scholarly journals Comparative Analysis of Extracellular and Intracellular Proteomes of Listeria monocytogenes Strains Reveals a Correlation between Protein Expression and Serovar

2008 ◽  
Vol 74 (23) ◽  
pp. 7399-7409 ◽  
Author(s):  
Emilie Dumas ◽  
Bruno Meunier ◽  
Jean-Louis Berdagué ◽  
Christophe Chambon ◽  
Mickaël Desvaux ◽  
...  
Keyword(s):  
Listeria Monocytogenes ◽  
Peptide Mass Fingerprinting ◽  
Etiologic Agent ◽  
Extracellular Proteins ◽  
Health Concern ◽  
Two Dimensional Gel Electrophoresis ◽  
Extracellular Proteome ◽  
Flight Mass Spectrometry ◽  
Peptide Mass ◽  
Phylogenetic Lineages

ABSTRACT Listeria monocytogenes, the etiologic agent of listeriosis, remains a serious public health concern, with its frequent occurrence in food environments coupled with a high mortality rate. Among the 13 serovars, human listeriosis is mostly associated with the serovar 4b, 1/2b, and 1/2a strains. To investigate the diversity of L. monocytogenes, the intracellular and extracellular proteins of 12 strains were analyzed by two-dimensional gel electrophoresis. These strains had different origins, belonged to different serovars (4b, 1/2a, and 1/2b), and presented with different levels of virulence in chicken embryos. The clustering of the strains in two groups based on proteomic patterns is in agreement with the L. monocytogenes phylogenetic lineages. Statistical analysis did not allow for identification of proteins specific to the isolate origin or the virulence level of the strains, but 26 and 21 protein spots were shown to be significantly overexpressed and underexpressed, respectively, in the six strains of serovar 1/2a (lineage II) compared to strains of serovar 1/2b or 4b. Moreover, a penicillin-binding protein was specific for serovar 1/2b and two protein spots identified as a serine protease were specific to serovar 4b. These protein spots, identified through peptide mass fingerprinting using matrix-assisted laser desorption ionization-time of flight mass spectrometry, were essentially found in the extracellular proteome and may have uses as potential markers for serotyping and risk analysis.

scholarly journals The surface (S)-layer gene cspB of Corynebacterium glutamicum is transcriptionally activated by a LuxR-type regulator and located on a 6 kb genomic island absent from the type strain ATCC 13032

Microbiology ◽  
2006 ◽  
Vol 152 (4) ◽  
pp. 923-935 ◽  
Author(s):  
Nicole Hansmeier ◽  
Andreas Albersmeier ◽  
Andreas Tauch ◽  
Thomas Damberg ◽  
Robert Ros ◽  
...  
Keyword(s):  
Corynebacterium Glutamicum ◽  
Affinity Purification ◽  
Regulatory Protein ◽  
Direct Repeat ◽  
Peptide Mass Fingerprinting ◽  
Gene Encoding ◽  
Force Microscopy ◽  
Flight Mass Spectrometry ◽  
Peptide Mass ◽  
Rapid Amplification

The surface (S)-layer gene region of the Gram-positive bacterium Corynebacterium glutamicum ATCC 14067 was identified on fosmid clones, sequenced and compared with the genome sequence of C. glutamicum ATCC 13032, whose cell surface is devoid of an ordered S-layer lattice. A 5·97 kb DNA region that is absent from the C. glutamicum ATCC 13032 chromosome was identified. This region includes cspB, the structural gene encoding the S-layer protomer PS2, and six additional coding sequences. PCR experiments demonstrated that the respective DNA region is conserved in different C. glutamicum wild-type strains capable of S-layer formation. The DNA region is flanked by a 7 bp direct repeat, suggesting that illegitimate recombination might be responsible for gene loss in C. glutamicum ATCC 13032. Transfer of the cloned cspB gene restored the PS2− phenotype of C. glutamicum ATCC 13032, as confirmed by visualization of the PS2 proteins by SDS-PAGE and imaging of ordered hexagonal S-layer lattices on living C. glutamicum cells by atomic force microscopy. Furthermore, the promoter of the cspB gene was mapped by 5′ rapid amplification of cDNA ends PCR and the corresponding DNA fragment was used in DNA affinity purification assays. A 30 kDa protein specifically binding to the promoter region of the cspB gene was purified. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry and peptide mass fingerprinting of the purified protein led to the identification of the putative transcriptional regulator Cg2831, belonging to the LuxR regulatory protein family. Disruption of the cg2831 gene in C. glutamicum resulted in an almost complete loss of PS2 synthesis. These results suggested that Cg2831 is a transcriptional activator of cspB gene expression in C. glutamicum.

Identification of Sulfur Activation Relevant Protein Genes of Extremely Thermophilic Acidianus manzaensis

2017 ◽  
Vol 262 ◽  
pp. 461-465 ◽  
Author(s):  
Hong Chang Liu ◽  
Jin Lan Xia ◽  
Zhen Yuan Nie ◽  
Ya Long Ma ◽  
Yun Yang ◽  
...  
Keyword(s):  
Gel Electrophoresis ◽  
Elemental Sulfur ◽  
Extracellular Proteins ◽  
Laser Desorption Ionization ◽  
Two Dimensional Gel Electrophoresis ◽  
Ionization Time ◽  
Real Time Quantitative Pcr ◽  
Flight Mass Spectrometry ◽  
Genes Encoding ◽  
Acidianus Manzaensis

The sulfur activation by extracellular proteins is considered as the crucial stage during biooxidation of elemental sulfur (S0). In order to study genes encoding sulfur-activation related extracellular proteins of extremely thermophilic Acidianus manzaensis, the extracellular proteins with higher abundance for the strain grown on S0 allotropes than that on Fe2+ were first screened by two-dimensional gel electrophoresis, and then identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Nine genes amplified with PCR were satisfactory according to their agarose gel electrophoresis. The differential expression of these nine genes when the strain grown on S0 allotropes and Fe2+ were analyzed with real-time quantitative PCR (RT-qPCR). Results showed that seven of them were higher expressed when the strain grown on S0 allotropes than on Fe2+, indicating they may be related with sulfur activation by A. manzaensis.

scholarly journals Hypoxic Response of Mycobacterium tuberculosis Studied by Metabolic Labeling and Proteome Analysis of Cellular and Extracellular Proteins

2002 ◽  
Vol 184 (13) ◽  
pp. 3485-3491 ◽  
Author(s):  
Ida Rosenkrands ◽  
Richard A. Slayden ◽  
Janne Crawford ◽  
Claus Aagaard ◽  
Clifton E. Barry ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Proteome Analysis ◽  
Peptide Mass Fingerprinting ◽  
Extracellular Proteins ◽  
Metabolic Labeling ◽  
Low Oxygen ◽  
Hypoxic Response ◽  
Peptide Mass ◽  
Oxygen Conditions

ABSTRACT The events involved in the establishment of a latent infection with Mycobacterium tuberculosis are not fully understood, but hypoxic conditions are generally believed to be the environment encountered by the pathogen in the central part of the granuloma. The present study was undertaken to provide insight into M. tuberculosis protein expression in in vitro latency models where oxygen is depleted. The response of M. tuberculosis to low-oxygen conditions was investigated in both cellular and extracellular proteins by metabolic labeling, two-dimensional electrophoresis, and protein signature peptide analysis by liquid chromatography-mass spectrometry. By peptide mass fingerprinting and immunodetection, five proteins more abundant under low-oxygen conditions were identified from several lysates of M. tuberculosis: Rv0569, Rv2031c (HspX), Rv2623, Rv2626c, and Rv3841 (BfrB). In M. tuberculosis culture filtrates, two additional proteins, Rv0363c (Fba) and Rv2780 (Ald), were found in increased amounts under oxygen limitation. These results extend our understanding of the hypoxic response in M. tuberculosis and potentially provide important insights into the physiology of the latent bacilli.

scholarly journals Altered Protein Expression of Streptococcus oralis Cultured at Low pH Revealed by Two-Dimensional Gel Electrophoresis

2001 ◽  
Vol 67 (8) ◽  
pp. 3396-3405 ◽  
Author(s):  
Joanna C. Wilkins ◽  
Karen A. Homer ◽  
David Beighton
Keyword(s):  
Gel Electrophoresis ◽  
Peptide Mass Fingerprinting ◽  
Low Ph ◽  
Ionization Mass ◽  
Two Dimensional ◽  
Two Dimensional Gel Electrophoresis ◽  
Altered Expression ◽  
Streptococcus Oralis ◽  
Peptide Mass

ABSTRACT Streptococcus oralis is the predominant aciduric nonmutans streptococcus isolated from the human dentition, but the role of this organism in the initiation and progression of dental caries has yet to be established. To identify proteins that are differentially expressed by S. oralis growing under conditions of low pH, soluble cellular proteins extracted from bacteria grown in batch culture at pH 5.2 or 7.0 were analyzed by two-dimensional (2-D) gel electrophoresis. Thirty-nine proteins had altered expression at low pH; these were excised, digested with trypsin using an in-gel protocol, and further analyzed by peptide mass fingerprinting using matrix-assisted laser desorption ionization mass spectrometry. The resulting fingerprints were compared with the genomic database forStreptococcus pneumoniae, an organism that is phylogenetically closely related to S. oralis, and putative functions for the majority of these proteins were determined on the basis of functional homology. Twenty-eight proteins were up-regulated following growth at pH 5.2; these included enzymes of the glycolytic pathway (glyceraldehyde-3-phosphate dehydrogenase and lactate dehydrogenase), the polypeptide chains comprising ATP synthase, and proteins that are considered to play a role in the general stress response of bacteria, including the 60-kDa chaperone, Hsp33, and superoxide dismutase, and three distinct ABC transporters. These data identify, for the first time, gene products that may be important in the survival and proliferation of nonmutans aciduric S. oralis under conditions of low pH that are likely to be encountered by this organism in vivo.

Comparative proteomic analysis on human L-02 liver cells treated with varying concentrations of trichloroethylene

2007 ◽  
Vol 23 (2) ◽  
pp. 91-101 ◽  
Author(s):  
Jianjun Liu ◽  
Haiyan Huang ◽  
Xiumei Xing ◽  
Renrong Xi ◽  
Zhixiong Zhuang ◽  
...  
Keyword(s):  
Proteomic Analysis ◽  
Underlying Mechanism ◽  
Liver Cells ◽  
Control Group ◽  
Two Dimensional Gel Electrophoresis ◽  
Comparative Proteomic Analysis ◽  
Maldi Tof ◽  
Flight Mass Spectrometry ◽  
Peptide Mass ◽  
Tof Ms

To determine the differential proteomic expressions in human L-02 liver cells induced by varying concentrations of trichloroethylene (TCE), comparative proteomic analysis was performed on human L-02 liver cells which were treated with varying concentrations of TCE. According to the result of MTT test, we designed four different groups, in which the cells were treated with 0 μM (control group), 3, 10 or 40 μM TCE for 24 h, respectively. Comparative analysis of approximately 800 spots resolved by two-dimensional gel electrophoresis (2DE) in the soluble proteomes of L-02 cells from the four different groups resulted in 10 differential proteins. To identify the differential spots, matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS) was carried out; if the results from the tool were insufficient, tandem MS (MALDI-TOF-TOF-MS) was then performed. The raw data of peptide mass fingerprints (PMFs) and MS/MS spectra were searched against the IPI human data base for exact matches. Then western blot was employed to verify the result of proteomic analysis, the following result confirmed that the results of proteomic analysis were reliable. These results might provide an insight into the underlying mechanism of TCE intoxication and find biological markers for diagnosis and therapy of TCE-induced diseases.

Proteomic analysis of multiple sclerosis cerebrospinal fluid

2004 ◽  
Vol 10 (3) ◽  
pp. 245-260 ◽  
Author(s):  
B N Hammack ◽  
K YC Fung ◽  
S W Hunsucker ◽  
M W Duncan ◽  
M P Burgoon ◽  
...  
Keyword(s):  
Multiple Sclerosis ◽  
Cerebrospinal Fluid ◽  
Calcium Binding ◽  
Cell Signalling ◽  
Protein A ◽  
Peptide Mass Fingerprinting ◽  
Two Dimensional Gel Electrophoresis ◽  
Ph Gradients ◽  
Peptide Mass ◽  
Normal Human

Two-dimensional gel electrophoresis and peptide mass fingerprinting were used to identify proteins in cerebrospinal fluid (C SF) pooled from three patients with multiple sclerosis (MS) and in C SF pooled from three patients with non-MS inflammatory central nervous system (C NS) disorders. Resolution of C SF proteins on three pH gradients (3-10, 4-7 and 6-11) enabled identification of a total of 430 spots in the MS C SF proteome that represented 61 distinct proteins. The gels containing MS C SF revealed 103 protein spots that were not seen on control gels. A ll but four of these 103 spots were proteins known to be present in normal human C SF. The four exceptio ns were: C RTAC -1B (cartilage acidic protein), tetranectin (a plasminogen-binding protein), SPARC -like protein (a calcium binding cell signalling glycoprotein), and autotaxin t (a phosphodiesterase). It remains unknown whether these four proteins are related to the cause and patho genesis of MS.

scholarly journals Identification and Phenotypic Characterization of Sphingomonas wittichii Strain RW1 by Peptide Mass Fingerprinting Using Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry

2005 ◽  
Vol 71 (5) ◽  
pp. 2442-2451 ◽  
Author(s):  
Rolf U. Halden ◽  
David R. Colquhoun ◽  
Eric S. Wisniewski
Keyword(s):  
Mass Spectrometry ◽  
Laser Desorption ◽  
Peptide Mass Fingerprinting ◽  
Phenotypic Characterization ◽  
Laser Desorption Ionization ◽  
Ionization Time ◽  
Flight Mass Spectrometry ◽  
Peptide Mass ◽  
Sphingomonas Wittichii

ABSTRACT Mass spectrometry is a potentially attractive means of monitoring the survival and efficacy of bioaugmentation agents, such as the dioxin-mineralizing bacterium Sphingomonas wittichii strain RW1. The biotransformation activity of RW1 phenotypes is determined primarily by the presence and concentration of the dioxin dioxygenase, an enzyme initiating the degradation of both dibenzo-p-dioxin and dibenzofuran (DF). We explored the possibility of identifying and characterizing putative cultures of RW1 by peptide mass fingerprinting (PMF) targeting this characteristic phenotypic biomarker. The proteome from cells of RW1—grown on various media in the presence and absence of DF—was partially purified, tryptically digested, and analyzed using matrix-assisted laser desorption ionization-time of flight mass spectrometry. Mascot online database queries allowed statistically significant identification of RW1 in disrupted, digested cells (P < 0.01 to 0.05) and in digested whole-cell extracts (P < 0.00001 to 0.05) containing hundreds of proteins, as determined by two-dimensional gel electrophoresis. Up to 14 peptide ions of the alpha subunit of the dioxin dioxygenase (43% protein coverage) were detected in individual samples. A minimum of 107 DF-grown cells was required to identify dioxin degradation-enabled phenotypes. The technique hinges on the detection of multiple characteristic peptides of a biomarker that can reveal at once the identity and phenotypic properties of the microbial host expressing the protein. The results demonstrate the power of PMF of minimally processed microbial cultures as a sensitive and specific technique for the positive identification and phenotypic characterization of certain microorganisms used in biotechnology and bioremediation.

scholarly journals Effects of Germination on Protein, γ-Aminobutyric Acid, Phenolic Acids, and Antioxidant Capacity in Wheat

Molecules ◽  
2018 ◽  
Vol 23 (9) ◽  
pp. 2244 ◽  
Author(s):  
Mi Kim ◽  
Han Kwak ◽  
Sang Kim
Keyword(s):  
Antioxidant Capacity ◽  
Phenolic Acids ◽  
Antioxidant Properties ◽  
Peptide Mass Fingerprinting ◽  
Aminobutyric Acid ◽  
Food Material ◽  
Two Dimensional Gel Electrophoresis ◽  
Potential Health ◽  
Peptide Mass ◽  
Close Relationship

Germinated wheat is a food material with potential health benefits due to its high phenolic and antioxidant content, but the reason why germination increases this content is unclear. The aim of this study was to investigate the relationships between protein changes (determined by two-dimensional gel electrophoresis (2-DE)), phenolics, γ-aminobutyric acid (GABA) levels, and antioxidant capacity of wheat germinated for various periods (24, 48, 72, and 96 h) compared to control. Each phenolic acid tended to increase with increasing germination time. The GABA content was highest (39.98 mg/100 g dwb) after 96 h of germination. The total oxygen radical absorbance capacity (ORAC) was 1.97 times higher after 96 h than in ungerminated seeds. Fifteen proteins, among 82 proteins separated by 2-DE, were highly related with ORAC and were identified by peptide mass fingerprinting (PMS). The PMS revealed strong expression of granule bound starch synthase (GBSS) and glutathione S-transferase (GSTF) after 96 h of germination. Overall, the ORAC at 96 h exhibited a close relationship with the levels of phenolic acids, GABA, and proteins such as GBSS and GSTF. In conclusion, these findings add to the existing knowledge of wheat protein changes and their relationship to the antioxidant properties of germinating wheat seeds.

scholarly journals Hyperoxia-Induced Protein Alterations in Renal Rat Tissue: A Quantitative Proteomic Approach to Identify Hyperoxia-Induced Effects in Cellular Signaling Pathways

2015 ◽  
Vol 2015 ◽  
pp. 1-12 ◽  
Author(s):  
Jochen Hinkelbein ◽  
Lennert Böhm ◽  
Oliver Spelten ◽  
David Sander ◽  
Stefan Soltész ◽  
...  
Keyword(s):  
Protein Expression ◽  
Rat Kidney ◽  
Peptide Mass Fingerprinting ◽  
Renal Tissue ◽  
Short Term ◽  
Two Dimensional Gel Electrophoresis ◽  
Normobaric Hyperoxia ◽  
Peptide Mass ◽  
Proteomic Approach ◽  
Hyperoxia Exposure

Introduction. In renal tissue as well as in other organs, supranormal oxygen pressure may lead to deleterious consequences on a cellular level. Additionally, hyperoxia-induced effect in cells and related free radicals may potentially contribute to renal failure. The aim of this study was to analyze time-dependent alterations of rat kidney protein expression after short-term normobaric hyperoxia using proteomics and bioinformatic approaches.Material and Methods.N=36Wistar rats were randomized into six different groups: three groups with normobaric hyperoxia (exposure to 100% oxygen for 3 h) and three groups with normobaric normoxia (NN; room air). After hyperoxia exposure, kidneys were removed immediately, after 3 days and after 7 days. Kidney lysates were analyzed by two-dimensional gel electrophoresis followed by peptide mass fingerprinting using tandem mass spectrometry. Statistical analysis was performed with DeCyder 2D software (p<0.01). Biological functions of differential regulated proteins were studied using functional network analysis (Ingenuity Pathways Analysis and PathwayStudio).Results. Expression of 14 proteins was significantly altered(p<0.01): eight proteins (MEP1A_RAT, RSSA_RAT, F16P1_RAT, STML2_RAT, BPNT1_RAT, LGMN_RAT, ATPA_RAT, and VDAC1_RAT) were downregulated and six proteins (MTUS1_RAT, F16P1_RAT, ACTG_RAT, ACTB_RAT, 2ABA_RAT, and RAB1A_RAT) were upregulated. Bioinformatic analyses revealed an association of regulated proteins with inflammation.Conclusions. Significant alterations in renal protein expression could be demonstrated for up to 7 days even after short-term hyperoxia. The identified proteins indicate an association with inflammation signaling cascades. MEP1A and VDAC1 could be promising candidates to identify hyperoxic injury in kidney cells.

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