Azospira inquinata sp. nov., a nitrate-reducing bacterium of the family Rhodocyclaceae isolated from contaminated groundwater.

Two novel Gram-stain-negative bacterial strains, Azo-3T and Azo-2, were isolated from a toluene-producing enrichment culture that originated from contaminated groundwater at a site in southeast Louisiana (USA). Cells are non-spore forming straight to curved rods with single polar flagella. Strains Azo-3T and Azo-2 are oxidase-positive, catalase-negative, use nitrate and nitrite as electron acceptors, and are able to fix nitrogen. Poly-ß-hydroxybutyrate storage granules are produced. Dominant fatty acids when grown in R2A medium at 37 °C are C16:0, summed feature 3 (C16:1 ω7c and/or C15:0 iso 2OH), C17:0 cyclo and C18:1 ω7c. 16S rRNA gene sequence based phylogenetic analysis indicated that the strains cluster within the family Rhodocyclaceae, class Betaproteobacteria, most closely related to but distinct from type strains of the species Azospira oryzae (96.94% similarity) and Azospira restricta (95.10% similarity). Complete genome sequences determined for strains Azo-3T and Azo-2 revealed DNA G+C content of 62.70 mol%. Genome-wide comparisons based on average nucleotide identity by orthology and estimated DNA-DNA hybridization values combined with phenotypic and chemotaxonomic traits and phylogenetic analysis indicate that strains Azo-3T and Azo-2 represent a novel species within the genus Azospira for which the name Azospira inquinata sp. nov. is proposed. The type strain of Azospira inquinata is Azo-3T (=NRRL B-65590T=DSM 112046T).

Niveibacterium microcysteis sp. nov., isolated from a cyanobacterial bloom sample.

A novel bacterial strain, named HC41T, was isolated from a cyanobacterial bloom sample and was characterized as Gram-stain-negative, rod-shaped and non-motile. According to 16S rRNA phylogenetic analyses, this strain HC41T belongs to the family Rhodocyclaceae and is most closely related to Niveibacterium umoris KACC 17062T (=MIC 2059T; 98.63 %) and Uliginosibacterium gangwonense 5YN10-9 T (=KACC 11603T; 93.64 %). The genome size and DNA G+C content of strain HC41T were 4.8 Mbp and 64.17 mol%, respectively. Moreover, the average nucleotide identity, digital DNA-DNA hybridization and amino acid identity values between strain HC41T and N. umoris KACC 17062T were 81.8, 43.1 and 90.89 %, respectively. Additionally, strain HC41T exhibited weak catalase and oxidase activities and had no motility (swimming and swarming motilities). The cells grew at 11-40 °C (optimum, 30 °C), pH 5.5-8.0 (optimum, pH 7) and with 0-1.0 % (w/v) NaCl (optimum, 0 % NaCl) in Reasoner's 2A medium. Its major respiratory quinone was ubiquinone-8 and its major polar lipids were diphosphatidylglycerol, phosphatidylglycerol and phosphatidylethanolamine. Furthermore, C16 : 0 and summed feature 3 (C16 : 1 ω7c and/or C16 : 1 ω6c; C16 : 1 ω6c and/or C16 : 1 ω7c) were the predominant cellular fatty acids in strain HC41T according to fatty acid methyl ester analysis. Based on its genotypic and phenotypic characteristics, strain HC41T was identified as representing a novel Niveibacterium species, for which the name Niveibacterium microcysteis sp. nov. is proposed (=KACC 22091T=DSM 111425T).

Comparative Genomics Provides Insights into the Taxonomy of Azoarcus and Reveals Separate Origins of Nif Genes in the Proposed Azoarcus and Aromatoleum Genera.

Among other attributes, the Betaproteobacterial genus Azoarcus has biotechnological importance for plant growth-promotion and remediation of petroleum waste-polluted water and soils. It comprises at least two phylogenetically distinct groups. The "plant-associated" group includes strains that are isolated from the rhizosphere or root interior of the C4 plant Kallar Grass, but also strains from soil and/or water; all are considered to be obligate aerobes and all are diazotrophic. The other group (now partly incorporated into the new genus Aromatoleum) comprises a diverse range of species and strains that live in water or soil that is contaminated with petroleum and/or aromatic compounds; all are facultative or obligate anaerobes. Some are diazotrophs. A comparative genome analysis of 32 genomes from 30 Azoarcus-Aromatoleum strains was performed in order to delineate generic boundaries more precisely than the single gene, 16S rRNA, that has been commonly used in bacterial taxonomy. The origin of diazotrophy in Azoarcus-Aromatoleum was also investigated by comparing full-length sequences of nif genes, and by physiological measurements of nitrogenase activity using the acetylene reduction assay. Based on average nucleotide identity (ANI) and whole genome analyses, three major groups could be discerned: (i) Azoarcus comprising Az. communis, Az. indigens and Az. olearius, and two unnamed species complexes, (ii) Aromatoleum Group 1 comprising Ar. anaerobium, Ar. aromaticum, Ar. bremense, and Ar. buckelii, and (iii) Aromatoleum Group 2 comprising Ar. diolicum, Ar. evansii, Ar. petrolei, Ar. toluclasticum, Ar. tolulyticum, Ar. toluolicum, and Ar. toluvorans. Single strain lineages such as Azoarcus sp. KH32C, Az. pumilus, and Az. taiwanensis were also revealed. Full length sequences of nif-cluster genes revealed two groups of diazotrophs in Azoarcus-Aromatoleum with nif being derived from Dechloromonas in Azoarcus sensu stricto (and two Thauera strains) and from Azospira in Aromatoleum Group 2. Diazotrophy was confirmed in several strains, and for the first time in Az. communis LMG5514, Azoarcus sp. TTM-91 and Ar. toluolicum TT. In terms of ecology, with the exception of a few plant-associated strains in Azoarcus (s.s.), across the group, most strains/species are found in soil and water (often contaminated with petroleum or related aromatic compounds), sewage sludge, and seawater. The possession of nar, nap, nir, nor, and nos genes by most Azoarcus-Aromatoleum strains suggests that they have the potential to derive energy through anaerobic nitrate respiration, so this ability cannot be usefully used as a phenotypic marker to distinguish genera. However, the possession of bzd genes indicating the ability to degrade benzoate anaerobically plus the type of diazotrophy (aerobic vs. anaerobic) could, after confirmation of their functionality, be considered as distinguishing phenotypes in any new generic delineations. The taxonomy of the Azoarcus-Aromatoleum group should be revisited; retaining the generic name Azoarcus for its entirety, or creating additional genera are both possible outcomes.

Complete Genomes of the Anaerobic Degradation Specialists Aromatoleum petrolei ToN1T and Aromatoleum bremense PbN1T.

The betaproteobacterial genus Aromatoleum comprises facultative denitrifiers specialized in the anaerobic degradation of recalcitrant organic compounds (aromatic and terpenoid). This study reports on the complete and manually annotated genomes of Ar. petrolei ToN1T (5.41 Mbp) and Ar. bremense PbN1T (4.38 Mbp), which cover the phylogenetic breadth of the genus Aromatoleum together with previously genome sequenced Ar. aromaticum EbN1T [Rabus et al., Arch Microbiol. 2005 Jan;183(1):27-36]. The gene clusters for the anaerobic degradation of aromatic and terpenoid (strain ToN1T only) compounds are scattered across the genomes of strains ToN1T and PbN1T. The richness in mobile genetic elements is shared with other Aromatoleum spp., substantiating that horizontal gene transfer should have been a major driver in shaping the genomes of this genus. The composite catabolic network of strains ToN1T and PbN1T comprises 88 proteins, the coding genes of which occupy 86.1 and 76.4 kbp (1.59 and 1.75%) of the respective genome. The strain-specific gene clusters for anaerobic degradation of ethyl-/propylbenzene (strain PbN1T) and toluene/monoterpenes (strain ToN1T) share high similarity with their counterparts in Ar. aromaticum strains EbN1T and pCyN1, respectively. Glucose is degraded via the ED-pathway in strain ToN1T, while gluconeogenesis proceeds via the reverse EMP-pathway in strains ToN1T, PbN1T, and EbN1T. The diazotrophic, endophytic lifestyle of closest related genus Azoarcus is known to be associated with nitrogenase and type-6 secretion system (T6SS). By contrast, strains ToN1T, PbN1T, and EbN1T lack nif genes for nitrogenase (including cofactor synthesis and enzyme maturation). Moreover, strains PbN1T and EbN1T do not possess tss genes for T6SS, while strain ToN1T does and facultative endophytic "Aromatoleum" sp. CIB is known to even have both. These findings underpin the functional heterogeneity among Aromatoleum members, correlating with the high plasticity of their genomes.

Aromatoleum gen. nov., a novel genus accommodating the phylogenetic lineage including Azoarcus evansii and related species, and proposal of Aromatoleum aromaticum sp. nov., Aromatoleum petrolei sp. nov., Aromatoleum bremense sp. nov., Aromatoleum toluolicum sp. nov. and Aromatoleum diolicum sp. nov.

Comparative 16S rRNA gene sequence analysis and major physiological differences indicate two distinct sublineages within the genus Azoarcus: the Azoarcus evansii lineage, comprising Azoarcusevansii (type strain KB740T=DSM 6898T=CIP 109473T=NBRC 107771T), Azoarcusbuckelii (type strain U120T=DSM 14744T=LMG 26916T), Azoarcusanaerobius (type strain LuFRes1T=DSM 12081T=LMG 30943T), Azoarcustolulyticus (type strain Tol-4T=ATCC 51758T=CIP 109470T), Azoarcustoluvorans (type strain Td21T=ATCC 700604T=DSM 15124T) and Azoarcustoluclasticus (type strain MF63T=ATCC 700605T), and the Azoarcus indigens lineage, comprising Azoarcusindigens (type strain VB32T=ATCC 51398T=LMG 9092T), Azoarcus communis (type strain SWub3T=ATCC 51397T=LMG 9095T) and Azoarcusolearius (type strain DQS-4T=BCRC 80407T=KCTC 23918T=LMG 26893T). Az. evansii lineage members have remarkable anaerobic degradation capacities encompassing a multitude of alkylbenzenes, aromatic compounds and monoterpenes, often involving novel biochemical reactions. In contrast, Az. indigens lineage members are diazotrophic endophytes lacking these catabolic capacities. It is proposed that species of the Az. evansii lineage should be classified in a novel genus, Aromatoleum gen. nov. Finally, based on the literature and new growth, DNA-DNA hybridization and proteomic data, the following five new species are proposed: Aromatoleum aromaticum sp. nov. (type strain EbN1T=DSM 19018T=LMG 30748T and strain pCyN1=DSM 19016=LMG 31004), Aromatoleum petrolei sp. nov. (type strain ToN1T=DSM 19019T=LMG 30746T), Aromatoleumbremense sp. nov. (type strain PbN1T=DSM 19017T=LMG 31005T), Aromatoleum toluolicum sp. nov. (type strain TT=DSM 19020T=LMG 30751T) and Aromatoleum diolicum sp. nov. (type strain 22LinT=DSM 15408T=LMG 30750T).

Evolution of a xenobiotic degradation pathway: formation and capture of the labile phthaloyl-CoA intermediate during anaerobic phthalate degradation.

Xenobiotic phthalates are industrially produced on the annual million ton scale. The oxygen-independent enzymatic reactions involved in anaerobic phthalate degradation have only recently been elucidated. In vitro assays suggested that phthalate is first activated to phthaloyl-CoA followed by decarboxylation to benzoyl-CoA. Here, we report the heterologous production and characterization of the enzyme initiating anaerobic phthalate degradation from 'Aromatoleum aromaticum': a highly specific succinyl-CoA:phthalate CoA transferase (SPT, class III CoA transferase). Phthaloyl-CoA formed by SPT accumulated only to sub-micromolar concentrations due to the extreme lability of the product towards intramolecular substitution with a half-life of around 7 min. Upon addition of excess phthaloyl-CoA decarboxylase (PCD), the combined activity of both enzymes was drastically shifted towards physiologically relevant benzoyl-CoA formation. In conclusion, a massive overproduction of PCD in phthalate-grown cells to concentrations >140 µM was observed that allowed for efficient phthaloyl-CoA conversion at concentrations 250-fold below the apparent Km -value of PCD. The results obtained provide insights into an only recently evolved xenobiotic degradation pathway where a massive cellular overproduction of PCD compensates for the formation of the probably most unstable CoA ester intermediate in biology.

Uliginosibacterium sediminicola sp. nov., isolated from freshwater sediment.

Strain M1-21T is a Gram-stain-negative, strictly aerobic and short-rod-shaped bacterium, motile by means of a single polar flagellum; it was isolated from freshwater sediment in Korea. It grew at 10-40 °C (optimum 25 °C), pH 6.0-8.0 (optimum pH 7.0) and with 0-0.75 % (w/v) NaCl (optimal growth occurred in the absence of NaCl) on R2A agar, and it accumulated poly-ß-hydroxybutyrate granules inside the cells. According to 16S rRNA gene sequence analysis, strain M1-21T showed highest sequence similarity with Uliginosibacterium gangwonense (94.7 %) and Uliginosibacterium paludis (94.4 %). Phylogenetic analysis of the 16S rRNA gene sequences revealed that strain M1-21T belongs to the genus Uliginosibacterium. The DNA G+C content of strain M1-21T was 61.9 mol%. The predominant respiratory quinone was ubiquinone-8. The major fatty acids (>10 % of the total) were C16 : 0 and summed feature 3 (C16 : 1ω6c and/or C16 : 1ω7c), and the major polar lipids were diphosphatidylglycerol, phosphatidylglycerol and phosphatidylethanolamine. Strain M1-21T showed distinct phenotypic characteristics that differentiated it from species of the genus Uliginosibacterium. Based on these results, strain M1-21T represents a novel species of the genus Uliginosibacterium, for which the name Uliginosibacterium sediminicola sp. nov. is proposed. The type strain is M1-21T (=KACC 19271T=JCM 32000T).

Identification of active denitrifiers by DNA-stable isotope probing and amplicon sequencing reveals Betaproteobacteria as responsible for attenuation of nitrate contamination in a low impacted aquifer.

Groundwater reservoirs constitute important freshwater resources. However, these ecosystems are highly vulnerable to contamination and have to rely on the resident microbiota to attenuate the impact of this contamination. Nitrate is one of the main contaminants found in groundwater, and denitrification is the main process that removes the compound. In this study, the response to nutrient load on indigenous microbial communities in groundwater from a low impacted aquifer in Uruguay was evaluated. Denitrification rates were measured in groundwater samples from three different sites with nitrate, acetate and pyrite amendments. Results showed that denitrification is feasible under in situ nitrate and electron donor concentrations, although the lack of readily available organic energy source would limit the attenuation of higher nitrate concentrations. DNA-stable isotope probing, combined with amplicon sequencing of 16S rRNA, nirS and nirK genes, was used to identify the active denitrifiers. Members of the phylum Betaproteobacteria were the dominant denitrifiers in two of three sites, with different families being observed; members of the genus Vogesella (Neisseriaceae) were key denitrifiers at one site, while the genera Dechloromonas (Rhodocyclaceae) and Comamonas (Comamonadaceae) were the main denitrifiers detected at the other sites.

Interaction of "Candidatus Accumulibacter" and nitrifying bacteria to achieve energy-efficient denitrifying phosphorus removal via nitrite pathway from sewage.

To achieve energy-efficient denitrifying phosphorus removal via nitrite pathway from sewage, interaction of "Candidatus Accumulibacter" and nitrifying bacteria was investigated in a continuous-flow process. When nitrite in returned sludge of secondary settler was above 13mg/L, nitrite inhibition on anaerobic P-release of poly-phosphate organisms (PAOs) occurred. Clades IIC and IID were dominant, reaching 3.1%-11.9% of total bacteria. Clade IIC was sensitive to nitrite. Under low concentration of nitrite (<8mg/L), clade IIC primarily contributed to anoxic P-uptake. Clade IID had a strong tolerance to nitrite exposure. At high nitrite level (above 16mg/L), anoxic P-uptake was mainly performed by clade IID due to its strong tolerance to nitrite exposure. Ammonia oxidizing bacteria (AOB), nitrite oxidizing bacteria (NOB) and Accumulibacter interacted through variations of nitrite accumulation. High AOB abundance coupled with inhibition of NOB favored denitrifying phosphorus removal by clade IID. All Accumulibacter lineages were sorted into four clades of Type II. The most dominant ppk1 gene homologs were affiliated with clade IID, accounting for 69% of ppk1 clone library, and thus played an important role in denitrifying phosphorus removal via nitrite pathway.

Viridibacterium curvum gen. nov., sp. nov., isolated from freshwater.

A Gram stain-negative, yellowish green-pigmented, facultatively anaerobic, motile, curved rod-shaped bacterium designated as strain JJ016T was isolated from an artificial lake in South Korea, and characterized using a polyphasic approach. The 16S rRNA gene sequence of strain JJ016T indicated that the isolate belonged to the family Rhodocyclaceae and exhibited 95.0% identity to Uliginosibacterium gangwonense 5YN10-9T. The major cellular fatty acids of the novel strain were summed feature 3 (C16:1 ω6c and/or C16:1 ω7c), C16:0, C14:0, and summed feature 8 (C18:1 ω7c and/or C18:1 ω6c). The DNA G+C content of strain JJ016T was 61.9 mol%. The major respiratory quinone and major polar lipid of strain JJ016T were ubiquinone-8 and phosphatidylethanolamine, respectively. Based on the morphological and physiological properties and the biochemical evidence presented, we concluded that strain JJ016T represents a novel species of a new genus in the family Rhodocyclaceae, for which the name Viridibacterium curvum gen. nov., sp. nov. is proposed. The type strain is JJ016T (=KACC 16899T =JCM 18715T).

Functional soil metagenomics: elucidation of polycyclic aromatic hydrocarbon degradation potential following 12 years of in situ bioremediation.

A culture-independent function-based screening approach was used to assess the microbial aerobic catabolome for polycyclic aromatic hydrocarbons degradation of a soil subjected to 12 years of in situ bioremediation. A total of 422 750 fosmid clones were screened for key aromatic ring-cleavage activities using 2,3-dihydroxybiphenyl as substrate. Most of the genes encoding ring-cleavage enzymes on the 768 retrieved positive fosmids could not be identified using primer-based approaches and, thus, 205 fosmid inserts were sequenced. Nearly two hundred extradiol dioxygenase encoding genes of three different superfamilies could be identified. Additional key genes of aromatic metabolic pathways were identified, including a high abundance of Rieske non-heme iron oxygenases that provided detailed information on enzymes activating aromatic compounds and enzymes involved in activation of the side chain of methylsubstituted aromatics. The gained insights indicated a complex microbial network acting at the site under study, which comprises organisms similar to recently identified Immundisolibacter cernigliae TR3.2 and Rugosibacter aromaticivorans Ca6 and underlined the great potential of an approach that combines an activity-screening, a cost-effective high-throughput sequencing of fosmid clones and a phylogenomic-routed and manually curated database to carefully identify key proteins dedicated to aerobic degradation of aromatic compounds.

Candidatus Dactylopiibacterium carminicum, a Nitrogen-Fixing Symbiont of Dactylopius Cochineal Insects (Hemiptera: Coccoidea: Dactylopiidae).

The domesticated carmine cochineal Dactylopius coccus (scale insect) has commercial value and has been used for more than 500 years for natural red pigment production. Besides the domesticated cochineal, other wild Dactylopius species such as Dactylopius opuntiae are found in the Americas, all feeding on nutrient poor sap from native cacti. To compensate nutritional deficiencies, many insects harbor symbiotic bacteria which provide essential amino acids or vitamins to their hosts. Here, we characterized a symbiont from the carmine cochineal insects, Candidatus Dactylopiibacterium carminicum (betaproteobacterium, Rhodocyclaceae family) and found it in D. coccus and in D. opuntiae ovaries by fluorescent in situ hybridization, suggesting maternal inheritance. Bacterial genomes recovered from metagenomic data derived from whole insects or tissues both from D. coccus and from D. opuntiae were around 3.6 Mb in size. Phylogenomics showed that dactylopiibacteria constituted a closely related clade neighbor to nitrogen fixing bacteria from soil or from various plants including rice and other grass endophytes. Metabolic capabilities were inferred from genomic analyses, showing a complete operon for nitrogen fixation, biosynthesis of amino acids and vitamins and putative traits of anaerobic or microoxic metabolism as well as genes for plant interaction. Dactylopiibacterium nif gene expression and acetylene reduction activity detecting nitrogen fixation were evidenced in D. coccus hemolymph and ovaries, in congruence with the endosymbiont fluorescent in situ hybridization location. Dactylopiibacterium symbionts may compensate for the nitrogen deficiency in the cochineal diet. In addition, this symbiont may provide essential amino acids, recycle uric acid, and increase the cochineal life span.

Oryzomicrobium terrae gen. nov., sp. nov., of the family Rhodocyclaceae isolated from paddy soil.

A polyphasic approach was used to characterize a novel bacterium, designated strain TPP412T, isolated from a paddy soil in Taiwan. Strain TPP412T was Gram-stain-negative, facultatively anaerobic, rod-shaped, motile with a single polar flagellum and lacked bacteriochlorophyll. Growth was observed at 24-45 °C (optimal 25 °C), at pH 5.0-10.0 (optimal pH 7.0) and with 0-0.75 % (w/v) NaCl. Strain TPP412T showed highest 16S rRNA gene sequence similarity to members of the genera Rhodocyclus (94.1-94.5 %), Azospira (93.9-94.5 %) and Propionivibrio (93.4-94.4 %) and established a discrete taxonomic lineage in phylogenetic analysis. The major fatty acids found in strain TPP412T were C12 : 0, C12 : 0 3-OH, iso-C15 : 0 3-OH, C16 : 0, C16 : 1ω7c/C16 : 1ω6c and C18 : 1ω7c/C18 : 1ω6c. The major polar lipids consisted of phosphatidylmonomethylethanolamine, diphosphatidylglycerol, phosphatidylglycerol and an unidentified lipid. The polyamine pattern showed a predominance of putrescine and a minor amount of spermidine. The DNA G+C content was 58.4 mol% and the predominant quinone system was ubiquinone-8 (Q-8). The low 16S rRNA gene sequence similarity values (≤94.5%) and distinct phylogenetic clustering clearly distinguished strain TPP412T from other representatives of the family Rhodocyclaceae. Based on the discrete phylogenetic, phenotypic and chemotaxonomic traits together with results of comparative 16S rRNA gene sequence analysis, strain TPP412T is considered to represent a novel species of a new genus in the family Rhodocyclaceae, for which the name Oryzomicrobium terrae gen. nov., sp. nov. is proposed. The type strain of Oryzomicrobium terrae is TPP412T (=BCRC 80905T=JCM 30814T).

Rugosibacter aromaticivorans gen. nov., sp. nov., a bacterium within the family Rhodocyclaceae, isolated from contaminated soil, capable of degrading aromatic compounds.

A bacterial strain designated Ca6T was isolated from polycyclic aromatic hydrocarbon (PAH)-contaminated soil from the site of a former manufactured gas plant in Charlotte, NC, USA, and linked phylogenetically to the family Rhodocyclaceae of the class Betaproteobacteria. Its 16S rRNA gene sequence was highly similar to globally distributed environmental sequences, including those previously designated 'Pyrene Group 1' demonstrated to grow on the PAHs phenanthrene and pyrene by stable-isotope probing. The most closely related described relative was Sulfuritalea hydrogenivorans strain sk43HT (93.6 % 16S rRNA gene sequence identity). In addition to a limited number of organic acids, Ca6T was capable of growth on the monoaromatic compounds benzene and toluene, and the azaarene carbazole, as sole sources of carbon and energy. Growth on the PAHs phenanthrene and pyrene was also confirmed. Optimal growth was observed aerobically under mesophilic temperature, neutral pH and low salinity conditions. Major fatty acids present included summed feature 3 (C16 : 1ω7c or C16 : 1ω6c) and C16 : 0. The DNA G+C content of the single chromosome was 55.14  mol% as determined by complete genome sequencing. Due to its distinct genetic and physiological properties, strain Ca6T is proposed as a member of a novel genus and species within the family Rhodocyclaceae, for which the name Rugosibacter aromaticivorans gen. nov., sp. nov. is proposed. The type strain of the species is Ca6T (=ATCC TSD-59T=DSM 103039T).

Culture-Dependent and -Independent Identification of Polyphosphate-Accumulating Dechloromonas spp. Predominating in a Full-Scale Oxidation Ditch Wastewater Treatment Plant.

The oxidation ditch process is one of the most economical approaches currently used to simultaneously remove organic carbon, nitrogen, and also phosphorus (P) from wastewater. However, limited information is available on biological P removal in this process. In the present study, microorganisms contributing to P removal in a full-scale oxidation ditch reactor were investigated using culture-dependent and -independent approaches. A microbial community analysis based on 16S rRNA gene sequencing revealed that a phylotype closely related to Dechloromonas spp. in the family Rhodocyclaceae dominated in the oxidation ditch reactor. This dominant Dechloromonas sp. was successfully isolated and subjected to fluorescent staining for polyphosphate, followed by microscopic observations and a spectrofluorometric analysis, which clearly demonstrated that the Dechloromonas isolate exhibited a strong ability to accumulate polyphosphate within its cells. These results indicate the potential key role of Dechloromonas spp. in efficient P removal in the oxidation ditch wastewater treatment process.

Uliginosibacterium paludis sp. nov., isolated from a marsh.

A novel bacterial strain, designated KBP-13T, was isolated from a water sample taken from the Banping Lake Wetland Park in Taiwan and characterized using a polyphasic taxonomic approach. Cells of strain KBP-13T were Gram-stain-negative, aerobic, poly-ß-hydroxybutyrate-accumulating, motile rods that formed light yellow colonies. Growth occurred at 15-40 °C (optimum, 30-40 °C), at pH 6.0-8.0 (optimum, pH 6.0) and with 0-2 % (w/v) NaCl (optimum, 0 %). Phylogenetic analyses based on 16S rRNA gene sequences showed that strain KBP-13T belonged to the genus Uliginosibacterium within the family Rhodocyclaceae of the class Betaproteobacteria and its most closely related neighbour was Uliginosibacterium gangwonense 5YN10-9T with sequence similarity of 96.0 %. Strain KBP-13T contained summed feature 3 (comprising C16 : 1ω7c and/or C16 : 1ω6c), C16 : 0 and C14 : 0 as predominant fatty acids. The major respiratory quinone was Q-8. The DNA G+C content of the genomic DNA was 65.1 mol%. The polar lipid profile consisted of a mixture of phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, one uncharacterized aminophospholipid, one uncharacterized aminolipid, two uncharacterized phospholipids and three uncharacterized glycolipids. On the basis of the genotypic, chemotaxonomic and phenotypic data, strain KBP-13T represents a novel species in the genus Uliginosibacterium, for which the name Uliginosibacterium paludis sp. nov. is proposed. The type strain is KBP-13T (=BCRC 80903T=LMG 28837T=KCTC 42655T).

Altererythrobacter terrae sp. nov., isolated from mountain soil.

A Gram-negative, non-motile, non-spore-forming, ovoid-shaped bacterium designated as SWU3(T) was isolated from mountain soil collected at Seoul Women's University, South Korea. Based on 16S rRNA sequence analysis, strain SWU3(T) was found to belong to the genus Altererythrobacter. It shares high sequence similarities with A. dongtanensis JM27(T) (96.6 %), A. epoxidivorans JCS350(T) (96.6 %), and A. troitsensis KMM 6042(T) (96.5 %). Growth was observed between 15 and 37 °C (optimum, 30 °C) with pH of 6-9 (optimum, pH 7.0). It could tolerate 0-2 % (w/v) NaCl. Its predominant quinone was found to be ubiquinone (Q-10). Its major cellular fatty acids were determined to be C17:1 ω6c, C18:1 ω7c, and summed featured 3 (C16:1 ω7c/C16:1 ω6c), all of which are similar characteristics to those of species within the genus Altererythrobacter. Its G + C molar content was found to be 58.4 mol%. Phylogenetic evidence, together with phenotypic characteristics showed that strain SWU3(T) represents a new species of the genus Altererythrobacter. The name Altererythrobacter terrae sp. nov. is proposed and the type strain is SWU3(T) (=KEMB 9004-128(T) = JCM 19177(T)).

Functional and genomic diversity of methylotrophic Rhodocyclaceae: description of Methyloversatilis discipulorum sp. nov.

Three strains of methylotrophic Rhodocyclaceae (FAM1(T), RZ18-153 and RZ94) isolated from Lake Washington sediment samples were characterized. Based on phylogenetic analysis of 16S rRNA gene sequences the strains should be assigned to the genus Methyloversatilis. Similarly to other members of the family, the strains show broad metabolic capabilities and are able to utilize a number of organic acids, alcohols and aromatic compounds in addition to methanol and methylamine. The main fatty acids were 16:1ω7c (49-59%) and 16:0 (32-29%). Genomes of all isolates were sequenced, assembled and annotated in collaboration with the DOE Joint Genome Institute (JGI). Genome comparison revealed that the strains FAM1T, RZ18-153 and RZ94 are closely related to each other and almost equally distant from two previously described species of the genus Methyloversatilis, Methyloversatilis universalis and Methyloversatilis thermotolerans. Like other methylotrophic species of the genus Methyloversatilis, all three strains possess one-subunit PQQ-dependent ethanol/methanol dehydrogenase (Mdh-2), the N-methylglutamate pathway and the serine cycle (isocitrate lyase/malate synthase, Icl/ms(+) variant). Like M. universalis, strains FAM1(T), RZ18-153 and RZ94 have a quinohemoprotein amine dehydrogenase, a tungsten-containing formaldehyde ferredoxin oxidoreductase, phenol hydroxylase, and the complete Calvin cycle. Similarly to M. thermotolerans, the three strains possess two-subunit methanol dehydrogenase (MxaFI), monoamine oxidase (MAO) and nitrogenase. Based on the phenotypic and genomic data, the strains FAM1(T), RZ18-153 and RZ94 represent a novel species of the genus Methyloversatilis, for which the name Methyloversatilis discipulorum sp. nov. is proposed. The type strain is FAM1(T) ( = JCM 30542(T) = VKM = B-2888(T)).

Methyloversatilis thermotolerans sp. nov., a novel thermotolerant facultative methylotroph isolated from a hot spring.

A newly isolated facultatively methylotrophic bacterium (strain 3t(T)) was investigated. Cells of the isolate were Gram-stain-negative, asporogenous, non-motile rods that multiplied by binary fission. The strain utilized methanol, methylamine and a variety of multicarbon compounds as carbon and energy sources. Growth occurred at pH 6.5-8.5 (optimally at 7.0-7.5) and at 10-45 °C (optimally at 30-37 °C). The major fatty acids of methanol-grown cells were C16 : 1ω7c and C16 : 0. The predominant phospholipids were phosphatidylethanolamine and phosphatidylglycerol. The major ubiquinone was Q-8. Strain 3t(T) possessed pyrroloquinoline quinone (PQQ)-linked methanol dehydrogenase and assimilated C1 units at the level of formaldehyde and CO2 via the serine cycle. The DNA G+C content of the strain was 63.6 mol% (Tm). On the basis of 16S rRNA gene sequence similarity (98.1 %) and rather low DNA-DNA relatedness (30 %) with the type strain of the type species of the genus Methyloversatilis (Methyloversatilis universalis FAM5(T)), and physiological and biochemical characteristics, the isolate was classified as a representative of a new species of the genus and named Methyloversatilis thermotolerans 3t(T) ( = VKM B-2692(T) = CCUG 61694(T) = DSM 25156(T)).

Cultivation-independent analysis of archaeal and bacterial communities of the formation water in an Indian coal bed to enhance biotransformation of coal into methane.

Biogenic origin of the significant proportion of coal bed methane has indicated the role of microbial communities in methanogenesis. By using cultivation-independent approach, we have analysed the archaeal and bacterial community present in the formation water of an Indian coal bed at 600-700 m depth to understand their role in methanogenesis. Presence of methanogens in the formation water was inferred by epifluorescence microscopy and PCR amplification of mcrA gene. Archaeal 16S rRNA gene clone library from the formation water metagenome was dominated by methanogens showing similarity to Methanobacterium, Methanothermobacter and Methanolinea whereas the clones of bacterial 16S rRNA gene library were closely related to Azonexus, Azospira, Dechloromonas and Thauera. Thus, microbial community of the formation water consisted of predominantly hydrogenotrophic methanogens and the proteobacteria capable of nitrogen fixation, nitrate reduction and polyaromatic compound degradation. Methanogenic potential of the microbial community present in the formation water was elucidated by the production of methane in the enrichment culture, which contained 16S rRNA gene sequences showing close relatedness to the genus Methanobacterium. Microcosm using formation water as medium as well as a source of inoculum and coal as carbon source produced significant amount of methane which increased considerably by the addition of nitrite. The dominance of Diaphorobacter sp. in nitrite amended microcosm indicated their important role in supporting methanogenesis in the coal bed. This is the first study indicating existence of methanogenic and bacterial community in an Indian coal bed that is capable of in situ biotransformation of coal into methane.