute to understanding AOA functions in ecosystems.

Complete ammonia-oxidizing (comammox) bacteria play key roles in environmental nitrogen cycling and all belong to the genus Nitrospira, which was originally believed to include only strict nitrite-oxidizing bacteria (sNOB). Thus, differential estimation of sNOB abundance from that of comammox Nitrospira has become problematic, since both contain nitrite oxidoreductase genes that serve as common targets for sNOB detection. Herein, we developed novel comammox Nitrospira clade A- and B-specific primer sets targeting the α-subunit of the ammonia monooxygenase gene (amoA) and a sNOB-specific primer set targeting the cyanase gene (cynS) for quantitative PCR (qPCR). The high coverage and specificity of these primers were checked by use of metagenome and metatranscriptome data sets. Efficient and specific amplification with these primers was demonstrated using various environmental samples. Using the newly designed primers, we successfully estimated the abundances of comammox Nitrospira and sNOB in samples from two chloramination-treated drinking water systems and found that, in most samples, comammox Nitrospira clade A was the dominant type of Nitrospira and also served as the primary ammonia oxidizer. Compared with other ammonia oxidizers, comammox Nitrospira had a higher abundance in process water samples in these two drinking water systems. We also demonstrated that sNOB can be readily misrepresented by an earlier method, calculated by subtracting the comammox Nitrospira abundance from the total Nitrospira abundance, especially when the comammox Nitrospira proportion is relatively high. The new primer sets were successfully applied to comammox Nitrospira and sNOB quantification, which may prove useful in understanding the roles of Nitrospira in nitrification in various ecosystems. IMPORTANCENitrospira is a dominant nitrite-oxidizing bacterium in many artificial and natural environments. The discovery of complete ammonia oxidizers in the genus Nitrospira prevents the use of previously identified primers targeting the Nitrospira 16S rRNA gene or nitrite oxidoreductase (nxr) gene for differential determination of strict nitrite-oxidizing bacteria (sNOB) in the genus Nitrospira and among comammox bacteria in this genus. We designed three novel primer sets that enabled quantification of comammox Nitrospira clades A and B and sNOB with high coverage, specificity, and accuracy in various environments. With the designed primer sets, sNOB and comammox Nitrospira were differentially estimated in drinking water systems, and we found that comammox clade A predominated over sNOB and other ammonia oxidizers in process water samples. Accurate quantification of comammox Nitrospira and sNOB by use of the newly designed primers will provide essential information for evaluating the contribution of Nitrospira to nitrification in various ecosystems.

Archaea have inhabited the earth for a long period of time and are ubiquitously distributed in diverse environments. However, few studies have focused on the interactions of archaea with other organisms, including eukaryotes such as plants, since it is difficult to cultivate sufficient numbers of archaeal cells for analysis. In this study, we investigated the interaction between soil archaea and Arabidopsis thaliana. We demonstrate for the first time that soil archaea promote plant growth and trigger induced systemic resistance (ISR) against the necrotrophic bacterium Pectobacterium carotovorum subsp. carotovorum SCC1 and biotrophic bacterium Pseudomonas syringae pv. tomato DC3000. Ammonia-oxidizing archaeon Nitrosocosmicus oleophilus MY3 cells clearly colonized the root surface of Arabidopsis plants, and increased resistance against both pathogenic species via the salicylic acid-independent signalling pathway. This mechanism of bacterial resistance resembles that underlying soil bacteria- and fungi-mediated ISR signalling. Additionally, volatile emissions from N. oleophilus MY3 were identified as major archaeal determinants that elicit ISR. Our results lay a foundation for archaea-plant interactions as a new field of research.

Salmonella enterica ser. Typhimurium is a foodborne pathogen that causes salmonellosis. Symptoms of salmonellosis include fever, diarrhea, and gastroenteritis. Conventional culture methods for detecting foodborne bacterial pathogens require long incubation time, expensive immunoassay methods, and sample enrichment steps. The objective of this study was to develop an aptamer-based sandwich assay to detect S. enterica ser. Typhimurium. First, S. enterica ser. Typhimurium specific binding aptamers (S11 and S24) were obtained by whole-cell SELEX for high sensitivity and specificity detection using live S. enterica ser. Typhimurium. S11 and S24 aptamers were able to capture S. enterica ser. Typhimurium selectively and distinguish it from other species of Salmonella (S. enterica ser. Typhimurium, S. enterica ser. Choleraesuis, S. enterica ser. Dublin, and S. enterica ser. Enteritidis) and food-borne bacterial pathogens (Escherichia coli K12, Listeria monocytogenes, Shigella sonnei and Staphylococcus aureus) with KD values of 4.41×10-12 M and 3.75×10-11 M, respectively. S. enterica ser. Typhimurium. aptamer-based sandwich assay exhibited a linear response for sensing S. enterica ser. Typhimurium. cells at concentration ranging from 2×101 to 2×105 CFU/mL. This aptamer-based sandwich assay can be used for on-site detection of S. enterica ser. Typhimurium.

A mesophilic, chemolithoautotrophic, neutrophilic and aerobic ammonia-oxidizing archaeon, designated strain MY1T, was isolated from agricultural soil. Microscopic observation revealed short, rod-shaped cells with a diameter of 0.3–0.5 µm and length of 0.6–1.0 µm. The isolate had no flagella and pili, and possessed no genes associated with archaeal flagella synthesis. The major membrane lipids consisted mainly of the glycerol dibiphytanyl glycerol tetraether (GDGT) lipids GDGT-0 to GDGT-4 and crenarchaeol. The major intact polar lipids (IPLs) were determined as hexose plus phosphohexose IPL and dihexose IPL. Strain MY1T obtains energy by aerobically oxidizing ammonia and carbon by fixing CO2. An optimal growth was observed at 25 °C, at pH 7 and with 0.2–0.4 % (w/v) salinity that corresponds with its terrestrial habitat. The addition of α-keto acids was necessary to stimulate growth. The strain tolerated ammonium and nitrite concentrations up to 10 and 5 mM, respectively. The MY1T genome has a DNA G+C content of 32.7 mol%. Phylogenetic analysis based on the 16S rRNA gene showed that strain MY1T belongs to the family Nitrosopumilaceae of the phylum Thaumarchaeota , sharing the highest 16S rRNA gene sequence similarity (96.6–97.1 %) with marine isolates of the genus Nitrosopumilus . The average nucleotide identity was 78 % between strain MY1T and Nitrosopumilus maritimus SCM1T, indicating distant relatedness. Based on the phenotypic, phylogenetic and genomic analyses, it was concluded that strain MY1T belongs to the novel genus Nitrosarchaeum, under which the name Nitrosarchaeum koreense sp. nov. is proposed as the type species. The type strain is MY1T (=JCM 31640T=KCTC 4249T).

Strain GI5T was isolated from a surface seawater sample collected from Garorim Bay (West Sea, Republic of Korea). The isolated strain was aerobic, Gram-stain-negative, rod-shaped, motile by means of a polar flagellum, negative for catalase and weakly positive for oxidase. The optimum growth pH, salinity and temperature were determined to be pH 7.5–8.0, 3 % NaCl (w/v) and 25 °C, respectively; the growth ranges were pH 6.0–9.0, 1–7 % NaCl (w/v) and 18–40 °C. The results of phylogenetic analysis of 16S rRNA gene sequences indicated that GI5T clustered within the family Alcanivoracaceae , and most closely with Alcanivorax dieselolei B-5T and Alcanivorax marinus R8-12T (91.9 % and 91.6 % similarity, respectively). The major cellular fatty acids in GI5T were C18 : 1ω7c/C18 : 1ω6c (44.45 %), C16 : 1ω6c/C16 : 1ω7c (14.17 %) and C16 : 0 (10.19 %); this profile was distinct from those of the closely related species. The major respiratory quinone of GI5T was Q-8. The main polar lipids were phosphatidylethanolamine and phosphatidylglycerol. Two putative alkane hydroxylase (alkB) genes were identified in GI5T. The G+C content of the genomic DNA of GI5T was determined to be 51.2 mol%. On the basis of the results of phenotypic, chemotaxonomic and phylogenetic studies, strain GI5T represents a novel species of a novel genus of the family Alcanivoracaceae, for which we propose the name Ketobacter alkanivorans gen. nov., sp. nov.; the type strain is GI5T (=KCTC 52659T=JCM 31835T).

Members of the family Clostridiaceae within phylum Firmicutes are ubiquitous in various iron-reducing environments. However, genomic data on iron-reducing bacteria of the family Clostridiaceae, particularly regarding their environmental distribution, are limited. Here, we report the analysis and comparison of the genomic properties of Geosporobacter ferrireducens IRF9, a strict anaerobe that ferments sugars and degrades toluene under iron-reducing conditions, with those of the closely related species, Geosporobacter subterraneus DSM 17957. Putative alkyl succinate synthase-encoding genes were observed in the genome of strain IRF9 instead of the typical benzyl succinate synthase-encoding genes. Canonical genes associated with iron reduction were not observed in either genome. The genomes of strains IRF9 and DMS 17957 harbored genes for acetogenesis, that encode two types of Rnf complexes mediating the translocation of H+ and Na+ ions, respectively. Strain IRF9 harbored two different types of ATPases (Na+-dependent F-type ATPase and H+-dependent V-type ATPase), which enable full exploitation of ion gradients. The versatile energy conservation potential of strain IRF9 promotes its survival in various environmental conditions.

Shigella sonnei isolate invasion plasmid antigen protein, IpaH, was successfully expressed in recombinant overexpression bacterial system. The soluble expression IpaH was enhanced with molecular chaperon co-expressed environment. Specific aptamer IpaH17 was isolated through the SELEX process and showed fM binding affinity. IpaH17-SPR biosensor platform was involved to verify the binding sensitivity and specificity. The IpaH concentration dependent IpaH17-SPR sensor response was highly linear with a linear regression constant of 99.4% in the range between 0 and 100 ng/mL. In addition, S. sonnei revealed the specific RU value and detected in a real-time manner within 1 hour. Our study indicated that IpaH17-SPR sensor can allow for rapid, sensitive and specific determination of Shigella sonnei virulent factor.

Aptamer-based paper strip sensor for detecting Vibrio fischeri was developed. Our method was based on the aptamer sandwich assay between whole live cells, V. fischeri and DNA aptamer probes. Following 9 rounds of Cell-SELEX and one of the negative-SELEX, V. fischeri Cell Aptamer (VFCA)-02 and -03 were isolated, with the former showing approximately 10-fold greater avidity (in the subnanomolar range) for the target cells when arrayed on a surface. The colorimetric response of a paper sensor based on VFCA-02 was linear in the range of 4 × 101 to 4 × 105 CFU/mL of target cell by using scanning reader. The linear regression correlation coefficient (R2) was 0.9809. This system shows promise for use in aptamer-conjugated gold nanoparticle probes in paper strip format for in-field detection of marine bioindicating bacteria.

In this paper, whole-bacteria SELEX (WB-SELEX) strategy was adopted to isolate specific aptamers against Vibrio parahaemolyticus. Round selection for V. parahaemolyticus was conducted 11 rounds, including two negative selection rounds. It was determined through real-time PCR amplification and post-SELEX experiment. The selected aptmers had high binding property and specificity to V. parahaemolyticus. Of 28 aptamers tested, VPCA-apta#1 had the highest binding affinity compared to other aptamer candidates obtained. To detect V. parahaemolyticus, aptamer based SPR biosensor platform was constructed and pathogenic bacteria sensing was conducted in two steps. The first step was to construct 5'-biotinylated VPCA-apta#1 binding probe. The second step was to incubate V. parahaemolyticus and test microbes in functionalized SA sensor chip in parallel. Our platform showed significant activity for detecting and discriminating V. parahaemolyticus from other enteric species such as Escherichia coli, Listeria monocytogenes, Sigella sonnei, and Vibrio fischeri. This is the first report on the use of whole-SELEX to isolate DNA aptamers specific for V. parahaemolyticus. We demonstrated the feasibility of using aptamer platform for the detection of V. parahaemolyticus in various food supplies. It might be used in multiple points of care for diagnosing Vibriosis.

Strain Hb3T was isolated from a tidal flat in Jeollabuk-do Gunsan, Republic of Korea. Cells were Gram-stain-negative, oxidase- and catalase-positive, rod-shaped and motile. The strain grew optimally at 25–35 °C, at pH 6.0–6.5 and with 3.0–10.0 % (w/v) NaCl. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain Hb3T belonged to the genus Halomonas . Strain Hb3T was related most closely to Halomonas ventosae Al12T (98.6 % 16S rRNA gene sequence similarity), Halomonas denitrificans M29T (98.6 %) and Halomonas saccharevitans AJ275T (98.4 %). Moreover, multilocus sequence analysis using the gyrB, rpoD and secA genes supported the phylogenetic position of strain Hb3T. The genomic G+C content of strain Hb3T was 67.9 mol%. DNA–DNA hybridization values for strain Hb3T versus H. ventosae Al12T, H. denitrificans M29T and H. saccharevitans AJ275T were 38.0, 54.5 and 47.4 %, respectively. The major quinone was ubiquinone Q-9 and the major fatty acids were C18 : 1ω7c, summed feature 3 (C16 : 1ω6c and/or C16 : 1ω7c), C16 : 0 and C19 : 0 cyclo ω8c. Diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, amino lipid, six unidentified phospholipids and an unidentified lipid comprised the polar lipid profile. On the basis of the data presented in this report, strain Hb3T represents a novel species of the genus Halomonas . The name Halomonas aestuarii sp. nov. is proposed for this novel species. The type strain is Hb3T (=KCTC 52253T=JCM 31415T).

A taxonomic study was conducted on strain soj2014T, which was isolated from the surface water of a polynya in the Antarctic Sea. Comparative 16S rRNA gene sequence analysis showed that strain soj2014T belongs to the family Kiloniellaceae and is closely related to Kiloniella spongiae MEBiC09566T, ‘ Kiloniella litopenaei ’ P1-1T and Kiloniella laminariae LD81T (98.0 %, 97.8 % and 96.2 % 16S rRNA gene sequence similarity, respectively). The DNA–DNA hybridization values between strain soj2014T and closely related strains were below 28.6 %. The G+C content of the genomic DNA of strain soj2014T was 45.5 mol%. The predominant cellular fatty acids were summed feature 8 (composed of C18 : 1ω6c/C18 : 1ω7c, 57.0 %) and summed feature 3 (composed of C16 : 1ω6c/C16 : 1ω7c, 23.5 %). Strain soj2014T was Gram-stain-negative, slightly curved, spiral-shaped, and motile with a single polar flagellum. The strain grew at 0–30 °C (optimum, 25 °C), in 1.5–5.1 % (w/v) NaCl (optimum, 2.1–2.4 %) and at pH 5.5–9.5 (optimum, 7.5–8.0). It also had differential carbohydrate utilization traits and enzyme activities compared with closely related strains. Based on these phylogenetic, phenotypic and chemotaxonomic analyses, strain soj2014T represents a distinct species, separable from the reference strains, and is, therefore, proposed as a novel species, Kiloniella antarctica sp. nov. The type strain is soj2014T (=KCTC 42186T=JCM 30386T).

Consistent with the observation that ammonia-oxidizing bacteria (AOB) outnumber ammonia-oxidizing archaea (AOA) in many eutrophic ecosystems globally, AOB typically dominate activated sludge aeration basins from municipal wastewater treatment plants (WWTPs). In this study, we demonstrate that the growth of AOA strains inoculated into sterile-filtered wastewater was inhibited significantly, in contrast to uninhibited growth of a reference AOB strain. In order to identify possible mechanisms underlying AOA-specific inhibition, we show that complex mixtures of organic compounds, such as yeast extract, were highly inhibitory to all AOA strains but not to the AOB strain. By testing individual organic compounds, we reveal strong inhibitory effects of organic compounds with high metal complexation potentials implying that the inhibitory mechanism for AOA can be explained by the reduced bioavailability of an essential metal. Our results further demonstrate that the inhibitory effect on AOA can be alleviated by copper supplementation, which we observed for pure AOA cultures in a defined medium and for AOA inoculated into nitrifying sludge. Our study offers a novel mechanistic explanation for the relatively low abundance of AOA in most WWTPs and provides a basis for modulating the composition of nitrifying communities in both engineered systems and naturally occurring environments.

A wide diversity of ammonia-oxidizing archaea (AOA) within the phylum Thaumarchaeota exists and plays a key role in the N cycle in a variety of habitats. In this study, we isolated and characterized an ammonia-oxidizing archaeon, strain MY3, from a coal tar-contaminated sediment. Phylogenetically, strain MY3 falls in clade 'Nitrosocosmicus' of the thaumarchaeotal group I.1b. The cells of strain MY3 are large 'walnut-like' cocci, divide by binary fission along a central cingulum, and form aggregates. Strain MY3 is mesophilic and neutrophilic. An assay of 13 C-bicarbonate incorporation into archaeal membrane lipids indicated that strain MY3 is capable of autotrophy. In contrast to some other AOA, TCA cycle intermediates, i.e. pruvate, oxaloacetate and α-ketoglutarate, did not affect the growth rates and yields of strain MY3. The attachment of cells of strain MY3 to XAD-7 hydrophobic beads and to the adsorbent vermiculite demonstrated the potential of strain MY3 to form biofilms. The cell surface was confirmed to be hydrophobic by the extraction of strain MY3 from an aqueous medium with p-xylene. Our finding of a strong potential for surface attachment by strain MY3 may reflect an adaptation to the selective pressures in hydrophobic terrestrial environments.

Figure S1. A) Satellite-based surface chlorophyll-a concentrations (mg/m3) and photosynthetically available radiance (PAR) in the polynya of the Amundsen Sea during the sampling cruises. B) Detailed satellite-based surface chlorophyll-a concentrations (mg/m3) and sampling day of this study. GPS position polynya center station, 73.25–73.75S, 114.25–113.75 W. The data constructed from multi ocean color sensors were obtained from the Globcolour webpage ( http://hermes.acri.fr/ ). Figure S2. Neighbor-joining tree of the most abundant OTUs in a) Polaribacter, b) Oceanospirillaceae, c) SAR92, and d) SAR11 from PK, DC, and SI. The representative sequences of each OTU selected by QIIME are highlighted in bold and coded as follows: OTU N (=OTU number), site (PK, DC, or SI), and percentage of the total read number. Bootstrap values of ≥ 50% are shown. Figure S3. NMDS plot showing relationships between bacterial compositions and environmental variables. Bray–Curtis stress 0.117. *indicates p

Supplementary Results and Methods. (DOCX 939 kb)

Methanotrophs mediate methane oxidation to produce CO2 which is facilitated by methane monooxygenase. Owing to this unique nature, methanotrophs are required in many industrial processes and have environmental applications. For this purpose, diverse aerobic proteobacterial and verrucomicrobial methanotrophs with different traits have been isolated and characterized. Members of the “NC10” phylum of bacteria and Archaea are involved in methane oxidation in anoxic conditions. These methanotrophs have been isolated from various environments including extreme environments, and thus, have diverse physiological and biochemical properties as regards to the required optimum pH, oxygen, temperature, salinity, and electron acceptors. To obtain novel methanotrophs with high potential of application, new cultivation technologies have been developed with the aid of advanced methodologies for monitoring the identity and activity of methanotrophs. In this chapter, we summarize the factors to be considered in the cultivation of methanotrophs and technologies developed for enrichment and isolation of diverse aerobic and anaerobic methanotrophs from various environments.

Table S1. Diversity and abundance of bacterial 16S rRNA gene sequences obtained by the pyrosequencing of PCR amplicons in this study. Taxa with frequencies of

Thaumarchaeota are responsible for a significant fraction of ammonia oxidation in the oceans and in soils that range from alkaline to acidic. However, the adaptive mechanisms underpinning their habitat expansion remain poorly understood. Here we show that expansion into acidic soils and the high pressures of the hadopelagic zone of the oceans is tightly linked to the acquisition of a variant of the energy-yielding ATPases via horizontal transfer. Whereas the ATPase genealogy of neutrophilic Thaumarchaeota is congruent with their organismal genealogy inferred from concatenated conserved proteins, a common clade of V-type ATPases unites phylogenetically distinct clades of acidophilic/acid-tolerant and piezophilic/piezotolerant species. A presumptive function of pumping cytoplasmic protons at low pH is consistent with the experimentally observed increased expression of the V-ATPase in an acid-tolerant thaumarchaeote at low pH. Consistently, heterologous expression of the thaumarchaeotal V-ATPase significantly increased the growth rate of E. coli at low pH. Its adaptive significance to growth in ocean trenches may relate to pressure-related changes in membrane structure in which this complex molecular machine must function. Together, our findings reveal that the habitat expansion of Thaumarchaeota is tightly correlated with extensive horizontal transfer of atp operons.

Here, we report the draft genome sequence of “Candidatus Izimaplasma sp.” strain ZiA1 (1.88 Mb and 29.6% G+C content)., Here, we report the draft genome sequence of “Candidatus Izimaplasma sp.” strain ZiA1 (1.88 Mb and 29.6% G+C content). Strain ZiA1 was cocultured with iron-reducing and toluene-degrading bacteria in an enrichment culture from tidal flat sediment. Like the genomes of other strains of “Ca. Izimaplasma,” the ZiA1 genome contained genes required for anaerobic fermentation.

The discovery of complete ammonia oxidizers (comammox) refutes the century-old paradigm that nitrification requires the activity of two types of microbes. Determining the distribution and abundance of comammox in various environments is important for revealing the ecology of microbial nitrification within the global nitrogen cycle. In this study, the ubiquity and diversity of comammox were analyzed for samples from different types of environments, including soil, sediment, sludge, and water. The results of a two-step PCR using highly degenerate primers (THDP-PCR) and quantitative real-time PCR (qPCR) supported the relatively high abundance of comammox in nearly half of all samples tested, sometimes even outnumbering canonical ammonia-oxidizing bacteria (AOB). In addition, a relatively high proportion of comammox in tap and coastal water samples was confirmed via analysis of metagenomic data sets in public databases. The diversity of comammox was estimated by comammox-specific partial nested PCR amplification of the ammonia monooxygenase subunit A (amoA) gene, and phylogenetic analysis of comammox AmoA clearly showed a split of clade A into clades A.1 and A.2, with the proportions of clades A.1, A.2, and B differing among the various environmental samples. Moreover, compared to the amoA genes of AOB and ammonia-oxidizing archaea (AOA), the comammox amoA gene exhibited higher diversity indices. The ubiquitous distribution and high diversity of comammox indicate that they are likely overlooked contributors to nitrification in various ecosystems. IMPORTANCE The discovery of complete ammonia oxidizers (comammox), which oxidize ammonia to nitrate via nitrite, refutes the century-old paradigm that nitrification requires the activity of two types of microbes and redefines a key process in the biogeochemical nitrogen cycle. Understanding the functional relationships between comammox and other nitrifiers is important for ecological studies on the nitrogen cycle. Therefore, the diversity and contribution of comammox should be considered during ecological analyses of nitrifying microorganisms. In this study, a ubiquitous and highly diverse distribution of comammox was observed in various environmental samples, similar to the distribution of canonical ammonia-oxidizing bacteria. The proportion of comammox was relatively high in coastal water and sediment samples, whereas it was nearly undetectable in open-ocean samples. The ubiquitous distribution and high diversity of comammox indicate that these microorganisms might be important contributors to nitrification.

Ammonia-oxidizing archaea (AOA) from the phylum Thaumarchaeota are ubiquitous in marine ecosystems and play a prominent role in carbon and nitrogen cycling. Previous studies have suggested that, like all microbes, thaumarchaea are infected by viruses and that viral predation has a profound impact on thaumarchaeal functioning and mortality, thereby regulating global biogeochemical cycles. However, not a single virus capable of infecting thaumarchaea has been reported thus far. Here we describe the isolation and characterization of three Nitrosopumilus spindle-shaped viruses (NSVs) that infect AOA and are distinct from other known marine viruses. Although NSVs have a narrow host range, they efficiently infect autochthonous Nitrosopumilus strains and display high rates of adsorption to their host cells. The NSVs have linear double-stranded DNA genomes of ∼28 kb that do not display appreciable sequence similarity to genomes of other known archaeal or bacterial viruses and could be considered as representatives of a new virus family, the “Thaspiviridae.” Upon infection, NSV replication leads to inhibition of AOA growth, accompanied by severe reduction in the rate of ammonia oxidation and nitrite reduction. Nevertheless, unlike in the case of lytic bacteriophages, NSV propagation is not associated with detectable degradation of the host chromosome or a decrease in cell counts. The broad distribution of NSVs in AOA-dominated marine environments suggests that NSV predation might regulate the diversity and dynamics of AOA communities. Collectively, our results shed light on the diversity, evolution, and potential impact of the virosphere associated with ecologically important mesophilic archaea.

Polynyas are a key ecosystem for carbon cycling in the Antarctic Ocean due to the intensive primary production. Most of the knowledge regarding the bacterioplankton community in the Antarctic Ocean that is responsible for re-mineralization of fixed carbon comes from metagenomic analyses. Here, the extinction-dilution method was used to obtain representative heterotrophs from a polynya in the Amundsen Sea, Antarctica, and their biochemical potential for carbon re-mineralization were assessed. All 23 strains have close relatives belonging to type strains within the following genera (number of strains; % 16S rRNA gene sequence similarity): Bizionia (4; >97.8%), Leeuwenhoekiella (1; 96.2%), Pseudoalteromonas (14; >98.5%), Pseudomonas (1; 99.4%) and Sulfitobacter (3; 100%), which were also observed in 454 pyrosequencing-based analysis of 16S rRNA gene sequences of the polynya. Although sequence reads related to Polaribacter were the most common, Polaribacter strains could only be obtained from colonies cultured on agar plates. The strain of Leeuwenhoekiella showed a prominent ability in hydrolyzing diverse esters, amides, and glycosides while the strains of Pseudoalteromonas, Polaribacter, and Bizionia showed extracellular enzyme activities only on a narrow range of amides. The strains of Leeuwenhoekiella, Pseudoalteromonas, and Sulfitobacter utilized various labile carbon sources: carbohydrates, organic acids, amino acids, and peptides. The most frequent isolates, strains of Pseudoaltermonas, showed marked differences in terms of their potential to utilize different types of labile carbon sources, which may reflect high genomic diversity. The strains of Bizionia and Pseudomonas did not utilize carbohydrates. Unique biochemical properties associated with extracellular hydrolase activities and labile carbon utilization were revealed for dominant culturable heterotrophs which gives insights into their roles in active re-mineralization of fixed carbons in polynya.