Chelativorans petroleitrophicus sp. nov., a paraffin oil-degrading bacterium isolated from a mixture of oil-based drill cuttings and paddy soil.

We isolated a paraffin oil-degrading bacterial strain from a mixture of oil-based drill cutting and paddy soil, and characterized the strain using a polyphasic approach. The Gram-positive, aerobic, rod-shaped and non-spore-forming strain (SCAU 2101T) grew optimally at 50 °C, pH 7.0 and 0.5 % (w/v) NaCl. Phylogenetic analysis based on 16S rRNA gene sequence indicated that the strain represented a distinct clade in the genus Chelativorans, neighbouring Chelativorans intermedius LMG 28482T (97.1 %). The genome size and DNA G+C content of the strain were 3 969 430 bp and 63.1 mol%, respectively. Whole genome based phylogenomic analyses showed that the average nucleotide identity and digital DNA-DNA hybridization values between strain SCAU 2101T and C. intermedius LMG 28482T were 77.5 and 21.2 %, respectively. The major respiratory quinone was Q-10. The dominant fatty acids were C19 : 0 cyclo ω8c (50.6 %), summed feature 8 (C18 : 1 ω7c and/or C18 : 1 ω6c; 22.5 %) and C18 : 0 (13.8 %). The polar lipids of the strain included phosphatidylethanolamine, phosphatidylmonomethylethanolamine, phosphatidylglycerol, phosphatidylcholine and diphosphatidylglycerol. Based on the results, strain SCAU 2101T was considered to represent a novel species in the genus Chelativorans, for which the name Chelativorans petroleitrophicus sp. nov. is proposed. The type strain is SCAU 2101T (= CCTCC AB 2021125T=KCTC 92067T).

Enhanced Effects of Iron on Mycelial Growth, Metabolism and In Vitro Antioxidant Activity of Polysaccharides from Lentinula edodes.

The polysaccharides found in Lentinula edodes have a variety of medicinal properties, such as anti-tumor and anti-viral effects, but their content in L. edodes sporophores is very low. In this study, Fe2+ was added to the liquid fermentation medium of L. edodes to analyze its effects on mycelial growth, polysaccharide and enzyme production, gene expression, and the activities of enzymes involved in polysaccharide biosynthesis, and in vitro antioxidation of polysaccharides. The results showed that when 200 mg/L of Fe2+ was added, with 7 days of shaking at 150 rpm and 3 days of static culture, the biomass reached its highest value (0.28 mg/50 mL) 50 days after the addition of Fe2+. Besides, Fe2+ addition also enhanced intracellular polysaccharide (IPS) and exopolysaccharide (EPS) productions, the levels of which were 2.98- and 1.79-fold higher than the control. The activities of the enzymes involved in polysaccharides biosynthesis, including phosphoglucomutase (PGM), phosphoglucose isomerase (PGI), and UDPG-pyrophosphorylase (UGP) were also increased under Fe2+ addition. Maximum PGI activity reached 1525.20 U/mg 30 days after Fe2+ addition, whereas PGM and UGP activities reached 3607.05 U/mg and 3823.27 U/mg 60 days after Fe2+ addition, respectively. The Pearson correlation coefficient showed a strong correlation (p < 0.01) between IPS production and PGM and UGP activities. The corresponding coding genes of the three enzymes were also upregulated. When evaluating the in vitro antioxidant activities of polysaccharides, EPS from all Fe2+-treated cultures exhibited significantly better capacity (p < 0.05) for scavenging -OH radicals. The results of the two-way ANOVA indicated that the abilities of polysaccharides to scavenge O2− radicals were significantly (p < 0.01) affected by Fe2+ concentration and incubation time. These results indicated that the addition of iron provided a good way to achieve desirable biomass, polysaccharide production, and the in vitro antioxidation of polysaccharides from L. edodes.

Role of rhizobia in promoting non-enzymatic antioxidants to mitigate nitrogen-deficiency and nickel stresses in Pongamia pinnata.

The contribution of rhizobia in the mitigation of non-enzymatic antioxidants against nitrogen deficiency and heavy metal toxicity for legume plant is not clear. Therefore, it is hypothesized that the inoculation of rhizobia could mitigate nitrogen deficiency and nickel (Ni) stresses in P. pinnata tissues by enhancing the formation of certain non-enzymatic antioxidants. The effect of symbiotic nitrogen-fixing rhizobia on the mitigation of nitrogen-deficiency and Ni stresses in P. pinnata was evaluated by inoculating two different rhizobia, i.e., Rhizobium pisi PZHK2 and Ochrobacterium pseudogrignonense PZHK4, around the rhizosphere of P. pinnata grown in soil containing 40 mg kg-1 Ni2+ and without nitrogen addition. The inoculation with both rhizobial strains promoted the growth of P. pinnata under nickel stress or nitrogen-deficiency condition, increased nitrogen content in all plant tissues and nickel content in shoots and leaves, but reduced nickel accumulation in roots. The four non-enzymatic antioxidants including glutathione (GSH), proanthocyanidin (OPC), ascorbic acid (ASA) and flavonoids (FLA) distributed in roots, shoots and leaves were followed in descending order: GSH > OPC > ASA > FLA. The four non-enzymatic antioxidants showed different levels of change under the nitrogen-deficiency and nickel stresses and in the non-stress control. The inoculation of PZHK2 and PZHK4 significantly (p < 0.05) increased the four non-enzymatic antioxidants in P. pinnata tissues, especially in roots. Some non-enzymatic antioxidants showed correlations with nickel or nitrogen in P. pinnata tissues, and the four non-enzymatic antioxidants also had correlations among each other. Therefore, this research revealed an excellent role of rhizobia in promoting non-enzymatic antioxidants to mitigate nitrogen-deficiency or nickel stress for P. pinnata.

Bio-Matrix Pot Addition Enhanced the Vegetation Process of Iron Tailings by Pennisetum giganteum.

The barrenness of large mine tailing sand reservoirs increases the risks for landslides and erosion that may be accompanied with transfer of contaminants into the surrounding environment. The tailing sand is poor in nutrients, which effectively complicates the vegetation process. We investigated direct planting of Pennisetum giganteum into tailing sand using two pit planting methods: the plants were transplanted either directly into pits filled with soil or into soil-filled bio-matrix pots made of organic material. After growing P. giganteum in iron tailing sand for 360 days, the dry weight of the plants grown in the bio-matrix pot (T2) was approximately twofold higher than that of the plants grown in soil placed directly into the sand (T1). At 360 days, the organic matter (OM) content in the soil below the pit was the lowest in the not-planted treatment (T0) and the highest in T2, the available N (AN) contents were higher in T1 and T2 than in T0, and the available P and K contents were the highest in T2. At 360 days, the Shannon diversity of the soil microbial communities was higher in T1 and T2 than in T0, and the community compositions were clearly separated from each other. The profiles of predicted C cycle catabolism functions and N fixation-related functions in T1 and T2 at 360 days were different from those in the other communities. The results showed that P. giganteum grew well in the iron tailing sand, especially in the bio-matrix pot treatment, and the increased nutrient contents and changes in microbial communities indicated that using the bio-matrix pot in planting had potential to improve the vegetation process in iron tailing sands effectively.

Nickel mine soil is a potential source for soybean plant growth promoting and heavy metal tolerant rhizobia.

Mine soil is not only barren but also contaminated by some heavy metals. It is unclear whether some rhizobia survived under extreme conditions in the nickel mine soil. Therefore, this study tries to isolate some effective soybean plant growth promoting and heavy metal resistant rhizobia from nickel mine soil, and to analyze their diversity. Soybean plants were used to trap rhizobia from the nickel mine soil. A total of 21 isolates were preliminarily identified as rhizobia, which were clustered into eight groups at 87% similarity level using BOXA1R-PCR fingerprinting technique. Four out of the eight representative isolates formed nodules on soybean roots with effectively symbiotic nitrogen-fixing and plant growth promoting abilities in the soybean pot experiment. Phylogenetic analysis of 16S rRNA, four housekeeping genes (atpD-recA-glnII-rpoB) and nifH genes assigned the symbiotic isolates YN5, YN8 and YN10 into Ensifer xinjiangense and YN11 into Rhizobium radiobacter, respectively. They also showed different tolerance levels to the heavy metals including cadmium, chromium, copper, nickel, and zinc. It was concluded that there were some plant growth promoting and heavy metal resistant rhizobia with the potential to facilitate phytoremediation and alleviate the effects of heavy metals on soybean cultivation in nickel mine soil, indicating a novel evidence for further exploring more functional microbes from the nickel mine soil.

Synergistic anti-oxidative effects of Pongamia pinnata against nickel mediated by Rhizobium pisi and Ochrobacterium pseudogrignonense.

Nickel is widely spread by different anthropogenic activities and shows toxicity for plant growth and development. Whether rhizobia symbiotically fix nitrogen can eliminate or reduce nickel toxic effect on plant or not is still unknown. This study was aimed to investigate the effect of different rhizobia genus inoculation on growth, nitrogen fixing ability, metal accumulation and enzymatic antioxidative balance of Pongamia pinnnaa. Inoculation with Rhizobium pisi and Ochrobacterium pseudogrignonense increased the all the growth parameters both in 0 and 40 mg/kg nickel as comparison with control. Only shoot length increased in presence of nitrogen as compared with no supply of nitrogen. Nitrogen content also increased both in rhizobia inoculation as compared to no nitrogen supply and non-inoculation control, respectively. Nickel uptake was higher in shoots and leaves but lower in roots in case of inoculation as compared to non-inoculation control. Rhizobia inoculation improved the plant antioxidant capacity by increasing the activity of enzymatic scavengers catalase (CAT), superoxide dismutase (SOD), peroxidase (POD) and ascorbate (GR). However, 40 mg/kg of nickel adding showed mostly effect on the activity CAT, SOD, POD in leaves. All the enzymatic activity showed a significant increase in absence of nitrogen supply as compared nitrogen supply. Our results suggested that rhizobia inoculation effectively mediated nickel stress for legume plants by increasing nitrogen supplement and inducing antioxidant capacity.

Effects of Co-application of Cadmium-Immobilizing Bacteria and Organic Fertilizers on Houttuynia cordata and Microbial Communities in a Cadmium-Contaminated Field.

Cadmium pollution is a serious threat to the soil environment. The application of bio-based fertilizers in combination with beneficial microbial agents is a sustainable approach to solving Cd pollution in farm soil. The present study investigated the effects of co-application of a Cd-immobilizing bacterial agent and two fermented organic fertilizers (fermentative edible fungi residue; fermentative cow dung) on Houttuynia cordata and its microbial communities in a Cd-polluted field. It showed that both the application of the Cd-immobilizing bacterial agent alone and the combined application of bio-based soil amendments and the bacterial agent effectively reduced >20% of the uptake of Cd by the plant. Soil nitrogen level was significantly raised after the combined fertilization. The multivariate diversity analysis and co-occurrence network algorithm showed that a significant shift of microbial communities took place, in which the microbial populations tended to be homogeneous with reduced microbial richness and increased diversity after the co-application. The treatment of fermentative cow dung with the addition of the bacterial agent showed a significant increase in the microbial community dissimilarity (R = 0.996, p = 0.001) compared to that treated with cow dung alone. The co-application of the bacterial agent with both organic fertilizers significantly increased the abundance of Actinobacteria and Bacteroidetes. The FAPROTAX soil functional analysis revealed that the introduction of the microbial agent could potentially suppress human pathogenic microorganisms in the field fertilized with edible fungi residue. It also showed that the microbial agent can reduce the nitrite oxidation function in the soil when applied alone or with the organic fertilizers. Our study thus highlights the beneficial effects of the Cd-immobilizing bacterial inoculant on H. cordata and provides a better understanding of the microbial changes induced by the combined fertilization using the microbial agent and organic soil amendments in a Cd-contaminated field.

Purification and Characterization of an ATPase GsiA from Salmonella enterica.

The coding sequence of Salmonella enterica gsiA was cloned and expressed in E. coli. The protein was purified and ATPase activity was characterized by NADH oxidation method. GsiA exhibited optimum activity at 30°C and at pH 8 in Tris/HCl buffer. GsiA protein was stable at 20°C. 66% and 44% activity remained after incubation at 30°C and 40°C for 30 min. pH 7 and pH 9 incubation would obviously reduce the ATPase activity. In vivo functionality of gsiA was determined by constructing gene deletion strains. gsiA was shown to be essential for GSI mediated glutathione uptake and gsiA deletion could decrease the virulence of Salmonella enterica. Interactions of glutathione import proteins GsiA, GsiB, GsiC, and GsiD were investigated by using bacterial two-hybrid system. GsiA could interact with itself and inner membrane proteins GsiC and GsiD. This report provides the first description of gsiA functions in Salmonella enterica. The results could help elucidating the glutathione uptake mechanism and glutathione functions in bacteria.

Structural and functional studies of conserved nucleotide-binding protein LptB in lipopolysaccharide transport.

Lipopolysaccharide (LPS) is the main component of the outer membrane of Gram-negative bacteria, which plays an essential role in protecting the bacteria from harsh conditions and antibiotics. LPS molecules are transported from the inner membrane to the outer membrane by seven LPS transport proteins. LptB is vital in hydrolyzing ATP to provide energy for LPS transport, however this mechanism is not very clear. Here we report wild-type LptB crystal structure in complex with ATP and Mg(2+), which reveals that its structure is conserved with other nucleotide-binding proteins (NBD). Structural, functional and electron microscopic studies demonstrated that the ATP binding residues, including K42 and T43, are crucial for LptB's ATPase activity, LPS transport and the vitality of Escherichia coli cells with the exceptions of H195A and Q85A; the H195A mutation does not lower its ATPase activity but impairs LPS transport, and Q85A does not alter ATPase activity but causes cell death. Our data also suggest that two protomers of LptB have to work together for ATP hydrolysis and LPS transport. These results have significant impacts in understanding the LPS transport mechanism and developing new antibiotics.

Identification and characterization of a multi-domain sulfurtransferase in Phanerochaete chrysosporium.

A sulfurtransferase gene (PcSft) with a coding region of 546 bp was cloned from the filamentous white-rot fungus Phanerochaere chrysosporium. The 181-amino acid protein contains a highly conserved "Rhodanese-like" domain and an ATP-binding site, with a molecular weight of 20.68 kDa. Semi-quantitative RT-PCR showed that the selective expression of PcSft was involved in secondary metabolism. The recombinant PcSFT protein was expressed in E. coli BL21 (DE3) and purified by Ni(2+)-chelating and size-exclusion chromatography. Its ATPase and sulfurtransferase (SFT) activities were indentified and characterized. PcSFT exhibited optimal SFT activity at pH 8 and 30 °C as well as stability at 20 °C and pH 8. The enzyme's stability under different temperature and pH P. indicates a potential usefulness for the detoxification of cyanide in the environment.

Optimizing expression and purification of an ATP-binding gene gsiA from Escherichia coli k-12 by using GFP fusion

The cloning, expression and purification of the glutathione (sulfur) import system ATP-binding protein (gsiA) was carried out. The coding sequence of Escherichia coli gsiA, which encodes the ATP-binding protein of a glutathione importer, was amplified by PCR, and then inserted into a prokaryotic expression vector pWaldo-GFPe harboring green fluorescent protein (GFP) reporter gene. The resulting recombinant plasmid pWaldo-GFP-GsiA was transformed into various E. coli strains, and expression conditions were optimized. The effect of five E. coli expression strains on the production of the recombinant gsiA protein was evaluated. E. coli BL21 (DE3) was found to be the most productive strain for GsiA-GFP fusion-protein expression, most of which was insoluble fraction. However, results from in-gel and Western blot analysis suggested that expression of recombinant GsiA in Rosetta (DE3) provides an efficient source in soluble form. By using GFP as reporter, the most suitable host strain was conveniently obtained, whereby optimizing conditions for overexpression and purification of the proteins for further functional and structural studies, became, not only less laborious, but also time-saving.