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Wild-type E. Coli (wild-type + e._coli)
Selected AbstractsBranching sites and morphological abnormalities behave as ectopic poles in shape-defective Escherichia coliMOLECULAR MICROBIOLOGY, Issue 4 2004Trine Nilsen Summary Certain mutants in Escherichia coli lacking multiple penicillin-binding proteins (PBPs) produce misshapen cells containing kinks, bends and branches. These deformed regions exhibit two structural characteristics of normal cell poles: the peptidoglycan is inert to dilution by new synthesis or turnover, and a similarly stable patch of outer membrane caps the sites. To test the premise that these aberrant sites represent biochemically functional but misplaced cell poles, we assessed the intracellular distribution of proteins that localize specifically to bacterial poles. Green fluorescent protein (GFP) hybrids containing polar localization sequences from the Shigella flexneri IcsA protein or from the Vibrio cholerae EpsM protein formed foci at the poles of wild-type E. coli and at the poles and morphological abnormalities in PBP mutants. In addition, secreted wild-type IcsA localized to the outer membrane overlying these aberrant domains. We conclude that the morphologically deformed sites in these mutants represent fully functional poles or pole fragments. The results suggest that prokaryotic morphology is driven, at least in part, by the controlled placement of polar material, and that one or more of the low-molecular-weight PBPs participate in this process. Such mutants may help to unravel how particular proteins are targeted to bacterial poles, thereby creating important biochemical and functional asymmetries. [source] Is the Rehydrin TrDr3 from Tortula ruralis Associated with Tolerance to Cold, Salinity, and Reduced pH?PLANT BIOLOGY, Issue 3 2005HdeD from Escherichia coli in Response to Abiotic Stress, Physiological Evaluation of the TrDr3 -Orthologue Abstract: We have employed EST analysis in the resurrection moss Tortula ruralis to discover genes that control vegetative desiccation tolerance and describe the characterization of the EST-derived cDNA TrDr3 (Tortula ruralis desiccation-stress related). The deduced polypeptide TRDR3 has a predicted molecular mass of 25.5 kDa, predicted pI of 6.7, and six transmembrane helical domains. Preliminary expression analyses demonstrate that the TrDr3 transcript ratio increases in response to slow desiccation relative to the hydrated control in both total and polysomal mRNA (mRNP fraction), which classifies TrDr3 as a rehydrin. Bioinformatic searches of the electronic databases reveal that Tortula TRDR3 shares significant similarities to the hdeD gene product (HNS-dependent expression) from Escherichia coli. The function of the HdeD protein in E. coli is unknown, but it is postulated to be involved in a mechanism of acid stress defence. To establish the role of E. coli HdeD in abiotic stress tolerance, we determined the log survival percentage from shaking cultures of wild-type bacteria and the isogenic hdeD deletion strain (,hdeD) in the presence of low temperature (28 °C), elevated NaCl (5 % (w/v)), or decreased pH (4.5), or all treatments simultaneously. The ,hdeD deletion strain was less sensitive, as compared to wild-type E. coli, in response to decreased pH (p > 0.009), and the combination of all three stresses (p > 0.0001). [source] Genetic analysis of G protein-coupled receptor expression in Escherichia coli: Inhibitory role of DnaJ on the membrane integration of the human central cannabinoid receptorBIOTECHNOLOGY & BIOENGINEERING, Issue 2 2009Georgios Skretas Abstract The overexpression of G protein-coupled receptors (GPCRs) and of many other heterologous membrane proteins in simple microbial hosts, such as the bacterium Escherichia coli, often results in protein mistargeting, aggregation into inclusion bodies or cytoplasmic degradation. Furthermore, membrane protein production is very frequently accompanied by severe cell toxicity. In this work, we have employed a genetic strategy to isolate E. coli mutants that produce markedly increased amounts of the human central cannabinoid receptor (CB1), a pharmacologically significant GPCR that expresses very poorly in wild-type E. coli. By utilizing a CB1 fusion with the green fluorescent protein (GFP) and fluorescence-activated cell sorting (FACS), we screened an E. coli transposon library and identified an insertion in dnaJ that resulted in a large increase in CB1-GFP fluorescence and a dramatic enhancement in bacterial production of membrane-integrated CB1. Furthermore, the dnaJ::Tn5 inactivation suppressed the severe cytotoxicity associated with CB1 production. This revealed an unexpected inhibitory role of the chaperone/ co-chaperone DnaJ in the protein folding or membrane insertion of bacterially produced CB1. Our strategy can be easily adapted to identify expression bottlenecks for different GPCRs or any other integral membrane protein, provide useful and unanticipated mechanistic insights, and assist in the construction of genetically engineered E. coli strains for efficient heterologous membrane protein production. Biotechnol. Bioeng. 2009;102: 357,367. © 2008 Wiley Periodicals, Inc. [source] Analysis of Escherichia coli anaplerotic metabolism and its regulation mechanisms from the metabolic responses to altered dilution rates and phosphoenolpyruvate carboxykinase knockoutBIOTECHNOLOGY & BIOENGINEERING, Issue 2 2003Chen Yang Abstract The gluconeogenic phosphoenolpyruvate (PEP) carboxykinase is active in Escherichia coli during its growth on glucose. The present study investigated the influence of growth rates and PEP carboxykinase knockout on the anaplerotic fluxes in E. coli. The intracellular fluxes were determined using the complementary methods of flux ratio analysis and metabolic flux analysis based on [U- 13C6]glucose labeling experiments and 2D nuclear magnetic resonance (NMR) spectroscopy of cellular amino acids and glycerol. Significant activity of PEP carboxykinase was identified in wild-type E. coli, and the ATP dissipation for the futile cycling via this reaction accounted for up to 8.2% of the total energy flux. Flux analysis of pck deletion mutant revealed that abolishment of PEP carboxykinase activity resulted in a remarkably reduced flux through the anaplerotic PEP carboxylase and the activation of the glyoxylate shunt, with 23% of isocitrate found being channeled in the glyoxylate shunt. The changes in intracellular metabolite concentrations and specific enzyme activities associated with different growth rates and pck deletion, were also determined. Combining the measurement data of in vivo fluxes, metabolite concentrations and enzyme activities, the in vivo regulations of PEP carboxykinase flux, PEP carboxylation, and glyoxylate shunt in E. coli are discussed. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 84: 129,144, 2003. [source] Expression of a Porphyromonas gingivalis lipid A palmitylacyltransferase in Escherichia coli yields a chimeric lipid A with altered ability to stimulate interleukin-8 secretionCELLULAR MICROBIOLOGY, Issue 1 2006Brian W. Bainbridge Summary In Escherichia coli the gene htrB codes for an acyltransferase that catalyses the incorporation of laurate into lipopolysaccharide (LPS) as a lipid A substituent. We describe the cloning, expression and characterization of a Porphyromonas gingivalis htrB homologue. When the htrB homologue was expressed in wild-type E. coli or a mutant strain deficient in htrB, a chimeric LPS with altered lipid A structure was produced. Compared with wild-type E. coli lipid A, the new lipid A species contained a palmitate (C16) in the position normally occupied by laurate (C12) suggesting that the cloned gene performs the same function as E. coli htrB but preferentially transfers the longer-chain palmitic acid that is known to be present in P. gingivalis LPS. LPS was purified from wild-type E. coli, the E. coli htrB mutant strain and the htrB mutant strain expressing the P. gingivalis acyltransferase. LPS from the palmitate bearing chimeric LPS as well as the htrB mutant exhibited a reduced ability to activate human embryonic kidney 293 (HEK293) cells transfected with TLR4/MD2. LPS from the htrB mutant also had a greatly reduced ability to stimulate interleukin-8 (IL-8) secretion in both endothelial cells and monocytes. In contrast, the activity of LPS from the htrB mutant bacteria expressing the P. gingivalis gene displayed wild-type activity to stimulate IL-8 production from endothelial cells but a reduced ability to stimulate IL-8 secretion from monocytes. The intermediate activation observed in monocytes for the chimeric LPS was similar to the pattern seen in HEK293 cells expressing TLR4/MD2 and CD14. Thus, the presence of a longer-chain fatty acid on E. coli lipid A altered the activity of the LPS in monocytes but not endothelial cell assays and the difference in recognition does not appear to be related to differences in Toll-like receptor utilization. [source] Antimicrobial susceptibility in Escherichia coli of human and avian origin,a comparison of wild-type distributionsCLINICAL MICROBIOLOGY AND INFECTION, Issue 5 2009M. Sjölund Abstract In the present study, the antimicrobial susceptibilities of 97 Escherichia coli isolates from birds, and 100 clinical isolates from blood cultures, were determined by disk diffusion. The wild-type distributions were defined by the normalized resistance interpretation method. It is shown that the avian and clinical inhibition zone diameter distributions of wild-type E. coli are indistinguishable. [source] | ||||||||||