Complete genome sequence of Sphingobium sp. strain PAMC 28499 reveals a potential for degrading pectin with comparative genomics approach.

BACKGROUND: Spingobium sp. PAMC 28499 is isolated from the glaciers of Uganda. Uganda is a unique region where hot areas and glaciers coexist, with a variety of living creatures surviving, but the survey on them is very poor. The genetic character and complete genome information of Sphingobium strains help with environmental studies and the development of better to enzyme industry. OBJECTIVE: In this study, complete genome sequence of Spingobium sp. PAMC 28499 and comparative analysis of Spingobium species strains isolated from variety of the region. METHODS: Genome sequencing was performed using PacBio sequel single-molecule real-time (SMRT) sequencing technology. The predicted gene sequences were functionally annotated and gene prediction was carried out using the program NCBI non-redundant database. And using dbCAN2 and KEGG data base were degradation pathway predicted and protein prediction about carbohydrate active enzymes (CAZymes). RESULTS: The genome sequence has 64.5% GC content, 4432 coding protein coding genes, 61 tRNAs, and 12 rRNA operons. Its genome encodes a simple set of metabolic pathways relevant to pectin and its predicted degradation protein an unusual distribution of CAZymes with extracellular esterases and pectate lyases. CAZyme annotation analyses revealed 165 genes related to carbohydrate active, and especially we have found GH1, GH2, GH3, GH38, GH35, GH51, GH51, GH53, GH106, GH146, CE12, PL1 and PL11 such as known pectin degradation genes from Sphingobium yanoikuiae. These results confirmed that this Sphingobium sp. strain PAMC 28499 have similar patterns to RG I pectin-degrading pathway. CONCLUSION: In this study, isolated and sequenced the complete genome of Spingobium sp. PAMC 28499. Also, this strain has comparative genome analysis. Through the complete genome we can predict how this strain can store and produce energy in extreme environment. It can also provide bioengineered data by finding new genes that degradation the pectin.

Development of the production of 2-pyrone-4,6-dicarboxylic acid from lignin extracts, which are industrially formed as by-products, as raw materials.

Lignosulfonate is a by-product of the cooking process by sulfite pulping for paper manufacturing. The treatment of wood chips by various salts of sulfurous acid solubilizes lignin to produce a cellulose-rich wood pulp. Developing a technique for the conversion of lignosulfonate by-product to high value materials has an important industrial utility. Sphingobium sp. strain SYK-6, which was isolated from pulping wastewater, is one of the best enzymatically or genetically characterized bacteria for degrading lignin-derived aromatics. We have previously established a system for the production of 2-pyrone-4,6-dicarboxylic acid (PDC), a novel platform chemical that can produce a variety of bio-based polymers, by introducing of ligA, ligB, and ligC genes from SYK-6 into a mutant strain of Pseudomonas putida PpY1100. In this study, extracts from lignosulfonates, which were desulphonated and depolymerized by alkaline oxidation, were evaluated as substrates for microbiological conversion to PDC by the transgenic bacteria.

Biodegradation of Tetralin: Genomics, Gene Function and Regulation.

Tetralin (1,2,3,4-tetrahydonaphthalene) is a recalcitrant compound that consists of an aromatic and an alicyclic ring. It is found in crude oils, produced industrially from naphthalene or anthracene, and widely used as an organic solvent. Its toxicity is due to the alteration of biological membranes by its hydrophobic character and to the formation of toxic hydroperoxides. Two unrelated bacteria, Sphingopyxis granuli strain TFA and Rhodococcus sp. strain TFB were isolated from the same niche as able to grow on tetralin as the sole source of carbon and energy. In this review, we provide an overview of current knowledge on tetralin catabolism at biochemical, genetic and regulatory levels in both strains. Although they share the same biodegradation strategy and enzymatic activities, no evidences of horizontal gene transfer between both bacteria have been found. Moreover, the regulatory elements that control the expression of the gene clusters are completely different in each strain. A special consideration is given to the complex regulation discovered in TFA since three regulatory systems, one of them involving an unprecedented communication between the catabolic pathway and the regulatory elements, act together at transcriptional and posttranscriptional levels to optimize tetralin biodegradation gene expression to the environmental conditions.

Sphingobium aquiterrae sp. nov., a toluene, meta- and para-xylene-degrading bacterium isolated from petroleum hydrocarbon-contaminated groundwater.

A Gram-negative, aerobic, slightly yellow-pigmented bacterium, designated as SKLS-A10T, was isolated from groundwater sample of the 'Siklós' petroleum hydrocarbon contaminated site (Hungary). Phylogenetic analysis based on 16S rRNA gene sequence revealed that strain SKLS-A10T formed a distinct phyletic lineage within the genus Sphingobium. It shared the highest 16S rRNA gene homology with Sphingobium abikonense DSM 23268T (97.29 %), followed by Sphingobium lactosutens DSM 23389T (97.23 %), Sphingobium phenoxybenzoativorans KCTC 42448T (97.16 %) and Sphingobium subterraneum NBRC 109814T (96.74 %). The predominant fatty acids (>5 % of the total) are C18 : 1ω7c, C14 : 0 2-OH, C16 : 1ω7c/iso C15 : 0 2-OH, C17 : 1ω6c and C16 : 0. The major ubiquinone is Q-10. The predominant polyamine is spermidine. The major polar lipids are sphingoglycolipid and diphosphatidylglycerol. The DNA G+C content of strain SKLS-A10T is 65.9 mol%. On the basis of evidence from this taxonomic study using a polyphasic approach, strain SKLS-A10T represents a novel species of the genus Sphingobium for which the name Sphingobiumaquiterrae sp. nov. is proposed. The type strain is SKLS-A10T (=DSM 106441T=NCAIM B. 02634T).

Colonization and Maize Growth Promotion Induced by Phosphate Solubilizing Bacterial Isolates.

Phosphorus (P) limits the production of maize, one of the major food crops in China. Phosphate-solubilizing bacteria (PSB) have the capacity to solubilize phosphate complexes into plant absorbable and utilizable forms by the process of acidification, chelation, and exchange reactions. In this study, six bacteria, including one Paenibacillus sp. B1 strain, four Pseudomonas sp. strains (B10, B14, SX1, and SX2) and one Sphingobium sp. SX14 strain, were those isolated from the maize rhizosphere and identified based on their 16S rRNA sequences. All strains could solubilize inorganic P (Ca3(PO4)2, FePO4 and AlPO4), and only B1 and B10 organic P (lecithin). All strains, except of SX1, produced IAA, and SX14 and B1 showed the highest level. B1 incited the highest increase in root length and the second increase in shoot and total dry weight, shoot length, and total P and nitrogen (N), along with increased root length. In addition, by confocal laser scanning microscopy (CLSM), we found that green fluorescent protein (GFP)-labeled B1 mainly colonized root surfaces and in epidermal and cortical tissue. Importantly, B1 can survive through forming spores under adverse conditions and prolong quality guarantee period of bio-fertilizer. Therefore, it can act as a good substitute for bio-fertilizer to promote agricultural sustainability.

Complete genome sequence of Sphingorhabdus sp. M41, a versatile hydrocarbon degrader, isolated from crude oil-contaminated costal sediment.

Sphingorhabdus sp. M41, capable of degrading aliphatic and aromatic hydrocarbons, was isolated from crude oil-contaminated costal sediment by an enrichment culture and its complete genome was sequenced. The genome of strain M41 has a chromosome with a size of 3,324,420bp, including 44 tRNAs, 6 rRNAs, and 3118 protein-coding genes. In addition, many potential genes responsible for the biodegradation of aliphatic and aromatic hydrocarbons were identified from the genome. This is the first complete genome of the genus Sphingorhabdus, which will provide insights into the bioremediation of crude oil-contaminated costal sediment by strain M41.

11-Hydroxylation of Protoberberine by the Novel Berberine-Utilizing Aerobic Bacterium Sphingobium sp. Strain BD3100.

Protoberberine alkaloids, including berberine, palmatine, and berberrubine, are produced by medicinal plants and are known to have various pharmacological effects. We isolated two berberine-utilizing bacteria, Sphingobium sp. strain BD3100 and Rhodococcus sp. strain BD7100, from soil collected at a natural medicine factory. BD3100 had the unique ability to utilize berberine or palmatine as the sole carbon and energy source. BD3100 produced demethyleneberberine in berberine-supplemented medium. In a resting-cell incubation with berberine, BD3100 produced 11-hydroxyberberine; the structure of 11-hydroxyberberine was determined by detailed analysis of NMR and MS spectroscopic data. α-Naphthoflavone, miconazole, and ketoconazole, which are known inhibitors of cytochrome P450, interfered with BD3100 metabolism of berberine in resting cells. Inhibition by miconazole led to the production of a new compound, 11-hydroxydemethyleneberberine. In a resting-cell incubation with palmatine, BD3100 generated 11-hydroxypalmatine. This work represents the first report of the isolation and characterization of novel berberine-utilizing aerobic bacteria for the production of 11-hydroxylation derivatives of berberine and palmatine.

Hephaestia caeni gen. nov., sp. nov., a novel member of the family Sphingomonadaceae isolated from activated sludge.

A Gram-staining-negative, rod-shaped and motile bacterium, designated strain ERB1-3(T), was isolated from a laboratory-scale activated sludge system treating coke plant effluent using thiocyanate-supplemented growth medium. Strain ERB1-3(T) was oxidase-positive and weakly catalase-positive. The predominant fatty acids were C18:1ω7c (35.6 %) and C17:1ω6c (29.2%), and the major respiratory quinone was Q-10. Polar lipids were dominated by sphingoglycolipid and phosphatidylglycerol. Major polyamines were spermidine and sym-homospermidine. The G+C content of the genomic DNA of strain ERB1-3(T) was 66.4 mol%. Based on the 16S rRNA gene, strain ERB1-3(T) exhibited the highest sequence similarity values to Sphingomonas sanxanigenens DSM 19645(T) (96.1%), Sphingobium scionense DSM 19371(T) (95.1%) and Stakelama pacifica LMG 24686(T) (94.8%) within the family Sphingomonadaceae. The novel isolate had some unique chemotaxonomic features that differentiated it from these closely related strains, contained much more C17 : 1ω6c, C15 : 0 2-OH, C17:0 and C17:1ω8c fatty acids and possessed diphosphatidylglycerol only in trace amounts. On the basis of the phenotypic, chemotaxonomic and molecular data, strain ERB1-3(T) is considered to represent a novel genus and species, for which the name Hephaestia caeni gen. nov., sp. nov. is proposed. The type strain is ERB1-3(T) ( = DSM 25527(T) = NCAIM B 02511(T)).

Sphingopyxis indica sp. nov., isolated from a high dose point hexachlorocyclohexane (HCH)-contaminated dumpsite.

A Gram-stain-negative, aerobic, non-motile, non-spore-forming, rod-shaped and light-yellow-pigmented bacterium, designated DS15(T), was isolated from a soil sample collected from a hexachlorocyclohexane dumpsite in Lucknow, Uttar Pradesh, India. Strain DS15(T) showed highest 16S rRNA gene sequence similarity to Sphingopyxis panaciterrulae DCY34(T) (98.7%) and Sphingopyxis soli BL03(T) (98.0%). The 16S rRNA gene sequence similarity between strain DS15(T) and species of genus Sphingopyxis with validly published names ranged from 92.5% to 98.7%. The DNA G+C content of strain DS15(T) was 67.5 mol%. The chemotaxonomic markers in strain DS15(T) were consistent with its classification in the genus Sphingopyxis, i.e. Q-10 as the major ubiquinone and summed feature 8 (C18:1ω7c/C18:1ω9c), C17:1ω6c, summed feature 3 (C16:1ω7c/C16:1ω6c), C14:0 2-OH, C15:0 2-OH, C16:0 and C17:1ω8c as the predominant fatty acids. The major polar lipids of strain DS15(T) were phosphatidylethanolamine (PE), diphosphatidylglycerol (DPG), phosphatidylcholine (PC), phosphatidylglycerol (PG) and sphingoglycolipids (SGL) and spermidine was detected as the major polyamine. Phylogenetic analysis, DNA-DNA hybridization, and chemotaxonomic and phenotypic analysis support the conclusion that strain DS15(T) represents a novel species within the genus Sphingopyxis, for which the name Sphingopyxis indica is proposed. The type strain is DS15(T) (=MTCC 9455(T)=CCM 7542(T)=MCC 2023(T)).

Characterization of polyhydroxyalkanoates (PHAs) biosynthesis by isolated Novosphingobium sp. THA_AIK7 using crude glycerol.

Biodiesel-contaminated wastewater was used to screen for PHAs-producing bacteria by using crude glycerol as the sole carbon source. A gram-negative THA_AIK7 isolate was chosen as a potential PHAs producer. The 16S rRNA phylogeny indicated that THA_AIK7 isolate is a member of Novosphingobium genus which is supported by a bootstrap percentage of 100% with Novosphingobium capsulatum. The 1,487 bp of 16S rRNA gene sequence of THA_AIK7 isolate has been deposited in the GenBank database under the accession number HM031593. Polymer content of 45% cell dry weight was achieved in 72 h with maximum product yield coefficient of 0.29 g PHAs g⁻¹ glycerol. Transmission electron micrograph results exhibited the PHAs granules accumulated inside the bacterial cell. PHAs polymer production in mineral salt media supplemented with 2% (w/v) of crude glycerol at initial pH 7 was extracted by the sodium hypochlorite method. Polymer film spectrographs from Nuclear magnetic resonance displayed a pattern of signal virtually identical to spectra of commercial PHB. Thermal analysis by Differential scanning calorimeter showed a melting temperature at 179°C. Molecular weight analysis by Gel permeation chromatography showed two main peaks of 133,000 and 700 g mol⁻¹ with weight-average molecular weight value of 23,800 and number-average molecular weight value of 755. Endotoxinfree of PHAs polymer was preliminarily assessed by a negative result of the gel-clot formation, Pyrotell® Single test vial, at sensitivity of 0.25 EU ml⁻¹. To our knowledge, this is the first reported test of endotoxin-free PHAs naturally produced from gram-negative bacteria which could be used for biomedical application.

Carbon metabolism limits recombinant protein production in Pichia pastoris.

The yeast Pichia pastoris enables efficient (high titer) recombinant protein production. As the molecular tools required are well established and gene specific optimizations of transcription and translation are becoming available, metabolism moves into focus as possible limiting factor of recombinant protein production in P. pastoris. To investigate the impact of recombinant protein production on metabolism systematically, we constructed strains that produced the model protein ß-aminopeptidase BapA of Sphingosinicella xenopeptidilytica at different production yields. The impact of low to high BapA production on cell physiology was quantified. The data suggest that P. pastoris compensates for the additional resources required for recombinant protein synthesis by reducing by-product formation and by increasing energy generation via the TCA cycle. Notably, the activity of the TCA cycle was constant with a rate of 2.1 ± 0.1 mmol g CDW-1 h(-1) irrespective of significantly reduced growth rates in high BapA producing strains, suggesting an upper limit of TCA cycle activity. The reduced growth rate could partially be restored by providing all 20 proteinogenic amino acids in the fermentation medium. Under these conditions, the rate of BapA synthesis increased twofold. The successful supplementation of the growth medium by amino acids to unburden cellular metabolism during recombinant protein production suggests that the metabolic network is a valid target for future optimization of protein production by P. pastoris.

Sphingopyxis panaciterrulae sp. nov., isolated from soil of a ginseng field.

A Gram-negative, rod-shaped, motile bacterium was isolated from the soil of a ginseng field in Daejeon, South Korea, and characterized in order to determine its taxonomic position. Phylogenetic analysis based on 16S rRNA gene sequence analysis revealed that strain DCY34(T) belonged to the family Sphingomonadaceae, and the highest degree of sequence similarity was found with Sphingopyxis witflariensis W-50(T) (97.1 %), Sphingopyxis ginsengisoli Gsoil 250(T) (97.0 %), Sphingopyxis chilensis S37(T) (96.9 %), Sphingopyxis macrogoltabida IFO 15033(T) (96.8 %), Sphingopyxis alaskensis RB2256(T) (96.7 %) and Sphingopyxis taejonensis JSS54(T) (96.7 %). Chemotaxonomic data revealed that strain DCY34(T) possessed ubiquinone Q-10 as the predominant respiratory lipoquinone, which is common to members of the genus Sphingopyxis. The predominant fatty acids were C18:1ω7c (27.5 %), summed feature 4 (C16:1ω7c and/or C15 :0 iso 2-OH; 18.6 %), C16:0 (15.6 %) and summed feature 8 (C19:1ω6c and/or unknown 18.864; 15.4 %). The major polar lipids were phosphatidylethanolamine, diphosphatidylglycerol, phosphatidylglycerol, phosphatidylcholine, sphingoglycolipid and an unknown polar lipid. The results of physiological and biochemical tests clearly demonstrated that strain DCY34(T) represented a separate species and supported its affiliation to the genus Sphingopyxis. Based on these data, the new isolate represents a novel species, for which the name Sphingopyxis panaciterrulae sp. nov. is proposed. The type strain is DCY34(T) (=KCTC 22112(T)=JCM 14844(T)).

Sphingopyxis ginsengisoli sp. nov., isolated from soil of a ginseng field in South Korea.

A Gram-negative, aerobic, motile, rod-shaped bacterium (strain Gsoil 250(T)) was isolated from soil of a ginseng field in Pocheon province (South Korea) and was characterized using a polyphasic approach in order to determine its taxonomic position. Comparative analysis of 16S rRNA gene sequences showed that strain Gsoil 250(T) belonged to the family Sphingomonadaceae of the class Alphaproteobacteria, and was related to Sphingopyxis macrogoltabida (98.7 %), Sphingopyxis chilensis (98.2 %), Sphingopyxis alaskensis (97.9 %), Sphingopyxis taejonensis (97.9 %) and Sphingopyxis witflariensis (97.8 %). The phylogenetic distance from any other species with validly published names within the genus Sphingopyxis was greater than 3.8 %. The G+C content of the genomic DNA of strain Gsoil 250(T) was 69.2 mol%. Strain Gsoil 250(T) contained Q-10 as the predominant respiratory lipoquinone and C(18 : 1)omega7c and summed feature 4 (C(16 : 1)omega7c and/or iso-C(15 : 0) 2-OH; 34.6 %) as the major fatty acids. No 3-hydroxy fatty acids were detected. Major polar lipids consisted of sphingoglycolipid, phosphatidylglycerol, phosphatidylcholine, phosphatidylethanolamine, diphosphatidylglycerol and two unknown glycolipids. Phenotypic and chemotaxonomic data supported the affiliation of strain Gsoil 250(T) to the genus Sphingopyxis. The results of DNA-DNA hybridization and physiological and biochemical tests allowed genotypic and phenotypic differentiation of strain Gsoil 250(T) from the eight recognized Sphingopyxis species. Strain Gsoil 250(T) therefore represents a novel species, for which the name Sphingopyxis ginsengisoli sp. nov. is proposed, with the type strain Gsoil 250(T) (=KCTC 12582(T)=LMG 23390(T)).

Sphingopyxis panaciterrae sp. nov., isolated from soil from ginseng field.

A Gram-negative, strictly aerobic, motile bacterial strain, designated Gsoil 124T, was isolated from a soil sample taken from a ginseng field in Pocheon Province (South Korea). The isolate contained Q-10 as the predominant lipoquinone, plus C18:1 7c and summed feature 4 (C16:1 6c and/or iso- C15:0 2-OH) as the major fatty acids. The G+C content of the genomic DNA was 68.1 mol%, and the major polar lipids consisted of sphingoglycolipid, phosphatidylglycerol, phosphatidylcholine, and phosphatidylethanolamine. A comparative 16S rRNA gene sequence analysis showed that strain Gsoil 124T was most closely related to Sphingopyxis chilensis (98.7%), Sphingopyxis alaskensis (98.2%), Sphingopyxis witflariensis (98.2%), Sphingopyxis taejonensis (98.0%), and Sphingopyxis macrogoltabida (97.6%). However, the DNA-DNA relatedness between strain Gsoil 124T and its phylogenetically closest neighbors was less than 22%. Thus, on the basis of its phenotypic properties and phylogenetic distinctiveness, strain Gsoil 124T should be classified as representing a novel species in the genus Sphingopyxis, for which the name Sphingopyxis panaciterrae sp. nov. is proposed. The type strain is Gsoil 124T (=KCTC 12580T=LMG 24003T).

Sphingobium olei sp. nov., isolated from oil-contaminated soil.

The taxonomic status of a yellow-coloured bacterial isolate from an oil-contaminated soil sample was determined using a polyphasic taxonomic approach. Comparative analysis of 16S rRNA gene sequences showed that the novel isolate formed a distinct phyletic line within the genus Sphingobium. The generic assignment was confirmed by chemotaxonomic data, which revealed: a fatty acid profile that is characteristic of the genus Sphingobium consisting of straight-chain saturated and unsaturated as well as 2-OH fatty acids; a ubiquinone with ten isoprene units (Q-10) as the predominant respiratory quinone; a polar lipid pattern consisting of diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylmonomethylethanolamine, phosphatidylcholine and sphingoglycolipid, and spermidine as the major polyamine component. Genotypic and phenotypic data show that the new isolate merits classification as a representative of a novel species of the genus Sphingobium, for which the name Sphingobium olei sp. nov. is proposed. The type strain is IMMIB HF-1T (=DSM 18999T=CCUG 54329T).

Sandarakinorhabdus limnophila gen. nov., sp. nov., a novel bacteriochlorophyll a-containing, obligately aerobic bacterium isolated from freshwater lakes.

Three strains (so36, so42T and wo26) representing a novel Gram-negative, obligately aerobic, bacteriochlorophyll a-containing species of the alpha-4 subgroup of the Proteobacteria were isolated from freshwater lakes using a high-throughput cultivation technique. The non-motile and slender rod-shaped cells formed orange-red-pigmented colonies. The main carotenoids were nostoxanthin and keto-nostoxanthin. According to the absorption spectrum, two different photosynthetic light-harvesting complexes, an LHI complex and a B800-830-type peripheral LHII complex, were present in the cells. The predominant fatty acids of strain so42T were hexadecenoic acid (16 : 1omega7c) and octadecenoic acid (18 : 1omega7c), whereas 17 : 1omega6c and 14 : 0 iso 2-OH were present in smaller amounts. The main polar lipids were phosphatidylethanolamine, phosphatidylglycerol, phosphatidylcholine, diphosphatidylglycerol, glycolipid and sphingoglycolipids. The major respiratory lipoquinone was ubiquinone-10, whereas ubiquinone-9 was present in smaller amounts. The three strains were cytochrome oxidase-negative and catalase-positive and formed alkaline and acid phosphatases. The strains grew chemoorganoheterotrophically in mineral media supplemented with various organic acids, amino acids or complex substrates such as peptone and yeast extract. The G+C content of the genomic DNA of strain so42T was 64.3 mol%. The three novel isolates contained the same 16S rRNA gene sequence. The 16S rRNA gene sequence similarity to the closest phylogenetic relative Sandaracinobacter sibiricus was only 92.8 %. Accordingly, the three strains represent a new genus and species, for which the name Sandarakinorhabdus limnophila gen. nov., sp. nov., is proposed, with strain so42T (=DSM 17366T = CECT 7086T) as the designated type strain.