Scutellaria baicalensis Georgi. These flavones can induce apoptosis in a variety of cancer cell lines but have no effect on normal cells. Furthermore, they have many additional benefits for human health, such as anti-oxidant, antiviral, and liver-protective properties. Here, we report the isolation and characterization of two CYP450 enzymes, SbCYP82D1.1 and SbCYP82D2, which function as the flavone 6-hydroxylase (F6H) and flavone 8-hydroxylase (F8H), respectively, in S. baicalensis. SbCYP82D1.1 has broad substrate specificity for flavones such as chrysin and apigenin and is responsible for biosynthesis of baicalein and scutellarein in roots and aerial parts of S. baicalensis, respectively. When the expression of SbCYP82D1.1 is knocked down, baicalin and baicalein levels are reduced significantly while chrysin glycosides accumulate in hairy roots. SbCYP82D2 is an F8H with high substrate specificity, accepting only chrysin as its substrate to produce norwogonin, although minor 6-hydroxylation activity can also be detected. Phylogenetic analysis suggested that SbCYP82D2 might have evolved from SbCYP82D1.1 via gene duplication followed by neofunctionalization, whereby the ancestral F6H activity is partially retained in the derived SbCYP82D2.Baicalein, wogonin, and their glycosides are major bioactive compounds found in the medicinal plant  Scutellaria baicalensis. Like the main 4′-deoxyflavone enzymes, these decorating enzymes have evolved their functionalities by convergence with the more ubiquitous 4′-hydroxyflavone pathway enzymes.We report the characterization of two CYP450 enzymes, which 6- and 8-hydroxylate chrysin to form the 4′-deoxyflavone bioactives in roots of  Scutellaria baicalensis Georgi or Huangqin is a medicinal plant used widely in China and many other Asian countries. Its dried roots have been used for treating lung infections, liver problems, inflammation, diarrhea, and dysentery for thousands of years : baicalin, wogonoside, and their aglycones, baicalein and wogonin . Elucidation of the biosynthetic pathways for the RSFs would lay a solid foundation for these applications.of years . The phaScutellaria RSFs are not produced from naringenin, the common intermediate for most flavones, but from pinocembrin, a 4′-deoxyflavanone  ligase (SbCLL-7) to form cinnamoyl-CoA. Cinnamoyl-CoA is condensed with malonyl-CoA by a specific isoform of chalcone synthase, SbCHS-2, and isomerized by chalcone isomerase (SbCHI) to form pinocembrin. A newly evolved isoform of flavone synthase II (FNSII), SbFNSII-2, which accepts only pinocembrin as a substrate, then converts pinocembrin to chrysin  activity was first detected in microsomes extracted from flavones . Additioflavones .Scutellaria belong to the Lamiaceae family, and might have genes encoding proteins from the same subfamily that perform similar enzymatic reactions to hydroxylate the 6-position of flavonoids. However, the 4′-deoxyflavone biosynthetic pathway appears to have evolved relatively recently, and the genes encoding enzymes involved in the biosynthesis of baicalein probably evolved independently of those involved in the synthesis of 7-O-methylflavones. Furthermore, the F8H of sweet basil is localized in plastids  databases of hairy roots and flowers  and 509 aa, respectively. Two cDNAs were isolated using primer pairs based on the SbCYP82D1 sequence (SbCYP82D1.1 and SbCYP82D1.2. SbCYP82D1.1 is 72 bp longer than the 1482-bp CDS (coding sequence) of SbCYP82D1.2 and encodes a protein 24 aa longer (517 aa) than the SbCYP82D1.2 protein of 493 aa. The 1581-bp coding sequence of SbCYP82D2 encoded a 526-aa protein. SbCYP82D1.1 was 64% identical to SbCYP82D2 at the amino acid level, and SbCYP82D1.1 and SbCYP82D2 had 60% and 67% identity to CYP82D33 from sweet basil, respectively.Based on the sequences from the RNA-seq databases, we successfully obtained the open reading frames (ORFs) of sequence , which min vivo yeast assay on strains expressing each CYP450 in Saccharomyces cerevisiae WAT11, an engineered strain overexpressing an Arabidopsis NADPH-cytochrome P450 reductase gene . We did not observe any substantial new products in the strains expressing SbCYP71D1, SbCYP71D2, SbCYP82D1.2, or SbCYP82D2 compared with the empty vector control, but a large, new peak was detected in extracts from yeast expressing SbCYP82D1.1  in line 2 compared with 4.7 mg g−1 DW in controls. Wogonin levels were also increased slightly in lines 2 and 6, although these changes were not statistically significant. A new peak, detected in CYP82D1.1 RNAi lines, with an m/z of 431, was identical to chrysin glucuronide , has been reported, which can 8-hydroxylate the flavones salvigenin or crisimaritin to produce 8-hydroxysalvigenin or 8-hydroxycrisimaritin . However, this gene had highest expression levels in leaves, as shown by qRT–PCR, with 1.37-, 2.14-, and 3.12-fold higher transcript levels than in flowers, stems, and roots, respectively  to enhance product formation. Interestingly, the product of SbCYP82D2 ran about 1 min before the baicalein standard on our high-performance liquid chromatography (HPLC) system and had the same retention time as the norwogonin standard, which is the 8-hydroxylated chrysin and the precursor of wogonin A. We anafragment C. Althouin vitro enzyme assay using chrysin as the substrate. In accordance with previous results, this enzyme could convert chrysin to norwogonin in the presence of NADPH with an apparent KM of 1.051 μM and Vmax of 37.453 pkat mg−1 protein  also diverged from the ancestor of SbCYP82D1. These proteins branched into two paths, suggesting that the gene encoding SbCYP82D1.1 underwent neofunctionalization from the ancestor of CYP82D33 or CYP82D62 in the Lamiaceae, since CYP82D33 or CYP82D62 have activity only on 7-O-methylflavones. The gene encoding SbCYP82D2 may also have undergone neofunctionalization from the gene encoding SbCYP82D1.1.The two SbCYP82Ds function as flavone 6- and 8-hydroxylases in amiaceae B and 1C,amiaceae . The Lamamiaceae . This trin vivo assays using Q-TOF to examine compounds with m/z 271, because baicalein and norwogonin have the same molecular mass. We traced these fragments and found that m/z 271 formed two peaks which ran at 5.703 min and 5.936 min, respectively, on our system -geranyllinalool to the insect-induced C16-homoterpene -4,8,12-trimethyltrideca-1,3,7,11-tetraene (TMTT)  and SbF8H (CYP82D2) were functional in planta, we overexpressed these two genes in transgenic Arabidopsis under the control of the CaMV35S promoter, respectively.Flavones are very difficult to detect in s genome . Three spsoralen . CYP82G1e (TMTT) . No enzy−1 DW, while no baicalein was found in empty vector controls  could use apigenin as a substrate in vivo to produce scutellarein. While there were plenty of flavonoid peaks in the UV spectra of extracts from these inoculated plants, there were clearly three novel peaks not seen in inoculations made with the GFP pEAQ vector alone. One peak, running at just over 7.5 min, ran in the same position as the baicalin standard. The second peak, which ran a little faster, was identified as apigenin. A third peak, not found in extracts inoculated with the GFP vector control, ran at 5.4 min. This was identical to the scutellarein standard. The new peak of scutellarein was present at substantially higher levels than baicalein. MS/MS spectra confirmed the identities of these products  to make wogonin. Among the five F6H candidate genes  analyzed in yeast strains, only SbCYP82D1.1 was able to convert chrysin to baicalein . The SbCYP82D1.1 protein identified from S. baicalensis is distinct from CYP82D33 and CYP82D62, and can hydroxylate chrysin efficiently, a flavone containing neither a 7-O-methyl group nor a 4′-OH group, to produce baicalein. SbCYP82D1.1 has similar or slightly higher binding affinity for chrysin (KM 0.12 μM) than CYP82D33 has for genkwanin (KM 0.20 μM). Furthermore, SbCYP82D1.1 exhibits a level of substrate promiscuity, because it can use apigenin, 7-O-methylchrysin, and chrysin as substrates with comparable turnover rates , confirming the broad substrate range of CYP82D2 in vivo  had its highest expression levels in leaves but the lowest in roots  in a tilted conformation in the binding pocket, placing C6 in close proximity (4.9 Å) to the catalytic protoporphyrin iron . In contS. baicalensis Georgi were bought from Northern Medicinal Seeds, Anguo county, Hebei province of China. Plants of S. baicalensis Georgi were grown in plant chambers at 25°C under a 16-h/8-h photoperiod.Seeds of http://www.sigmaaldrich.com/). Norwogonin was purchased from Extrasynthese (http://www.extrasynthese.com). Wogonoside was purchased from Carbosynthe (http://www.carbosynth.com/). 7-O-Methylchrysin was purchased from Yuanye (http://www.shyuanye.com/). The compounds were dissolved in methanol to obtain standard stock solutions (1 mg ml−1).Baicalin, baicalein, scutellarein, wogonin, pinocembrin, chrysin, and apigenin were purchased from Sigma-Aldrich  were isolated using the primers listed in http://www.thermofisher.com).The sequences of CYP82D33, CYP71D9, and ObF8H were used for BLAST searches against SbCYP82D1.1, SbCYP82D1.2, SbCYP82D2, SbCYP71D1, and SbCYP71D2 were cloned into the yeast expression vector pYesdest52. The full-length CDSs of SbCYP82D1.1 and SbCYP82D2 were also cloned into plasmid pK7WG2R (Arabidopsis transformation. All constructs were made using Gateway LR Clonase II Enzyme Mix (http://www.thermofisher.com) according to the manufacturer's protocols.Full-length CDSs of  pK7WG2R  for ArabSbCYP82D1.1, SbCYP82D1.2, SbCYP82D2, SbCYP71D1, SbCYP71D2, or an empty vector were transformed into yeast S. cerevisiae WAT11  or 24 h (for F8H), the cells were centrifuged, ice-dried, and extracted with 70% MeOH for LC–MS analysis.The yeast expression vector pYesdest52 constructs with ae WAT11  for expr−1 galactose for 16 h. Microsomal proteins were isolated according to the protocol described by O-methylchrysin, or pinocembrin). The assays were initiated by adding NADPH at 1 mM and were incubated for 30 min for F6H or 6 h for F8H at 28°C. MeOH was then added to a final concentration of 70% into the assays to quench the reactions. The samples were filtered through 0.22-μm nylon column and analyzed by LC–MS. Microsomal proteins extracted from yeast harboring the empty vector were assayed as a negative control.Recombinant yeast strains were grown as described previously . Target KM and Vmax values were evaluated using an Eadie–Hofstee plot.For kinetics measurements, chrysin at varying concentrations from 0.1 to 5 μM was added to the reaction system as above and incubated at 28°C for 3 min for F6H or 30 min for F8H. SbCYP82D1.1, SbCYP82D2, and SbRTO cDNAs were amplified using primers listed in Agrobacterium rhizogenes A4 strain was transformed with the RNAi constructs by electroporation. The reconstructed strains were used for inducing hairy roots from leaf explants of S. baicalensis.Non-conserved regions of sequence from S. baicalensis plants were firstly treated with 75% methanol for 30 s, then sterilized with 10% bleach for 10 min, and washed with sterile water five times. The explants were then scratched using a knife dipped with A. rhizogenes suspension solutions , dried on sterile filter-paper, and co-cultured on B5 medium containing 50 μM acetosyringone for 3 days in the dark at 25°C. The explants were then transferred to B5 medium supplemented with 500 mg l−1 cefotaxime (Sigma) to remove bacteria. After 2 weeks, hairy roots could be found at the wound site on the explants, and successful transformants were screened for expression of dsRed using a fluorescence microscope and removed from explants. The dsRed-positive hairy roots were kept on B5 medium containing 400 mg/l cefotaxime (Sigma) as separate independent lines at 25°C in the dark.Young leaf explants collected from −1 cefotaxime, and maintained by shaking (90 rpm) at 25°C. Liquid medium was added gradually up to 50 ml with growth of the roots. After 50 days, the hairy roots were collected and ground in liquid N2. The samples were then freeze-dried and extracted in a sonicator with 70% methanol for 2 h. The extracts were filtered using 0.22-μm columns before HPLC analysis.An elongated hairy root was cut from each line, cultured in a 100-ml flask containing 10 ml of B5 liquid with 400 mg l−1 with the following gradient: 0–3 min, 20% B; 20 min, 50% B; 20–30 min, 50% B; 36 min, 30% B; 37 min, 20% B; and 37–43 min, 20% B. The column was maintained at 35°C and absorption was detected at 280 nm with a diode array detector (Agilent). Metabolites were measured by comparing the area of the individual peaks with standard curves obtained from standard compounds.An Agilent 1260 Infinity II HPLC system was used for metabolite analysis. Separation was carried out on a 100 × 2-mm 3μ Luna C18(2) column using 0.1% formic acid in water (A) versus 1:1 ACN/MeOH + 0.1% formic acid (B) and run at 260 μL minm/z 200–2000 . The instrument also collected automatic (data-dependent) MS2 spectra of the most abundant precursor ions, at an isolation width of m/z 3.0, 50% collision energy, and 50% collision gas, with an ion accumulation time of 10 ms. Spray chamber conditions were 250°C curved desorbation line, 300°C heatblock, 1.5 l min−1 nebulizing gas, and drying gas “on.” The instrument was calibrated using sodium trifluoroacetate before use.LC–MS/MS was carried out on an ion-trap TOF mass spectrometer attached to a Prominence/Nexera UHPLC system (Shimadzu). Separation was on a 100 × 2-mm 3μ Luna C18(2) column using the same gradient described above. Flavonoids were detected by UV absorption, collecting spectra from 200 to 600 nm from which we extracted chromatograms at the desired wavelength, 280 nm. They were also detected by positive electrospray MS, collecting spectra from Protein sequences were aligned using the Clustal X 2 . The phyhttp://www.qiagen.com/). First-strand cDNA was synthesized using SuperScript III  with oligo(dT)17 primer plus random primers (sigma). Real-time qRT–PCR was performed based on the corresponding cDNA samples using gene-specific primers as shown in Plant samples were collected and frozen in liquid nitrogen immediately. The samples were ground into fine power and total RNA was extracted from them using RNeasy plant mini kits  , as this returned the highest sequence identity of all proteins in the RCSB PDB (csb.org) . The coocsb.org) .Arabidopsis thaliana Columbia-0 was used for transformation. Seeds were sown in soil and stratified at 4°C for 2 days before transfer to a growth chamber. The plants were maintained under long-day  conditions. Temperature and humidity were kept at 23°C and 50%. Plant binary vector pH7WG2R was used for expression of SbCYP82D1.1 and SbCYP82D2, which were driven by CaMV 35S promoter. The constructs as well as an empty vector control were introduced into Agrobacterium tumefaciens GV3101 pMP90 by electroporation. The transformed bacteria were used for transformation of A. thaliana by in planta infiltration  were used for LC–MS analysis.Two-week-old sterile GFP, SbCLL-7, SbCHS-2, SbCHI, SbFNSII-2, and SbCYP82D1.1 were cloned into pEAQ-HT-DEST1 and transformed into A. tumefaciens GV3101 pMP90 , the Chenshan Special Fund for Shanghai Landscaping Administration Bureau Program G172402 and G162409, and by the CAS/JIC and Center of Excellence for Plant and Microbial Sciences (CEPAMS) joint foundation support to Q.Z., D.Y., X.-Y.C., and C.M. Q.Z., C.M., L.H., and D.Y. were also supported by the Institute Strategic Program Understanding and Exploiting Plant and Microbial Secondary Metabolism (BB/J004596/1) from the BBSRC to JIC. D.Y. was supported by a CSC fellowship from the State Scholarship fund of China. J. Li was supported by a John Innes Foundation Rotation Studentship during the course of this work. This work was also supported by the Pew Scholar Program in the Biomedical Sciences and the Searle Scholars Program to J.-K.W.This work was supported by Q.Z. and C.M. designed and managed the project; Q.Z. isolated the genes and characterized the enzymes; Q.Z., M.-Y.C., and J. Liu performed silencing of genes, gene expression assays, and bioinformatics analysis; O.L. and J.-K.W. modeled the hydroxylases; D.Y. and J. Li reconstructed the pathway in tobacco leaf; L.H. helped in LC–MS analysis; C.M., Q.Z., L.Y., Y.H., J.-K.W., and X.-Y.C. analyzed and interpreted the data; Q.Z. and C.M. wrote the paper with significant input from all authors.


Psychonomic Bulletin and & Review, 10(3), 703-–710, Neural evidence for the strategic retrieval of task-relevant ‘target’ memories at the expense of less relevant ‘nontarget’ memories has been demonstrated across a wide variety of studies. In ERP studies, this evidence consists of the ERP correlate of recollection  being evident for targets and attenuated for nontargets. It is not yet known, however, whether this degree of strategic control can be extended to emotionally valenced words, or whether these items instead reactivate associated memories. The present study used a paradigm previously employed to demonstrate the strategic retrieval of neutral words (Herron & Rugg,  Episodic memory is memory for personally experienced events and is often accompanied by the recollection of contextual information such as time, place, and sensory details. It is widely accepted that control processes help us to navigate episodic memory, enabling us to constrain retrieval attempts in order to selectively recollect memories relevant to current goals. This concept has been expressed within cognitive theories of memory in various ways, such as ‘descriptors’ that guide a memory search , it is worth briefly revisiting the strength of association between recollection and the left parietal old/new effect  if the recollection of negative items is subject to the same degree of strategic control as that previously observed for neutral items, then the left parietal old/new effect will show equivalent levels of attenuation for both classes of nontargets with respect to targets; (ii) if source monitoring is impaired for negative items, then ERP correlates of postretrieval monitoring will be smaller for these items at right frontal sites post-800 ms; (iii) if the recollection of extrinsic contextual information is unaffected by valence, then no effects of valence will be observed for behavioral and ERP measures of target recollection. The findings will inform models of strategic retrieval by providing novel information regarding the kind of memory contents that are subject to retrieval control. They will also inform models of emotional memory by providing new behavioral and neural evidence regarding the impact of stimulus valence on the retrieval of extrinsic contextual information for both goal-relevant and nontarget stimuli.N = 16 in each of two experiments) who employed the same experimental design. Ethical approval was for the study granted by Cardiff University’s School of Psychology ethics committee.Participants were drawn from the undergraduate population studying psychology at Cardiff University and participated on a voluntary basis in return for course credit after giving informed consent. Eighteen right-handed native English speakers ages 18 to 28 years  participated in the experiment and all participants contributed data to the analyses reported below. The sample size was based upon that used by Herron and Rugg (SD = 0.46), whereas the mean valence rating for neutral words was 5.49 (SD = 0.52), a difference shown to be significant t = 53.58, p < .001, Cohen’s dz = 6.92. The mean arousal rating for negative words was 5.86 (SD = 0.94), whereas the mean arousal rating for neutral words was 4.18 (SD = 0.70), a difference again shown to be significant t = 15.73, p < .001, Cohen’s dz = 2.03. All stimuli were presented in white letters on a black background, on a monitor located 1.2 m from the participant. All stimuli were presented at central fixation and subtended maximum visual angles of 0.5°  and 2.2° . The experiment consisted of Study 1, Study 2, and an exclusion memory test. Forty neutral and 40 negative words were randomly intermixed in Study 1. Participants were required to verbally generate a meaningful sentence incorporating each word before proceeding to the next item. A further 40 neutral and 40 negative words were randomly intermixed and presented in Study 2. Participants made ‘very pleasant’/’fairly pleasant’/’fairly unpleasant’/’very unpleasant’ judgments to each item by button press.Stimuli consisted of 240 words selected from the ANEW database  or on a second key both to new items and to items from Study 1 (i.e. those that had been encoded in the sentence generation task). The response to each item initiated the next trial after an interval of 1,500 ms during which the screen remained blank. Two-minute intervals separated Study 1 and Study 2, and Study 2 and test, during which instructions were given to the participant for the upcoming task. Items were rotated across participants so that they served as Study 1 items, Study 2 items, and unstudied items an equal number of times. The hands used for target and nontarget/new responses were also counterbalanced across participants.EEG was recorded using a Biosemi active electrode system from 32 recording locations based on the International 10-20 system Jasper,  includinAll analyses included the Greenhouse–Geisser correction for nonsphericity where necessary  of negative stimuli attracted ‘fairly unpleasant’ or ‘very unpleasant’ responses, while 86% (SD = 11) of neutral items attracted ‘fairly pleasant’ or ‘very pleasant’ responses. It should be noted here that there was no neutral response option. The four response options were scored  and analysed for each stimulus type. Negative items received a mean valence score of 1.52 (SD = 0.26) whereas neutral items received a mean valence score of 3.04 (SD = 0.17). A paired t test revealed this difference to be significant t = 17.49, p < .001, Cohen’s dz = 4.12.Analysis of self-report pleasantness judgments during Study 2 showed that an average of 97% .Table r) between targets and new items  was .80 and .68 for neutral and negative items, respectively, and measures of response bias ) between targets and new items were .15 and .41 for neutral and negative items respectively  = 12.68, p < .001, ηp2 = 0.43, a main effect of Valence, F = 28.80, p < 0.001, η2p = 0.63, and a Response Type x Valence interaction F = 5.03, p < .05, ηp2 = 0.23. Subsidiary pairwise comparisons  revealed that the hit rate was statistically equivalent for negative and neutral targets, whereas the correct rejection rate was higher for neutral words than for their negative equivalents for both nontargets, F = 20.98, p < .001, ηp2 = 0.55, and for new items, F = 13.79, p = .002, ηp2 = 0.45. The parallel ANOVA of RTs associated with correct responses gave a main effect of Response Type, F = 31.88, p < .001, ηp2 = 0.65, a main effect of Valence, F = 19.62, p < .001, ηp2 = 0.54, and a Response Type × Valence interaction, F = 5.47, p < .05, ηp2 = 0.24. Subsidiary pairwise comparisons  revealed statistically equivalent RTs for negative and neutral target hits, whereas RTs were faster for neutral than for negative items for both nontarget CRs, F = 17.39, p = .001, ηp2 = 0.51, and new item CRs, F = 7.28, p = .015, ηp2 = 0.30.Discrimination  = 5.49, p < .05, ηp2 = 0.24. Pairwise comparison of target hits and new CRs revealed a main effect of Response Type, F = 9.11, p < .01, ηp2 = 0.35, and an interaction between Response Type × Site, F = 4.55, p < .05, ηp2 = 0.21, reflecting greater positivity for targets maximal towards the midline  = 10.55, p < .01, ηp2 = 0.37. A main effect of Response Type, F = 4.51, p < .05, ηp2 = 0.21, was also obtained in the pairwise comparison of nontarget and new CRs which was moderated by a Response Type × Valence × Hemisphere × Site interaction, F = 3.50, p < .05, ηp2 = 0.17, indicating a left-lateralised old/new effect (greater positivity for nontargets) maximal towards the midline. Although the effect at left hemisphere midline sites  was larger in magnitude for negative items, a main effect of Response Type obtained at these sites, F = 17.68, p < .05, ηp2 = 0.30, was not significantly moderated by Valence. A topographic analysis motivated by the Response Type × Valence × Hemisphere × Site interaction examined whether the nontarget old/new effects elicited by neutral and negative items had different scalp distributions. This was conducted on difference scores obtained by subtracting mean amplitudes of new CRs from nontarget CRs for neutral and negative items, respectively. The data were rescaled using the max-min method to avoid confounding changes in amplitude with changes in the shape of scalp distributions  = 10.26, p < .01, ηp2 = 0.38, and interactions between Response Type × Anterior/Posterior × Hemisphere, F = 4.51, p < .05, ηp2 = 0.21, and Response Type × Anterior/Posterior × Site, F = 2.49, p < .05, ηp2 = 0.13. Pairwise comparison of target hits and new CRs showed a main effect of Response Type, F = 17.53, p < .01, ηp2 = 0.51, and interactions between Response Type × Site, F = 10.99, p < .001, ηp2 = 0.39, and Response Type × Anterior/Posterior × Hemisphere, F = 9.88, p < .001, ηp2 = 0.37, reflecting greater positivity for targets maximal at left parietal sites towards the midline. A main effect of Response Type was obtained in a confirmatory analysis of data from this maxima (P1), F = 20.08, p < .001, ηp2 = 0.53. Pairwise comparison of nontargets and new CRs showed interactions between Response Type × Anterior/Posterior × Hemisphere, F = 7.39, p < .01, ηp2 = 0.30, Response Type × Anterior/Posterior × Site, F = 3.15, p < .05, ηp2 = 0.16, and Response Type × Valence × Hemisphere, F = 6.66, p < .05, ηp2 = 0.28. These reflected a small positivity for negative and neutral nontargets in the left hemisphere maximal at posterior sites, and greater negativity for neutral nontargets in the right hemisphere  = 4.60, p < .05, ηp2 = 0.21, reflecting a significant effect of Response Type for neutral items only, Response Type, F = 5.38, p < .05, ηp2 = 0.24. The pairwise comparison of targets and nontargets revealed a main effect of Response Type, F = 28.27, p < .001, ηp2 = .062, reflecting greater positivity for targets than for nontargets.The global analysis between 500 and 800 ms revealed a main effect of Response Type, F.4, 24.6 F = 4.11, p < .05, ηp2 = 0.19, and interactions between Response Type × Anterior/Posterior × Hemisphere, F = 4.17, p < .01, ηp2 = 0.20, and Response Type × Anterior/Posterior × Site, F = 3.48, p < .01, ηp2 = 0.17. Pairwise comparison of target hits and new CRs revealed crossover interactions between Response Type × Anterior/Posterior × Hemisphere, F = 6.44, p < .01, ηp2 = 0.27, and Response Type × Anterior/Posterior × Site, F = 4.00, p < .01, ηp2 = 0.19, reflecting greater positivity for targets maximal at right anterior sites and greater negativity for targets at right posterior sites maximal towards the midline. Post hoc tests revealed a main effect of Response Type at right frontal sites, F = 4.68, p < .05, ηp2 = 0.21, and no effect of Response Type at the right posterior midline site (P2). Pairwise comparison of nontargets and new CRs revealed interactions between Response Type × Anterior/Posterior × Hemisphere, F = 8.24, p < .01, ηp2 = 0.33, and Response Type × Anterior/Posterior × Site, F = 5.07, p < .01, ηp2 = 0.23, reflecting greater negativity for nontargets maximal at right posterior sites towards the midline. A post hoc test conducted at the right posterior midline site (P2) confirmed that this late posterior negativity was significant, F = 9.12, p < .01, ηp2 = 0.34. Pairwise comparison of targets and nontargets revealed a main effect of Response Type, F = 13.51, p < .01, ηp2 = 0.44, reflecting generally greater positivity for targets than for nontargets.The global analysis between 800 and 1,400 ms showed a main effect of Response Type, F = 14.53, p < .001, ηp2 = 0.46. Subsidiary pairwise comparisons between each pair of Response Types  revealed main effects of Response Type between target hits and new CRs F = 25.11, p < .001, ηp2 = 0.60 and between target hits and nontarget CRs F = 16.82, p = .001, ηp2 = 0.50, but not between nontarget CRs and new CRs  between 500 and 800 ms incorporated the factors of Response Type (target hits/nontarget CRs/new CRs), Valence , and Site . This analysis revealed a main effect of Response Type, , P1 betwF = 3.66, p < .05, ηp2 = 0.18, and a Response Type × Valence interaction, F = 4.27, p < .05, ηp2 = 0.20. The following six post hoc tests  were conducted: (i) targets/new CRs, (ii) nontargets/new CRs, (iii) targets/nontargets , and (iv) negative/neutral targets, (v) negative/neutral nontargets, (vi) negative/neutral new CRs . The only significant right frontal effects obtained in these analyses were a main effect of Valence in the negative/neutral nontargets contrast, F = 9.03, p = .0079, ηp2 = 0.35  = 8.65, p = .009, ηp2 = 0.34, in the contrast between target hits and nontarget CRs, reflecting a significant target/nontarget effect for neutral items only, F = 16.64, p < .001, ηp2 = 0.49  between 800 and 1,400 ms incorporated the factors of Response Type (target hits/nontarget CRs/new CRs), Valence , and Site . This analysis revealed a main effect of Response Type, , F2 betwF1.9, 2.1 = 3.6This experiment employed the paradigm used in the high target accuracy condition from , the behavioural data would point towards the superior recollection of source information for neutral words given the higher levels of accuracy for neutral than for negative nontargets. At first sight this seems surprising given previous reports of superior source judgments for emotionally arousing stimuli when participants were asked to recall the colour or location of stimuli during encoding  or ‘don’t think’ of the paired associate of each item presented at test, with the repeated suppression of ‘don’t think’ items reducing their memorability during subsequent recall  or evaluate nontarget source information for neutral items, whereas the behavioural and ERP data suggest that negative nontargets engaged source monitoring processes even in the absence of robust ERP indices of recollection. Whether the enhanced monitoring evident for negative nontargets was due to negative valence per se or to the greater semantic relatedness of negative words (Maratos et al., Supporting the view that nontargets were recognised on the basis of familiarity, it was found that nontarget old/new effects were significant in the 300–500-ms epoch. ERP memory effects within this time window are widely considered to reflect familiarity-based recognition (Rugg & Curran One constraint of the present study was that the list lengths required to obtain desirable levels of memory performance while maintaining an adequate signal-to-noise ratio precluded the inclusion of overtly positively valenced stimuli, and it therefore remains unknown whether the strategic control of emotional memory extends to this class of stimuli. On a related note, there were insufficient trials to perform a four-way separation of nontarget ERPs according to the precise valence rating assigned by participants during study. Negative nontargets were instead classified on the basis of ANEW ratings in the present study, and while 97% of these items attracted subjective ‘fairly unpleasant’ or ‘very unpleasant’ ratings, it would be interesting to investigate whether the present findings extend to nontargets attracting extremely negative subjective valence ratings and whether there is a graded effect according to subjective levels of valence. Finally, it would also be of interest to determine whether neutral nontargets embedded in a negative context at encoding (e.g. Maratos & Rugg, In conclusion, it was found that healthy young adults were able to prioritise their recollection of targets over nontargets for both neutral and negatively valenced words. This was demonstrated by left parietal old/new effects for both neutral and negative targets relative to nontargets and new words. Participants achieved this so effectively that no significant left parietal old/new effects were detected for either neutral or negative nontargets, despite the fact that these items were encoded in a deeply elaborative task and equivalent items had elicited very high levels of recollection when they served as targets in a previous behavioural study. This extends the strategic retrieval literature by showing that memories associated with negatively valenced test stimuli are subject to strategic control, and that these items do not automatically reactivate recollection via a bottom-up mechanism despite the higher arousal values associated with these items. These findings also extend the directed forgetting literature by demonstrating that the recollection of negatively valenced words can be attenuated without recourse to explicit direct suppression or thought substitution instructions. The finding that behavioural and ERP measures of target recollection were unaffected by valence supports the view that recollection of extrinsic contextual information is not moderated by stimulus valence. Finally, although target right frontal old/new effects were unaffected by valence, these were detected between targets and nontargets for neutral items only. The finding that negative nontargets also elicited enhanced positivity at right frontal sites relative to neutral nontargets indicates that negative nontargets required a greater degree of source monitoring than their neutral counterparts.


T, was isolated from a nodule of the leguminous tree Erythrina brucei native to Ethiopia. The type strain 10.1601/nm.146310.1601/strainfinder?urlappend=%3Fid%3DCCBAU+10071T, was isolated from the nodules of Lespedeza cuneata in Beijing, China. The genomes of ERR11T and 10.1601/strainfinder?urlappend=%3Fid%3DCCBAU+10071T were sequenced by DOE–JGI and deposited at the DOE–JGI genome portal as well as at the European Nucleotide Archive. The genome of ERR11T is 9,163,226 bp in length and has 102 scaffolds, containing 8548 protein–coding and 86 RNA genes. The 10.1601/strainfinder?urlappend=%3Fid%3DCCBAU+10071T genome is arranged in 108 scaffolds and consists of 8,201,522 bp long and 7776 protein–coding and 85 RNA genes. Both genomes contain symbiotic genes, which are homologous to the genes found in the complete genome sequence of 10.1601/nm.2449810.1601/strainfinder?urlappend=%3Fid%3DUSDA+110T. The genes encoding for nodulation and nitrogen fixation in ERR11T showed high sequence similarity with homologous genes found in the draft genome of peanut–nodulating 10.1601/nm.2738610.1601/strainfinder?urlappend=%3Fid%3DLMG+26795T. The nodulation genes nolYA-nodD2D1YABCSUIJ-nolO-nodZ of ERR11T and 10.1601/strainfinder?urlappend=%3Fid%3DCCBAU+10071T are organized in a similar way to the homologous genes identified in the genomes of 10.1601/strainfinder?urlappend=%3Fid%3DUSDA+110T,10.1601/nm.2580610.1601/strainfinder?urlappend=%3Fid%3DUSDA+4 and 10.1601/nm.146210.1601/strainfinder?urlappend=%3Fid%3DCCBAU+05525. The genomes harbor hupSLCFHK and hypBFDE genes that code the expression of hydrogenase, an enzyme that helps rhizobia to uptake hydrogen released by the N2-fixation process and genes encoding denitrification functions napEDABC and norCBQD for nitrate and nitric oxide reduction, respectively. The genome of ERR11T also contains nosRZDFYLX genes encoding nitrous oxide reductase. Based on multilocus sequence analysis of housekeeping genes, the novel species, which contains eight strains formed a unique group close to the 10.1601/nm.25806 branch. Genome Average Nucleotide Identity (ANI) calculated between the genome sequences of ERR11T and closely related sequences revealed that strains belonging to 10.1601/nm.25806 branch (10.1601/strainfinder?urlappend=%3Fid%3DUSDA+4 and 10.1601/strainfinder?urlappend=%3Fid%3DCCBAU+15615), were the closest strains to the strain ERR11T with 95.2% ANI. Type strain ERR11T showed the highest DDH predicted value with 10.1601/strainfinder?urlappend=%3Fid%3DCCBAU+15615 (58.5%), followed by 10.1601/strainfinder?urlappend=%3Fid%3DUSDA+4 (53.1%). Nevertheless, the ANI and DDH values obtained between ERR11T and 10.1601/strainfinder?urlappend=%3Fid%3DCCBAU+15615 or 10.1601/strainfinder?urlappend=%3Fid%3DUSDA+4 were below the cutoff values  for strains belonging to the same species, suggesting that ERR11T is a new species. Therefore, based on the phylogenetic analysis, ANI and DDH values, we formally propose the creation of 10.1601/nm.30737 sp. nov. with strain ERR11T (10.1601/strainfinder?urlappend=%3Fid%3DHAMBI+3532T=10.1601/strainfinder?urlappend=%3Fid%3DLMG+30162T) as the type strain.The type strain of the prospective 10.1601/nm.30737 sp. nov. ERR11The online version of this article (10.1186/s40793-017-0283-x) contains supplementary material, which is available to authorized users. In addition, unique 10.1601/nm.1459 groups isolated from diverse legume species might represent new species [Biological nitrogen fixation is a vital process in ecosystem functioning, offering a nitrogen for plant growth. Legume plants form a nitrogen–fixing symbiotic association with soil bacteria known as rhizobia. The symbiotic association results in the formation of nodules, shelter and powerhouse of nitrogen fixation for the rhizobia, on the roots or stems of host legumes . The rhi species .In rhizobial taxonomic studies, polyphasic approaches such as phenotypic features, analysis of the 16S rRNA genetic marker, and DDH were for years used as standard criteria for the description of new bacterial species. Nevertheless, the 16S rRNA gene sequence difference between closely related species, particularly in the genus 10.1601/nm.1459 is low for differentiation of closely related species , 13. Bac-Móra [Multilocus sequence analysis (MLSA) of housekeeping protein–coding genes has become a common practice in bacterial taxonomic studies. The method offers high resolution and hence, has been used in rhizobial taxonomic studies for species identification and differentiating strains at the species level , 20, 21.-Móra  and Kim -Móra , the ANI-Móra  by inclu-Móra .E. brucei [Crotalaria spp. [Indigofera spp. [Crotalaria spp. [Crotalaria spp. [Indigofera spp. [E. brucei [Crotalaria spp. [Indigofera spp. [T) was selected for genome sequencing. The sequencing was done under the DOE–JGI 2014 Genomic Encyclopedia of Type Strains, Phase III, a project designed for sequencing of soil and plant–associated and newly described type strains [T. In addition, the genome sequence and annotation of reference type strain 10.1601/nm.146310.1601/strainfinder?urlappend=%3Fid%3DCCBAU+10071T [In Ethiopia, an endemic multipurpose legume tree . brucei  is used . brucei  and it a. brucei . Crotalaria spp.  and Indiera spp.  are amonera spp. . Crotalaria spp.  are usedria spp.  can be uria spp. –34. Indiera spp.  are usedera spp. . A group. brucei  and the ria spp.  and Indiera spp.  growing era spp. . To desc strains . Therefo+10071T  sequenceT is the type strain of newly proposed 10.1601/nm.30737 sp. nov. This novel species includes strains isolated from nodules of E. brucei [Indigofera spp. [Crotalaria spp. [recA, glnII, and rpoB single gene sequence analysis and on the phylogenetic tree constructed based concatenated recA–glnII–rpoB gene sequences. On the phylogenetic tree, the strains in the novel group formed their own cluster exclusive of validly published species, and consequently, this group were designated as 10.1601/nm.1459 genosp ETH1 [recA–glnII–rpoB sequences by including more and recently published reference sequences from the public database. In this phylogenetic tree, the bacterial grouping was consistent with our previous tree produced from concatenated recA, rpoB and glnII gene sequences [Glycine max) [. The average recA–glnII–rpoB gene sequences (1411 bp) similarity between the type strain ERR11T and other strains in the novel species was in the range 99–100% (data not shown). The closest species was 10.1601/nm.25806 [. The similarity between strains in the novel group and strains in the closest species was 96% and they all showed 95% average gene sequence similarity with strains in 10.1601/nm.1462   grown inine max)  nm.25806  followednm.25806 . The simquences 11 bp simiT [T [T , 43 and [T [T [T .Fig. 1MaT is a rod–shaped Gram–negative strain and has a dimension of 1.0–2.3 μm length and 0.7–1.0 μm width  saline solution. Then, each of the wells of the Biolog GN2 Microplate was filled with 150 μl of the suspension. The results were recorded as positive when the wells turned purple after 4, 24, 48 h or 96 h incubation at 28 °C [T [Minimum Information about the Genome Sequence is provided in Table escribed  using Biescribed . Conciseat 28 °C . The car28 °C [T , only thT and other strains in 10.1601/nm.1463 were first described as distinct species using phenotypic features, SDS–PAGE analysis of whole–cell proteins, DNA–DNA hybridization and16S rRNA gene sequence analyses [recA–glnII–rpoB sequence analysis, the strains belonging to 10.1601/nm.1463 formed a district branch in Fig. T is motile and Gram–negative. The rod–shaped form  [I. arrecta [C. juncea [E. brucei [E. brucei [T and other strains obtained from E. brucei [,Crotalaria spp. [Indigofera spp. [E. brucei [, I. arrecta [E. brucei, I. arrecta, soybean or peanut. Only the strains from Crotalaria spp. [Indigofera spp. [C. juncea plants [E. brucei [T was isolated from the nodules of the Lespedeza cuneata [Medicago sativa [Melilotus albus [The symbiotic properties of the strains in 10.1601/nm.30737 sp. nov. was studied in our previous study . The strria spp.  formed aria spp. . The typ. brucei , Indigof arrecta  and Crota juncea  as well ypogaea) . All the arrecta  and C. j. juncea  seeds we. juncea . Seeds f. brucei , soybean. brucei  seeds we. brucei ,Crotalaria spp.  and Indi. brucei , I. arre arrecta , soybeanria spp.  and India plants . Strains. brucei  includin cuneata  legume io sativa  and Melius albus .T and 10.1601/strainfinder?urlappend=%3Fid%3DCCBAU+10071T were sequenced at the DOE–JGI as part of the Genomic Encyclopedia of Bacterial and Archaeal Type Strains, Phase III: the genomes of soil and plant–associated and newly described type strains sequencing project. The plant and soil associated bacteria were considered for sequencing to understand better their environmental and agricultural importance from the sequence information. The sequencing project was also designed to produce genome sequence data that can be used for bacterial classification studies and for a description of a new species using ANI and Genome–to–Genome–Distance values [T together with other strains formed a distinctive phylogenetic group without including any known 10.1601/nm.1459 species, and this group representing most likely a new species [T was to describe the group as a new species by comparing the genome sequence data of ERR11T with the genome sequences of other 10.1601/nm.1459 species present in public databases. For this purpose, the type strain 10.1601/strainfinder?urlappend=%3Fid%3DCCBAU+10071T [T genome project is deposited at the DOE–JGI genome portal [FMAI01000001–FMAI01000102. The genome sequence of 10.1601/strainfinder?urlappend=%3Fid%3DCCBAU+10071T is also available at DOE–JGI genome portal [FMAE01000001–FMAE01000108. The Sequencing, assembling, finishing, and annotation were performed by the DOE–JGI [Type strains ERR11e values . Based o+10071T  was alsoe portal  as well e portal  under ace portal  and at Ee portal  under ac DOE–JGI . The genT =10.1601/strainfinder?urlappend=%3Fid%3DHAMBI+3532T and 10.1601/strainfinder?urlappend=%3Fid%3DCCBAU+10071T = 10.1601/strainfinder?urlappend=%3Fid%3DLMG+21827T were grown on YEM agar plates at 28 °C for 7–10 days and a pure colony of the cultures was transferred and grown in YEM broth till the culture reached late–logarithmic phase. The genomic DNAs were extracted from cell pellets following the CTAB DNA extraction protocol of the DOE–JGI [The growth conditions and DNA isolation methods were as previously described . In brie DOE–JGI .T and 10.1601/strainfinder?urlappend=%3Fid%3DCCBAU+10071T were sequenced at the DOE–JGI by using the Illumina technology [T and 9,923,442 reads counting 1498.4 Mb of 10.1601/strainfinder?urlappend=%3Fid%3DCCBAU+10071T. Details regarding the general aspects of library construction and sequencing methods can be found at the DOE–JGI website [T; 109 contigs in 108 scaffolds with a total size of 8.2 Mb for 10.1601/strainfinder?urlappend=%3Fid%3DCCBAU+10071T. The final assembly was based on 1399.7 Mb Illumina data and 225.2X input read coverage for the strain ERR11T; 1399.7 Mb Illumina data and 279.9× input read coverage for the strain 10.1601/strainfinder?urlappend=%3Fid%3DCCBAU+10071T.Strains ERR11chnology . An Illu website . Artifac website . The fil website  and 1–3  website . The finUniProt, TIGRFam, Pfam, KEGG, COG, and InterPro databases. The tRNAScanSE tool [Genes were first predicted by the Prodigal  program nSE tool  was usednSE tool . Other nnSE tool . AdditionSE tool  developenSE tool .T consists 102 scaffolds with a total size of 9,163,226 bp and a 63.2% G + C content. From a total 8634 genes, 8548 were protein–coding genes and 86 RNA encoding genes. The genome of 10.1601/strainfinder?urlappend=%3Fid%3DCCBAU+10071T is arranged in 108 scaffolds and has a size of 6,928,453 bp with a 63.8% G + C content and of the 7861 predicted genes 7776 were protein–coding genes and 85 were RNAs–coding genes (Table T (72.8%) and 10.1601/strainfinder?urlappend=%3Fid%3DCCBAU+10071T (72.6%) were annotated to functions and the remaining 2266 (26.3%) and 2073 (26.4%) genes were without a functional prediction for ERR11T and 10.1601/strainfinder?urlappend=%3Fid%3DCCBAU+10071T, respectively. About 62% CDSs of ERR11T and 63% CDSs of 10.1601/strainfinder?urlappend=%3Fid%3DCCBAU+10071T were assigned to COG functional categories. The distribution of the genes assigned into COGs functional categories is presented in Table The genome of ERR11recA–glnII–rpoB concatenated gene sequences  followed by 10.1601/strainfinder?urlappend=%3Fid%3DUSDA+4 (53.1%) is below the threshold of 70%, which is commonly used value for species delineation [recA–glnII–rpoB gene sequence analysis, both ANI and DDH results revealed that the closest strains for ERR11T were strains belong to 10.1601/nm.25806 group (10.1601/strainfinder?urlappend=%3Fid%3DCCBAU+15615 and 10.1601/strainfinder?urlappend=%3Fid%3DUSDA+4). Nevertheless, both the ANI and DDH values between ERR11T and 10.1601/strainfinder?urlappend=%3Fid%3DCCBAU+15615 or 10.1601/strainfinder?urlappend=%3Fid%3DUSDA+4 were below the cutoff values of the strains of the same species, suggesting that ERR11T belong to the novel group.The ANI values and DDH estimated results are presented in Table  species . The cloineation , 79. In T and the closest reference strains 10.1601/strainfinder?urlappend=%3Fid%3DUSDA+4 and 10.1601/strainfinder?urlappend=%3Fid%3DCCBAU+83689 were identified using an OrthoVenn program [T, 10.1601/strainfinder?urlappend=%3Fid%3DUSDA+4 and 10.1601/strainfinder?urlappend=%3Fid%3DCCBAU+83689 was 6850, 5897 and 6923, respectively. In the genome of ERR11T, 99 of the clusters were identified as unique protein clusters without homologs in the other genomes. In 10.1601/strainfinder?urlappend=%3Fid%3DUSDA+4 and 10.1601/strainfinder?urlappend=%3Fid%3DCCBAU+83689, 44 and 77 protein clusters respectively, were also identified as unique clusters with no detectable homologous with other genomes. Of the total proteins used in the analysis 1456, 2028, 1102 were single copy gene clusters in ERR11T, 10.1601/strainfinder?urlappend=%3Fid%3DUSDA+4 and 10.1601/strainfinder?urlappend=%3Fid%3DCCBAU+83689, respectively. Of the clusters, in total 5310 homologous protein clusters were shared in common by all of the three genomes. Strain ERR11T shares about 76.7% (6560) of its proteins with 10.1601/strainfinder?urlappend=%3Fid%3DUSDA+4 and 64.4% (5501) clusters with 10.1601/strainfinder?urlappend=%3Fid%3DCCBAU+83689. Based on the pairwise comparison, ERR11T shared the highest number with strain 10.1601/strainfinder?urlappend=%3Fid%3DUSDA+4 with 1250 protein clusters and ERR11T shared only 191 protein clusters with 10.1601/strainfinder?urlappend=%3Fid%3DCCBAU+83689. This result is in accordance with the phylogenetic tree .Shared orthologous protein clusters between the genomes of ERR11 program  as descr program . The ortnod, nol, noe) for the synthesis of the backbone of LCO Nod factors and substituent groups and genes coding for nitrogen fixation  are required in rhizobia–legume symbiosis [T and 10.1601/strainfinder?urlappend=%3Fid%3DCCBAU+10071T, the genomes were assembled against completely sequenced 10.1601/strainfinder?urlappend=%3Fid%3DUSDA+110T and 10.1601/strainfinder?urlappend=%3Fid%3DUSDA+6T using the Genome Gene Best Homologs package from program IMG–ER [T, 10.1601/strainfinder?urlappend=%3Fid%3DCGMCC+1.10947T, 10.1601/strainfinder?urlappend=%3Fid%3DCGMCC+1.10948T, 10.1601/strainfinder?urlappend=%3Fid%3DUSDA+4, and 10.1601/strainfinder?urlappend=%3Fid%3DCCBAU+05525. To see the arrangement of symbiotic genes, the genome of ERR11T and references were aligned using the progressive Mauve alignment method [T and 10.1601/strainfinder?urlappend=%3Fid%3DCCBAU+10071T and their locations in the genomes and resemblance with genes in the reference genomes are shown in Additional file nolY–nolA–nodD2–nodD1YABCSUIJ–nolO–nodZ were identified in scaffolds Ga0061098_1039 and Ga0061099_1014, in the genome of ERR11T and 10.1601/strainfinder?urlappend=%3Fid%3DCCBAU+10071T, respectively. The result of the Mauve alignment , Ga0061098_1016 (nodU), Ga0061098_1006 (nodT) and Ga0061098_1031 . These genes are also identified in the genome of 10.1601/strainfinder?urlappend=%3Fid%3DCCBAU+10071T in Ga0061099_1013 and Ga0061099_1018, Ga0061099_1014, Ga0061099_1005 and Ga0061099_1022, respectively.The nodulation genes , and Ga0061098_1039 (fixR–nifA–fixA). The nif/fix genes in the genome of 10.1601/strainfinder?urlappend=%3Fid%3DCCBAU+10071T are distributed in scaffolds Ga0061099_1013 (nifDKENX), Ga0061099_1041 (nifT–nifB–nifZ), Ga0061099_1036 (nifHQV–fixBCX), and Ga0061099_1014 (fixR–nifA–fixA). The fix genes (fixK2–fixJL–fixNOPGHIS), which are required for creating microoxic respiration for the rhizobia during symbiosis, are also conserved in the genomes of ERR11T (in scaffold Ga0061098_1024) and 10.1601/strainfinder?urlappend=%3Fid%3DCCBAU+10071T (in scaffold Ga0061099_10014) in a similar fashion as the homologous genes found in 10.1601/strainfinder?urlappend=%3Fid%3DUSDA+110T. Generally, the nodulation and nitrogen fixation genes of ERR11T and 10.1601/strainfinder?urlappend=%3Fid%3DCCBAU+10071T showed 70.0–100% sequence similarity with homologous genes found in the reference genomes of 10.1601/strainfinder?urlappend=%3Fid%3DUSDA+110T, 10.1601/strainfinder?urlappend=%3Fid%3DUSDA+6T, 10.1601/strainfinder?urlappend=%3Fid%3DUSDA+4, 10.1601/strainfinder?urlappend=%3Fid%3DCCBAU+23303T, 10.1601/strainfinder?urlappend=%3Fid%3DCGMCC+1.10947T, and 10.1601/strainfinder?urlappend=%3Fid%3DCCBAU+05525  specifically with homologous genes found in the genome of peanut–nodulating strain 10.1601/strainfinder?urlappend=%3Fid%3DLMG+26795T, suggesting that these strains may have a similar origin of symbiotic genes.In the genome of ERR11 complex  are main2 as a result of the reduction of N2 by nitrogenase. The rhizobia which have hydrogen–uptake systems are capable of recycling the released H2 in the rhizobia–legume symbiosis [hupNCUVSLCDFGHIJK, hypABFCDE, and hoxXA genes [. Clusters of hupSLCFHK and hypBFDE genes were identified in the genomes of ERR11T in scaffold Ga0061098_1005 and in 10.1601/strainfinder?urlappend=%3Fid%3DCCBAU+10071T in scaffold Ga0061099_1013  was not found in ERR11T. Therefore, the nitrite reductase activity may be lacking in ERR11T and denitrification in this strain may depend only on nitrate, nitric oxide, and nitrous oxide reductase reactions. The genome of 10.1601/strainfinder?urlappend=%3Fid%3DCCBAU+10071T harbors only denitrifying genes napEDABC and norCBQD for nitrate and nitric oxide reduction, respectively  belonging to the novel species formed a unique group within the genus 10.1601/nm.1459. In order to verdict this result, comparative genomic analyses based on ANI calculation and DDH methods were done. The results from both ANI and DDH supported the result from the phylogenetic analysis, in which the genome of the type strain ERR11T showed 95.2% ANI and 53.1 DDH similarity with the closest reference strain 10.1601/strainfinder?urlappend=%3Fid%3DUSDA+4. These values are lower than the 96.5% ANI and 70% DDH cutoff values designed for strains of the same species. These results confirm that 10.1601/nm.30737 sp. nov. should be considered as a new 10.1601/nm.1459 species. Therefore, based on the phylogenetic analysis, ANI and DDH results and by including phenotypic characteristics, we formally propose the creation of 10.1601/nm.30737 sp. nov. that contains the strain ERR11T (= 10.1601/strainfinder?urlappend=%3Fid%3DHAMBI+3532T=10.1601/strainfinder?urlappend=%3Fid%3DLMG+30162T). The type strain forms an effective nitrogen–fixing symbiosis with E. brucei [, I. arrecta (47) and peanut.In this study, we present the genome sequences of 10.1601/nm.30737 sp. nov. strain ERR11+26795T , 43 The shewense of Shewa, pertaining to Shewa, the region in Ethiopia, where the type strain was obtained). The bacteria are non–spore–forming, Gram–negative rods with a size of 1.0–2.4 μM in length and 0.7–1.0 μm width. Strains included in species are slow–growing, forming creamy, raised and smooth margin colonies of 1–2 mm in diameter after 7 to 10 days of incubation on YEMA plate containing Congo red at 28 °C and pH 7 optimal growth conditions. The strains are able to grow at 15 °C–30 °C, in 0.0–0.5 NaCl and at 5–10 pH ranges. They do not grow at pH 4, at 4 °C and at 35 °C and in 1–5% NaCl. In general, the type and the other strains in this species could ferment the following substrates as carbon sources in Biolog GN2 microplates; Tween 40, Tween 80, adonitol, L–arabinose, D–arabitol, glycogen, N–acetyl–D–glucosamine, adonitol, L–arabinose, D–arabitol, D–cellobiose, I–erythritol, D–fructose, L–fucose, D–galactose, α–D–glucose, α–D–lactose, lactulose, D–mannitol, D–mannose, D–melibiose, β–methyl–D–glucoside, D–raffinose, L–rhamnose, D–sorbitol, sucrose, turanose, xylitol, pyruvic acid methyl ester, acetic acid, succinic acid mono–methyl–ester, cis–aconitic acid, citric acid, formic acid, D–galactonic acid lactone, D–galacturonic acid, D–gluconic acid, D–glucosaminic acid, D–glucuronic acid, α–Hydroxybutyric acid, β–hydroxybutyric acid, γ–hydroxybutyric acid, p–hydroxy phenylacetic acid, itaconic acid, α–keto butyric acid, α–keto glutaric acid, α–keto valeric acid, D,L–lactic acid, propionic acid, quinic acid, D–saccharic acid, sebacic acid, succinic acid, bromosuccinic acid, succinamic acid, glucuronamide, L–alaninamide, D–alanine, L–alanine, L–alanyl–glycine, L–asparagine, L–aspartic Acid, L–glutamic acid, glycyl–L–aspartic acid, glycyl–L–glutamic acid, L–leucine, L–phenylalanine, L–proline, L–pyroglutamic acid, D–serine, L–threonine, urocanic acid, and glycerol. However, all the strains included in this test failed to oxidize α–cyclodextrin, glycogen, N–acetyl–D–galactosamine, N–acetyl–D–glucosamine, D–cellobiose, I–erythritol, gentiobiose, M–inositol, α–D–lactose, lactulose, D–melibiose, β–methyl–D–glucoside, D–raffinose, melonic acid, L–histidine, hydroxy–L–proline, L–ornithine, D,L–carnitine, γ–amino butyric acid, uridine, thymidine, phenyethyl–amine, putrescine, 2–aminoethanol, 2,3–butanediol, D,L–α–glycerol phosphate, α–D–glucose–1–phosphate, and D–glucose–6–phosphate. The type strain ERR11T was obtained from root nodules of E. brucei [T is available at the DOE–JGI genome portal [FMAI01000001 to FMAI01000102. The type strain has been deposited in the HAMBI (10.1601/strainfinder?urlappend=%3Fid%3DHAMBI+3532T) and LMG (10.1601/strainfinder?urlappend=%3Fid%3DLMG+30162T) culture collections.10.1601/nm.30737 Carbon sources utilization response between Additional file 3: Table S3.Bradyrhizobium shewense. sp. nov. ERR11T and B. yuanmingense CCBAU 10071T. (XLSX 121 kb)Symbiotic and denitrifying genes identified in the genomes of


CYP353D1v2 is a cytochrome P450 related to imidacloprid resistance in Laodelphax striatellus. This work was conducted to examine the ability of CYP353D1v2 to metabolize other insecticides. Carbon monoxide difference spectra analysis indicates that CYP353D1v2 was successfully expressed in insect cell Sf9. The catalytic activity of CYP353D1v2 relating to degrading buprofezin, chlorpyrifos, and deltamethrin was tested by measuring substrate depletion and analyzing the formation of metabolites. The results showed the nicotinamide–adenine dinucleotide phosphate (NADPH)-dependent depletion of buprofezin (eluting at 8.7 min) and parallel formation of an unknown metabolite (eluting 9.5 min). However, CYP353D1v2 is unable to metabolize deltamethrin and chlorpyrifos. The recombinant CYP353D1v2 protein efficiently catalyzed the model substrate p-nitroanisole with a maximum velocity of 9.24 nmol/min/mg of protein and a Michaelis constant of Km = 6.21 µM. In addition, imidacloprid was metabolized in vitro by the recombinant CYP353D1v2 microsomes  0.064 pmol/min/pmol P450, Km = 6.41 µM. The mass spectrum of UPLC-MS analysis shows that the metabolite was a product of buprofezin, which was buprofezin sulfone. This result provided direct evidence that L. striatellus cytochrome P450 CYP353D1v2 is capable of metabolizing imidacloprid and buprofezin. N-acetyl-d-glucosamine (2-(acetylamino)-2-deoxy-d-glucose) into chitin. Buprofezin kills the insect by its vapour phase activity through inhalation and by direct contact through adsorption by the integument +: 338.39 (+ and [205.13]+ (Samples from the imidacloprid metabolism tests were subjected to an ultra-performance liquid chromatograph tandem mass spectrometry ultra-performance liquid chromatograph (UPLC-MS/MS). The positive ion mode mass spectrum of the major detectable metabolite was buprofezin sulfone generated from buprofezin with a molecular ion peak at : 338.39 . The MS/205.13]+ .L. striatellus [L. striatellus [The extensive applications of buprofezin and imidacloprid resulted in cross-resistance between imidacloprid and buprofezin in iatellus . Cytochriatellus . CYP353Diatellus . HoweverBemisia tabaci [Drosophila melanogaster [A. gambiae [A. gambiae were found to degrade a juvenile hormone analogue, pyriproxyfen [CYP genes in resistant insects could result in cross-resistance among chemically unrelated insecticides.Our previous study demonstrated that CYP353D1v2 expressed in Sf9 cells could degrade imidacloprid. Besides this, in the current study, insecticide degradation experiments confirmed that CYP353D1v2 is also able to catalyze the oxidation of buprofezin to buprofezin sulfone. The first evidence of a single P450 enzyme metabolizing chemically unrelated insecticide was provided when neonicotinoid-metabolizing cytochrome P450 CYP6CM1 was reported as being capable of degrading pymetrozine in a tabaci ,22. Furtnogaster  and CYP6 gambiae . Recentlproxyfen . TherefoL. striatellus have reported that CYP6AY3v2 and CYP353D1v2 are associated with cross-resistance among imidacloprid, deltamethrin, and chlorpyrifos [Indeed, previous studies in rpyrifos . Howeverrpyrifos .p-nitroanisole were 6.41 ± 1.27 μM and 6.21 ± 0.65 µM, respectively. The higher Km value of imidacloprid suggests that CYP353D1v2 has a lower binding affinity to imidacloprid than p-nitroanisole. This result calls for further research on the molecular pharmacology of buprofezin, imidacloprid, and p-nitroanisole metabolism.Moreover, the Michaelis constant values for imidacloprid and p-Nitroanisole was obtained from AnaSpec INC, Fremont, CA, USA. All consumables for recombinant expression were obtained from .Buprofezin 97%, chlorpyrifos 96.5%, and deltamethrin 98% were purchased from Invitrogen Biotechnology Co., Ltd., Shanghai, China. Nicotinamide–adenine dinucleotide phosphate (NADPH), NADP+, Glucose-6-phosphate, and Glucose-6-phosphate dehydrogenase were purchased from Sigma . L. striatellus were obtained from NCBI . Forward and reverse primer for CYP353D1v2 was BamHI: CGCGGATCCGCCACCATGGTGAAAATAAATGGAAAG, and xho1: CGGGGTACCTTATCTATCATTGAAAAAAAT, respectively, and for CPR were BamHI: GGATCCATGGAGGTGGAGGCTGAT, and Hind111: GACAAGCTTTTAGCTCCAGACATCGGCCG. Both CYP353D1v2 and CPR were inserted into the pFastBacTM expression vector (Invitrogen Life Technologies). The constructed pFastBacTM was transformed into DH10Bac cells according to the manufacturer’s instructions (Invitrogen Life Technologies). The white colonies were used for isolating bacmid DNA according to the manufacturer’s protocol. Sf9 insect cells were transfected with recombinant bacmid DNA of CYP353D1v2 and CPR. The cells were grown to a density of 2 × 106 cells/mL and were co-infected with recombinant baculoviruses containing CYP353D1v2 and CPR with various multiplicity of infection ratios to identify optimal conditions. After further culture for 48 h, the infected cells were collected and washed with 0.1 M potassium phosphate buffer, and the microsome of the membrane fraction was prepared according to standard procedures and stored at −80 °C [CYP353D1v2 was expressed in Sf9 cells by using a baculovirus expression system according to Elzaki et al. (2017) . Brieflyt −80 °C . The prot −80 °C . P450 cot −80 °C . The actt −80 °C .p-nitroanisole [p-nitroanisole concentrations (0–100 µM). As described by Elzaki et al. (2017), the reaction was initiated by the addition of 10 µL of 9.6 mM NADPH. The absorbance was read with a Spectra Max M2 reader  at 405 nm and 30 °C for 15 min. The Michaelis constant (Km) and maximum velocity (Vmax) were established from the plot of p-nitroanisole concentrations against the initial velocities through a non-linear regression, by fitting the data to the Michaelis–Menten equation using GraphPad Prism  [Since in previous work CYP353D1v2 recorded high activity against oanisole , the kinCA, USA) . 2, 0.4 U mL−1 glucose-6-phosphate dehydrogenase) and 100 µM insecticide at 30 °C for 4 h and a different time course. The total assay volume was 1000 µL. The corresponding microsome-insecticide incubation without the NADPH-regenerating system served as the control.The metabolism of buprofezin, chlorpyrifos, and deltamethrin was assayed by incubation of the recombinant CYP353D1v2/CPR microsomes (30 pmol) in 0.1 M potassium phosphate buffer with an NADPH-regenerating system . Reactions were carried out with 400 rpm shaking, were stopped at different time intervals using 100 µL of acetonitrile, and were incubated for a further 20 min to ensure that all insecticide was dissolved. The quenched reactions were centrifuged at 20,000× v/v) over the first 4 min; followed by 90% acetonitrile for 6 min; and finally, returning to 20% acetonitrile for 4 min.Buprofezin, chlorpyrifos, and deltamethrin were separated on an Acclaim C18  reverse phase analytical column and detected at 245, 290, and 230 nm, respectively. The maximum running conditions were established for different insecticides. For separating metabolites of buprofezin, time-course reactions were run with a linear gradient from 20% to 90% acetonitrile in water  were used. The rates of substrate turnover from three independent reactions were plotted against substrate concentration. Km, Vmax, and Kcat were determined.m/z. The HPLC system was a Waters Acquity UPLC consisting of a binary solvent manager, column manager, and sample manager. The samples were run on a Waters Atlantis BEHC18 1.7 μm column (2.1 × 100 mm) running in a reversed-phase gradient mode with acetonitrile 80% as the eluent. For MS/MS analysis, the collision energy was set at 25 eV.Samples were measured on an UPLC/XEVO TQ-S MS/MS system run with Electrospray Ionization (ESI) in positive-ionization mode with a capillary voltage of 5 kV and Turbo V gas temperature of 500 °C. The mass scan ranged from 100 to 500 L. striatellus.This study has proved that imidacloprid-metabolizing cytochrome P450 CYP353D1v2 is also capable of metabolizing insect growth regulator insecticide, buprofezin. However, CYP353D1v2 is unable to metabolize deltamethrin and chlorpyrifos. These findings provide a better understanding of the insecticide resistance molecular mechanism in


There is much literature devoted to the problem of drug resistance and decreased susceptibility of fungi to commonly used antifungals.Candida albicans and non-Candida albicans strains isolated from the hands of people without any symptoms of disease over a 16-year period.To analyze drug susceptibility of Candida-type strains isolated from the hands of people without any symptoms of disease, including: in 1999, 432 strains; in 2004, 368; and in 2015, 454 strains. Biological monitoring of hand surface contamination was performed using the Count-TactTM applicator with Count-Tact plates (bioMerieux). Drug susceptibility was evaluated using FUNGITEST®.The study included a total of 1,274 p < 0.001). In 1999, the most strains showed resistance to fluconazole (50.6%), in 2004 to itraconazole (52.9%), and in 2015 to fluconazole (44.9%). Resistance to more than one drug was 52.9% in 1999, 64.3% in 2004, and 88.1% in 2015. Mean resistance to azole antifungals significantly increased from a mean of 76 ± 9.7 strains in 1999, to 95.3 ± 24.2 in 2004, and to 97.3 ± 16.6 in 2015 (p < 0.001).In 1999, the most strains showed resistance to fluconazole (53.2%), in 2004 to itraconazole (52.9%), and in 2015 to fluconazole (85.8%). Resistance to more than one drug was 35.8% in 1999, 64.7% in 2004, and 92% in 2015. Mean resistance to azole antifungals significantly increased from 98 ± 39.7 strains in 1999 to 118.3 ± 29.6 in 2015 (C. albicans and non-Candida albicans strain resistance to commonly used antifungal chemotherapeutics, mainly imidazole. We found a clear rise in susceptibility of C. albicans and non-Candida albicans strains to several studied antifungals.We showed increased  Candida glabrata and Candida krusei, for example, show primary resistance to fluconazole  or changes in their transcriptional regulatory system   . Mitochoe levels . In addie common . There iCandida is the most common cause of healthcare-associated bloodstream infections in the United States . The dominant hands of the participants without clinical symptoms were sampled in the morning.Candida-type strains isolated from the hands of people without any symptoms of disease, including the following. In 1999, 432 strains from 229 students; in 2004, 368 from 198 students; and in 2015, 454 strains from 240 students were isolated. To facilitate the analysis of statistical dependence, the studied strains were divided into two groups: I—C. albicans and II—non-Candida albicans. Group I had 630 C. albicans strains, including 282 strains from 1999, 172 from 2004, and 176 from 2015. Group II had 644 non-Candida albicans strains, including 170 from 1999, 196 from 2004, and 278 from 2015.The study included a total of 1,274 C. albicans and the presumptive identification of C. tropicalis, C. glabrata and C. krusei.Biological monitoring of hand surface contamination was performed using the Count-Tact™ applicator with Count-Tact plates  containing a medium complying with the Draft European Standard CEN/TC 243/WG2 requirements. CandiSelect  was used to identify yeast-like fungi. It is a selective chromogenic medium designed for the isolation of yeasts, the direct identification of C. albicans produced pink to purple colored colonies; C. glabrata produced turquoise colonies that were shiny and flat with a regular outline; C. tropicalis produced intense turquoise colored colonies that were spherical with a regular outline; C. krusei produced large turquoise colonies with a dry appearance and an irregular outline.After sampling, Count-Tact plates were incubated at 37C up to 48 h. Next, the fungal culture was inoculated into CandiSelect. The plates were incubated for 72 h to allow for identification. The color and intensity of colonies was assessed every 24 h. ®  ; 5-fluorocytosine (2–32 µg/ml), amphotericin B (2–8 µg/ml), miconazole (0.5–8 µg/ml) kétoconazole (0.5–4 µg/ml), itraconazole (0.5–4 µg/ml), fluconazole (8–64 µg/ml); two negative control wells. The breakpoints have been chosen following the study of the distribution of the antifungal agents M.I.C’s obtained with prototype microplates used with the same procedure as Fungitest. Results were interpreted in accordance with the manufacturer’s instructions, always by the same person, and always with reference to the color of two wells containing the same drug: a blue color in both wells indicated an in vitro susceptible strain; a pink color at lower concentrations and a blue color at higher concentrations indicated an in vitro strain with low susceptibility; and a pink color in both wells indicated an in vitro resistant strain.Drug susceptibility was assessed using FUNGITEST France)  to analyThe study has been accepted by ethic committee of the Medical University of Białystok, Poland, approval numbers: R-I-003/64/99; R-I-003/222/2004, and RI-002/489/2010.2 test and the Kruskal–Wallis test on Statistica 10.0 software.Statistical analysis of the results was done using the chiC. albicans strains were most susceptible to 5-fluorocytosine (67.8%) and least susceptible to itraconazole (14.1%). The C. albicans strains were most resistant to fluconazole (52.9%) and least resistant to amphotericin B (6.2%). Generally, the most non-Candida albicans strains were most susceptible to 5-fluorocytosine (69.7%), and least susceptible to ketoconazole (27.6%). The non-Candida albicans were most resistant to ketoconazole (50.5%) and least susceptible to amphotericin B (5.9%). Details are presented in Generally, C. albicans were most resistant to fluconazole (53.2%), and the least resistant to amphotericin B (1.1%). In 2004, we found the C. albicans were most resistant to itraconazole (52.9%), and least resistant to amphotericin B (3.5%). In 2015, the most C. albicans showed resistance to fluconazole (85.8%), and least resistance to amphotericin B (17%). Results are presented in C. albicans strains:In 1999, the  •p = 0.011), ketoconazole (p = 0.016), itraconazole (p < 0.001), and fluconazole (p < 0.001)in susceptibility to amphotericin B , and least resistant to amphotericin B (10.6%). In 2004, we detected that non-Candida albicans were most resistant to itraconazole (58.2%), and least resistant to 5-fluorocytosine (5.1%). In 2015, the non-Candida albicans showed most resistance to fluconazole (44.9%), and the least resistant to amphotericin B (1.4%). Results are presented in In 1999, the non-Candida albicans strains:We observed statistically significant differences between the analyzed years pertaining to  •p < 0.05) and ketoconazole (p < 0.001)in susceptibility to miconazole ( •p < 0.001) and ketoconazole (p < 001)in decreased susceptibility to 5-fluorocytosine ( •p < 0.001), amphotericin B (p < 0.001), and ketoconazole (p < 0.05).in resistance to 5-fluorocytosine . Resistance to more than one drug was 35.8% in 1999, 64.7% in 2004, and 92% in 2015. Results are presented in Generally, 60.2% of non-Candida albicans strains showed resistance to one or more of the studied antifungals, and most often to one (26%) or two drugs (25.7%). Resistance to more than one drug was 52.9% in 1999, 64.3% in 2004, and 88.1% in 2015. Results are presented in Generally, 71.8% of C. albicans and non-Candida albicans strains to one or more antifungal drugs (p < 0001). Generally, 295 ± 71.3 C. albicans strains were resistant to all studied antifungals. The mean resistance of C. albicans strains to antifungals significantly increased: 98 ± 39.7 in 1999, 118.3 ± 29.6 in 2015 (p < 0.001). Details are shown in We found significant differences between the analyzed years and resistance of the studied non-Candida albicans strains were resistant to the studied antifungals. The mean resistance of non-Candida albicans to the studied antifungals significantly increased: 85.5 ± 23.6 of strains in 1999, 95.3 ± 24.2 in 2004, and 97.3 ± 16.6 in 2015 (<0.001). Details are shown in Generally, 259 ± 50.2 of p = 0.399), Ketoconazole 72.9% of resistant strains in 1999, 56.6% in 2004, and decreased to 32.4% in 2015 (p < 0.05), Itraconazole 47.1% of resistant strains in 1999, 58.2% in 2004, and decreased to 33.5% in 2015 (p = 0.07) and Fluconazole 45.9% of resistant strains in 2004, 52% in 2015, and decreased to 44.9% in 2015 (p = 0.999). We found significant (p < 0.001) differences in resistance to the studied antifungals between C. albicans and non-Candida albicans strains.We observed an increase of resistance to: Miconazole: 35.3% of resistant strains in 1999, 56.3% in 2004, and decreased to 29.6% in 2015 , nystatin (91.6%), and flucytosine (94%). The authors found low susceptibility of the investigated strains to econazole, ketoconazole, miconazole, and fluconazole. According to various authors, the percentage of strains resistant to amphotericin B oscillates from 29 to 100 , and resistance to more than one drug rose from 52.9% in 1999 to 88.1% in 2015.It is alarming that 60.2% in vitro study on antifungal drug resistance of 186 non-Candida albicans and 61 C. albicans strains during three time periods: 2005–2007, 2009–2011, 2012–2014. The authors found a high resistance (35%–79%) to itraconazole. C. albicans strains exhibited a high susceptibility to fluconazole and voriconazole.C. albicans from 15.1% in 1994 to 27.7% in 1999, and a decline in drug resistance of C. species from 26.2% in 1994 to 12.2% in 1999. In 1999, the development of resistance of C. albicans to 5-fluorocytosine was an interesting phenomenon. C. albicans strains showed a two-fold increase in resistance to two or more antimycotics over a five year period, as opposed to C. species isolates, which showed a nearly two-fold decrease in their resistance to two or more drugs.In an earlier study , we condC. albicans strain susceptibility to amphotericin B, itraconazole, and fluconazole as well as reduced susceptibility and resistance to all drugs; and in the case of non-C. albicans strains to itraconazole and fluconazole.In the present study, we demonstrated statistically significant differences between the studied years in  1.Candida albicans and non-Candida albicans strain resistance to commonly used antifungal chemotherapeutics, mainly imidazole.We showed increased  2.Candida albicans and non-Candida albicans strains to several studied antifungals.We found a clear rise in susceptibility of 10.7717/peerj.3038/supp-1Data S1® to analyze yeast-like fungal growth in the presence of six drugs: 5-fluorocytosine, amphotericin B, miconazole, ketoconazole, itraconazole and fluconazole.Drug susceptibility was assessed using FUNGITESTClick here for additional data file.10.7717/peerj.3038/supp-2Data S2Candida and non-Candida resistance, susceptibility to antifungals in excel file.Raw data of Click here for additional data file.10.7717/peerj.3038/supp-3File S1Click here for additional data file.


Corynebacterium glutamicum is an important oxidation process generating excess NADH. In this study, we proposed a novel strategy to couple the process of 1,3-PDO synthesis with glutamate production for cofactor regeneration. With the optimization of 1,3-PDO synthesis route, C. glutamicum can efficiently convert glycerol into 1,3-PDO with the yield of ~ 1.0 mol/mol glycerol. Co-production of 1,3-PDO and glutamate was also achieved which increased the yield of glutamate by 18% as compared to the control. Since 1,3-PDO and glutamate can be easily separated in downstream process, this study provides a potential green route for coupled production of 1,3-PDO and glutamate to enhance the economic viability of biorefinery process.Production of 1,3-propanediol  from glycerol is a promising route toward glycerol biorefinery. However, the yield of 1,3-PDO is limited due to the requirement of NADH regeneration via glycerol oxidation process, which generates large amounts of undesired byproducts. Glutamate fermentation by  Escherichia coli which can directly utilize glucose for 1,3-PDO production with high titer and yield6791,3-Propanediol  is an important platform chemical which is used in a wide range of areas, including the textile industry, solvent, food, lubricants, and medicine123KlebsiellaClostridiaEnterobacterCitrobacterLactobacilli45K. pneumoniae with an overall yield of 0.66 mol 1,3-PDO plus 3-HP per mol glycerolSeveral species of microorganisms, including Corynebacterium glutamicum is a gram-positive bacterium which has been widely used for amino acids production in industry20212223C. glutamicum is a well-established industrial process which produces about 2.5 million tons of glutamate per year28+-ATPase significantly enhanced glutamate productionC. glutamicum for efficient cofactor regeneration C. glutamicum MB001. The pduCDEGH gene from K. penumoniae, encoding diol dehydratase and its activator, was co-expressed with dhaT gene encoding 1,3-PDO dehydrogenase in plasmid pEC-K18mob2 under the control of constitutive lac promoter. The recombinant strain PT01 still cannot utilize glycerol as carbon source, indicating that there is no active glycerol oxidation pathway in C. glutamicum. When cultured in LPG2 medium using glucose and glycerol as co-substrates, strain PT01 can grow and simultaneously utilize glucose and glycerol  were also observed during the fermentation  and 1,3-PDO production rate (0.23 g/L/h) were relatively low. To improve the production rate of 1,3-PDO, we attempted to further optimize 1,3-PDO synthesis pathway. 1,3-PDO dehydrogenase is the terminal enzyme of 1,3-PDO synthesis pathway which catalyzes the reduction of 3-HPA. There are two enzymes which have been reported to be able to catalyze this reaction: the NADH-dependent alcohol dehydrogenase encoded by yqhD-pdu . The resyqhD-pdu . The yiepduCDEGH  at 200 rpm. For micro-aerobic condition, the cultivation was performed in 500 ml baffled flasks containing 100 ml modified LPG2 medium (20 g/L of glycerol) at 100 rpm. As shown in C. glutamicum. The specific activities of glycerol dehydratase under micro-aerobic condition and aerobic condition were comparable (5.34 ± 0.17 U/mg vs 4.23 ± 0.15 U/mg). However, since the cell grew much faster under aerobic condition, the higher biomass may contribute to the higher glycerol consumption rate.After pathway optimization, we attempted to examine the effect of culture conditions for 1,3-PDO production. Since strain PY02 showed the highest glycerol consumption rate and 1,3-PDO production rate, it was selected for further optimization. Since the activity of glycerol dehydratase is affected by oxygenK. pneumoniae337383940C. glutamicum PY02. With the initial concentration of 20 g/L or 30 g/L, glycerol can be completely consumed and the yield of 1,3-PDO is close to 1.0 mol/mol glycerol  strain, was engineered for the first time to produce 1,3-PDO from glycerol. C. glutamicum is a widely used industrial workhorse which is currently utilized for production of million tons of amino acids including glutamate and lysine20212223C. glutamicum cannot oxidize glycerolC. glutamicum strains can convert almost all of glycerol into 1,3-PDO (~1.0 mol PDO/mol glycerol). The conversion of glycerol to 1,3-PDO is only catalyzed by two enzymes. However, the balance of enzyme activities between the two enzymes is very important. Increasing the expression of diol dehydratase accelerated glycerol consumption rate and 1,3-PDO formation rate. However, the toxic intermediate 3-HPA was accumulated at high initial glycerol concentration which caused the abnormal cessation of fermentation. 3-HPA accumulation was also observed in Enterobacterial agglomerans and K. pneumoniae3373833738et al. reported that application of UTR (untranslated region) engineering to precisely control the expression of glycerol dehydrogenase for optimal metabolic balance could significantly enhance the production of 3-HPMicrobial production of 1,3-PDO from renewable resources has been widely investigated in recent years459When glucose was utilized as carbon source for cell growth and NADH generation, about ~50 g/L of glucose was consumed for the conversion of 30 g/L of glycerol . To incrE. coli DH5α was used for routine cloning procedures. C. glutamicum MB001, a prophage-free strain derived from C. glutamicum ATCC 13032, was used as a background strainC. glutamicum OD01, which can produce glutamate without induction, is derived from strain MB001 with the deletion of odhA gene encoding a subunit of α-ketoglutarate dehydrogenaseE. coli/C. glutamicum shuttle vector used for gene overexpressionStrains and plasmids used in this study are listed in yqhD gene encoding alcohol dehydrogenase and pduCDEGH gene encoding diol dehydratase and its activator were amplified from the genome of E. coli K12 and K. pneumoniae DSM 2026 using primers 11-F/11-R and 12-F/12-R. The two fragments were inserted into the restriction site of EcoRI/XbaI of pEC-K18mob2 by Gibson Assembly Master Kit (NEB)dhaT gene encoding 1,3-PDO dehydrogenase was PCR amplified from the genome of K. pneumoniae DSM 2026 using primers 13-F/13-R. The backbone of pEC-yqhD-pdu was also PCR amplified using primers 14-F/14-R. The dhaT fragment and pEC-yqhD-pdu backbone were assembled into plasmid pEC-dhaT-pdu by Gibson Assembly Master Kit (NEB). To insert an artificial constitutive promoter H36pduCDEGH, the H36 promoter was amplified from plasmid pEC-H36glpF gene encoding glycerol facilitator was PCR amplified from the genome of K. pneumoniae DSM 2026 using primers 17-F/17-R. The fragment was inserted into the restriction site of EcoRI of pEC-yqhD-H36-pdu by Gibson Assembly Master Kit (NEB).The C. glutamicum strains MB001 and OD01 using electroporation as described by van der Rest et al.The recombinant plasmids were transformed into C. glutamicum strains. Production of 1,3-PDO by C. glutamicum was performed in 500 ml baffled flasks. For aerobic cultivation, the culture was carried out in 500 ml baffled flasks containing 50 ml modified LPG2 medium at 30 °C and 200 rpm. For micro-aerobic cultivation, the culture was carried out in 500 ml baffled flasks containing 100 ml modified LPG2 medium at 30 °C and 100 rpm. The modified LPG2 medium consists (per liter): 80 g glucose, 10–50 g glycerol, 10 g corn steep liquor, 20 g (NH4)2SO4, 4.5 g urea, 0.5 g MgSO4·7H2O, 0.5 g KH2PO4, 10 mg FeSO4·7H2O, 10 mg MnSO4·H2O, 0.2 mg biotin, 5 mg thiamine-HCl, 50 μM of vitamin B12, and 30 g CaCO3. When appropriate, the medium was supplemented with 25 μg/ml kanamycin. For the coproduction of 1,3-PDO and glutamate, the pH decreased dramatically during the cultivation. Thus, we manually adjusted the pH to 7.0 every 4 h by adding 5 M NaOH. All experiments were repeated for three times.Lysogenic broth (LB) medium was used for the routine culture of et al.The activity of glycerol dehydratase was assayed by the 3-methyl-2-benzothiazolinone hydrazone (MBTH) method as described by Toraya The activity of 1,3-PDO dehydrogenase (DhaT and YqhD) was assayed using the reverse reaction34All of the enzyme assays were repeated for three times.2SO4 as the mobile phase with a flow rate of 0.6 mL/min, and detection via refractive index or UV absorption at 210 nmet al.The cell density was determined by monitoring the absorbance at 600 nm using a spectrophotometer. Glucose, glycerol, 1,3-PDO and other organic acids were quantified by using High performance liquid chromatography (HPLC) equipped with an Aminex HPX-87H Column (300 × 7.8 mm) using 0.005 M HHow to cite this article: Huang, J. et al. Cofactor recycling for coproduction of 1,3-propanediol and glutamate by metabolically engineered Corynebacterium glutamicum. Sci. Rep.7, 42246; doi: 10.1038/srep42246 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Current prevalence and genotype distribution of hepatitis C virus (HCV) infection remain unknown in Ningxia, northwest China.From June to December 2013, 13,022 individuals were screened in Ningxia HIV/AIDS Sentinel Surveillance System, with their demographic features collected and serum samples tested for HCV antibody. Sero-positive drug users were further subjected to sequencing of NS5B and Core regions of HCV.The anti-HCV prevalence was 0.34 % among individuals without history of drug use, while it was 15.80 % among drug users. Of 79 NS5B sequences amplified from drug users, 64 (81.0 %) were male and 51 (64.0 %) were injection drug users (IDUs). Subtype 3a (40.5 %) and 1b (25.3 %) were the most predominant subtypes, followed in frequency by 3b (10.1 %) and 2a (7.6 %). Subtype distribution has no significant difference between injection and non-injection drug users. Based on phylogeographic analysis, HCV strains in Ningxia IDUs were mainly originated from two sites, Yunnan province  and Xinjiang Autonomous Region , which are the two major drug trafficking originates in China. Previously reported drug-resistance mutations were also scanned in this treatment-naïve population. Amino acid substitutions (C316N) associated with direct anti-viral agents (DAA) resistance were identified in the NS5B region in seven samples.This study is the first to reveal the existence of multiple genotypes of HCV in Ningxia, an inland province in northwest China, suggesting the rapid spreading of the virus.The online version of this article (doi:10.1186/s12985-016-0635-y) contains supplementary material, which is available to authorized users. Hepatitis C is the second leading cause of chronic hepatitis, liver cirrhosis and hepatocellular carcinoma (HCC) in China. The seroprevalence of hepatitis C infection in China is about 0.43 % in general population, corresponding to 5.6 million people . HoweverFlaviviridae, is a positive single-stranded RNA virus. HCV is mainly divided into 7 genotypes and more than 80 subtypes. HCV genotype 1, 2, and 3 are commonly distributed around the world [HCV, a member of the family he world . Severalhe world , 12. Thehe world . Han is he world .Fig. 1ThAs HIV and HCV share the same routes of transmission, we recruited patients from Ningxia HIV/AIDS Sentinel Surveillance System, part of the nationwide disease management program in China launched in 1995 , 16. TheHCV genotyping and phylogenetic analysis provide opportunity to reveal the recent HCV epidemic in Ningxia and the possible transmission networks in China. Such epidemiological and molecular information can also inform the efficacy of current regimens with Peginterferion and Ribavirin, and the upcoming highly effective direct anti-viral agents (DAAs) as well. Besides, few drug-resistance mutations have been reported in China, where no DAAs have not been approved yet. Therefore, pre-treatment testing for individuals may benefit in regimen choosing more effective regimens in the new DAA era.Serum samples were collected from Ningxia HIV/AIDS Sentinel Surveillance System, where nine groups of key affecting populations (KAPs) were tested for HCV routinely, including 1) drug users (IDU and non-injection drug users), 2) blood donors, 3) female sex workers (FSW), 4) men who have sex with men (MSM), 5) sexual transmitted disease (STD) outpatients, 6) long-distance truck drivers, 7) migratory populations, 8) pregnant women and new mothers, and 9) young students, for HIV, HCV and syphilis screening  using NS5B and Core specific external primers with the following protocol: 45 °C 30 min, 94 °C 2 min,  × 35 cycles, and 72 °C 10 min; the second round PCR was performed using Promega Taq PCR Mastermix kit , using 5ul product from the first round as the template, and NS5B and Core specific internal primers  with HCV-IgG ELISA kit , and positive results were repeated twice for accuracy. HCV RNA was quantified by real-time PCR, using Roche light cycle 2.0 Diagnostic Kit for hepatitis C virus RNA (PCR-Fluorescence Probing) . HCV RNA positive was defined as RNA copies over 0.5 × 10Four hundred fifteen NS5B reference sequences of subtype 1a, 1b, 2a, 2b, 3a, 3b, 6a, 6b, 6n, 6u, 6v were downloaded from GenBank, of which accession numbers were KF585503 - KF585907, GQ206087, EU256071, AB661379, JQ745651, KC844048, EU408332, EU408331, EU408330, D84262, D37855. We performed sequence comparison using the software MEGA 6.0, with the method of Clustal W1.8, and constructed phylogenetic trees using maximum-likelihood (ML) method and GTR+I+Γ model –19. The −4 substitution/site*year was set as the prior rate [Bayesian coalescent analysis was performed in BEAST software (v1.8.3). The GTR+I+gamma substitution model, uncorrected exponential clock model and Bayesian skygrid coalescent model were selected , and 5.0ior rate . The XMLp-value less than 0.05 were considered as statistically significant. Kappa values were calculated to estimate the agreement of NS5B and Core genotyping methods , 87.3 % had middle school or less education, and 69.6 % were unemployed. All drug users have a history of drug use longer than one year, and 64.6 % of them were IDUs. Nine patients (11.4 %), all IDUs, were also HIV co-infected. Detailed demographic characteristics and drug use information were summarized in Table p < 0.05), which indicates an emerging prevalence of subtype 3a infection among the high-risk population in Ningxia. In addition, subtype 1a, 2b, 6u and 6b were exclusively found in IDUs. Genotyping results based on Core region were present in Additional file Seventy-nine of those 116 RNA positive drug users were sent for sequencing for NS5B region of HCV, and nine subtypes were identified in total. Subtype 3a was the most common one, accounting for 40.5 % patients Fig. , followeBayesian phylogenetic trees by subtypes were reconstructed for NS5B sequences to explore the possible transmission patterns of HCV infections, based on a large number of references from 17 provinces or municipalities and a few references of subtype 6u, 6b, 1a, and 2b strains from other provinces or countries outside China  Subtype 1b used to be the most prevalent subtype in China, and strains from different regions were crossly distributed, suggesting simultaneous dissemination. In Fig. Two major groups of 2a were reported: Group A contained sequences mainly from the northwest China, and Group B from various regions in China  Fig. 3b3b. Five Subtype 3a sequences formed three separated geographic groups Fig. . Group AThe phylogeny of genotype 6 was reconstructed on the basis of nine Ningxia sequences and 55 reference sequences Fig. . Three gThe homology of every cluster of subtype was as follows: 1b 95.14 %, 2a 96.91 %, 2b 92.90 %, 3a 95.69 %, 3b 96.72 %, 6a 98.27 %, 6b 98.30 %. Highest homology was associated with subtype 6a and 6b, which suggested the late introduction of these subtypes to Ningxia; and the lowest homology (89.68 %) was associated with subtype 2b, indicating its long endemic history .We scanned for previously reported DAA-resistant mutations on NS5B, including S282T and C316N/Y in 79 NS5B sequences , 28. No We found that seroprevalence of HCV was 0.34 % among individuals without drug use history and 15.8 % for drug users in Ningxia, based on a screening on individuals from Ningxia HIV/AIDS Sentinel Surveillance System. Ningxia was conventionally considered as a low-risk area, with the seroprevalence about 0.10 % among general population . SpecialPrevious genotyping studies provided evidences to the hypothesis of two epidemics of HCV in China. Blood transfusion contributed to the first epidemic in China . SubtypeStudies on drug trafficking routes suggested the correlation of HIV transmission network and the drug smuggling routes from two major heroin-producing regions, “The Golden Triangle”  and “The Golden Crescent” (Afghanistan and Pakistan) . HCV shaWe found 11 co-infected patients of both HIV and HCV (13.9 %), which is much higher than the prevalence of 1.88 % among drug users in prior studies . This inWith regard to the naturally occurring drug-resistance mutations, several single nucleotide polymorphisms (SNPs) of NS5B protease resistance have been previously reported in treatment-naïve hepatitis C patients , 42. As The findings of this study may impact the HCV controlling approaches in terms of screening, treatment, and patients management. First, several studies have suggested that interventions targeting drug users were effective approaches for HCV control , 49. ConThis study has several limitations. First, all serum samples were collected from individuals presented in HIV/AIDS Sentinel Surveillance System in 2013, and therefore the prevalence could not reflect that of general population. Further studies on the phylogeny on sequences from general population may present with a different pattern. Second, only NS5B and Core sequences were analyzed in our study. Whole genome sequencing performed on deep sequencing would provide more information on genotype distribution and mixed infection of various strains within one host . Third, In conclusion, the overall seroprevalence of HCV was 0.34 % among people with no history of drug use in Ningxia, while it was 15.8 % among drug users and 47.25 % among IDUs, based on a screening on individuals from Ningxia HIV/AIDS Sentinel Surveillance System. Nine subtypes, 1a, 1b, 2a, 2b, 3a, 3b, 6a, 6b and 6u were found among 79 isolates from drug users. 3a (41.38 %) followed by 1b (25.29 %) were the most predominant. Phylogenetic analysis suggested two possible originates of HCV transmission, accompanying the drug trafficking route in China. Our study suggested that drug use, especial IDU, is the most important risk factor for HCV infection, and management of drug users in Ningxia may play a crucial role in controlling the ongoing HCV epidemic.