Genome mining revealed polyhydroxybutyrate biosynthesis by Ramlibacter agri sp. nov., isolated from agriculture soil in Korea

A white-colony-forming, facultative anaerobic, motile and Gram-stain-negative bacterium, designated G-1-2-2 ^T was isolated from soil of agriculture field near Kyonggi University, Republic of Korea. Strain G-1-2-2 ^T synthesized the polyhydroxybutyrate and could grow at 10–35 °C. The phylogenetic analysis based on 16S rRNA gene sequence showed that, strain G-1-2-2 ^T formed a lineage within the family Comamonadaceae and clustered as a member of the genus Ramlibacter. The 16S rRNA gene sequence of strain G-1-2-2 ^T showed high sequence similarities with Ramlibacter ginsenosidimutans BXN5-27 ^T (97.9%), Ramlibacter monticola G-3-2 ^T (97.9%) and Ramlibacter alkalitolerans CJ661^T (97.5%). The sole respiratory quinone was ubiquinone-8 (Q-8). The major polar lipids were phosphatidylethanolamine, diphosphatidylglycerol, phosphatidylglycerol, and an unidentified phospholipid. The principal cellular fatty acids were C_16:0, cyclo-C_17:0, summed feature 3 (C_16:1ω7c and/or C_16:1ω6c) and summed feature 8 (C_18:1ω7c and/or C_18:1ω6c). The genome of strain G-1-2-2 ^T was 7,200,642 bp long with 13 contigs, 6,647 protein-coding genes, and DNA G?+?C content of 68.9%. The average nucleotide identity and in silico DNA–DNA hybridization values between strain G-1-2-2 ^T and close members were?≤?81.2 and 24.1%, respectively. The genome of strain G-1-2-2 ^T showed eight putative biosynthetic gene clusters responsible for various secondary metabolites. Genome mining revealed the presence of atoB, atoB2, phaS, phbB, phbC, and bhbD genes in the genome which are responsible for polyhydroxybutyrate biosynthesis. Based on these data, strain G-1-2-2 ^T represents a novel species in the genus Ramlibacter, for which the name Ramlibacter agri sp. nov. is proposed. The type strain is G-1-2-2 ^T (=?KACC 21616 ^T?=?NBRC 114389 ^T).

     

Rhizoremediation of diesel-contaminated soil using the plant growth-promoting rhizobacterium <Emphasis Type="Italic">Gordonia</Emphasis> sp. S2RP-17

A plant growth-promoting rhizobacterium (PGPR) was isolated and identified as Gordonia sp. S2RP-17, which showed ACC deaminase and siderophore synthesizing activities. Its maximum specific growth rate was 0.54 ± 0.12 d⁻¹ at 5,000 mg L⁻¹ of total petroleum hydrocarbon (TPH), and its maximum diesel degradation rate was 2,434.0 ± 124.4 mg L⁻¹ d⁻¹ at 20,000 mg L⁻¹ of TPH. The growth of Zea mays was significantly promoted by the inoculation of Gordonia sp. S2RP-17 in the diesel-contaminated soil. Measured TPH removal efficiencies by various means were 13% by natural attenuation, 84.5% by planting Zea mays, and 95.8% by the combination of Zea mays and Gordonia sp. S2RP-17. The S2RP-17 cell counts were maintained at 1 × 10⁶ CFU g-soil⁻¹ during the remediation period, although they slightly decreased from their initial numbers (2.94 × 10⁷ CFU g-soil⁻¹). These results indicate that rhizoremediation using both Zea mays and Gordonia sp. S2RP-17 is a promising strategy for enhancing remediation efficiency of diesel-contaminated soils.     

Experimental study of solute transport and extraction by a single root in soil

The fate of ¹⁴C-2,4,6-trinitrotoluene ([U-¹⁴C]TNT) in soil/plant systems was studied using onion (Allium cepa L.) plants with only a single root. It was found that the single roots grew exponentially and that the rate of water uptake of the onion plants increased exponentially, as well. The concentration of [U-¹⁴C] in the roots at first increased and then appeared to reach a steady state, while the [U-¹⁴C] concentration in the leaves was found to increase linearly with time. The [U-¹⁴C] concentration in the rhizosphere increased gradually, while in the bulk soil it decreased slowly. The accumulation of [U-¹⁴C] in the rhizosphere is likely to difference between movement into the rhizosphere (through advective mass flow of soil water by root uptake) and its uptake into the roots. The distribution of ¹⁴C in the soil/plant system was found to be 60–85% in the soil solid phase, 7–11% in the soil liquid phase, <1% in the soil air phase, <1% in the root compartment, and <0.01% in the leaf compartment. The maximum RCF (root concentration factor) value for TNT and its derivates was found to be about 20, and the maximum TSCF (transpiration stream concentration factor) was 0.18. These values can be changed by a variety of factors in soil-plant systems     

Niabella thaonhiensis sp. nov., Isolated From the Forest Soil of Kyonggi University in Korea

Strain NHI-24^T was isolated from forest soil by a polycarbonate membrane transwell plate. It is a Gram-negative, rod-shaped, non-motile, non-spore-forming bacterium. Phylogenetic analysis based on 16S rRNA gene sequence data indicated that strain NHI-24^T is closely related to members of the genus Niabella: N. drilacis E90^T (97.7 %), N. tibetensis 15-4^T (96.7 %), N. aurantiaca R2A15-11^T (96.5 %), N. hirudinis E96^T (95.8 %), N. soli JS13-8^T (94.7 %), N. ginsengisoli NBRC106414^T (94.4 %), and N. yanshanensis CCBAU 05354^T (94.2 %). Growth temperatures range widely, from 15 to 37 °C, with 30 °C as the optimum. Salt tolerance ranges from 0 to 2 %. The strain grows at pH 6.5–11.0, with an optimal range of pH 7.0–9.5. Cells produce flexirubin-type pigments, and the predominant menaquinone is MK-7. The major fatty acids of strain NHI-24^T are iso-C_15:0 (36.72 %), iso-C_15:1 G (20.8 %), and summed feature 3 (C_16:1 ω7c/C_16:1 ω6c; 15.2 %). DNA–DNA hybridization values between strain NHI-24^T and members of the genus Niabella range from 37 to 53 %. Based on these results, it is proposed that strain NHI-24^T represents a novel species of the genus Niabella with the name Niabella thaonhiensis (= KACC 17215^T = KEMB 9005-018^T = JCM 18864^T).     

Characterization of <Emphasis Type="Italic">Flavobacterium aquimarinum</Emphasis> sp. nov., a halotolerant bacterium isolated from seawater

A novel, aerobic, Gram-stain-negative, non-motile, non-spore forming, rod-shaped bacterium, designated strain Dol 15-39^T, was isolated from a seawater sample near Geoje Island in the South Sea, Republic of Korea. The strain was found to be oxidase-negative and catalase-positive. The isolate was observed to grow at temperatures from 4 to 37°C, at salinities of up to 7%, and at pH levels from 6 to 9; moreover, it was not able to degrade starch, DNA, esculin, or tyrosine. Phylogenetic analysis based on 16S rRNA gene sequences showed that Dol 15-39^T was most closely related to Flavobacterium jumunjinense HME7102^T with a sequence similarity of 97.3%. However, the levels of DNA-DNA relatedness between Dol 15-39^T and the most closely related species were much lower than 70%, confirming that they represented distinct genomic species. The genomic DNA G + C content of Dol 15-39^T was calculated to be 32.6 mol%. MK-6 was the predominant respiratory quinine, while iso-C_15:0 (25.0%), iso-C_15:1 G (17.0%), and iso-C_17:0 3-OH (10.4%) were the major cellular fatty acids. Phosphatidylethanolamine was identified as a major polar lipid, while various unidentified aminolipids and polar lipids were also detected. Based on polyphasic taxonomic data, Dol 15-39^T represents a novel species of the genus Flavobacterium, for which the name F. aquimarinum sp. nov. is proposed. The type strain is accessible under the culture collection numbers (KEMB 9005-617^T = JCM 31930^T).     

Proposal of three novel species of soil bacteria, <Emphasis Type="Italic">Variovorax ureilyticus,Variovorax rhizosphaerae</Emphasis>, and <Emphasis Type="Italic">Variovorax robiniae</Emphasis>, in the family <Emphasis Type="Italic">Comamonadaceae</Emphasis>

Three novel bacterial strains (UCM-2^T, UCM-G28^T, and UCM-G35^T) were obtained while isolating soil bacteria for the development of antibiotics. Cells of these strains were Gram-negative, non-spore forming, motile by means of a single flagellum, and rod shaped. In all strains, the predominant isoprenoid quinone was ubiquinone-8 (Q-8). Cells contained C_16:0, summed feature 3 (C_16:1ω7c and/or C_16:1ω6c), summed feature 8 (C_18:1ω7c and/or C_18:1ω6c), and C_17:0 cyclo as the major fatty acids, and C_10:0 3-OH as the major hydroxy fatty acid. The polar lipid profiles of the three novel strains were dominated by diphosphatidylglycerol, phosphatidylethanolamine, and phosphatidylglycerol. The genomic DNA G + C contents of strains UCM-2^T, UCM-G28^T, and UCMG35^T were 67.5, 65.9, and 66.4 mol%, respectively. Phylogenetic analyses based on 16S rRNA sequences showed that strain UCM-2^T was most closely related to Variovorax soli NBRC 106424^T, whereas strains UCM-G28^T and UCM-G35^T were most similar to Variovorax ginsengisoli Gsoil 3165^T. Values indicating DNA-DNA hybridization between the novel isolates and closely related species in the genus Variovorax were lower than the 70% cut-off point. These phenotypic, chemotaxonomic, and phylogenetic data indicate that the three isolates should be classified as new members of the genus Variovorax, for which the names Variovorax ureilyticus sp. nov., Variovorax rhizosphaerae sp. nov., and Variovorax robiniae sp. nov. are proposed. The type strains are UCM-2^T (= KACC 18899^T = NBRC 112306^T), UCMG28^T (= KACC 18900^T = NBRC 112307^T), and UCM-G35^T (= KACC 18901^T = NBRC 112308^T), respectively.     

<Emphasis Type="Italic">Acidovorax monticola</Emphasis> sp. nov., isolated from soil

A novel strain K-4-16^T was isolated from forest soil of Namsan Mountain, Seoul, South Korea, and was taxonomically characterized by a polyphasic approach. Strain K-4-16^T was observed to be a Gram-staining negative, grayish white-coloured, motile with peritrichous flagella, and rod shaped bacterium. It was able to grow at 15–45 °C, at pH 4.5–10.5, and at 0–4% (w/v) NaCl concentration. Based on the 16S rRNA gene sequence analysis, strain K-4-16^T belongs to the genus Acidovorax and is closely related to Acidovorax anthurii CFBP 3232^T (98.3% sequence identity), Acidovorax konjaci K2^T (97.9% sequence identity), Acidovorax valerianellae CFBP 4730^T (97.8% sequence identity), and Acidovorax caeni R-24608^T (97.8% sequence identity). The only respiratory quinone was ubiquinone-8. The major polar lipids were phosphatidylethanolamine, phosphatidylglycerol, and diphosphatidylglycerol. The predominant fatty acids of strain K-4-16^T were summed feature 3 (C_16:1ω7c and/or C_16:1ω6c), C_16:0, and summed feature 8 (C_18:1ω7c and/or C_18:1ω6c). The genomic DNA G+C content of this novel strain was 64.7 mol%. The DNA–DNA relatedness between strain K-4-16^T and its reference strains were below the threshold value of 70%. The morphological, physiological, chemotaxonomic, and phylogenetic analyses clearly distinguished this strain from its close phylogenetic neighbors. Thus, strain K-4-16^T represents a novel species of the genus Acidovorax, for which the name Acidovorax monticola sp. nov. is proposed. The type strain is K-4-16^T (=?KEMB 9005-570^T?=?KACC 19171^T?=?NBRC 113141^T).     

<Emphasis Type="Italic">Mesorhizobium soli</Emphasis> sp. nov., a novel species isolated from the rhizosphere of <Emphasis Type="Italic">Robinia pseudoacacia</Emphasis> L. in South Korea by using a modified culture method

Strain NHI-8^T was isolated from a forest soil sample, collected in South Korea, by using a modified culture method. Comparative analysis of its nearly full-length 16S rRNA gene sequence showed that strain NHI-8^T belongs to the genus Mesorhizobium and to be closely related to Mesorhizobium chacoense PR5^T (97.32 %). The levels of DNA–DNA relatedness between strain NHI-8^T and reference type strains of the genus Mesorhizobium were 32.28–53.65 %. SDS-PAGE of total soluble proteins and the sequences of the housekeeping genes recA, glnII, and atpD were also used to support the clade grouping in rhizobia. The new strain contained summed feature 8 (57.0 %), cyclo-C_19:0ω8c (17.3 %), and C_18:0 (11.0 %) as the major fatty acids, as in genus Mesorhizobium. The strain contained cardiolipin, phosphatidylglycerol, ornithine-containing lipid, phosphatidylethanolamine, phosphatidyl-N-dimethylethanolamine, and phosphatidylcholine. Morphological and physiological analyses were performed to compare the characteristics of our strain with those of the reference type strains. Based on the results, strain NHI-8^T was determined to represent a novel member of the genus Mesorhizobium, and the name Mesorhizobium soli is proposed. The type strain is NHI-8^T (=KEMB 9005-153^T = KACC 17916^T = JCM 19897^T).     

<Emphasis Type="Italic">Azohydromonas riparia</Emphasis> sp. nov. and <Emphasis Type="Italic">Azohydromonas ureilytica</Emphasis> sp. nov. isolated from a riverside soil in South Korea

White and pale yellow coloured bacteria were isolated from the riverside soil, Daejeon, South Korea, and were designated UCM-11^T, UCM-F25, and UCM-80^T. We found that all strains were able to reduce nitrate, and the cells were aerobic and motile. The DNA G+C contents of UCM-11^T, UCM-F25, and UCM-80^T were between 68.9 to 71.2 mol% and the main ubiquinone was observed as Q-8. Based on16S rRNA gene sequences, strains UCM-11^T and UCM-F25 were found to closely match with Azohydromonas australica IAM 12664^T (98.48–98.55%), and the strain UCM-80^T was the closest match with Azohydromonas lata IAM 12599^T (98.34%). The presence of summed feature 3 (C_16:1 ω7c and/or C_16:1 ω6c), C_16:0, summed feature 8 (C_18:1 ω7c and/or C_18:1 ω6c) as well as twokinds of hydroxyfatty acids consisting of C_10:0 3-OH and C_12:0 2-OH, and branched fatty acids containing C_16:0 iso and C_17:0 cyclo were detected in all the strains. Phosphatidylethanolamine was a major polar lipid. DNA–DNA relatedness confirmed UCM-11^T, UCM-F25 and UCM-80^T as novel members of the genus Azohydromonas. Based on the morphological, physiological, biochemical and genotypic characteristics, we suggest that strains UCM-11^T, UCM-F25, and UCM-80^T represent novel species within the genus Azohydromonas. The names Azohydromonas riparia sp. nov., and Azohydromonas ureilytica sp. nov. are proposed for the type strains UCM-11^T (=KACC 18570^T =NBRC 111646^T) and UCM-80^T (=KACC 18576^T =NBRC 111658^T), respectively.     

Characterization of a diesel-degrading bacterium, <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> IU5, isolated from oil-contaminated soil in Korea

A diesel-degrading bacterium (strain IU5) isolated from oil-contaminated soil was characterized in this study. Fatty acid and 16s rDNA sequence analysis identified IU5 as a strain of Pseudomonas aeruginosa, and growth curve experiments identified the bacterium’s optimum conditions as pH 7 and 30 °C. P. aeruginosa IU5 degraded up to 60 of applied diesel (8500 mg/kg) over 13 days in a soil-slurry phase. In addition, this strain was able to grow on many other petroleum hydrocarbons as sole carbon sources, including crude oil, gasoline, benzene, toluene, xylene, and even PAHs such as naphthalene, phenanthrene and pyrene. Therefore, P. aeruginosa IU5 may be useful for bioremediation of soils and groundwater contaminated with a variety of hydrocarbons.