Expanding the Diversity of Accumulibacter with a Novel Type and Deciphering the Transcriptional and Morphological Features among Co-Occurring Strains.

"Candidatus Accumulibacter" is the major polyphosphate-accumulating organism (PAO) in global wastewater treatment systems, and its phylogenetic and functional diversity have expanded in recent years. In addition to the widely recognized type I and II sublineages, we discovered a novel type enriched in laboratory bioreactors. Core gene and machine learning-based gene feature profiling supported the assertion that type III "Ca. Accumulibacter" is a potential PAO with the unique function of using dimethyl sulfoxide as an electron acceptor. Based on the correlation between ppk1 and genome similarity, the species-level richness of Accumulibacter was estimated to be over 100, suggesting that the currently recognized species are only the tip of the iceberg. Meanwhile, the interstrain transcriptional and morphological features of multiple "Ca. Accumulibacter" strains co-occurring in a bioreactor were investigated. Metatranscriptomics of seven co-occurring strains indicated that the expression level and interphasic dynamics of PAO phenotype-related genes had minimal correlation with their phylogeny. In particular, the expression of denitrifying and polyphosphate (poly-P) metabolism genes exhibited higher interstrain and interphasic divergence than expression of glycogen and polyhydroxyalkanoate metabolic genes. A strategy of cloning rRNA genes from different strains based on similar genomic synteny was successfully applied to differentiate their morphology via fluorescence in situ hybridization. Our study further expands the phylogenetic and functional diversity of "Ca. Accumulibacter" and proposes that deciphering the function and capability of certain "Ca. Accumulibacter" should be tailored to the environment and population in question. IMPORTANCE In the last 2 decades, "Ca. Accumulibacter" has garnered significant attention as the core functional but uncultured taxon for enhanced biological phosphorus removal due to its phylogenetic and functional diversity and intragenus niche differentiation. Since 2002, it has been widely known that this genus has two sublineages (type I and II). However, in this study, a metagenomic approach led to the discovery of a novel type (type III) with proposed novel functional features. By comparing the average nucleotide identity of "Ca. Accumulibacter" genomes and the similarity of ppk1, a phylogenetic biomarker largely deposited in databases, the global species-level richness of "Ca. Accumulibacter" was estimated for the first time to be over 100. Furthermore, we observed the co-occurrence of multiple "Ca. Accumulibacter" strains in a single bioreactor and found the simultaneous transcriptional divergence of these strains intriguing with regard to their niche differentiation within a single community. Our results indicated a decoupling feature between transcriptional pattern and phylogeny for co-occurring strains.

Phylogenetic analyses of Candidatus Branchiomonas cysticola refine the taxonomic classification of Betaproteobacteria associated with epitheliocystis in fish.

Candidatus Branchiomonas cysticola is recognized as the most prevalent bacterial agent causing epitheliocystis in Atlantic salmon (Salmo salar). Based on its partial 16S rRNA sequence, the bacterium has previously been found to be a member of Burkholderiales in the class Betaproteobacteria. Multilocus Sequence Analysis (MLSA) of the bacterium and 60 type strains of Betaproteobacteria using newly identified housekeeping genes (dnaK, rpoC, and fusA) and ribosomal subunit sequences (16S and 23S), instead supported the bacterium's affiliation to Nitrosomodales. Taxonomic rank normalization by Relative Evolutionary Divergence (RED) showed the phylogenetic distinction between Cand. B. cysticola and its closest related type strain to be at the family level. A novel bacterial family named Branchiomonaceae has thus been proposed to include a monophyletic clade of Betaproteobacteria exclusively associated with epitheliocystis in fish.

Paludibacterium denitrificans sp. nov., a Novel Denitrifying Bacterium Isolated from Activated Sludge.

A Gram-reaction-negative, facultatively aerobic, motile, non-spore-forming, rod-shaped, and denitrifying bacterium, designated dN18-1T, was isolated from activated sludge, Republic of Korea. This bacterium was investigated via a polyphasic approach to reveal its taxonomic position. Phylogenetic analysis based on 16S rRNA gene sequence indicated that strain dN18-1T belongs to the genus Paludibacterium and is most closely related to P. purpuratum KCTC 42852T (96.2% sequence similarity), P. yongneupense KACC 11601T (96.1%), and P. paludis BCRC 80514T (95.2%). The average nucleotide identity values and digital DNA-DNA hybridization values calculated between strain dN18-1T and the closely related strains were 72.5-73.1% and 19.0-19.6%. The genome comprises of 3,347,996 bp with a G + C content of 57.3 mol%. Strain dN18-1T possesses ubiquinone Q-8 as a predominant respiratory quinone, and summed feature 3 (C16:1 ω6c and/or C16:1 ω7c), summed feature 8 (C18:1 ω6c/C18:1 ω7c), C16:0 and C12:0, as its major fatty acids (>5%). The polar lipids consisted of diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, two unidentified phospholipids and four unidentified aminophospholipids. The results of ANI calculation, digital DNA-DNA hybridization, physiological and biochemical tests allowed phenotypic differentiation of strain dN18-1T from rephrase other genus Paludibacterium species with validly published names. Therefore, this isolate represents a novel species, for which the name Paludibacterium denitrificans sp. nov. (type strain dN18-1T = KACC 19537T = CGMCC 1.16961T) is proposed.

Accumulibacter diversity at the sub-clade level impacts enhanced biological phosphorus removal performance.

Accumulibacter is a well-known group of organisms, typically considered to be polyphosphate accumulating organisms (PAOs), but potentially capable of glycogen accumulating organism (GAO) metabolism under limiting influent phosphate levels. Metabolic features of Accumulibacter are typically linked to its phylogenetic identity at the Type or clade level, though it is unclear the extent to which Accumulibacter diversity can correlate with its capacity to perform P removal. This paper investigates the fine-scale diversity of Accumulibacter and its link with enhanced biological phosphorus removal (EBPR) performance under various operating conditions, to understand the conditions and community structure leading to successful and unsuccessful EBPR operation. For this purpose, the organic carbon feeding rate and total organic carbon concentration were varied during three distinct operational periods, where influent phosphate was never limiting. Accumulibacter was always the dominant microbial group (>80% of all bacteria according to quantitative fluorescence in situ hybridisation - FISH) and low levels of Competibacter and other GAOs were consistently observed (<15% of all bacteria). Steady state was achieved in each of the three periods, with average phosphorus removal levels of 36%, 99% and >99%, respectively. Experimentally determined stoichiometric activity supported the expression of a mixed PAO/GAO metabolism in the first steady state period and the typical PAO metabolism in the other two steady state periods. FISH quantification and amplicon sequencing of the polyphosphate kinase (ppk1) functional gene indicated that Accumulibacter clade IIC was selected in the first steady state period, which shifted to clade IA after decreasing the carbon feeding rate in steady state period 2, and finally shifted back to clade IIC in the third steady state period. Fine-resolution Ppk-based phylogenetic analysis revealed three different clusters within Accumulibacter clade IIC, where clusters IICii and IICiii were linked to poor EBPR performance in period 1, and cluster IICi was linked to good EBPR performance in period 3. This study shows that the deterioration of EBPR processes through GAO activity at non-limiting P concentrations can be linked to organisms that are typically classified as PAOs, not only to known GAOs such as Competibacter. Intra-clade phylogenetic diversity within Accumulibacter showed that some clusters actually behave similarly to GAOs even without influent phosphate limitation. This study highlights the need to closely re-examine traditional interpretations regarding the link between the microbial community composition and identity with the performance and metabolism of EBPR systems.

Differential effect of silver nanoparticles on the microbiome of adult and developing planaria.

Silver nanoparticles (AgNPs) are widely incorporated in household, consumer and medical products. Their unintentional release via wastewaters raises concerns on their environmental impact, particularly for aquatic organisms and their associated bacterial communities. It is known that the microbiome plays an important role in its host's health and physiology, e.g. by producing essential nutrients and providing protection against pathogens. A thorough understanding of the effects of AgNPs on bacterial communities and on their interactions with the host is crucial to fully assess AgNP toxicity on aquatic organisms. Our results indicate that the microbiome of the invertebrate Schmidtea mediterranea, a freshwater planarian, is affected by AgNP exposure at the tested 10 µg/ml concentration. Using targeted amplification of the bacterial 16S rRNA gene V3-V4 region, two independent experiments on the microbiomes of adult worms revealed a consistent decrease in Betaproteobacteriales after AgNP exposure, mainly attributed to a decrease in Curvibacter and Undibacterium. Although developing tissues and organisms are known to be more sensitive to toxic compounds, three independent experiments in regenerating worms showed a less pronounced effect of AgNP exposure on the microbiome, possibly because underlying bacterial community changes during development mask the AgNP induced effect. The presence of a polyvinyl-pyrrolidone (PVP) coating did not significantly alter the outcome of the experiments compared to those with uncoated particles. The observed variation between the different experiments underlines the highly variable nature of microbiomes and emphasises the need to repeat microbiome experiments, within and between physiological states of the animal.

ATP-dependent hydroxylation of an unactivated primary carbon with water.

Enzymatic hydroxylation of unactivated primary carbons is generally associated with the use of molecular oxygen as co-substrate for monooxygenases. However, in anaerobic cholesterol-degrading bacteria such as Sterolibacterium denitrificans the primary carbon of the isoprenoid side chain is oxidised to a carboxylate in the absence of oxygen. Here, we identify an enzymatic reaction sequence comprising two molybdenum-dependent hydroxylases and one ATP-dependent dehydratase that accomplish the hydroxylation of unactivated primary C26 methyl group of cholesterol with water: (i) hydroxylation of C25 to a tertiary alcohol, (ii) ATP-dependent dehydration to an alkene via a phosphorylated intermediate, (iii) hydroxylation of C26 to an allylic alcohol that is subsequently oxidised to the carboxylate. The three-step enzymatic reaction cascade divides the high activation energy barrier of primary C-H bond cleavage into three biologically feasible steps. This finding expands our knowledge of biological C-H activations beyond canonical oxygenase-dependent reactions.

Steel slag amendment impacts on soil microbial communities and activities of rice (Oryza sativa L.).

With the increase in iron/steel production, the higher volume of by-products (slag) generated necessitates its efficient recycling. Because the Linz-Donawitz (LD) slag is rich in silicon (Si) and other fertilizer components, we aim to evaluate the impact of the LD slag amendment on soil quality (by measuring soil physicochemical and biological properties), plant nutrient uptake, and strengthens correlations between nutrient uptake and soil bacterial communities. We used 16 S rRNA illumine sequencing to study soil bacterial community and APIZYM assay to study soil enzymes involved in C, N, and P cycling. The LD slag was applied at 2 Mg ha-1 to Japonica and Indica rice cultivated under flooded conditions. The LD slag amendment significantly improved soil pH, plant photosynthesis, soil nutrient availability, and the crop yield, irrespective of cultivars. It significantly increased N, P, and Si uptake of rice straw. The slag amendment enhanced soil microbial biomass, soil enzyme activities and enriched certain bacterial taxa featuring copiotrophic lifestyles and having the potential role for ecosystem services provided to the benefit of the plant. The study evidenced that the short-term LD slag amendment in rice cropping systems is useful to improve soil physicochemical and biological status, and the crop yield.

Retroconversion of estrogens into androgens by bacteria via a cobalamin-mediated methylation.

Steroid estrogens modulate physiology and development of vertebrates. Conversion of C19 androgens into C18 estrogens is thought to be an irreversible reaction. Here, we report a denitrifying Denitratisoma sp. strain DHT3 capable of catabolizing estrogens or androgens anaerobically. Strain DHT3 genome contains a polycistronic gene cluster, emtABCD, differentially transcribed under estrogen-fed conditions and predicted to encode a cobalamin-dependent methyltransferase system conserved among estrogen-utilizing anaerobes; an emtA-disrupted DHT3 derivative could catabolize androgens but not estrogens. These data, along with the observed androgen production in estrogen-fed strain DHT3 cultures, suggested the occurrence of a cobalamin-dependent estrogen methylation to form androgens. Consistently, the estrogen conversion into androgens in strain DHT3 cell extracts requires methylcobalamin and is inhibited by propyl iodide, a specific inhibitor of cobalamin-dependent enzymes. The identification of the cobalamin-dependent estrogen methylation thus represents an unprecedented metabolic link between cobalamin and steroid metabolism and suggests that retroconversion of estrogens into androgens occurs in the biosphere.

Profiling population-level diversity and dynamics of Accumulibacter via high throughput sequencing of ppk1.

As the key organism for enhanced biological phosphorus removal, Accumulibacter has shown high intragenus diversity based on the phylogeny of polyphosphate kinase1 gene (ppk1) and many clade-specific features related to performance of wastewater treatment. However, the widely used molecular approaches are deficient or cost-inefficient in providing a comprehensive and quantitative population-level profile for Accumulibacter in complex community. In this study, we introduced a pipeline to analyze the population-level diversity and dynamics of Accumulibacter via high throughput sequencing (HTS) of ppk1 and 16S rRNA gene simultaneously. The HTS approach was assessed by testing primer coverage, performing sample replication, and comparing with a traditional clone library. Based on survey on full-scale activated sludge samples, unexpected high microdiversity in Accumulibacter and a tendency of exclusivity between two phylogenetic types were discovered. Moreover, the pipeline facilitated monitoring the population-level dynamics and co-occurrence pattern under various laboratory enriching conditions. The results revealed previously uncharacterized intraclade dynamics during enrichment, little effect of denitrifying process on the Accumulibacter diversity, and the niche adaption of Clade IIC on propionate as sole carbon source. Co-occurrence of Accumulibacter populations further partially supported the exclusivity of two types. A few bacterial taxa, including Cytophagaceae-, Prosthecobacter-, and Compteibacter-related taxa, showed co-occurrence with many Accumulibacter populations, suggesting their niche co-selection or potential metabolic interactions with Accumulibacter. The present pipeline is transplantable for studying microdiversity and niche differentiation of other functional microorganisms in complex microbial systems.

Seasonal blooms of neutrophilic Betaproteobacterial Fe(II) oxidizers and Chlorobi in iron-rich coal mine drainage sediments.

Waters draining from flooded and abandoned coal mines in the South Wales Coalfield (SWC) are substantial sources of pollution to the environment characterized by circumneutral pH and elevated dissolved iron concentrations (>1 mg L-1). The discharged Fe precipitates to form Fe(III) (oxyhydr)oxides which sustain microbial communities. However, while several studies have investigated the geochemistry of mine drainage in the SWC, less is known about the microbial ecology of the sites presenting a gap in our understanding of biogeochemical cycling and pollutant turnover. This study investigated the biogeochemistry of the Ynysarwed mine adit in the SWC. Samples were collected from nine locations within sediment at the mine entrance from the upper and lower layers three times over one year for geochemical and bacterial 16S rRNA gene sequence analysis. During winter, members of the Betaproteobacteria bloomed in relative abundance (>40%) including the microaerophilic Fe(II)-oxidizing genus Gallionella. A concomitant decrease in Chlorobi-associated bacteria occurred, although by summer the community composition resembled that observed in the previous autumn. Here, we provide the first insights into the microbial ecology and seasonal dynamics of bacterial communities of Fe(III)-rich deposits in the SWC and demonstrate that neutrophilic Fe(II)-oxidizing bacteria are important and dynamic members of these communities.

Widespread detection of Candidatus Accumulibacter phosphatis, a polyphosphate-accumulating organism, in sediments of the Columbia River estuary.

Enhanced biological phosphorus removal (EBPR) exploits the metabolism of polyphosphate-accumulating organisms (PAOs) to remove excess phosphorus (P) from wastewater treatment. Candidatus Accumulibacter phosphatis (Accumulibacter) is the most abundant and well-studied PAO in EBPR systems. In a previous study, we detected polyphosphates throughout peripheral bay sediments, and hypothesized that an estuary is an ideal setting to evaluate PAOs in a natural system, given that estuaries are characterized by dynamic dissolved oxygen fluctuations that potentially favour PAO metabolism. We detected nucleotide sequences attributable to Accumulibacter (16S rRNA, ppk1) in sediments within three peripheral bays of the Columbia River estuary at abundances rivalling those observed in conventional wastewater treatment plants (0.01%-2.6%). Most of the sequences attributable to Accumulibacter were Type I rather than Type II, despite the fact that the estuary does not have particularly high nutrient concentrations. The highest diversity of Accumulibacter was observed in oligohaline peripheral bays, while the greatest abundances were observed at the mouth of the estuary in mesohaline sediments in the spring and summer. In addition, an approximately 70% increase in polyphosphate concentrations observed at one of the sites between dawn and dusk suggests that PAOs may play an important role in P cycling in estuary sediments.

Resolving the individual contribution of key microbial populations to enhanced biological phosphorus removal with Raman-FISH.

Enhanced biological phosphorus removal (EBPR) is a globally important biotechnological process and relies on the massive accumulation of phosphate within special microorganisms. Candidatus Accumulibacter conform to the classical physiology model for polyphosphate accumulating organisms and are widely believed to be the most important player for the process in full-scale EBPR systems. However, it was impossible till now to quantify the contribution of specific microbial clades to EBPR. In this study, we have developed a new tool to directly link the identity of microbial cells to the absolute quantification of intracellular poly-P and other polymers under in situ conditions, and applied it to eight full-scale EBPR plants. Besides Ca. Accumulibacter, members of the genus Tetrasphaera were found to be important microbes for P accumulation, and in six plants they were the most important. As these Tetrasphaera cells did not exhibit the classical phenotype of poly-P accumulating microbes, our entire understanding of the microbiology of the EBPR process has to be revised. Furthermore, our new single-cell approach can now also be applied to quantify storage polymer dynamics in individual populations in situ in other ecosystems and might become a valuable tool for many environmental microbiologists.

Population Structure and Morphotype Analysis of "Candidatus Accumulibacter" Using Fluorescence In Situ Hybridization-Staining-Flow Cytometry.

"Candidatus Accumulibacter" is the dominant polyphosphate-accumulating organism (PAO) in denitrifying phosphorus removal (DPR) systems. In order to investigate the community structure and clade morphotypes of "Candidatus Accumulibacter" in DPR systems through flow cytometry (FCM), denitrifying phosphorus removal of almost 100% using nitrite and nitrate as the electron acceptor was achieved in sequencing batch reactors (SBRs). An optimal method of flow cytometry combined with fluorescence in situ hybridization and SYBR green I staining (FISH-staining-flow cytometry) was developed to quantify PAOs in DPR systems. By setting the width value of FCM, bacterial cells in a sludge sample were divided into three groups in different morphotypes, namely, coccus, coccobacillus, and bacillus. Average percentages that the three different PAO populations accounted for among total bacteria from SBR1 (SBR2) were 42% (45%), 14% (13%), and 4% (2%). FCM showed that the ratios of PAOs to total bacteria in the two reactors were 61% and 59%, and the quantitative PCR (qPCR) results indicated that IIC was the dominant "Candidatus Accumulibacter" clade in both denitrifying phosphorus removal systems, reaching 50% of the total "Candidatus Accumulibacter" bacteria. The subdominant clade in the reactor with nitrite as the electron acceptor was IID, accounting for 31% of the total "Candidatus Accumulibacter" bacteria. The FCM and qPCR results suggested that clades IIC and IID were both coccus, clade IIF was coccobacillus, and clade IA was bacillus. FISH analysis also indicated that PAOs were major cocci in the systems. An equivalence test of FCM-based quantification confirmed the accuracy of FISH-staining-flow cytometry, which can meet the quantitative requirements for PAOs in complex activated sludge samples.IMPORTANCE As one group of the most important functional phosphorus removal organisms, "Candidatus Accumulibacter," affiliated with the Rhodocyclus group of the Betaproteobacteria, is a widely recognized and studied PAO in the field of biological wastewater treatment. The morphotypes and population structure of clade-level "Candidatus Accumulibacter" were studied through novel FISH-staining-flow cytometry, which involved denitrifying phosphorus removal (DPR) achieving carbon and energy savings and simultaneous removal of N and P, thus inferring the different denitrifying phosphorus removal abilities of these clades. Additionally, based on this method, in situ quantification for specific polyphosphate-accumulating organisms (PAOs) enables a more efficient process and more accurate result. The establishment of FISH-staining-flow cytometry makes cell sorting of clade-level noncultivated organisms available.

Iron targeted transcriptome study draws attention to novel redox protein candidates involved in ferrous iron oxidation in "Ferrovum" sp. JA12.

The response of the acidophilic iron oxidizer "Ferrovum" sp. JA12 to elevated concentrations of ferrous iron was targeted at transcriptome level in order to assess models on oxidative stress management and ferrous iron oxidation. Overall transcriptome profiles indicate a high cellular activity of "Ferrovum" sp. JA12 up to 50 mM of ferrous iron with genes predicted to be involved in iron oxidation, carbon fixation and ribosome formation showing the highest transcript levels. The data support the iron oxidation pathway inferred from genome analysis and draws attention to further redox proteins potentially associated with iron oxidation. The restriction of homologous proteins to iron oxidizing beta- and zetaproteobacteria underlines the previous notion of a common origin of iron oxidation in these phyla. Detoxification of reactive oxygen species and primary products of oxidative damage of membrane lipids appears to be of permanent relevance under conditions mimicking those of the original habitat of "Ferrovum" sp. JA12. Also the maintenance of a reverse membrane potential appears to be its most important strategy to withstand the acidic external pH.

Acidovorax monticola sp. nov., isolated from soil.

A novel strain K-4-16T was isolated from forest soil of Namsan Mountain, Seoul, South Korea, and was taxonomically characterized by a polyphasic approach. Strain K-4-16T was observed to be a Gram-staining negative, grayish white-coloured, motile with peritrichous flagella, and rod shaped bacterium. It was able to grow at 15-45 °C, at pH 4.5-10.5, and at 0-4% (w/v) NaCl concentration. Based on the 16S rRNA gene sequence analysis, strain K-4-16T belongs to the genus Acidovorax and is closely related to Acidovorax anthurii CFBP 3232T (98.3% sequence identity), Acidovorax konjaci K2T (97.9% sequence identity), Acidovorax valerianellae CFBP 4730T (97.8% sequence identity), and Acidovorax caeni R-24608T (97.8% sequence identity). The only respiratory quinone was ubiquinone-8. The major polar lipids were phosphatidylethanolamine, phosphatidylglycerol, and diphosphatidylglycerol. The predominant fatty acids of strain K-4-16T were summed feature 3 (C16:1ω7c and/or C16:1ω6c), C16:0, and summed feature 8 (C18:1ω7c and/or C18:1ω6c). The genomic DNA G+C content of this novel strain was 64.7 mol%. The DNA-DNA relatedness between strain K-4-16T and its reference strains were below the threshold value of 70%. The morphological, physiological, chemotaxonomic, and phylogenetic analyses clearly distinguished this strain from its close phylogenetic neighbors. Thus, strain K-4-16T represents a novel species of the genus Acidovorax, for which the name Acidovorax monticola sp. nov. is proposed. The type strain is K-4-16T (= KEMB 9005-570T = KACC 19171T = NBRC 113141T).

Aquincola amnicola sp. nov., isolated from a freshwater river.

Strain TTM-94T, isolated from a water sample taken from the Caohu River in Taiwan, was characterized using a polyphasic taxonomic approach. Cells of strain TTM-94T were Gram-staining-negative, aerobic, poly-ß-hydroxybutyrate-accumulating, motile by a single polar flagellum, short rod-shaped and surrounded by a thick capsule and it formed cream colored colonies. Growth occurred at 20-30 °C (optimum, 30 °C), at pH 6.0-8.0 (optimum, pH 6.0), and in the presence of 0-2% NaCl (optimum 0.5%). Phylogenetic analyses based on 16S rRNA gene sequences showed that strain TTM-94T belonged to the genus Aquincola in the Rubrivivax-Roseateles-Leptothrix-Ideonella-Aquabacterium branch of the class Betaproteobacteria and its most closely related neighbour was Aquincola tertiaricarbonis L10T with sequence similarity of 97.0%. Strain TTM-94T contained summed feature 3 (comprising C16:1ω7c and/or C16:1ω6c), C16:0 and C18:1ω7c as the predominant fatty acids. The polar lipid profile consisted of phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol and several uncharacterized lipids. The major respiratory quinone was Q-8. Genomic DNA G + C content of strain TTM-94T was 70.7 mol%. Strain TTM-94T exhibited less than 30% DNA-DNA relatedness with A. tertiaricarbonis L10T. Differential phenotypic properties, together with the phylogenetic inference, demonstrate that strain TTM-94T should be classified as a novel species of the genus Aquincola, for which the name Aquincola amnicola sp. nov. is presented. The type strain is TTM-94T (= BCRC 80890T = LMG 28709T).

Uliginosibacterium flavum sp. nov. isolated from an artificial lake.

A Gram-negative, facultative anaerobic, rod-shaped, motile by means of a polar flagellum, greenish-yellow-pigmented bacterial strain (designated strain JJ3220T) was isolated from an artificial lake in South Korea and characterized using a polyphasic approach. The 16S rRNA gene sequence of strain JJ3220T indicated that the isolate belongs to the family Rhodocyclaceae, and that it exhibits 96.4% similarity to Uliginosibacterium paludis KBP-13T. The major cellular fatty acids of the novel strain were C14:0, C16:0, and summed feature 3 (C16:1 ω6c and/or C16:1 ω7c). Strain JJ3220T had flexirubin-type pigments. The DNA G+C content of the strain was 62.8%. The major respiratory quinone and major polar lipid of strain JJ3220T were ubiquinone-8 and phosphatidylethanolamine, respectively. Based on the morphological and physiological properties and biochemical evidence presented, it can be concluded that strain JJ3220T represents a novel species of the genus Uliginosibacterium. The type strain Uliginosibacterium flavum is JJ3220T (=KACC 17644T =JCM 19465T).

A thermophilic-like ene-reductase originating from an acidophilic iron oxidizer.

Ene-reductases originating from extremophiles are gaining importance in the field of biocatalysis due to higher-stability properties. The genome of the acidophilic iron-oxidizing bacterium "Ferrovum" sp. JA12 was found to harbor a thermophilic-like ene-reductase (FOYE-1). The foye-1 gene was ligated into a pET16bp expression vector system, and the enzyme was produced in Escherichia coli BL21 (DE3; pLysS) cells in yields of 10 mg L-1. FOYE-1 showed remarkable activity and rates on N-phenylmaleimide and N-phenyl-2-methylmaleimide (up to 89 U mg-1, >97 % conversion, 95 % (R)-selective) with both nicotinamide cofactors, NADPH and NADH. The catalytic efficiency with NADPH was 27 times higher compared to NADH. At the temperature maximum (50 °C) and pH optimum (6.5), activity was almost doubled to 160 U mg-1. These findings accomplish FOYE-1 for a valuable biocatalyst in the synthesis of succinimides. The appearance of a thermophilic-like ene-reductase in an acidic habitat is discussed with respect to its phylogenetic placement and to the genomic neighborhood of the encoding gene, awarding FOYE-1 a putative involvement in a quorum-sensing process.

Piscinibacterium candidicorallinum gen. nov., sp. nov., a member of the order Burkholderiales isolated from a fish pond.

A bacterial strain designated LYH-15T was isolated from a freshwater fish pond in Taiwan and characterized using a polyphasic taxonomy approach. Cells of LYH-15T were Gram-staining-negative, aerobic, motile by means of a single polar flagellum, poly-ß-hydroxybutyrate-containing, non-spore forming, straight rods and formed light-coral-colored colonies. Growth occurred at 15-40 °C (optimum, 30 °C), at pH 5.0-9.0 (optimum, pH 7.0) and with 0-0.5 % NaCl (optimum, 0 %). Phylogenetic analyses based on 16S rRNA gene sequences showed that LYH-15T forms a distinct phyletic line within the order Burkholderiales, with less than 94 % sequence similarity to its closest relatives with validly published names. The predominant fatty acids were summed feature 3 (comprising C16 : 1ω7c and/or C16 : 1ω6c), C16 : 0 and C18 : 1ω7c. The major isoprenoid quinone was Q-8 and the DNA G+C content was 63.8 mol%. The major polar lipids were phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol and several uncharacterized lipids. The major polyamines were 2-hydroxyputrescine and putrescine. On the basis of the genotypic and phenotypic data, LYH-15T represents a novel species of a new genus in the order Burkholderiales, for which the name Piscinibacterium candidicorallinum gen. nov., sp. nov. is proposed. The type strain is LYH-15T (=BCRC 80969T=LMG 29480T=KCTC 52168T).

Microbial phylogenetic and functional responses within acidified wastewater communities exhibiting enhanced phosphate uptake.

Acid stimulated accumulation of insoluble phosphorus within microbial cells is highly beneficial to wastewater treatment but remains largely unexplored. Using single cell analyses and next generation sequencing, the response of active polyphosphate accumulating microbial communities under conditions of enhanced phosphorus uptake under both acidic and aerobic conditions was characterised. Phosphorus accumulation activities were highest under acidic conditions (pH 5.5>8.5), where a significant positive effect on bioaccumulation was observed at pH 5.5 when compared to pH 8.5. In contrast to the Betaproteobacteria and Actinobacteria dominated enhanced biological phosphorus removal process, the functionally active polyP accumulators at pH 5.5 belonged to the Gammaproteobacteria, with key accumulators identified as members of the families Aeromonadaceae and Enterobacteriaceae. This study demonstrated a significant enrichment of key polyphosphate kinase and exopolyphosphatase genes within the community metagenome after acidification, concomitant with an increase in P accumulation kinetics.