期刊：
International Journal of Biological Macromolecules,2021年167:1406-1413 ISSN：0141-8130
通讯作者：
Wu, Yunhua
作者机构：
[He, Zheng; Deng, Huan; Li, Yong; Wu, Yunhua; Liang, Xiaosheng] South Cent Univ Nationalities, Coll Life Sci, Wuhan 430074, Peoples R China.;[Wang, Qiang] South Cent Univ Nationalities, Coll Pharm, Wuhan 430074, Peoples R China.;[Liu, Deli] Cent China Normal Univ, Coll Life Sci, Wuhan 430079, Peoples R China.
通讯机构：
[Wu, Yunhua] S;South Cent Univ Nationalities, Coll Life Sci, Wuhan 430074, Peoples R China.
关键词：
Cytochrome P450 55A3;Nitric oxide;Electrochemical and spectrometric method
摘要：
Cytochrome P450 55A3 (CYP55A3) is an enzyme with the catalytic activity of nitric oxide (NO) to nitrous oxide using NADH or NADPH as the electron donor. Herein CYP55A3 has been expressed in E. coli and purified by His-tag columns. The electrochemical and spectroscopic characteristic of CYP55A3 and its interaction with NO has been studied. The direct electrochemistry of Fe3+/Fe2+ redox peaks in CYP55A3 was realized on the pyrolitic graphite electrode with the redox potential of -475 mV in pH 7.0 phosphate buffer. With the addition of NO a ferric nitroxyl complex (Fe3+-NO) formed with a new reduction peak at -0.78 V. The reduction peak current increased with the concentration of NO and showed typical Michaelis-Menten kinetic characteristics with the apparent Michaelis constant Kmapp 9.78 mu M. The binding constant K calculated to be 3.93 x 10(4) M by UV-vis method. The fluorescence emission spectra of iron porphyrin in CYP55A3 showed with the peak wavelength 633 nm, and its fluorescence intensity increased after binding with NO. The fluorescence analysis demonstrated that NADH can relay electrons to iron porphyrin and reduce NO. The reductive product of NO released and the iron porphyrin in CYP55A3 turned back to the original form. (C) 2020 Elsevier B.V. All rights reserved.
摘要：
Penicillium italicum (blue mold) is one of citrus pathogens causing undesirable citrus fruit decay even at strictly-controlled low temperatures (< 10 °C) during shipping and storage. P. italicum isolates with considerably high resistance to sterol demethylation inhibitor (DMI) fungicides have emerged; however, mechanism(s) underlying such DMI-resistance remains unclear. In contrast to available elucidation on anti-DMI mechanism for P. digitatum (green mold), how P. italicum DMI-resistance develops has not yet been clarified. The present study prepared RNA-sequencing (RNA-seq) libraries for two P. italicum strains (highly resistant (Pi-R) versus highly sensitive (Pi-S) to DMI fungicides), with and without prochloraz treatment, to identify prochloraz-responsive genes facilitating DMI-resistance. After 6 h prochloraz-treatment, comparative transcriptome profiling showed more differentially expressed genes (DEGs) in Pi-R than Pi-S. Functional enrichments identified 15 DEGs in the prochloraz-induced Pi-R transcriptome, simultaneously up-regulated in P. italicum resistance. These included ATP-binding cassette (ABC) transporter-encoding genes, major facilitator superfamily (MFS) transporter-encoding genes, ergosterol (ERG) anabolism component genes ERG2, ERG6 and EGR11 (CYP51A), mitogen-activated protein kinase (MAPK) signaling-inducer genes Mkk1 and Hog1, and Ca2+/calmodulin-dependent kinase (CaMK) signaling-inducer genes CaMK1 and CaMK2. Fragments Per Kilobase per Million mapped reads (FPKM) analysis of Pi-R transcrtiptome showed that prochloraz induced mRNA increase of additional 4 unigenes, including the other two ERG11 isoforms CYP51B and CYP51C and the remaining kinase-encoding genes (i.e., Bck1 and Slt2) required for Slt2-MAPK signaling. The expression patterns of all the 19 prochloraz-responsive genes, obtained in our RNA-seq data sets, have been validated by quantitative real-time PCR (qRT-PCR). These lines of evidence in together draw a general portrait of anti-DMI mechanisms for P. italicum species. Intriguingly, some strategies adopted by the present Pi-R were not observed in the previously documented prochloraz-resistant P. digitatum transcrtiptomes. These included simultaneous induction of all major EGR11 isoforms (CYP51A/B/C), over-expression of ERG2 and ERG6 to modulate ergosterol anabolism, and concurrent mobilization of Slt2-MAPK and CaMK signaling processes to overcome fungicide-induced stresses. The present findings provided transcriptomic evidence on P. italicum DMI-resistance mechanisms and revealed some diversity in anti-DMI strategies between P. italicum and P. digitatum species, contributing to our knowledge on P. italicum DMI-resistance mechanisms.
摘要：
Penicillium sp. are damaging to a range of foods and fruits including citrus. To date, double-stranded (ds)RNA viruses have been reported in most Penicillium species but not in citrus pathogen P. crustosum. Here we report a novel dsRNA virus, designated as Penicillium crustosum chrysovirus 1 (PcCV1) and isolated from P. crustosum strain HS-CQ15. PcCV1 genome comprises four dsRNA segments, referred to as dsRNA1, dsRNA2, dsRNA3, and dsRNA4, which are 3600, 3177, 3078, and 2808 bp in length, respectively. Sequence analysis revealed the presence of four open reading frames (ORFs) in the PcCV1 genome. ORF1 in dsRNA1 encodes a putative RNA-dependent RNA polymerase (RdRp) and ORF2 in dsRNA2 encodes a putative coat protein (CP). The two remaining ORFs, ORF3 in dsRNA3 and ORF4 in dsRNA4, encode proteins of unknown function. Phylogenetic analysis based on RdRp sequences showed that PcCV1 clusters with other members of the genus Chrysovirus, family Chrysoviridae. Transmission electron microscope (TEM) analysis revealed that the PcCV1 visions are approximately 40 nm in diameter. Regarding biological effects of PcCV1, HS-CQ15 harboring the chrysovirus exhibited no obvious difference in colony morphology under fungicide-free conditions but decreased resistance to demethylation inhibitor (DMI)-fungicide prochloraz, as compared to PcCV1-cured strain. Here we provide the first evidence of a virus present in citrus pathogenic fungus P. crustosum and the chrysovirus-induced change in fungicide-resistance of its host fungus.
摘要：
Mycoviruses have been found to infect more than 12 species of Penicillium, but have not been isolated from Penicillium italicum (P. italicum). In this study, we isolated and characterized a new double-stranded RNA (dsRNA) virus, designated Penicillium italicum chrysovirus 1 (PiCV1), from the citrus pathogen P. italicum HSPi-YN1. Viral genome sequencing and molecular characterization indicated that PiCV1 was highly homologous to the previously described Penicillium chrysogenum virus. We further constructed the mutant HSPi-YN1DeltapksP defective in the polyketide synthase gene (pksP), which is involved in pigment biosynthesis, and these mutants formed albino (white) colonies. Then we applied hyphal anastomosis method to horizontally transmit PiCV1 from the white virus-donors (i.e., HSPi-YN1 mutants) to wild-type recipients (i.e., P. italicum strains HSPi-CQ54, HSPi-HB4, and HSPi-HN1), and the desirable PiCV1-infected isogenic recipients, a certain part of blue wild-type strains, can be eventually selected and confirmed by viral genomic dsRNA profile analysis. This blue-white colony screening would be an easier method to select virus-infected P. italicum recipients, according to distinguishable color phenotypes between blue virus-recipients and white virus-donors. In summary, the current work newly isolated and characterized PiCV1, verified its horizontal transmission among dually cultured P. italicum isolates, and based on these, established an effective and simplified approach to screen PiCV1-infected isogenic recipients.
作者机构：
[Yuan, Yongze; Liu, Deli; Niu, Yuhui; Mao, Jiali; Wang, Menglan; Zhang, Tingfu; Yang, Zhu; Geng, Hui; Liu, DL] Cent China Normal Univ, Sch Life Sci, Hubei Key Lab Genet Regulat & Integrat Biol, Wuhan 430079, Hubei, Peoples R China.;[Zheng, Yongliang] Huanggang Normal Univ, Coll Life Sci, Huanggang 438000, Hubei, Peoples R China.
通讯机构：
[Yuan, YZ; Liu, DL] C;Cent China Normal Univ, Sch Life Sci, Hubei Key Lab Genet Regulat & Integrat Biol, Wuhan 430079, Hubei, Peoples R China.
摘要：
To date, partitiviruses, including gammapartitiviruses, have been extensively studied in various fungal hosts but have not been reported in Penicillium digitatum (also called green mold, the pathogenic fungus infecting citrus). In the present work, we isolated and molecularly characterized a double-stranded RNA (dsRNA) partitivirus from citrus green mold, which we have named "Penicillium digitatum gammapartitivirus 1" (PdGV1). The bisegmented genome of PdGV1 contains two dsRNA segments (dsRNA1 and dsRNA2) with a length of 1795 bp and 1622 bp, respectively. Each of the two genomic dsRNAs contains a single open reading frame encoding a putative RNA-dependent RNA polymerase (RdRp) and a coat protein (CP), respectively. Phylogenetic analysis based on RdRp and CP sequences showed that PdGV1 clustered with mycoviruses belonging to the genus Gammapartitivirus, family Partitiviridae, e.g., Penicillium stoloniferum virus S. The 5'- and 3'-untranslated regions (UTRs) of the PdGV1 genomic dsRNAs both contained unique conserved RNA motifs that have never been found in any other partitivirus. This is the first report of a new gammapartitivirus that infects the citrus-pathogenic fungus P. digitatum.
作者机构：
[Yuan, Yongze; Cao, Qianwen; Liu, Deli; Wang, Shengqiang; Niu, Yuhui; Mao, Jiali; Zhang, Tingfu; Yang, Zhu] Cent China Normal Univ, Sch Life Sci, Hubei Key Lab Genet Regulat & Integrat Biol, Wuhan 430079, Hubei, Peoples R China.
通讯机构：
[Liu, Deli] C;Cent China Normal Univ, Sch Life Sci, Hubei Key Lab Genet Regulat & Integrat Biol, Wuhan 430079, Hubei, Peoples R China.
摘要：
Pathogenic fungi including Penicillium digitatum and Penicillium italicum are the main destructive pathogens in the citrus industry, causing great losses during postharvest process. To our knowledge, only one mycovirus from P. digitatum has been reported, and the prevalence of such mycoviruses against citrus postharvest pathogenic fungi and their genotyping were still under investigation. In the present study, we showed that 39 of 152 Penicillium isolates from main citrus-growing areas in China were infected with various mycoviruses belonging to polymycoviruses, Narna-like viruses, and families Totiviridae, Partitivirdae and Chrysoviridae. The next generation sequencing (NGS) towards virus genome library and the following molecular analysis revealed two novel mycoviruses Penicillium digitatum polymycovirus 1 (PdPmV1) and Penicillium digitatum Narna-like virus 1 (PdNLV1), coexisting in P. digitatum strain HS-RH2. The fungicide-resistant P. digitatum strains HS-F6 and HS-E9 coinfected by PdPmV1 and PdNLV1 exhibited obvious reduction in triazole drug prochloraz resistance by mycelial growth analysis on both PDA plates and citrus fruit epidermis with given prochloraz concentration. This report at the first time characterized two novel mycoviruses from P. digitatum and revealed the mycovirus-induced reduction of fungicide resistance.
摘要：
Sterol 14α-demethylases from Cytochrome P450 family (CYP51s) are essential enzymes in sterol biosynthesis and well-known as the target of antifungal drugs. The 3D structure of CYP51A from Penicillium italicum (PiCYP51A) was constructed through homology modeling based on the crystal structure of human CYP51A (PDB: 3LD6). Molecular dynamics (MD) simulation was operated to relax the initial model and followed by quality assessment using PROCHECK program. On the basis of the docking information on the currently available CYP51s with the patent demethylase inhibitors (DMIs), pharmacophore-based virtual screening combined with docking analysis was performed to pick out twelve new compounds from ZINC database. Six hits revealed in the ligand database suggested potential ability to inhibit PiCYP51A. Compared to patent fungicide triazolone, the top three lead compounds had similar or higher affinity with the target enzyme, and accordingly, exhibited comparable or lower EC50 values to P. italicum isolates. The results could provide references for de novo antifungal drug design.
摘要：
Aminoalcohols have been addressed as activating buffers for alkaline phosphatase. However, there is no record on the buffer activation regarding organophosphorus hydrolase (OPH). Here we reported the activating effects of aminoalcohols on OPH-catalyzed hydrolysis of diisopropylfluorophosphate (DFP), an analog molecule of G-type warfare agents. The kinetic parametors kcat, Vmax and kcat/Km in the OPH reaction were remarkably increased in the buffers (pH 8.0, 25°C) containing aminoalcohols with C2 between nitrogen (N) and oxygen (O) in their structures, including triethanolamine (TEA), diethanolamine, monoethanolamine, 1-amino-2-propanol, 2-amino-2-methyl-1-propanol, and triisopropanolamine. In contrast, much lower or no rate-enhancing effects were observed in the adding of amines, alcohols, amine/alcohol mixtures, or 3-amino-1-propanol (C3 between N and O). The 300 mM TEA further increased DFP-degrading activities of OPH mutants F132Y and L140Y, the previously reported OPH mutants with desirable activities towards DFP. However, the treatment of ethylenediaminetetraacetate (EDTA) markedly abolished the TEA-induced activation of OPH. The product fluoride effectively inhibited OPH-catalyzed hydrolysis of DFP by a linear mixed inhibition (inhibition constant Ki ~ 3.21 mM), which was partially released by TEA adding at initial or later reaction stage. The obtained results indicate the activation of OPH by aminoalcohol buffers could be attributed to the reduction of fluoride inhibition, which would be beneficial to the hydrolase-based detoxification of organophosphofluoridate.
期刊：
CANADIAN JOURNAL OF MICROBIOLOGY,2017年63(11):895-908 ISSN：0008-4166
通讯作者：
Liu, Deli;Zhu, Derui
作者机构：
[Liu, Deli; Han, Rui] Cent China Normal Univ, Sch Life Sci, Hubei Key Lab Genet Regulat & Integrat Biol, Wuhan 430079, Hubei, Peoples R China.;[Chen, Laisheng; Han, Rui] Qinghai Univ, Acad Agr & Forestry Sci, Qinghai Key Lab Vegetable Genet & Physiol, Xining 810016, Qinghai, Peoples R China.;[Zhang, Xin; Liu, Jing; Zhu, Derui; Long, Qifu] Qinghai Univ, Res Ctr Basic Med Sci, Med Coll, Xining 810016, Qinghai, Peoples R China.
通讯机构：
[Liu, Deli] C;[Zhu, Derui] Q;Cent China Normal Univ, Sch Life Sci, Hubei Key Lab Genet Regulat & Integrat Biol, Wuhan 430079, Hubei, Peoples R China.;Qinghai Univ, Res Ctr Basic Med Sci, Med Coll, Xining 810016, Qinghai, Peoples R China.
关键词：
hypersaline environment;Bacteria;Archaea;community structure;microbial diversity;environnement hypersalin;Bacteria;Archaea;structure de la communauté;diversité microbienne
摘要：
Keke Salt Lake is located in the Qaidamu Basin of China. It is a unique magnesium sulfate-subtype hypersaline lake that exhibits a halite domain ecosystem, yet its microbial diversity has remained unstudied. Here, the microbial community structure and diversity was investigated via high-throughput sequencing of the V3-V5 regions of 16S rRNA genes. A high diversity of operational taxonomic units was detected for Bacteria and Archaea (734 and 747, respectively), comprising 21 phyla, 43 classes, and 201 genera of Bacteria and 4 phyla, 4 classes, and 39 genera of Archaea. Salt-saturated samples were dominated by the bacterial genera Bacillus (51.52%-58.35% relative abundance), Lactococcus (9.52%-10.51%), and Oceanobacillus (8.82%-9.88%) within the Firmicutes phylum (74.81%-80.99%), contrasting with other hypersaline lakes. The dominant Archaea belonged to the Halobacteriaceae family, and in particular, the genera (with an abundance of >10% of communities) Halonotius, Halorubellus, Halapricum, Halorubrum, and Natronomonas. Additionally, we report the presence of Nanohaloarchaeota and Woesearchaeota in Qinghai-Tibet Plateau lakes, which has not been previously documented. Total salinity (especially Mg2+, Cl-, Na+, and K+) mostly correlated with taxonomic distribution across samples. These results expand our understanding of microbial resource utilization within hypersaline lakes and the potential adaptations of dominant microorganisms that allow them to inhabit such environments.
作者机构：
[Yuan, Y. Z.; Li, D. D.; Chen, S. L.; Zhang, Y. Z.; Liu, D. L.; Niu, Y. H.; Li, S. X.; Liu, J.; Geng, H.; Li, N.] Cent China Normal Univ, Sch Life Sci, Hubei Key Lab Genet Regulat & Integrat Biol, Wuhan 430079, Peoples R China.;[Li, N.] Honghe Univ, Key Lab Crops High Qual & Efficient Cultivat & Se, Yunnan Higher Educ Inst, Coll Life Sci & Technol, Mengzi 661100, Peoples R China.
通讯机构：
[Liu, D. L.] C;Cent China Normal Univ, Sch Life Sci, Hubei Key Lab Genet Regulat & Integrat Biol, Wuhan 430079, Peoples R China.
作者机构：
[Yuan, Y. Z.; Li, D. D.; Zhang, YZ; Liu, DL; Chen, S. L.; Zhang, Y. Z.; Liu, D. L.; Niu, Y. H.; Li, S. X.; Liu, J.; Geng, H.; Li, N.] Cent China Normal Univ, Sch Life Sci, Hubei Key Lab Genet Regulat & Integrat Biol, Wuhan 430079, Peoples R China.;[Li, N.] Honghe Univ, Coll Life Sci & Technol, Yunnan Higher Educ Inst, Key Lab Crops High Qual & Efficient Cultivat & Se, Mengzi 661100, Peoples R China.
通讯机构：
[Zhang, YZ; Liu, DL] C;Cent China Normal Univ, Sch Life Sci, Hubei Key Lab Genet Regulat & Integrat Biol, Wuhan 430079, Peoples R China.
摘要：
Cytochrome P450nor catalyzes an unusual reaction that transfers electrons from NADP/NADPH to bound heme directly. To improve the expression level of P450nor2 from Cylindrocarpon tonkinense (C.P450nor2), Escherichia coli system was utilized to substitute the yeast system we constructed for expression of the P450nor2 gene, and the protein was purified in soluble form using Ni+-NTA affinity chromatography. In contrast to P450nor from Fusarium oxysporum (F.P450nor) and P450nor1 from Cylindrocarpon tonkinense (C.P450nor1), C.P450nor2 shows a dual specificity for using NADH or NADPH as electron donors. The present study developed a computational approach in order to illustrate the coenzyme specificity of C.P450nor2 for NADH and NADPH. This study involved homology modeling of C.P450nor2 and docking analyses of NADH and NADPH into the crystal structure of F.P450nor and the predictive model of C.P450nor2, respectively. The results suggested that C.P450nor2 and F.P450nor have different coenzyme specificity for NADH and NADPH; whilst the space around the B'-helix of the C.P450nor2, especially the Ser79 and Gly81, play a crucial role for the specificity of C.P450nor2. In the absence of the experimental structure of C.P450nor2, we hope that our model will be useful to provide rational explanation on coenzyme specificity of C.P450nor2.
摘要：
In this work, strain HS-NH1 which utilized phthalate acid esters (PAEs) as sole carbon and energy sources for growth, was isolated and identified as Gordonia sp. Phthalate acid (PA) and protocatechuate acid (PCA) were the major intermediate products by HPLC analysis. The phthalate acid catabolic gene cluster (phtBAabcdCR) which is responsible for the conversion of PA into PCA in strain HS-NH1 was obtained by genome analysis. The phtB, phtAab, phtAcd and phtC genes were expressed in Escherichia coli. Mixture of PhtAab and PhtAcd were able to convert PA into a product, which was then transformed to PCA by mixture of PhtB and PhtC. The enzymatic results as well as homology analysis of phtBAabcdCR gene sequences demonstrated that phtAabcd encodes the 3,4-phthalate dioxygenase which consists of three components: a hetero-oligomeric oxygenase, a [3Fe-4S]-type ferredoxin, and a GR-type reductase. PA was oxidized by 3,4-phthalate dioxygenase, subsequently transformed into PCA by dihydrodiol dehydrogenase (PhtB) and dihydroxyphthalate decarboxylase (PhtC). This study firstly reports the characterization of pht operon in Gordonia sp. (C) 2015 Elsevier Ltd. All rights reserved.
