Seasonal Dynamics of Methanotrophic Bacteria in a Boreal Oil Sands End Pit Lake.

Base Mine Lake (BML) is the first full-scale demonstration end pit lake for the oil sands mining industry in Canada. We examined aerobic methanotrophic bacteria over all seasons for 5 years in this dimictic lake. Methanotrophs comprised up to 58% of all bacterial reads in 16S rRNA gene amplicon sequencing analyses (median 2.8%), and up to 2.7 × 104 cells mL-1 of water (median 0.5 × 103) based on qPCR of pmoA genes. Methanotrophic activity and populations in the lake water were highest during fall turnover and remained high through the winter ice-covered period into spring turnover. They declined during summer stratification, especially in the epilimnion. Three methanotroph genera (Methylobacter, Methylovulum, and Methyloparacoccus) cycled seasonally, based on both relative and absolute abundance measurements. Methylobacter and Methylovulum populations peaked in winter/spring, when methane oxidation activity was psychrophilic. Methyloparacoccus populations increased in the water column through summer and fall, when methane oxidation was mesophilic, and also predominated in the underlying tailings sediment. Other, less abundant genera grew primarily during summer, possibly due to distinct CH4/O2 microniches created during thermal stratification. These data are consistent with temporal and spatial niche differentiation based on temperature, CH4 and O2. This pit lake displays methane cycling and methanotroph population dynamics similar to natural boreal lakes. IMPORTANCE The study examined methanotrophic bacteria in an industrial end pit lake, combining molecular DNA methods (both quantitative and descriptive) with biogeochemical measurements. The lake was sampled over 5 years, in all four seasons, as often as weekly, and included sub-ice samples. The resulting multiseason and multiyear data set is unique in its size and intensity, and allowed us to document clear and consistent seasonal patterns of growth and decline of three methanotroph genera (Methylobacter, Methylovulum, and Methyloparacoccus). Laboratory experiments suggested that one major control of this succession was niche partitioning based on temperature. The study helps to understand microbial dynamics in engineered end pit lakes, but we propose that the dynamics are typical of boreal stratified lakes and widely applicable in microbial ecology and limnology. Methane-oxidizing bacteria are important model organisms in microbial ecology and have implications for global climate change.

Methylicorpusculum oleiharenae gen. nov., sp. nov., an aerobic methanotroph isolated from an oil sands tailings pond.

An aerobic methane oxidizing bacterium, designated XLMV4T, was isolated from the oxic surface layer of an oil sands tailings pond in Alberta, Canada. Strain XLMV4T is capable of growth on methane and methanol as energy sources. NH4Cl and sodium nitrate are nitrogen sources. Cells are Gram-negative, beige to yellow-pigmented, motile (via a single polar flagellum), short rods 2.0-3.3 µm in length and 1.0-1.6 µm in width. A thick capsule is produced. Surface glycoprotein or cup shape proteins typical of the genera Methylococcus, Methylothermus and Methylomicrobium were not observed. Major isoprenoid quinones are Q-8 and Q-7 at an approximate molar ratio of 71 : 22. Major polar lipids are phosphoglycerol and ornithine lipids. Major fatty acids are C16 : 1 ω8+C16 : 1 ω7 (34 %), C16 : 1 ω5 (16 %), and C18 : 1 ω7 (11 %). Optimum growth is observed at pH 8.0 and 25 °C. The DNA G+C content based on a draft genome sequence is 46.7 mol%. Phylogenetic analysis of 16S rRNA genes and a larger set of conserved genes place strain XLMV4T within the class Gammaproteobacteria and family Methylococcaceae, most closely related to members of the genera Methylomicrobium and Methylobacter (95.0-97.1 % 16S rRNA gene sequence identity). In silico genomic predictions of DNA-DNA hybridization values of strain XLMV4T to the nearest phylogenetic neighbours were all below 26 %. On the basis of the data presented, strain XLMV4T is considered to represent a new genus and species for which the name Methylicorpusculum oleiharenae is proposed. Strain XLMV4T (=DSMZ DSM 27269=ATCC TSD-186) is the type strain.

Oleiharenicola alkalitolerans gen. nov., sp. nov., a new member of the phylum Verrucomicrobia isolated from an oilsands tailings pond.

A novel member of the phylum Verrucomicrobia was isolated from an oilsands tailings pond in Alberta, Canada. Cells of isolate NVTT are Gram-negative, strictly aerobic, non-pigmented, non-motile cocci to diplococci 0.5-1.0 µm in diameter. The bacterium is neutrophilic (optimum pH 6.0-8.0) but alkalitolerant, capable of growth between pH 5.5 and 11.0. The temperature range for growth is 15-40 °C (optimum 25-37 °C). Carbon and energy sources include sugars and organic acids. Nitrogen sources include nitrate, urea, l-glycine, l-alanine, l-proline and l-serine. Does not fix atmospheric nitrogen. Does not require NaCl and is inhibited at NaCl concentrations above 3.0 % (w/v). The DNA G+C content of strain NVTT, based on a draft genome sequence, is 66.1 mol%. MK-6 and MK-7 are the major respiratory quinones. Major cellular fatty acids are anteiso-C15 : 0 and iso-C15 : 0. Phylogenetic analysis of 16S rRNA gene sequences revealed that the strain belongs to the family Opitutaceae of the phylum Verrucomicrobia. The most closely related validated species is Opitutus terrae (93.7 % 16S rRNA gene sequence identity to its type strain PB90-1T). Based on genotypic, phenotypic and chemotaxonomic characteristics, it was concluded that this strain represents a novel genus and species, for which the name Oleiharenicola alkalitolerans gen. nov., sp. nov. is proposed. The type strain of this novel species is NVTT (=ATCC BAA-2697T;=DSM 29249T).

Actinocrinis puniceicyclus gen. nov., sp. nov., an actinobacterium isolated from an acidic spring.

An aerobic, mildly acidophilic actinobacterium was isolated from the Ochre Beds bog in Kootenay National Park, Canada. Cells of isolate OB1T were Gram-stain-positive, non-motile, pink- to purple-pigmented filaments. The pH range for growth was pH 3.5-6.5 (optimum pH 5.5), and the temperature range was 13-30°C. The major cellular fatty acids were i-C16 : 0 (28.5 %), i-C15 : 0 (14.6 %) and ai-C15 : 0 (14.3 %), and the major polar lipid was phosphohexose. The major quinone was menaquinone-11 (MK-11), and the peptidoglycan type was A1γ. The DNA G+C content was 70.2 %. Along with growth on complex media including yeast extract, proteose peptone, casamino acids and tryptic soy broth, growth occured on mono- and disaccharides (glucose, sucrose, galactose and xylose) and polysaccharides (starch, gellan, pectin, xylan and alginate). Anaerobic growth was not observed. The cells did not fix atmospheric nitrogen. On the basis of comparative 16S rRNA gene sequence analysis, this isolate belonged to the family Actinospicaceae, in the suborder Catenulisporineae of the order Actinomycetales. The most closely related species was Actinospica robiniae. However, the 16S rRNA gene sequence identity to this bacterium was low (92.8 %) and there were several chemotaxonomic differences from this species. We therefore propose a novel genus and species, Actinocrinis puniceicyclus gen. nov., sp. nov., with strain OB1T (=DSM 45618T=ATCC BAA-2771T) as the type strain.

Chryseolinea serpens gen. nov., sp. nov., a member of the phylum Bacteroidetes isolated from soil.

An aerobic chemoheterotrophic gliding bacterium, designated RYG(T), was isolated from a soil in Germany. Cells were Gram-stain-negative, thin rods (0.4-0.6 µm in width and 2.0-5.5 µm in length). Cells multiplied by normal cell division and no resting stages were observed. Colonies were yellow and displayed swarming edges. Gliding motility was observed in wet mounts. Strain RYG(T) grew at pH 5.6-7.7 (optimum pH 6.6-7.0), at 13-37 °C (optimum 25-30 °C) and with 0-1.0 % NaCl (optimum 0-0.1 %). The isolate was incapable of atmospheric nitrogen fixation and grew on most mono- and disaccharides as well as a few polysaccharides and organic acids. The predominant menaquinone was MK-7, the major cellular fatty acids were C(16 : 1)ω5c and iso-C(15 : 0) and the major intact polar lipids were composed of phosphatidylethanolamine derivatives and two unknown series. The DNA G+C content was 49.9 mol%. Based on 16S rRNA gene sequence analysis, the isolate belonged to the phylum Bacteroidetes, class Cytophagia, order Cytophagales, but was only distantly related to any cultured bacteria. The closest relatives were Ohtaekwangia koreensis 3B-2(T) and Ohtaekwangia kribbensis 10AO(T) (both 93 % 16S rRNA gene sequence similarity). We propose a novel genus and species, Chryseolinea serpens gen. nov., sp. nov.. Strain RYG(T) ( = DSM 24574(T) = ATCC BAA-2075(T)) is the type strain.

Isolation of a novel cutinase homolog with polyethylene terephthalate-degrading activity from leaf-branch compost by using a metagenomic approach.

The gene encoding a cutinase homolog, LC-cutinase, was cloned from a fosmid library of a leaf-branch compost metagenome by functional screening using tributyrin agar plates. LC-cutinase shows the highest amino acid sequence identity of 59.7% to Thermomonospora curvata lipase. It also shows the 57.4% identity to Thermobifida fusca cutinase. When LC-cutinase without a putative signal peptide was secreted to the periplasm of Escherichia coli cells with the assistance of the pelB leader sequence, more than 50% of the recombinant protein, termed LC-cutinase*, was excreted into the extracellular medium. It was purified and characterized. LC-cutinase* hydrolyzed various fatty acid monoesters with acyl chain lengths of 2 to 18, with a preference for short-chain substrates (C(4) substrate at most) most optimally at pH 8.5 and 50°C, but could not hydrolyze olive oil. It lost activity with half-lives of 40 min at 70°C and 7 min at 80°C. LC-cutinase* had an ability to degrade poly(ε-caprolactone) and polyethylene terephthalate (PET). The specific PET-degrading activity of LC-cutinase* was determined to be 12 mg/h/mg of enzyme (2.7 mg/h/µkat of pNP-butyrate-degrading activity) at pH 8.0 and 50°C. This activity is higher than those of the bacterial and fungal cutinases reported thus far, suggesting that LC-cutinase* not only serves as a good model for understanding the molecular mechanism of PET-degrading enzyme but also is potentially applicable for surface modification and degradation of PET.

Flavobacterium compostarboris sp. nov., isolated from leaf-and-branch compost, and emended descriptions of Flavobacterium hercynium, Flavobacterium resistens and Flavobacterium johnsoniae.

A strictly aerobic, Gram-negative, yellow-pigmented, non-spore-forming rod, designated 15C3(T), was isolated from aerobic leaf-and-branch compost at EXPO Park in Osaka, Japan. Growth was observed at 9-33 °C (optimum 25 °C) and pH 5.6-7.9 (optimum pH 6.1-7.0). No growth occurred with >2% (w/v) NaCl. Strain 15C3(T) reduced nitrate to nitrogen and showed catalase activity but not oxidase activity. The predominant fatty acids were iso-C(15:0), iso-C(17:0) 3-OH and summed feature 3 (comprising C(16:1)ω7c and/or iso-C(15:0) 2-OH). The isolate contained phosphatidylethanolamine as the major polar lipid and menaquinone-6 as the major respiratory quinone. The G+C content of the genomic DNA of strain 15C3(T) was 33.6 mol%. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain 15C3(T) belonged to the genus Flavobacterium and was most closely related to Flavobacterium hercynium WB 4.2-33(T) (96.9% sequence similarity). On the basis of phenotypic and phylogenetic distinctiveness, strain 15C3(T) is considered to represent a novel species in the genus Flavobacterium, for which the name Flavobacterium compostarboris sp. nov. is proposed. The type strain is 15C3(T) ( = KACC 14224(T)  = JCM 16527(T)). Emended descriptions of F. hercynium, Flavobacterium resistens and Flavobacterium johnsoniae are also given.

Flavobacterium banpakuense sp. nov., isolated from leaf-and-branch compost.

A strictly aerobic, Gram-stain-negative, yellow-pigmented, non-spore-forming, motile (by gliding), rod-shaped bacterium, designated strain 15F3(T), was isolated from leaf-and-branch compost. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain 15F3(T) was most closely related to Flavobacterium reichenbachii WB 3.2-61(T) and formed a distinct phyletic lineage within the genus Flavobacterium, the type genus of the family Flavobacteriaceae. Growth was observed at 10-34 °C (optimum, 30 °C) and pH 6.0-8.0 (optimum, pH 7.0). No growth occurred in the presence of ≥2 % (w/v) NaCl. Strain 15F3(T) reduced nitrate to nitrogen and showed catalase activity but no oxidase activity. The predominant cellular fatty acids were iso-C(15 : 0) and summed feature 3 (comprising C(16 : 1)ω7c and/or iso-C(15 : 0) 2-OH). The major isoprenoid quinone was menaquinone-6. The G+C content of the genomic DNA was 31.1 mol%. On the basis of data from this polyphasic study, strain 15F3(T) may be classified as a representative of a novel species within the genus Flavobacterium, for which the name Flavobacterium banpakuense sp. nov. is proposed; the type strain is 15F3(T) ( = KACC 14225(T)  = JCM 16466(T)).

Characterization of growth-supporting factors produced by Geobacillus toebii for the commensal thermophile Symbiobacterium toebii.

Symbiobacterium toebii is a commensal symbiotic thermophile that cannot grow without support from a partner bacterium. We investigated the properties of Symbiobacterium growth-supporting factors (SGSFs) produced by the partner bacterium Geobacillus toebii. SGSFs occurred in both the cell-free extract (CFE) and culture supernatant of G. toebii and might comprise multifarious materials because of their different biological properties. The heavy SGSF contained in the cytosolic component exhibited heat- and proteinase-sensitive proteinaceous properties and had a molecular mass of >50 kDa. In contrast, the light SGSF contained in the extracellular component exhibited heat-stable, proteinase-resistant, nonprotein properties and had a molecular mass of <10 kDa. Under morphological examination using light microscopy, S. toebii cultured with the culture supernatant of G. toebii was filamentous, whereas S. toebii cultured with the CFE of G. toebii was rod-shaped. These results strongly suggest that the SGSFs produced by G. toebii comprise two or more types that differ in their growth-supporting mechanisms, although all support the growth of S. toebii. Upon the examination of the distribution of SGSFs in other bacteria, both cytosolic and extracellular components of Geobacillus kaustophilus, Escherichia coli, and Bacillus subtilis had detectable growth-supporting effects for S. toebii, indicating that common SGSF materials are widely present in various bacterial strains.

Isolation of uncultivable anaerobic thermophiles of the family Clostridiaceae requiring growth-supporting factors.

Novel groups of uncultivable anaerobic thermophiles were isolated from compost by enrichment cultivation in medium with a cell-free extract of Geobacillus toebii. The cell-free extract of G. toebii provided the medium with growth-supporting factors (GSF) needed to cultivate the previously uncultured microorganisms. Twenty-nine GSF-requiring candidates were successfully cultivated, and 16 isolated novel bacterial strains were classified into three different groups of uncultivable bacteria. The similarity among these 16 isolates and a phylogenetic analysis using 16S rRNA gene sequences revealed that these GSF-requiring strains represented novel groups within the family Clostridiaceae.

Corrigendum to "Physiological indices for the categorization of Mibyeong severity" [Integr. Med. Res. 2017; 6: 88-92].

[This corrects the article DOI: 10.1016/j.imr.2017.01.005.].

Lower cellular metabolic power can be an explanation for obesity trend in Tae-Eum type: hypothesis and clinical observation.

BACKGROUND: Those classified as Tae-Eum (TE)-type people in Sasang constitutional medicine (SCM) are prone to obesity. Although extensive clinical observations have confirmed this tendency, the underlying physiological mechanisms are unknown. Here, we propose a novel hypothesis using integrative physiology to explain this phenomenon. METHODS: Hypoactive lung function in the TE type indicates that respiration is attenuated at the cellular level-specifically, mitochondrial oxygen consumption. Because a functional reduction in cellular energy metabolism is suggestive of intrinsic hypoactivity in the consumption (or production) of metabolic energy, we reasoned that this tendency can readily cause weight gain via an increase in anabolism. Thus, this relationship can be derived from the graph of cellular metabolic power plotted against body weight. We analyzed the clinical data of 548 individuals to test this hypothesis. RESULTS: The statistical analysis revealed that the cellular metabolic rate was lower in TE-type individuals and that their percentage of obesity (body mass index >25) was significantly higher compared to other constitutional groups. CONCLUSION: Lower cellular metabolic power can be an explanation for the obesity trend in TE type people.

Physiological indices for the categorization of Mibyeong severity.

BACKGROUND: Individuals with Mibyeong are difficult to identify. Although extensive research has attempted to introduce an easy and clear method for Mibyeong diagnosis, the indices used to categorize Mibyeong severity are unclear. We hypothesized that individuals with severe Mibyeong have reduced physiological function, thus activating homeostatic regulatory functions and inducing alterations in vascular resistance and capacitance. METHODS: Novel indices used to categorize Mibyeong severity based on the cardiovascular system model are described. We analyzed resistance and capacitance values using a simple cardiovascular system model optimally satisfying the measured systolic and diastolic pressures, heart rate, and age. RESULTS: Clinical data from 509 individuals were examined to test our hypothesis. A statistical analysis revealed that the vascular resistance was lower in individuals with severe Mibyeong symptoms and decreased with increasing Mibyeong severity, whereas the vascular capacitance showed an opposite trend. CONCLUSION: We derived indices to categorize Mibyeong severity and tested 509 individuals. An epidemiological analysis revealed that the vascular resistance decreased while the capacitance increased with increasing Mibyeong severity, indicating the validity of the values as Mibyeong indices.

Benzene and Naphthalene Degrading Bacterial Communities in an Oil Sands Tailings Pond.

Oil sands process-affected water (OSPW), produced by surface-mining of oil sands in Canada, is alkaline and contains high concentrations of salts, metals, naphthenic acids, and polycyclic aromatic compounds (PAHs). Residual hydrocarbon biodegradation occurs naturally, but little is known about the hydrocarbon-degrading microbial communities present in OSPW. In this study, aerobic oxidation of benzene and naphthalene in the surface layer of an oil sands tailings pond were measured. The potential oxidation rates were 4.3 µmol L-1 OSPW d-1 for benzene and 21.4 µmol L-1 OSPW d-1 for naphthalene. To identify benzene and naphthalene-degrading microbial communities, metagenomics was combined with stable isotope probing (SIP), high-throughput sequencing of 16S rRNA gene amplicons, and isolation of microbial strains. SIP using 13C-benzene and 13C-naphthalene detected strains of the genera Methyloversatilis and Zavarzinia as the main benzene degraders, while strains belonging to the family Chromatiaceae and the genus Thauera were the main naphthalene degraders. Metagenomic analysis revealed a diversity of genes encoding oxygenases active against aromatic compounds. Although these genes apparently belonged to many phylogenetically diverse taxa, only a few of these taxa were predominant in the SIP experiments. This suggested that many members of the community are adapted to consuming other aromatic compounds, or are active only under specific conditions. 16S rRNA gene sequence datasets have been submitted to the Sequence Read Archive (SRA) under accession number SRP109130. The Gold Study and Project submission ID number in Joint Genome Institute IMG/M for the metagenome is Gs0047444 and Gp0055765.

Structure and stability of metagenome-derived glycoside hydrolase family 12 cellulase (LC-CelA) a homolog of Cel12A from Rhodothermus marinus.

Ten genes encoding novel cellulases with putative signal peptides at the N-terminus, termed pre-LC-CelA-J, were isolated from a fosmid library of a leaf-branch compost metagenome by functional screening using agar plates containing carboxymethyl cellulose and trypan blue. All the cellulases except pre-LC-CelG have a 14-29 residue long flexible linker (FL) between the signal peptide and the catalytic domain. LC-CelA without a signal peptide (residues 20-261), which shows 76% amino acid sequence identity to Cel12A from Rhodothermus marinus (RmCel12A), was overproduced in Escherichia coli, purified and characterized. LC-CelA exhibited its highest activity across a broad pH range (pH 5-9) and at 90 °C, indicating that LC-CelA is a highly thermostable cellulase, like RmCel12A. The crystal structure of LC-CelA was determined at 1.85 Å resolution and is nearly identical to that of RmCel12A determined in a form without the FL. Both proteins contain two disulfide bonds. LC-CelA has a 16-residue FL (residues 20-35), most of which is not visible in the electron density map, probably due to structural disorder. However, Glu34 and Pro35 form hydrogen bonds with the central region of the protein. ΔFL-LC-CelA (residues 36-261) and E34A-LC-CelA with a single Glu34 â†’ Ala mutation were therefore constructed and characterized. ΔFL-LC-CelA and E34A-LC-CelA had lower melting temperatures (T m) than LC-CelA by 14.7 and 12.0 °C respectively. The T m of LC-CelA was also decreased by 28.0 °C in the presence of dithiothreitol. These results suggest that Glu34-mediated hydrogen bonds and the two disulfide bonds contribute to the stabilization of LC-CelA.

A commensal symbiotic interrelationship for the growth of Symbiobacterium toebii with its partner bacterium, Geobacillus toebii.

BACKGROUND: Symbiobacterium toebii is a commensal symbiotic thermophile that absolutely requires its partner bacterium Geobacillus toebii for growth. Despite development of an independent cultivation method using cell-free extracts, the growth of Symbiobacterium remains unknown due to our poor understanding of the symbiotic relationship with its partner bacterium. Here, we investigated the interrelationship between these two bacteria for growth of S. toebii using different cell-free extracts of G. toebii. RESULTS: Symbiobacterium toebii growth-supporting factors were constitutively produced through almost all growth phases and under different oxygen tensions in G. toebii, indicating that the factor may be essential components for growth of G. toebii as well as S. toebii. The growing conditions of G. toebii under different oxygen tension dramatically affected to the initial growth of S. toebii and the retarded lag phase was completely shortened by reducing agent, L-cysteine indicating an evidence of commensal interaction of microaerobic and anaerobic bacterium S. toebii with a facultative aerobic bacterium G. toebii. In addition, the growth curve of S. toebii showed a dependency on the protein concentration of cell-free extracts of G. toebii, demonstrating that the G. toebii-derived factors have nutrient-like characters but not quorum-sensing characters. CONCLUSIONS: Not only the consistent existence of the factor in G. toebii during all growth stages and under different oxygen tensions but also the concentration dependency of the factor for proliferation and optimal growth of S. toebii, suggests that an important biosynthetic machinery lacks in S. toebii during evolution. The commensal symbiotic bacterium, S. toebii uptakes certain ubiquitous and essential compound for its growth from environment or neighboring bacteria that shares the equivalent compounds. Moreover, G. toebii grown under aerobic condition shortened the lag phase of S. toebii under anaerobic and microaerobic conditions, suggests a possible commensal interaction that G. toebii scavengers ROS/RNS species and helps the initial growth of S. toebii.

Characterization of Symbiobacterium toebii, an obligate commensal thermophile isolated from compost.

A symbiotic thermophilic bacterium, strain SC-1, was isolated from hay compost (toebi) in Korea. The new isolate exhibited an obligate commensal interaction with a thermophilic Geobacillus strain and required crude extracts and/or culture supernatant from Geobacillus sp. SK-1 for axenic growth. The growth factors from Geobacillus sp. SK-1 were irreversibly inactivated by phenol or protease treatment, suggesting that they might be proteins. The cells of strain SC-1 were non-spore forming, nonmotile rods that were stained Gram-negatively. The isolate was a microaerophilic heterotroph. Growth was observed between 45 degrees and 70 degrees C (optimum: 60 degrees C; 2.4-h doubling time) and pH 6.0 and 9.0 (optimum: pH 7.5). The G+C content of the genomic DNA was 65 mol%, and the major quinones were MK-6 and MK-7. A phylogenetic analysis of its 16S rDNA sequence indicated that strain SC-1 is closely related to Symbiobacterium thermophilum and so was named Symbiobacterium toebii on the basis of its physiological and molecular properties.