Genomic analysis of Cobetia sp. D5 reveals its role in marine sulfur cycling.

Dimethylsulfoniopropionate (DMSP) is one of the most abundant sulfur-containing organic compounds on the earth, which is an important carbon and sulfur source and plays an important role in the global sulfur cycle. Marine microorganisms are an important group involved in DMSP metabolism. The strain Cobetia sp. D5 was isolated from seawater samples in the Yellow Sea area of Qingdao during an algal bloom. There is still limited knowledge on the capacity of DMSP utilization of Cobetia bacteria. The study reports the whole genome sequence of Cobetia sp. D5 to understand its DMSP metabolism pathway. The genome of Cobetia sp. D5 consists of a circular chromosome with a length of 4,233,985 bp and the GC content is 62.56%. Genomic analysis showed that Cobetia sp. D5 contains a set of genes to transport and metabolize DMSP, which can cleave DMSP to produce dimethyl sulphide (DMS) and 3-Hydroxypropionyl-Coenzyme A (3-HP-CoA). DMS diffuses into the environment to enter the global sulfur cycle, whereas 3-HP-CoA is catabolized to acetyl CoA to enter central carbon metabolism. Thus, this study provides genetic insights into the DMSP metabolic processes of Cobetia sp. D5 during a marine algal bloom, and contributes to the understanding of the important role played by marine bacteria in the global sulfur cycle.

Novel PTCH1 Mutation Causes Gorlin-Goltz Syndrome.

OBJECTIVE: To analyse the aetiology and pathogenesis of Gorlin-Goltz syndrome (GS; also known as nevoid basal cell carcinoma syndrome [NBCCS] or basal cell nevus syndrome [BCNS]) in a Chinese family. METHODS: Whole-exome sequencing (WES) was performed on genomic DNA samples from the subjects in a family, followed by the investigation of pathogenesis via bioinformatic approaches and conformational analysis. RESULTS: A novel heterozygous non-frameshift deletion patched 1 (PTCH1) [NM_000264: c.3512_3526del (p.1171_1176del)] was identified by WES and further validated by Sanger sequencing. Bioinformatic and conformational analysis showed that the mutation caused altered PTCH1 protein structure, which may be related to functional abnormalities. CONCLUSION: This study expands the mutation spectrum of PTCH1 in GS and facilitates the early diagnosis and screening of GS. PTCH1 [c.3512_3526del (p.1171_1176del)] may cause structural abnormalities and functional disabilities, leading to GS in families.

Variability of BMP-2 content in DBM products derived from different long bone.

Demineralized bone matrix (DBM) has been regarded as an ideal bone substitute as a native carrier of bone morphogenetic proteins (BMPs) and other growth factors. However, the osteoinductive properties diverse in different DBM products. We speculate that the harvest origin further contributing to variability of BMPs contents in DBM products besides the process technology. In the study, the cortical bone of femur, tibia, humerus, and ulna from a signal donor were prepared and followed demineralizd into DBM products. Proteins in bone martix were extracted using guanidine-HCl and collagenase, respectively, and BMP-2 content was detected by sandwich enzyme-linked immunosorbent assay (ELISA). Variability of BMP-2 content was found in 4 different DBM products. By guanidine-HCl extraction, the average concentration in DBMs harvested from ulna, humerus, tibia, and femur were 0.613 ± 0.053, 0.848 ± 0.051, 3.293 ± 0.268, and 21.763 ± 0.344, respectively (p < 0.05), while using collagenase, the levels were 0.089 ± 0.004, 0.097 ± 0.004, 0.330 ± 0.012, and 1.562 ± 0.008, respectively (p < 0.05). In general, the content of BMP-2 in long bones of Lower limb was higher than that in long bones of upper limb, and GuHCl had remarkably superior extracted efficiency for BMP-2 compared to collagenase. The results suggest that the origin of cortical bones harvested to fabricate DBM products contribute to the variability of native BMP-2 content, while the protein extracted method only changes the measured values of BMP-2.

[Application of Fe3[KG-*2/5]O4/Mulberry Stem Biochar Effects on Soil Arsenic Species and Rice Arsenic Content].

The passivation effect of Fe3O4/mulberry pole biochar (Fe-MBC) prepared at different carbonization temperatures on soil available arsenic content was studied through soil culture experiments, and Fe-MBC-800 (prepared by carbonization at 800℃) with good passivation effect was selected and characterized. The effects of 1%-7% (mass fraction of biochar to soil) Fe-MBC-800, MBC-800, and Fe3O4 on soil pH value, soil electrical conductivity, soil arsenic form, rice biomass, and total arsenic (As) content in rice were studied using a pot experiment. The results showed that:①Fe-MBC-800 successfully loaded Fe3O4, and its main functional groups were C=O double bond, O-H bond, C-O bond, and Fe-O bond. The specific surface areas of Fe-MBC-800, MBC-800, and Fe3O4 were 209.659 m2·g-1, 517.714 m2·g-1, and 68.025 m2·g-1, respectively. ②The addition of Fe-MBC-800 could increase the soil pH value, decrease the soil EC value, increase the content of residual arsenic in soil, and reduce the content of water-soluble arsenic and available arsenic in the soil. Under the treatment using 7% Fe-MBC-800 (ω) amendments, the content of water-soluble arsenic and available arsenic in the soil decreased by 81.6% and 56.33%, respectively. ③When the addition ratio of Fe-MBC-800 in the soil was 5%-7%, it could promote the growth of rice plants, increase rice biomass, and reduce the bioaccumulation of arsenic by between 62.5% and 68.75%.

A novel class of xylanases specifically degrade marine red algal ß1,3/1,4-mixed-linkage xylan.

Xylans are polysaccharides composed of xylose and include ß1,4-xylan, ß1,3-xylan, and ß1,3/1,4-mixed-linkage xylan (MLX). MLX is widely present in marine red algae and constitutes a significant organic carbon in the ocean. Xylanases are hydrolase enzymes that play an important role in xylan degradation. While a variety of ß1,4-xylanases and ß1,3-xylanases involved in the degradation of ß1,4-xylan and ß1,3-xylan have been reported, no specific enzyme has yet been identified that degrades MLX. Herein, we report the characterization of a new MLX-specific xylanase from the marine bacterium Polaribacter sp. Q13 which utilizes MLX for growth. The bacterium secretes xylanases to degrade MLX, among which is Xyn26A, an MLX-specific xylanase that shows low sequence similarities (<27%) to ß1,3-xylanases in the glycoside hydrolase family 26 (GH26). We show that Xyn26A attacks MLX precisely at ß1,4-linkages, following a ß1,3-linkage toward the reducing end. We confirm that Xyn26A and its homologs have the same specificity and mode of action on MLX, and thus represent a new xylanase group which we term as MLXases. We further solved the structure of a representative MLXase, AlXyn26A. Structural and biochemical analyses revealed that the specificity of MLXases depends critically on a precisely positioned ß1,3-linkage at the -2/-1 subsite. Compared to the GH26 ß1,3-xylanases, we found MLXases have evolved a tunnel-shaped cavity that is fine-tuned to specifically recognize and hydrolyze MLX. Overall, this study offers a foremost insight into MLXases, shedding light on the biochemical mechanism of bacterial degradation of MLX.

Dimethylsulfoniopropionate and its catabolites are important chemical signals mediating marine microbial interactions.

Dimethylsulfoniopropionate (DMSP) is a ubiquitous organosulfur compound with key ecological roles in marine environments. This paper offers a brief insight into the mechanisms, environmental diversity, and importance of DMSP-mediated marine microbial interactions, including algae-microzooplankton interactions, bacteria-microzooplankton interactions, and algae-bacteria interactions. We also highlight current challenges that warrant further investigation.

Structural and molecular basis for urea recognition by Prochlorococcus.

Nitrogen (N) is an essential element for microbial growth and metabolism. The growth and reproduction of microorganisms in more than 75% of areas of the ocean are limited by N. Prochlorococcus is numerically the most abundant photosynthetic organism on the planet. Urea is an important and efficient N source for Prochlorococcus. However, how Prochlorococcus recognizes and absorbs urea still remains unclear. Prochlorococcus marinus MIT 9313, a typical Cyanobacteria, contains an ABC-type transporter, UrtABCDE, which may account for the transport of urea. Here, we heterologously expressed and purified UrtA, the substrate-binding protein of UrtABCDE, detected its binding affinity toward urea, and further determined the crystal structure of the UrtA/urea complex. Molecular dynamics simulations indicated that UrtA can alternate between "open" and "closed" states for urea binding. Based on structural and biochemical analyses, the molecular mechanism for urea recognition and binding was proposed. When a urea molecule is bound, UrtA undergoes a state change from open to closed surrounding the urea molecule, and the urea molecule is further stabilized by the hydrogen bonds supported by the conserved residues around it. Moreover, bioinformatics analysis showed that ABC-type urea transporters are widespread in bacteria and probably share similar urea recognition and binding mechanisms as UrtA from P. marinus MIT 9313. Our study provides a better understanding of urea absorption and utilization in marine bacteria.

Aerobic methylation of hydrogen sulfide to dimethylsulfide in diverse microorganisms and environments.

Dimethylsulfide (DMS) is the major biosulfur source emitted to the atmosphere with key roles in global sulfur cycling and potentially climate regulation. The main precursor of DMS is thought to be dimethylsulfoniopropionate. However, hydrogen sulfide (H2S), a widely distributed and abundant volatile in natural environments, can be methylated to DMS. The microorganisms and the enzymes that convert H2S to DMS, and their importance in global sulfur cycling were unknown. Here we demonstrate that the bacterial MddA enzyme, previously known as a methanethiol S-methyltransferase, could methylate inorganic H2S to DMS. We determine key residues involved in MddA catalysis and propose the mechanism for H2S S-methylation. These results enabled subsequent identification of functional MddA enzymes in abundant haloarchaea and a diverse range of algae, thus expanding the significance of MddA mediated H2S methylation to other domains of life. Furthermore, we provide evidence for H2S S-methylation being a detoxification strategy in microorganisms. The mddA gene was abundant in diverse environments including marine sediments, lake sediments, hydrothermal vents and soils. Thus, the significance of MddA-driven methylation of inorganic H2S to global DMS production and sulfur cycling has likely been considerably underestimated.

Muyocoxanthones O-S: Undescribed xanthones with antioxidative damage bioactivity to cardiomyocytes from the endophytic fungus Muyocopron laterale.

The metabolites from the endophytic fungus Muyocopron laterale hosted in the medicinal plant Tylophora ovata were investigated, and five undescribed xanthones, muyocoxanthones O-S, along with seven known compounds were isolated. Their structures were elucidated by HR-ESI-MS, NMR, and ECD calculations. Compounds were evaluated for their anti-cardiomyocyte oxidative damage activity using a model of oxidative damage induced by cell hypoxia incubation. Muyocoxanthones O-Q and blennolide L exhibited moderate activity against oxidative damage to cardiomyocytes with relative viabilities of 62.4, 54.8, 60.3 and 54.9%, respectively.

A new dimethylsulfoniopropionate lyase of the cupin superfamily in marine bacteria.

Dimethylsulfoniopropionate (DMSP) is a marine organosulfur compound with important roles in stress protection, marine biogeochemical cycling, chemical signalling and atmospheric chemistry. Diverse marine microorganisms catabolize DMSP via DMSP lyases to generate the climate-cooling gas and info-chemical dimethyl sulphide. Abundant marine heterotrophs of the Roseobacter group (MRG) are well known for their ability to catabolize DMSP via diverse DMSP lyases. Here, a new DMSP lyase DddU within the MRG strain Amylibacter cionae H-12 and other related bacteria was identified. DddU is a cupin superfamily DMSP lyase like DddL, DddQ, DddW, DddK and DddY, but shares <15% amino acid sequence identity with these enzymes. Moreover, DddU proteins forms a distinct clade from these other cupin-containing DMSP lyases. Structural prediction and mutational analyses suggested that a conserved tyrosine residue is the key catalytic amino acid residue in DddU. Bioinformatic analysis indicated that the dddU gene, mainly from Alphaproteobacteria, is widely distributed in the Atlantic, Pacific, Indian and polar oceans. For reference, dddU is less abundant than dddP, dddQ and dddK, but much more frequent than dddW, dddY and dddL in marine environments. This study broadens our knowledge on the diversity of DMSP lyases, and enhances our understanding of marine DMSP biotransformation.

Novel D-glutamate catabolic pathway in marine Proteobacteria and halophilic archaea.

D-glutamate (D-Glu) is an essential component of bacterial peptidoglycans, representing an important, yet overlooked, pool of organic matter in global oceans. However, little is known on D-Glu catabolism by marine microorganisms. Here, a novel catabolic pathway for D-Glu was identified using the marine bacterium Pseudoalteromonas sp. CF6-2 as the model. Two novel enzymes (DgcN, DgcA), together with a transcriptional regulator DgcR, are crucial for D-Glu catabolism in strain CF6-2. Genetic and biochemical data confirm that DgcN is a N-acetyltransferase which catalyzes the formation of N-acetyl-D-Glu from D-Glu. DgcA is a racemase that converts N-acetyl-D-Glu to N-acetyl-L-Glu, which is further hydrolyzed to L-Glu. DgcR positively regulates the transcription of dgcN and dgcA. Structural and biochemical analyses suggested that DgcN and its homologs, which use D-Glu as the acyl receptor, represent a new group of the general control non-repressible 5 (GCN5)-related N-acetyltransferases (GNAT) superfamily. DgcA and DgcN occur widely in marine bacteria (particularly Rhodobacterales) and halophilic archaea (Halobacteria) and are abundant in marine and hypersaline metagenome datasets. Thus, this study reveals a novel D-Glu catabolic pathway in ecologically important marine bacteria and halophilic archaea and helps better understand the catabolism and recycling of D-Glu in these ecosystems.

Mechanistic Insight Into the Production of Collagen Oligopeptides by the S8 Family Protease A4095.

Collagen oligopeptides have wide applications in foods, pharmaceuticals, cosmetics, and others due to their high bioactivities and bioavailability. The S8 family is the second-largest family of serine proteases. Several collagenolytic proteases from this family have been reported to have good potential in the preparation of collagen oligopeptides, however, the underlying mechanism remains unknown. A4095 was the most abundant S8 protease secreted by the protease-producing bacterium Anoxybacillus caldiproteolyticus 1A02591. Here, we characterized A4095 as an S8 collagenolytic protease and illustrated its structural basis to produce collagen oligopeptides. Protease A4095 preferentially hydrolyzed the Y-Gly peptide bonds in denatured bovine bone collagen, leading to high production (62.48% <1000 Da) of collagen oligopeptides. Structural and mutational analyses indicated that A4095 has a unique S1' substrate-binding pocket to preferentially bind Gly, which is the structural determinant for the high production of collagen oligopeptides. This study provides mechanistic insight into the advantage of the S8 collagenolytic proteases in preparing collagen oligopeptides.

Antibacterial activity of peptaibols from Trichoderma longibrachiatum SMF2 against gram-negative Xanthomonas oryzae pv. oryzae, the causal agent of bacterial leaf blight on rice.

Bacterial leaf blight caused by Gram-negative pathogen Xanthomonas oryzae pv. oryzae (Xoo) is one of the most destructive bacterial diseases on rice. Due to the resistance, toxicity and environmental issues of chemical bactericides, new biological strategies are still in need. Although peptaibols produced by Trichoderma spp. can inhibit the growth of several Gram-positive bacteria and plant fungal pathogens, it still remains unclear whether peptaibols have anti-Xoo activity to control bacterial leaf blight on rice. In this study, we evaluated the antibacterial effects of Trichokonins A (TKA), peptaibols produced by Trichoderma longibrachiatum SMF2, against Xoo. The in vitro antibacterial activity analysis showed that the growth of Xoo was significantly inhibited by TKA, with a minimum inhibitory concentration of 54 µg/mL and that the three TKs in TKA all had remarkable anti-Xoo activity. Further inhibitory mechanism analyses revealed that TKA treatments resulted in the damage of Xoo cell morphology and the release of intracellular substances, such as proteins and nucleic acids, from Xoo cells, suggesting the damage of the permeability of Xoo cell membrane by TKA. Pathogenicity analyses showed that the lesion length on rice leaf was significantly reduced by 82.2% when treated with 27 µg/mL TKA. This study represents the first report of the antibacterial activity of peptaibols against a Gram-negative bacterium. Thus, TKA can be of a promising agent in controlling bacterial leaf blight on rice.

Undescribed meleagrin alkaloids from the endophytic fungus Penicillium commune.

Six undescribed meleagrin analogues, isomeleagrin, meleagrin F, meleagrin G, methylmeleagrin G, isomethylmeleagrin G and meleagrin H, were isolated from the endophytic fungus Penicillium commune, which was obtained from the fresh leaves of a toxic medicinal plant, Tylophora ovata. The structures of these analogues were elucidated through extensive spectroscopic data analysis, and their absolute configurations were characterized by calculated electronic circular dichroism (ECD). Structurally, meleagrin F features an undescribed skeleton with an aniline moiety, which is linked to meleagrin through a C-C bond at C8-C26. Connecting N19-C3' through the C-N bond in meleagrin G, methylmeleagrin G, isomethylmeleagrin G and meleagrin H was rare for amino acid condensation. The cytotoxicity activity of these undescribed compounds was evaluated, and isomeleagrin exhibited a selective cytotoxicity activity against HGC27 cells with an IC50 value of 2.01 µM.

Two sydowic acid derivatives and a sulfonyl metabolite from the endophytic fungus Aspergillus sydowii.

Two new sydowic acid derivatives, a pair of enantiomers, involving (+)-sydowiccal (1a) and (-)-sydowiccal (1b), a new sulfonyl metabolite of 2-methoxy-5-methyl-3-(methylsulfonyl)phenol (2), as well as three known sydowic acid derivatives, were isolated from Aspergillus sydowii, an endophytic fungus of Rhododendron mole. The structures of these new compounds were elucidated by analyzing their NMR and HRESIMS data, and the absolute configurations of enantiomers were determined on the basis of the CD spectrum. Three new metabolites showed weak anti-inflammation on nitric oxide (NO) production in LPS-induced RAW 264.7 cells.

Mechanistic Insight into the Fragmentation of Type I Collagen Fibers into Peptides and Amino Acids by a Vibrio Collagenase.

Vibrio collagenases of the M9A subfamily are closely related to Vibrio pathogenesis for their role in collagen degradation during host invasion. Although some Vibrio collagenases have been characterized, the collagen degradation mechanism of Vibrio collagenase is still largely unknown. Here, an M9A collagenase, VP397, from marine Vibrio pomeroyi strain 12613 was characterized, and its fragmentation pattern on insoluble type I collagen fibers was studied. VP397 is a typical Vibrio collagenase composed of a catalytic module featuring a peptidase M9N domain and a peptidase M9 domain and two accessory bacterial prepeptidase C-terminal domains (PPC domains). It can hydrolyze various collagenous substrates, including fish collagen, mammalian collagens of types I to V, triple-helical peptide [(POG)10]3, gelatin, and 4-phenylazobenzyloxycarbonyl-Pro-Leu-Gly-Pro-o-Arg (Pz-peptide). Atomic force microscopy (AFM) observation and biochemical analyses revealed that VP397 first assaults the C-telopeptide region to dismantle the compact structure of collagen and dissociate tropocollagen fragments, which are further digested into peptides and amino acids by VP397 mainly at the Y-Gly bonds in the repeating Gly-X-Y triplets. In addition, domain deletion mutagenesis showed that the catalytic module of VP397 alone is capable of hydrolyzing type I collagen fibers and that its C-terminal PPC2 domain functions as a collagen-binding domain during collagenolysis. Based on our results, a model for the collagenolytic mechanism of VP397 is proposed. This study sheds light on the mechanism of collagen degradation by Vibrio collagenase, offering a better understanding of the pathogenesis of Vibrio and helping in developing the potential applications of Vibrio collagenase in industrial and medical areas. IMPORTANCE Many Vibrio species are pathogens and cause serious diseases in humans and aquatic animals. The collagenases produced by pathogenic Vibrio species have been regarded as important virulence factors, which occasionally exhibit direct pathogenicity to the infected host or facilitate other toxins' diffusion through the digestion of host collagen. However, our knowledge concerning the collagen degradation mechanism of Vibrio collagenase is still limited. This study reveals the degradation strategy of Vibrio collagenase VP397 on type I collagen. VP397 binds on collagen fibrils via its C-terminal PPC2 domain, and its catalytic module first assaults the C-telopeptide region and then attacks the Y-Gly bonds in the dissociated tropocollagen fragments to release peptides and amino acids. This study offers new knowledge regarding the collagenolytic mechanism of Vibrio collagenase, which is helpful for better understanding the role of collagenase in Vibrio pathogenesis and for developing its industrial and medical applications.

Secondary metabolites with diversified structures from an endophytic fungus Colletotrichum gloeosporioides associated with a toxic medicinal plant Tylophora ovata.

Six new secondary metabolites, including two new nor-triterpenes (1 and 2), one new sesquiterpene (4), two new α-pyrone derivatives (6 and 7), and one new natural product (5) along with two known compounds (3 and 8) were isolated from an endophytic fungus Colletotrichum gloeosporioides obtained from a toxic medicinal plant Tylophora ovata. Their structures were elucidated by spectroscopic data analyses, while their absolute configurations were determined by CD and X-ray diffraction analyses. The in vitro anti-inflammatory activities of these compounds were evaluated.

Structure of Vibrio collagenase VhaC provides insight into the mechanism of bacterial collagenolysis.

The collagenases of Vibrio species, many of which are pathogens, have been regarded as an important virulence factor. However, there is little information on the structure and collagenolytic mechanism of Vibrio collagenase. Here, we report the crystal structure of the collagenase module (CM) of Vibrio collagenase VhaC and the conformation of VhaC in solution. Structural and biochemical analyses and molecular dynamics studies reveal that triple-helical collagen is initially recognized by the activator domain, followed by subsequent cleavage by the peptidase domain along with the closing movement of CM. This is different from the peptidolytic mode or the proposed collagenolysis of Clostridium collagenase. We propose a model for the integrated collagenolytic mechanism of VhaC, integrating the functions of VhaC accessory domains and its collagen degradation pattern. This study provides insight into the mechanism of bacterial collagenolysis and helps in structure-based drug design targeting of the Vibrio collagenase.

Value of Contrast-Enhanced Ultrasound in Adjusting the Classification of Chinese-TIRADS 4 Nodules.

OBJECTIVES: To explore the value of applying contrast-enhanced ultrasound (CEUS) in adjusting the classification of category 4 nodules in the Chinese-Thyroid Imaging Report and Data System (C-TIRADS). METHODS: The data of preoperative conventional ultrasound and CEUS examinations of 125 C-TIRADS 4 nodules in 109 patients were retrospectively analyzed. We divided the thyroid nodules into two groups based on whether recommend by the guide fine-needle aspiration (FNA). Group I included C-TIRADS 4A nodules with a maximum diameter ≤15 mm and C-TIRADS 4B and 4C nodules with a maximum diameter ≤10 mm, and Group II included C-TIRADS 4A nodules with a maximum diameter >15 mm and C-TIRADS 4B and 4C nodules with a maximum diameter >10 mm. In CEUS, thyroid nodules showing suspicious malignant features such as hypoenhancement or early washout were adjusted to a level higher in the C-TIRADS classification; thyroid nodules showing possible benign features such as iso- or hyperenhancement were adjusted to a level lower; and thyroid nodules showing no enhancement were adjusted to C-TIRADS 3. Taking the pathological results as the gold standard, the receiver operating characteristic (ROC) curves of the C-TIRADS classification before and after the adjustment based on CEUS were plotted, and the diagnostic efficiency was compared. RESULTS: The sensitivity, specificity, accuracy, and positive and negative predictive values of the C-TIRADS classification for the diagnosis of thyroid nodule malignancy before the adjustment based on the CEUS results were 83.6%, 63.8%, 74.4%, 72.7%, and 77.1%, respectively, and these values were 91.0%, 82.8%, 87.2%, 85.9%, and 88.9%, respectively, after the adjustment. The area under the ROC curve (AUC) was 0.737 and 0.869, respectively, showing a significant difference (Z = 3.288, P=0.001). The diagnostic efficiency of C-TIRADS classification after the adjustment based on the CEUS results in both groups was improved compared with the result before the adjustment, and the difference in Group II was significant (Z = 2.931, P=0.003). CONCLUSIONS: CEUS significantly improved the diagnostic performance in the adjustment of C-TIRADS 4 nodule classification, especially for the nodules which needs FNA recommended by the C-TIRADS.