Identification of a Broad-Spectrum Peptidoglycan Hydrolase Associated with the Particle of Xanthomonas oryzae Phage Xop411

2018 ◽  
Vol 28 (2) ◽  
pp. 78-86
Author(s):  
Shu-Fen Weng ◽  
Yung-Chieh Fu ◽  
Juey-Wen Lin ◽  
Tsai-Tien Tseng
Keyword(s):  
Western Blotting ◽  
Inclusion Bodies ◽  
Protein S ◽  
Bioinformatic Analysis ◽  
Immunogold Labelling ◽  
Xanthomonas Oryzae ◽  
Opportunistic Bacteria ◽  
Peptidoglycan Hydrolases ◽  
Related Plant ◽  
Zymogram Assay

Virion-associated peptidoglycan hydrolases (VAPGH) in bacteriophages are potential antimicrobials. Xop411 is a syphophage infecting the Gram-negative <i>Xanthomonas oryzae</i> pv. oryzae that causes bacterial leaf blight in rice plants. The Xop411 gp21 protein was identified here as a peptidoglycan glycohydrolase by Western blotting and zymogram assay, and localized to the phage tail by immunogold-labelling electron microscopy. This protein showed an apparent molecular mass of 17 kDa in SDS-polyacrylamide gels, larger than that calculated from the amino acid sequence, 15 kDa with 130 residues. The recombinant gp21 expressed in <i>Escherichia coli</i> formed inclusion bodies, which gained enzyme activity after in-gel renaturation. In contrast, the secreted recombinant protein (s-gp21His) expressed in <i>Pichia pastoris</i> was soluble and enzymatically active. Plate assays showed that s-gp21His was capable of killing 3 species of <i>Xanthomonas</i>, a genus containing 27 closely related plant pathogenic species, as well as the opportunistic <i>Pseudomonas aeruginosa</i> and <i>Stenotrophomonas maltophilia</i> causing nosocomial infections. These results indicate that the Xop411 gp21 has possible wide applications as an antimicrobial against xanthomonads and at least 2 opportunistic bacteria. Several other VAPGH from <i>Xanthomonas</i> phages were also identified by bioinformatic analysis, with 1 being confirmed by Western blotting.

scholarly journals Characteristic Dissection of Xanthomonas oryzae pv. oryzae Responsive MicroRNAs in Rice

2020 ◽  
Vol 21 (3) ◽  
pp. 785
Author(s):  
Yanfeng Jia ◽  
Chunrong Li ◽  
Quanlin Li ◽  
Pengcheng Liu ◽  
Dongfeng Liu ◽  
...  
Keyword(s):  
Rice Variety ◽  
Bioinformatic Analysis ◽  
Infection Process ◽  
Xanthomonas Oryzae ◽  
Differentially Expressed ◽  
Small Rna Sequencing ◽  
Sequencing Data ◽  
Stem Loop ◽  
Differentially Expressed Mirnas ◽  
Plant Pathogen Interaction

MicroRNAs (miRNAs) are crucial player in plant-pathogen interaction. While the evidence has demonstrated that rice miRNAs mediate immune response to pathogens invasion, the roles of miRNAs on Xanthomonas oryzae pv. oryzae (Xoo) attack remain be in place. Herein, we monitored the responsive changes of rice miRNAs at 0, 8, 24 h across Xoo strain PXO86 infection in its compatible rice variety IR24 and incompatible variety IRBB5 by small RNA sequencing, and the genes targeted by miRNAs were also detected via degradome technology. The faithfulness of sequencing data was validated through quantitative real-time stem-loop reverse transcription-polymerase chain reaction assay. Bioinformatic analysis showed that the differentially expressed miRNAs could be divided into three immunity-related clusters, and 80 regulatory units were emerged in infection process, which comprises 29 differentially expressed known miRNAs and 38 cleaved targets. Furthermore, the miRNA presumptive function of separate immunity cluster in rice-Xoo interplay was confirmed through overexpressing osa-miR164a, osa-miR167d and osa-miR159b, and the disruption of regulatory units, osa-miR164a/OsNAC60, osa-miR167d-5p/OsWD40-174 and osa-miR159b/OsMYBGA, OsLRR-RLK2, OsMPK20-4, may reset rice defense response to Xoo infestation in a controllable manner. These findings provide new insights into the complex roles of characteristic miRNAs and their targets in rice-Xoo interactions.

scholarly journals Mutations in the Predicted Active Site of Xanthomonas oryzae pv. oryzae XopQ Differentially Affect Virulence, Suppression of Host Innate Immunity, and Induction of the HR in a Nonhost Plant

2015 ◽  
Vol 28 (2) ◽  
pp. 195-206 ◽  
Author(s):  
Mahesh Kumar Gupta ◽  
Rajkanwar Nathawat ◽  
Dipanwita Sinha ◽  
Asfarul S. Haque ◽  
Rajan Sankaranarayanan ◽  
...  
Keyword(s):  
Innate Immunity ◽  
Active Site ◽  
Plant Cell Wall ◽  
Structural Model ◽  
Bioinformatic Analysis ◽  
Xanthomonas Oryzae ◽  
Active Site Residues ◽  
Callose Deposition ◽  
Nonhost Plant ◽  
Bacterial Blight Pathogen

Xanthomonas oryzae pv. oryzae, the bacterial blight pathogen of rice, secretes a number of effectors through a type 3 secretion system. One of these effectors, called XopQ, is required for virulence and suppression of rice innate immune responses induced by the plant cell-wall-degrading enzyme lipase/esterase A (LipA). Bioinformatic analysis suggested that XopQ is homologous to inosine-uridine nucleoside hydrolases (NH). A structural model of XopQ with the protozoan Crithidia fasciculata purine NH suggested that D116 and Y279 are potential active site residues. X. oryzae pv. oryzae xopQ mutants (xopQ−/pHM1::xopQD116A and xopQ−/pHM1::xopQY279A) show reduced virulence on rice compared with xopQ−/pHM1::xopQ. The two predicted XopQ active site mutants (xopQ−/pHM1::xopQD116A and xopQ−/pHM1::xopQY279A) exhibit a reduced hypersensitive response (HR) on Nicotiana benthamiana, a nonhost. However, Arabidopsis lines expressing either xopQ or xopQY279A are equally proficient at suppression of LipA-induced callose deposition. Purified XopQ does not show NH activity on standard nucleoside substrates but exhibits ribose hydrolase activity on the nucleoside substrate analogue 4-nitrophenyl β-D-ribofuranoside. The D116A and Y279A mutations cause a reduction in biochemical activity. These results indicate that mutations in the predicted active site of XopQ affect virulence and induction of the HR but do not affect suppression of innate immunity.

scholarly journals ACTB and GAPDH proteins appear at multiple positions of SDS-PAGE and may not be suitable for serving as reference genes for the protein level determination in such techniques as Western blotting

2020 ◽  
Author(s):  
Yan He ◽  
Ju Zhang ◽  
Jiayuan Qu ◽  
Lucas Zellmer ◽  
Yan Zhao ◽  
...  
Keyword(s):  
Western Blotting ◽  
Protein Level ◽  
Reference Genes ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Bioinformatic Analysis ◽  
Protein Isoforms ◽  
Leading Role ◽  
Non Coding Rna ◽  
Sds Page

AbstractMost human genes can produce multiple protein isoforms that should appear at multiple positions of polyacrylamide gel electrophoresis (PAGE) with sodium dodecyl sulfate (SDS), but most published results of Western blotting show only one protein. We performed SDS-PAGE of proteins from several human cell lines, isolated the proteins at the 72-, 55-, 48-, 40-, and 26-kD positions, and used liquid chromatography and tandem mass spectrometry (LC-MS/MS) to determine the protein identities. Although ACTB and GAPDH are 41.7-kD and 36-kD proteins, respectively, LC-MS/MS identified peptides of ACTB and GAPDH at all of these SDS-PAGE positions, making us wonder whether they produce some unknown protein isoforms. The NCBI (National Center for Biotechnology Information, USA) database lists only one ACTB mRNA but five GAPDH mRNAs and one non-coding RNA. The five GAPDH mRNAs encode three protein isoforms, while our bioinformatic analysis identified a 17.6-kD isoform encoded by the non-coding RNA. All LC-MS/MS-identified GAPDH peptides at all positions studied are unique, but some of the identified ACTB peptides are shared by ACTC1, ACTBL2, POTEF, POTEE, POTEI, and POTEJ. ACTC1 and ACTBL2 belong to the ACT family with great similarities to ACTB in protein sequence, whereas the four POTEs are ACTB-containing chimeric genes with the C-terminus of their proteins highly similar to ACTB. These data collectively disqualify GAPDH and ACTB from serving as the reference genes for determination of the protein level in such techniques as Western blotting, a leading role these two genes have been playing for decades in the biomedical research.

scholarly journals First evidence showing that Pepper vein yellows virus P4 protein is a movement protein

2020 ◽  
Author(s):  
Sangsang Li ◽  
Xianyang Su ◽  
Xiangwen Luo ◽  
Yu Zhang ◽  
Deyong Zhang ◽  
...  
Keyword(s):  
Transmembrane Domain ◽  
Movement Protein ◽  
Protein S ◽  
Bioinformatic Analysis ◽  
Plant Viruses ◽  
Specific Protein ◽  
Virus Movement ◽  
Genomic Rnas ◽  
Pepper Vein Yellows Virus ◽  
Substitution Mutant

Abstract Background Plant viruses move through plasmodesmata (PD) to infect new cells. To overcome the PD barrier, plant viruses have developed specific protein(s) to guide their genomic RNAs or DNAs to path through the PD. Results In the present study, we analyzed the function of Pepper vein yellows virus P4 protein. Our bioinformatic analysis showed that the P4 protein contains an transmembrane domain, encompassing the amino acid residue 117-138. The P4 protein was found to target PD and form small punctates near walls. The P4 deletion mutant or the substitution mutant lost their function to produce punctates near the walls inside the fluorescent loci. The P4-YFP fusion was found to move from cell to cell in infiltrated leaves, and P4 could complement Cucumber mosaic virus movement protein deficiency mutant to move between cells. Conclustion Taking together, we consider that the P4 protein is a movement protein of Pepper vein yellows virus.

Rabbit Plasma, unlike Its Human Counterpart, Contains no Complex between Protein S and C4b-Binding Protein

1994 ◽  
Vol 71 (04) ◽  
pp. 446-451 ◽  
Author(s):  
Xuhua He ◽  
Björn Dahlbäck
Keyword(s):  
Molecular Weight ◽  
Monoclonal Antibody ◽  
Complex Formation ◽  
Human Plasma ◽  
High Molecular Weight ◽  
Western Blotting ◽  
Vitamin K ◽  
Protein S ◽  
Rabbit Plasma ◽  
Free Protein

SummaryIn human plasma, the anticoagulant vitamin K-dependent protein S exists in two molecular forms, as free protein and complexed to C4b- binding protein (C4BP), a complement regulatory protein. It has been suggested that rabbit plasma also contains two forms of protein S and that the interaction between protein S and C4BP m rabbits can be modulated by synthetic peptides corresponding to a sequence (residues 605-614) in the carboxy-lerminal part of protein S. In this report, we provide itsulls which challenge the conclusion that rabbit plasma contains the complexed form of protein S. The two forms of protein S in human plasma were separated by gel filtration chromatography on Sephacryl S-300 and the presence of protein S in the various fractions analyzed by Western blotting using a monoclonal antibody (HPS 21) directed against the γ-carboxyglutamic acid rich module of human protein S. This antibody, which was found to cross-react with rabbit protein S on Western blotting, was used in affinity purification of protein S from rabbit plasma as well as of recombinant rabbit protein S. HPS 21 specifically recognized protein S in rabbit plasma and did not cross-react with the other vitamin K-depeudenl plasma proteins. To elucidate whether rabbit plasma contained two forms of protein S, rabbit plasma was subjected to gelfiltration chromatography followed by Western blotting of the fractions with monoclonal antibody HPS 21. Protein S was found only in fractions eluting at a position corresponding to that of free protein S. A radiolabelled trace amount of recombinant rabbit protein S added to rabbit plasma chromatographed as free protein S and no high molecular weight form corresponding to a C4BP-protein S complex was detected. Rabbit protein S had the capacity to bind human C4BP and the addition of human C4BP to rabbit plasma changed the elution profile, of rabbit plasma protein S. After the addition of human C4BP, rabbit plasma protein S quantitatively eluted as a high molecular weight complex, suggesting that all the protein S in rabbit plasma was bound to human C4BP. The anticoagulant activity of human protein S is modulated by the complex formation with C4BP. Our results demonstrate that this function of C4BP and the protein S-C4BP complex formation has not been conserved throughout the evolution even though protein S has a preserved C4BP binding site.

MicroRNAs 155, -221 and -222 Control Megakaryopoiesis at Progenitor and Precursor Level through Ets-1 Multitargeting.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1187-1187
Author(s):  
Giovanna Marziali ◽  
Valentina Lulli ◽  
Simona Coppola ◽  
Paolo Romania ◽  
Laura Fontana ◽  
...  
Keyword(s):  
Expression Profiles ◽  
Direct Interaction ◽  
Protein S ◽  
Firefly Luciferase ◽  
Bioinformatic Analysis ◽  
Luciferase Assay ◽  
Hematopoietic Progenitor Cell ◽  
Functional Studies ◽  
Cell Proliferation And Differentiation ◽  
Enhanced Expression

Abstract It is generally accepted that microRNAs (miRs) control basic biological functions, such as cell proliferation and differentiation. However, only few targets of the ~ 300 known mammalian miRs have been validated so far, thus hampering delineation of miR-based control circuitries. Particularly, little is known on miR function in mammalian hematopoiesis. We have investigated by microarray and Northern blot evalutation miR expression profiles in human megakarypoiesis, as evaluated in cord blood hematopoietic progenitor cell (HPC) unilineage culture through the megakaryopoietic (MK) differentiation-maturation pathway. These studies indicated that miR-155, -221 and -222 are abundant in HPCs, but sharply decline during megakaryopoiesis. The decline may favour megakaryopoiesis by unblocking translation of key functional target protein(s). Bioinformatic analysis predicted that miR-155, -221 and -222 target Ets-1, a transcription factor up-regulated in Mk differentiation, which transactivates relevant Mk genes (e.g., TPO receptor, PF4, CD42 and von Willebrand factor). Consistently, in megakaryocytic cells the increase of Ets-1 protein expression coincides with the miR-155, -221 and -222 decrease. To find out whether Ets-1 mRNA is a possible target of miR-155, -221 and -222, we cloned segments of the 3′UTR of the Ets-1 gene downstream of a firefly luciferase ORF. Luciferase assay confirmed a direct interaction between each of these miRs and the 3′UTR of Ets-1. Functional studies showed that enhanced expression of these three miRs impairs proliferation, differentiation and maturation of MK cells, at least in part via enhanced degradation of Ets1 mRNA and down-modulation of Ets-1 protein. Similar results were obtained by RNA interference against Ets-1. Finally, HPCs transfected with miR-155, -221, and -222 showed a significant reduction of their Mk clonogenic capacity, suggesting that down-modulation of these miRs favours MK progenitor recruitment and commitment. Altogether, these studies indicate that a novel regulatory circuitry, based on miR-155, -221, -222 multitargeting Ets-1 mRNA leading in turn to transactivation of Mk-specific genes, plays a key role in the control of megakaryopoiesis at both progenitor and precursor level.

scholarly journals Identification and Characterization of a Novel Gene, hshB, in Xanthomonas oryzae pv. oryzicola Co-Regulated by Quorum Sensing and clp

2012 ◽  
Vol 102 (3) ◽  
pp. 252-259 ◽  
Author(s):  
Yancun Zhao ◽  
Guoliang Qian ◽  
Jiaqin Fan ◽  
Fangqun Yin ◽  
Yijin Zhou ◽  
...  
Keyword(s):  
Quorum Sensing ◽  
Extracellular Polysaccharide ◽  
Bioinformatic Analysis ◽  
Xanthomonas Oryzae ◽  
Pcr Analysis ◽  
Homologous Gene ◽  
Upstream Gene ◽  
Novel Gene ◽  
Putative Signal Peptide ◽  
Diffusible Signal Factor

Virulence factors of Xanthomonas oryzae pv. oryzicola, the causal agent of bacterial leaf streak in rice, are regulated by a diffusible signal factor (DSF)-dependent quorum-sensing (QS) system. In this study, a novel pathogenicity-related gene, Xoryp_010100018570 (named hshB), of X. oryzae pv. oryzicola was characterized. hshB encodes a hydrolase with a putative signal peptide, which is a homolog of imidazolonepropionase. Bioinformatic analysis showed that hshB is relatively conserved in the genus Xanthomonas but the homologous gene of hshB was not found in X. oryzae pv. oryzae. Reverse-transcription polymerase chain reaction (PCR) analysis showed that hshB and its upstream gene, Xoryp_010100018565 (named hshA), are co-transcribed in X. oryzae pv. oryzicola. Subsequent experimental results indicated that mutation of hshB remarkably impaired the virulence, extracellular protease activity, extracellular polysaccharide production, growth in minimal medium, and resistance to oxidative stress and bismerthiazol of X. oryzae pv. oryzicola. Mutation of clp, encoding a global regulator, resulted in similar phenotypes. Real-time PCR assays showed that hshB transcription is positively regulated by clp and DSF, and induced by poor nutrition. Our study not only found a novel gene hshB regulated by DSF-dependent QS system and clp but also showed that hshB was required for virulence of X. oryzae pv. oryzicola.

scholarly journals First evidence showing that Pepper vein yellows virus P4 protein is a movement protein

2020 ◽  
Author(s):  
Sangsang Li ◽  
Xianyang Su ◽  
Xiangwen Luo ◽  
Yu Zhang ◽  
Deyong Zhang ◽  
...  
Keyword(s):  
Transmembrane Domain ◽  
Movement Protein ◽  
Protein S ◽  
Bioinformatic Analysis ◽  
Plant Viruses ◽  
Specific Protein ◽  
Virus Movement ◽  
Genomic Rnas ◽  
Pepper Vein Yellows Virus ◽  
Substitution Mutant

Abstract Background Plant viruses move through plasmodesmata (PD) to infect new cells. To overcome the PD barrier, plant viruses have developed specific protein(s) to guide their genomic RNAs or DNAs to path through the PD. Results In the present study, we analyzed the function of Pepper vein yellows virus P4 protein. Our bioinformatic analysis showed that the P4 protein contains an transmembrane domain, encompassing the amino acid residue 117-138. The P4 protein was found to target PD and form small punctates near walls. The P4 deletion mutant or the substitution mutant lost their function to produce punctates near the walls inside the fluorescent loci. The P4-YFP fusion was found to move from cell to cell in infiltrated leaves, and P4 could complement Cucumber mosaic virus movement protein deficiency mutant to move between cells. Conclustion Taking together, we consider that the P4 protein is a movement protein of Pepper vein yellows virus.

The ordered macromolecular surface of polyester inclusion bodies inPseudomonas oleovotans

1995 ◽  
Vol 41 (13) ◽  
pp. 84-93 ◽  
Author(s):  
Elizabeth S. Stuart ◽  
R. Clinton Fuller ◽  
Robert W. Lenz
Keyword(s):  
Inclusion Bodies ◽  
Amorphous State ◽  
Immunogold Labelling ◽  
Boundary Region ◽  
Pseudomonas Oleovorans ◽  
Sds Page ◽  
Degradative Enzymes ◽  
Layer Chromatography ◽  
Intracellular Inclusion

Intracellular inclusion bodies of poly(β-hydroxyalkanoates) (PHAs) have been studied in various microorganisms since Lemoigne's discovery of PHAs in 1925. Recently, the research in several laboratories, including our own, has addressed the role of proteins, lipids, and water associated with these accumulations. Our research has examined the boundary of polymer inclusion bodies. Electron microscopy demonstrated that the polymer is encompassed by two paracrystalline arrays. SDS-PAGE, Western blot, or immunogold labelling demonstrated that both contain a 43-kDa protein as a major component. Immunogold labelling also demonstrated that 55- and 59-kDa proteins are located, exclusively, on the array associated with the accumulating polymer. Results from microelemental analysis and preliminary thin-layer chromatography of released lipids were consistent with the suggestion that phospholipids also had a role in this organized assembly. A model has been suggested, aimed at focusing attention on this organized boundary region. It is consistent with maintenance of the amorphous state of the polymer both intracellularly and after isolation, provides sites for biosynthetic and degradative enzymes, and accounts for the polyester, protein, and lipid components known to be present. Interestingly, the anti-43-kDa antibody also recognized a 43-kDa species released from the outer surface of this microbe. The research presented here and the model developed from it, suggest that microbial synthesis, containment, and degradation of polyester are carried out in association with a highly organized and complex intracellular assembly that may provide, within the bacterial cytosol, a unique microenvironment for biochemical activities.Key words: polyester, inclusion granule, Pseudomonas oleovorans.

scholarly journals Formononetin Suppresses Interleukin-6 Production in Lipopolysaccharide-Stimulated Macrophages Via Activation of the AMP-Activated Protein Kinase

2020 ◽  
Author(s):  
Lanfang Zhang ◽  
Xiaoyan Zhang ◽  
Liying Wang ◽  
Wanxia Yang ◽  
Yuanbiao Qiao ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Western Blotting ◽  
Interleukin 6 ◽  
Kinase Inhibitor ◽  
Herbal Medicines ◽  
Bioinformatic Analysis ◽  
Immunofluorescence Assay ◽  
Nuclear Staining ◽  
Ampk Phosphorylation ◽  
Amp Activated Protein Kinase

Abstract Background: Interleukin-6 (IL-6), an inflammatory cytokine, plays a majoy role in the pathogenesis of inflammation and serves as a key marker in the diagnosis and treatment of inflammation and related diseases. Formononetin (FMN), an isoflavone ingredient in various Chinese herbal medicines, inhibits the production of IL-6 in some cells. However, its mechanism of action has not been clearly established. In this study, we aimed to identify whether FMN inhibits IL-6 production in Lipopolysaccharide (LPS)-stimulated macrophages via AMPK activation.Methods: In the current study, the anti-IL-6 mechanism of FMN was evaluated using a macrophage model with LPS stimulation. An inverted microscope was utilized to obtain images of cells. Nuclear staining assay and CCK-8 assay were used to identify the viability of ANA-1 cells. The expression of IL-6 in cells was investigated by Enzyme-linked immunosorbent (ELISA) and Western blotting. The expression of AMP-activated protein kinase (AMPK) was determined by Western blotting and phosphorylation of AMPK was determined by Western blotting and Immunofluorescence assay. Bioinformatic analysis was used to predict potential targets of FMN. Results: we found that FMN reduced the expression of IL-6 in ANA-1 cells and increased the phosphorylation of AMPK. The effect of FMN was similar to that of acadesine, an AMPK activator, which also reduced IL-6 expression in LPS-induced ANA-1 cells and increased AMPK phosphorylation. Its combination with dorsomorphin (an AMPK inhibitor), however, reversed the effects of FMN on AMPK phosphorylation and IL-6 expression. The target of FMN was identified as the cAMP-dependent protein kinase inhibitor alpha, as searched in Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform and further verified with the STRING database. Conclusions: In conclusion, our findings suggest that FMN effectively inhibits IL-6 production by activating AMPK in LPS-stimulated macrophages.

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