Sulfur and methane oxidation by a single microorganism.

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 S4I system. Strain HY1 employed the Calvin-Benson-Bassham cycle for CO2 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.

Methylococcus mesophilus sp. nov., the first non-thermotolerant methanotroph of the genus Methylococcus, from a rice field.

Strain 16-5T, a mesophilic methanotroph of the genus Methylococcus, was isolated from rice field soil sampled in Chungcheong Province, Republic of Korea. Strain 16-5T had both particulate and soluble methane monooxygenases and could only grow on methane and methanol as electron donors. Strain 16-5 T cells are Gram-negative, white to light tan in color, non-motile, non-flagellated, diplococcoid to cocci, and have the typical type I intracytoplasmic membrane system. Strain 16-5T grew at 18-38 °C (optimum, 27 °C) and at pH 5.0-8.0 (optimum, pH 6.5-7.0). C16 : 1 ω7c (38.8%), C16 : 1 ω5c (18.8%), C16 : 1 ω6c (16.8%) and C16 : 0 (16.9%) were the major fatty acids, and phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol and an unidentified phospholipid were the major polar lipids. The main respiratory quinone was methylene-ubiquinone-8. Strain 16-5T displayed the highest 16S rRNA gene sequence similarities to other taxonomically recognized members of the genus Methylococcus, i.e. Methylococcus capsulatus TexasT (98.62%) and Methylococcus geothermalis IM1T (98.49 %), which were its closest relatives. It did, however, differ from all other taxonomically described Methylococcus species due to some phenotypic differences, most notably its inability to grow at temperatures above 38 °C, where other Methylococcus species thrive. Its 4.34 Mbp-sized genome has a DNA G+C content of 62.47 mol%, and multiple genome-based properties such as average nucleotide identity and digital DNA-DNA hybridization value distanced it from its closest relatives. Based on the data presented above, this strain represents the first non-thermotolerant species of the genus Methylococcus. The name Methylococcus mesophilus sp. nov. is proposed, and 16-5T (=JCM 35359T=KCTC 82050T) is the type strain.

Methylacidiphilum caldifontis gen. nov., sp. nov., a thermoacidophilic methane-oxidizing bacterium from an acidic geothermal environment, and descriptions of the family Methylacidiphilaceae fam. nov. and order Methylacidiphilales ord. nov.

Strain IT6T, a thermoacidophilic and facultative methane-oxidizing bacterium, was isolated from a mud-water mixture collected from Pisciarelli hot spring in Pozzuoli, Italy. The novel strain is white when grown in liquid or solid media and forms Gram-negative rod-shaped, non-flagellated, non-motile cells. It conserves energy by aerobically oxidizing methane and hydrogen while deriving carbon from carbon dioxide fixation. Strain IT6T had three complete pmoCAB operons encoding particulate methane monooxygenase and genes encoding group 1d and 3b [NiFe] hydrogenases. Simple carbon-carbon substrates such as ethanol, 2-propanol, acetone, acetol and propane-1,2-diol were used as alternative electron donors and carbon sources. Optimal growth occurred at 50-55°C and between pH 2.0-3.0. The major fatty acids were C18 : 0, C15 : 0 anteiso, C14 : 0 iso, C16 : 0 and C14 : 0, and the main polar lipids were phosphatidylethanolamine, aminophospholipid, phosphatidylglycerol, diphosphatidylglycerol, some unidentified phospholipids and glycolipids, and other unknown polar lipids. Strain IT6T has a genome size of 2.19 Mbp and a G+C content of 40.70 mol%. Relative evolutionary divergence using 120 conserved single-copy marker genes (bac120) and phylogenetic analyses based on bac120 and 16S rRNA gene sequences showed that strain IT6T is affiliated with members of the proposed order 'Methylacidiphilales' of the class Verrucomicrobiia in the phylum Verrucomicrobiota. It shared a 16S rRNA gene sequence identity of >96 % with cultivated isolates in the genus 'Methylacidiphilum' of the family 'Methylacidiphilaceae', which are thermoacidophilic methane-oxidizing bacteria. 'Methylacidiphilum sp.' Phi (100 %), 'Methylacidiphilum infernorum' V4 (99.02 %) and 'Methylacidiphilum sp.' RTK17.1 (99.02 %) were its closest relatives. Its physiological and genomic properties were consistent with those of other isolated 'Methylacidiphilum' species. Based on these results, we propose the name Methylacidiphilum caldifontis gen. nov., sp. nov. to accommodate strain IT6T (=KCTC 92103T=JCM 39288T). We also formally propose that the names Methylacidiphilaceae fam. nov. and Methylacidiphilales ord. nov. to accommodate the genus Methylacidiphilum gen. nov.

Proposed minimal standards for description of methanogenic archaea.

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.

Spindle-shaped viruses infect marine ammonia-oxidizing thaumarchaea.

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.

ICTV Virus Taxonomy Profile: Thaspiviridae 2021.

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.

Bacterial Viruses Subcommittee and Archaeal Viruses Subcommittee of the ICTV: update of taxonomy changes in 2021.

In this article, we - the Bacterial Viruses Subcommittee and the Archaeal Viruses Subcommittee of the International Committee on Taxonomy of Viruses (ICTV) - summarise the results of our activities for the period March 2020 - March 2021. We report the division of the former Bacterial and Archaeal Viruses Subcommittee in two separate Subcommittees, welcome new members, a new Subcommittee Chair and Vice Chair, and give an overview of the new taxa that were proposed in 2020, approved by the Executive Committee and ratified by vote in 2021. In particular, a new realm, three orders, 15 families, 31 subfamilies, 734 genera and 1845 species were newly created or redefined (moved/promoted).

Use of Newly Designed Primers for Quantification of Complete Ammonia-Oxidizing (Comammox) Bacterial Clades and Strict Nitrite Oxidizers in the Genus Nitrospira.

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.

Methylococcus geothermalis sp. nov., a methanotroph isolated from a geothermal field in the Republic of Korea.

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.

Plant growth-promoting archaea trigger induced systemic resistance in Arabidopsis thaliana against Pectobacterium carotovorum and Pseudomonas syringae.

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.

Sphingorhabdus pulchriflava sp. nov., isolated from a river.

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).

Hydrogen peroxide detoxification is a key mechanism for growth of ammonia-oxidizing archaea.

Ammonia-oxidizing archaea (AOA), that is, members of the Thaumarchaeota phylum, occur ubiquitously in the environment and are of major significance for global nitrogen cycling. However, controls on cell growth and organic carbon assimilation by AOA are poorly understood. We isolated an ammonia-oxidizing archaeon (designated strain DDS1) from seawater and used this organism to study the physiology of ammonia oxidation. These findings were confirmed using four additional Thaumarchaeota strains from both marine and terrestrial habitats. Ammonia oxidation by strain DDS1 was enhanced in coculture with other bacteria, as well as in artificial seawater media supplemented with α-keto acids (e.g., pyruvate, oxaloacetate). α-Keto acid-enhanced activity of AOA has previously been interpreted as evidence of mixotrophy. However, assays for heterotrophic growth indicated that incorporation of pyruvate into archaeal membrane lipids was negligible. Lipid carbon atoms were, instead, derived from dissolved inorganic carbon, indicating strict autotrophic growth. α-Keto acids spontaneously detoxify H2O2 via a nonenzymatic decarboxylation reaction, suggesting a role of α-keto acids as H2O2 scavengers. Indeed, agents that also scavenge H2O2, such as dimethylthiourea and catalase, replaced the α-keto acid requirement, enhancing growth of strain DDS1. In fact, in the absence of α-keto acids, strain DDS1 and other AOA isolates were shown to endogenously produce H2O2 (up to ∼4.5 µM), which was inhibitory to growth. Genomic analyses indicated catalase genes are largely absent in the AOA. Our results indicate that AOA broadly feature strict autotrophic nutrition and implicate H2O2 as an important factor determining the activity, evolution, and community ecology of AOA ecotypes.

Ubiquity and Diversity of Complete Ammonia Oxidizers (Comammox).

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.

Nitrosarchaeum koreense gen. nov., sp. nov., an aerobic and mesophilic, ammonia-oxidizing archaeon member of the phylum Thaumarchaeota isolated from agricultural soil.

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 Nitrosopumilaceaeof 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).

Ketobacter alkanivorans gen. nov., sp. nov., an n-alkane-degrading bacterium isolated from seawater.

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 dieseloleiB-5T and Alcanivorax marinusR8-12T (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).

Aptamer-Immobilized Surface Plasmon Resonance Biosensor for Rapid and Sensitive Determination of Virulence Determinant.

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.

Surface Plasmon Resonance Aptamer Biosensor for Discriminating Pathogenic Bacteria Vibrio parahaemolyticus.

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.

Ammonia-oxidising archaea living at low pH: Insights from comparative genomics.

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.

Marinobacter salinus sp. nov., a moderately halophilic bacterium isolated from a tidal flat environment.

Two Gram-stain-negative, aerobic, motile, halophilic, rod-shaped bacteria, designated Hb8T and Hb20, were isolated from a tidal flat environment located on the South-West Korean peninsula. The isolates grew at 10-37 °C, at pH 5.0-9.0 and in NaCl concentrations of 0.5-15 % (w/v; optimum, 3.0-6.0 %). Sequence analysis of the 16S rRNA indicated that the isolates belong to the genus Marinobacter and are most closely related to Marinobacter sediminumR65T (98.3 %), followed by Marinobacter lipolyticus SM19T, Marinobacter salsuginis SD-14BT and Marinobacter similis A3d10T. The overall 16S rRNA gene sequence similarity with these species was 97.9 %, but Hb8T and Hb20 showed 100 % sequence similarity with each other. DNA-DNA relatedness values of H8T and Hb20 suggested that these isolates represent a single species, while DNA-DNA relatedness values of the two novel isolates with M. sediminum DSM 27079T and M. similis DSM 15400T were only 21.3 and 22.9 %, respectively. The major fatty acids present in strain Hb8T were identified as C16 : 0, C16 : 1ω9c, C18 : 1ω9c, C18 : 0 3-OH and summed feature 3 (C16 : 1ω6c and/or C16 : 1ω7c). Ubiquinone-9 was the main respiratory quinone in both the novel strains. The polar lipids found to be present included diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, four unidentified phospholipids and five unidentified lipids. The genomic DNA G+C content of Hb8T and Hb20 was 54.5 mol%. Polyphasic analysis indicated that the two isolates are representatives of a novel species of the genus Marinobacte, for which the name Marinobacter salinus sp. nov. is proposed. The type strain is Hb8T (=KCTC 52255T=JCM 31416T).