scholarly journals Diminished Exoproteome of Frankia spp. in Culture and Symbiosis

2009 ◽  
Vol 75 (21) ◽  
pp. 6721-6728 ◽  
Author(s):  
J. E. Mastronunzio ◽  
Y. Huang ◽  
D. R. Benson
Keyword(s):  
Root Nodules ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Hydrolytic Enzymes ◽  
Extracellular Proteins ◽  
Secreted Proteins ◽  
Signal Peptides ◽  
Cytoplasmic Proteins ◽  
Host Microbe Interactions ◽  
Strain Cci3

ABSTRACT Frankia species are the most geographically widespread gram-positive plant symbionts, carrying out N2 fixation in root nodules of trees and woody shrubs called actinorhizal plants. Taking advantage of the sequencing of three Frankia genomes, proteomics techniques were used to investigate the population of extracellular proteins (the exoproteome) from Frankia, some of which potentially mediate host-microbe interactions. Initial two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of culture supernatants indicated that cytoplasmic proteins appeared in supernatants as cells aged, likely because older hyphae lyse in this slow-growing filamentous actinomycete. Using liquid chromatography coupled to tandem mass spectrometry to identify peptides, 38 proteins were identified in the culture supernatant of Frankia sp. strain CcI3, but only three had predicted export signal peptides. In symbiotic cells, 42 signal peptide-containing proteins were detected from strain CcI3 in Casuarina cunninghamiana and Casuarina glauca root nodules, while 73 and 53 putative secreted proteins containing signal peptides were identified from Frankia strains in field-collected root nodules of Alnus incana and Elaeagnus angustifolia, respectively. Solute-binding proteins were the most commonly identified secreted proteins in symbiosis, particularly those predicted to bind branched-chain amino acids and peptides. These direct proteomics results complement a previous bioinformatics study that predicted few secreted hydrolytic enzymes in the Frankia proteome and provide direct evidence that the symbiosis succeeds partly, if not largely, because of a benign relationship.

scholarly journals Influence of Gemini Surfactants on Biochemical Profile and Ultrastructure of Aspergillus brasiliensis

2019 ◽  
Vol 9 (2) ◽  
pp. 245 ◽  
Author(s):  
Anna Koziróg ◽  
Anna Otlewska ◽  
Magdalena Gapińska ◽  
Sylwia Michlewska
Keyword(s):  
Gemini Surfactants ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Control Sample ◽  
Extracellular Proteins ◽  
Practical Applications ◽  
Aspergillus Brasiliensis ◽  
Cationic Gemini Surfactants ◽  
Wide Range ◽  
Cationic Compounds

In this study, we investigated the activities of hexamethylene-1,6-bis-(N,N-dimethyl-N-dodecylammonium bromide) (C6), pentamethylene-1,5-bis-(N,N-dimethyl-N-dodecylammonium bromide) (C5), and their two neutral analogues: hexamethylene-1,6-bis-(N-methyl-N-dodecylamine) (A6) and pentamethylene-1,5-bis-(N-methyl-N-dodecylamine) (A5) at concentrations of ½ MIC, MIC, and 2 MIC (minimal inhibitory concentration) against hyphal forms of Aspergillus brasiliensis ATCC 16404. Enzymatic profiles were determined using the API-ZYM system. Extracellular proteins were extracted from the mycelia and analyzed using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The ultrastructure was evaluated using a transmission electron microscope (TEM). Both groups of surfactants caused changes in the enzyme profiles. Larger changes in the number and concentration of enzymes were noted after the action of non-ionic gemini surfactants, which may have been due to the 100× higher concentration of neutral compounds. Larger differences between the protein profiles of the control sample and the biocide samples were observed following the use of cationic compounds. On the basis of TEM analyses, we found that, with increasing concentrations of compound C6, the mycelium cells gradually degraded. After treatment at 2 MIC, only membranous structures, multiform bodies, and dense electron pellets remained. Based on these results, we concluded that cationic gemini surfactants, in comparison with their non-ionic analogues, could have a wide range of practical applications as active compounds.

scholarly journals Biological Control of Fusarium Wilt of Cucumber by Chitinolytic Bacteria

1999 ◽  
Vol 89 (1) ◽  
pp. 92-99 ◽  
Author(s):  
Pushpinder Paul Singh ◽  
Yong Chul Shin ◽  
Chang Seuk Park ◽  
Young Ryun Chung
Keyword(s):  
Fusarium Wilt ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Hydrolytic Enzymes ◽  
Partial Purification ◽  
Streptomyces Sp ◽  
Chitinolytic Bacteria ◽  
Potting Medium ◽  
Paenibacillus Sp ◽  
Molecular Masses

Two chitinolytic bacterial strains, Paenibacillus sp. 300 and Streptomyces sp. 385, suppressed Fusarium wilt of cucumber (Cucumis sativus) caused by Fusarium oxysporum f. sp. cucumerinum in nonsterile, soilless potting medium. A mixture of the two strains in a ratio of 1:1 or 4:1 gave significantly (P < 0.05) better control of the disease than each of the strains used individually or than mixtures in other ratios. Several formulations were tested, and a zeolite-based, chitosan-amended formulation (ZAC) provided the best protection against the disease. Dose-response studies indicated that the threshold dose of 6 g of formulation per kilogram of potting medium was required for significant (P < 0.001) suppression of the disease. This dose was optimum for maintaining high rhizosphere population densities of chitinolytic bacteria (log 8.1 to log 9.3 CFU/g dry weight of potting medium), which were required for the control of Fusarium wilt. The ZAC formulation was suppressive when added to pathogen-infested medium 15 days before planting cucumber seeds. The formulation also provided good control when stored for 6 months at room temperature or at 4°C. Chitinase and β-1,3-glucanase enzymes were produced when the strains were grown in the presence of colloidal chitin as the sole carbon source. Partial purification of the chitinases, followed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and activity staining, revealed the presence of five bands with molecular masses of 65, 62, 59, 55, and 52 kDa in the case of Paenibacillus sp. 300; and three bands with molecular masses of 52, 38, and 33 kDa in the case of Streptomyces sp. 385. Incubation of cell walls of F. oxysporum f. sp. cucumerinum with partially purified enzyme fractions led to the release of N-acetyl-D-glucosamine (NAGA). NAGA content was considerably greater when pooled enzyme fractions (64 to 67) from Paenibacillus sp. were used, because they contained high β-1,3-glucanase activity in addition to chitinase activity. Suppression of Fusarium wilt of cucumber by a combination of these two bacteria may involve the action of these hydrolytic enzymes.

scholarly journals Hemolysin, Protease, and EPS Producing PathogenicAeromonas hydrophilaStrain An4 Shows Antibacterial Activity against Marine Bacterial Fish Pathogens

2010 ◽  
Vol 2010 ◽  
pp. 1-9 ◽  
Author(s):  
Anju Pandey ◽  
Milind Naik ◽  
Santosh Kumar Dubey
Keyword(s):  
Antibacterial Activity ◽  
Aeromonas Hydrophila ◽  
Pathogenic Bacteria ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Antibacterial Properties ◽  
Cell Extract ◽  
Extracellular Proteins ◽  
Butylated Hydroxytoluene ◽  
Crude Cell Extract

A pathogenicAeromonas hydrophilastrain An4 was isolated from marine catfish and characterized with reference to its proteolytic and hemolytic activity along with SDS-PAGE profile (sodium dodecyl sulphate-Polyacrylamide gel electrophoresis) of ECPs (extracellular proteins) showing hemolysin (approximately 50 kDa). Agar well diffusion assay using crude cell extract of the bacterial isolate clearly demonstrated antibacterial activity against indicator pathogenic bacteria,Staphylococcus arlettaestrain An1,Acinetobactersp. strain An2,Vibrio parahaemolyticusstrain An3, andAlteromonas aurentiaSE3 showing inhibitory zone >10 mm well comparable to common antibiotics. Further GC-MS analysis of crude cell extract revealed several metabolites, namely, phenolics, pyrrolo-pyrazines, pyrrolo-pyridine, and butylated hydroxytoluene (well-known antimicrobials). Characterization of EPS using FTIR indicated presence of several protein-related amine and amide groups along with peaks corresponding to carboxylic and phenyl rings which may be attributed to its virulent and antibacterial properties, respectively. Besides hemolysin, EPS, and protease,Aeromonas hydrophilastrain An4 also produced several antibacterial metabolites.

Application of metaproteomic analysis for studying extracellular polymeric substances (EPS) in activated sludge flocs and their fate in sludge digestion

2008 ◽  
Vol 57 (12) ◽  
pp. 2009-2015 ◽  
Author(s):  
C. Park ◽  
R. F. Helm
Keyword(s):  
Activated Sludge ◽  
Protein Separation ◽  
Extracellular Polymeric Substances ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Cation Exchange Resin ◽  
Methanosarcina Barkeri ◽  
Extracellular Proteins ◽  
Sludge Digestion ◽  
Sds Page

Metaproteomic analysis, comprising protein separation and identification, was applied to study extracellular proteins in activated sludges and to track their fate in sludge digestion under both anaerobic and aerobic conditions. The complex sludge proteins were first separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and further analysed by liquid chromatography tandem mass spectrometry (LC-MS/MS) to search their identification. Base extraction and cation exchange resin (CER) method were used to extract EPS from sludges at 0, 12 and 30 days of batch digestion. Several important observations were made during this study. Firstly, protein bands were well separated by extraction/SDS-PAGE protocol used in this study. Secondly, numerous protein bands remained after digestion, indicating that these proteins are not easily degradable in sludge digestion. Thirdly, protein bands detected following anaerobic and aerobic digestion differed, suggesting that proteins degraded in two different digestion environments are not the same. Finally, protein bands that emerged distinctively following anaerobic digestion was found to be subunits of methyl-coenzyme M reductase, the enzyme involved in methane generation, in Methanosarcina barkeri. These results demonstrated that metaproteomic investigation on activated sludge EPS is useful for studying floc formation in activated sludges and their degradation in various digestion environments.

scholarly journals Cymbopogon nardus Essential Oil as Protein Inducer in Bacillus subtilis ATCC21332

2018 ◽  
Vol 1 (1) ◽  
Author(s):  
Hairul Shahril Muhamad ◽  
Ismatul Nurul Asyikin Ismail ◽  
Nabilah Ahmad Alhadi ◽  
Salina Mat Radzi ◽  
Maryam Mohamed Rehan ◽  
...  
Keyword(s):  
Antimicrobial Activity ◽  
Bacillus Subtilis ◽  
Essential Oil ◽  
Protein Production ◽  
Antimicrobial Agents ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Extracellular Proteins ◽  
Specific Chemical ◽  
Cymbopogon Nardus

Protein production by bacteria might be increased in stressful conditions such as in the presence of antimicrobial agents. Many studies have proven that antibiotics or antimicrobial agents at low concentration are able to activate or repress gene transcription process in bacteria. However, there have been comparatively few studies on the potential of natural compounds in nature as a specific chemical signal that can trigger a variety of biological functions. An attempt was made to study the effect of essential oil from Cymbopogon nardus in regulating protein production by Bacillus subtilis ATCC21332. The bacterial cells were further exposed to the C. nardus essential oil at concentration of 0.02 % for 48 h at 37°C. The intracellular proteins were then isolated and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Proteins profile showed that a band with approximate size of 180 kDa appeared for the treated bacteria with C. nardus essential oil. An alignment of peptide sequences to the NCBI BLAST database revealed that B. subtilis ATCC21332 in stressful condition tend to produce intracellular protein recognized as respiratory nitrate reductase ? subunit enzyme. Besides, the extracellular proteins secreted by B. subtilis ATCC21332 after being subjected to 0.02% of C. nardus essential oil for 48 and 72 h at 30°C, were further analyzed on antimicrobial activity. The extracellular proteins secreted by B. subtilis ATCC21332 prior to enhancing with 0.02 % C. nardus essential oil at 30°C for 72 h exhibited antimicrobial activity towards two strains of bacteria, which are Bacillus cereus and Escherichia coli.

scholarly journals MPB70 and MPB83 as Indicators of Protein Localization in Mycobacterial Cells

1998 ◽  
Vol 66 (1) ◽  
pp. 289-296 ◽  
Author(s):  
Morten Harboe ◽  
Harald G. Wiker ◽  
Gunni Ulvund ◽  
Bent Lund-Pedersen ◽  
Åse Bengård Andersen ◽  
...  
Keyword(s):  
Protein Localization ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Culture Fluid ◽  
Secreted Proteins ◽  
Enzyme Linked Immunosorbent Assay ◽  
Sds Page ◽  
Shock Proteins ◽  
Triton X

ABSTRACT Culture fluids after growth of Mycobacterium bovis BCG on Sauton medium contain actively secreted proteins and proteins released by bacterial lysis. BCG culture fluids and sonicates ofMycobacterium tuberculosis and Mycobacterium paratuberculosis were tested after separation by gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The localization of marker proteins was determined by enzyme-linked immunosorbent assay and Western blotting with selected monoclonal antibodies of known specificities. Soluble secreted proteins (MPB64 and proteins of the antigen 85 complex) and three heat shock proteins (DnaK, GroEL, and GroES) were recovered in a single peak after gel filtration, indicating their occurrence as a free monomer in the culture fluid and cytosol, respectively. Other constituents eluted in two distinct peaks during gel filtration. The first peak corresponded to the void volume, indicating complex formation between several proteins or attachment to lipids in the surface layer or the cytoplasmic membrane; the second peak corresponded to the expected monomer size indicated by SDS-PAGE under conditions that separate proteins from each other during sample preparation. The two-peak group contained constituents with known lipid contents, the 19- and 38-kDa lipoproteins and lipoarabinomannan. The 26-kDa form of MPB83 behaved similarly. After extraction with Triton X-114, these constituents entered into the detergent phase, confirming the lipoprotein nature of 26-kDa MPB83. The MPB83 molecule was shown to be available on the surface of BCG Tokyo bacilli for reaction with monoclonal antibody MBS43 by flow cytometry.

scholarly journals COMPARISON OF EXTRACELLULAR SECRETION OF RECOMBINANT HUMAN EPIDERMAL GROWTH FACTOR USING TORA AND PELB SIGNAL PEPTIDES IN ESCHERICHIA COLI BL21 (DE3)

2019 ◽  
pp. 81-84 ◽  
Author(s):  
RIMA MELATI ◽  
ANNISA INDRIYANI ◽  
SHABARNI GAFFAR ◽  
SRIWIDODO ◽  
IMAN PERMANA MAKSUM
Keyword(s):  
Escherichia Coli ◽  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Signal Peptides ◽  
Extracellular Expression ◽  
E Coli ◽  
Human Epidermal Growth Factor ◽  
Epidermal Growth

Objective: The objective of this study was to evaluate two signal peptides (TorA and PelB), representing the most common secretion pathways in Escherichia coli, for their ability to secrete recombinant human epidermal growth factor (rhEGF) protein in the extracellular expression. Methods: E. coli BL21 (DE3) as the host cell to be transformed using recombinant plasmid pD881-TorA the consensus already containing hEGF gene and the signal peptide TorA or PelB, then expressed by L-rhamnose induction. rhEGF purified by heat treatment and ion-exchange chromatography. The hEGF protein was characterized using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and ELISA. Results: The result showed that PelB was secreting more hEGF protein compared to TorA with protein expression results of 48.2 μg/L and purification results of 0.360 μg/L, with a purity level of 83%. Conclusion: The results of this study explain in extracellular expression of hEGF protein in E. coli, PelB helps hEGF protein secretion to culture media better than TorA.

scholarly journals Role of interleukin-15 and interleukin-18 in the secretion of sIL-6R and sgp130 by human neutrophils

2003 ◽  
Vol 12 (3) ◽  
pp. 179-183 ◽  
Author(s):  
E. Jablonska ◽  
M. Marcinczyk
Keyword(s):  
Monoclonal Antibodies ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Human Neutrophils ◽  
Enzyme Linked Immunosorbent Assay ◽  
Cytoplasmic Proteins ◽  
Wide Range ◽  
Interleukin 15 ◽  
Culture Supernatants

Background:Available data indicate that neutrophils (PMN) produce a wide range of cytokines with the potential to modulate immune response. Recent investigation have shown that interleukin (IL)-15 and IL-18 potentiated several functions of normal neutrophils. It has been reported that IL-18-induced cytokine production may be significantly enhanced by coincident addition of IL-15.Aims:In the present study we compared the effect of recombinant human (rh)IL-15 and rhIL-18 as well as effect of a rhIL-15 and rhIL-18 combination on the induction secretion of sIL-6Rα and sgp130 by human neutrophils. Methods: PMN were isolated from heparinized whole blood of healthy persons. The PMN were cultured for 18 h at 37°C in a humidified incubator with 5% CO2. rhIL-15 and/or rhIL-18 and lipopolysaccharide were tested to PMN stimulation. The culture supernatants of PMN were removed and examined for the presence of sIL-6R and sgp130 by human enzyme-linked immunosorbent assay kits. Cytoplasmic protein fractions of PMN were analysed for the presence of sIL-6R and sgp130 by western blotting using monoclonal antibodies capable of detecting these proteins. Cells were lysed and cytoplasmic proteins were electrophoresed on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The resolved proteins were transferred onto nitrocellulose and incubated with the primary monoclonal antibodies anti-sIL-6R and anti-sgp130. The membranes were incubated at room temperature with alkaline phosphatase anti-mouse immunoglobulin G. Immunoreactive protein bans were visualized by an AP Conjugate Substrate Kit.Results and conclusion:The results of our investigation revealed that IL-15 alone, similarly to IL-18, has no significant ability for the regulation of both soluble IL-6 receptors, sIL-6R and sgp130, released by human neutrophils. It is interesting to note that the secretion of sgp130 was changed after PMN stimulation with rhIL-15 in the presence of rhIL-18. The combination of rhIL-15 and rhIL-18 was shown to induce PMN to secretion relatively higher amounts of sgp130 compared with the stimulation of PMN with rhIL-15 alone and rhIL-18 alone. The results obtained suggest that IL-15 and IL-18, belonging to the inflammatory cytokines, through the regulation of sgp130 secretion must be also considered as anti-inflammatory mediators that may influence the balance reactions mediated by the IL-6 cytokine family.

scholarly journals Lectin-Like Glycoprotein PsNLEC-1 Is Not Correctly Glycosylated and Targeted in Boron-Deficient Pea Nodules

2001 ◽  
Vol 14 (5) ◽  
pp. 663-670 ◽  
Author(s):  
Luis Bolaños ◽  
Arancha Cebrián ◽  
Miguel Redondo-Nieto ◽  
Rafael Rivilla ◽  
Ildefonso Bonilla
Keyword(s):  
Escherichia Coli ◽  
Gel Electrophoresis ◽  
Root Nodules ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Host Cells ◽  
Immunogold Localization ◽  
Electrophoretic Mobilities ◽  
Cytoplasmic Vesicles ◽  
Infected Cells

Symbiosome development was studied in pea root nodules from plants growing in the absence of boron (B). Rhizobia released into the host cells of nodules from B-deficient plants developed to abnormal endophytic forms with an altered electrophoretic lipopolysaccharide pattern. Immunostaining after sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electroblotting of nodule homogenates with antibodies that recognize glycoprotein components showed that two previously described lectin-like glycoproteins (PsNLEC-1A and PsNLEC-1B) did not harbor the carbohydrate epitope normally recognized by specific monoclonal antibodies. Material derived from B-deficient nodules, however, still contained three antigenic isoforms with similar electrophoretic mobilities to PsNLEC-1 isoforms A, B, and C. These could be detected following immunoblotting and immunostaining with a specific antiserum originating from the purified PsNLEC protein that had been heterologously expressed in Escherichia coli. Immunogold localization of PsNLEC-1 sugar epitopes in B-deficient nodules showed that they were associated mostly with cytoplasmic vesicles rather than normal localization in the symbiosome compartment of mature infected cells. These results suggest that a modification of the glycosyl-moieties of PsNLEC-1 and an alteration of vesicle targeting occur during the development of pea nodules in the absence of B, and that these changes are associated with the development of aberrant nonfunctional symbiosomes.

scholarly journals The Role of Subinhibitory Concentrations of Daptomycin and Tigecycline in Modulating Virulence in Staphylococcus aureus

Antibiotics ◽  
2021 ◽  
Vol 10 (1) ◽  
pp. 39
Author(s):  
Salman Sahab Atshan ◽  
Rukman Awang Hamat ◽  
Marco J. L. Coolen ◽  
Gary Dykes ◽  
Zamberi Sekawi ◽  
...  
Keyword(s):  
Gene Expression ◽  
Staphylococcus Aureus ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Extracellular Proteins ◽  
Clinical Isolates ◽  
Sds Page ◽  
Strain Type ◽  
2D Gel ◽  
Culture Supernatants

Staphylococcus aureus (S. aureus) infections are notoriously complicated by the ability of the organism to grow in biofilms and are difficult to eradicate with antimicrobial therapy. The purpose of the current study was to clarify the influence of sub-inhibitory concentrations (sub-MICs) of daptomycin and tigecycline antibiotics on biofilm adhesion factors and exoproteins expressions by S. aureus clinical isolates. Six clinical isolates representing positive biofilm S. aureus clones (3 methicillin-sensitive S. aureus (MSSA) and 3 methicillin-resistant S. aureus (MRSA)) were grown with sub-MICs (0.5 MIC) of two antibiotics (daptomycin and tigecycline) for 12 h of incubation. RNA extracted from culture pellets was used via relative quantitative real-time-PCR (qRT-PCR) to determine expression of specific adhesion (fnbA, fnbB, clfA, clfB, fib, ebps, cna, eno) and biofilm (icaADBC) genes. To examine the effect of sub-MIC of these antibiotics on the expression of extracellular proteins, samples from the culture supernatants of six isolates were collected after 12 h of treatment with or without tigecycline in order to profile protein production via 2D gel sodium dodecyl sulfate-polyacrylamide gel electrophoresis (2D gel-SDS-PAGE). Sub-MIC treatment of all clinical MRSA and MSSA strains with daptomycin or tigecycline dramatically induced or suppressed fnbA, fnbB, clfA, clfB, fib, ebps, cna, eno, and icaADBC gene expression. Furthermore, sub-MIC use of tigecycline significantly reduced the total number of separated protein spots across all the isolates, as well as decreasing production of certain individual proteins. Collectively, this study showed very different responses in terms of both gene expression and protein secretion across the various isolates. In addition, our results suggest that sub-MIC usage of daptomycin and tigecycline could signal virulence induction by S. aureus via the regulation of biofilm adhesion factor genes and exoproteins. If translating findings to the clinical treatment of S. aureus, the therapeutic regimen should be adapted depending on antibiotic, the virulence factor and strain type.

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