Solubilization of the Cytoplasmic Membrane of
            <i>Escherichia coli</i>
            by the Ionic Detergent Sodium-Lauryl Sarcosinate

Filip, Camille; Fletcher, Gail; Wulff, Judith L; Earhart, Charles F.

doi:10.1128/jb.115.3.717-722.1973

Cited by 931 publications

(329 citation statements)

References 18 publications

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“…Briefly, the crude lysate of S typhimurium was centrifuged at 105 000 g for 1 h to obtain the cytoplasmic supernatant and membrane pellet. The membrane pellet was treated with 20 mM Tris-HCl (pH 7·5) containing 2·0% (wt/vol) sodium lauryl sarcosine (SLS) for 30 min at 37°C [20]. It was then centrifuged at 105 000 g to obtain the Omp pellet and washed three times with distilled water.…”

Section: Preparation and Fractionation Of Outer Membrane Proteins (Ommentioning

confidence: 99%

Low molecular weight proteins of outer membrane of Salmonella typhimurium are immunogenic in Salmonella induced reactive arthritis revealed by proteomics

Singh

Shasany

Aggarwal

et al. 2007

Clinical and Experimental Immunology

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SummaryIn patients with reactive arthritis (ReA)/undifferentiated spondyloarthropathy (uSpA), synovial fluid mononuclear cells (SFMC) show proliferation to bacterial antigens that trigger ReA, i.e. Chlamydia, Yersinia, Campylobactor, Shigella and Salmonella species. We have shown previously that SFMC proliferate significantly to outer membrane proteins of S typhimurium in Salmonella induced ReA. In the present study we characterized the immunoreactive fractions of outer membrane protein (Omp) of S typhimurium in Salmonella induced ReA. Omp of Salmonella was isolated and fractionated by continuous elution sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) using Prep-Cell into eight Omp fractions based on molecular weight. Twenty-three patients with ReA were screened for the bacterial trigger using the SFMC proliferative response to crude lysates of Y enterocolitica, S flexneri, C jejuni and S typhimurium using thymidine uptake assay. SFMC from patients with salmonella induced ReA were tested against eight fractions. Seven of 23 patients with ReA had S typhimuriuminduced ReA. Of these seven patients, five patients SFMC had a significant stimulation index (SI) against < 22, 22-26, 25-35 and 28-40 kDa fractions of Omp. These fractions were analysed by SDS-PAGE and matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry, which revealed 10 proteins. These proteins were 37 kDa OmpA, 33 kDa TsX, 28 kDa putative Omp, 28 kDa Vac J, 39 kDa OmpD, 18 kDa OmpX, 23 kDa OmpW, 43 kDa OmpS1 and 19 kDa peptidoglycan-associated lipoprotein. In conclusion, for the first time we have identified some low molecular weight proteins in the Omps of Salmonella which are T cells immunoreactive in patients with salmonella induced ReA/uSpA.

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Section: Preparation and Fractionation Of Outer Membrane Proteins (Ommentioning

confidence: 99%

Low molecular weight proteins of outer membrane of Salmonella typhimurium are immunogenic in Salmonella induced reactive arthritis revealed by proteomics

Singh

Shasany

Aggarwal

et al. 2007

Clinical and Experimental Immunology

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“…Immunodetection of proteins in total cell lysates or in membrane fractions was performed with polyclonal PilA4 antiserum (dilution 1 : 5000), PilM antiserum (dilution 1 : 5000), PilN antiserum (dilution 1 : 2500), PilO antiserum (dilution 1 : 2000), PilW antiserum (dilution 1 :10 000) or PilQ antiserum (dilution 1 : 10 000) obtained from Davids Biotechnologie (Regensburg, Germany). ProteinA-horse radish [38]. After ultracentrifugation of the membranes for 2 h at 120 000 g (4°C; rotor type Ti70, Beckman Coulter), the outer membrane pellet was washed once with 10 mL of 10 mm Tris ⁄ HCl (pH 8.0) (120 000 g, 1 h, 4°C; rotor type Ti70, Beckman Coulter), resuspended in H 2 O and stored at ) 20°C.…”

Section: Western Blot Analysesmentioning

confidence: 99%

Identification, subcellular localization and functional interactions of PilMNOWQ and PilA4 involved in transformation competency and pilus biogenesis in the thermophilic bacterium Thermus thermophilus HB27

2006

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Members of the extremely thermophilic genus Thermus belong to one of the oldest branches of bacterial evolution and, together with the genus Deinococcus, form a distinctive group within the Bacteria deserving the taxonomic status of a phylum [1,2]. Thermus representatives, such as Thermus thermophilus strain HB27, Thermus thermophilus HB8, Thermus flavus AT62, Thermus caldophilus, and Thermus aquaticus YT1, exhibit the extraordinary trait of high transformation competence [3,4]. The high transformation frequencies, together with the high thermotolerance, suggest a significant impact of the Thermus transformation system on DNA transfer in extreme environments and therefore on the evolution of life. This is supported by recent data from comparative genomics and phylogenetic analyses in the thermophilic bacterium T. thermophilus HB27. This strain seems to have acquired numerous genes from (hyper)thermophilic bacteria and archaea, suggesting that horizontal gene transfer was probably decisive in its thermophilic adaptation [5]. Despite the significance of natural transformation systems of thermophiles, information about transformation The natural transformation system of the thermophilic bacterium Thermus thermophilus HB27 comprises at least 16 distinct competence proteins encoded by seven distinct loci. In this article, we present for the first time biochemical analyses of the Thermus thermophilus competence proteins PilMNOWQ and PilA4, and demonstrate that the pilMNOWQ genes are each essential for natural transformation. We identified three different forms of PilA4, one with an apparent molecular mass of 14 kDa, which correlates with that of the deduced protein, an 18-kDa form and a 23-kDa form; the last was found to be glycosylated. We demonstrate that PilM, PilN and PilO are located in the inner membrane, whereas PilW, PilQ and PilA4 are located in the inner and outer membranes. These data show that PilMNOWQ and PilA4 are components of a DNA translocator structure that spans the inner and outer membranes. We further show that PilA4 and PilQ both copurify with pilus structures. Possible functions of PilQ and PilA4 in DNA translocation and in pilus biogenesis are discussed. Comparative mutant studies revealed that mutations in either pilW or pilQ significantly affect the location of the other protein in the outer membrane. Furthermore, no PilA4 was present in the outer membranes of these mutants. From these findings, we conclude that the abilities of PilW, PilQ and PilA4 to stably localize or accumulate in the outer membrane fraction are strongly dependent on one another, which is in accord with an outer membrane DNA translocator complex comprising PilW, PilQ, and PilA4.Abbreviations IPTG, isopropyl thio-b-D-galactoside; TFMS, trifluoromethanesulfonic acid; TM, Thermus medium.

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“…Outer membranes were prepared by the Sarkosyl solubilization method (Filip et al, 1973) as previously described (Cornelis et al, 1989). The proteins were solubilized in sample buffer, heated at 99°C for 5 min and separated by SDS-PAGE (12% precast criterion gels, Bio-Rad).…”

Section: Outer Membrane Protein Preparation Sds-page and Western Blomentioning

confidence: 99%

Analysis of the Pseudomonas aeruginosa oprD gene from clinical and environmental isolates

Pirnay

Vos

Mossialos

et al. 2002

Environmental Microbiology

112

104

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Genomes are constantly evolving. Our report highlights the wide mutational diversity of clinical as well as environmental isolates, compared with the laboratory strain(s), through the systematic genetic analysis of a chromosomal porin gene (oprD) in relation to a specific antibiotic resistance. Mutational inactivation of the oprD gene is associated with carbapenem resistance in Pseudomonas aeruginosa. The sequence of the oprD gene of 55 Pseudomonas aeruginosa natural isolates obtained from across the world--from sources as diverse as patients and rhizospheres--was analysed. A microscale mosaic structure for this gene--resulting from multiple intra- and possibly interspecies recombinational events--is reported. An array of independent and seemingly fast-occurring defective oprD mutations were found, none of which had been described before. A burn wound isolate demonstrated unusually high overall sequence variability typical of mutator strains. We also present evidence for the existence of OprD homologues in other fluorescent pseudomonads.

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Solubilization of the Cytoplasmic Membrane of Escherichia coli by the Ionic Detergent Sodium-Lauryl Sarcosinate

Cited by 931 publications

References 18 publications

Low molecular weight proteins of outer membrane of Salmonella typhimurium are immunogenic in Salmonella induced reactive arthritis revealed by proteomics

Low molecular weight proteins of outer membrane of Salmonella typhimurium are immunogenic in Salmonella induced reactive arthritis revealed by proteomics

Identification, subcellular localization and functional interactions of PilMNOWQ and PilA4 involved in transformation competency and pilus biogenesis in the thermophilic bacterium Thermus thermophilus HB27

Analysis of the Pseudomonas aeruginosa oprD gene from clinical and environmental isolates

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