Refining the taxonomy of the order Hyphomicrobiales (Rhizobiales) based on whole genome comparisons of over 130 type strains.

The alphaproteobacterial order Hyphomicrobiales consists of 38 families comprising at least 152 validly published genera as of January 2024. The order Hyphomicrobiales was first described in 1957 and underwent important revisions in 2020. However, we show that several inconsistencies in the taxonomy of this order remain and we argue that there is a need for a consistent framework for defining families within the order. We propose a common genome-based framework for defining families within the order Hyphomicrobiales, suggesting that families represent monophyletic groups in core-genome phylogenies that share pairwise average amino acid identity values above ~75 % when calculated from a core set of 59 proteins. Applying this framework, we propose the formation of four new families and to reassign the genera Salaquimonas, Rhodoblastus, and Rhodoligotrophos into Salaquimonadaceae fam. nov., Rhodoblastaceae fam. nov., and Rhodoligotrophaceae fam. nov., respectively, and the genera Albibacter, Chenggangzhangella, Hansschlegelia, and Methylopila into Methylopilaceae fam. nov. We further propose to unify the families Bartonellaceae, Brucellaceae, Phyllobacteriaceae, and Notoacmeibacteraceae as Bartonellaceae; the families Segnochrobactraceae and Pseudoxanthobacteraceae as Segnochrobactraceae; the families Lichenihabitantaceae and Lichenibacteriaceae as Lichenihabitantaceae; and the families Breoghaniaceae and Stappiaceae as Stappiaceae. Lastly, we propose to reassign several genera to existing families. Specifically, we propose to reassign the genus Pseudohoeflea to the family Rhizobiaceae; the genera Oricola, Roseitalea, and Oceaniradius to the family Ahrensiaceae; the genus Limoniibacter to the emended family Bartonellaceae; the genus Faunimonas to the family Afifellaceae; and the genus Pseudochelatococcus to the family Chelatococcaceae. Our data also support the recent proposal to reassign the genus Prosthecomicrobium to the family Kaistiaceae.

The effect of lanthanum on growth and gene expression in a facultative methanotroph.

The biological importance of lanthanides has only recently been identified, initially as the active site metal of the alternative methanol dehydrogenase (MDH) Xox-MDH. So far, the effect of lanthanide (Ln) has only been studied in relatively few organisms. This work investigated the effects of Ln on gene transcription and protein expression in the facultative methanotroph Methylocella silvestris BL2, a widely distributed methane-oxidizing bacterium with the unique ability to grow not just on methane but also on other typical components of natural gas, ethane and propane. Expression of calcium- or Ln-dependent MDH was controlled by Ln (the lanthanide switch) during growth on one-, two- or three-carbon substrates, and Ln imparted a considerable advantage during growth on propane, a novel result extending the importance of Ln to consumers of this component of natural gas. Two Xox-MDHs were expressed and regulated by Ln in M. silvestris, but interestingly Ln repressed rather than induced expression of the second Xox-MDH. Despite the metabolic versatility of M. silvestris, no other alcohol dehydrogenases were expressed, and in double-mutant strains lacking genes encoding both Ca- and Ln-dependent MDHs (mxaF and xoxF5 or xoxF1), growth on methanol and ethanol appeared to be enabled by expression of the soluble methane monooxygenase.

Extracellular and Intracellular Lanthanide Accumulation in the Methylotrophic Beijerinckiaceae Bacterium RH AL1.

Recent work with Methylorubrum extorquens AM1 identified intracellular, cytoplasmic lanthanide storage in an organism that harnesses these metals for its metabolism. Here, we describe the extracellular and intracellular accumulation of lanthanides in the Beijerinckiaceae bacterium RH AL1, a newly isolated and recently characterized methylotroph. Using ultrathin-section transmission electron microscopy (TEM), freeze fracture TEM (FFTEM), and energy-dispersive X-ray spectroscopy, we demonstrated that strain RH AL1 accumulates lanthanides extracellularly at outer membrane vesicles (OMVs) and stores them in the periplasm. High-resolution elemental analyses of biomass samples revealed that strain RH AL1 can accumulate ions of different lanthanide species, with a preference for heavier lanthanides. Its methanol oxidation machinery is supposedly adapted to light lanthanides, and their selective uptake is mediated by dedicated uptake mechanisms. Based on transcriptome sequencing (RNA-seq) analysis, these presumably include the previously characterized TonB-ABC transport system encoded by the lut cluster but potentially also a type VI secretion system. A high level of constitutive expression of genes coding for lanthanide-dependent enzymes suggested that strain RH AL1 maintains a stable transcript pool to flexibly respond to changing lanthanide availability. Genes coding for lanthanide-dependent enzymes are broadly distributed taxonomically. Our results support the hypothesis that central aspects of lanthanide-dependent metabolism partially differ between the various taxa. IMPORTANCE Although multiple pieces of evidence have been added to the puzzle of lanthanide-dependent metabolism, we are still far from understanding the physiological role of lanthanides. Given how widespread lanthanide-dependent enzymes are, only limited information is available with respect to how lanthanides are taken up and stored in an organism. Our research complements work with commonly studied model organisms and showed the localized storage of lanthanides in the periplasm. This storage occurred at comparably low concentrations. Strain RH AL1 is able to accumulate lanthanide ions extracellularly and to selectively utilize lighter lanthanides. The Beijerinckiaceae bacterium RH AL1 might be an attractive target for developing biorecovery strategies to obtain these economically highly demanded metals in environmentally friendly ways.

Linking prokaryotic community composition to carbon biogeochemical cycling across a tropical peat dome in Sarawak, Malaysia.

Tropical peat swamp forest is a global store of carbon in a water-saturated, anoxic and acidic environment. This ecosystem holds diverse prokaryotic communities that play a major role in nutrient cycling. A study was conducted in which a total of 24 peat soil samples were collected in three forest types in a tropical peat dome in Sarawak, Malaysia namely, Mixed Peat Swamp (MPS), Alan Batu (ABt), and Alan Bunga (ABg) forests to profile the soil prokaryotic communities through meta 16S amplicon analysis using Illumina Miseq. Results showed these ecosystems were dominated by anaerobes and fermenters such as Acidobacteria, Proteobacteria, Actinobacteria and Firmicutes that cover 80-90% of the total prokaryotic abundance. Overall, the microbial community composition was different amongst forest types and depths. Additionally, this study highlighted the prokaryotic communities' composition in MPS was driven by higher humification level and lower pH whereas in ABt and ABg, the less acidic condition and higher organic matter content were the main factors. It was also observed that prokaryotic diversity and abundance were higher in the more oligotrophic ABt and ABg forest despite the constantly waterlogged condition. In MPS, the methanotroph Methylovirgula ligni was found to be the major species in this forest type that utilize methane (CH4), which could potentially be the contributing factor to the low CH4 gas emissions. Aquitalea magnusonii and Paraburkholderia oxyphila, which can degrade aromatic compounds, were the major species in ABt and ABg forests respectively. This information can be advantageous for future study in understanding the underlying mechanisms of environmental-driven alterations in soil microbial communities and its potential implications on biogeochemical processes in relation to peatland management.

Genome Scale Metabolic Model of the versatile methanotroph Methylocella silvestris.

BACKGROUND: Methylocella silvestris is a facultative aerobic methanotrophic bacterium which uses not only methane, but also other alkanes such as ethane and propane, as carbon and energy sources. Its high metabolic versatility, together with the availability of tools for its genetic engineering, make it a very promising platform for metabolic engineering and industrial biotechnology using natural gas as substrate. RESULTS: The first Genome Scale Metabolic Model for M. silvestris is presented. The model has been used to predict the ability of M. silvestris to grow on 12 different substrates, the growth phenotype of two deletion mutants (ΔICL and ΔMS), and biomass yield on methane and ethanol. The model, together with phenotypic characterization of the deletion mutants, revealed that M. silvestris uses the glyoxylate shuttle for the assimilation of C1 and C2 substrates, which is unique in contrast to published reports of other methanotrophs. Two alternative pathways for propane metabolism have been identified and validated experimentally using enzyme activity tests and constructing a deletion mutant (Δ1641), which enabled the identification of acetol as one of the intermediates of propane assimilation via 2-propanol. The model was also used to integrate proteomic data and to identify key enzymes responsible for the adaptation of M. silvestris to different substrates. CONCLUSIONS: The model has been used to elucidate key metabolic features of M. silvestris, such as its use of the glyoxylate shuttle for the assimilation of one and two carbon compounds and the existence of two parallel metabolic pathways for propane assimilation. This model, together with the fact that tools for its genetic engineering already exist, paves the way for the use of M. silvestris as a platform for metabolic engineering and industrial exploitation of methanotrophs.

Lanthanide-Dependent Methylotrophs of the Family Beijerinckiaceae: Physiological and Genomic Insights.

Methylotrophic bacteria use methanol and related C1 compounds as carbon and energy sources. Methanol dehydrogenases are essential for methanol oxidation, while lanthanides are important cofactors of many pyrroloquinoline quinone-dependent methanol dehydrogenases and related alcohol dehydrogenases. We describe here the physiological and genomic characterization of newly isolated Beijerinckiaceae bacteria that rely on lanthanides for methanol oxidation. A broad physiological diversity was indicated by the ability to metabolize a wide range of multicarbon substrates, including various sugars, and organic acids, as well as diverse C1 substrates such as methylated amines and methylated sulfur compounds. Methanol oxidation was possible only in the presence of low-mass lanthanides (La, Ce, and Nd) at submicromolar concentrations (>100 nM). In a comparison with other Beijerinckiaceae, genomic and transcriptomic analyses revealed the usage of a glutathione- and tetrahydrofolate-dependent pathway for formaldehyde oxidation and channeling methyl groups into the serine cycle for carbon assimilation. Besides a single xoxF gene, we identified two additional genes for lanthanide-dependent alcohol dehydrogenases, including one coding for an ExaF-type alcohol dehydrogenase, which was so far not known in Beijerinckiaceae Homologs for most of the gene products of the recently postulated gene cluster linked to lanthanide utilization and transport could be detected, but for now it remains unanswered how lanthanides are sensed and taken up by our strains. Studying physiological responses to lanthanides under nonmethylotrophic conditions in these isolates as well as other organisms is necessary to gain a more complete understanding of lanthanide-dependent metabolism as a whole.IMPORTANCE We supplemented knowledge of the broad metabolic diversity of the Beijerinckiaceae by characterizing new members of this family that rely on lanthanides for methanol oxidation and that possess additional lanthanide-dependent enzymes. Considering that lanthanides are critical resources for many modern applications and that recovering them is expensive and puts a heavy burden on the environment, lanthanide-dependent metabolism in microorganisms is an exploding field of research. Further research into how isolated Beijerinckiaceae and other microbes utilize lanthanides is needed to increase our understanding of lanthanide-dependent metabolism. The diversity and widespread occurrence of lanthanide-dependent enzymes make it likely that lanthanide utilization varies in different taxonomic groups and is dependent on the habitat of the microbes.

Widespread soil bacterium that oxidizes atmospheric methane.

The global atmospheric level of methane (CH4), the second most important greenhouse gas, is currently increasing by ∼10 million tons per year. Microbial oxidation in unsaturated soils is the only known biological process that removes CH4 from the atmosphere, but so far, bacteria that can grow on atmospheric CH4 have eluded all cultivation efforts. In this study, we have isolated a pure culture of a bacterium, strain MG08 that grows on air at atmospheric concentrations of CH4 [1.86 parts per million volume (p.p.m.v.)]. This organism, named Methylocapsa gorgona, is globally distributed in soils and closely related to uncultured members of the upland soil cluster α. CH4 oxidation experiments and 13C-single cell isotope analyses demonstrated that it oxidizes atmospheric CH4 aerobically and assimilates carbon from both CH4 and CO2 Its estimated specific affinity for CH4 (a0s) is the highest for any cultivated methanotroph. However, growth on ambient air was also confirmed for Methylocapsa acidiphila and Methylocapsa aurea, close relatives with a lower specific affinity for CH4, suggesting that the ability to utilize atmospheric CH4 for growth is more widespread than previously believed. The closed genome of M. gorgona MG08 encodes a single particulate methane monooxygenase, the serine cycle for assimilation of carbon from CH4 and CO2, and CO2 fixation via the recently postulated reductive glycine pathway. It also fixes dinitrogen and expresses the genes for a high-affinity hydrogenase and carbon monoxide dehydrogenase, suggesting that atmospheric CH4 oxidizers harvest additional energy from oxidation of the atmospheric trace gases carbon monoxide (0.2 p.p.m.v.) and hydrogen (0.5 p.p.m.v.).

Facultative methanotrophs are abundant at terrestrial natural gas seeps.

BACKGROUND: Natural gas contains methane and the gaseous alkanes ethane, propane and butane, which collectively influence atmospheric chemistry and cause global warming. Methane-oxidising bacteria, methanotrophs, are crucial in mitigating emissions of methane as they oxidise most of the methane produced in soils and the subsurface before it reaches the atmosphere. Methanotrophs are usually obligate, i.e. grow only on methane and not on longer chain alkanes. Bacteria that grow on the other gaseous alkanes in natural gas such as propane have also been characterised, but they do not grow on methane. Recently, it was shown that the facultative methanotroph Methylocella silvestris grew on ethane and propane, other components of natural gas, in addition to methane. Therefore, we hypothesised that Methylocella may be prevalent at natural gas seeps and might play a major role in consuming all components of this potent greenhouse gas mixture before it is released to the atmosphere. RESULTS: Environments known to be exposed to biogenic methane emissions or thermogenic natural gas seeps were surveyed for methanotrophs. 16S rRNA gene amplicon sequencing revealed that Methylocella were the most abundant methanotrophs in natural gas seep environments. New Methylocella-specific molecular tools targeting mmoX (encoding the soluble methane monooxygenase) by PCR and Illumina amplicon sequencing were designed and used to investigate various sites. Functional gene-based assays confirmed that Methylocella were present in all of the natural gas seep sites tested here. This might be due to its ability to use methane and other short chain alkane components of natural gas. We also observed the abundance of Methylocella in other environments exposed to biogenic methane, suggesting that Methylocella has been overlooked in the past as previous ecological studies of methanotrophs often used pmoA (encoding the alpha subunit of particulate methane monooxygenase) as a marker gene. CONCLUSION: New biomolecular tools designed in this study have expanded our ability to detect, and our knowledge of the environmental distribution of Methylocella, a unique facultative methanotroph. This study has revealed that Methylocella are particularly abundant at natural gas seeps and may play a significant role in biogeochemical cycling of gaseous hydrocarbons.

Qingshengfania soli Zhang et al. 2015 is a later heterotypic synonym of Pseudochelatococcus lubricantis Kämpfer et al. 2015.

Qingshengfania soli DSM 103870T was compared with Pseudochelatococcus lubricantis MPA 1113T to clarify the taxonomic relationship of both species because of their high phylogenetic relationship. 16S rRNA gene sequence comparisons demonstrated that these species share 100  % sequence similarity. Investigation of fatty acid patterns, substrate utilization, and matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) profiles displayed no striking differences between the type strains of both species. DNA-DNA hybridization between both strains showed a 95 % (reciprocal 82 %) similarity, which clearly demonstrated that both strains are members of the same species. Due to priority of publication and validation of the name, Qingshengfania soli is reclassified as Pseudochelatococcus lubricantis, based on the estimated phylogenetic position derived from 16S rRNA gene sequence data, fatty acid, biochemical data, MALDI-TOF, and DNA-DNA hybridization results.

Chelatococcus composti sp. nov., isolated from penicillin fermentation fungi residue with pig manure co-compost.

A novel Gram-stain-negative bacterium, designated strain PC-2T, was isolated from penicillin fermentation fungi residue with pig manure co-compost in China. Phylogenetic analysis, based on 16S rRNA gene sequence comparisons, revealed that strain PC-2T should be assigned to the genus Chelatococcus and that it had 98.9 % similarity with Chelatococcus daeguensis, 98.8 % with Chelatococcus sambhunathii, 98.4 %, with Chelatococcus caeni and 96.0 % with Chelatococcus asaccharovorans. The G+C content of genomic DNA was 70.9 mol%. On the basis of the phylogenetic analysis, DNA-DNA relatedness values, phenotypic characteristics and chemotaxonomic data, strain PC-2 T represents a novel species of the genus Chelatococcus, for which the name Chelatococcus composti sp. nov. is proposed. The type strain is PC-2T (=DSM 101465T=CGMCC 1.15283T).

O2 -independent demethylation of trimethylamine N-oxide by Tdm of Methylocella silvestris.

Bacterial trimethylamine N-oxide (TMAO) demethylase, Tdm, carries out an unusual oxygen-independent demethylation reaction, resulting in the formation of dimethylamine and formaldehyde. In this study, site-directed mutagenesis, homology modelling and metal analyses by inorganic mass spectrometry have been applied to gain insight into metal stoichiometry and underlying catalytic mechanism of Tdm of Methylocella silvestris BL2. Herein, we demonstrate that active Tdm has 1 molar equivalent of Zn2+ and 1 molar equivalent of non-haem Fe2+ . We further investigated Zn2+ - and Fe2+ -binding sites through homology modelling and site-directed mutagenesis and found that Zn2+ is coordinated by a 3-sulfur-1-O motif. An aspartate residue (D198) likely bridges Fe2+ and Zn2+ centres, either directly or indirectly via H-bonding through a neighbouring H2 O molecule. H276 contributes to Fe2+ binding, mutation of which results in an inactive enzyme, and the loss of iron, but not zinc. Site-directed mutagenesis of Tdm also led to the identification of three hydrophobic aromatic residues likely involved in substrate coordination (F259, Y305, W321), potentially through a cation-π interaction. Furthermore, a crossover experiment using a substrate analogue gave direct evidence that a trimethylamine-alike intermediate was produced during the Tdm catalytic cycle, suggesting TMAO has a dual role of being both a substrate and an oxygen donor for formaldehyde formation. Together, our results provide novel insight into the role of Zn2+ and Fe2+ in the catalysis of TMAO demethylation by this unique oxygen-independent enzyme.

Chelatococcus reniformis sp. nov., isolated from a glacier.

A Gram-stain-negative, non-motile, reniform bacterial strain, B2974T, was isolated from an ice core of the Muztagh Glacier, on the Tibetan Plateau, China. Strain B2974T grew optimally at pH 7.0-7.5 and 25-30 °C in the presence of 0-2.0 % (w/v) NaCl. 16S rRNA gene sequence similarity analysis indicated that strain B2974T was closely related to Chelatococcus asaccharovorans LMG 25503T at a level of 97.1 %. The major quinone of strain B2974T was ubiquinone Q10. The predominant fatty acids were summed feature 8 (C18 : 1ω7c and/or C18 : 1ω6c) and C19 : 0 cyclo ω8c. sym-Homospermidine was the major polyamine. The genomic DNA G+C content of the strain was 64 mol%. In DNA-DNA hybridization tests, strain B2974T shared 49.32 % DNA-DNA relatedness with the type strain of Chelatococcus asaccharovorans LMG 25503T. Based on the results of phenotypic and chemotaxonomic characteristics, strain B2974T was considered as a novel species of the genus Chelatococcus, for which the name Chelatococcus reniformis sp. nov. is proposed. The type strain is B2974T (=JCM 30308T=CGMCC 1.12919T).

Isolation and identification of biocellulose-producing bacterial strains from Malaysian acidic fruits.

UNLABELLED: Biocellulose (BC) is pure extracellular cellulose produced by several species of micro-organisms that has numerous applications in the food, biomedical and paper industries. However, the existing biocellulose-producing bacterial strain with high yield was limited. The aim of this study was to isolate and identify the potential biocellulose-producing bacterial isolates from Malaysian acidic fruits. One hundred and ninety-three bacterial isolates were obtained from 19 local acidic fruits collected in Malaysia and screened for their ability to produce BC. A total of 15 potential bacterial isolates were then cultured in standard Hestrin-Schramm (HS) medium statically at 30°C for 2 weeks to determine the BC production. The most potent bacterial isolates were identified using 16S rRNA gene sequence analysis, morphological and biochemical characteristics. Three new and potent biocellulose-producing bacterial strains were isolated from soursop fruit and identified as Stenotrophomonas maltophilia WAUPM42, Pantoea vagans WAUPM45 and Beijerinckia fluminensis WAUPM53. Stenotrophomonas maltophilia WAUPM42 was the most potent biocellulose-producing bacterial strain that produced the highest amount of BC 0·58 g l(-1) in standard HS medium. Whereas, the isolates P. vagans WAUPM45 and B. fluminensis WAUPM53 showed 0·50 and 0·52 g l(-1) of BC production, respectively. SIGNIFICANCE AND IMPACT OF THE STUDY: Biocellulose (BC) is pure extracellular cellulose that is formed by many micro-organisms in the presence of carbon source and acidic condition. It can replace plant-based cellulose in multifarious applications due to its unique characteristics. In this study, three potential biocellulose-producing bacterial strains were obtained from Malaysian acidic fruits and identified as Stenotrophomonas maltophilia WAUPM42, Pantoea vagans WAUPM45 and Beijerinckia fluminensis WAUPM53. This study reports for the first time the new biocellulose-producing bacterial strains isolated from Malaysian acidic fruits.

Methylocapsa palsarum sp. nov., a methanotroph isolated from a subArctic discontinuous permafrost ecosystem.

An aerobic methanotrophic bacterium was isolated from a collapsed palsa soil in northern Norway and designated strain NE2T. Cells of this strain were Gram-stain-negative, non-motile, non-pigmented, slightly curved thick rods that multiplied by normal cell division. The cells possessed a particulate methane monooxygenase enzyme (pMMO) and utilized methane and methanol. Strain NE2T grew in a wide pH range of 4.1­8.0 (optimum pH 5.2­6.5) at temperatures between 6 and 32 °C (optimum 18­25 °C), and was capable of atmospheric nitrogen fixation under reduced oxygen tension. The major cellular fatty acids were C18 : 1ω7c, C16 : 0 and C16 : 1ω7c, and the DNA G+C content was 61.7 mol%. The isolate belonged to the family Beijerinckiaceae of the class Alphaproteobacteria and was most closely related to the facultative methanotroph Methylocapsa aurea KYGT (98.3 % 16S rRNA gene sequence similarity and 84 % PmoA sequence identity). However, strain NE2T differed from Methylocapsa aurea KYGT by cell morphology, the absence of pigmentation, inability to grow on acetate, broader pH growth range, and higher tolerance to NaCl. Therefore, strain NE2T represents a novel species of the genus Methylocapsa, for which we propose the name Methylocapsa palsarum sp. nov. The type strain is NE2T ( = LMG 28715T = VKM B-2945T).

Pseudochelatococcus lubricantis gen. nov., sp. nov. and Pseudochelatococcus contaminans sp. nov. from coolant lubricants.

Two Gram-negative, rod-shaped, non-spore-forming bacteria, isolated from metal working fluids were investigated to determine their taxonomic positions. On the basis of 16S rRNA gene sequence phylogeny, both strains (MPA 1113(T) and MPA 1105(T)) formed a distinct cluster with 97.7 % sequence similarity between them, which was in the vicinity of members of the genera Methylobacterium, Camelimonas, Chelatococcus, Bosea, Salinarimonas and Microvirga to which they showed low sequence similarities (below 94 %). The predominant compounds in the polyamine pattern and in the quinone system of the two strains were spermidine and ubiquinone Q-10, respectively. The polar lipid profiles were composed of the major compounds: phosphatidylmonomethylethanolamine, phosphatidylglycerol, phosphatidylcholine, major or moderate amounts of diphosphatidylglycerol, two unidentified glycolipids and three unidentified aminolipids. Several minor lipids were also detected. The major fatty acids were either C19 : 0 cyclo ω8c or C18 : 1ω7c. The results of fatty acid analysis and physiological and biochemical tests allowed both, the genotypic and phenotypic differentiation of the isolates from each other, while the chemotaxonomic traits allowed them to be differentiated from the most closely related genera. In summary, low 16S rRNA gene sequence similarities and marked differences in polar lipid profiles, as well as in polyamine patterns, is suggestive of a novel genus for which the name Pseudochelatococcus gen. nov. is proposed. MPA 1113(T) ( = CCM 8528(T) = LMG 28286(T) = CIP 110802(T)) and MPA 1105(T) ( = CCM 8527(T) = LMG 28285(T)) are proposed to be the type strains representing two novel species within the novel genus, Pseudochelatococcus gen. nov., for which the names Pseudochelatococcus lubricantis sp. nov. and Pseudochelatococcus contaminans sp. nov. are suggested, respectively.

Chelatococcus caeni sp. nov., isolated from a biofilm reactor sludge sample.

A polyphasic taxonomic study was carried out on strain EBR-4-1(T), which was isolated from a biofilm reactor in the Republic of Korea. The cells of the strain were Gram-stain-negative, non-spore-forming, motile and rod-shaped. Comparative 16S rRNA gene sequence studies showed a clear affiliation of this strain to the Alphaproteobacteria, and it was most closely related to Chelatococcus daeguensis CCUG 54519(T), Chelatococcus sambhunathii HT4(T), and Chelatococcus asaccharovorans DSM 6462(T) with 16S rRNA gene sequence similarities to the type strains of these species of 98.8 %, 98.7 %, and 96.3 %, respectively. The G+C content of the genomic DNA of strain EBR-4-1(T) was 68.7 mol%. Phenotypic and chemotaxonomic data [Q-10 as the major ubiquinone; C19 : 0cycloω8c, C18 : 1 2-OH, and summed feature 8 (C18 : 1ω7c and/or C18 : 1ω6c) as the major fatty acids] supported the affiliation of strain EBR-4-1(T) to the genus Chelatococcus. On the basis of the polyphasic evidence, it is proposed that strain EBR-4-1(T) should be assigned to a new species, Chelatococcus caeni sp. nov. The type strain is EBR-4-1(T) ( = KCTC 32487(T) = JCM 30181(T)).

Isolation and characterization of two novel strains capable of using cyclohexane as carbon source.

Two strains capable of degrading cyclohexane were isolated from the soil and sludge of the wastewater treatment plant of the University of Stuttgart and a biotrickling filter system. The strains were classified as gram negative and identified as Acidovorax sp. CHX100 and Chelatococcus sp. CHX1100. Both strains have demonstrated the capability to degrade cycloalkanes (C5-C8), while only strain CHX1100 used as well short linear n-alkanes (C5-C8) as the sole source of carbon and energy. The growth of Acidovorax sp. CHX100 using cyclohexane was much faster compared to Chelatococcus sp. CHX1100. Degenerated primers were optimized from a set sequences of cyclohexanol dehydrogenase genes (chnA) as well as cyclohexanone monooxygenases (chnB) and used to amplify the gene cluster, which encodes the conversion of cyclohexanol to caprolactone. Phylogenetic analysis has indicated that the two gene clusters belong to different groups. The cyclohexane monooxygenase-induced activity which oxidizes also indole to 5-hydroxyindole has indicated the presence of a CYP-type system monooxygenase involved in the transformation of cyclohexane to cyclohexanol.

Trace-gas metabolic versatility of the facultative methanotroph Methylocella silvestris.

The climate-active gas methane is generated both by biological processes and by thermogenic decomposition of fossil organic material, which forms methane and short-chain alkanes, principally ethane, propane and butane. In addition to natural sources, environments are exposed to anthropogenic inputs of all these gases from oil and gas extraction and distribution. The gases provide carbon and/or energy for a diverse range of microorganisms that can metabolize them in both anoxic and oxic zones. Aerobic methanotrophs, which can assimilate methane, have been considered to be entirely distinct from utilizers of short-chain alkanes, and studies of environments exposed to mixtures of methane and multi-carbon alkanes have assumed that disparate groups of microorganisms are responsible for the metabolism of these gases. Here we describe the mechanism by which a single bacterial strain, Methylocella silvestris, can use methane or propane as a carbon and energy source, documenting a methanotroph that can utilize a short-chain alkane as an alternative to methane. Furthermore, during growth on a mixture of these gases, efficient consumption of both gases occurred at the same time. Two soluble di-iron centre monooxygenase (SDIMO) gene clusters were identified and were found to be differentially expressed during bacterial growth on these gases, although both were required for efficient propane utilization. This report of a methanotroph expressing an additional SDIMO that seems to be uniquely involved in short-chain alkane metabolism suggests that such metabolic flexibility may be important in many environments where methane and short-chain alkanes co-occur.

The (d)evolution of methanotrophy in the Beijerinckiaceae--a comparative genomics analysis.

The alphaproteobacterial family Beijerinckiaceae contains generalists that grow on a wide range of substrates, and specialists that grow only on methane and methanol. We investigated the evolution of this family by comparing the genomes of the generalist organotroph Beijerinckia indica, the facultative methanotroph Methylocella silvestris and the obligate methanotroph Methylocapsa acidiphila. Highly resolved phylogenetic construction based on universally conserved genes demonstrated that the Beijerinckiaceae forms a monophyletic cluster with the Methylocystaceae, the only other family of alphaproteobacterial methanotrophs. Phylogenetic analyses also demonstrated a vertical inheritance pattern of methanotrophy and methylotrophy genes within these families. Conversely, many lateral gene transfer (LGT) events were detected for genes encoding carbohydrate transport and metabolism, energy production and conversion, and transcriptional regulation in the genome of B. indica, suggesting that it has recently acquired these genes. A key difference between the generalist B. indica and its specialist methanotrophic relatives was an abundance of transporter elements, particularly periplasmic-binding proteins and major facilitator transporters. The most parsimonious scenario for the evolution of methanotrophy in the Alphaproteobacteria is that it occurred only once, when a methylotroph acquired methane monooxygenases (MMOs) via LGT. This was supported by a compositional analysis suggesting that all MMOs in Alphaproteobacteria methanotrophs are foreign in origin. Some members of the Beijerinckiaceae subsequently lost methanotrophic functions and regained the ability to grow on multicarbon energy substrates. We conclude that B. indica is a recidivist multitroph, the only known example of a bacterium having completely abandoned an evolved lifestyle of specialized methanotrophy.

Removal of nitric oxide in a biotrickling filter under thermophilic condition using Chelatococcus daeguensis.

UNLABELLED: The development of a thermophilic biotrickling filter (BTF) system to inoculate a newly isolated strain of Chelatococcus daeguensis TAD1 for the effective treatment of nitric oxide (NO) is described. A bench-scale BTF was run under high concentrations of NO and 8% O2 in thermophilic aerobic environment. A novel aerobic denitrifier Chelatococcus daeguensis TAD1 was isolated from the biofilm of an on-site biotrickling filter and it showed a denitrifying capability of 96.1% nitrate removal rate in a 24 h period in aerobic environment at 50 degrees C, with no nitrite accumulation. The inlet NO concentration fluctuated between approximately 133.9 and 669.6 mg m-3 and kept on a steady NOx removal rate above 80% in an oxygen stream of 8%. The BTF system was able to consistently remove 80-93.7% NO when the inlet NO was 535.7 mg m-3 in an oxygen stream of 2-20%. The biological removal efficiency of NO at 50 degrees C is higher than that at 25 degrees C, suggesting that the aerobic denitrifier TAD1 display well denitrification performance under thermophilic condition. Starvation for 2, 4 and 8 days resulted in the re-acclimation times of Chelatococcus daeguensis TAD1 ranging between 4 and 16 hours. A longer recovery time than that for weekend shutdown will be required when a longer starvation occurs. The results presented here demonstrate the feasibility of biotrickling filter for the thermophilic removal of NOx from gas streams. IMPLICATIONS: A novel denitrifier Chelatococcus daeguensis TAD1 was isolated from an on-site biotrickling filter in aerobic environment at 50 degrees C. To date, C. daeguensis has not been previously reported to be an aerobic denitrifier. In this study, a thermophilic biotrickling filter system inoculated with Chelatococcus daeguensis TADI for treatment of nitric oxide is developed. In coal-fired power plants, influent flue gas stream for nitrogen oxides (NOx) removal typically exhibit temperatures between 50 and 60 degrees C. Traditionally, cooling gases to below 40 degrees C prior to biological treatment is inevitable, which is costly. Therefore, the application ofthermophilic microorganisms for the removal of nitric oxide (NO) at this temperature range would offer great savings and would greatly extend the applicability ofbiofilters and biotrickling filters. Until now there has not been any study published about thermophilic biological treatment of NO under aerobic condition.