Characterization of three rapidly growing novel Mycobacterium species with significant polycyclic aromatic hydrocarbon bioremediation potential.

Mycobacterium species exhibit high bioremediation potential for the degradation of polycyclic aromatic hydrocarbons (PAHs) that are significant environmental pollutants. In this study, three Gram-positive, rapidly growing strains (YC-RL4T, MB418T, and HX176T) were isolated from petroleum-contaminated soils and were classified as Mycobacterium within the family Mycobacteriaceae. Genomic average nucleotide identity (ANI; < 95%) and digital DNA-DNA hybridization (dDDH; < 70%) values relative to other Mycobacterium spp. indicated that the strains represented novel species. The morphological, physiological, and chemotaxonomic characteristics of the isolates also supported their affiliation with Mycobacterium and their delineation as novel species. The strains were identified as Mycobacterium adipatum sp. nov. (type strain YC-RL4T = CPCC 205684T = CGMCC 1.62027T), Mycobacterium deserti sp. nov. (type strain MB418T = CPCC 205710T = KCTC 49782T), and Mycobacterium hippophais sp. nov. (type strain HX176T = CPCC 205372T = KCTC 49413T). Genes encoding enzymes involved in PAH degradation and metal resistance were present in the genomes of all three strains. Specifically, genes encoding alpha subunits of aromatic ring-hydroxylating dioxygenases were encoded by the genomes. The genes were also identified as core genes in a pangenomic analysis of the three strains along with 70 phylogenetically related mycobacterial strains that were previously classified as Mycolicibacterium. Notably, strain YC-RL4T could not only utilize phthalates as their sole carbon source for growth, but also convert di-(2-ethylhexyl) phthalate into phthalic acid. These results indicated that strains YC-RL4T, MB418T, and HX176T were important resources with significant bioremediation potential in soils contaminated by PAHs and heavy metals.

Transcriptome analysis and cytochrome P450 monooxygenase reveal the molecular mechanism of Bisphenol A degradation by Pseudomonas putida strain YC-AE1.

BACKGROUND: Bisphenol A (BPA) is a rapid spreading organic pollutant that widely used in many industries especially as a plasticizer in polycarbonate plastic and epoxy resins. BPA reported as a prominent endocrine disruptor compound that possesses estrogenic activity and fulminant toxicity. Pseudomonas putida YC-AE1 was isolated in our previous study and exerted a strong degradation capacity toward BPA at high concentrations; however, the molecular degradation mechanism is still enigmatic. RESULTS: We employed RNA sequencing to analyze the differentially expressed genes (DEGs) in the YC-AE1 strain upon BPA induction. Out of 1229 differentially expressed genes, 725 genes were positively regulated, and 504 genes were down-regulated. The pathways of microbial metabolism in diverse environments were significantly enriched among DEGs based on KEGG enrichment analysis. qRT-PCR confirm the involvement of BPA degradation relevant genes in accordance with RNA Seq data. The degradation pathway of BPA in YC-AE1 was proposed with specific enzymes and encoded genes. The role of cytochrome P450 (CYP450) in BPA degradation was further verified. Sever decrease in BPA degradation was recorded by YC-AE1 in the presence of CYP450 inhibitor. Subsequently, CYP450bisdB deficient YC-AE1 strain △ bisdB lost its ability toward BPA transformation comparing with the wild type. Furthermore, Transformation of E. coli with pET-32a-bisdAB empowers it to degrade 66 mg l-1 of BPA after 24 h. Altogether, the results showed the role of CYP450 in biodegradation of BPA by YC-AE1. CONCLUSION: In this study we propose the molecular basis and the potential role of YC-AE1cytochrome P450 monooxygenase in BPA catabolism.

Characterization of the phosphotriesterase capable of hydrolyzing aryl-organophosphate flame retardants.

A related group of phosphotriesters known as organophosphate flame retardants (OPFRs) has become emerging contaminants due to its worldwide use. The lack of an easily hydrolysable bond renders OPFRs inert to the well-known phosphotriesterases capable of hydrolyzing the neurotoxic organophosphates. An OPFRs phosphotriesterase gene stpte was cloned from plasmid pStJH of strain Sphingopyxis terrae subsp. terrae YC-JH3 and was heterologously expressed in Escherichia coli. The recombinant protein St-PTE was purified and analyzed. St-PTE showed the highest catalytic activity at pH 8.5 and 35 °C. The optimal substrate for St-PTE is triphenyl phosphate, with kcat/Km of 5.03 × 106 M-1 s-1, two orders of magnitude higher than those of tricresyl phosphate (4.17 × 104 M-1 s-1), 2-ethylhexyl diphenyl phosphate (2.03 × 104 M-1 s-1) and resorcinol bis(diphenyl phosphate) (6.30 × 104 M-1 s-1). St-PTE could break the P-O bond of tri-esters and convert aryl-OPFRs into their corresponding di-ester metabolites, including polymers of resorcinol bis(diphenyl phosphate). Mediated by transposase, the gene of OPFRs phosphotriesterase could be transferred horizontally among closely related strains of Sphingomonas, Sphingobium and Sphingopyxis. KEY POINTS: • St-PTE from Sphingopyxis terrae subsp. terrae YC-JH3 could hydrolyze aryl-OPFRs. • Metabolites of RBDPP hydrolyzed by phosphotriesterase were identified. • St-PTE could hydrolyze the P-O cleavage of dimer and trimer of RBDPP. • Phosphotriesterase genes transfer among Sphingomonadaceae mediated by transposase.

First Report of Postharvest Blue Mold decay caused by Penicillium expansum on Lemon (Citrus limon) fruit in China.

Lemon (Citrus limon) is one of the most important commercial (both dried and fresh) citrus fruits in China. In the spring of 2019, postharvest blue mold decay was observed at an incidence of 3-5% on lemon fruit at the local markets in Beijing, China. Fruit lesions were circular, brown, soft, and watery, and rapidly expanded at 25°C. To isolate the causal organism, small pieces (2 mm3) were cut from the lesions, surface-sterilized for 1 min in 1.5% NaOCl, rinsed three times with sterilized water, dried with sterile filter paper, placed onto potato dextrose agar (PDA) medium, and incubated at 25°C for 6 days. Eight morphologically similar single-colony fungal isolates were recovered from six lemon fruit. Colony surfaces were bluish-green on the upper surface and cream to yellow-brown one the reverse. Hyphae on colony margins were entirely subsurface and cream in color. Mycelium was highly branched, septate, and colorless, and conidiophores were 250 to 450 × 3.0 to 4.0 µm in size. Stipe of conidiophores were smooth-walled, bearing terminal penicilli, typically terverticillate or less commonly birverticillate, rami occurring singly, 16 to 23 × 3.0 to 4.0 µm, metulae in 3 to 6, measuring 12 to 15 × 3.0 to 4.0 µm. Phialides were ampulliform to almost cylindrical, in verticils of 5 to 8, measuring 8 to 11 × 2.5 to 3.2 µm with collula. Conidia were smooth-walled, ellipsoidal, measuring 3.0 to 3.5 × 2.5 to 3.0 µm. According to morphological characteristics, the fungus was identified as Penicillium expansum (Visagie et al. 2014). For molecular identification, genomic DNA of eight fungal isolates was extracted, regions of the beta-tubulin (TUB), and calmodulin (CAL) genes and ITS region, were amplified using Bt2a/Bt2b, CAL-228F/ CAL-737, and ITS1/ITS4 primers respectively. Obtained sequences of all isolates were identical to sequences of the representative isolate YC-IK12, which was submitted in the GenBank. BLAST results of YC-IK12 sequences (ITS; MT856700: TUB; MT856958: CAL; MT856959) showed 98 to 100% similarity with P. expansum accessions (NR-077154, LN896428, JX141581). For pathogenicity tests, 10 µl of conidial suspension (10 × 105 conidia/ml) from seven-day-old YC-IK12 culture was inoculated using a sterilized needle into the surface of each five asymptomatic disinfected lemons. As a control, three lemons were inoculated using sterile distilled water. All inoculated lemons were placed in plastic containers and incubated at 25°C for 7 days. Decay lesions, identical to the original observations, developed on all inoculated lemons, while control lemons remained asymptomatic. Fungus re-isolated from the inoculated lemon was identified as P. expansum on the basis morphology and Bt2a/Bt2b, CAL-228F/ CAL-737, and ITS1/ITS4 sequences. Previously, Penicillium spp. including P. expansum have been reported as post-harvest pathogens on various Citrus spp. (Louw & Korsten 2015). However, P. digitatum has been reported on lemons and P. expansum has been reported on stored Kiwifruit (Actinidia arguta), Malus, and Pyrus species in China (Tai, 1979; Wang et al. 2015). To our knowledge, this is the first report of blue mold caused by P. expansum on lemons in China. References Louw, J. P., Korsten, L. 2015. Plant Dis. 99:21-30. Tai, F.L. 1979. Sylloge Fungorum Sinicorum. Sci. Press, Acad. Sin., Peking, 1527 pages. 8097 Visagie, C.M. et al. 2014. Studies. Mycol.78: 343. Wang, C. W. et al. 2015. Plant Dis. 99:1037.

Primary biodegradation and mineralization of aryl organophosphate flame retardants by Rhodococcus-Sphingopyxis consortium.

In this study, the biodegradation towards aryl organophosphate flame retardants (aryl-OPFRs) was investigated by the Rhodococcus-Sphingopyxis consortium, mixture of strain Rhodococcus sp. YC-JH2 and Sphingopyxis sp. YC-JH3. The optimal ratio between the two composition strains was determined as 1:1. Under the optimum condition (pH 8, 35 °C and 0% salinity), the consortium could utilize aryl-OPFRs as sole carbon source and degrade them rapidly with half-life of 4.53, 21.11 and 23.0 h for triphenyl phosphate (TPhP), tricresyl phosphate (TCrP) and 2-ethylhexyl diphenyl phosphate (EHDPP) respectively. The consortium maintained high degrading efficiency under a wide of range of pH (6-10), temperature (20-40 °C) and salinity (0-6%). Besides, the consortium could rapidly degrade high concentration of TPhP and no inhibitory effect towards degradation speed was observed up to 500 mg/L. The effect of metal ions and surfactants was estimated. Most metal ions exhibited significant inhibition, except Zn2+ and Pb2+, which showed no effect or slight promotion. Ionic surfactants could severely reduce the degrading capacity, while nonionic surfactants showed no effect. With abundant inoculation of the consortium, mineralization higher than 75% could be achieved within a week. This study provides efficient microorganisms for bioremediation of aryl-OPFRs contamination.

Biodegradation of Di (2-Ethylhexyl) Phthalate by a novel Enterobacter spp. Strain YC-IL1 Isolated from Polluted Soil, Mila, Algeria.

Di-(2-ethylhexyl) phthalate (DEHP) is one of the phthalic acid ester representatives and is mainly used as a plasticizer to endow polyvinyl chloride plastics with desirable physical properties. It is synthesized in massive amounts worldwide. Many studies have proved the adverse effects of DEHP on human health and wildlife. DEHP is labeled as an endocrine disruptor which causes human reproductive problems. Enterobacter spp. YC-IL1, a novel isolated strain from contaminated soil, was identified by 16S rRNA gene analysis and electronic microscope. It is capable of efficiently degrading DEHP (100%) and a wide range of phthalic acid ester PAEs, particularly those containing side chains with branches, or ring structures such as dutylbenzyl phthalate and dicyclohexyl phthalate, which are hard to degrade, with, respectively, 81.15% and 50.69% degradation after 7 days incubation. YC-IL1 is an acido-tolerant strain which remained in pH values lower than pH 5.0 with the optimum pH 7.0 and temperature 30 °C. The DEHP metabolites were detected using HPLC-QQQ and then the degradation pathway was tentatively proposed. Strain YC-IL1 showed high DEHP degradation rate in artificially contaminated soil with 86% removed in 6 days. These results indicate the application potential of YC-IL1 in bioremediation of PAE-polluted sites, even the acidic ones.

Cometabolic biodegradation of quizalofop-p-ethyl by Methylobacterium populi YC-XJ1 and identification of QPEH1 esterase

BACKGROUND: Quizalofop-p-ethyl (QPE), a unitary R configuration aromatic oxyphenoxypropionic acid ester (AOPP) herbicide, was widely used and had led to detrimental environmental effects. For finding the QPEdegrading bacteria and promoting the biodegradation of QPE, a series of studies were carried out. RESULTS: A QPE-degrading bacterial strain YC-XJ1 was isolated from desert soil and identified as Methylobacterium populi, which could degrade QPE with methanol by cometabolism. Ninety-seven percent of QPE (50 mg/L) could be degraded within 72 h under optimum biodegradation condition of 35°C and pH 8.0. The maximum degradation rate of QPE was 1.4 mg/L/h, and the strain YC-XJ1 exhibited some certain salinity tolerance. Two novel metabolites, 2-hydroxy-6-chloroquinoxaline and quinoxaline, were found by high-performance liquid chromatography/mass spectroscopy analysis. The metabolic pathway of QPE was predicted. The catalytic efficiency of strain YC-XJ1 toward different AOPPs herbicides in descending order was as follows: haloxyfop-pmethyl ≈ diclofop-methyl ≈ fluazifop-p-butyl N clodinafop-propargyl N cyhalofop-butyl N quizalofop-p-ethyl N fenoxaprop-p-ethyl N propaquizafop N quizalofop-p-tefuryl. The genome of strain YC-XJ1 was sequenced using a combination of PacBio RS II and Illumina platforms. According to the annotation result, one α/ß hydrolase gene was selected and named qpeh1, for which QPE-degrading function has obtained validation. Based on the phylogenetic analysis and multiple sequence alignment with other QPE-degrading esterases reported previously, the QPEH1 was clustered with esterase family V. CONCLUSION: M. populi YC-XJ1 could degrade QPE with a novel pathway, and the qpeh1 gene was identified as one of QPE-degrading esterase gene.

The Genome Analysis of Methylobacterium populi YC-XJ1 with Diverse Xenobiotics Biodegrading Capacity and Degradation Characteristics of Related Hydrolase.

Methylobacterium populi YC-XJ1 isolated from desert soil exhibited a diverse degrading ability towards aromatic oxyphenoxypropionic acid esters (AOPPs) herbicide, phthalate esters (PAEs), organophosphorus flame retardants (OPFRs), chlorpyrifos and phoxim. The genome of YC-XJ1 was sequenced and analyzed systematically. YC-XJ1 contained a large number of exogenous compounds degradation pathways and hydrolase resources. The quizalofop-p-ethyl (QPE) degrading gene qpeh2 and diethyl phthalate (DEP) degrading gene deph1 were cloned and expressed. The characteristics of corresponding hydrolases were investigated. The specific activity of recombinant QPEH2 was 0.1 ± 0.02 U mg-1 for QPE with kcat/Km values of 1.8 ± 0.016 (mM-1·s-1). The specific activity of recombinant DEPH1 was 0.1 ± 0.02 U mg-1 for DEP with kcat/Km values of 0.8 ± 0.02 (mM-1·s-1). This work systematically illuminated the metabolic versatility of strain YC-XJ1 via the combination of genomics analysis and laboratory experiments. These results suggested that strain YC-XJ1 with diverse xenobiotics biodegrading capacity was a promising candidate for the bioremediation of polluted sites.

Biodegradation of Bisphenol A by Sphingobium sp. YC-JY1 and the Essential Role of Cytochrome P450 Monooxygenase.

Bisphenol A (BPA) is a widespread pollutant threatening the ecosystem and human health. An effective BPA degrader YC-JY1 was isolated and identified as Sphingobium sp. The optimal temperature and pH for the degradation of BPA by strain YC-JY1 were 30 °C and 6.5, respectively. The biodegradation pathway was proposed based on the identification of the metabolites. The addition of cytochrome P450 (CYP) inhibitor 1-aminobenzotriazole significantly decreased the degradation of BPA by Sphingobium sp. YC-JY1. Escherichia coli BL21 (DE3) cells harboring pET28a-bisdAB achieved the ability to degrade BPA. The bisdB gene knockout strain YC-JY1ΔbisdB was unable to degrade BPA indicating that P450bisdB was an essential initiator of BPA metabolism in strain YC-JY1. For BPA polluted soil remediation, strain YC-JY1 considerably stimulated biodegradation of BPA associated with the soil microbial community. These results point out that strain YC-JY1 is a promising microbe for BPA removal and possesses great application potential.

Biodegradation of endocrine disruptor Bisphenol A by Pseudomonas putida strain YC-AE1 isolated from polluted soil, Guangdong, China.

BACKGROUND: Bisphenol A is an important organic chemical as an intermediate, final and inert ingredient in manufacturing of many important products like polycarbonate plastics, epoxy resins, flame retardants, food-drink packaging coating, and other. BPA is an endocrine disruptor compound that mimics the function of estrogen causing damage to reproductive organs. Bacterial degradation has been consider as a cost effective and eco-friendly method for BPA degradation compared with physical and chemical methods. This study aimed to isolate and identify bacterial strain capable to degrade and tolerate high concentrations of this pollutant, studying the factors affecting the degradation process and study the degradation mechanism of this strain. RESULTS: YC-AE1 is a Gram negative bacterial strain isolated from soil and identified as Pseudomonas putida by 16S rRNA gene sequence and BIOLOG identification system. This strain found to have a high capacity to degrade the endocrine disruptor Bisphenol A (BPA). Response surface methodology using central composite design was used to statistically optimize the environmental factors during BPA degradation and the results obtained by significant model were 7.2, 30 °C and 2.5% for optimum initial pH, temperature and inoculum size, respectively. Prolonged incubation period with low NaCl concentration improve the biodegradation of BPA. Analysis of variance (ANOVA) showed high coefficient of determination, R2 and Adj-R2 which were 0.9979 and 0.9935, respectively. Substrate analysis found that, strain YC-AE1 could degrade a wide variety of bisphenol A-related pollutants such as bisphenol B, bisphenol F, bisphenol S, Dibutyl phthalate, Diethylhexyl phthalate and Diethyl phthalate in varying proportion. Pseudomonas putida YC-AE1 showed high ability to degrade a wide range of BPA concentrations (0.5-1000 mg l- 1) with completely degradation for 500 mg l- 1 within 72 h. Metabolic intermediates detected in this study by HPLC-MS were identified as 4,4-dihydroxy-alpha-methylstilbene, p-hydroxybenzaldeyde, p-hydroxyacetophenone, 4-hydroxyphenylacetate, 4-hydroxyphenacyl alcohol, 2,2-bis(4-hydroxyphenyl)-1-propanol, 1,2-bis(4-hydroxyphenyl)-2-propanol and 2,2-bis(4-hydroxyphenyl) propanoate. CONCLUSIONS: This study reports Pseudomonas putida YC-AE1 as BPA biodegrader with high performance in degradation and tolerance to high BPA concentration. It exhibited strong degradation capacity and prominent adaptability towards a wide range of environmental conditions. Moreover, it degrades BPA in a short time via two different degradation pathways.

[Characterization and 16S rRNA gene-based metagenomic analysis of the organophosphorous flame retardants degrading consortium YC-BJ1].

As flame retardants, organophosphate is recognized as a global environmental contaminant because of its wide application. This contaminant is hardly degradable by hydrolysis in the environment due to its special physicochemical properties. Therefore, it is of urgent needs to study the microbial degradation of organophosphate. Through continuous enrichment, we isolated one bacterial consortium, named YC-BJ1, from leachate of waste treatment plant in Beijing. The bacterial consortium YC-BJ1 could efficiently degrade 99.8% of triphenyl phosphate (TPhP) and 91.9% of tricresyl phosphate (TCrP) with the concentration of 100 mg/L within 4 days. Besides aryl phosphates, it could degrade chloro-phosphates, tris(1,3-dichloroisopropyl) phosphate (TDCPP) and tris(2-chloroethyl) phosphate (TCEP) by 16.5% and 22.0% respectively. The degradation of the consortium on TPhP was optimized through a broad range of temperature (15-40 ℃), pH (5.0-12.0) and salinity (0%-4%). 16S rRNA gene-based metagenomic analysis revealed that Hyphomicrobium (38.80%), Chryseobacterium (17.57%) and Sphingopyxis (17.46%) were the dominant genera of the consortium YC-BJ1. Compared with the reported organophosphorus flame retardants (OPFRs) degrading bacteria and microflora, the mixed microflora YC-BJ1 exhibited great advantages in degradation efficiency and environmental adaptability, demonstrating its wide application potential. The enrichment and isolation of highly efficient degrading flora can provide abundant microbial resources for the degradation of OPFRs and the bioremediation towards OPFRs-contaminated environments, and also lay a solid foundation for the exploration of its degradation mechanism.

Characterization and 16S metagenomic analysis of organophosphorus flame retardants degrading consortia.

Three bacterial consortia, named YC-SY1, YC-BJ1 and YC-GZ1, were enriched from different areas of China. Bacterial consortia YC-SY1, YC-BJ1 and YC-GZ1 could efficiently degrade triphenyl phosphate (TPhP) (100 mg/L) by approximately 79.4%, 99.8% and 99.6%, tricresyl phosphate (TCrP) by 90.6%, 91.9% and 96.3%, respectively, within 4 days. And they could retain high degrading efficiency under a broad range of temperature (15-40 ℃), pH (6.0-10.0) and salinity (0-4%). A total of 10 bacterial isolates were selected and investigated their degradation capacity. Among these isolates, two were significantly superior to the others. Strain Rhodococcus sp. YC-JH2 could utilize TPhP (50 mg/L) as sole carbon source for growth with 37.36% degradation within 7 days. Strain Sphingopyxis sp. YC-JH3 could efficiently degrade 96.2% of TPhP (50 mg/L) within 7 days, except that no cell growth was observed. Combined with 16S diversity analysis, our results suggest that the effective components of three bacterial consortia responsible for TPhP and TCrP degradation were almost the same, that is, bacteria capable of degrading TPhP and TCrP are limited, in this study, the most efficient component is Sphingopyxis. This study provides abundant microorganism sources for research on organophosphorus flame retardants (OPFRs) metabolism and bioremediation towards OPFRs-contaminated environments.

Identification and characterization of a meta-cleavage product hydrolase involved in biphenyl degradation from Arthrobacter sp. YC-RL1.

Polychlorinated biphenyls (PCBs) are a group of persistent organic pollutants (POPs) widely existing in the environment. Arthrobacter sp. YC-RL1 is a biphenyl-degrading bacterium that shows metabolic versatility towards aromatic compounds. A 2-hydroxy-6-oxo-6-phenylhexa-2, 4-dienoate (HOPDA) hydrolase (BphD) gene involved in the biodegradation of biphenyl was cloned from strain YC-RL1 and heterologously expressed in Escherichia coli BL21 (DE3). The recombinant BphDYC-RL1 was purified and characterized. BphDYC-RL1 showed the highest activity at 45 °C and pH 7. It was stable under a wide range of temperature (20-50 °C). The enzyme had a Km value of 0.14 mM, Kcat of 11.61 s-1, and Vmax of 0.027 U/mg. Temperature dependence catalysis exhibited a biphasic Arrhenius Plot with a transition at 20 °C. BphDYC-RL1 was inactivated by SDS, Tween 20, Tween 80, Trition X-100, DTT, CHAPS, NBS, PMSF, and DEPC, but insensitive to EDTA. Site-directed mutagenesis of the active-site residues revealed that the catalytic triad residues (Ser115, His275, and Asp247) of BphDYC-RL1 were necessary for its activity. The investigation of BphDYC-RL1 not only provides new potential enzyme resource for the biodegradation of biphenyl but also helps deepen our understanding on the catalytic process and mechanism.

In silico genome analysis reveals the metabolic versatility and biotechnology potential of a halotorelant phthalic acid esters degrading Gordonia alkanivorans strain YC-RL2.

Members of genus Gordonia are known to degrade various xenobitics and produce secondary metabolites. The genome of a halotorelant phthalic acid ester (PAEs) degrading actinobacterium Gordonia alkanivorans strain YC-RL2 was sequenced using Biosciences RS II platform and Single Molecular Real-Time (SMRT) technology. The reads were assembled de novo by hierarchical genome assembly process (HGAP) algorithm version 2. Genes were annotated by NCBI Prokaryotic Genome Annotation Pipeline. The generated genome sequence was 4,979,656 bp with an average G+C content of 67.45%. Calculation of ANI confirmed previous classification that strain YC-RL2 is G. alkanivorans. The sequences were searched against KEGG and COG databases; 3132 CDSs were assigned to COG families and 1808 CDSs were predicted to be involved in 111 pathways. 95 of the KEGG annotated genes were predicted to be involved in the degradation of xenobiotics. A phthalate degradation operon could not be identified in the genome indicating that strain YC-RL2 possesses a novel way of phthalate degradation. A total of 203 and 22 CDSs were annotated as esterase/hydrolase and dioxygenase genes respectively. A total of 53 biosynthetic gene clusters (BGCs) were predicted by antiSMASH (antibiotics & Secondary Metabolite Analysis Shell) bacterial version 4.0. The genome also contained putative genes for heavy metal metabolism. The strain could tolerate 1 mM of Cd2+, Co2+, Cu2+, Ni2+, Zn2+, Mn2+ and Pb2+ ions. These results show that strain YC-RL2 has a great potential to degrade various xenobiotics in different environments and will provide a rich genetic resource for further biotechnological and remediation studies.

Draft Genome Sequence of Halomonas urumqiensis BZ-SZ-XJ27T, an Aerobic Halophilic Bacterium Isolated from a Salt Lake.

The aerobic halophilic bacterium Halomonas urumqiensis BZ-SZ-XJ27T, growing optimally at 1.42 M Na+, with a range of 0.22 to 4.32 M Na+, was isolated from a salt lake in the Xinjiang Uyghur Autonomous Region of China. Here, we report the draft genome sequence of strain BZ-SZ-XJ27T, which consists of approximately 3.97 Mb and contains 3,588 predicted genes. Some of the genes that maintain intracellular osmotic balance were identified, offering valuable insights into specific adaptations to the hypersaline environment.

Insights into Xylan Degradation and Haloalkaline Adaptation through Whole-Genome Analysis of Alkalitalea saponilacus, an Anaerobic Haloalkaliphilic Bacterium Capable of Secreting Novel Halostable Xylanase.

The obligately anaerobic haloalkaliphilic bacterium Alkalitalea saponilacus can use xylan as the sole carbon source and produce propionate as the main fermentation product. Using mixed carbon sources of 0.4% (w/v) sucrose and 0.1% (w/v) birch xylan, xylanase production from A. saponilacus was 3.2-fold greater than that of individual carbon sources of 0.5% (w/v) sucrose or 0.5% (w/v) birch xylan. The xylanse is halostable and exhibits optimal activity over a broad salt concentration (2⁻6% NaCl). Its activity increased approximately 1.16-fold by adding 0.2% (v/v) Tween 20. To understand the potential genetic mechanisms of xylan degradation and molecular adaptation to saline-alkali extremes, the complete genome sequence of A. saponilacus was performed with the pacBio single-molecule real-time (SMRT) and Illumina Misseq platforms. The genome contained one chromosome with a total size of 4,775,573 bps, and a G+C genomic content of 39.27%. Ten genes relating to the pathway for complete xylan degradation were systematically identified. Furthermore, various genes were predicted to be involved in isosmotic cytoplasm via the "compatible-solutes strategy" and cytoplasmic pH homeostasis though the "influx of hydrogen ions". The halostable xylanase from A. saponilacus and its genomic sequence information provide some insight for potential applications in industry under double extreme conditions.

Toxicity Evaluation and Biomarker Selection with Validated Reference Gene in Embryonic Zebrafish Exposed to Mitoxantrone.

Notwithstanding the widespread use and promising clinical value of chemotherapy, the pharmacokinetics, toxicology, and mechanism of mitoxantrone remains unclear. To promote the clinical value in the treatment of human diseases and the exploration of potential subtle effects of mitoxantrone, zebrafish embryos were employed to evaluate toxicity with validated reference genes based on independent stability evaluation programs. The most stable and recommended reference gene was gapdh, followed by tubα1b, for the 48 h post fertilization (hpf) zebrafish embryo mitoxantrone test, while both eef1a1l1 and rpl13α were recommended as reference genes for the 96 hpf zebrafish embryo mitoxantrone test. With gapdh as an internal control, we analyzed the mRNA levels of representative hepatotoxicity biomarkers, including fabp10a, gclc, gsr, nqo1, cardiotoxicity biomarker erg, and neurotoxicity biomarker gfap in the 48 hpf embryo mitoxantrone test. The mRNA levels of gclc, gsr, and gfap increased significantly in 10 and 50 µg/L mitoxantrone-treated 48 hpf embryos, while the transcript levels of fabp10a decreased in a dose-dependent manner, indicating that mitoxantrone induced hepatotoxicity and neurotoxicity. Liver hematoxylin⁻eosin staining and the spontaneous movement of embryos confirmed the results. Thus, the present research suggests that mitoxantrone induces toxicity during the development of the liver and nervous system in zebrafish embryos and that fabp10a is recommended as a potential biomarker for hepatotoxicity in zebrafish embryos. Additionally, gapdh is proposed as a reference gene for the 48 hpf zebrafish embryo mitoxantrone toxicity test, while eef1a1l1 and rpl13α are proposed as that for the 96 hpf test.

Insight Into Metabolic Versatility of an Aromatic Compounds-Degrading Arthrobacter sp. YC-RL1.

The genus Arthrobacter is ubiquitously distributed in different natural environments. Many xenobiotic-degrading Arthrobacter strains have been isolated and described; however, few have been systematically characterized with regard to multiple interrelated metabolic pathways and the genes that encode them. In this study, the biodegradability of seven aromatic compounds by Arthrobacter sp. YC-RL1 was investigated. Strain YC-RL1 could efficiently degrade p-xylene (PX), naphthalene, phenanthrene, biphenyl, p-nitrophenol (PNP), and bisphenol A (BPA) under both separated and mixed conditions. Based on the detected metabolic intermediates, metabolic pathways of naphthalene, biphenyl, PNP, and BPA were proposed, which indicated that strain YC-RL1 harbors systematic metabolic pathways toward aromatic compounds. Further, genomic analysis uncovered part of genes involved in the proposed pathways. Both intradiol and extradiol ring-cleavage dioxygenase genes were identified in the genome of strain YC-RL1. Meanwhile, gene clusters predicted to encode the degradation of biphenyl (bph), para-substituted phenols (npd) and protocatechuate (pca) were identified, and bphA1A2BCD was proposed to be a novel biphenyl-degrading gene cluster. The complete metabolic pathway of biphenyl was deduced via intermediates and functional gene analysis (bph and pca gene clusters). One of the these genes encoding ring-cleavage dioxygenase in bph gene cluster, a predicted 2,3-dihydroxybiphenyl 1,2-dioxygenase (BphC) gene, was cloned and its activity was confirmed by heterologous expression. This work systematically illuminated the metabolic versatility of aromatic compounds in strain YC-RL1 via the combination of metabolites identification, genomics analysis and laboratory experiments. These results suggested that strain YC-RL1 might be a promising candidate for the bioremediation of aromatic compounds pollution sites.

Chloroplast proteome analysis of Nicotiana tabacum overexpressing TERF1 under drought stress condition.

BACKGROUND: Chloroplast is indispensable for plant response to environmental stresses, growth and development, whose function is regulated by different plant hormones. The chloroplast proteome is encoded by chloroplast genome and nuclear genome, which play essential roles in plant photosynthesis, metabolism and other biological processes. Ethylene response factors (ERFs) are key transcription factors in activating the ethylene signaling pathway and plant response to abiotic stress. But we know little about how ethylene regulates plastid function under drought stress condition. In this study we utilized tobacco overexpressing tomato ethylene responsive factor 1 (TERF1), an ERF transcription factor isolated from tomato, to investigate its effects on the plastid proteome under drought stress condition by method of iTRAQ technology. RESULTS: Results show that TERF1 represses the genes encoding the photosynthetic apparatus at both transcriptional and translational level, but the genes involved in carbon fixation are significantly induced by TERF1. TERF1 regulates multiple retrograde signaling pathways, providing a new mechanism for regulating nuclear gene expression. TERF1 also regulates plant utilization of phosphorus (Pi) and nitrogen (N). We find that several metabolic and signaling pathways related with Pi are significantly repressed and gene expression analysis shows that TERF1 significantly represses the Pi transport from root to shoot. However, the N metabolism is upregulated by TERF1 as shown by the activation of different amino acids biosynthesis pathways due to the induction of glutamine synthetase and stabilization of nitrate reductase although the root-to-shoot N transport is also reduced. TERF1 also regulates other core metabolic pathways and secondary metabolic pathways that are important for plant growth, development and response to environmental stresses. Gene set linkage analysis was applied for the upregulated proteins by TERF1, showing some new potential for regulating plant response to drought stress by TERF1. CONCLUSIONS: Our research reveals effects of ethylene signaling on plastid proteome related with two key biological processes, including photosynthesis and nutrition utilization. We also provide a new mechanism to regulate nuclear gene expression by ERF1 transcription factor through retrograde signals in chloroplast. These results can enrich our knowledge about ERF1 transcription factor and function of ethylene signaling pathway.