N. A. Kostrikina

Nitrification expanded: discovery, physiology and genomics of a nitrite-oxidizing bacterium from the phylum Chloroflexi

Nitrite-oxidizing bacteria (NOB) catalyze the second step of nitrification, a major process of the biogeochemical nitrogen cycle, but the recognized diversity of this guild is surprisingly low and only two bacterial phyla contain known NOB. Here, we report on the discovery of a chemolithoautotrophic nitrite oxidizer that belongs to the widespread phylum Chloroflexi not previously known to contain any nitrifying organism. This organism, named Nitrolancetus hollandicus, was isolated from a nitrifying reactor. Its tolerance to a broad temperature range (25–63 °C) and low affinity for nitrite (Ks=1 mM), a complex layered cell envelope that stains Gram positive, and uncommon membrane lipids composed of 1,2-diols distinguish N. hollandicus from all other known nitrite oxidizers. N. hollandicus grows on nitrite and CO2, and is able to use formate as a source of energy and carbon. Genome sequencing and analysis of N. hollandicus revealed the presence of all genes required for CO2 fixation by the Calvin cycle and a nitrite oxidoreductase (NXR) similar to the NXR forms of the proteobacterial nitrite oxidizers, Nitrobacter and Nitrococcus. Comparative genomic analysis of the nxr loci unexpectedly indicated functionally important lateral gene transfer events between Nitrolancetus and other NOB carrying a cytoplasmic NXR, suggesting that horizontal transfer of the NXR module was a major driver for the spread of the capability to gain energy from nitrite oxidation during bacterial evolution. The surprising discovery of N. hollandicus significantly extends the known diversity of nitrifying organisms and likely will have implications for future research on nitrification in natural and engineered ecosystems.

Carboxydothermus hydrogenoformans gen. nov., sp. nov., a CO-utilizing Thermophilic Anaerobic Bacterium from Hydrothermal Environments of Kunashir Island

Summary A new extremely thermophilic, anaerobic, obligately carboxydotrophic, chemolithoautotrophic freshwater bacterium, strain Z-2901, was isolated from a hot swamp of Kunashir Island. This isolate is characterized by a novel type of chemotrophic metabolism. It grows rapidly on CO (100% CO in the gas phase) as sole carbon and energy source with the production of equimolar quantities of H2 according to the equation CO + H2O → H2 + CO2. Other products are not formed and other inorganic and organic substrates are not used. Growth temperatures are between 40 and 78°C with an optimum at 70–72°C. The pH optimum is 6.8–7.0. The generation time under optimal conditions is 120 min. The cells of the new isolate are Gram-positive non-sporeforming short rods with an S-layer. They often occur in pairs, chains and aggregates and are motile by one or two lateral flagella. The GC-content of the DNA is 39.0 mol%. Growth and CO-utilization are inhibited by penicillin, chloramphenicol and streptomycin (100 μg/ml). On the basis of its unique physiological features and thermophilic nature of the new bacterium the isolate is proposed to represent a new genus, Carboxydothermus with the type species C. hydrogenoformans; the type strain is Z-2901 (DSM 6008).

Desulfonatronovibrio hydrogenovorans gen. nov., sp. nov., an alkaliphilic, sulfate-reducing bacterium.

A new alkaliphilic, sulfate-reducing bacterium, strain Z-7935T (T = type strain), was isolated from a soda-depositing lake, Lake Magadi in Kenya. This organism is a motile vibrio which utilizes only hydrogen and formate as electron donors and sulfate, sulfite, and thiosulfate, but not sulfur, as electron acceptors. Thiosulfate is dismutated. Strain Z-7935T is an obligately sodium-dependent alkaliphile which grows in sodium carbonate medium and does not grow at pH 7; the maximum pH for growth is more than pH 10, and the optimum pH is 9.5 to 9.7. The optimum NaCl concentration for growth is 3% (wt/vol). The optimum temperature for growth is 37 degrees C. The G + C content of the DNA is 48.6 mol%. 16S ribosomal DNA sequence analysis revealed that strain Z-7935T represents a new lineage with genus status in the delta subclass of the Proteobacteria. The name Desulfonatronovibrio hydrogenovorans gen. nov., sp. nov. is proposed for this organism; the type strain of D. hydrogenovorans is strain Z-7935 (= DSM 9292).

The first evidence of anaerobic CO oxidation coupled with H2 production by a hyperthermophilic archaeon isolated from a deep-sea hydrothermal vent

From 24 samples of hydrothermal venting structures collected at the East Pacific Rise (13°N), 13 enrichments of coccoid cells were obtained which grew on CO, producing H2 and CO2 at 80°C. A hyperthermophilic archaeon capable of lithotrophic growth on CO coupled with equimolar production of H2 was isolated. Based on its 16S rRNA sequence analysis, this organism was affiliated with the genus Thermococcus. Other strains of Thermococcales species (Pyrococcus furiosus, Thermococcus peptonophilus, T. profundus, T. chitonophagus, T. stetteri, T. gorgonarius, T. litoralis, and T. pacificus) were shown to be unable to grow on CO. Searches in sequence databases failed to reveal deposited sequences of genes related to CO metabolism in Thermococcales. Our work provides the first evidence of anaerobic CO oxidation coupled with H2 production performed by an archaeon as well as the first documented case of lithotrophic growth of a Thermococcales representative.

Thermoanaerobacter siderophilus sp. nov., a novel dissimilatory Fe(III)-reducing, anaerobic, thermophilic bacterium

A thermophilic, anaerobic, spore-forming, dissimilatory Fe(III)-reducing bacterium, designated strain SR4T, was isolated from sediment of newly formed hydrothermal vents in the area of the eruption of Karymsky volcano on the Kamchatka peninsula. Cells of strain SR4T were straight-to-curved, peritrichous rods, 0.4-0.6 micron in diameter and 3.5-9.0 microns in length, and exhibited a slight tumbling motility. Strain SR4T formed round, refractile, heat-resistant endospores in terminally swollen sporangia. The temperature range for growth was 39-78 degrees C, with an optimum at 69-71 degrees C. The pH range for growth was 4.8-8.2, with an optimum at 6.3-6.5. Strain SR4T grew anaerobically with peptone as carbon source. Amorphous iron(III) oxide present in the medium stimulated the growth of strain SR4T; cell numbers increased with the concomitant accumulation of Fe(II). In the presence of Fe(III), strain SR4T grew on H2/CO2 and utilized molecular hydrogen. Strain SR4T reduced 9,10-anthraquinone-2,6-disulfonic acid, sulfite, thiosulfate, elemental sulfur and MnO2. Strain SR4T did not reduce nitrate or sulfate and was not capable of growth with O2. The fermentation products from glucose were ethanol, lactate, H2 and CO2. The G + C content of DNA was 32 mol%. 16S rDNA sequence analysis placed the organism in the genus Thermoanaerobacter. On the basis of physiological properties and phylogenetic analysis, it is proposed that strain SR4T (= DSM 12299T) should be assigned to a new species, Thermoanaerobacter siderophilus sp. nov.

Desulfurella acetivorans gen. nov. and sp. nov. —a new thermophilic sulfur-reducing eubacterium

A new type of thermophilic cyanobacterial mat, rich in elemental sulfur and containing large numbers of sulfur-reducing bacteria able to utilize different growth substrates at 55° C, was found in the Uzon caldere (Kamchatka). One of the largest groups among these organisms were acetate-oxidizing sulfur-reducing bacteria, numbering 106 cells · cm−3 of mat. The pure culture of a sulfur-reducing eubacterium growing on acetate was isolated. Cells of the new isolate are Gram-negative short rods, often in pairs, motile, with a single polar flagellum. The optimal temperature for growth is 52 to 57° C, with no growth observed at 42 or 73° C. The pH optimum is 6.8 to 7.0. The new isolate is demonstrated to be a true dissimilatory sulfur reducer: it is an obligate anaerobe, it is unable to ferment organic substrates and it can use no electron acceptors other than elemental sulfur. Acetate is the only energy and carbon source, and H2S and CO2 are growth products. No cytochromes were detected. The G+C content of DNA is rather low, only 31.4 mol%. Thus, morphological and physiological features of the new isolate are quite close to those of Desulfuromonas. But on the grounds of a significant difference in the G+C content of DNA, the absence of cytochromes and because of its thermophilic nature, a new genus Desulfurella is proposed with the type species Desulfurella acetivorans.

Nautilia lithotrophica gen. nov., sp. nov., a thermophilic sulfur-reducing epsilon-proteobacterium isolated from a deep-sea hydrothermal vent.

A novel, strictly anaerobic, thermophilic sulfur-reducing bacterium, strain 525T, was isolated from tubes of the deep-sea hydrothermal vent polychaete Alvinella pompejana, collected on the East Pacific Rise (13 degrees N). This organism grew in the temperature range 37-68 degrees C, the optimum being 53 degrees C, and in the pH range 6.4-7.4, the optimum being 6.8-7.0. The NaCl range for growth was 0.8-5.0%, the optimum being 3.0%. Strain 525T grew lithoautotrophically with H2 as energy source, S0 as electron acceptor and CO2 as carbon source. Alternatively, strain 525T was able to use formate as an energy source. The G+C content of the genomic DNA was 34.7 mol%. Phylogenetic analysis of the 16S rDNA gene sequence placed strain 525T in the epsilon-subclass of the Proteobacteria, where it forms a deep cluster with recently isolated relatives. On the basis of phenotypic and phylogenetic differences between strain 525T and its closest phylogenetic relatives, it is proposed that the new isolate should be described as a member of a new genus, Nautilia, for which the name Nautilia lithotrophica gen. nov., sp. nov. is proposed. The type strain is strain 525T (= DSM 13520T).

Carboxydocella thermautotrophica gen. nov., sp. nov., a novel anaerobic, CO-utilizing thermophile from a Kamchatkan hot spring.

A novel anaerobic, thermophilic, CO-utilizing bacterium, strain 41(T), was isolated from a terrestrial hot vent on the Kamchatka Peninsula. Strain 41(T) was found to be a Gram-positive bacterium, its cells being short, straight, motile rods. Chains of three to five cells were often observed. The isolate grew only chemolithoautotrophically on CO, producing equimolar quantities of H2 and CO2 (according to the equation CO+H2O --> CO2+H2). Growth was observed in the temperature range 40-68 degrees C, with an optimum at 58 degrees C, and in the pH range 6.5-7.6, with an optimum at pH 7.0. The generation time under optimal conditions for chemolithotrophic growth was 1.1 h. The DNA G+C content was 46 +/- 1 mol%. Growth was completely inhibited by penicillin, ampicillin, streptomycin, kanamycin and neomycin. On the basis of the phenotypic and phylogenetic features, it is proposed that this isolate represents a new genus and species, Carboxydocella thermautotrophica gen. nov., sp. nov. (type strain 41(T) = DSM 12356(T) = VKM B-2282(T)).

Thermococcus stetteri sp. nov., a New Extremely Thermophilic Marine Sulfur-Metabolizing Archaebacterium

Summary Four strains of new extremely thermophilic anaerobic archaebacteria were isolated from marine solfataric fields of Kraternaya cove (Ushishir archipelago, Northern Kurils). The cells are irregular cocci 1 to 2 μm in diameter. Two strains are motile due to a tuft of flagella. Two strains are non-motile. The cell envelope consists of two layers of subunits. Two strains (non-motile) grow at temperatures from 55 to 94°C (opt. 75 °C) and two (motile) from 75 to 98 °C. The pH range for growth of all strains is 5.7 to 7.2 (opt. around 6.5). Salt (opt. 2.5% NaCl) and elemental sulfur are obligately required for growth. Peptides and polysaccharides are utilized. During growth more than 20 μmol/ml H 2 S are formed and CO 2 , acetate, isobutyrate and isovalerate are produced. The G+C content of DNA of isolate K-3 is 50.2 mol.%. The four isolates exhibited high DNA homology among each other, indicating that they belong to the same species. Partial 16S rRNA sequencing of isolate K-3 indicated that it belongs to the genus Thermococcus . It shows no significant DNA homology with Thermococcus celer . In view of all differences between the isolates and Thermococcus celer , they are considered as representatives of a new species described as Thermococcus stetteri . Type strain is isolate K-3, DSM 5262.

Geoalkalibacter ferrihydriticus gen. nov. sp. nov., the first alkaliphilic representative of the family Geobacteraceae, isolated from a soda lake

Investigation of iron reduction in bottom sediments of alkaline soda lakes resulted in the isolation of a new obligately anaerobic iron-reducing bacterium, strain Z-0531, from Lake Khadyn (Tuva, Russia) sediment samples. The cells of strain Z-0531 are short (1.0–1.5 by 0.3–0.5 µm), motile, non-spore-forming, gram-negative rods. The isolate is an obligate alkaliphile, developing in the pH range of 7.8–10.0, with an optimum at pH 8.6. It does not require NaCl but grows at NaCl concentrations of 0–50 g/l. It can oxidize acetate with such electron acceptors as amorphous Fe(III) hydroxide (AFH), EDTA-Fe(III), anthraquinone-2,6-disulfonate (quinone), Mn(IV), and S0. On medium with EDTA-Fe(III), the isolate can oxidize, apart from acetate, ethanol, pyruvate, oxalate, arginine, tartrate, lactate, propionate, and serine. H2 is not utilized. The reduced products formed during growth with AFH are siderite or magnetite, depending on the growth conditions. The isolate is incapable of fermenting sugars, peptides, and amino acids. Yeast extract or vitamins are required as growth factors. The organism is capable of dinitrogen fixation and harbors the nifH gene. The DNA G+C content is 55.3 mol %. 16S rRNA analysis places strain Z-0531 into the family Geobacteraceae. Its closest relative (93% similarity) is Desulfuromonas palmitatis. Based on phenotypic distinctions and phylogenetic position, it is proposed that this strain be assigned to the new genus and species Geoalkalibacter ferrihydriticus gen. nov., sp. nov. (Z-0531T-DSMZ-17813-VKMB-2401).