Leucobacter chromiisoli sp. nov., isolated from chromium-containing chemical plant soil.

A Gram-stain-positive, rod-shaped, aerobic, non-motile, non-sporulating bacterial strain, designated CSA1T, was isolated from chromium-containing soil sampled at a chemical plant. Growth of strain CSA1T occurred at pH 6-10 (optimum, pH 7), 15-45 °C (optimum, 30 °C) and in the presence of 0.5-6.5 % (w/v) NaCl (optimum, 2 %). The 16S rRNA gene sequence of strain CSA1T revealed the highest similarity to Leucobacter ruminantium A2T (97.5 %), Leucobacter tardus K 70/01T (97.3 %), Leucobacter humi Re6T (96.6 %), Leucobacter kyeonggiensis F3-P9T (96.2 %), Leucobacter zeae CC-MF41T (96.1 %) and Leucobacter weissii S27T (96.0 %). The draft genome of CSA1T was approximately 3 350 931 bp in size with a G+C content of 70.6 mol%. The average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values among strain CSA1T and the selected Leucobacter species were 74.0-79.2 % (ANIb), 84.3-87.1 % (ANIm) and 21.5-25.4 % (dDDH), which are below the recommended cutoff values for species delineation. The major fatty acids were anteiso-C15 : 0, iso-C16 : 0 and anteiso-C17 : 0. The polar lipids were diphosphatidylglycerol, phosphatidylglycerol and an unknown glycolipid. The predominant menaquinones were MK-11, MK-8 and MK-6. The cell-wall amino acids were 2,4-diaminobutyric acid, alanine, glycine, glutamic acid and threonine. From the phenotypic, chemotaxonomic and molecular features, strain CSA1T was considered to represent a novel species of the genus Leucobacter, for which the name Leucobacter chromiisoli sp. nov. is proposed. The type strain is CSA1T (=JCM 34359T=CGMCC 1.18746T).

Cloning, heterologous expression and characterization of a novel streptomyces trypsin in Bacillus subtilis SCK6.

A novel streptomyces trypsin GM2938 was selected as the object of study. The active GM2938 contains 223 amino acid residues. Constructing recombinant plasmid and transforming Bacillus subtilis SCK6, the heterogenous expression of GM2938 was achieved. Through optimization of fermentation conditions, the expression level of GM2938 reached 1622.2 U/mL (esterase activity) and 33.8 U/mL (amidase activity). The recombinant trypsin was purified and measured: the specific activity of esterase was 5.6 × 103 U/mg, and the specific activity of amidase was 1.1 × 103 U/mg. Furthermore, the enzymatic properties of GM2938 were explore: the optimal reaction temperature and pH were 50 °C and 9.0, respectively; the recombinant enzyme show high stability at 25 °C and range of pH 5.0-9.0; Ca2+, K+, Mg2+, EDTA, DTT, DMSO, methanol, glycerin and ethanediol could promote the esterase and amidase activities at the investigated concentrations, while Fe2+, SDS, tritonx-100, acetone, chloroform and n-hexane inhibited the trypsin activities. Kinetic parameters of GM2938 were calculated: the Km of BAEE was 3.15 × 10-5 mol·L-1, Vmax value was 2.87 × 10-4 mol·L-1·min-1; the Km of BAPAN was 2.20 × 10-4 mol·L-1, the Vmax was 2.40 × 10-4 mol·L-1·min-1. These properties give trypsin GM2938 a potential application prospect.

High-expression keratinase by Bacillus subtilis SCK6 for enzymatic dehairing of goatskins.

An extracellular keratinase gene from Bacillus sp. LCB12, isolated from saline-alkali soil, was cloned and high-effectively expressed in Bacillus subtilis SCK6. The enzyme was purified by ammonium sulphate precipitation and cation-exchange chromatography. Its molecular mass was estimated to be 30.95 kDa by SDS-PAGE. The optimum conditions for catalytic activity of the enzyme were pH 10.0 and 60 °C. The enzyme activity was completely inhibited by PMSF, while it was slightly inhibited by EDTA. Moreover, the surfactants Tween 20, Tween 80 and Triton X-100, showed little effect on enzyme activity respectively. The crude enzyme was used as an alternative to sodium sulfide for dehairing of goat skins. The goatskin was dehaired by the enzyme at 33-35 °C in 6 h. The enzymatic dehaired pelt showed better general appearance and better whiteness by visual tests, and the grain surface of enzymatic dehaired pelt revealed absence of hair shaft with empty follicles by stereoscopic observation. Meanwhile, the epidermis was completely removed and the collagen fiber structure of enzymatic dehaired pelt was more opened, regular and even in dermis comparing with conventional dehaired pelt by histological analysis.

Glycine betaine enhances biodegradation of phenol in high saline environments by the halophilic strain Oceanobacillus sp. PT-20.

The halophilic bacterial strain PT-20, isolated from saline alkali soil samples and identified as a member of the genus Oceanobacillus, exhibited a robust ability to degrade phenol under high salt conditions. It was determined that strain PT-20 was capable of degrading 1000 mg L-1 phenol completely in the presence of 10% NaCl within 120 h. Under the optimal degradation conditions, pH 8.0, 3% NaCl and 30 °C, 1000 mg L-1 phenol could be completely degraded in 48 h. Interestingly, the biodegradation rate of phenol was dramatically improved in the presence of glycine betaine. When glycine betaine was added, the time required to degrade 1000 mg L-1 phenol completely was significantly reduced from 120 h to 72 h, and the corresponding average degradation rate increased from 8.43 to 14.28 mg L-1 h-1 with 10% NaCl. Furthermore, transcriptome analysis was performed to investigate the effects of phenol and glycine betaine on the transcriptional levels of strain PT-20. The results indicated that the addition of glycine betaine enhanced the resistance of cells to phenol, increased the growth rate of strain PT-20 and upregulated the expression of related enzyme genes. In addition, the results of enzyme activity assays indicated that strain PT-20 degraded phenol mainly through a meta-fission pathway.

Enhanced extracellular recombinant keratinase activity in Bacillus subtilis SCK6 through signal peptide optimization and site-directed mutagenesis.

Keratinase has a great commercial value owing to its applications in the enzymatic dehairing of goatskins. In this study, we adopted a combined strategy to enhance the extracellular recombinant keratinase activity in Bacillus subtilis SCK6. First, nine signal peptides were screened to enhance the expression of extracellular keratinase. The recombinant strain with SPLipA exhibited the highest extracellular keratinase activity of 739.03 U per mL, which was two-fold higher activity of the wild type. Second, based on the multiple sequence alignment with the bacterial alkaline proteases, the mutant (M123L/V149I/A242N) was introduced into the keratinase. Comparing with the wild type of keratinase, the mutant M123L/V149I/A242N showed an increase in the extracellular keratinase activity, which was about 1.2-fold higher activity of the wild type. Finally, the keratinase expression vector with SPLipA and mutant M123L/V149I/A242N was constructed, and the extracellular keratinase activity reported at 830.91 U per mL was a 2.2-fold activity of the wild type. Then, the mutant keratinase was purified and characterized. The mutant exhibited properties similar to those of the wild type at an optimal temperature of 60 °C and pH 10.0. Conclusively, the extracellular expression of keratinase was enhanced via a combined strategy, and the mutant keratinase demonstrated properties similar to that of the wild type of keratinase.

Culturable epiphytic bacteria isolated from Teleogryllus occipitalus crickets metabolize insecticides.

The development of insecticide resistance is attributed to evolutionary changes in pest insect genomes, such as alteration of drug target sites, upregulation of degrading enzymes, and enhancement of drug excretion. Beyond these well-known mechanisms, symbiotic bacteria may confer insecticide resistance to host crickets. The current study was designed to screen all possible culturable bacterial groups found living in and on the bodies of Teleogryllus occipitalis crickets. We recovered 263 visible bacterial colonies and cultured them individually. After identifying the colonies based on morphology and phylogenetic analysis, we shortlisted 55 bacterial strains belonging to 28 genera. Of these 55 bacterial strains, 18 degraded at least 50% of the original amount of 400 mg/L chlorpyrifos (CP) after 24 hr of coculture. Six of these strains degraded more than 70% of the original amount of 400 mg/L CP. Three strains had antagonistic effects on Bacillus thuringiensis growth. Additionally, the ability of the isolates to degrade glyphosate, phoxim, and esfenvalerate was assessed. We also detected extracellular hydrolase enzyme activities in these isolates. We propose that epiphytic bacterial strains play multiple roles in cricket biology, one of which contributes to chemical and biological pesticide resistance.

Streptomyces dioscori sp. nov., a Novel Endophytic Actinobacterium Isolated from Bulbil of Dioscorea bulbifera L.

A novel endophytic actinobacterium strain, A217T, was isolated from the bulbil of Dioscorea bulbifera L. Its taxonomic position was characterized using a polyphasic study. Morphological and chemotaxonomic characteristics of strain A217T were consistent with those of members of the genus Streptomyces: long straight to flexuous spore chain; cellular components contained LL-diaminopimelic acid, ribose, and small traces of glucose in whole-cell hydrolysates; MK-9(H6) and MK-9(H8) as predominant menaquinones. The patterns of major fatty acids are C16:0, anteiso-C15:0, C15:0, iso-C14:0, C16:1 ω7c, anteiso-C17:0, and iso-C17:1 ω5c. The polar lipids comprised diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol mannoside, and glycolipid, as well as two unidentified phospholipids, one unidentified aminolipid, and one unidentified aminophospholipid. The DNA G + C content of draft genome is 70.7 mol%. Analysis of the 16S rRNA gene sequence and phylogenetic trees revealed that the isolate was most closely related to S. aurantiacus JCM 4453T (99.0%), S. glomeroaurantiacus JCM 4677T (99.0%), and S. tauricus JCM 4837T (98.8%). The DNA-DNA hybridization values between the strain A217T and three reference strains ranged from 34.6% to 51.7%. On the basis of phenotypic and genotypic differentiation from all tested strains, isolate A217T is proposed to represent a novel species of the genus Streptomyces, named Streptomyces dioscori sp. nov. The type strain is A217T (= CGMCC 4.7415T = JCM 32173T).

Actinocorallia populi sp. nov., an endophytic actinomycete isolated from a root of Populus adenopoda (Maxim.).

An endophytic actinobacterium, strain A251T, was isolated from the root of Populus adenopoda Maxim and subjected to characterization using polyphasic taxonomy. Analysis of the 16S rRNA gene sequence revealed that the isolate represented a member of the phylogenetic cluster of the genus Actinocorallia and was most closely related to Actinocorallia aurantiaca JCM 8201T (98.0 %) and Actinocorallia libanotica IFO 10495T (98.0 %). DNA-DNA hybridization values between A251T and these strains were 41.2 % and 45.0 %, respectively. The G+C content of the DNA was 71.5 mol%. Major fatty acids were C16 : 0, C16 : 1ω7c and C18 : 1ω9c. The peptidoglycan diamino acid of A251T was meso-diaminopimelic acid and the whole-cell hydrolysates contained glucose and ribose. The major menaquinones were MK-9(H4) and MK-9(H6). The phospholipid profile included diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol, an undefined aminophospholipid and two undefined phospholipids. DNA-DNA hybridization data in combination with differences in the biochemical and physiological properties, indicated that A251T should be classified as representing a novel species within the genus Actinocorallia, for which the name Actinocorallia populi sp. nov. is proposed, with A251T (=CGMCC 4.7421T=JCM 32178T) as the type strain.

Halomonas saliphila sp. nov., a moderately halophilic bacterium isolated from a saline soil.

A novel, Gram-stain-negative, aerobic, rod-shaped, motile and moderately halophilic bacterium, designated strain LCB169T, was isolated from a saline soil sample from Gansu Province, PR China. The cells of LCB169T grew at 10-52 °C (optimum 30 °C), at pH 6.0-10.0 (optimum pH 8.0) and in the presence of 0-17 % (w/v) NaCl (optimum 10-15 %). Phylogenetic analysis based on 16S rRNA gene sequences and concatenated 16S rRNA, gyrB and rpoD genes sequences revealed that LCB169T represented a member of the genus Halomonas in the class Gammaproteobacteria. The most closely related species were Halomonas daqingensis DQD2-30T (98.0 % 16S rRNA gene sequence similarity), Halomonas kenyensis AIR-2T (97.8 %) and Halomonas desiderata FB2T (97.5 %). DNA-DNA relatedness values between LCB169T and H. daqingensis CGMCC 1.6443T, H. desiderata DSM 9502T and H. kenyensis DSM 17331T were 33, 35 and 38 %, respectively. The polar lipids were identified as diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylcholine and three unidentified phospholipids. The major fatty acids were C18 : 1ω7c, C16 : 0, C16 : 1ω7c and C12 : 0 3-OH. The genomic DNA G+C content was 66.1 mol% and the predominant respiratory quinone was Q-9. On the basis of the results of phenotypic, phylogenetic and chemotaxonomic analyses and DNA-DNA hybridization relatedness values, LCB169T is considered to represent a novel species of the genus Halomonas, for which the name Halomonas saliphila sp. nov. is proposed. The type strain is LCB169T (=CGMCC 1.15818T=KCTC 52618T).

Screening cyhalothrin degradation strains from locust epiphytic bacteria and studying Paracoccus acridae SCU-M53 cyhalothrin degradation process.

All locust epiphytic bacteria were screened and a total of 62 epiphytic bacteria were obtained from samples of Acrida cinerea. Via phylogenetic analysis, the 62 epiphytic bacteria were allocated to 27 genera, 18 families, 13 orders, six classes, and four phylums. Then, cyhalothrin degradation experiments were conducted, and the 10 strains that degraded more than 30% cyhalothrin and Paracoccus acridae SCU-M53 showed the highest cyhalothrin degradation rate of 70.5%. Furthermore, Paracoccus acridae SCU-M53 was selected for optimal cyhalothrin biodegradation conditions via the response surface method (Design-Expert). Under the optimum conditions (28 °C, 75 mg/L, and 180 rpm), the cyhalothrin degradation rate reached 79.84% after 2 days. This suggests the possibility that isolating biodegradation cyhalothrin strains from Acrida cinerea is feasible.

Ornithinibacillus salinisoli sp. nov., a moderately halophilic bacterium isolated from a saline-alkali soil.

A taxonomic study was performed on strain LCB256T, which was isolated from a saline-alkali soil sample taken from northwestern China. Cells of strain LCB256T were Gram-stain-positive, aerobic, rod-shaped and grew at 3-17 % (w/v) NaCl (optimum 10-15 %), 10-52 °C (optimum 25-30 °C) and pH 7.0-9.0 (optimum 8.0). Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain LCB256T was most closely related to the two genera of Ornithinibacillus and Oceanobacillus, showing highest sequence similarity to Oceanobacillus limi KCTC 13823T (97.8 %) and Ornithinibacillus bavariensis WSBC 24001T (97.2 %). The peptidoglycan amino acid type was found to be A4ß and the major respiratory quinone was determined to be MK-7. The polar lipid profile of strain LCB256T contained diphosphatidylglycerol, phosphatidylglycerol, one unidentified phospholipid and two unidentified aminolipids. The dominant cellular fatty acids were anteiso-C15 : 0 and iso-C15 : 0. The G+C content of genomic DNA was 39.3 mol%. DNA-DNA relatedness values between strain LCB256T and Ornithinibacillus halophilus KCTC 13822T and Oceanobacillus limi KCTC 13823T were 46.2 and 34.8 %, respectively. Based on this polyphasic taxonomic study, a novel species of the genus Ornithinibacillus, Ornithinibacillussalinisoli sp. nov. is proposed. The type strain is LCB256T (=CGMCC 1.15809T=KCTC 33862T).

Planococcus salinus sp. nov., a moderately halophilic bacterium isolated from a saline-alkali soil.

A novel aerobic, Gram-stain-positive, motile, moderately halophilic and coccoid bacterial strain, designated LCB217T, was isolated from a saline-alkali soil in north-western China and identified using a polyphasic taxonomic approach. Growth occurred with 3-15 % (w/v) NaCl (optimum 3-5 %), at 10-45 °C (optimum 30 °C) and at pH 7.0-9.0 (optimum pH 9.0). Strain LCB217T contained MK-7 and MK-8 as the predominant menaquinones and anteiso-C15 : 0, iso-C14 : 0 and iso-C16 : 0 as the major fatty acids. The polar lipids from strain LCB217T consisted of diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylcholine, one unidentified phospholipid, one unidentified aminophospholipid and one unidentified lipid. The peptidoglycan type was A4α (l-Lys-d-Glu). Phylogenetic analysis of the 16S rRNA gene sequence showed that strain LCB217T belonged to the genus Planococcus and was closely related to the type strains Planococcus plakortidis AS/ASP6 (II)T (98.2 % similarity), Planococcus maitriensis S1T (97.7 %) and Planococcus salinarum ISL-16T (97.2 %). The G+C content of the genomic DNA was 49.4 mol%. DNA-DNA relatedness values between strain LCB217T andPlanococcusplakortidis AS/ASP6 (II)T, Planococcusmaitriensis S1T andPlanococcussalinarum ISL-16T were 29.5, 38.1 and 39.5 %, respectively. On the basis of the phenotypic, phylogenetic and genomic data, strain LCB217T represents a novel species of the genus Planococcus, for which the name Planococcus salinus sp. nov. is proposed. The type strain is LCB217T (=CGMCC 1.15685T=KCTC 33861T).

Adaptations of Bacillus shacheensis HNA-14 required for long-term survival under osmotic challenge: a multi-omics perspective.

Genomic sequence, transcriptomic, metabolomic and fatty acid analyses of strain HNA-14 were performed to understand the mechanism of salt tolerance for long-term survival. The results indicated that strain HNA-14 has different osmotic resistance mechanisms for long-term survival and short-term salt stress. The cells mainly synthesized compatible solutes to resist osmotic pressure when cultured under nutrient deficient conditions, while they can slow down the synthesis rate and uptake from the environment when cultured under a nutritionally rich environment. Also, the amounts of branched and unsaturated fatty acids in the cell membrane are maintained to a high degree (>50%) to maintain the fluidity of the cell membrane; when the cells are cultured in a high osmotic environment for long-term survival, they may increase the content of branched fatty acids and phosphoric fatty acids to increase the fluidity of the cell membrane to resist the high osmotic pressure.

Diversity, bioactivities, and metabolic potentials of endophytic actinomycetes isolated from traditional medicinal plants in Sichuan, China.

The present study was designed to determine the taxonomic diversity and metabolic activity of the actinomycetes community, including 13 traditional medicinal plants collected in Sichuan province, China, using multiple approaches such as morphological and molecular identification methods, bioactivity assays, and PCR screening for genes involved in antibiotics biosynthesis. 119 endophytic actinomycetes were recovered; 80 representative strains were chosen for 16S rRNA gene partial sequence analyses, with 66 of them being affiliated to genus Streptomyces and the remaining 14 strains being rare actinomycetes. Antimicrobial tests showed that 12 (15%) of the 80 endophytic actinomycetes displayed inhibitory effects against at least one indicator pathogens, which were all assigned to the genus Streptomyces. In addition, 87.5% and 58.8% of the isolates showed anticancer and anti-diabetic activities, respectively. Meanwhile, the anticancer activities of the isolates negatively correlated with their anti-diabetic activities. Based on the results of PCR screening, five genes, PKS-I, PKS-II, NRPS, ANSA, and oxyB, were detected in 55.0%, 58.8%, 90.0%, 18.8% and 8.8% of the 80 actinomycetes, respectively. In conclusion, the PCR screening method employed in the present study was conducive for screening and selection of potential actinomycetes and predicting potential secondary metabolites, which could overcome the limitations of traditional activity screening models.

Bacillus shacheensis sp. nov., a moderately halophilic bacterium isolated from a saline-alkali soil.

A moderately halophilic bacterium, strain HNA-14(T), was isolated from a saline-alkali soil sample collected in Shache County, Xinjiang Province. On the basis of the polyphasic taxonomic data, the isolate was considered to be a member of the genus Bacillus. The organism grew optimally at 30 °C and pH 8.0. It was moderately halophilic and its optimum growth occurred at 5-10% NaCl. The diamino acid found in the cell-wall peptidoglycan was meso-diaminopimelic acid and the predominant menaquinone was MK-7. The major cellular fatty acids were anteiso-C15:0 and iso-C15:0 and the polar lipid profile consisted of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol, phosphatidylinositol mannosides and two unknown phospholipids. The G+C content of the genomic DNA was 48.6 mol%. Strain HNA-14(T) exhibited a low 16S rRNA gene sequence similarity of 96% with its nearest neighbors [Bacillus clausii KSM-K16 (96.5%), Bacillus xiaoxiensis DSM 21943(T)(96.2%), Bacillus clausii DSM 8716(T) (96.1%), Bacillus patagoniensis PAT05(T) (96.1%), Bacillus lehensis MLB-2(T) (96.0%), Bacillus oshimensis K11(T) (95.9%) and Bacillus hunanensis DSM 23008(T) (95.8%)] and the phenotypic characteristics indicate that strain HNA-14(T) can be distinguished from them. Therefore, a novel species of the genus Bacillus, Bacillus shacheensis sp. nov. (type strain, HNA-14(T) = KCTC 33145 = DSM 26902) is proposed.