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.).

Study of extracellular polymeric substances in the biofilms of a suspended biofilter for nitric oxide removal.

The extraction and quantitative analysis of extracellular polymeric substances (EPS) have been frequently reported in studies of activated sludge. However, little is currently known about the EPS in the biofilms of biofilter systems. This study investigates the EPS in biofilms of Chelatococcus daeguensis TAD1 established in a suspended biofilter for nitric oxide (NO) removal under thermophilic conditions. Polysaccharide was the main EPS component under all experimental operation conditions of the aerobic biofilter, although the EPS contents and components varied under different operating conditions. As the concentration of the inlet NO varied from 200 to 2000 mg/m3, the EPS and protein contents generally increased. At the highest inlet concentration (2000 mg/m3), the EPS and protein contents reached 0.118 and 0.055 mg/g, respectively (representing increases of 7.3 and 35 %, respectively, over the inlet concentration of 200 mg/m3). In contrast, the polysaccharide content was quite stable against inlet NO concentration. Decreasing the empty bed residence time increased the EPS and polysaccharide contents, but exerted little effect on the protein content. Varying the pH of the circulating fluid from 4 to 8 changed the EPS and its components in complex ways. We also found a strong correlation between the total EPS content and the NO removal efficiency. Therefore, it is possible to take EPS into consideration for biofilter control.

Methylorosula polaris gen. nov., sp. nov., an aerobic, facultatively methylotrophic psychrotolerant bacterium from tundra wetland soil.

Three strains of Gram-negative, aerobic, motile bacteria with bipolar flagella were isolated from acidic tundra wetland soils near the city of Vorkuta and from the Chukotka and Yugorsky Peninsulas and designated strains V-022(T), Ch-022 and Ju-022. The cells were rod-shaped, 0.5-0.6 µm in width and 1.3-4.5 µm in length and reproduced by irregular fission. These bacteria were facultative methylotrophs that used methanol, methylamines and a wide range of other sources of carbon and energy such as sugars and polysaccharides, ethanol and amino acids. The isolates used the Calvin-Benson pathway for the assimilation of one-carbon compounds and were unable to fix nitrogen. The new strains were moderately acidophilic and psychrotolerant, capable of growth over a pH range of 4.0 to 7.8, with optimum growth at pH 5.5-6.0. Growth occurred between 4 and 30 °C (optimum 20-25 °C). The principal phospholipid fatty acid was C(18:1)ω7c. The DNA G+C content of strain V-022(T) was 65.2 mol%. Analysis of the 16S rRNA gene sequences revealed that all three isolates V-022(T), Ch-022 and Yu-022 exhibited almost identical 16S rRNA gene sequences (99.9% gene sequence similarity) and formed a new lineage within the class Alphaproteobacteria. The name Methylorosula polaris is suggested to accommodate this new genus and novel species with strain V-022(T) (=DSM 22001(T)=VKM V-2485(T)) as the type strain of the type species.

Methyloferula stellata gen. nov., sp. nov., an acidophilic, obligately methanotrophic bacterium that possesses only a soluble methane monooxygenase.

Two strains of aerobic methanotrophic bacteria, AR4(T) and SOP9, were isolated from acidic (pH 3.8-4.0) Sphagnum peat bogs in Russia. Another phenotypically similar isolate, strain LAY, was obtained from an acidic (pH 4.0) forest soil in Germany. Cells of these strains were Gram-negative, non-pigmented, non-motile, thin rods that multiplied by irregular cell division and formed rosettes or amorphous cell conglomerates. Similar to Methylocella species, strains AR4(T), SOP9 and LAY possessed only a soluble form of methane monooxygenase (sMMO) and lacked intracytoplasmic membranes. Growth occurred only on methane and methanol; the latter was the preferred growth substrate. mRNA transcripts of sMMO were detectable in cells when either methane or both methane and methanol were available. Carbon was assimilated via the serine and ribulose-bisphosphate (RuBP) pathways; nitrogen was fixed via an oxygen-sensitive nitrogenase. Strains AR4(T), SOP9 and LAY were moderately acidophilic, mesophilic organisms capable of growth between pH 3.5 and 7.2 (optimum pH 4.8-5.2) and at 4-33 °C (optimum 20-23 °C). The major cellular fatty acid was 18 : 1ω7c and the quinone was Q-10. The DNA G+C content was 55.6-57.5 mol%. The isolates belonged to the family Beijerinckiaceae of the class Alphaproteobacteria and were most closely related to the sMMO-possessing methanotrophs of the genus Methylocella (96.4-97.0 % 16S rRNA gene sequence similarity), particulate MMO (pMMO)-possessing methanotrophs of the genus Methylocapsa (96.1-97.0 %), facultative methylotrophs of the genus Methylovirgula (96.1-96.3 %) and non-methanotrophic organotrophs of the genus Beijerinckia (96.5-97.0 %). Phenotypically, strains AR4(T), SOP9 and LAY were most similar to Methylocella species, but differed from members of this genus by cell morphology, greater tolerance of low pH, detectable activities of RuBP pathway enzymes and inability to grow on multicarbon compounds. Therefore, we propose a novel genus and species, Methyloferula stellata gen. nov., sp. nov., to accommodate strains AR4(T), SOP9 and LAY. Strain AR4(T) ( = DSM 22108(T)  = LMG 25277(T)  = VKM B-2543(T)) is the type strain of Methyloferula stellata.

Isolation and characterization of new poly(3HB)-accumulating star-shaped cell-aggregates-forming thermophilic bacteria.

AIMS: This study aimed at isolating thermophilic bacteria that utilize cheap carbon substrates for the economically feasible production of poly(3-hydroxybutyrate), poly(3HB), at elevated temperatures. METHODS AND RESULTS: Thermophilic bacteria were enriched from an aerobic organic waste treatment plant in Germany, and from hot springs in Egypt. Using the viable colony staining method for hydrophobic cellular inclusions with Nile red in mineral salts medium (MSM) containing different carbon sources, six Gram-negative bacteria were isolated. Under the cultivation conditions used in this study, strains MW9, MW11, MW12, MW13 and MW14 formed stable star-shaped cell-aggregates (SSCAs) during growth; only strain MW10 consisted of free-living rod-shaped cells. The phylogenetic relationships of the strains as derived from 16S rRNA gene sequence comparisons revealed them as members of the Alphaproteobacteria. The 16S rRNA gene sequences of the isolates were very similar (>99% similarity) and exhibited similarities ranging from 93 to 99% with the most closely related species that were Chelatococcus daeguensis, Chelatococcus sambhunathii,Chelatococcus asaccharovorans, Bosea minatitlanensis, Bosea thiooxidans and Methylobacterium lusitanum. Strains MW9, MW10, MW13 and MW14 grew optimally in MSM with glucose, whereas strains MW11 and MW12 preferred glycerol as sole carbon source for growth and poly(3HB) accumulation. The highest cell density and highest poly(3HB) content attained were 4·8g l(-l) (cell dry weight) and 73% (w/w), respectively. Cells of all strains grew at temperatures between 37 and 55°C with the optimum growth at 50°C. CONCLUSIONS: New PHA-accumulating thermophilic bacterial strains were isolated and characterized to produce poly(3HB) from glucose or glycerol in MSM at 50°C. SSCAs formation was reported during growth. SIGNIFICANCE AND IMPACT OF THE STUDY: To the best of our knowledge, this is the first report on the formation of SSCAs by PHA-accumulating bacteria and also by thermophilic bacteria. PHA-producing thermophiles can significantly reduce the costs of fermentative PHA production.

Chelatococcus sambhunathii sp. nov., a moderately thermophilic alphaproteobacterium isolated from hot spring sediment.

A moderately thermophilic bacterial strain (HT4(T)) isolated from a hot spring sediment was characterized phenotypically and phylogenetically. Cells were Gram-negative, aerobic, non-sporulating, rod-shaped and motile by means of a single polar flagellum. Both oxidase and catalase activities were positive. Heterotrophic growth was observed at pH 6.0-8.5 and at 20-50 degrees C; optimum growth occurred at pH 7.5-8.0 and 37-42 degrees C. The major cellular fatty acids were C( 14 : 0) 3-OH, C(18 : 0) 3-OH, C( 18 : 1) 2-OH, C(18 : 1)omega 7c and C(19 : 0) cyclo omega8 c. The DNA G+C content of strain HT4(T) was 67.8 mol%. 16S rRNA gene sequence analysis indicated that strain HT4(T) clustered within the radiation of the genus Chelatococcus and showed 99.0 % similarity with Chelatococcus daeguensis CCUG 54519(T) and 96 % similarity with Chelatococcus asaccharovorans DSM 6462(T). However, levels of DNA-DNA relatedness between strain HT4(T) and Chelatococcus daeguensis CCUG 54519(T) and Chelatococcus asaccharovorans DSM 6462(T) were 52 and 20%, respectively. On the basis of the phenotypic, physiological and chemotaxonomic data, 16S rRNA gene sequence analysis and DNA-DNA hybridization results, strain HT4(T) is considered to represent a novel species of the genus Chelatococcus, for which the name Chelatococcus sambhunathii sp. nov. is proposed. The type strain is HT4(T) (=DSM 18167(T)=JCM 14988(T)).

Chelatococcus daeguensis sp. nov., isolated from wastewater of a textile dye works, and emended description of the genus Chelatococcus.

A Gram-negative, non-spore-forming, rod-shaped bacterial strain, K106(T), was isolated from wastewater collected from a textile dye works in Korea. Strain K106(T) grew optimally at pH 7.0-7.5 and 30-37 degrees C in the presence of 0-1.0% (w/v) NaCl. A phylogenetic tree based on 16S rRNA gene sequences showed that strain K106(T) joined the type strain of Chelatococcus asaccharovorans with a bootstrap resampling value of 99.9%. The predominant ubiquinone of strain K106(T) was Q-10. The fatty acid profile of strain K106(T) was similar to that of C. asaccharovorans DSM 6462(T). Major polar lipids of strain K106(T) and C. asaccharovorans DSM 6462(T) were phosphatidylcholine, phosphatidylglycerol, phosphatidylethanolamine, two aminolipids and two unidentified phospholipids. sym-Homospermidine, spermidine and putrescine were major polyamines. The DNA G+C content was 68.3 mol%. Strain K106(T) exhibited 16S rRNA gene sequence similarity of 96.6% to the type strain of C. asaccharovorans. DNA-DNA relatedness data and differential phenotypic properties, particularly differences in cell morphology and the ability to utilize nitrilotriacetate, demonstrated that strain K106(T) can be differentiated from C. asaccharovorans. On the basis of phenotypic, phylogenetic and genetic data, strain K106(T) represents a novel species of the genus Chelatococcus, for which the name Chelatococcus daeguensis sp. nov. is proposed. The type strain is K106(T) (=KCTC 12979(T) =CCUG 54519(T)).