The critical roles of propanethiol oxidoreductase and sulfide-quinone oxidoreductase in the propanethiol catabolism pathway in Pseudomonas putida S-1.

Propanethiol (PT) is a hazardous pollutant that poses risks to both the environment and human well-being. Pseudomonas putida S-1 has been identified as a microorganism capable of utilizing PT as its sole carbon source. However, the metabolic pathway responsible for PT degradation in P. putida S-1 has remained poorly understood, impeding its optimization and practical application. In this study, we investigated the catabolic network involved in PT desulfurization with P. putida S-1 and identified key gene modules crucial to this process. Notably, propanethiol oxidoreductase (PTO) catalyzes the initial degradation of PT, a pivotal step for P. putida S-1's survival on PT. PTO facilitates the oxidation of PT, resulting H2S, H2O2, and propionaldehyde (PA). Catalase-peroxidase catalyzes the conversion of H2O2 to oxygen and water, while PA undergoes gradual conversion to Succinyl-CoA, which is subsequently utilized in the tricarboxylic acid cycle. H2S is digested in a comprehensive desulfurization network where sulfide-quinone oxidoreductase (SQOR) predominantly converts it to sulfane sulfur. The transcriptome analysis suggests that sulfur can be finally converted to sulfite or sulfate and exported out of the cell. The PT degradation capacity of P. putida S-1 was enhanced by increasing the transcription level of PTO and SQOR genes in vivo.IMPORTANCEThis work investigated the PT catabolism pathway in Pseudomonas putida S-1, a microorganism capable of utilizing PT as the sole carbon source. Critical genes that control the initiation of PT degradation were identified and characterized, such as pto and sqor. By increasing the transcription level of pto and sqor genes in vivo, we have successfully enhanced the PT degradation efficiency and growth rate of P. putida S-1. This work does not only reveal a unique PT degradation pathway but also highlights the potential of enhancing the microbial desulfurization process in the bioremediation of thiol-contaminated environment.

Assimilation of phthalate esters in bacteria.

The massive usage of phthalate esters (PAEs) has caused serious pollution. Bacterial degradation is a potential strategy to remove PAE contamination. So far, an increasing number of PAE-degrading strains have been isolated, and the catabolism of PAEs has been extensively studied and reviewed. However, the investigation into the bacterial PAE uptake process has received limited attention and remains preliminary. PAEs can interact spontaneously with compounds like peptidoglycan, lipopolysaccharides, and lipids on the bacterial cell envelope to migrate inside. However, this process compromises the structural integrity of the cells and causes disruptions. Thus, membrane protein-facilitated transport seems to be the main assimilation strategy in bacteria. So far, only an ATP-binding-cassette transporter PatDABC was proven to transport PAEs across the cytomembrane in a Gram-positive bacterium Rhodococcus jostii RHA1. Other cytomembrane proteins like major facilitator superfamily (MFS) proteins and outer membrane proteins in cell walls like FadL family channels, TonB-dependent transporters, and OmpW family proteins were only reported to facilitate the transport of PAEs analogs such as monoaromatic and polyaromatic hydrocarbons. The functions of these proteins in the intracellular transport of PAEs in bacteria await characterization and it is a promising avenue for future research on enhancing bacterial degradation of PAEs. KEY POINTS: • Membrane proteins on the bacterial cell envelope may be PAE transporters. • Most potential transporters need experimental validation.

Comparison of anti-tumor activities and underlying mechanisms of glucuronomannan oligosaccharides and its sulfated derivatives on the hepatocarcinoma Huh7.5 cells.

Hepatocellular carcinoma (HCC) is an aggressive tumor triggered by various factors such as virus infection and alcohol abuse. Glucuronomannan polysaccharide (Gx) is a subtype of fucoidans that possesses many bioactivities, but its anti-tumor activities in HCC have not been reported. In this paper, the anti-tumor effects of glucuronomannan oligosaccharides (Gx) and its sulfated derivatives (GxSy) on hepatocarcinoma Huh7.5 cells were investigated. The anti-proliferation, anti-metastasis activities, and underlying mechanism of Gx and GxSy on Huh7.5 cells were analyzed and compared by MTT, wound healing, transwell, and western blotting assays, respectively. Results showed that the best anti-proliferation effects were G4S1 and G4S2 among 13 drugs, which were 38.67% and 30.14%, respectively. The cell migration rates were significantly inhibited by G2S1, G4S2, G6S2, and unsulfated Gn. In addition, cell invasion effects treated with G4S1, G4S2, and G6S1 decreased to 48.62%, 36.26%, and 42.86%, respectively. Furthermore, sulfated G4 regulated the expression of (p-) FAK and MAPK pathway, and sulfated G6 down-regulated the MAPK signaling pathway while activating the PI3K/AKT pathway. On the contrary, sulfated G2 and unsulfated Gx had no inhibited effects on the FAK-mTOR pathway. These results indicated that sulfated Gx derivatives have better anti-tumor activities than unsulfated Gx in cell proliferation and metastasis process in vitro, and those properties depend on the sulfation group levels. Moreover, degrees of polymerization of Gx also played a vital role in mechanisms and bioactivities. This finding shows the structure-activity relationship for developing and applying the marine oligosaccharide candidates.

Regulation Mechanism of Nicotine Catabolism in Sphingomonas melonis TY by a Dual Role Transcriptional Regulator NdpR.

A gene cluster ndp, responsible for nicotine degradation via a variant of the pyridine and pyrrolidine pathways, was previously identified in Sphingomonas melonis TY, but the regulation mechanism remains unknown. The gene ndpR within the cluster was predicted to encode a TetR family transcriptional regulator. Deletion of ndpR resulted in a notably shorter lag phase, higher maximum turbidity, and faster substrate degradation when cultivated in the presence of nicotine. Real-time quantitative PCR and promoter activity analysis in wild-type TY and TYΔndpR strains revealed that genes in the ndp cluster were negatively regulated by NdpR. However, complementation of ndpR to TYΔndpR did not restore transcription repression, but, instead, the complemented strain showed better growth than TYΔndpR. Promoter activity analysis indicates that NdpR also functions as an activator in the transcription regulation of ndpHFEGD. Further analysis through electrophoretic mobility shift assay and DNase I footprinting assay revealed that NdpR binds five DNA sequences within ndp and that NdpR has no autoregulation. These binding motifs overlap with the -35 or -10 box or are located distal upstream of the corresponding transcriptional start site. Multiple sequence alignment of these five NdpR-binding DNA sequences found a conserved motif, with two of the binding sequences being partially palindromic. 2,5-Dihydroxypyridine acted as a ligand of NdpR, preventing NdpR from binding to the promoter region of ndpASAL, ndpTB, and ndpHFEGD. This study revealed that NdpR binds to three promoters in the ndp cluster and is a dual-role transcriptional regulator in nicotine metabolism. IMPORTANCE Gene regulation is critical for microorganisms in the environment in which they may encounter various kinds of organic pollutants. Our study revealed that transcription of ndpASAL, ndpTB, and ndpHFEGD is negatively regulated by NdpR, and NdpR also exhibits a positive regulatory effect on PndpHFEGD. Furthermore, 2,5-dihydroxypyridine was identified as the effector molecular for NdpR and can both prevent the binding of free NdpR to the promoter and release NdpR from the promoters, which is different from previously reported NicR2. Additionally, NdpR was found to have both negative and positive transcription regulatory effects on the same target, PndpHFEGD, while only one binding site was identified, which is notably different from the previously reported TetR family regulators. Moreover, NdpR was revealed to be a global transcriptional regulator. This study provides new insight into the complex gene expression regulation of the TetR family.

Effects of the multi-stress-resistant strain Zygosaccharomyces parabailii MC-5K3 bioaugmentation on microbial communities and metabolomics in tobacco waste extract.

Industrial tobacco waste was mainly treated via a reconstituted tobacco process using the paper-making method, which involves aqueous concentrated tobacco waste extract (cTWE) fermentation (aging). The fermentation was done to improve the quality of reconstituted tobacco. However, cTWE is a multi-stress environment that is characterized by low pH (about 4), as well as high sugar (above 150 g/L) and nicotine (above 15 g/L) content. In this study, a specific selection strategy was used to successfully isolate multi-stress-resistant bacterial or fungal strains, that exhibited positive effects on cTWE fermentation, thereby improving the quality of final products. A potential strain Zygosaccharomyces parabailii MC-5K3 was used for the bioaugmentation of cTWE fermentation and it significantly influenced the microbial diversity of the fermented cTWE. Zygosaccharomyces was observed to be the only dominant fungal genus instead of some pathogenic bacterial genera, with an abundance of over 95% after four days, and still more than 80% after a week. Meanwhile, metabolomics profiling showed significant concentration decrease with regard to some flavor-improving relative metabolites, such as 3-hydroxybenzoic acid (log2FC = - 5.25) and sorbitol (log2FC = - 5.54). This finding is extrapolated to be the key influence factor on the quality of the fermented cTWE. The correlation analysis also showed that the alterations in microbial diversity in the fermented cTWE led to some important differential metabolite changes, which finally improved various properties of tobacco products.

Synthetic bacterial consortia enhanced the degradation of mixed priority phthalate ester pollutants.

Dimethyl phthalate (DMP), diethyl phthalate (DEP), di-n-butyl phthalate (DBP), butyl benzyl phthalate (BBP), bis(2-ethylhexyl) phthalate (DEHP), and di-n-octyl phthalate (DOP) are hazardous chemicals listed as priority pollutants that disrupt endocrine systems. According to available reports, these six priority phthalate esters (PAEs) are considered the most polluting; however, no studies have been conducted on the efficient remediation of these PAEs. We therefore designed and constructed a synthetic bacterial consortium capable of the simultaneous and efficient degradation of six priority PAEs in minimal inorganic salt medium (MSM) and soil. The consortium comprised Glutamicibacter sp. ZJUTW, which demonstrates priority for degrading short-chain PAEs; Cupriavidus sp. LH1, which degrades phthalic acid (PA) and protocatechuic acid (PCA), intermediates of the PAE biodegradation process; and Gordonia sp. GZ-YC7, which efficiently degrades long-chain priority PAEs, including DEHP and DOP. In MSM containing the six mixed PAEs (250 mg/L each), the ZJUTW + YC + LH1 consortium completely degraded the four short-chain PAEs within 48 h, and DEHP (100%) and DOP (62.5%) within 72 h. In soil containing the six mixed PAEs (DMP, DEP, BBP, and DOP, 400 mg/kg each; DBP and DEHP, 500 mg/kg, each), the ZJUTW + YC + LH1 consortium completely degraded DMP, DEP, BBP, and DBP within 6 days, and 70.84% of DEHP and 66.24% of DOP within 2 weeks. The consortium efficiently degraded the six mixed PAEs in both MSM and soil. We thus believe that this synthetic microbial consortium is a strong candidate for the bioremediation of environments contaminated with mixed PAE pollutants.

De novo biosynthesis of sakuranetin from glucose by engineered Saccharomyces cerevisiae.

Sakuranetin is a plant-natural product, which has increasingly been utilized in cosmetic and pharmaceutical industries for its extensive anti-inflammatory, anti-tumor, and immunomodulatory effects. Sakuranetin was mostly produced by extraction technology from plants, which is limited to natural conditions and biomass supply. In this study, a de novo biosynthesis pathway of sakuranetin by engineered S. cerevisiae was constructed. After a series of heterogenous gene integration, a biosynthetic pathway of sakuranetin from glucose was successfully constructed in S. cerevisiae whose sakuranetin yield reached only 4.28 mg/L. Then, a multi-module metabolic engineering strategy was applied for improving sakuranetin yield in S. cerevisiae: (1) adjusting the copy number of sakuranetin synthesis genes, (2) removing the rate-limiting factor of aromatic amino acid pathway and optimizing the synthetic pathway of aromatic amino acids to enhance the supply of carbon flux for sakuranetin, and (3) introducing acetyl-CoA carboxylase mutants ACC1S659A,S1157A and knocking out YPL062W to strengthen the supply of malonyl-CoA which is another synthetic precursor of sakuranetin. The resultant mutant S. cerevisiae exhibited a more than tenfold increase of sakuranetin titer (50.62 mg/L) in shaking flasks. Furthermore, the sakuranetin titer increased to 158.65 mg/L in a 1-L bioreactor. To our knowledge, it is the first report on the sakuranetin de novo synthesis from glucose in S. cerevisiae. KEY POINTS: • De novo biosynthesis of sakuranetin was constructed by engineered S. cerevisiae. • Sakuranetin production was enhanced by multi-module metabolic engineering strategy. • It is the first report on the sakuranetin de novo synthesis in S. cerevisiae.

Comparative analyses of five complete chloroplast genomes from the endemic genus Cremanthodium (Asteraceae) in Himalayan and adjacent areas.

Cremanthodium Benth. is an endemic genus in the Himalayas and adjacent areas. Some plants of the genus are traditional medicinal plants in Tibetan medicine. In this study, the chloroplast genomes of five species (Cremanthodium arnicoides (DC. ex Royle) Good, Cremanthodium brunneopilosum S. W. Liu, Cremanthodium ellisii (Hook. f.) Kitam., Cremanthodium nervosum S. W. Liu, and Cremanthodium rhodocephalum Diels) were collected for sequencing. The sequencing results showed that the size of the chloroplast genome ranged from 150,985 to 151,284 bp and possessed a typical quadripartite structure containing one large single copy (LSC) region (83,326-83,369 bp), one small single copy (SSC) region (17,956-18,201 bp), and a pair of inverted repeats (IR) regions (24,830-24,855 bp) in C. arnicoides, C. brunneopilosum, C. ellisii, C. nervosum, and C. rhodocephalum. The chloroplast genomes encoded an equal number of genes, of which 88 were protein-coding genes, 37 were transfer ribonucleic acid genes, and eight were ribosomal ribonucleic acid genes, and were highly similar in overall size, genome structure, gene content, and order. In comparison with other species in the Asteraceae family, their chloroplast genomes share similarities but show some structural variations. There was no obvious expansion or contraction in the LSC, SSC or IR regions among the five species, indicating that the chloroplast gene structure of the genus was highly conserved. Collinearity analysis showed that there was no gene rearrangement. The results of the phylogenetic tree showed that the whole chloroplast genomes of the five species were closely related, and the plants of this genus were grouped into one large cluster with Ligularia Cass. and Farfugium Lindl.

MOF-Enabled Ion-Regulating Gel Electrolyte for Long-Cycling Lithium Metal Batteries Under High Voltage.

High-voltage lithium metal batteries (LMBs) are a promising high-energy-density energy storage system. However, their practical implementations are impeded by short lifespan due to uncontrolled lithium dendrite growth, narrow electrochemical stability window, and safety concerns of liquid electrolytes. Here, a porous composite aerogel is reported as the gel electrolyte (GE) matrix, made of metal-organic framework (MOF)@bacterial cellulose (BC), to enable long-life LMBs under high voltage. The effectiveness of suppressing dendrite growth is achieved by regulating ion deposition and facilitating ion conduction. Specifically, two hierarchical mesoporous Zr-based MOFs with different organic linkers, that is, UiO-66 and NH2 -UiO-66, are embedded into BC aerogel skeletons. The results indicate that NH2 -UiO-66 with anionphilic linkers is more effective in increasing the Li+ transference number; the intermolecular interactions between BC and NH2 -UiO-66 markedly increase the electrochemical stability. The resulting GE shows high ionic conductivity (≈1 mS cm-1 ), high Li+ transference number (0.82), wide electrochemical stability window (4.9 V), and excellent thermal stability. Incorporating this GE in a symmetrical Li cell successfully prolongs the cycle life to 1200 h. Paired with the Ni-rich LiNiCoAlO2 (Ni: Co: Al = 8.15:1.5:0.35, NCA) cathode, the NH2 -UiO-66@BC GE significantly improves the capacity, rate performance, and cycle stability, manifesting its feasibility to operate under high voltage.

Sulfated modification of hyaluronan tetrasaccharide enhances its antitumor activity on human lung adenocarcinoma A549 cells in vitro and in vivo.

Hyaluronan (HA) is a glycosaminoglycan polymer involved in cell phenotype change, inflammation modulation, and tumor metastasis progression. HA oligosaccharides have a higher solubility and drug-forming ability than polysaccharides. HA tetrasaccharide was reported as the smallest fragment required for inhibiting triple-negative breast cancer, but the anti-tumor activity of HA tetrasaccharide (HA4) and its sulfated derivatives in lung cancer is still unknown. In this study, HA4 was prepared via HA degradation by chondroitinase ABC (CSABC), while its sulfated derivatives were prepared by sulfur pyridine trioxide complex in N, N-dimethylformamide (DMF). Then, the anti-tumor activity was detected via MTT assay and xenograft tumor experiments, while the expression level change of apoptosis genes was analyzed by qRT-PCR. Electrospray mass spectrometry (ESI-MS) analysis showed several HA4 sulfated derivatives, GlcA2GlcNAc2 (SO3H)n contains 0-6 sulfation groups, which mainly contain 3-6, 2-3, and 0-1 sulfation groups were classified as HA4S1, HA4S2, and HA4S3, respectively. After the addition of 1.82 mg/mL HA4, HA4S1, HA4S2, and HA4S3, the cell viability of A549 cells was reduced to 81.2 %, 62.1 %, 50.3 %, and 65.9 %, respectively. Thus, HA4S2 was chosen for further measurement, the qRT-PCR results showed it significantly up-regulated the expression of genes in the apoptosis pathway. Moreover, HA4S2 exhibited stronger antitumor activity than HA4 in vivo and the tumor inhibition rate reached 36.90 %. In summary, this study indicated that the CSABC enzyme could effectively degrade HA into oligosaccharides, and sulfation modification was an effective method to enhance the antitumor activity of HA tetrasaccharides.

Nicotine metabolism pathway in bacteria: mechanism, modification, and application.

Nicotine is a harmful pollutant mainly from the waste of tobacco factories. It is necessary to remove nicotine via high efficient strategies such as bioremediation. So far, an increasing number of nicotine degrading strains have been isolated. However, their degrading efficiency and tolerance to high content nicotine is still not high enough for application in real environment. Thus, the modification of nicotine metabolism pathway is obligated and requires comprehensive molecular insights into whole cell metabolism of nicotine degrading strains. Obviously, the development of multi-omics technology has accelerated the mechanism study on microbial degradation of nicotine and supplied more novel strategy of strains modification. So far, three pathways of nicotine degradation, pyridine pathway, pyrrolidine pathway, and the variant of pyridine and pyrrolidine pathway (VPP pathway), have been clearly identified in bacteria. Muti-omics analysis further revealed specific genome architecture, regulation mechanism, and specific genes or enzymes of three pathways, in different strains. Especially, muti-omics analysis revealed that functional modules coexisted in different genome loci and played additional roles on enhanced degradation efficiency in bacteria. Based on the above discovery, genomic editing strategy becomes more feasible to greatly improve bacterial degrading efficiency of nicotine.

Carpelipines C and D, Two Anti-Inflammatory Germacranolides from the Flowers of Carpesium lipskyi Winkl. (Asteraceae).

Two new germacranolides, carpelipine C (1) and carpelipine D (2), together with four known ones (3-6), were isolated from Carpesium lipskyi Winkl. flowers, a folk Tibetan herbal medicine with antipyretic-analgesic and anti-inflammatory effects. The chemical structures of new structure were illuminated by diversified spectroscopic and X-ray crystallographic analyses. Compounds 1 and 3 dramatically suppressed the synthesis of NO and decreased pre-inflammatory protein expression of iNOS and COX-2 in LPS-induced RAW264.7 cells. Furthermore, it was revealed that NF-κB/MAPK signaling pathway were involved in the anti-inflammatory process of 1 and 3, and their effects on reducing oxidative stress by activating Nrf2/HO-1 pathway were also measured. This article indicated that the traditional use of C. lipskyi to treat inflammatory diseases has a certain rationality.

Lipskynoids A-G, New Acyclic Diterpenes from the Flowers of Carpesium lipskyi.

Seven new acyclic diterpenes, namely lipskynoids A-G (1-7), were isolated from the flowers of Carpesium lipskyi, a traditional Tibetan herbal medicine with anti-inflammatory and antipyretic-analgesic effects. These new compounds were elucidated by analysis of extensive spectroscopic data including ESI-MS, 1D, 2D NMR, and DP4+ analyses. Biological assays showed that 1-7 display significant inhibitory effects against the NO production in LPS-induced RAW264.7 cells with its IC50 values from 9.9 to 18.47 µM, however, no cytotoxicity effect was observed of these isolates against the growth of HePG2, PC3, DU145, and A549 cells.

A Type VI Secretion System Facilitates Fitness, Homeostasis, and Competitive Advantages for Environmental Adaptability and Efficient Nicotine Biodegradation.

Gram-negative bacteria employ secretion systems to translocate proteinaceous effectors from the cytoplasm to the extracellular milieu, thus interacting with the surrounding environment or microniche. It is known that bacteria can benefit from the type VI secretion system (T6SS) by transporting ions to combat reactive oxygen species (ROS). Here, we report that T6SS activities conferred tolerance to nicotine-induced oxidative stress in Pseudomonas sp. strain JY-Q, a highly active nicotine degradation strain isolated from tobacco waste extract. AA098_13375 was identified to encode a dual-functional effector with antimicrobial and anti-ROS activities. Wild-type strain JY-Q grew better than the AA098_13375 deletion mutant in nicotine-containing medium by antagonizing increased intracellular ROS levels. It was, therefore, tentatively designated TseN (type VI secretion system effector for nicotine tolerance), homologs of which were observed to be broadly ubiquitous in Pseudomonas species. TseN was identified as a Tse6-like bacteriostatic toxin via monitoring intracellular NAD+ TseN presented potential antagonism against ROS to fine tune the heavy traffic of nicotine metabolism in strain JY-Q. It is feasible that the dynamic tuning of NAD+ driven by TseN could satisfy demands from nicotine degradation with less cytotoxicity. In this scenario, T6SS involves a fascinating accommodation cascade that prompts constitutive biotransformation of N-heterocyclic aromatics by improving bacterial robustness/growth. In summary, the T6SS in JY-Q mediated resistance to oxidative stress and promoted bacterial fitness via a contact-independent growth competitive advantage, in addition to the well-studied T6SS-dependent antimicrobial activities.IMPORTANCE Mixtures of various pollutants and the coexistence of numerous species of organisms are usually found in adverse environments. Concerning biodegradation of nitrogen-heterocyclic contaminants, the scientific community has commonly focused on screening functional enzymes that transform pollutants into intermediates of attenuated toxicity or for primary metabolism. Here, we identified dual roles of the T6SS effector TseN in Pseudomonas sp. strain JY-Q, which is capable of degrading nicotine. The T6SS in strain JY-Q is able to deliver TseN to kill competitors and provide a growth advantage by a contact-independent pattern. TseN could monitor the intracellular NAD+ level by its hydrolase activity, causing cytotoxicity in competitive rivals but metabolic homeostasis on JY-Q. Moreover, JY-Q could be protected from TseN toxicity by the immunity protein TsiN. In conclusion, we found that TseN with cytotoxicity to bacterial competitors facilitated the nicotine tolerance of JY-Q. We therefore reveal a working model between T6SS and nicotine metabolism. This finding indicates that multiple diversified weapons have been evolved by bacteria for their growth and robustness.

Additional Role of Nicotinic Acid Hydroxylase for the Transformation of 3-Succinoyl-Pyridine by Pseudomonas sp. Strain JY-Q.

Nicotine and nicotinic acid (NA) are both considered to be representatives of N-heterocyclic aromatic compounds, and their degradation pathways have been revealed in Pseudomonas species. However, the cooccurrence of these two pathways has only been observed in Pseudomonas sp. strain JY-Q. The nicotine pyrrolidine catabolism pathway of strain JY-Q consists of the functional modules Nic1, Spm, and Nic2. The module enzyme, 3-succinoylpyridine monooxygenase (Spm), catalyzes transformation of 3-succinoyl-pyridine (SP) to 6-hydroxy-3-succinoyl-pyridine (HSP). There exist two homologous but not identical Spm enzymes (namely, Spm1 and Spm2) in JY-Q. However, when spm1 and spm2 were both in-frame deleted, the mutant still grew well in basic salt medium (BSM) supplemented with nicotine as the sole carbon/nitrogen nutrition, suggesting that there exists an alternative pathway responsible for SP catabolism in JY-Q. NicAB, an enzyme accounting for NA hydroxylation, contains reorganized domains similar to those of Spm. When the JY-Q_nicAB gene (nicAB in strain JY-Q) was introduced into another Pseudomonas strain, one that is unable to degrade NA, the resultant recombinant strain exhibited the ability to transform SP to HSP, but without the ability to metabolize NA. Here, we conclude that NicAB in strain JY-Q exhibits an additional role in SP transformation. The other genes in the NA cluster, NicXDFE (Nic2 homolog), then also exhibit a role in subsequent HSP metabolism for energy yield. This finding also suggests that the cooccurrence of nicotine and NA degradation genes in strain JY-Q represents an advantage for JY-Q, making it more effective and flexible for the degradation of nicotine.IMPORTANCE 3-Succinoyl-pyridine (SP) and 6-hydroxy-3-succinoyl-pyridine (HSP) are both valuable chemical precursors to produce insecticides and hypotensive agents. SP and HSP could be renewable through the nicotine microbial degradation pathway, in which 3-succinoylpyridine monooxygenases (Spm) account for transforming SP into HSP in Pseudomonas sp. strain JY-Q. However, when two homologous Spm genes (spm1 and spm2) were knocked out, the mutant retained the ability to degrade nicotine. Thus, in addition to Spm, JY-Q should have an alternative pathway for SP conversion. In this research, we showed that JY-Q_NicAB was responsible for this alternative SP conversion. Both of the primary functions for nicotinic acid dehydrogenation and the additional function for SP metabolism were detected in a recombinant strain harboring JY-Q_NicAB. As a result, both nicotinic acid and nicotine degradation pathways in JY-Q contribute to its remarkable nicotine tolerance and nicotine degradation availability. These findings also provide one more metabolic engineering strategy for accumulation for value-added intermediates.

Differential Effects of Homologous Transcriptional Regulators NicR2A, NicR2B1, and NicR2B2 and Endogenous Ectopic Strong Promoters on Nicotine Metabolism in Pseudomonas sp. Strain JY-Q.

Nicotine is a toxic environmental pollutant that widely exists in tobacco wastes. As a natural nicotine-degrading strain, Pseudomonas sp. strain JY-Q still has difficulties degrading high concentrations of nicotine. In this study, we investigated the effect of two homologous transcriptional regulators and endogenous ectopic strong promoters on the efficiency of nicotine degradation. Comparative genomics analysis showed that two homologous transcriptional regulators, namely, NicR2A and NicR2Bs (NicR2B1 plus NicR2B2), can repress nicotine degradation gene expression. When both nicR2A and nicR2Bs were deleted, the resulting mutant JY-Q ΔnicR2A ΔnicR2B1 ΔnicR2B2 (QΔABs) exhibits a 17% higher nicotine degradation efficiency than wild-type JY-Q. Transcriptome sequencing (RNA-seq) analysis showed that the transcription levels (fragments per kilobase per million [FPKM] value) of six genes were higher than those of the other genes in JY-Q. Based on the genetic organization of these genes, three putative promoters, PRS28250 , PRS09985 , and PRS24685 , were identified. Their promoter activities were evaluated by comparing their expression levels using reverse transcriptase quantitative PCR (RT-qPCR). We found that the transcription levels of RS28250, RS09985, and RS24685 were respectively 16.8, 2.6, and 1.6 times higher than that of hspB2, encoding 6-hydroxy-3-succinylpyridine hydroxylase, which is involved in nicotine degradation. Thus, two strong endogenous promoters, namely, PRS28250 and PRS09985 , were selected to replace the original promoters of nic2 gene clusters. The effect of the endogenous ectopic promoter was also related to the position of target gene clusters. When the promoter PRS28250 replaced the promoter of hspB2, the resultant mutant QΔABs-ΔPhspB2 ::PRS28250 exhibited nicotine-degrading efficiency 69% higher than that of JY-Q. This research suggests a feasible strategy to enhance strains' capacity for nicotine degradation by removal of repressing regulatory proteins and replacing the target promoter with strong endogenous ectopic promoters.IMPORTANCE This study evaluated the differential effects of homologous NicR2A and NicR2Bs and endogenous ectopic strong promoters on nicotine metabolism in Pseudomonas sp. strain JY-Q. Based on our differential analysis, a feasible strategy is presented to modify wild-type (WT) strain JY-Q by removing repressing regulatory proteins NicR2A and NicR2Bs and replacing the target promoter with strong endogenous ectopic promoters. The resulting mutants exhibited high tolerance and degradation of nicotine. These findings should be beneficial for improving the pollutant-degrading capacity of natural strains through genomic modification.

In-Situ Synthesis of N, O, P-Doped Hierarchical Porous Carbon from Poly-bis(phenoxy)phosphazene for Polysulfide-Trapping Interlayer in Lithium-Sulfur Batteries.

The shuttling of polysulfides is the most detrimental contribution to degrading the capacity and cycle stability of lithium-sulfur (Li-S) batteries. Adding a carbon interlayer to prevent the polysulfides from migrating is feasible, and a rational design of the structures and surface properties of the carbon layer is essential to increasing its effectiveness. Herein, we report a hierarchical porous carbon (HPC) created by carbonization of bis(phenoxy)phosphazene and in-situ doping of triple heteroatoms into the carbon lattice to fabricate an effective polysulfide-trapping interlayer. The generated carbon integrates the advantages of a hierarchical porous structure, a high specific area and rich dopants (N, O and P), to yield chemisorption and physical confinement for polysulfides and fast ion-transport synergistically. The HPC interlayer significantly improves the electrochemical performance of Li-S batteries, including an exceptional discharge capacity of 1509 mA h/g at 0.06 C and a high capacity retention of 83.7 % after 250 cycles at 0.3 C. This work thus proposes a facile in-situ synthesis of heteroatom-doped carbon with rational porous structures for suppressing the shuttle effect.

Expression and functional identification of two homologous nicotine dehydrogenases, NicA2 and Nox, from Pseudomonas sp. JY-Q.

Nicotine contamination in tobacco waste effluent (TWE) from tobacco industry is a serious threat to public health and environment. Microbial degradation is an impending approach to remove nicotine and transform it into some other high value chemicals. Pseudomonas sp. JY-Q exhibits high efficiency of degradation, which can degrade 5 g/L of nicotine within 24 h. In strain JY-Q, we found the co-occurrence of two homologous key enzymes NicA2 and Nox, which catalyze nicotine to N-methylmyosmine, and then to pseudooxylnicotine via simultaneous hydrolysis. In this study, recombinant NicA2 and Nox were expressed in E. coli BL21(DE3) and purified. In vitro, the activity of recombinant NicA2 and Nox was accelerated by adding co-factor NAD+, suggesting that they worked as dehydrogenases. The optimal reaction conditions, substrate affinity, catabolism efficiency, pH-stability and thermal-stability were determined. Nox showed lower efficiency, but at a higher stability level than NicA2. Nox exhibited wider pH range and higher temperature as optimal conditions for the enzymatic reaction. In addition, The Nox showed higher thermo-stability and acid-stability than that of NicA2. The study on enzymatic reaction kinetics showed that Nox had a lower Km and higher substrate affinity than NicA2. These results suggest that Nox plays more significant role than NicA2 in nicotine degradation in TWE, which usually is processed at low pH (4-5) and high temperature (above 40 °C). Genetic engineering is required to enhance the affinity and suitability of NicA2 for an increased additive effect on homologous NicA2 and Nox in strain JY-Q.

Superresilient Hard Carbon Nanofabrics for Sodium-Ion Batteries.

Developing supermechanically resilient hard carbon materials that can quickly accommodate sodium ions is highly demanded in fabricating durable anodes for wearable sodium-ion batteries. Here, an interconnected spiral nanofibrous hard carbon fabric with both remarkable resiliency (e.g., recovery rate as high as 1200 mm s-1 ) and high Young's modulus is reported. The hard carbon nanofabrics are prepared by spinning and then carbonizing the reaction product of polyacrylonitrile and polar molecules (melamine). The resulting unique hard carbon possesses a highly disordered carbonaceous structure with enlarged interlayer spacing contributed from the strong electrostatic repulsion of dense pyrrolic nitrogen atoms. Its excellent resiliency remains after intercalation/deintercalation of sodium ions. The outstanding sodium-storage performance of the derived anode includes excellent gravimetric capacity, high-power capability, and long-term cyclic stability. More significantly, with a high loading mass, the hard carbon anode displays a high-power capacity (1.05 mAh cm-2 at 2 A g-1 ) and excellent cyclic stability. This study provides a unique strategy for the design and fabrication of new hard carbon materials for advanced wearable energy storage systems.

VRprofile: gene-cluster-detection-based profiling of virulence and antibiotic resistance traits encoded within genome sequences of pathogenic bacteria.

VRprofile is a Web server that facilitates rapid investigation of virulence and antibiotic resistance genes, as well as extends these trait transfer-related genetic contexts, in newly sequenced pathogenic bacterial genomes. The used backend database MobilomeDB was firstly built on sets of known gene cluster loci of bacterial type III/IV/VI/VII secretion systems and mobile genetic elements, including integrative and conjugative elements, prophages, class I integrons, IS elements and pathogenicity/antibiotic resistance islands. VRprofile is thus able to co-localize the homologs of these conserved gene clusters using HMMer or BLASTp searches. With the integration of the homologous gene cluster search module with a sequence composition module, VRprofile has exhibited better performance for island-like region predictions than the other widely used methods. In addition, VRprofile also provides an integrated Web interface for aligning and visualizing identified gene clusters with MobilomeDB-archived gene clusters, or a variety set of bacterial genomes. VRprofile might contribute to meet the increasing demands of re-annotations of bacterial variable regions, and aid in the real-time definitions of disease-relevant gene clusters in pathogenic bacteria of interest. VRprofile is freely available at http://bioinfo-mml.sjtu.edu.cn/VRprofile.