An improved genome editing system for Sphingomonadaceae.

The sphingomonads encompass a diverse group of bacteria within the family Sphingomonadaceae, with the presence of sphingolipids on their cell surface instead of lipopolysaccharide as their main common feature. They are particularly interesting for bioremediation purposes due to their ability to degrade or metabolise a variety of recalcitrant organic pollutants. However, research and development on their full bioremediation potential has been hampered because of the limited number of tools available to investigate and modify their genome. Here, we present a markerless genome editing method for Sphingopyxis granuli TFA, which can be further optimised for other sphingomonads. This procedure is based on a double recombination triggered by a DNA double-strand break in the chromosome. The strength of this protocol lies in forcing the second recombination rather than favouring it by pressing a counterselection marker, thus avoiding laborious restreaking or passaging screenings. Additionally, we introduce a modification with respect to the original protocol to increase the efficiency of the screening after the first recombination event. We show this procedure step by step and compare our modified method with respect to the original one by deleting ecfG2, the master regulator of the general stress response in S. granuli TFA. This adds to the genetic tool repertoire that can be applied to sphingomonads and stands as an efficient option for fast genome editing of this bacterial group.

Sphingomonas flavescens sp. nov., isolated from soil.

An aerobic bacterium, designated as PT-12T, was isolated from soil collected from agriculture field, and its taxonomic position was validated through a comprehensive polyphasic methodology. The strain was identified as Gram-stain-negative, non-motile, rod-shaped, and catalase- and oxidase-positive. The yellow-colored colonies showed growth ability at temperature range of 18-37 °C, NaCl content of 0-1.0% (w/v), and at a pH of 6.0-8.0. The 16S rRNA gene and phylogenetic analysis showed that strain PT-12T affiliated with the genus Sphingomonas in the family Sphingomonadaceae, and displayed the highest 16S rRNA nucleotide sequence similarity with Sphingomonas limnosediminicola 03SUJ6T (98.4%). The genome size of strain PT-12T was 2,656,862 bp and the DNA G + C content estimated from genome was 63.5%. The highest values of average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) were observed between strain PT-12T and Sphingomonas segetis YJ09T, accounting to 76.2% and 20.2%, respectively. In addition, both ANI and dDDH values between strain PT-12T and other phylogenetically related neighbors ranged between 69.6% and 76.2% and 18.4% and 20.2%, respectively. Chemotaxonomic features exhibited Q-10 as the only ubiquinone; homospermidine as the major polyamine; summed feature 8 (C18:1ω7c and/or C18:1ω6c), C16:0, and 10-methyl C18:0 as the notable fatty acids; and phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, phosphatidylcholine, and sphingoglycolipid as dominating polar lipids. Overall, the comprehensive polyphasic data supported that strain PT-12T represents a novel bacterial species within the genus Sphingomonas. Accordingly, we propose the name Sphingomonas flavescens sp. nov. The type strain is PT-12T (= KCTC 92114T = NBRC 115717T).

Agromyces chromiiresistens sp. nov., Novosphingobium album sp. nov., Sphingobium arseniciresistens sp. nov., Sphingomonas pollutisoli sp. nov., and Salinibacterium metalliresistens sp. nov.: five new members of Microbacteriaceae and Sphingomonadaceae from polluted soil.

There are many unidentified microbes in polluted soil needing to be explored and nominated to benefit the study of microbial ecology. In this study, a taxonomic research was carried out on five bacterial strains which were isolated and cultivated from polycyclic aromatic hydrocarbons, and heavy metals polluted soil of an abandoned coking plant. Phylogenetical analysis showed that they belonged to the phyla Proteobacteria and Actinobacteria, and their 16S rRNA gene sequence identities were lower than 98.5% to any known and validly nominated bacterial species, suggesting that they were potentially representing new species. Using polyphasic taxonomic approaches, the five strains were classified as new species of the families Microbacteriaceae and Sphingomonadaceae. Genome sizes of the five strains ranged from 3.07 to 6.60 Mb, with overall DNA G+C contents of 63.57-71.22 mol%. The five strains had average nucleotide identity of 72.38-87.38% and digital DNA-DNA hybridization of 14.0-34.2% comparing with their closely related type strains, which were all below the thresholds for species delineation, supporting these five strains as novel species. Based on the phylogenetic, phylogenomic, and phenotypic characterizations, the five novel species are proposed as Agromyces chromiiresistens (type strain H3Y2-19aT = CGMCC 1.61332T), Salinibacterium metalliresistens (type strain H3M29-4T = CGMCC 1.61335T), Novosphingobium album (type strain H3SJ31-1T = CGMCC 1.61329T), Sphingomonas pollutisoli (type strain H39-1-10T = CGMCC 1.61325T), and Sphingobium arseniciresistens (type strain H39-3-25T = CGMCC 1.61326T). Comparative genome analysis revealed that the species of the family Sphingomonadaceae represented by H39-1-10T, H39-3-25T, and H3SJ31-1T possessed more functional protein-coding genes for the degradation of aromatic pollutants than the species of the family Microbacteriaceae represented by H3Y2-19aT and H3M29-4T. Furthermore, their capacities of resisting heavy metals and metabolizing aromatic compounds were investigated. The results indicated that strains H3Y2-19aT and H39-3-25T were robustly resistant to chromate (VI) and/or arsenite (III). Strains H39-1-10T and H39-3-25T grew on aromatic compounds, including naphthalene, as carbon sources even in the presence of chromate (VI) and arsenite (III). These features reflected their adaptation to the polluted soil environment.

Comparative Genomic Analysis of Sphingomonas morindae sp. NBD5 and Sphingopyxis sp. USTB-05 for Producing Macular Pigment.

Sphingomonas morindae sp. NBD5, which we previously identified and tested, is a new bacterial strain for producing lutein. Here, based on the next-generation sequencing technology, we analyzed high throughput genomic sequences and compared related functional genes of Sphingomonas morindae sp. NBD5 and Sphingopyxis sp. USTB-05. The genome of Sphingomonas morindae sp. NBD5 has two sets of chromosomes, which is 4,239,716 bp and harbors 3882 protein coding genes. There are 59 protein-coding genes related to the macular pigment (MP) biosynthesis, of which four genes (ackA, pgm, gpmI and pckA) are unique. These genes, pckG, porB, meh, and fldA, are unique in Sphingopyxis sp. USTB-05. The analysis of Sphingomonas morindae sp. NBD5 and Sphingopyxis sp. USTB-05 genomes gives an insight into the new pathway for MP production. These genes for the transformation of glucose to MP were also found in Sphingomonas morindae sp. NBD5 and Sphingopyxis sp. USTB-05. This study expands the understanding of the pathway for complete biosynthesis of MP by Sphingomonas morindae sp. NBD5 and Sphingopyxis sp. USTB-05.

Molecular Mechanism of Chloramphenicol and Thiamphenicol Resistance Mediated by a Novel Oxidase, CmO, in Sphingomonadaceae.

Antibiotic resistance mediated by bacterial enzyme inactivation plays a crucial role in the degradation of antibiotics in the environment. Chloramphenicol (CAP) resistance by enzymatic inactivation comprises nitro reduction, amide bond hydrolysis, and acetylation modification. However, the molecular mechanism of enzymatic oxidation of CAP remains unknown. Here, a novel oxidase gene, cmO, was identified and confirmed biochemically. The encoded CmO oxidase could catalyze the oxidation at the C-1' and C-3' positions of CAP and thiamphenicol (TAP) in Sphingobium sp. strain CAP-1. CmO is highly conserved in members of the family Sphingomonadaceae and shares the highest amino acid similarity of 41.05% with the biochemically identified glucose methanol choline (GMC) oxidoreductases. Molecular docking and site-directed mutagenesis analyses demonstrated that CAP was anchored inside the protein pocket of CmO with the hydrogen bonding of key residues glycine (G) 99, asparagine (N) 518, methionine (M) 474, and tyrosine (Y) 380. CAP sensitivity tests demonstrated that the acetyltransferase and CmO could enable a higher level of resistance to CAP than the amide bond-hydrolyzing esterase and nitroreductase. This study provides a better theoretical basis and a novel diagnostic gene for understanding and assessing the fate and resistance risk of CAP and TAP in the environment. IMPORTANCE Rising levels of antibiotic resistance are undermining ecological and human health as a result of the indiscriminate usage of antibiotics. Various resistance mechanisms have been characterized-for example, genes encoding proteins that degrade antibiotics-and yet, this requires further exploration. In this study, we report a novel gene encoding an oxidase involved in the inactivation of typical amphenicol antibiotics (chloramphenicol and thiamphenicol), and the molecular mechanism is elucidated. The findings provide novel data with which to understand the capabilities of bacteria to tackle antibiotic stress, as well as the complex function of enzymes in the contexts of antibiotic resistance development and antibiotic removal. The reported gene can be further employed as an indicator to monitor amphenicol's fate in the environment, thus benefiting risk assessment in this era of antibiotic resistance.

Triterpenoid and Steroidal Saponins Differentially Influence Soil Bacterial Genera.

Plant specialized metabolites (PSMs) are secreted into the rhizosphere, i.e., the soil zone surrounding the roots of plants. They are often involved in root-associated microbiome assembly, but the association between PSMs and microbiota is not well characterized. Saponins are a group of PSMs widely distributed in angiosperms. In this study, we compared the bacterial communities in field soils treated with the pure compounds of four different saponins. All saponin treatments decreased bacterial α-diversity and caused significant differences in ß-diversity when compared with the control. The bacterial taxa depleted by saponin treatments were higher than the ones enriched; two families, Burkholderiaceae and Methylophilaceae, were enriched, while eighteen families were depleted with all saponin treatments. Sphingomonadaceae, which is abundant in the rhizosphere of saponin-producing plants (tomato and soybean), was enriched in soil treated with α-solanine, dioscin, and soyasaponins. α-Solanine and dioscin had a steroid-type aglycone that was found to specifically enrich Geobacteraceae, Lachnospiraceae, and Moraxellaceae, while soyasaponins and glycyrrhizin with an oleanane-type aglycone did not specifically enrich any of the bacterial families. At the bacterial genus level, the steroidal-type and oleanane-type saponins differentially influenced the soil bacterial taxa. Together, these results indicate that there is a relationship between the identities of saponins and their effects on soil bacterial communities.

Description and functional testing of four species of the novel phototrophic genus Chioneia gen. nov., isolated from different East Antarctic environments.

Seven Gram-negative, aerobic, non-sporulating, motile strains were isolated from terrestrial (R-67880T, R-67883, R-36501 and R-36677T) and aquatic (R-39604, R-39161T and R-39594T) East Antarctic environments (i.e. soil and aquatic microbial mats), between 2007 and 2014. Analysis of near-complete 16S rRNA gene sequences revealed that the strains potentially form a novel genus in the family Sphingomonadaceae (Alphaproteobacteria). DNA-DNA reassociation and average nucleotide identity values indicated distinction from close neighbors in the family Sphingomonadaceae and showed that the seven isolates form four different species. The main central pathways present in the strains are the glycolysis, tricarboxylic acid cycle and pentose phosphate pathway. The strains can use only a limited number of carbon sources and mainly depend on ammonia and sulfate as a nitrogen and sulfur source, respectively. The novel strains showed the potential of aerobic anoxygenic phototrophy, based on the presence of bacteriochlorophyll a pigments, which was corroborated by the presence of genes for all building blocks for a type 2 photosynthetic reaction center in the annotated genomes. Based on the results of phenotypic, genomic, phylogenetic and chemotaxonomic analyses, the strains could be assigned four new species in the novel genus Chioneia gen. nov. in the family Sphingomonadaceae, for which the names C. frigida sp. nov. (R-67880T, R-67883 and R-36501), C. hiemis sp. nov. (R-36677T), C. brumae sp. nov. (R-39161T and R-39604) and C. algoris sp. nov. (R-39594T) are proposed.

Sphingomonas aliaeris sp. nov., a new species isolated from pork steak packed under modified atmosphere.

Species belonging to the genus Sphingomonas have been isolated from environments such as soil, water and plant tissues. Many strains are known for their capability of degrading aromatic molecules and producing extracellular polymers. A Gram-stain-negative, strictly aerobic, motile, red-pigmented, oxidase-negative, catalase-positive, rod-shaped strain, designated DH-S5T, has been isolated from pork steak packed under CO2-enriched modified atmosphere. Cell diameters were 1.5×0.9 µm. Growth optima were at 30 °C and at pH 6.0. Phylogenetic analyses based on both complete 16S rRNA gene sequence and whole-genome sequence data revealed that strain DH-S5T belongs to the genus Sphingomonas, being closely related to Sphingomonas alpina DSM 22537T (97.4 % gene sequence similarity), followed by Sphingomonas qilianensis X1T (97.4 %) and Sphingomonas hylomeconis GZJT-2T (97.3 %). The DNA G+C content was 64.4 mol%. The digital DNA-DNA hybridization value between the isolate strain and S. alpina DSM 22537T was 21.0 % with an average nucleotide identity value of 77.03 %. Strain DH-S5T contained Q-10 as the ubiquinone and major fatty acids were C18 : 1 cis 11 (39.3 %) and C16 : 1 cis 9 (12.5 %), as well as C16 : 0 (12.1 %) and C14 : 0 2-OH (11.4 %). As for polar lipids, phosphatidylcholine, phosphatidylglycerol, diphosphatidylglycerol, phosphatidylethanolamine, dimethylphosphatidylethanolamine and sphingoglycolipid could be detected, alongside traces of monomethylphosphatidylethanolamine. Based on its phenotypic, chemotaxonomic and phylogenetic characteristics, strain DH-S5T (=DSM 110829T=LMG 31606T) is classified as a representative of the genus Sphingomonas, for which the name Sphingomonas aliaeris sp. nov. is proposed.

Sphingomonas sabuli sp. nov., a carotenoid-producing bacterium isolated from beach sand.

A Gram-stain-negative, aerobic and non-motile bacterium, strain sand1-3T, was isolated from beach sand collected from Haeundae Beach located in Busan, Republic of Korea. Based on the results of 16S rRNA gene sequence and phylogenetic analyses, Sphingomonas daechungensis CH15-11T (97.0 %), Sphingomonas edaphi DAC4T (96.8 %), Sphingomonas xanthus AE3T (96.5 %) and Sphingomonas oryziterrae YC6722T (96.0 %) were selected for comparing phenotypic and chemotaxonomic characteristics. Cells of strain sand1-3T grew at 7-50 °C (optimum, 30-35 °C), pH 5.0-8.0 (optimum, pH 7.0-8.0) and in the presence of 0-0.5 % (w/v) NaCl (optimum, 0 %). Major polar lipids included diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, sphingoglycolipid, one unidentified glycolipid and one unidentified phosphoglycolipid. The major fatty acids were summed feature 8 (C18 : 1 ω6c and/or C18 : 1 ω7c) and C18 : 1 2-OH. Moreover, the sole respiratory quinone and major polyamine were identified as ubiquinone-10 and homospermidine, respectively. The genomic DNA G+C content was 65.9 mol%. The digital DNA-DNA hybridization, average nucleotide identity and average amino acid identity values of strain sand1-3T and its reference strains with publicly available genomes were 17.9-18.9 %, 72.0-75.3 % and 63.3-76.5 % respectively. Based on polyphasic evidence, we propose Sphingomonas sabuli sp. nov. as a novel species within the genus Sphingomonas. The type strain is sand1-3T (=KCTC 82358T=NBRC 114538T).

Proteome, Bioinformatic, and Functional Analyses Reveal a Distinct and Conserved Metabolic Pathway for Bile Salt Degradation in the Sphingomonadaceae.

Bile salts are amphiphilic steroids with digestive functions in vertebrates. Upon excretion, bile salts are degraded by environmental bacteria. Degradation of the bile salt steroid skeleton resembles the well-studied pathway for other steroids, like testosterone, while specific differences occur during side chain degradation and the initiating transformations of the steroid skeleton. Of the latter, two variants via either Δ1,4- or Δ4,6-3-ketostructures of the steroid skeleton exist for 7-hydroxy bile salts. While the Δ1,4 variant is well known from many model organisms, the Δ4,6 variant involving a 7-hydroxysteroid dehydratase as a key enzyme has not been systematically studied. Here, combined proteomic, bioinformatic, and functional analyses of the Δ4,6 variant in Sphingobium sp. strain Chol11 were performed. They revealed a degradation of the steroid rings similar to that of the Δ1,4 variant except for the elimination of the 7-OH as a key difference. In contrast, differential production of the respective proteins revealed a putative gene cluster for the degradation of the C5 carboxylic side chain encoding a CoA ligase, an acyl-CoA dehydrogenase, a Rieske monooxygenase, and an amidase but lacking most canonical genes known from other steroid-degrading bacteria. Bioinformatic analyses predicted the Δ4,6 variant to be widespread among the Sphingomonadaceae, which was verified for three type strains which also have the predicted side chain degradation cluster. A second amidase in the side chain degradation gene cluster of strain Chol11 was shown to cleave conjugated bile salts while having low similarity to known bile salt hydrolases. This study identifies members of the Sphingomonadaceae that are remarkably well adapted to the utilization of bile salts via a partially distinct metabolic pathway. IMPORTANCE This study highlights the biochemical diversity of bacterial degradation of steroid compounds, in particular bile salts. Furthermore, it substantiates and advances knowledge of a variant pathway for degradation of steroids by sphingomonads, a group of environmental bacteria that are well known for their broad metabolic capabilities. Biodegradation of bile salts is a critical process due to the high input of these compounds from manure into agricultural soils and wastewater treatment plants. In addition, these results may also be relevant for the biotechnological production of bile salts or other steroid compounds with pharmaceutical functions.

Novosphingobium aureum sp. nov., a marine bacterium isolated from salt flat sediment.

A Gram-stain-negative, aerobic, pale yellow-coloured, rod-shaped marine bacterium designated strain YJ-S2-02T was isolated from salt flat sediment sampled in Yongyu-do, Republic of Korea. Strain YJ-S2-02T grew at pH 6.0-9.0 (optimum, pH 7.0), 10-40 °C (optimum, 30 °C) and with optimum 1 % (w/v) NaCl. The 16S rRNA gene sequence analysis indicated that strain YJ-S2-02T was closely related to Novosphingobium naphthalenivorans NBRC 102051T (97.8 %) followed by Novosphingobium mathurense SM117T (97.5 %), Novosphingobium indicum H25T (97.3 %), Novosphingobium pentaromativorans US6-1T (96.8 %), Novosphingobium fontis STM-14T (96.6 %), Novosphingobium endophyticum EGI60015T (96.5 %), Novosphingobium naphthae D39T (96.5 %) and Novosphingobium malaysiense MUSC 273T (95.9 %). The average nucleotide identity and estimated DNA-DNA hybridization values between YJ-S2-02T and related type strains were 77.0-77.9 % and 19.1-24.0 %. Strain YJ-S2-02T was characterized as having Q-10 as the predominant respiratory quinone and the principal fatty acids (>10 %) were summed feature 8 (C18 : 1 ω6c/ω7c, 20.7 %), C18 : 3 ω6c (16.3 %) and C17 : 1 ω6c (11.8 %). The polar lipids consisted of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, sphingolipids and two unidentified lipids. The DNA G+C content of strain YJ-S2-02T was 65.6 mol%. On the basis of the polyphasic taxonomic evidence presented in this study, YJ-S2-02T should be classified as representing a novel species within the genus Novosphingobium, for which name Novosphingobium aureum is proposed, with the type strain YJ-S2-02T (=KACC 21677T =KCTC 72891T=JCM 33996T).

Characterization of the Aerobic Anoxygenic Phototrophic Bacterium Sphingomonas sp. AAP5.

An aerobic, yellow-pigmented, bacteriochlorophyll a-producing strain, designated AAP5 (=DSM 111157=CCUG 74776), was isolated from the alpine lake Gossenköllesee located in the Tyrolean Alps, Austria. Here, we report its description and polyphasic characterization. Phylogenetic analysis of the 16S rRNA gene showed that strain AAP5 belongs to the bacterial genus Sphingomonas and has the highest pairwise 16S rRNA gene sequence similarity with Sphingomonas glacialis (98.3%), Sphingomonas psychrolutea (96.8%), and Sphingomonas melonis (96.5%). Its genomic DNA G + C content is 65.9%. Further, in silico DNA-DNA hybridization and calculation of the average nucleotide identity speaks for the close phylogenetic relationship of AAP5 and Sphingomonas glacialis. The high percentage (76.2%) of shared orthologous gene clusters between strain AAP5 and Sphingomonas paucimobilis NCTC 11030T, the type species of the genus, supports the classification of the two strains into the same genus. Strain AAP5 was found to contain C18:1ω7c (64.6%) as a predominant fatty acid (>10%) and the polar lipid profile contained phosphatidylglycerol, diphosphatidylglycerol, phosphatidylethanolamine, sphingoglycolipid, six unidentified glycolipids, one unidentified phospholipid, and two unidentified lipids. The main respiratory quinone was ubiquinone-10. Strain AAP5 is a facultative photoheterotroph containing type-2 photosynthetic reaction centers and, in addition, contains a xathorhodopsin gene. No CO2-fixation pathways were found.

Simultaneous Presence of Bacteriochlorophyll and Xanthorhodopsin Genes in a Freshwater Bacterium.

Photoheterotrophic bacteria represent an important part of aquatic microbial communities. There exist two fundamentally different light-harvesting systems: bacteriochlorophyll-containing reaction centers or rhodopsins. Here, we report a photoheterotrophic Sphingomonas strain isolated from an oligotrophic lake, which contains complete sets of genes for both rhodopsin-based and bacteriochlorophyll-based phototrophy. Interestingly, the identified genes were not expressed when cultured in liquid organic media. Using reverse transcription quantitative PCR (RT-qPCR), RNA sequencing, and bacteriochlorophyll a quantification, we document that bacteriochlorophyll synthesis was repressed by high concentrations of glucose or galactose in the medium. Coactivation of photosynthesis genes together with genes for TonB-dependent transporters suggests the utilization of light energy for nutrient import. The photosynthetic units were formed by ring-shaped light-harvesting complex 1 and reaction centers with bacteriochlorophyll a and spirilloxanthin as the main light-harvesting pigments. The identified rhodopsin gene belonged to the xanthorhodopsin family, but it lacks salinixanthin antenna. In contrast to bacteriochlorophyll, the expression of xanthorhodopsin remained minimal under all experimental conditions tested. Since the gene was found in the same operon as a histidine kinase, we propose that it might serve as a light sensor. Our results document that photoheterotrophic Sphingomonas bacteria use the energy of light under carbon-limited conditions, while under carbon-replete conditions, they cover all their metabolic needs through oxidative phosphorylation.IMPORTANCE Phototrophic organisms are key components of many natural environments. There exist two main phototrophic groups: species that collect light energy using various kinds of (bacterio)chlorophylls and species that utilize rhodopsins. Here, we present a freshwater bacterium Sphingomonas sp. strain AAP5 which contains genes for both light-harvesting systems. We show that bacteriochlorophyll-based reaction centers are repressed by light and/or glucose. On the other hand, the rhodopsin gene was not expressed significantly under any of the experimental conditions. This may indicate that rhodopsin in Sphingomonas may have other functions not linked to bioenergetics.

Sphingomonas lacunae sp. nov., isolated from a freshwater pond.

A novel bacterial strain, designated CSW-10T, isolated from a freshwater pond in Taiwan, was characterized using a polyphasic taxonomic approach. Cells were Gram-stain-negative, aerobic, non-motile, rod-shaped and formed yellow-coloured colonies. Optimal growth occurred at 30 °C, pH 7, and in the absence of NaCl. Phylogenetic analyses based on 16S rRNA gene sequences and coding sequences of 92 protein clusters indicated that strain CSW-10T formed a phylogenetic lineage in the genus Sphingomonas. The 16S rRNA gene sequence similarity indicated that strain CSW-10T was most closely related to Sphingomonas fonticola TNR-2T (97.6%). Strain CSW-10T showed 69.8-70.7% average nucleotide identity and 19.0-23.0% digital DNA-DNA hybridization identity with the strains of other related Sphingomonas species. The major fatty acids of strain CSW-10T were summed feature 8 (C18:1 ω7c and/or C18:1 ω6c) and C17:1 ω6c. The polar lipid profile consisted of phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, phosphatidyldimethylethanolamine, phosphatidylcholine, one uncharacterized sphingoglycolipid, five uncharacterized aminophospholipids, one uncharacterized phospholipid and one uncharacterized lipid. The predominant polyamines were homospermidine and spermidine. The major isoprenoid quinone was Q-10. Genomic DNA G+C content of strain CSW-10T was 62.0 mol%. On the basis of phenotypic and genotypic properties and phylogenetic inference, strain CSW-10T should represent a novel species of the genus Sphingomonas, for which the name Sphingomonas lacunae sp. nov. is proposed. The type strain is CSW-10T (=BCRC 81190T =LMG 31340T).

Novosphingobium ovatum sp. nov., isolated from a freshwater mesocosm.

A bacterial strain, designated FSY-8T, was isolated from a freshwater mesocosm in Taiwan and characterized using the polyphasic taxonomy approach. Cells of strain FSY-8T were aerobic, Gram-stain-negative, rod-shaped, non-motile and formed yellow coloured colonies on Reasoner's 2A agar. Growth occurred at 20-40 °C (optimum, 30-37 °C) and pH 5-7 (optimum, pH 6) and in the presence of 0-0.5 % NaCl (optimum, 0 %, w/v). The major fatty acids (>10 %) of strain FSY-8T were summed feature 8 (C18 : 1 ω7c and/or C18 : 1 ω6c) and summed feature 3 (C16 : 1 ω7c and/or C16 : 1 ω6c). The polar lipid profile consisted of a mixture of phosphatidylethanolamine, phosphatidylglycerol, phosphatidylcholine, phosphatidylmonomethylethanolamine, phosphatidyldimethylethanolamine, sphingoglycolipid, diphosphatidylglycerol, an uncharacterized aminophospholipid, an uncharacterized glycolipid and an uncharacterized lipid. The major polyamine was spermidine. The major isoprenoid quinone was Q-10. The DNA G+C content was 64.8 mol %. Phylogenetic analyses based on 16S rRNA gene sequences and coding sequences of 92 protein clusters indicated that strain FSY-8T formed a phylogenetic lineage in the genus Novosphingobium. Strain FSY-8T showed 71.6-77.2 % average nucleotide identity and 19.9-22.8 % digital DNA-DNA hybridization identity with the strains of other Novosphingobium species. On the basis of phenotypic and genotypic properties and phylogenetic inference, strain FSY-8T should be classified in a novel species of the genus Novosphingobium, for which the name Novosphingobium ovatum sp. nov. is proposed. The type strain is FSY-8T (=BCRC 81051T=LMG 30053T=KCTC 52812T).

Sphingomonas parva sp. nov., isolated from soil in Jeju Island.

A Gram staining-negative, yellow-colored, rod-shaped, non-motile and aerobic, designated strain 17J27-24T was isolated from a soil sample in Korea. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain 17J27-24T was related to the members of the family Sphingomonadaceae and formed a distinct monophyletic cluster within the genus Sphingomonas with Sphingomonas deserti (98.3% 16S rRNA gene sequence similarity). Growth was observed at 30 °C (optimum), at pH 7.0 (optimum), and in the absence of NaCl (%). The predominant cellular fatty acids were summed feature 8 (18:1 ω7c and/or 18:1 ω6c) and C17:1 ω6c. The major respiratory quinone was Q10. The polar lipids profile comprised of diphosphatidylglycerol (DPG), phosphatidylglycerol (PG), phosphatidylethanolamine (PE), and sphingoglycolipid (SGL). The DNA G + C content was 77.8 mol%. The average nucleotide identity (ANI) and digital DNA-DNA hybridisation (dDDH) values between 17J27-24T and its phylogenetically closest Sphingomonas deserti (KCTC 62411T) were below the established cut-off < 94% (ANI) and < 70% (dDDH) for species delineation. Moreover, the results of the polyphasic approach confirmed that strain 17J27-24T represents a novel species of the genus Sphingomonas within the family Sphingomonadaceae, for which the name Sphingomonas parva sp. nov. is proposed. The type strain of this species is 17J27-24T (= KCTC 62208T = JCM 3896T). An emended description of the species Sphingomonas parva is provided.

Reclassification of Sphingomonas aeria as a later heterotypic synonym of Sphingomonas carotinifaciens based on whole-genome sequence analysis.

The 16S rRNA gene sequences of Sphingomonas carotinifaciens L9-754T and Sphingomonas aeria B093034T possess 99.71 % sequence similarity. Further studies were undertaken to clarify the taxonomic assignments of these species. Whole-genome comparisons showed that S. aeria B093034Tand S. carotinifaciens L9-754T shared 96.9 % average nucleotide identity, 98.4 % average amino acid identity and 76.1 % digital DNA-DNA hybridization values. These values exceeded or approached the recommended species delineation threshold values. Furthermore, a phylogenetic tree based on 41 of the most conserved genes provided additional evidence that S. aeria B093034T and S. carotinifaciens L9-754T are very closely related. Based on this evidence we propose the reclassification of S. aeria Xue et al. 2018 as a later heterotypic synonym of S. carotinifaciens Madhaiyan et al. 2017.

Novosphingobium umbonatum sp. nov., isolated from a freshwater mesocosm.

A bacterial strain designated FSY-9T was isolated from a freshwater mesocosm in Taiwan and characterized to determine its taxonomic affiliation. Phylogenetic analyses based on 16S rRNA gene sequences and coding sequences of 92 protein clusters indicated that strain FSY-9T formed a phylogenetic lineage in the genus Novosphingobium. Strain FSY-9T was most closely related to Novosphingobium humi R1-4T with a 97.2 % 16S rRNA gene sequence similarity. Strain FSY-9T showed 71.3-72.6 % average nucleotide identity and 17.7-23.0 % digital DNA-DNA hybridization identity with the strains of other Novosphingobium species. Cells of strain FSY-9T were facultatively anaerobic, Gram-negative, rod-shaped, non-motile and formed light yellow coloured colonies. Growth occurred at 15-37 °C and pH 5.5-7, and in the presence of 0-0.5 % NaCl. The major fatty acids (>10 %) of strain FSY-9T were summed feature 3 (C16 : 1ω7c and/or C16 : 1ω6c), C18 : 1ω7c and C16 : 0. The polar lipid profile consisted of a mixture of phosphatidylethanolamine, phosphatidylglycerol, phosphatidylcholine, phosphatidyldimethylethanolamine, sphingoglycolipid, an uncharacterized aminophospholipid, an uncharacterized glycolipid and an uncharacterized lipid. The major polyamine was spermidine. The major isoprenoid quinone was Q-10. The DNA G+C content was 61.5 mol%. On the basis of phenotypic and genotypic properties and phylogenetic inference, strain FSY-9T should be classified as a novel species of the genus Novosphingobium, for which the name Novosphingobium umbonatum sp. nov. is proposed. The type strain is FSY-9T (=BCRC 81052T=LMG 30054T=KCTC 52813T).

Sphingobium algorifonticola sp. nov., isolated from a cold spring.

Strain TLA-22T, isolated from a cold spring in Taiwan, was characterized using a polyphasic taxonomy approach. Cells were Gram-stain-negative, aerobic, poly-ß-hydroxybutyrate-accumulating, motile by means of a single polar flagellum, rod-shaped and formed bright yellow colonies. Optimal growth occurred at 20-25 °C, pH 6-6.5, and in the presence of 0.5 % NaCl. The major fatty acids of TLA-22T were C18 : 1 ω7 c and C17 : 1ω6c. The predominant hydroxy fatty acids were C15 : 0 2-OH and C14 : 0 2-OH. The polar lipid profile consisted of a mixture of phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, phosphatidyldimethylethanolamine, sphingoglycolipid, an unidentified aminophospholipid, an unidentified phospholipid and three unidentified lipids. TLA-22T contained spermidine as the major polyamine and putrescine as the minor component. The only isoprenoid quinone was Q-10. The genomic DNA G+C content of TLA-22T was 63.2 mol%. Phylogenetic analyses based on 16S rRNA gene sequences and coding sequences of 92 protein clusters indicated that TLA-22T was a mem,ber of a phylogenetic lineage including members of the genus Sphingobium. TLA-22T was most closely related to Sphingobium aromaticiconvertens RW16T, with a 97.4 % 16S rRNA gene sequence similarity. TLA-22T showed 74.8-75.7 % average nucleotide identity and 20.1-22.0 % digital DNA-DNA hybridization identity with the strains of other species of the genus Sphingobium. On the basis of phenotypic and genotypic properties and phylogenetic inference, strain TLA-22T should be classified as representing a novel species of the genus Sphingobium, for which the name Sphingobium algorifonticola sp. nov. is proposed. The type strain is TLA-22T (=BCRC 81097T =LMG 30309T=KCTC 62189T).

Sphingobium fluviale sp. nov., isolated from a river.

A Gram-stain-negative, rod-shaped, non-motile, poly-ß-hydroxybutyrate-accumulating and aerobic bacterial strain, designated CHR27T, was isolated and characterized by using the polyphasic taxonomy approach. The results of phylogenetic analyses based on 16S rRNA gene sequences and coding sequences of an up-to-date bacterial core gene set (92 protein clusters) indicated that strain CHR27T is affiliated with species in the genus Sphingobium. 16S rRNA gene sequence similarity results indicated that strain CHR27T was closely related to species of the genus Sphingobium (94.3-97.0 %), and had the highest sequence similarity to Sphingobium qiguonii X23T (97.0 %). Strain CHR27T showed 19.4-22.1 % digital DNA-DNA hybridization values and 73.2-74.8 % average nucleotide identity values with the strains of other Sphingobium species. Optimal growth occurred at 25 °C, pH 7.5 and in the absence of NaCl. The major fatty acids of strain CHR27T were C18 : 1ω7c and summed feature 3 (C16 : 1ω7c and/or C16 : 1ω6c). The predominant hydroxy fatty acid was C14 : 0 2-OH. The polar lipid profile consisted of a mixture of phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, phosphatidylmonomethylethanolamine, phosphatidyldimethylethanolamine, two unidentified sphingoglycolipids and an unidentified aminophospholipid. Strain CHR27T contained spermidine as the major polyamine and putrescine as a minor component. The only isoprenoid quinone was ubiquinone-10. The genomic DNA G+C content of strain CHR27Twas 61.8 mol%. On the basis of the phylogenetic inference and phenotypic data, strain CHR27T was considered a representative of a novel species within the genus Sphingobium. The name Sphingobium fluviale sp. nov. is proposed, with strain CHR27T (=BCRC 81121T=LMG 30596T=KCTC 62510T) as the type strain.