Joo-Han Gwak

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.

Calculibacillus koreensis gen. nov., sp. nov., an anaerobic Fe(III)-reducing bacterium isolated from sediment of mine tailings

A strictly anaerobic bacterium, strain B5T, was isolated from sediment of an abandoned coal mine in Taebaek, Republic of Korea. Cells of strain B5T were non-spore-forming, straight, Gram-positive rods. The optimum pH and temperature for growth were pH 7.0 and 30°C, respectively, while the strain was able to grow within pH and temperature ranges of 5.5–7.5 and 25–45°C, respectively. Growth of strain B5T was observed at NaCl concentrations of 0 to 6.0% (w/v) with an optimum at 3.0–4.0% (w/v). The polar lipids consisted of phosphatidylethanolamine, phosphatidylglycerol, an unknown phospholipid and three unknown polar lipids. Strain B5T grew anaerobically by reducing nitrate, nitrite, ferric-citrate, ferric-nitrilotriacetate, elemental sulfur, thiosulfate, and anthraquinone-2-sulfonate in the presence of proteinaceous compounds, organic acids, and carbohydrates as electron donors. The isolate was not able to grow by fermentation. Strain B5T did not grow under aerobic or microaerobic conditions. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain B5T is most closely related to the genus Tepidibacillus (T. fermentans STGHT; 96.3%) and Vulcanibacillus (V. modesticaldus BRT; 94.6%). The genomic DNA G+C content (36.9 mol%) of strain B5T was higher than those of T. fermentans STGHT (34.8 mol%) and V. modesticaldus BRT (34.5 mol%). Based on its phenotypic, chemotaxonomic, and phylogenetic properties, we describe a new species of a novel genus Calculibacillus, represented by strain B5T (=KCTC 15397T =JCM 19989T), for which we propose the name Calculibacillus koreensis gen. nov., sp. nov.

Comparative genomic analysis of Geosporobacter ferrireducens and its versatility of anaerobic energy metabolism

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.

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).

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).

Genomic Insights Into the Acid Adaptation of Novel Methanotrophs Enriched From Acidic Forest Soils

Soil acidification is accelerated by anthropogenic and agricultural activities, which could significantly affect global methane cycles. However, detailed knowledge of the genomic properties of methanotrophs adapted to acidic soils remains scarce. Using metagenomic approaches, we analyzed methane-utilizing communities enriched from acidic forest soils with pH 3 and 4, and recovered near-complete genomes of proteobacterial methanotrophs. Novel methanotroph genomes designated KS32 and KS41, belonging to two representative clades of methanotrophs (Methylocystis of Alphaproteobacteria and Methylobacter of Gammaproteobacteria), were dominant. Comparative genomic analysis revealed diverse systems of membrane transporters for ensuring pH homeostasis and defense against toxic chemicals. Various potassium transporter systems, sodium/proton antiporters, and two copies of proton-translocating F1F0-type ATP synthase genes were identified, which might participate in the key pH homeostasis mechanisms in KS32. In addition, the V-type ATP synthase and urea assimilation genes might be used for pH homeostasis in KS41. Genes involved in the modification of membranes by incorporation of cyclopropane fatty acids and hopanoid lipids might be used for reducing proton influx into cells. The two methanotroph genomes possess genes for elaborate heavy metal efflux pumping systems, possibly owing to increased heavy metal toxicity in acidic conditions. Phylogenies of key genes involved in acid adaptation, methane oxidation, and antiviral defense in KS41 were incongruent with that of 16S rRNA. Thus, the detailed analysis of the genome sequences provides new insights into the ecology of methanotrophs responding to soil acidification.