Disruption of reducing pathways is not essential for efficient disulfide bond formation in the cytoplasm of E. coli

<p>Abstract</p> <p>Background</p> <p>The formation of native disulfide bonds is a complex and essential post-translational modification for many proteins. The large scale production of these proteins can be difficult and depends on targeting the protein to a compartment in which disulfide bond formation naturally occurs, usually the endoplasmic reticulum of eukaryotes or the periplasm of prokaryotes. It is currently thought to be impossible to produce large amounts of disulfide bond containing protein in the cytoplasm of wild-type bacteria such as <it>E. coli </it>due to the presence of multiple pathways for their reduction.</p> <p>Results</p> <p>Here we show that the introduction of Erv1p, a sulfhydryl oxidase and FAD-dependent catalyst of disulfide bond formation found in the inter membrane space of mitochondria, allows the efficient formation of native disulfide bonds in heterologously expressed proteins in the cytoplasm of <it>E. coli </it>even without the disruption of genes involved in disulfide bond reduction, for example <it>trxB </it>and/or <it>gor</it>. Indeed yields of active disulfide bonded proteins were higher in BL21 (DE3) pLysSRARE, an <it>E. coli </it>strain with the reducing pathways intact, than in the commercial Δ<it>gor </it>Δ<it>trxB </it>strain rosetta-gami upon co-expression of Erv1p.</p> <p>Conclusions</p> <p>Our results refute the current paradigm in the field that disruption of at least one of the reducing pathways is essential for the efficient production of disulfide bond containing proteins in the cytoplasm of <it>E. coli </it>and open up new possibilities for the use of <it>E. coli </it>as a microbial cell factory.</p

L’Irenophile de Jacques Saint-Germain d’Apchon – la deuxième vie d’un pamphlet

Obukowicz Natalia. L’Irenophile de Jacques Saint-Germain d’Apchon – la deuxième vie d’un pamphlet. In: Réforme, Humanisme, Renaissance, n°69, 2009. pp. 145-156

Properties of Three Plasmids Isolated From Plant Pathogenic Pseudomonads

149 p.Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 1983.Indigenous plasmids isolated from Pseudomonas tabaci ATCC 11528 (pJP1), P. tabaci BR2 (pBPW1), and P. angulata 45 (pJP30) were labeled with Tn3 using RSF1010::Tn3 as a vector and the host strains were subsequently cured of their Tn3-containing plasmid. Plasmid-containing and respective cured strains produced nearly identical symptoms on host and nonhost plants when serial dilutions of bacteria were injected directly into leaf tissue. Also, cured and plasmid-containing isolates of P. tabaci 11528 and P. tabaci BR2 produced approximately the same amount of tabtoxin. These data indicate that pJP1, pBPW1, and pJP30 do not encode genes essential for pathogenicity and/or tabtoxin synthesis.The cryptic plasmids pJP1, pBPW1, and pJP30 were then examined for four possible plasmid-encoded functions; (1) conjugative ability, (2) synthesis of Pseudomonas phage-specific receptor(s), (3) carbon compound catabolism, and (4) a competitive advantage conferred to host bacteria in their ability to multiply on leaf surfaces. Results showed that; (1) pBPW1::Tn3 mobilized itself and RSF1010 at high frequencies into P. mellea recipients. Mobilization of each plasmid was a separate event in that pBPW1::Tn3 and RSF1010 were found singly in nearly half of the transconjugants. No detectable homology by Southern blotting was found between pBPW1 and RSF1010 in donors prior to conjugation or in recipients containing both plasmids after conjugation. These data indicated that pBPW1 and RSF1010 did not recombine during mobilization. Rather, pBPW1 donates RSF1010 by a yet unknown mechanism. (2) pJP1 and pJP30, like pBPW1, are conjugative and pJP1 is also able to mobilize RSF1010. (3) pBPW1 encodes phage receptor(s) for the phages PRD1 and PR3. (4) No differences in carbon compound utilization by plasmid-containing and cured strains were detected. (5) No differences in epiphytic growth of cured and plasmid-containing strains of P. tabaci 11528 or P. tabaci BR2 on host and nonhost plants were observed.U of I OnlyRestricted to the U of I community idenfinitely during batch ingest of legacy ETD