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

When simple sequence comparison fails: the cryptic case of the shared domains of the bacterial replication initiation proteins DnaB and DnaD

DnaD and DnaB are essential DNA-replication-initiation proteins in low-G+C content Gram-positive bacteria. Here we use sensitive Hidden Markov Model-based techniques to show that the DnaB and DnaD proteins share a common structure that is evident across all their structural domains, termed DDBH1 and DDBH2 (DnaD DnaB Homology 1 and 2). Despite strong sequence divergence, many of the DNA-binding and oligomerization properties of these domains have been conserved. Although eluding simple sequence comparisons, the DDBH2 domains share the only strong sequence motif; an extremely highly conserved YxxxIxxxW sequence that contributes to DNA binding. Sequence alignments of DnaD alone fail to identify another key part of the DNA-binding module, since it includes a poorly conserved sequence, a solvent-exposed and somewhat unstable helix and a mobile segment. We show by NMR, in vitro mutagenesis and in vivo complementation experiments that the DNA-binding module of Bacillus subtilis DnaD comprises the YxxxIxxxW motif, the unstable helix and a portion of the mobile region, the latter two being essential for viability. These structural insights lead us to a re-evaluation of the oligomerization and DNA-binding properties of the DnaD and DnaB proteins

The Transcriptional Regulator Rok Binds A+T-Rich DNA and Is Involved in Repression of a Mobile Genetic Element in Bacillus subtilis

The rok gene of Bacillus subtilis was identified as a negative regulator of competence development. It also controls expression of several genes not related to competence. We found that Rok binds to extended regions of the B. subtilis genome. These regions are characterized by a high A+T content and are known or believed to have been acquired by horizontal gene transfer. Some of the Rok binding regions are in known mobile genetic elements. A deletion of rok resulted in higher excision of one such element, ICEBs1, a conjugative transposon found integrated in the B. subtilis genome. When expressed in the Gram negative E. coli, Rok also associated with A+T-rich DNA and a conserved C-terminal region of Rok contributed to this association. Together with previous work, our findings indicate that Rok is a nucleoid associated protein that serves to help repress expression of A+T-rich genes, many of which appear to have been acquired by horizontal gene transfer. In these ways, Rok appears to be functionally analogous to H-NS, a nucleoid associated protein found in Gram negative bacteria and Lsr2 of high G+C Mycobacteria

横隔神経と伴走血管による胸部X線写真における心大血管辺縁不明瞭化

Purpose: Our aim was to clarify the frequency of cardiovascular border obliteration on frontal chest radiography and to prove that the phrenic nerve with accompanying vessels can be considered as a cause of obliteration of cardiovascular border on an otherwise normal chest radiography. Materials and methods: Two radiologists reviewed chest radiographs and computed tomography (CT) images of 100 individuals. CT confirmed the absence of intrapulmonary or extrapulmonary abnormalities in all of them. We examined the frequency of cardiovascular border obliteration on frontal chest radiography and summarized the causes of obliteration as pericardial fat pad, phrenic nerve, intrafissure fat, pulmonary vessels, and others, comparing them with CT in each case. Results: Cardiovascular border was obliterated on frontal chest radiography in 46 cases on the right and in 61 on the left. The phrenic nerve with accompanying vessels was found to be a cause of obliteration in 34 of 46 cases (74 %) on the right and 29 of 61 (48 %) cases on the left. The phrenic nerve was the most frequent cause of cardiovascular border obliteration on both sides. Conclusion: The phrenic nerve with accompanying vessels, forming a prominent fold of parietal pleura, can be attributed as a cause of cardiovascular border obliteration on frontal chest radiography.長崎大学学位論文 学位記番号:博(医歯薬)甲第822号 学位授与年月日:平成28年2月3日Author: Shiri Farhana, Kazuto Ashizawa , Hideyuki Hayashi, Yukihiro Ogihara, Nobuya Aso, Kuniaki Hayashi, Masataka UetaniCitation: Japanese Journal of Radiology, 33(12), pp.734-740; 2015Nagasaki University (長崎大学)課程博