Jatropha biodiesel waste residues as carbon rich source by marine bacterial strain (Bacillus licheniformis PL26), isolated from west coast of India. The synthesized ε-polylysine and polyhydroxyalkanoate PHA by Bacillus licheniformis PL26 was characterized by thermogravimetric analysis (TGA), differential scanning colorimetry (DSC), Fourier transform infrared spectroscopy (FTIR), and 1H Nuclear magnetic resonance spectroscopy (NMR). The PHA produced by Bacillus licheniformis was found to be poly-3-hydroxybutyrate-co-3-hydroxyvalerate (P3HB-co-3HV). The developed process needs to be statistically optimized further for gaining still better yield of both the products in an efficient manner.Crude glycerol is generated as a by-product during transesterification process and during hydrolysis of fat in the soap-manufacturing process, and poses a problem for waste management. In the present approach, an efficient process was designed for simultaneous production of 0.2 g/L extracellular ε-polylysine and 64.6% (w/w) intracellular polyhydroxyalkanoate (PHA) in the same fermentation broth (1 L shake flask) utilizing  Most of the global economy is driven by petroleum fuels as the main source of energy. However, due to market fluctuation, it is moving towards a sustainable bio based economy as fossil reserves are projected to decline completely by 2050 ,2,3. In For sustainable development and commercialization of biodiesel production, effective utilization of crude glycerol into value added products are desired. However, conversion of crude glycerol will also promote the accretion of integrated biorefineries. 1,3-propanediol, citric acid, PHA’s, ε-polylysine, butanol, hydrogen, ethanol, phytase, lipase, succinic acid, docosahexaenoic acid, eicosapentanoic acid, monoglycerides, lipids and syngas can be produced from crude glycerol through microbial strains ,29,30,31The utilization of low quality glycerol obtained as by-product of biodiesel production is a big challenge as this glycerol cannot be used for direct food and cosmetic uses. An effective usage for conversion of crude glycerol to specific products may cut down the biodiesel production costs. The process for biodiesel preparation and generating value added products simultaneously through the use of waste generated during the biodiesel production is a very effective approach . ClearlyStreptomyces albulus (CCRC 11814) utilizing crude glycerol as carbon source through aerobic fermentation yielding 0.2 g/L ε-polylysine. However, growth rate of Streptomyces albulus (CCRC 11814) was slow due to other impurities such as methanol and other salts present in crude glycerol.Various studies on tuning the material properties of the polyhydroxyalkanoate (PHA) polymer are carried out for its higher applicability in diverse areas . Nerve 2ε-polylysine and polyhydroxyalkanoates are important biopolymers which can be conjugated with other biopolymers for its various applications e.g., ε-polylysine may be utilized as water absorbable hydrogels, drug carriers and anticancer agents. Simultaneously, PHA may be used in drug delivery systems, atrial septal defect repair and cardiovascular stents. ε-polylysine and polyhydroxyalkanoates are important biopolymers which are synthesized through microbe in an efficient and eco-friendly manner. However, these biopolymers may be conjugated with other biopolymers for its application as water absorbable hydrogels, drug carriers and anticancer agents. The hydrogels prepared from these complex biopolymers may be used for its application in quick peritoneal repair and prevention of post-surgical intraabdominal adhesions.In the present study, for sustainable development of biodiesel production, efforts have been made for effective utilization of crude glycerol as the carbon source for simultaneous production of ε-polylysine and PHA. The present bioconversion route will effectively convert the waste stream of biodiesel production into value added products. In addition, metabolic engineering may used in future for improving product yield of such strains.2, Na2SO4, CaCl2, KCl, NaHCO3, KBr, SrCl2, H3BO3, Na2O3Si, NaF, (NH4)(NO3) and Na2HPO4 were purchased from M/S Hi-Media Limited Mumbai, and were of the highest purity available. ε-PL was procured from Handary SA, Brussels, Belgium, Polyhydroxybutyrate and 3-hydroxyvalerate from Sigma, Bangalore, India.Peptone, yeast extract, iron(III) citrate, NaCl, MgClBacillus licheniformis was isolated from sea brine of experimental salt farm, Bhavnagar, India. It was maintained on Zobell marine agar plates containing (g/L) peptone 5.0; yeast extract 1.0; iron(III) citrate 0.1; NaCl 19.45; MgCl2 8.8; Na2SO4 3.24; CaCl2 1.8; KCl 0.55; NaHCO3 0.16; KBr 0.08; SrCl2 0.034; H3BO3 0.022; Na2O3Si 0.004; NaF 0.0024; (NH4)(NO3) 0.0016; Na2HPO4 0.008; agar, 1.5, at pH 7.6 ± 0.2. 5 mL of glycerol was added to the above medium. The slants were incubated at 37 °C for 4 days and then stored at 4 °C.4)2SO4, 15; MgSO4, 0.5; K2HPO4, 0.8; KH2PO4, 1.4; FeSO4, 0.04; ZnSO4, 0.04. The pH of the medium was adjusted to 6.8 with 1 N NaOH before sterilization [8 cells/mL) was used as inoculum. Shake flask cultures of the organism were incubated at temperature 37 ± 2 °C with continuous agitation at 150 rpm for 96 h. These fermentation parameters were kept uniform for all the studies conducted. All experiments were carried out in triplicates.Experiments were carried out in 250 mL Erlenmeyer flasks with 100 mL of production medium with following components (g/L): yeast extract, 10; glucose, 50;  peptone 5.0; yeast extract 1.0; iron(III) citrate 0.1; NaCl 19.45; MgCl2 8.8; Na2SO4 3.24; CaCl2 1.8; KCl 0.55; NaHCO3 0.16; KBr 0.08; SrCl2 0.034; H3BO3 0.022; Na2O3Si 0.004; NaF 0.0024; (NH4)(NO3) 0.0016; Na2HPO4 0.008 in one liter of the medium maintained at pH 7.6 ± 0.2. 20% seed culture was inoculated in the production medium which contained 20 g crude glycerol, yeast extract 5 g, (NH4)2SO4 10 g, K2HPO4 0.8 g, KH2PO4 1.36 g, MgSO4 0.5 g, ZnSO4 0.04 g, FeSO4 0.03 g in one litre of the medium maintained at pH 8.9 ± 0.2.The marine bacteria was cultured in Zobell marine broth to obtain seed culture having O.D.g rcf for 10 min in refrigerated centrifuge. 1 mL of supernatant was added to 1 mL of 1 mM methyl orange, mixed thoroughly under shaking condition along with incubating it at 37 °C for 60 min [g rcf for 10 min in refrigerated centrifuge and absorbance of the supernatant was measured at 465 nm on UV-vis spectrophotometer . A standard curve was derived from measurements with known amounts (0.1–2 mg/mL) of standard ε-PL procured from Handary S.A. [The culture broth was harvested after fermentation and cells were separated by centrifugation at 15,296× r 60 min . Furtherary S.A. .Bacillus licheniformis PL26 was calculated aPercentage carbon utilized by Glycerol estimation was carried out by using “Waters Alliance” high performance chromatographic system equipped with RI detector  and separation module . Chromatographic separations were performed on an “Aminex HPX-87H” column (300 × 7.8 mm)  with a precolumn (30 × 4.6 mm) of the same stationary phase . Isocratic elution at a flow rate of 0.6 mL/min was carried out using a mixture of 5 mM sulfuric acid. Peak detection was made by keeping the cells of the RI detector at 30 °C. The samples were appropriately degassed, twice diluted with double-distilled water, filtered through a “Whatman” 0.45-μm filter membrane , and then injected (50-μL loop volume). Data were obtained and processed by using “Waters EMPOWER” software . Peak identification was carried out by spiking the sample with pure standards and comparing the retention times with those of pure compounds.g rcf for 10 min in refrigerated centrifuge. The cell pellets were oven dried overnight at 60 °C. Cellular digestion of dried cell pellet was carried out by re-suspending it in 6% (v/v) sodium hypochlorite solution followed by centrifugation at 10,000 rpm for 5 min. Further, the digested cell pellets were washed twice with methanol followed by distilled water to remove the traces of impurities resulting in a purified product, which was further dissolved in chloroform and weighed after air drying [After completion of 96 h fermentation, culture broth was centrifuged at 15,296× r drying .− anion. The polycationic ε-PL salt with the TPB− anion was further purified by washing the mixed precipitate with acetone to remove triphenylborate and benzene. Thereafter, the precipitate reacted with 1 M HCl for obtaining ε-PL hydrochloride.After removal of cells through centrifugation, the supernatant obtained was treated with sodium tetraphenylborate for precipitating ε-PL as a polyelectrolyte salt with the TPB1H Nuclear magnetic resonance spectroscopy of PHA was determined on Bruker Avance-II 500 (Ultra shield) spectrometer, Bangalore, India, at 500 MHz, in CDCl3. Proton 1H NMR spectroscopy was also used to determine copolymer composition through running standards of 3HB and 3HV. Differential scanning calorimetry (DSC) of PHA was carried out using a DSC 204 F1 phoenix instrument with Netzsch software . The PHA samples were scanned from −20 °C to 500 °C with the heating rate of 10 °C/min. Glass transition temperature and onset melting points were determined in the scan between −20 °C and 500 °C in DSC analysis. Thermo-gravimetric analysis (TGA) of PHA was carried out in temperature range of 27–500 °C using TG 209 F1 instrument .FT-IR spectra of obtained PHA were recorded on a Perkin-Elmer Spectrum GX  instrument. After complete submerged fermentation of 96 h at an agitation of 220 rpm, fermentation broth was centrifuged to obtain supernatant containing ε-polylysine and biomass for PHA extraction.B. licheniformis PL26 was obtained utilizing crude glycerol as the carbon source. However, B. licheniformis is able to produce 0.2 g/L ε-polylysine extracellularly in the fermentation broth along with 64.59% PHA with respect to dry cell weight i.e., 1.1 g/L P(3HB-co-3HV) having 96 h production age at 37 °C  is reported to produce ε-polylysine utilizing crude glycerol [S. albulus being an Actinomycetes, it possesses a relatively slower growth rate as compared to Bacillus sp. As previously reported, S. albulus produces ε-polylysine after 120 h in M3G medium containing glucose as the carbon source, 0.2 g/L of ε-polylysine was produced by Streptomyces albulus (CCRC 11814) after 168 h [B. licheniformis at a concentration of 0.2 g/L. In addition, fast growth rate and less production age of B. licheniformis which is producing 64.59% PHA with respect to dry cell weight i.e., 1.1 g/L P(3HB-co-3HV), which may be considered as the additional advantage of the process. Simultaneously, crude glycerol was replaced with analytical grade pure glycerol with similar concentration of the carbon source and it was found that pure glycerol yielded 0.06 g/L ε-polylysine and 0.4 g/L P(3HB-co-3HV).In the present case, glycerol . S. albul-lysine produced from Bacillus licheniformis PL26 is in similar range with respect to its production from a wild strain Streptomyces albulus CCRC 11814 as reported in the literature [Haloferax mediterranei, simultaneously poly was produced [0.2 g/L of ε-Poly-terature . Howeverterature . Similarproduced . Few micBacillus licheniformis PL26 is shown in Bacillus licheniformis isolated from salt pan has a 30% total carbon utilization percentage as it utilizes 0.21% total carbon from 0.7% total carbon present in the production medium.Carbon utilization percentage of isolated B. licheniformis PL26 grown in production media was characterized through TGA, DSC, NMR and Fourier transform infrared spectroscopy (FTIR).The polymer extracted from The DSC analysis shown in 1H NMR spectra of the extracted polymer, standard PHB and standard 3 hydroxy valerate were found to be comparable with respect to each other. Prominent peaks were observed at δ = 1.6 ppm for CH3, δ = 2.4 ppm for CH2 and δ = 5.2 ppm for CH group  spectra  showed ia, Hc) attached to α-amino groups arrived together as broad singlet at δ 3.76 ppm and protons  attached to ε-amino groups arrived together as broad singlet at δ 3.14 ppm. Protons attached to β and β’ carbons come at δ 1.75 ppm as broad singlet. While the other protons attached to carbons come at δ 1.47 ppm  and δ 1.30 ppm ) as well as ε-polylysine (0.2 g/L) using crude glycerol as the carbon source. In addition, there are no such reports as per our knowledge wherein two polymers are produced at a time in the same fermentation broth. Previously, Bera et al., 2014 reported microbial synthesis of such polymer (P(3HB-co-3HV)) by Halomonas hydrothermalis (MTCC accession no. 5445) from seaweed derived levulinic acid at a concentration of 57.5% PHA/dry cell weight and Ghosh et al., 2011 reported microbial synthesis of PHA from biodiesel by-products i.e., from crude glycerol and Jatropha deoiled cake hydrolysate at a concentration of 75% PHA/dry cell weight. However, further optimization will be required for efficient production of PHA and ε-polylysine at larger scale.The described process showed the potential of utilizing The most important parameter responsible for cost of the production in PHA is substrate for carbon and energy source. The economic feasibility of PHA would depend on few important factors like growth rate of microbe for generation of biomass, substrate cost and recovery process including the solvent involved. However, production of other biopolymer along with PHA in the fermentation medium will have additional advantage in further reducing the production cost. PHA production from biodiesel waste stream can reduce production cost and at the same time solves the problem of waste disposal. In order to increase the PHA yield, the concentration of carbon source and other nutrient source desired for the microbial production may be optimized.Bacillus licheniformis) isolated from west coast of India utilizing Jatropha biodiesel waste residues as carbon rich source. A maximum of 0.2 g/L ε-polylysine content and 64.6% (w/w) P(3HB-co-3HV) production with respect to dry biomass was obtained in the fermentation broth using Bacillus licheniformis. ε-polylysine and PHA are important class of biopolymers which have various applications in food, agriculture, medicine, pharmacy, controlled drug release, tissue engineering, etc. Although, the present approach provides a solution for the effective utilization of biodiesel by-product, still, the developed process needs to be optimized further for gaining still better yield of both the products for its acclamation as cost effective and sustainable process.In the present study, an integrated process for simultaneous production of extracellular ε-polylysine and intracellular P(3HB-co-3HV) developed through marine bacterial strain (


This pathway has attracted much interest because of its roles in carbon and nitrogen metabolism, stress responses, and signaling in higher plants. The aim of this study was to isolate and characterize genes encoding GADs from Caragana intermedia, an important nitrogen-fixing leguminous shrub.Glutamate decarboxylase (GAD), as a key enzyme in the CiGAD1 and CiGAD2) were isolated and characterized. Multiple alignment and phylogenetic analyses were conducted to evaluate their structures and identities to each other and to homologs in other plants. Tissue expression analyses were conducted to evaluate their transcriptional responses to stress  and exogenous abscisic acid.Two full-length cDNAs encoding GADs -binding domain in the C-terminal region. The phylogenetic analysis showed that they were more closely related to the GADs of soybean, another legume, than to GADs of other model plants. According to Southern blotting analysis, CiGAD1 had one copy and CiGAD2-related genes were present as two copies in C. intermedia. In the tissue expression analyses, there were much higher transcript levels of CiGAD2 than CiGAD1 in bark, suggesting that CiGAD2 might play a role in secondary growth of woody plants. Several stress treatments  significantly increased the transcript levels of both CiGADs, except for CiGAD2 under Cd stress. The CiGAD1 transcript levels strongly increased in response to Zn stress (74.3-fold increase in roots) and heat stress (218.1-fold increase in leaves). The transcript levels of both CiGADs significantly increased as GABA accumulated during a 24-h salt treatment. Abscisic acid was involved in regulating the expression of these two CiGADs under salt stress.The CiGADs cloned from C. intermedia are closely related to homologs in another legume, soybean. CiGAD2 expression was much higher than that of CiGAD1 in bark, indicating that CiGAD2 might participate in the process of secondary growth in woody plants. Multiple stresses, interestingly, showed that Zn and heat stresses had the strongest effects on CiGAD1 expression, suggesting that CiGAD1 plays important roles in the responses to Zn and heat stresses. Additionally, these two genes might be involved in ABA dependent pathway during stress. This result provides important information about the role of GADs in woody plants’ responses to environmental stresses.This study showed that two  We analyzed changes in their transcript levels in response to different stress factors and exogenous abscisic acid (ABA). These results allowed us to explore the relationship between GAD expression and GABA accumulation, which will increase our understanding of the roles of GABA in stress adaptation in plants.Our previous studies showed that salt stress induced significant expression of EST sequences  homologois shrub . TherefoC. intermedia were collected from a desert habitat in Inner Mongolia, China. Seedlings were cultivated as described by 4 (100  µM), CdCl2 (500  µM), high temperature (40 °C), low temperature (4 °C), or dehydration (for 3 h); (2) NaCl (200 mM) for different durations ; and (3) exogenous ABA  combined with the 200 mM NaCl treatment for 0, 3, and 24 h. Roots and leaves were harvested, frozen immediately in liquid nitrogen, and then stored at −80 °C. Also, different tissues including roots, stems, leaves, seeds, testae, and bark were collected from mature individuals growing at the Chinese Academy of Forestry, Beijing, frozen immediately in liquid nitrogen, and then stored at −80 °C. Every experimental treatment had three biological replicates.Seeds of CiGAD1 and CiGAD2 have been deposited in GenBank under the accession numbers KU586714 and KU586715, respectively.Total RNA was extracted from pooled roots and leaves with Trizol reagent , and complementary DNA (cDNA) was generated with the SMARTer™ RACE cDNA amplification kit  according to the manufacturer’s instructions. Then, 5′ and 3′ RACE were performed using nested gene-specific primers . The obtCiGAD1 and CiGAD2 were analyzed with ORF Finder (https://www.ncbi.nlm.nih.gov/orffinder/), and the isoelectric point (pI) and molecular weight (MW) of the predicted protein were estimated using ExPASy (http://web.expasy.org/compute_pi/). Sequences for the protein homology analysis were obtained from phytozome , and aligned using DNAMAN 8.0. The phylogenetic tree was constructed using MEGA 6.06 software by the maximum-likelihood algorithm with 1,000 bootstrap replicates.The two full-length cDNA sequences of C. intermedia using CTAB method . Briefly, the DIG-labeled DNA probe was prehybridized at 60 °C for 2 h and then hybridized 37 °C for 12 h. Following hybridization, the blot was washed twice in 2 × SSC containing 0.1% SDS at 25 °C for 5 min and was washed twice in 0.1 × SSC containing 0.1% SDS at 50 °C for 15 min. The immunodetection of the DIG-labeled probe was performed with 1:10,000 anti DIG-AP. The blot was exposed to X-ray film . DNA probes were amplified from genomic DNA by using the primers designed in the sequenced DNA fragments based on the cDNA sequences of CiGAD1 and CiGAD2, while the probe for CiGAD1 was mainly in the region from 1,076–1,370 bp in the full-text cDNA, and the probe for CiGAD2 was mainly in the region from 639–1,031 bp in the full-text cDNA. Primers are listed in Genomic DNA was isolated from leaves of B method , and 20 About 0.1 g sample  was ground into a powder in liquid nitrogen and transferred to a 2-mL centrifuge tube. Then, 1 mL pre-cooled extraction buffer  polyvinylpyrrolidone, 3 mM 2-mercaptoethanol, 0.2 mM PLP , 10% (v/v) glycerol, 1 mM phenylmethylsulfonyl fluoride, pH 5.8) was added, and the homogenate was mixed well. After 15 min incubation on ice, the homogenate was centrifuged at 10,000 g for 15 min at 4 °C. The supernatant was collected into a 2-mL volumetric flask and the volume was adjusted to 1 mL. This was used as the crude enzyme solution CES; .To measure GAD activity, the assay mixture (1 mL) contained 0.4 mL 0.1% sodium glutamate, 0.2 mL 0.25 mM PLP, and 0.4 mL CES. After mixing well, the reaction mixture was incubated at 40 °C for 5 h and then boiled for 5 min to end the reaction. Sodium glutamate was replaced by 0.4 mL distilled water in the blank control. After cooling, the absorbance of the mixture was measured at 640 nm using a Multi-Mode Detection Platform . A GABA standard curve was constructed and used to calculate the GABA content in the samples based on absorption value, as described by β-NADP+, 5 mM 2-ketoglutarate and 0.02 units of GABase (Sigma). The increase of OD340 nm was recorded using 96-well microplate reader. The amount of GABA was calculated according to external calibration curve of GABA . Real-time qRT-PCR was performed in quadruplicate using the SYBR Premix Ex Taq™ II kit (Takara) on a Roche lightCycler 480  according to the manufacturer’s instructions. The 2−ΔCt method was adopted to analyze the qRT-PCR result, and ΔCt = Cttarget-Ct αEF1. Values shown were representative of three biological experiments.Total RNAs were extracted from a pool of roots and leaves with Trizol reagent (Invitrogen), and then cDNAs were synthesized with 0.5 µg RNAs using the Prime Script™ RT reagent kit  according to the manufacturer’s instructions. Specific RT-PCR primers  were desP < 0.05.Data were evaluated by Duncan’s multiple test in SPSS v.19. Differences were considered significant at GAD) genes were isolated from C. intermedia by 5′- and 3′-RACE, as shown in CiGAD1 (GenBank ID: KU586714) and CiGAD2 (GenBank ID: KU586715). Bioinformatics analyses showed that CiGAD1 had a 1922-bp full-length mRNA sequence containing a 1521-bp open reading frame (ORF), a 107-bp 5′ puntranslated region (UTR), and a 294-bp 3′-UTR; and that CiGAD2 had a 1797-bp full-length mRNA sequence containing a 1797-bp ORF, a 60-bp 5′-UTR, and a 237-bp 3′-UTR. The alignment showed that the nucleotide identity between CiGAD1 and CiGAD2 was 69.0%. Theoretically, CiGAD1 encodes a 57.3-kDa peptide consisting of 506 amino acids with the theoretical isoelectric point (pI) of 5.5, and CiGAD2 encodes a 56.1-kDa peptide consisting of 499 amino acids with the theoretical isoelectric point (pI) of 5.6.Two full-length cDNAs of glutamate decarboxylase , followed by testae and roots . The GAD activity was highest in testaes (9.9 U), followed by seeds (3.2 U). But its activity was lower, and similar, among the other tested tissues (1.1–1.9 U)  .CiGADs were expressed differently in response to various stress conditions, total RNA was extracted from roots and leaves of young seedlings under several different stress treatments. Both CiGADs showed dramatic increases in their transcript levels in roots and leaves in response to stress , but ABA at other concentrations drastically reduced the transcript levels of both genes, except for CiGAD1 at 10 µM ABA  were cloned based on specific EST sequences  to GADs from three model plants: soybean, Arabidopsis, and poplar, so CiGADs were classified into the GAD family  and nitrogen metabolism   stress treatments including salinity (Na), heavy metals (Zn and Cd), heat (40 °C), cold (4 °C), and drought (3 h dehydration), the transcript levels of CiGAD1 and CiGAD2 increased in the roots and leaves (2+ induced by each stress treatment (CiGAD2 transcript levels did not increase in response to Cd2+ (2+) from proteins or compete with Ca2+ to bind to Ca2+-binding proteins (CiGAD1 and CiGAD2 expression more strongly in leaves (20.6–36.0 fold) than in roots (4.4–5.5 fold)  . These rpathways . When Cad leaves , possiblreatment . The onl to Cd2+ , 4B, whiproteins . The sal.5 fold) , 5B. Forreatment . This reCiGADs in leaves and roots, and induced a substantial increase in CiGAD1 expression (a 74.0-fold increase) in roots (CiGAD1 expression (4.4–6.0 fold), but no change or a decrease in CiGAD2 expression  of the salt treatment  Lane 1, 2, 3, 4: PCR amplification of open reading frame (ORF) of CiGAD1; Lane 5, 6, 7, 8: PCR amplification of ORF in CiGAD2. Red arrows were targeted bands; M: 2,000 bp marker.(A) Lane 1, 2: 5′ RACE of Click here for additional data file.10.7717/peerj.3439/supp-2Figure S2AT5G17330), AtGAD2 (AT1G65960). , AtGAD3 (AT2G02000), AtGAD4 (AT2G02010), AtGAD5 (AT3G17760), PtGAD1 (Potri.T059200), , PtGAD2 (Potri.004G075200), PtGAD3 (Potri.004G075300), PtGAD4 (Potri.010G100500),, PtGAD5 (Potri.012G039000), , GmGAD1 (Glyma02g40840), , GmGAD2 (Glyma09g29900), GmGAD3 (Glyma14g39170), GmGAD4 (Glyma16g34450), and GmGAD5 (Glyma18g04940). The abbreviation of gene names are as follows: At, Arabidopsis thaliana; Pt, Populus trichocarpa; Gm, Glycine max.Accession number: AtGAD1 (Click here for additional data file.10.7717/peerj.3439/supp-3Table S1Click here for additional data file.10.7717/peerj.3439/supp-4Table S2Click here for additional data file.10.7717/peerj.3439/supp-5Supplemental Information 1C. intermedia under salt stress by RNA-seq.Two EST sequences were identified in Click here for additional data file.10.7717/peerj.3439/supp-6Supplemental Information 2CiGAD1 and CiGAD2 were KU586714 and KU586715 respectively in GenBank.Note: the accession numbers of Click here for additional data file.10.7717/peerj.3439/supp-7Supplemental Information 3Click here for additional data file.


The crystal structure of bis(tri-2-pyridylamine)iron(II) bis(perchlorate) has been redetermined, and that of the isotypic bis(tri-2-pyridylamine)nickel(II) bis(perchlorate) complex has been rerefined. In each case, the perchlorate anion is disordered over four sets of atomic sites, and the ions are linked by C—H⋯O hydrogen bonds to form a supramolecular three-dimensional framework. 15H12N4)2](ClO4)2, (I), confirms the structure previously reported (ClO4)2, (III), has been refined using the original data set , again using a four-component disorder model for the anion, rather than a two-component model as in the original report, leading to more satisfactory Cl—O distances and O—Cl—O angles.The redetermination of the structure of the title compound, [Fe(Cl. 1978a. Aust. J al. 2011. Acta Cr In the se Fig. 3 is very et al., 1978bAs noted above, the cobalt analogue (II) of compounds (I)P21/n with Z′ = 0.5  and 2.006 (3) Å; the usual (4 + 2) coordination of CuII is completed by two weakly-coordinated perchlorato ligands with a Cu—O distance of 2.593 (8) Å. By contrast, in the corresponding bis(trifluoromethanesulfonate) salt the anion plays no role in the metal coordination, where the bidentate amine ligands form a distorted tetrahedral geometry v/v, 20 ml) was placed in a Teflon-lined autoclave and heated at 423 K for 48 h. The autoclave was then allowed to cool to ambient temperature. Red prismatic crystals of the title compound were collected by filtration, washed with water and dried in air (yield 25%).For the synthesis of compound (I)Uiso(H) = 1.2Ueq(C). For the minor disorder components of the perchlorate anion in each compound the bonded distances and the 1,2 non-bonded distances were restrained to be the same as the corresponding distances in the dominant component, subject to s.u.s of 0.005 Å and 0.01°, respectively: in addition, the anisotropic displacement parameters for corresponding atom sites were constrained to be the same. Subject to these conditions, the refined values of the anion occupancies were 0.415 (3), 0.267 (3), 0.256 (3) and 0.061 (3) in (I)K = [mean(Fo2)/mean(Fc2)], 11.399 for the group of 368 very weak reflections having Fc/Fc(max) in the range 0.000 < Fc/Fc(max) < 0.007, and 3.057 for the group of 312 very weak reflections having Fc/Fc(max) in the range 0.008 < Fc/Fc(max) < 0.0014; the corresponding value for (III)Fc/Fc(max) in the range 0.000 < Fc/Fc(max) < 0.007.Crystal data, data collection and structure refinement details are summarized in Table 310.1107/S2056989018005601/su5436sup1.cifCrystal structure: contains datablock(s) global, I, III. DOI: 10.1107/S2056989018005601/su5436Isup2.hklStructure factors: contains datablock(s) I. DOI: 10.1107/S2056989018005601/su5436IIIsup3.hklStructure factors: contains datablock(s) III. DOI: 1836078, 1556391CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Phenols are chemical compounds which are included in the high priority of pollutants by environmental protection agency (USEPA). The presence of high concentrations of phenols in wastewaters like oil refineries, petrochemical plants, olive oil, pesticide production and oil field operations contain high soluble solids (TDS) and in an olive oil plant, wastewater is acidic, high salty and phenol concentrations are in the range of 0.1- 1%./l.Kinetic parameters were calculated according to Monod, Modified Stover- Kincannon, Hamoda and Haldane models. The influence of different initial phenol concentrations on the biodegradation rate was performed. The concentrations of phenol varied from 0 to 500 mgi in saline phenolic wastewater in attached growth systems was higher than suspended growth systems that represented a higher phenol inhibition in suspended growth systems. It was obvious that the best model fitting the obtained data are Hamoda model and the Modified Stover-Kincannon model, having highest R2 values of 0.991 and 1, respectively. The value of Ki in saline phenolic wastewater in attached growth system was higher than suspended growth systems which represented a higher phenol inhibition in suspended growth systems.The value of KHamoda model and the Modified Stover-Kincannon model having highest R2 value of 0.991 and 1, respectively, and also predicting reasonable kinetic coefficient values. Phenols are chemical compounds which have been categorized in the high priority of pollutants by environmental protection agency (USEPA) . PhenolsKinetic models which related to culture growth and substrate utilization in presence of some inhibitory materials are showed in Table Results of Gaudy study are shown with the above given equation . Hamoda Primitive equation equation  of ModifIf we replace the area disc with the effective volume of the reactor, in that case, we will have the equation below equation  15]:3\d:3\d15]:3This equation can also be written as a linear response as follows equation :4\documeHussain et al. performed some experiments to study the kinetic study of aerobic treatment of phenolic wastewater. Kinetics of phenol degradation has also been studied using Haldane model . In addimax, Ks and Ki were determined as 1.296 d−1, 19.23 mg/l,1571 mg/l, respectively ) for determination of the inhibitory coefficient (Ki). In Hamoda model, K and Ks were calculated by plotting 1/S vs. [A 0-S)]. The slope of regression line was equal to K/Ks and the intercept of regression line was equal to 1/Ks by plotting of [(S0-S)/X] vs.  vs. V/QS0.The principal aim of the present research was to define the growth and biodegradation kinetics of microorganisms at phenol concentration of 500 mg/l and salt concentration of 3% with HRT of 6, 12, 18, 24 and 36 h. For determination of the amount of biomass concentration at each stage, some number of media were removed randomly from the MBBR reactor which was followed by weighting. By measuring the weight difference of media with initial weight of media at each stage, biomass concentration was calculated. For conduction a comprehensive research, various kinetic models for degradation of phenol were applied. The kinetic parameters were also calculated according to Monod, Modified Stover- Kincannon, Hamoda and Haldane models. In Monod model, K and K0-S ]) was calculated as 1641 mg/l. The kinetic coefficients of K, Ks, Y and Kd in Hamoda model, is shown in Figs. s, Y and Kd were calculated 1.1 d−1, 91.74 mg/l, 0.482 mg/mg and 0.011 d−1, respectively. The kinetic parameters of Umax and KB in modified Stover - Kincannon model, according to Fig. Assuming a specific decay rate, KS, Kd, Y and μmax were obtained as 3.2 d−1, 71.7 mg/l, 0.077 d−1, 0.48 mg/mg, 1.5 d−1, respectively ) was calculated 1641 mg/l. The growth kinetics parameter values obtained from different models are shown in Table Kinetic studies are very important for industrial laboratory, in order to justify kinetic studies to generalize why using their results in the industrial scale . In the ectively . KineticS, Kd and Y were also determined as 9.39 mg/l, 169.3 mg/l, 0.1071 d−1, 0.882 mg/mg, respectively [max, K and Y were calculated as 5.28- 9.36 d−1, (33.4- 27.7) and 0.19- 0.28 mg/mg, respectively. By increasing phenol concentration up to 300 mg/l, the amount of μmax, Y, K were decreased to 5.28- 2.16, 0.2-0.29, 10.2- 18.2, respectively [max and amount of Ks were 1.92-8.64 d−1 (by average: 4.56 d−1) and 75 mg/l in their investigation, respectively [max and Ks were calculated as 4.432 d−1 and, 87.4 mg/l, respectively. The findings of that research were different from present one [s, Ki and μmax were also calculated as 2.256 d−1, 77.7 mg/l, 319.4 mg/l and 1.128 d−1, respectively [max, Ks, Ki were calculated as 1.296 d−1, 19.23 mg/l, 1571 mg/l, respectively. The inhibitor constant, in this study, was found 1641 mg/l [R2). According to Figs. R2 value and predicting reasonable kinetic coefficient values. The literature reported that performance of both Hamoda and Modified Stover-Kincannon models were more suitable for designing the aerobic submerged attached growth biological reactors [Tallaei et al. studied the kinetic parameters of crude oil decomposition by means of pseudomonas aeruginosa bacteria, The amount of K, Kectively . For finectively . Moreove d−1, 33.- 27.7 anreactors , 31, 32.s, Kd, Y and μmax were calculated 16.147 d−1, 130.32 mg/l, 0.0282 d−1, 0.4391 mg/mg and 7.23 d−1, respectively. Kinetic parameters like K, Ks, Y and Kd, according to Hamoda model, were calculated as 1.1 d−1, 91.47 mg/l, 0.482 mg/mg, 0.011 d−1, respectively. Kinetic parameters of the modified Stover- kincanon model (Umax and KB) were calculated as 47.61 g/l.d and 13.47 g/l.d, respectively. The value of Ki in saline phenolic wastewater in attached growth system was higher than suspended growth systems which represented a higher phenol inhibition in suspended growth systems. Hamoda model and the Modified Stover-Kincannon model having highest R2 value of 0.991 and 1, respectively, and also predicting reasonable kinetic coefficient values.In this research, we studied the growth and biodegradation kinetics of microorganisms at 500 mg/l phenol concentration and salinity of 3%. According to Monod equation, K, K


In vitro studies using autologous human NK cells show that DENV-infected monocyte-derived dendritic cells (MDDCs), but not monocytes, activate NK cells in a contact-dependent manner, resulting in upregulation of CD69 and CD25 and secretion of IFN-γ. Blocking adhesion molecules  on NK cells abolishes NK cell activation, IFN-γ secretion, and the control of DENV replication. NK cells activated by infected MDDCs also inhibit DENV infection in monocytes. These findings show the essential role of human NK cells in protection against acute DENV infection in vivo, identify adhesion molecules and dendritic cells required for NK cell activation, and delineate the sequence of events for NK cell activation and protection against DENV infection.Natural killer (NK) cells play a protective role against dengue virus (DENV) infection, but the cellular and molecular mechanisms are not fully understood. Using an optimized humanized mouse model, we show that human NK cells, through the secretion of gamma interferon (IFN-γ), are critical in the early defense against DENV infection. Depletion of NK cells or neutralization of IFN-γ leads to increased viremia and more severe thrombocytopenia and liver damage in humanized mice.  in vivo and reveal the sequence of molecular and cellular events that activate NK cells to control dengue virus infection.Dengue is a mosquito-transmitted viral disease with a range of symptoms, from mild fever to life-threatening dengue hemorrhagic fever. The diverse disease manifestation is thought to result from a complex interplay between viral and host factors. Using mice engrafted with a human immune system, we show that human NK cells inhibit virus infection through secretion of the cytokine gamma interferon and reduce disease pathogenesis, including depletion of platelets and liver damage. During a natural infection, DENV initially infects dendritic cells in the skin. We find that NK cells interact with infected dendritic cells through physical contact mediated by adhesion molecules and become activated before they can control virus infection. These results show a critical role of human NK cells in controlling DENV infection  Dengue virus (DENV) infection is the most important arthropod-borne human viral disease worldwide. An estimated 390 million infections occur each year, of which 500,000 cases develop severe dengue and 20,000 die . DENV isNatural killer (NK) cells are a critical component of the innate immune system and provide early defense against viral infections , 5. Clin6–During a natural DENV infection by an infective mosquito, viral particles are introduced into the subcutaneous space, where resident dendritic cells (DCs) are probably the first cell type to be infected . A subse+ hematopoietic stem/progenitor cells into immunodeficient NOD-scid IL2rg−/− (NSG) mice, it is possible to reconstitute the human immune system in the recipient mice  in uninjected humice to 8.6% ± 3.8% (n = 8) in injected humice (n = 8) or injected humice . Humice injected with cytokine plasmids are referred to as NK-optimized humice hereinafter, whereas humice without cytokine plasmid injection are referred to control humice.To study human NK cells in the control of acute DENV infection, we boosted the human NK cell levels in humanized mice by expressing human IL-15 and Flt3L through hydrodynamic injection of cytokine-encoding plasmids . Seven dd humice  and B. TP < 0.0001). The serum levels of human IFN-γ on day 3 postinfection were significantly higher in NK-optimized humice  than in control humice (50 ± 52 pg/ml) or mock-infected NK-optimized humice (38 ± 51 pg/ml)  . Of note1 pg/ml) .P < 0.05). Consistent with a previous report  for 2 h. After adsorption, cells were cultured and viral titers were measured by plaque assay in culture supernatants at 12, 24, 48, and 72 h. (A to E) Viral titers in culture supernatants from 5 different donors. Each dot represents a technical replicate, with median values indicated by horizontal lines. *, P < 0.05; ns, not statistically significant. Download FIG S1, TIF file, 0.4 MB.Kinetics of DENV-2 infection in monocyte-derived dendritic cells from heathy donors. Human monocytes were purified by negative selection, and monocyte-derived dendritic cells (MDDCs) were generated Copyright © 2017 Costa et al.2017Costa et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mBio.00741-17.2FIG S2 P < 0.05; ND, not detectable. Download FIG S2, TIF file, 0.1 MB.Cytokine levels in culture supernatants of monocyte-derived dendritic cells following DENV-2 infection. MDDCs were infected with DENV-2 (MOI = 1) for 2 h, and cytokine levels were measured by ELISA in culture supernatants at 0, 12, 24, 48, and 72 h. Mean values ± SD for 4 different donors are shown. *, Copyright © 2017 Costa et al.2017Costa et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mBio.00741-17.3FIG S3 P < 0.05. Download FIG S3, TIF file, 0.02 MB.Effects of different doses of recombinant IFN-γ on DENV-2 infection of monocyte-derived dendritic cells. MDDCs were infected with DENV-2 (MOI of 1) for 2 h. After adsorption, cells were incubated in the presence of medium or different doses of recombinant IFN-γ. Viral titers in culture supernatants were assayed by plaque assay 48 h after infection. Each dot represents a different donor, with median values indicated by horizontal lines. *, Copyright © 2017 Costa et al.2017Costa et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the P < 0.01)  . Further < 0.01)  and elev < 0.01) . These r10.1128/mBio.00741-17.4FIG S4 in vitro. MDDCs were infected with DENV-1, DENV-3, or DENV-4 (MOI or 1) for 2 h and then incubated with autologous purified human NK cells at a ratio of 1:5 for 48 h. (A) Viral titers in the culture supernatants, measured by plaque assay. Each dot represents a different donor. (B) IFN-γ levels  in culture supernatants, determined by ELISA. *, P < 0.05; ND, not detectable. Download FIG S4, TIF file, 0.1 MB.Naive NK cells control DENV-1, DENV-3, and DENV-4 infection of MDDCs Copyright © 2017 Costa et al.2017Costa et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the We determined whether cell-cell contact was required for NK cell activation by infected MDDCs via a transwell system. NK cells were either cocultured with infected autologous MDDCs in the same chamber or separated by a 0.4-μm membrane in a different chamber where NK cells did not have direct contact with infected MDDCs . As expeGiven the requirement for direct cell-cell contact in NK cell activation and control of dengue infection, we examined the involvement of specific NK cell surface molecules, including adhesion molecules  and natural cytotoxicity-triggering receptors . NK cells expressed the various adhesion molecules, but their levels of expression were not changed significantly following coculture with infected MDDCs . NK cell10.1128/mBio.00741-17.5FIG S5 FIG S5, TIF file, 0.2 MB.Expression of adhesion molecules by NK cells before and after coculture with infected MDDCs. Human NK cells were cocultured with DENV-2-infected autologous MDDCs for 48 h. Cells were stained with antibodies specific for CD56, CD1a, and the indicated adhesion molecules. Shown are representative staining profiles of CD56 versus the indicated adhesion molecules, obtained by gating on CD1a-negative cells (left). Numbers are percentages of positive cells in gated regions. Percentages of positive cells  from four donors are shown on the left. Each dot represents a different donor. Download Copyright © 2017 Costa et al.2017Costa et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mBio.00741-17.6FIG S6 FIG S6, TIF file, 0.2 MB.Surface expression of NK cell receptors before and after coculture with infected MDDCs. Human NK cells were cocultured with DENV-2-infected autologous MDDCs for 48 h. Cells were stained with antibodies specific for CD56, CD1a, and the indicated NK cell receptors. Shown are representative staining profiles of CD56 versus the indicated receptors, obtained by gating on CD1a-negative cells (left). Numbers are percentages of positive cells in gated regions. Percentages of positive cells  from five donors are shown on the right. Each dot represents a different donor. Download Copyright © 2017 Costa et al.2017Costa et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the To block each of these receptors, purified NK cells were incubated with receptor-specific antibodies individually for 2 h and then cocultured with infected MDDCs for 48 h. NK cells were assayed for CD69 expression, and the culture supernatants for virus titers and IFN-γ. As shown by the results in Monocytes and macrophages can also be infected by DENV . We alsoDuring a natural DENV infection in humans, dendritic cells in the skin are infected first, followed by infection of monocytes and macrophages in the peripheral blood. We investigated whether NK cells activated by infected MDDCs could control DENV infection in human monocytes. NK cells were cocultured with infected MDDCs for 48 h, purified by cell sorting, and cultured with DENV-infected autologous monocytes at a ratio of 1:5 for another 48 h. As with IL-2-activated NK cells, iMDDC-activated NK cells upregulated CD69 expression  and B, r10.1128/mBio.00741-17.7FIG S7 in vitro. Human monocytes and NK cells were purified by negative selection. NK cells were activated by culturing with IL-2 for 5 days. Monocytes were infected with DENV-2 (MOI of 10) for 2 h and incubated with either naive NK cells or IL-2-activated NK cells for 48 h in the same chamber or in a transwell. (A) Viral titers in culture supernatants. (B) IFN-γ levels in culture supernatants. Bar graph shows mean values ± SD. Each dot in a dot plot represents a different donor. *, P < 0.05; ND, not detectable. Download FIG S7, TIF file, 0.1 MB.Cell-cell contact is required for control of DENV-2 infection in monocytes Copyright © 2017 Costa et al.2017Costa et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mBio.00741-17.8FIG S8 P < 0.05; ND, not detectable; ns, not statistically significant. Download FIG S8, TIF file, 0.1 MB.Adhesion molecules are involved in NK cell-monocyte interactions and control of infection. Human monocytes and NK cells were purified from PBMC by negative selection. NK cells were activated by culturing with IL-2 for 5 days. Monocytes were infected with DENV-2 (MOI of 10) for 2 h and incubated with IL-2-activated NK cells for 48 h in the presence or absence of various neutralizing antibodies. (A) Viral titers in culture supernatants. (B) IFN-γ levels in culture supernatants. Bar graphs show mean values ± SD. Each dot in a dot plot represents a different donor, and horizontal bars show median values. *, Copyright © 2017 Costa et al.2017Costa et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the in vivo (NK-optimized humice) and the cellular and molecular events required for NK cell activation and IFN-γ secretion. Although studies have suggested an important role of NK cells and IFN-γ in protection against DENV infection, previous studies were carried out either in vitro using purified NK cells due to the lack of a small animal model of dengue infection , consistent with a similar report by Lim et al. . In addit manner . These fIn contrast to the case for adhesion molecules, blocking NKp44, NKp46, NKp30, and NKG2D did not have any effect on NK cell activation and IFN-γ secretion and control of DENV infection. The levels of NKp44 and NKp30 on NK cells are minimal, and this could explain the absence of any effect. Both NKG2D and NKp46 are expressed by NK cells. In fact, genome-wide associated studies have identified MICA and MICB, which are ligands for NKG2D, as susceptibility loci for dengue shock syndrome , 27. Howin vivo. Lim et al. have reported that the combination of cell-cell contact and type I interferon and TNF-α is required for activating NK cell cytotoxicity . All women were informed of the risks of participation in the study, and a written consent for fetal tissue donation to research was obtained from each woman. SingHealth and National Health Care Group Research Ethics Committees Singapore specifically approved this study (CIRB reference number 2012/064/B).This study was carried out in strict accordance with the ethical and animal experiment regulations of the National Advisory Committee for Laboratory Animal Research (NACLAR) . The expnull (NSG) mice were purchased from Jackson Laboratories  and maintained under pathogen-free conditions in National University of Singapore animal facilities. To generate humice, 2 × 105 human CD34+ cells were injected intracardially into each sublethally irradiated NSG pup within 24 to 48 h of birth as previously described  in RPMI 1640 medium (Gibco) with 10% fetal bovine serum (FBS) (Lonza) at 28°C. To concentrate the virus, 50 ml of the cell culture supernatant was loaded onto a VivaCell 100 centrifugal concentrator (Sartorius) and centrifuged at 2,000 × g for 10 min, and the supernatant remaining in the concentrator was aliquoted and stored at −80°C. For quantification of virus, BHK-21 cells (C-13)  were grown to a confluent monolayer in RPMI 1640 medium with 10% FBS (Lonza) and 1% penicillin-streptomycin-glutamine (Gibco) in 24-well plates at 37°C. The virus was serially diluted in serum-free medium and then inoculated into the cells at 37°C with gentle shaking every 15 min for 1 h. Next, the medium was replaced with RPMI 1640 medium containing 2% carboxymethylcellulose (CMC) and 2% FBS (Lonza) (overlay medium) and the culture kept at 37°C. After 5 days, the cells were fixed in 3% formalin in phosphate-buffered saline (PBS) for 1 h, washed, and stained with 0.1% crystal violet in 10% formalin solution for 1 h. Then, the plates were washed with water, and the plaques were counted. One PFU of the cell culture supernatant was observed to have close to 1,000 copies of viral RNA.DENV-2 (Eden-07K2861), DENV-1 (Eden-2402DK1), DENV-3 (Eden-863DK1), and DENV-4 (Eden-2270DK1) strains were propagated in mosquito cells  and then injected into the humanized mice as the control. Blood, liver, spleen, lymph node (axillary), and bone marrow (femur) were collected from control and infected mice at different days postinfection (dpi) for flow cytometry assays, viral RNA quantification, alanine aminotransferase (ALT) assays, platelet counts, and histology.Humanized mice were infected by injecting 1 × 10ALT levels in the sera from mice at 7 dpi were measured using a Cobas C111 analyzer (Roche) in the NUS comparative medicine in-house veterinary diagnostic laboratory. Histopathological analysis was performed as previously described by Costa et al. . Briefly+ CD209+ CD14− CD83−/low. Infection of MDDCs by DENV-2 virus was performed at an MOI of 1 in RPMI medium (RPMI supplemented with 3% FBS) for 2 h, followed by 3 washes to remove free virus. Autologous NK cells were purified from PBMC using the EasySep human NK cell enrichment kit (Stemcell Technologies) and cocultured with infected or uninfected MDDCs at ratios of 1:1, 1:5, and 1:10 in RPMI medium.Monocytes were negatively selected from total PBMC using the EasySep human monocyte enrichment kit without CD16 depletion (Stemcell Technologies). To generate MDDCs, monocytes were cultured for 7 days in RPMI 1640 (Sigma) supplemented with 10% FBS (Gibco), IL-4 , and GM-CSF . The MDDC phenotype was verified by flow cytometry as CD1aAlternatively, monocytes were infected with DENV (MOI of 10) in RPMI medium (RPMI supplemented with 3% FBS) for 2 h, followed by 3 washes to remove free viruses. A higher MOI was used for monocyte infection than for MDDC infection because monocytes are more resistant to DENV infection. Autologous NK cells were cocultured with infected or uninfected monocytes at ratios of 1:1, 1:5, and 1:10 in RPMI medium. For transwell experiments, culture plates with a 0.4-μm membrane from Millipore were used. For receptor blocking experiments, the following neutralizing antibodies (BioLegend) were used at 1 μl/ml: anti-CD11a (HI111), anti-CD18 (TS1/18), anti-CD2 (TS1/8), anti-DNAM (11A8), anti-2B4 (C1.7), anti-NKp46 (9E2), anti-NKp30 (P30-15), anti-NKp44 (P44-8), and anti-NKG2D (1D11) antibodies.The concentrations of cytokines  in serum samples or culture supernatants were measured using commercially available antibodies and according to the procedures supplied by the manufacturer . The results are expressed as picograms per milliliter. The detection limits of the ELISA assays were in the range of 4 to 8 pg/ml.Antibodies specific for human CD45 (2D1), CD14 (HCD14), CD56 (HCD56), CD56 (MEM188), CD19 (HIB19), CD3 (HIT3a), CD69 (FN50), and CD25 (FN50) and for mouse CD45.1 (A20) were obtained from BioLegend. Cells were stained with appropriate antibodies in 50 μl of fluorescence-activated cell sorting (FACS) buffer  for 30 min on ice. Stained cells were analyzed by flow cytometry using an LSRII instrument, and data were analyzed by using FACSDiva (BD Biosciences) or FlowJo .Mouse and human platelets were evaluated as previously described by Sridharan et al. . Brieflyt test was used to determine whether the means of the two samples were significantly different. For ranked data, such as the histological scoring of liver damage, the Mann-Whitney test was used to determine whether the scores were significantly different. Statistical analysis was performed using GraphPad Prism .For numerical continuous data, such as mean fluorescence intensities (MFI), cell percentages, DENV viremia, cytokine concentrations, liver enzymes, and platelet counts, the unpaired


Thalassiosira pseudonana, the substitution dynamics of lipid headgroups are well described, but those of the individual lipids, differing in fatty acid composition, are unknown. Moreover, the behavior of lipids outside the common headgroup classes and the relationship between lipid substitution and cellular particulate organic P (POP) have yet to be reported. We investigated these through the mass spectrometric lipidomics of P-replete (P+) and P-depleted (P−) T. pseudonana cultures. Nonlipidic POP was depleted rapidly by the initiation of P stress, followed by the cessation of P-lipid biosynthesis and per-cell reductions in the P-lipid levels of successive generations. Minor P-lipid degradative breakdown was observed, releasing P for other processes, but most P-lipids remained intact. This may confer an advantage on efficient heterotrophic lipid consumers in P-limited oceans. Glycerophosphatidylcholine (PC), the predominant P-lipid, was similar in composition to its betaine substitute lipid. During substitution, PC was less abundant per cell and was more highly unsaturated in composition. This may reflect underlying biosynthetic processes or the regulation of membrane biophysical properties subject to lipid substitution. Finally, levels of several diglycosylceramide lipids increased as much as 10-fold under P stress. These represent novel substitute lipids and potential biomarkers for the study of P limitation in situ, contributing to growing evidence highlighting the importance of sphingolipids in phycology. These findings contribute much to our understanding of P-lipid substitution, a powerful and widespread adaptation to P limitation in the oligotrophic ocean.Phytoplankton replace phosphorus-containing lipids (P-lipids) with non-P analogues, boosting growth in P-limited oceans. In the model diatom IMPORTANCE Unicellular organisms replace phosphorus (P)-containing membrane lipids with non-P substitutes when P is scarce, allowing greater growth of populations. Previous research with the model diatom species Thalassiosira pseudonana grouped lipids by polar headgroups in their chemical structures. The significance of the research reported here is threefold. (i) We described the individual lipids within the headgroups during P-lipid substitution, revealing the relationships between lipid headgroups and hinting at the underlying biochemical processes. (ii) We measured total cellular P, placing P-lipid substitution in the context of the broader response to P stress and yielding insight into the implications of substitution in the marine environment. (iii) We identified lipids previously unknown in this system, revealing a new type of non-P substitute lipid, which is potentially useful as a biomarker for the investigation of P limitation in the ocean. Diatoms are a diverse group of eukaryotic microalgae responsible for as much as 25% of global and 50% of oceanic annual primary production . Lipids,43−) concentrations   , 11, 12.liter−1) , 13.Thalassiosira pseudonana has been used as a model organism to study the effects of P starvation on lipid remodeling (+) growth conditions, T. pseudonana synthesizes glycerophosphatidylcholine (PC), glycerophosphatidylglycerol (PG), and glycerophosphatidylethanolamine (PE) , which is normally undetectable under P+ conditions   , 9. In cnditions , 9, 14. nditions –18. In al (SQDG) .T. pseudonana has been published , and digalactosyldiacylglycerol (DGDG) are enriched in the plastid thylakoid membranes . Further20–T. pseudonana are rapid, resulting in the exchange of the majority of cellular glycerophospholipids with DGCC and SQDG within 48 h of the initiation of P stress  substitution, reflecting a contrasting system  subject to P stress remain unknown. Revealing these dynamics has the potential to yield insight into mechanisms of P-lipid substitution, such as the synthesis of DGCC. Second, the effects of P-lipid substitution on particulate organic P (POP) content per cell have not been reported. Consequently, the relationship between the responses associated with lipidic and extralipidic POP remains unknown. Third, previous work has not provided a comprehensive discussion of the relative contributions of two potential underlying mechanisms for P-lipid substitution, i.e., active breakdown of glycerophospholipids and replacement with non-P alternatives versus a simple switch in biosynthesis to the production of non-P lipids . Exponential growth in P− cultures during the same period was marginally slower, at 0.026 ± 0.0016 h−1 .  Both P+ and P− cultures appeared to be transitioning into stationary phase at 96 h, with maximum populations of 1.08 × 106 ± 4.11 × 104 cells ml−1 and 1.14 × 106 ± 7.35 × 104 cells ml−1, respectively. Culture viability was high throughout, at >90%, in both P+ and P− cultures, except for the P− cultures at 48 h (87.95% ± 1.78%) ; the cultures were therefore considered to be healthy and not subject to stresses other than P up to 72 h.During exponential growth  between 43−])  in phosphorus-replete (P+) control cultures was in excess (>10 μmol liter−1) throughout the 96-h course of the experiment. [PO43−] in phosphorus-stressed (P−) cultures was 0.80 ± 0.00 μmol liter−1 at 0 h and dropped to 0.20 ± 0.00 μmol liter−1 after 6 h. This indicates a minor carryover of P from the P-replete seed cultures to the P− culture treatment, which was depleted around 6 to 12 h, subjecting the P− cultures to P stress. The dissolved nitrate concentration ([NO3−]) and dissolved silicate (orthosilicate ions) concentration ([SiO44−]) remained in excess throughout in both P+ and P− cultures , except for the P+ cultures after 72 h, in which [SiO44−] was depleted. The cultures were, therefore, not subject to confounding macronutrient stress, with the possible exception of the P+ 96-h samples. Taken together, the growth and nutrient data suggest that the 96-h P+ samples were entering an Si-limited stationary-growth phase. These data will, therefore, not be considered as a basis for drawing conclusions about P stress below, but they are included for context.The dissolved phosphate concentration  than the P+ control samples at 96 h. The POP quantities per cell determined for the P+ cultures, between 1.79 ± 0.07 and 2.84 ± 0.22 fmol cell−1, are approximately in line with previous observations of 2.8 to 6.2 fmol cell−1 for T. pseudonana (The quantity of particulate organic phosphorus (POP) per cell  fell witeudonana .+ and P− cultures between 0 and 6 h, from 0.73 ± 0.08 to 1.25 ± 0.05 fmol cell−1 in P− cultures, and declined rapidly thereafter to a minimum of 0.17 ± 0.02 fmol cell−1 at 96 h, 0.08-fold ± 0.01-fold less (P < 0.005) than the P+ control cultures at that time.Total phospholipid  represen+ cultures throughout and from P− cultures before 12 h. Concomitantly with the decline in P-lipid quantities, DGCC quantities increased over time in P− cultures after 12 h to reach a maximum of 0.85 ± 0.075 fmol cell−1 at 48 h and 0.67 ± 0.036 fmol cell−1 at 96 h.The betaine lipid diacylglycerylcarboxyhydroxymethylcholine (DGCC)  was abseP < 0.05) in P− cultures than in P+ control cultures at 6 h. SQDG quantities were statistically similar in P+ and P− cultures between 12 and 72 h, followed by a 0.71-fold ± 0.045-fold reduction (P < 0.005) in P− cultures relative to P+ cultures at 96 h. SQDG, therefore, did not change significantly in a consistent manner in response to P stress and did not appear to act as a substitute lipid for PG in this case. This finding contrasts with a previous report on the response of T. pseudonana and other phytoplankton, including cyanobacteria, isolated after longer periods of P starvation (The quantity of the sulfolipid sulfoquinovosyldiacylglycerol (SQDG)  was 1.31-fold ± 0.14-fold greater  in P− cultures at 96 h. To our knowledge, DAG has not been characterized in P-stressed T. pseudonana cultures previously and did not differ significantly and consistently in response to P stress.The quantity of diacylglycerol (DAG)  per cell did not differ significantly between PP. tricornutum, subject to P stress  storage lipids subject to P stress . This leP stress .− cultures, the quantity of nonlipidic POP declined rapidly from 1.21 ± 0.27 to 0.083 ± 0.17 fmol cell−1 over the course of the first 12 h, remaining approximately constant thereafter. Nonlipid POP in P+ cultures, in comparison, remained statistically constant during the first 12 h, decreasing over the course of 72 h from 1.19 ± 0.38 to 0.19 ± 0.12 fmol cell−1.The difference between the quantities of total POP and total P-lipids  was used−1, was 32.29% ± 10.91% higher (P < 0.005) than the dilution rate by culture growth, −0.023 ± 0.0015 h−1  and 12 h in the P− cultures (ΔPLt − 12 h) was also used to assess the total quantity of lipid per culture, regardless of per-cell quantities . At 48 and 72 h in P− cultures, ΔPLt − 12 h values were −42.27 ± 18.19 pmol ml−1 (P < 0.05) and −80.47 ± 20.03 pmol ml−1 (P < 0.005), respectively, indicating a net degradation of the total phospholipids at these times. The amount of P liberated from P-lipids between 12 and 72 h was 7.66 × 107 ± 1.95 × 107 atoms per daughter cell grown during that time, equivalent to 1.06 ± 0.55 diploid genomes per cell  at 72 h  corresponded to a loss of 34.10% ± 8.75% of the maximum total phospholipid observed at 12 h in P− cultures. This value is consistent with the P-lipid degradative loss rate as calculated above (32.29% ± 10.91% higher than the dilution rate by culture growth [P < 0.005]) over the same interval. In comparison, the change in total DGCC (ΔDGCCt − 12 h) showed a rapid increase over time, reaching +464.90 ± 50.05 pmol ml−1 at 72 h. Taking these findings together, the molar degradative decrease in PLt − 12 h was equivalent to 17.31% ± 4.69% of the increase in DGCC.The maximum degradative decrease (ΔPL+ and P− cultures at 72 h are shown in − cultures than in P+ control cultures. Some differences in the magnitude of this decrease were evident from the data: PC(18:4/16:0) and PC(20:5/18:4) showed the largest decreases, 0.018-fold ± 0.0035-fold (P < 0.005) and 0.023-fold ± 0.037-fold (P < 0.005), respectively. PC(16:1/16:0), PC(20:5/16:1), PC(20:5/16:0), and PC(38:6) showed highly significant, intermediate decreases between 0.053-fold ± 0.011-fold and 0.15-fold ± 0.043-fold. In contrast, the highly unsaturated PC(22:6/20:5) and PC(20:5/20:5) species displayed the smallest decreases, 0.28-fold ± 0.054-fold (P < 0.005) and 0.30-fold ± 0.052-fold (P < 0.005), respectively.Differences in the quantities of individual lipid species between P− cultures; levels were below detection in P+ control cultures. At 72 h, the five most abundant DGCC species in P− cultures were DGCC(20:5/16:0), DGCC(22:6/20:5), DGCC(20:5/20:5), DGCC(38:6), and DGCC(20:5/16:1), with quantities of 84.28 ± 14.14, 60.08 ± 9.39, 56.56 ± 11.87, 50.56 ± 7.40, and 33.05 ± 6.16 amol cell−1, respectively.Betaine lipid DGCC species  occurred− cultures than in P+ control cultures. Levels of the predominant PE lipid species PE(22:6/20:5), PE(20:5/20:5), and PE(20:5/16:1) decreased by 0.19-fold ± 0.065-fold (P < 0.005), 0.18-fold ± 0.060-fold (P < 0.05), and 0.07-fold ± 0.028-fold (P < 0.005), respectively, under P stress.The quantities of glycerophospholipid PE species  were low− cultures than in P+ control cultures. The predominant chemotypes, PG(16:1/16:0) (P < 0.005), PG(20:5/16:0) (P < 0.05), PG(20:5/16:1) (P < 0.005), PG(16:1/14:0) (P < 0.005), and PG(32:2) (P < 0.005), all exhibited decreases between 0.062-fold ± 0.027-fold and 0.11-fold ± 0.048-fold subject to P stress.The glycerophospholipid PG  behaved r) of 0.68 ± 0.25. In a second cluster, comprising PG, DAG, MGDG, and DGDG, fatty acid distributions were highly cross-correlated (r = 0.87 ± 0.10). In addition, these two clusters were highly distinct from each other (r = 0.074 ± 0.18). Interestingly, DGCC was moderately correlated with DAG at 12 h (r = 0.53 ± 0.023) but showed a weak relationship or none thereafter. DGCC abundance at 12 h was, however, negligible compared to that at 24 h and later.Bivariate analyses  were used to compare the relative abundances of individual lipid species within pairs of lipid classes in order to provide an indication of similarity in their fatty acid distributions. Overall, in considering the distributional similarity between all the lipid classes measured in both treatments, at all time points between 12 and 72 h, two primary clusters were observed. The first cluster, which comprised PC, its substitute DGCC, and PE, showed similar fatty acid distributions, with an average pairwise correlation coefficient  between 0.3 (P < 0.005) and 0.17 (P < 0.05), respectively. The linear regression coefficients were consistent with 18 and 20%, respectively, of the DGCC synthesized during these time intervals originating as recycled diglyceride moieties from degraded PC. This estimate is also consistent with the cumulative degradation of PC between 12 and 72 h, presented above, of 17.31% ± 4.69% of the increase in DGCC at the total class level .The absolute quantities of degradative loss of individual PC lipid species were weakly correlated with DGCC gains  for time intervals between 24 and 48 h and between 48 and 72 h in P+ control cultures were considered (−1 at 0 h to 168.57 ± 9.83 amol cell−1 at 24 h (P < 0.005) before dropping back to 39.67 ± 7.28 amol cell−1 by 72 h (P < 0.005). In contrast, the moderately unsaturated species PC(18:4/16:0), PC(20:5/18:1), and PC(20:5/18:4) displayed a consistently increasing trend with the progression of time in P+ control cultures. PC(20:5/18:4), for example, increased from 36.21 ± 7.44 amol cell−1 at 0 h to 159.26 ± 39.81 amol cell−1 at 96 h (P < 0.05).In order to study the temporal dynamics of P-lipids in the context of P stress at the individual lipid species level, the five most abundant PC lipids, as the predominant components of the total P-lipid pool, were analyzed in isolation. First, the underlying dynamics of the Pnsidered . The top− cultures at 12 h, was determined . The response of PC(22:6/20:5) was an exception, showing an initial 1.46-fold ± 0.11-fold increase between 12 and 24 h, followed by a large 0.33-fold ± 0.073-fold decrease (relative to 12 h) (P < 0.005). The least unsaturated of the major PC lipids, PC(18:4/16:0) showed the most rapid initial decrease between 12 and 24 h, dropping by 0.64-fold ± 0.070-fold.To decouple the P-stress-driven dynamics from the growth-driven dynamics, the change in PC lipid quantity over time, relative to the initiation of P stress in Ptermined . Overall+ control and P− cultures displayed sharp increases between 0 and 12 h, from 5.73 ± 0.098 to 7.53 ± 0.24 double bonds in P+ cultures. After 12 h, the results diverged for the two treatments, concomitantly with the initiation of P stress. The P+ cultures then displayed a decrease in average PC unsaturation to 5.63 ± 0.077 double bonds at 72 h, remaining constant thereafter. The P− cultures, in contrast, continued increasing to a maximum of 7.95 ± 0.14 double bonds at 24 h, followed by a decline until 96 h to 6.37 ± 0.25 double bonds, but remained more highly unsaturated than the P+ control cultures throughout.The variability in the responses of these PC lipids to P stress was reflected in the changing average unsaturation of the PC lipid pool over time . Average− cultures, described above ] with a formic acid adduct, displayed an absence-presence response to P stress. The chemical structure, and its determination by tandem mass spectrometry (MS2) fragmentation in both the negative- and positive-ion modes, is displayed in Fig. S5 in the supplemental material.Most remarkably, a 1,014.7112-Da species, corresponding to a diglycosylceramide with a dihydroxy(18:3) long-chain base and a 24:0 fatty amide ) and was stored at 4°C for <24 h before analysis. Cell size distributions were generated with a Beckman Coulter Multisizer 3 Coulter counter. A 70-μm aperture and 3% NaCl electrolyte were used, and the samples were diluted to ensure <10% aperture coincidence concentration. The Coulter counter was calibrated with 5.023-μm polystyrene latex standard beads prior to use . Size distributions were used to generate cell concentration values, between particle diameter limits of 3 and 9 μm.An aliquot (950 μl) of culture was mixed with a freshly prepared paraformaldehyde solution  was incubated with Sytox Green dye  at a concentration of 0.5 μM for 5 min in the dark. An 18-μl volume of this solution was then imaged with a Cellometer Vision Duo system . All cells were counted manually in the bright-field mode and stained, nonviable cells were counted in the fluorescence mode .g, 10 min, 18°C) before analysis to remove particulates. Nutrient samples were diluted 1/60 (dissolved nutrients [filtrate]) and 3/20 (oxidized particulate nutrients) in Milli-Q dH2O and were characterized by segmented flow autoanalysis on an AutoAnalyzer 3 system  for phosphorus, nitrate/nitrite, and silicon. POP quantities were corrected for carryover of inorganic phosphate from the growth medium based on the difference between the P+ and P− POP quantities measured at 0 h, normalized to the [PO4]3− concentration in the P+ growth medium for a given sample (as measured by the dissolved phosphate [DIP] assay): Experimental culture (10 ml) was syringe filtered over precombusted  GF/F filters. The filtrate was stored at −20°C. Filters were dried at 60°C for 24 h and were stored in a desiccator. Particulate phosphorus was determined by following an oxidation procedure described in reference −1; PG(12:0/12:0), 77.0 amol cell−1; PE(12:0/12:0), 47.6 amol cell−1; DAG(20:4/18:0), 99.7 amol cell−1; SQDG (mixed extract), 1.27 fmol cell−1. Phospholipid/DAG standards were acquired from Avanti Polar Lipids , and SQDG spinach leaf extract was provided by Lipid Products .Lipid samples (20 ml) from the experimental cultures were isolated by syringe filtration as described above. The filtrate was discarded and the filters stored at −78°C until extraction. Total-lipid extracts were prepared using a Bligh-Dyer extraction procedure  as modif6 cells (including standards as described above). This fraction was dried under N2 and was stored at −20°C until analysis.A variable volume of the lipid-containing phase was isolated per sample, ensuring that a constant quantity of lipid (including standards), as estimated by cell concentration in a given sample, was infused into the mass spectrometer during analysis . The optimal lipid quantity for analysis as outlined below was equivalent to the quantity derived from 0.8 × 102O). The sample solution was directly infused into the instrument at 6 μl min−1 and was analyzed by tandem MS (MS2) analysis of each lipid class . DGCC extract (purified by preparative high-performance liquid chromatography [HPLC] from T. pseudonana under P starvation) was provided by Benjamin A. S. Van Mooy . External-standard calibrations were generated from the addition of DGCC extract  to P-replete-culture-grown (hence with no intrinsic DGCC) T. pseudonana total-lipid extracts prepared at optimal, constant cellular lipid/standard concentrations as discussed above. Total DGCC counts were normalized to the chemically similar PC internal standard for the quantification of experimental samples according to the following linear calibration equation: DGCC counts/PC counts = 18.594 × DGCC quantity (R2 = 0.992).Spectra were processed by despiking, baseline subtraction, isotopic correction, and assignment by a Visual Basic macro . The spe2 analysis was performed on a Dionex UltiMate 3000 UPLC system coupled to a Bruker maXis 3G quadrupole–time of flight (Q-ToF) mass spectrometer with an electrospray ionization source. Lipid samples were dissolved in methanol (200 μl) prior to analysis. A 20-μl injection was taken by autosampler from vials in a cooled sample tray at 5°C. The sample was then chromatographically separated over 30 min with a Waters Acquity UPLC BEH C8 column . A constant flow rate of 0.3 ml min−1 was used, resulting in back pressures between 26,000 and 46,000 kPa.Ultraperformance LC (UPLC)-ESI-AutoMS−1: 35% eluent B at 0 min, increasing to 80% eluent B at 2 min, increasing to 95% eluent B at 12 min, and holding for a further 18 min until the end of the run. Eluent B was decreased to 35% over 0.5 min after the run, and the column was allowed to equilibrate for 4.5 min prior to the next run.Eluent A was water with 0.2% formic acid and 1% 1 M ammonium acetate, and eluent B was methanol with 0.2% formic acid and 1% 1 M ammonium acetate. The column was heated to 50°C, and the postcolumn eluent was cooled to 21°C, throughout the analysis. The following multistep linear gradient was applied, with a constant flow rate of 0.3 ml min+ in positive-ion mode. Full-scan MS was acquired in the positive and negative modes between 30 and 1,500 Da.The mass spectrometer was calibrated by direct infusion of a sodium formate solution prior to use (10 mM sodium hydroxide plus 0.2% formic acid in isopropanol-water [1:1]). The observed mass accuracy was 0.4 ppm, determined from the standard deviation from the quadratic calibration curve for calibrant ions up to 1,000 Da, in positive-ion mode. The Q-ToF mass spectrometer yielded a mass resolving power of 21,463.70, determined from full width at half maximum (FWHM) of the internal-standard peak, dilauroylphosphatidylcholine, at 622.4470 Da [M+H]2 fragmentation. The thresholds were set at 2,000 and 1,000 counts in the positive- and negative-ion modes, respectively. Ions with a mass between 500 and 1,500 Da or between 300 and 1,500 Da were subjected to MS2 fragmentation in the positive- and negative-ion modes, respectively. The two most abundant precursor ions eluting during an MS scan were fragmented, and after two MS2 spectra were acquired for a given ion, they were actively excluded from further MS2 for 1 min. Fragmentation for MS2 was achieved by collision-induced dissociation (CID) by impact with argon gas, with stepped collision energies for precursor ions of increasing mass. Ions of 300 to 500, 500 to 800, and >800 Da were fragmented with collision energies of 25, 40, and 50 eV, respectively, in positive-ion mode and 25, 30, and 40 eV in negative-ion mode.During each run , ions above the noise level threshold were subjected to data-dependent MSBruker CompassXport was used to export the raw data prior to processing with the MZMine 2 software package . MZMine m/z values based on a list of common molecular ion adducts in ESI-MS .The MS


Type I modular polyketide synthases assemble diverse bioactive natural products. Such multienzymes typically use malonyl and methylmalonyl-CoA building blocks for polyketide chain assembly. However, in several cases more exotic alkylmalonyl-CoA extender units are also known to be incorporated. In all examples studied to date, such unusual extender units are biosynthesized via reductive carboxylation of α, β-unsaturated thioesters catalysed by crotonyl-CoA reductase/carboxylase (CCRC) homologues. Here we show using a chemically-synthesized deuterium-labelled mechanistic probe, and heterologous gene expression experiments that the unusual alkylmalonyl-CoA extender units incorporated into the stambomycin family of polyketide antibiotics are assembled by direct carboxylation of medium chain acyl-CoA thioesters. X-ray crystal structures of the unusual β-subunit of the acyl-CoA carboxylase (YCC) responsible for this reaction, alone and in complex with hexanoyl-CoA, reveal the molecular basis for substrate recognition, inspiring the development of methodology for polyketide bio-orthogonal tagging via incorporation of 6-azidohexanoic acid and 8-nonynoic acid into novel stambomycin analogues. Polyketides are typically assembled from a starter unit and malonyl- and/or methylmalonyl-CoA-derived extender units, but the macrolide antibiotics stambomycins incorporate non-standard alkylmalonyl-CoA extender units. Here, the authors describe the biosynthetic pathway responsible for this unusual synthesis. The exer units 2. SeveraoA) ref. , filipinoA) ref. , antimycoA) ref. , chlorizoA) ref. , and theoA) ref. . In all Streptomyces species the YCCs acetyl-CoA carboxylase (ACC) and propionyl-CoA carboxylase (PCC) assemble malonyl and methylmalonyl-CoA via carboxylation of acetyl- and propionyl-CoA, respectively16in vitrosamR0483 gene within the stambomycin biosynthetic gene cluster encodes a protein with 72% similarity to PccB from S. coelicolor. In partnership with AccA2, the putative YCC β-subunit encoded by samR0483 has been proposed to be responsible for assembly of the unusual extender units incorporated into the stambomycins  generate the malonyl and methylmalonyl-CoA extender units commonly utilized by modular PKSs, in addition to carrying out a variety of other carboxylation reactionsbomycins 2. Howevenyl-CoAs . Thus, t2H2]heptanoic acid to S. ambofaciens rules out the involvement of a CCRC in unusual extender unit biosynthesis. Direct evidence for the involvement of samR0483 in this process is provided by expression of this gene in a revT mutant of Streptomyces sp. SN-593, in which the CCRC-dependent pathway for the biosynthesis of n-pentylmalonyl-CoA (and related) extender units incorporated into the reveromycins is abolished. X-ray crystal structures of SamR0483 alone and in complex with hexanoyl-CoA reveal the molecular basis for medium chain acyl-CoA recognition by this unusual YCC β-subunit, prompting us to rename it medium chain acyl-CoA carboxylase beta subunit (MccB). Taken together, these results establish the existence of a unique YCC-dependent pathway for unusual polyketide extender unit biosynthesis. Finally, the potential of this pathway to be harnessed for selective polyketide derivatization is demonstrated by feeding 6-azidohexanoic acid and 8-nonynoic acid to S. ambofaciens, resulting in production of the corresponding azide and alkyne-tagged stambomycin analogues.Here we report a series of experiments that elucidate the biosynthetic origin and mechanism of assembly of the unusual stambomycin PKS extender units. Incorporation of stable isotope-labelled precursors indicate that they originate from fatty acid metabolism. Retention of both deuterium atoms in the stambomycin analogue derived from feeding of heptanoic acid (23) as a mechanistic probe to determine whether the unusual extender units incorporated into the stambomycins are assembled by a CCRC or YCC-dependent pathway. Incorporation of [3-2H2]heptanoic acid (23) into the stambomycin analogue (22) via a CCRC-dependent pathway would be expected to proceed via initial SamR0482-catalysed conversion to the CoA thioester (24), followed by β-oxidation to yield the corresponding α, β-unsaturated thioester (25) and reductive carboxylation to yield n-pentylmalonyl-CoA (26) containing only a single deuterium label (2H2]heptanoyl-CoA (24), resulting in n-pentylmalonyl-CoA (26) containing two deuterium labels , a family of structurally-related polyketides that differ in the nature of their C-18 side chains  as a search model. Similar to other bacterial YCC β-subunits, MccB has 3–2 symmetry and is composed of three individual dimers that form a ring-like hexamer  and acyl moieties of hexanoyl-CoA quite differently (see below).The residues that interact with hexanoyl-CoA are localized in three regions at the MccB dimer interface. The first region is located at the entrance of the substrate binding pocket, which is characterized by a network of hydrogen bonds between the adenosine moiety of hexanoyl-CoA and the protein. The exocyclic amine and N-1 of adenine form hydrogen bond contacts with the peptide backbone of Ile147, Ala145 and Gly143 monomer B . The adeThe structures of MccB with hexanoyl-CoA bound were compared with the previously-reported structure of PccB bound to propionyl-CoA 21. In thFurther insight into the molecular basis for the unique substrate specificity of MccB was gleaned from sequence alignments and detailed structural comparisons with other YCC β-subunits . StructuInspired by the insights into the molecular basis for substrate specificity generated by our structural analysis of MccB, we sought to further probe the enzyme's substrate tolerance by investigating the precursor-directed biosynthesis of additional stambomycin analogues. In particular, we targeted the production of analogues bearing an alkyne or azide group at the terminus of the C-26 substituent, with the aim of carrying out click chemistry on these functional groups to produce biotinylated or fluorescently-labelled stambomycin derivatives for future mechanism of action studies.36 to cultures of S. ambofaciens W130. UHPLC-ESI-TOF-MS analyses of mycelial extracts identified a novel metabolite with the molecular formula C70H128N4O22 , corresponding to the stambomycin analogue 37 , corresponding to the stambomycin analogue 39 derived from 8-nonynoic acid  (BLAST searches identified two other polyketide biosynthetic gene clusters that contain genes encoding an YCC β-subunit with the same specificity-conferring residues as ens MccB . These cureus41) , and othAmycolatopsis species. The results presented herein suggest that it might be possible to produce an acetylene-tagged derivative of the metabolic product of this gene cluster which may facilitate purification and subsequent identification via a click-mediated biotinylation and avidin affinity purification strategy.The other gene cluster encoding MccB and SamR0482 homologues is cryptic (that is its metabolic product is unknown). It encodes only a single PKS module, and is widely conserved in Several examples of unusual type I modular PKS extender units have been reported over the past half-decade. In all cases, these are assembled via a common final biosynthetic step, involving reductive carboxylation of an α, β-unsaturated CoA thioester by a CCRC homologue. The data reported here reveal an alternative mechanism for assembly of unusual type I modular PKS extender units. This employs a unique YCC β-subunit (MccB) that, in partnership with the primary metabolic YCC α-subunit, is able to directly carboxylate medium chain acyl-CoA thioesters. X-ray crystallographic analysis revealed the structural basis for substrate recognition by MccB, allowing a homologue likely involved in the biosynthesis of butylmalonyl-CoA and other unusual extender units incorporated into the primycin complex of clinically-used antibiotics to be identified.S. ambofaciens resulted in the production of a stambomycin analogue containing a 9-carbon alkyne-terminated extender unit in comparable quantities to the natural products. Thus, MccB, the AT domain from module 12 of the stambomycin PKS and the medium chain acyl-CoA synthetase encoded by samR0482 are useful additions to the synthetic biology toolkit for bio-orthogonal tagging of polyketides.These findings inspired the precursor-directed biosynthesis of novel azide- and alkyne-containing stambomycin analogues. Several biosynthetic engineering approaches have recently been utilized to introduce alkyne-terminated extender units containing 5–8 carbons into erythromycin and antimycin analogues2627The bacterial strains and plasmids used in the study are described in samR0484, was PCR amplified from genomic DNA of S. ambofaciens ATCC23877 using the primers pOSV_samR0484_FW and pOSV_samR0484_RV and cloned into the ClaI/HindIII restriction sites of pOSV556t, placing samR0484 under the control of the constitutive ermE* promoter. pOSV556t-samR0484 was transferred into S. ambofaciens ATCC 23877 via conjugation from E. coli ET12567/pUZ8002 . A 4 μl aliquot of S. ambofaciens W130 spore stock was spread onto each plate and they were incubated at 30 °C for 4 days. The mycelia were scraped off the membranes and extracted with methanol. The extracts were analysed by UHPLC-ESI-TOF-MS on a Dionex 3,000 RS UHPLC attached to a Bruker MaXis mass spectrometer  through a loop injection of 20 μl at the beginning of each run. Source conditions were as follows. End plate offset: −500 V; capillary: −4,500 V; nebulizer gas (N2): 1.4 bar; dry gas (N2): 8 l/min; dry temperature: 180 °C. Ion tranfer conditions were as follows. Ion funnel 1 RF: 200 Vpp; ion funnel 2 RF: 200 vpp; hexapole RF: 200 Vpp; quadruple ion energy: 5 ev; quadrupole low mass: 55 m/z; collision energy: 5.0 ev; collision RF: ramped from 800 to 1,500 Vpp; transfer time: from 100 to 155 μs; pre-pulse storage time: 5 μs.MP5 agar plates containing 5 mM n-pentane 4-methylbenzenesulfonate29. To a stirred solution of [1-2H2]n-pentanol 28  in 23 ml of dry DCM, under argon, was added 4-dimethylamino-pyridine  and triethylamine  followed by p-toluene sulfonyl chloride . The reaction was stirred at room temperature under argon for 17 h, after which TLC indicated that it had gone to completion. Ethanolamine  was added and the resulting mixture was stirred for a further 2 h. 125 ml of water was added, the layers were separated and the aqueous layer was extracted with 4 × 50 ml of DCM. The organics were combined, washed with brine and dried over magnesium sulfate. The solvent was removed in vacuo and the residue was purified via flash column chromatography  to give the product as a colourless oil . 1H NMR : δ 7.79 , 7.34 , 2.44 , 1.62 , 1.28–1.23 , 0.84 ; 13C NMR : δ 144.7, 133.3, 129.8, 127.9, 70.2 , 28.3, 27.4, 22.1, 21.7, 13.8. HRMS (m/z) [M+H]+ calculated for C12H15D2NaO3S, 267.0994; found, 267.0990.dimethyl ([1-2H2]n-pentyl)malonate 30. To a stirred suspension of sodium hydride  in 10 ml of dry DCM, under argon, was added distilled dimethyl malonate  in 15 ml dry of THF drop wise. The resulting mixture was stirred at 60 °C for 1 h and [1-2H2]n-pentyl-4-methylbenzenesulfonate 29  in 10 ml of dry THF was added. Stirring was continued at 60 °C under argon for a further 36 h, after which TLC indicated that the reaction had gone to completion. 125 ml of water was added cautiously and the aqueous layer was extracted with 4 × 100 ml of ethyl acetate. The organics were combined, washed with brine and dried over magnesium sulfate. The solvent was removed in vacuo and the residue was purified by flash column chromatography , yielding the product as a colourless oil . 1H NMR : δ 3.69 , 3.30 , 1.29–1.22 , 0.84 ; 13C NMR : δ 170.0, 52.4, 51.6, 31.3, 28.2 , 26.8, 22.3, 13.9; HRMS (m/z): [M+Na]+ calculated for C10H16D2NaO4, 227.1223; found, 227.1221.[3-2H223]n-heptanoic acid . To a stirred solution of dimethyl ([1-2H2]n-pentyl)malonate 30  in 10 ml of methanol, was added 5 M sodium hydroxide  drop wise. The resulting mixture was stirred under reflux for 18 h, after which time TLC indicated that no starting material remained. The mixture was cooled to room temperature, acidified with 2 M HCl and the methanol was removed in vacuo. 100 ml of 20% brine was added to the residue and the resulting solution was extracted with 3 × 75 ml of ethyl acetate. The organics were combined, dried over magnesium sulfate and the solvent was removed in vacuo to yield diacid 31 as a crystalline solid. 15 ml of toluene was added and the resulting mixture was heated under reflux, at 125 °C for 96 h. The mixture was cooled to room temperature and the toluene was removed in vacuo to give the product . 1H NMR : δ 2.25 , 1.29–1.24  0.80 ; 13C NMR : δ 180.5, 34.1, 31.5, 28.7, 24.1 , 22.6, 14.2; HRMS (m/z): [M-H]−calculated for C7H12D2O2, 131.1047; found, 131.1046. The 1H and 13C NMR spectra for labelled and unlabelled n-heptanoic acid are compared in 8-nonyn-1-ol. Sodium hydride  was added to ethylenediamine (200 ml) under argon and the resulting mixture was heated to 60 °C for 60 min. 3-Nonyl-1-ol  was added and the resulting deep blue solution was stirred for 4 h, then cooled to 0 °C and carefully quenched by addition of 2 M HCl until the blue colour disappeared. The mixture was extracted with EtOAc, and the combined organics were dried (MgSO4) and concentrated under vacuum. The crude product was purified on silica gel (25% EtOAc in hexanes) to give the product  as a colourless oil.1H NMR : δ 3.64 , 2.18 , 1.96 , 1.57 , 1.53 , 1.44–1.31 . 13C NMR : δ 84.7, 68.1, 63.0, 32.7, 28.9, 28.7, 28.4, 25.6, 18.4; HRMS (m/z): [M+Na]+ calculated for C9H15ONa, 163.1099; found, 163.1096.8-nonynal. To a solution of oxalyl chloride  in dry DCM (110 ml), cooled to −78 °C under argon, was added DMSO  in dry DCM (25 ml) drop wise. The reaction mixture was stirred for 5 min, then 8-nonyn-1-ol  in dry DCM (50 ml) was added and stirring was continued for a further 15 min. Dry triethylamine  was added and the reaction mixture was allowed to warm to room temperature over 30 min. Water was added and the mixture was extracted with DCM. The combined organics were washed with 2 M HCl and saturated NaHCO3, dried (MgSO4), and concentrated under vacuum. The crude product was purified on silica gel (10% EtOAc in hexanes) to give the product  as a colourless oil.1H NMR : δ 9.55 , 2.42 , 2.17 , 1.92 , 1.62 , 1.57–1.28 ; 13C NMR : δ 202.8, 84.5, 68.3, 43.8, 28.6, 28.4, 28.2, 21.9, 18.3; HRMS (m/z): [M+Na]+ calculated for C9H13ONa, 161.0937; found, 161.0939.8-nonynoic acid 40. To a solution of 8-nonynal  in DMF (30 ml) was added Oxone . The resulting suspension was stirred for 15 min, then poured into water, acidified with 2 M HCl and extracted with EtOAc. The combined organics were dried (MgSO4) and concentrated under vacuum to give the product  as a colourless oil.1H-NMR : δ 2.29 , 2.12 , 1.87 , 1.59 , 1.47 , 1.40–1.27 . 13C-NMR : δ 179.6, 84.5, 68.3, 33.9, 28.5, 28.3, 28.2, 24.5, 18.3; HRMS (m/z): [M+Na]+ calculated for C9H13O2Na, 177.0886; found, 177.0884.2H2]n-heptanoic acid 23 was fed to cultures of S. ambofaciens W130 and the mycelial extract was prepared as described above. UHPLC-ESI-Q-TOF-MS analysis of the extract, as described above, showed that both of the deuterium atoms had been retained in the resulting 22  NaCl, 0.03% (w/v) MgSO4·7H2O, 0.01% (w/v) NaNO3, 0.005% (w/v) ZnSO4·7H2O and 0.005% (w/v) CuSO4·5H2O; pH 6.5 before autoclaving). After 2 days growth at 28 °C and 150 r.p.m., 0.3 mM of [3-2H2]n-heptanoic acid 23 was added and the culture was grown for three further days. 4 ml of culture broth was extracted with an equal volume of acetone and residual acetone was removed by evaporation. The pH was adjusted to 4 by adding acetic acid and the culture broth was extracted twice with an equal volume of ethyl acetate. The organic layer was concentrated in vacuo and dissolved in 1.2 ml of methanol. The resulting sample from the ΔrevR mutant culture was analysed by UPLC-ESI-MS on an Applied Biosystems API 3200 connected to a Waters ACQUITY UPLC and a Waters ACQUITY UPLC BEH C18 column . The conditions were as follows: flow rate 0.7 ml min−1; solvent A, water containing 0.05% formic acid; and solvent B, acetonitrile. After injection of 1 μl of the sample into a column that was equilibrated with 30% solvent B, the column was developed with a linear gradient from 30% to 100% solvent B in 1.9 min and kept at 100% solvent B for 0.96 min. Mass spectra were recorded in negative ion mode.The Δ mutants  were gro32 from other reveromycin derivatives, the ethyl acetate extracts from the ΔrevR and ΔrevT::samR0483 mutants were applied to a PEGASIL ODS column . The HPLC conditions were as follows: flow rate, 1 ml min−1; solvent A, water containing 0.05% formic acid; and solvent B, MeOH, isocratic system of 78% MeOH. The fraction containing reveromycin D 32 from the ΔrevT::samR0483 mutant cultures with and without supplementation of (3-2H2)-heptanoic acid 23 was concentrated and analysed by UPLC-ESI-MS on an Applied Biosystems API 3200 connected to a Waters ACQUITY UPLC and a Waters ACQUITY UPLC BEH C18 column . The flow rate was 0.2 ml min−1 and the column was eluted with isocratic 50% water containing 0.05% formic acid and 50% acetonitrile. Mass spectra were recorded in negative ion mode . pTYM19-Paph-samR0483 was transferred by intergenic conjugation from E. coli GM2929 hsdS::Tn10 (pUB307::Tn7) to Streptomyces sp. SN-593  following the manufacturer's instructions to give pET151-samR0483. It was then amplified from pET151-samR0483 using the primers pET28a-samR0483_FW and pET28a-samR0483_RV and the resulting amplimer was cloned into the NheI and NotI sites of pET28a (Novagen) to produce pET28a-samR0483 encoding an N-terminal hexahistidine MccB fusion protein.The MccB-encoding gene, E. coli BL21(DE3) cells containing pET28a-samR0483 were grown in 4 × 1L of LB medium supplemented with kanamycin (50 μg ml−1) to A600=0.6–0.8 at 37 °C and 180 r.p.m. Cultures were induced by the addition of 0.7 mM IPTG and incubated for 16 h at 19 °C and 180 r.p.m. Cells were harvested by centrifugation at 5,500 RCF for 10 min, re-suspended in lysis buffer , sonicated and the cell debris was removed by centrifugation . The clarified cell lysate was batch bound to 5 ml of Ni-IMAC resin (Bio-Rad Laboratories) for 1 h at 4 °C. The resulting slurry was transferred to a gravity-flow column and washed twice with 50 ml of lysis buffer. His6-MccB was eluted with 5 ml fractions containing 40, 100, 150, 250 and 450 mM imidazole in lysis buffer. Protein purity was assessed by SDS–polyacrylamide gel electrophoresis (SDS–PAGE). Fractions containing the highest purity (≥90%) were collected and dialyzed overnight at 4 °C against 50 mM Tris–HCl pH 8.0, 50 mM NaCl, 10% glycerol and 1 mM DTT. Dialyzed protein was concentrated to 5–10 mg ml−1 using a 30,000 Da molecular weight cutoff centrifugal filter (Millipore) and injected onto a Superdex 200 column  pre-equilibrated with 50 mM Tris pH 8.0 and 1 mM DTT. Fractions were collected, analysed by SDS–PAGE for purity, concentrated to 9 mg ml−1 and flash frozen for storage at −80 °C.6-MccB was treated with TEV protease (10 U per mg of MCC) post nickel affinity chromatography and dialyzed into 50 mM Tris·HCl pH 8.0, 50 mM NaCl, 10% glycerol, 1 mM DTT, and 1 mM EDTA at 18 °C for 18 h. Complete digestion was verified using MALDI-TOF mass spectrometry. The cleaved protein was dialyzed into crystallization buffer (50 mM Tris pH 8.0 and 1 mM DTT) and subjected to anion exchange chromatography  to remove excess TEV protease. Cleaved MccB was eluted using a linear gradient of 0–1 M NaCl and was dialyzed into 50 mM Tris pH 8.0 and 1 mM DTT for co-crystallization screens.For co-crystal studies, native His-derivatized His6-MccB was produced in E. coli BL21(DE3) cells harbouring pET28a-samR0483. Cells were grown in 4 × 1 l of LB medium supplemented with kanamycin (50 μg ml−1) at 37 °C and 180 rpm until A600 reached 0.6. Before induction, the cells were harvested by centrifugation at 5,500 RCF for 10 min, re-suspended and washed three times with 100 ml of M9 medium then transferred to 4 × 1 l flasks containing M9 medium with the following additives: kanamycin (50 μg ml−1); lysine, phenylalanine, threonine (100 mg L−1); isoleucine, leucine, valine (50 mg L−1); and L-SeMet (80 mg L−1)(Sigma). The temperature was then lowered to 19 °C and protein expression was induced upon the addition of 0.7 mM IPTG. The SeMet substituted protein was purified following the same procedure as native His6-MccB. Selenomethionine incorporation was confirmed using MALDI-TOF mass spectrometry.Selenomethionine (SeMet)-MccB crystallization. Frozen SeMET aliquots of His6-MccB were thawed on ice and incubated with 5 mM biotin and 5 mM heptanoic acid  (Sigma-Aldrich) at 4 °C for 1 h before crystallization screens. Small crystals of apo-MccB formed overnight in 30% methyl propanediol (MPD) (Qiagen), 0.2 M magnesium acetate and 0.1 M MES pH 6.5. Diffraction-quality crystals were obtained by mixing 2 μl of protein solution with 2 μl of well solution  using the hanging drop vapour diffusion method. Apo-MccB crystals were further optimized via four consecutive rounds of seeding using a Seed Bead (Hampton Research). The crystals were harvested, cryo-protected with 25% ethylene glycol and flash frozen in liquid nitrogen for subsequent X-ray diffraction data collection.ApoMccB Hexanoyl-CoA co-crystallization. Before crystallization, cleaved native MccB at 7 mg ml−1 was incubated with 5 mM biotin and either 5 mM or 10 mM hexanoyl-CoA (50 mM stock in 0.1 M Hepes pH 7.2) (Sigma-Aldrich). Crystals of the MccB-hexanoyl-CoA complex were obtained by mixing 2 μl of protein solution with 2 μl of mother liquor  using the hanging drop vapour diffusion method. MccB co-crystals grew at 25 °C over a period of 5 days. MccB co-crystals were cryo-protected in well solution plus 25% glycerol and flash frozen in liquid nitrogen for subsequent X-ray diffraction data collection.apo and mono-hexanoyl-CoA co-crystal forms of MccB were indexed, integrated, and scaled using Mosflm or HKL2000, respectively30apo-MccB was accomplished via molecular replacement (Phaserapo-PccB D422A structure (PDB ID: 3IBB) as a search model. An initial model was built using Autobuild, and this model was further improved through iterative rounds of model building , Lawrence Berkeley National Laboratory, using beamlines 8.2.1 or 8.2.2. Data sets corresponding to the nt Phaser using thOOT ref. ) and refOOT ref. . For the36, 6-heptynoic acid 38 and 8-nonynoic acid 40 were fed to cultures of S. ambofaciens W130 as described for precursor directed biosynthesis of stambomycin analogue 22. UHPLC-ESI-QTOF-MS analysis of the methanolic mycelial extracts, as described for precursor directed biosynthesis of stambomycin analogue 22, showed that new metabolites with molecular formulae corresponding to stambomycin analogues 37  suspended in degassed methanol (600 μl) and butanol (400 μl) under argon was added copper sulfate  and sodium ascorbate  in degassed water (1 ml), followed by azide-PEG3-conjugate  in degassed butanol (200 μl). Following 64 h stirring at 21 °C, the solvent was removed in vacuo and the biotinylated stambomycin derivative was purified by semi-preparative HPLC on an Agilent C18 column . Elution conditions were as follows: flow rate, 5 ml min−1; solvent A, water containing 0.1% formic acid; solvent B, methanol containing 0.1% formic acid; gradient initiated after 5 min from 40% B to 100% B over 25 min. The biotinylated stambomycin derivative eluted between 18.6 and 19.2 min. 2 mg (34%) of the biotinylated stambomycin derivative was recovered and analysed by HRMS  and 1H NMR spectroscopy .Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Supplementary Figures 1-28, Supplementary Tables 1-4 and Supplementary References.