So-Jeong Kim

Isotopic signatures of N2O produced by ammonia-oxidizing archaea from soils

N2O gas is involved in global warming and ozone depletion. The major sources of N2O are soil microbial processes. Anthropogenic inputs into the nitrogen cycle have exacerbated these microbial processes, including nitrification. Ammonia-oxidizing archaea (AOA) are major members of the pool of soil ammonia-oxidizing microorganisms. This study investigated the isotopic signatures of N2O produced by soil AOA and associated N2O production processes. All five AOA strains (I.1a, I.1a-associated and I.1b clades of Thaumarchaeota) from soil produced N2O and their yields were comparable to those of ammonia-oxidizing bacteria (AOB). The levels of site preference (SP), δ15Nbulk and δ18O -N2O of soil AOA strains were 13–30%, −13 to −35% and 22–36%, respectively, and strains MY1–3 and other soil AOA strains had distinct isotopic signatures. A 15N-NH4+-labeling experiment indicated that N2O originated from two different production pathways (that is, ammonia oxidation and nitrifier denitrification), which suggests that the isotopic signatures of N2O from AOA may be attributable to the relative contributions of these two processes. The highest N2O production yield and lowest site preference of acidophilic strain CS may be related to enhanced nitrifier denitrification for detoxifying nitrite. Previously, it was not possible to detect N2O from soil AOA because of similarities between its isotopic signatures and those from AOB. Given the predominance of AOA over AOB in most soils, a significant proportion of the total N2O emissions from soil nitrification may be attributable to AOA.

Metabolic versatility of toluene-degrading, iron-reducing bacteria in tidal flat sediment, characterized by stable isotope probing-based metagenomic analysis

DNA stable isotope probing and metagenomic sequencing were used to assess the metabolic potential of iron-reducing bacteria involved in anaerobic aromatic hydrocarbon degradation in oil spill-affected tidal flats. In a microcosm experiment, (13) C-toluene was degraded with the simultaneous reduction of Fe(III)-NTA, which was also verified by quasi-stoichiometric (13) C-CO2 release. The metabolic potential of the dominant member affiliated with the genus Desulfuromonas in the heavy DNA fraction was inferred using assembled scaffolds (designated TF genome, 4.40 Mbp with 58.8 GC mol%), which were obtained by Illumina sequencing. The gene clusters with peripheral pathways for toluene and benzoate conversion possessed the features of strict and facultative anaerobes. In addition to the class II-type benzoyl-CoA reductase (Bam) of strict anaerobes, the class I-type (Bcr) of facultative anaerobes was encoded. Genes related to the utilization of various anaerobic electron acceptors, including iron, nitrate (to ammonia), sulfur and fumarate, were identified. Furthermore, genes encoding terminal oxidases (caa3 , cbb3 and bd) and a diverse array of genes for oxidative stress responses were detected in the TF genome. This metabolic versatility may be an adaptation to the fluctuating availability of electron acceptors and donors in tidal flats.

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

Significance Ammonia-oxidizing archaea (AOA) are major players in global nitrogen cycling, but the AOA carbon-nutrition paradigm is poorly understood. Once considered strict autotrophs, AOA also have been reported to assimilate organic carbon. We used a marine AOA isolate to test hypotheses about the role of fixed carbon in AOA nutrition. Results were confirmed with tests with four additional marine and terrestrial AOA. We discovered that α-keto acids (pyruvate, oxaloacetate) were not directly incorporated into AOA cells. Instead, the α-keto acids functioned as chemical scavengers that detoxified intracellularly produced H2O2 during ammonia oxidation. H2O2 toxicity was also counteracted by co-inoculating the AOA with bacteria harboring catalases. Thus, H2O2 toxicity in AOA may be an evolutionary force controlling AOA communities and global ammonia cycling. 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.

Isolation, characterization, and abundance of filamentous members of Caldilineae in activated sludge

Chloroflexi are currently believed to serve as backbone forming agents in the activated sludge of wastewater treatment plants (WWTPs). In this study, we isolated and characterized filamentous bacteria in the class Caldilineae of the phylum Chloroflexi in municipal WWTPs. Diversity analysis using Chloroflexi-specific 16S rRNA gene clone libraries showed that 97% of the clones belonged to the subdivision Anaerolineae comprising the two classes Anaerolineae (95%) and Caldilineae (2%). Clones of Caldilineae were related to a thermophilic filament Caldilinea aerophila with 93% 16S rRNA gene sequence similarity. We obtained filamentous isolates classified into the class Caldilineae showing the best match to C. aerophila with 89% 16S rRNA gene sequence similarity. Isolates showed no ability to assimilate glucose or N-acetylglucosamine or to degrade biopolymers which were observed in filamentous Chloroflexi of WWTPs. The assessment of relative abundance based on quantitative PCR of the 16S rRNA gene indicated that members of the class Caldilineae comprised 12–19% of the Chloroflexi in the activated sludge. Additionally, fluorescence in situ hybridization experiments showed that diverse filamentous Caldilineae inhabit the activated sludge of municipal WWTPs. These findings yield insight into the role of filamentous mesophilic Caldilinea in stabilizing flocs of activated sludge in a wide range of WWTPs.

A Mesophilic, Autotrophic, Ammonia-Oxidizing Archaeon of Thaumarchaeal Group I.1a Cultivated from a Deep Oligotrophic Soil Horizon

ABSTRACT Soil nitrification plays an important role in the reduction of soil fertility and in nitrate enrichment of groundwater. Various ammonia-oxidizing archaea (AOA) are considered to be members of the pool of ammonia-oxidizing microorganisms in soil. This study reports the discovery of a chemolithoautotrophic ammonia oxidizer that belongs to a distinct clade of nonmarine thaumarchaeal group I.1a, which is widespread in terrestrial environments. The archaeal strain MY2 was cultivated from a deep oligotrophic soil horizon. The similarity of the 16S rRNA gene sequence of strain MY2 to those of other cultivated group I.1a thaumarchaeota members, i.e., Nitrosopumilus maritimus and “Candidatus Nitrosoarchaeum koreensis,” is 92.9% for both species. Extensive growth assays showed that strain MY2 is chemolithoautotrophic, mesophilic (optimum temperature, 30°C), and neutrophilic (optimum pH, 7 to 7.5). The accumulation of nitrite above 1 mM inhibited ammonia oxidation, while ammonia oxidation itself was not inhibited in the presence of up to 5 mM ammonia. The genome size of strain MY2 was 1.76 Mb, similar to those of N. maritimus and “Ca. Nitrosoarchaeum koreensis,” and the repertoire of genes required for ammonia oxidation and carbon fixation in thaumarchaeal group I.1a was conserved. A high level of representation of conserved orthologous genes for signal transduction and motility in the noncore genome might be implicated in niche adaptation by strain MY2. On the basis of phenotypic, phylogenetic, and genomic characteristics, we propose the name “Candidatus Nitrosotenuis chungbukensis” for the ammonia-oxidizing archaeal strain MY2.

Unveiling abundance and distribution of planktonic Bacteria and Archaea in a polynya in Amundsen Sea, Antarctica.

Polynyas, areas of open water surrounded by sea ice, are sites of intense primary production and ecological hotspots in the Antarctic Ocean. This study determined the spatial variation in communities of prokaryotes in a polynya in the Amundsen Sea using 454 pyrosequencing technology, and the results were compared with biotic and abiotic environmental factors. The bacterial abundance was correlated with that of phytoplankton, Phaeocystis spp. and diatoms. A cluster analysis indicated that the bacterial communities in the surface waters of the polynya were distinct from those under the sea ice. Overall, two bacterial clades, Polaribacter (20-64%) and uncultivated Oceanospirillaceae (7-34%), dominated the surface water in the polynya while the Pelagibacter clade was abundant at all depths (7-42%). The archaeal communities were not as diverse as the bacterial communities in the polynya, and marine group I was dominant (> 80%). Canonical correspondence analysis indicated that the oceanographic properties facilitated the development of distinct prokaryotic assemblages in the polynya. This analysis of the diversity and composition of the psychrophilic prokaryotes associated with high phytoplankton production provides new insights into the roles of prokaryotes in biogeochemical cycles in high-latitude polynyas.

Thioalbus denitrificans gen. nov., sp. nov., a chemolithoautotrophic sulfur-oxidizing gammaproteobacterium, isolated from marine sediment

A mesophilic, facultatively anaerobic, autotrophic bacterium, designated strain Su4(T), was isolated from marine sediment. The isolate was able to utilize reduced sulfur compounds including thiosulfate, tetrathionate, sulfur and sulfide but not sulfite as the energy source. Growth occurred under aerobic and denitrifying chemolithoautotrophic conditions in the presence of thiosulfate as an electron donor and bicarbonate as a carbon source. The G+C content of the genomic DNA was 64.5 mol%. Comparative 16S rRNA gene sequence studies showed that strain Su4(T) was clearly affiliated with the class Gammaproteobacteria. The isolate was Gram-negative-staining and rod-shaped, lacked flagella and grew in artificial seawater medium at 10-40 °C (optimum 28-32 °C) and in 1-5 % (w/v) NaCl (optimum 3 % NaCl). Strain Su4(T) possessed C₁₆:₀, C₁₆:₁ω7c/iso-C₁₅:₀ 2-OH and C₁₈:₁ω7c/ω9t/ω12t as the major fatty acids. On the basis of phenotypic and phylogenetic analysis, the isolate represents a novel species of a novel genus, for which the name Thioalbus denitrificans is proposed. The type strain is Su4(T) ( = KCTC 5699(T)  = JCM 15568(T)).

Marinobacterium maritimum sp. nov., a marine bacterium isolated from Arctic sediment.

A Gram-negative, aerobic, rod-shaped, motile, marine bacterium, strain AR11(T), was isolated from Arctic marine sediment. Strain AR11(T) grew with 0.5-7 % NaCl and at 7-37 degrees C and pH 5.5-9.0. It utilized propionate, 3-hydroxybenzoate, l-proline, acetate, d- and l-lactate, l-alanine, malate and phenylacetic acid. Alkaline phosphatase, esterase lipase (C8), leucine arylamidase and acid phosphatase activity tests were positive. Acid was produced from 5-ketogluconate and aesculin. Strain AR11(T) possessed C(16 : 0) (22.0 %), summed feature 4 (C(16 : 1)omega7c and/or iso-C(15 : 0) 2-OH; 28.1 %) and summed feature 7 (one or more of C(18 : 1)omega7c, omega9t and omega12t; 34.0 %) as the major cellular fatty acids. The major ubiquinone was Q-8. Comparative 16S rRNA gene sequence studies showed that strain AR11(T) belonged to the Gammaproteobacteria and was most closely related to Marinobacterium stanieri DSM 7027(T), Marinobacterium halophilum mano11(T) and Marinobacterium georgiense KW-40(T) (97.8, 97.0 and 96.7 % similarity, respectively). The G+C content of the genomic DNA of strain AR11(T) was 57.9 mol%. DNA-DNA relatedness data indicated that strain AR11(T) represented a distinct species that was separated from M. stanieri DSM 7027(T), M. halophilum KCTC 12240(T) and M. georgiense JCM 21667(T). On the basis of evidence from this polyphasic study, it is proposed that strain AR11(T) (=KCTC 22254(T)=JCM 15134(T)) represents the type strain of a novel species, Marinobacterium maritimum sp. nov.

Molecular analysis of spatial variation of iron-reducing bacteria in riverine alluvial aquifers of the Mankyeong River.

Alluvial aquifers are one of the mainwater resources in many countries. Iron reduction in alluvial aquifers is often a major anaerobic process involved in bioremediation or causing problems, including the release of As trapped in Fe(III) oxide. We investigated the distribution of potential iron-reducing bacteria (IRB) in riverine alluvial aquifers (B1, B3, and B6 sites) at the Mankyeong River, Republic of Korea. Inactive iron reduction zones, the diversity and abundance of IRB can be examined using a clone library and quantitative PCR analysis of 16S rRNA genes. Geobacter spp. are potential IRB in the iron-reducing zone at the B6 (9 m) site, where high Fe(II) and arsenic (As) concentrations were observed. At the B3 (16 m) site, where low iron reduction activity was predicted, a dominant clone (10.6%) was 99% identical in 16S rRNA gene sequence with Rhodoferax ferrireducens. Although a major clone belonging to Clostridium spp. was found, possible IRB candidates could not be unambiguously determined at the B1 (18 m) site. Acanonical correspondence analysis demonstrated that, among potential IRB, only the Geobacteraceae were well correlated with Fe(II) and As concentrations. Our results indicate high environmental heterogeneity, and thus high spatial variability, in thedistribution of potential IRB in the riverine alluvial aquifersnear the Mankyeong River.

An uncultivated nitrate-reducing member of the genus Herminiimonas degrades toluene

ABSTRACT Stable isotope probing (SIP) is a cultivation-free methodology that provides information about the identity of microorganisms participating in assimilatory processes in complex communities. In this study, a Herminiimonas-related bacterium was identified as the dominant member of a denitrifying microcosm fed [13C]toluene. The genome of the uncultivated toluene-degrading bacterium was obtained by applying pyrosequencing to the heavy DNA fraction. The draft genome comprised ∼3.8 Mb, in 131 assembled contigs. Metabolic reconstruction of aromatic hydrocarbon (toluene, benzoate, p-cresol, 4-hydroxybenzoate, phenylacetate, and cyclohexane carboxylate) degradation indicated that the bacterium might specialize in anaerobic hydrocarbon degradation. This characteristic is novel for the order Burkholderiales within the class Betaproteobacteria. Under aerobic conditions, the benzoate oxidation gene cluster (BOX) system is likely involved in the degradation of benzoate via benzoyl coenzyme A. Many putative genes for aromatic hydrocarbon degradation were closely related to those in the Rhodocyclaceae (particularly Aromatoleum aromaticum EbN1) with respect to organization and sequence similarity. Putative mobile genetic elements associated with these catabolic genes were highly abundant, suggesting gene acquisition by Herminiimonas via horizontal gene transfer.