Semi-quantitative colony immunoassay for determining and optimizing protein expression in Saccharomyces cerevisiae and Escherichia coli

This work describes a quick semi-quantitative colony immunoassay (QSCI) method for immunoblot detection of intracellularly expressed proteins in both yeast and bacterial cells. After induction of protein expression, only 4.5 h is required for cell breakage, protein detection, and data analysis. This protocol was used to screen and unambiguously identify Saccharomyces cerevisiae cells efficiently overexpressing glutathione S-transferase (GST)-tagged Yih1 in addition to cells expressing the myc-tagged large 297-kDa Gcn1 protein. In addition, the method was used to identify Escherichia coli cells efficiently expressing His6-tagged Yih1 and a GST-tagged Gcn1 fragment, respectively. The protocol allows the use of both epitope-specific and protein-specific antibodies. The same colony immunoassay can also be used to determine the minimal concentration of inducing agent sufficient for induction of optimal protein expression (e.g., galactose for yeast, isopropyl β-d-1-thiogalactopyranoside [IPTG] for E. coli). To our knowledge, this is the first report on a rapid low-cost procedure that allows the calibration of inducing agent on solid medium.     

Escherichia coli as a production host for novel enzymes from basidiomycota

Many enzymes from basidiomycota have been identified and more recently characterized on the molecular level. This report summarizes the potential biotechnological applications of these enzymes and evaluates recent advances in their heterologous expression in Escherichia coli. Being one of the most widely used hosts for the production of recombinant proteins, there are, however, recurrent problems of recovering substantial yields of correctly folded and active enzymes. Various strategies for the efficient production of recombinant proteins from basidiomycetous fungi are reviewed including the current knowledge on vectors and expression strains, as well as methods for enhancing the solubility of target expression products and their purification. Research efforts towards the refolding of recombinant oxidoreductases and hydrolases are presented to illustrate successful production strategies.     

Assembly and overexpression of membrane proteins in Escherichia coli

The bacterium Escherichia coli is one of the most popular model systems to study the assembly of membrane proteins of the so-called helix-bundle class. Here, based on this system, we review and discuss what is currently known about the assembly of these membrane proteins. In addition, we will briefly review and discuss how E. coli has been used as a vehicle for the overexpression of membrane proteins.     

An in vivo, label-free quick assay for xylose transport in Escherichia coli

Efficient use of xylose is necessary for economic production of biochemicals and biofuels from lignocellulosic materials. Current studies on xylose uptake for various microorganisms have been hampered by the lack of a facile assay for xylose transport. In this work, a rapid in vivo, label-free method for measuring xylose transport in Escherichia coli was developed by taking advantage of the Bacillus pumilus xylosidase (XynB), which cleaved a commercially available xylose analog, p-nitrophenyl-β-d-xylopyranoside (pNPX), to release a chromogenic group, p-nitrophenol (pNP). XynB was expressed alone or in conjunction with a Zymomonas mobilis glucose facilitator protein (Glf) capable of transporting xylose. This XynB-mediated transport assay was demonstrated in test tubes and 96-well plates with submicromolar concentrations of pNPX. Kinetic inhibition experiments validated that pNPX and xylose were competitive substrates for the transport process, and the addition of glucose (20 g/L) in the culture medium clearly diminished the transmembrane transport of pNPX and, thus, mimicked its inhibitory action on xylose uptake. This method should be useful for engineering of the xylose transport process in E. coli, and similar assay schemes can be extended to other microorganisms.     

High-level heterologous expression of fungal cytochrome P450s in Escherichia coli

A thorough understanding of the sequence–structure–function relationships of cytochrome P450 (P450) is necessary to better understand the metabolic diversity of living organisms. Significant amounts of pure enzymes are sometimes required for biochemical studies, and their acquisition often relies on the possibility of their heterologous expression. In this study, we performed extensive heterologous expression of fungal P450s in Escherichia coli using 304 P450 isoforms. Using large-scale screening, we confirmed that at least 27 P450s could be expressed with/without simple sequence deletion at the 5′ end of cDNAs, which encode the N-terminal hydrophobic domain of the enzyme. Moreover, we identified N-terminal amino acid sequences that can potentially be used to construct chimeric P450s, which could dramatically improve their expression levels even when the expression of the wild-type sequence was unpromising. These findings will help increase the chance of heterologous expression of a variety of fungal and other eukaryotic membrane-bound P450s in E. coli.     

Identification of sources of Escherichia coli in South Carolina estuaries using antibiotic resistance analysis

Fecal pollution from nonhuman (pets, livestock or wildlife) and human sources is often one of the major factors associated with urbanization that contribute to the degradation of water quality. Methods to differentiate animal from human sources of fecal coliform contamination could assist resource managers in developing strategies to protect shellfish harvesting areas and recreational waters. In this study, surface water samples were collected from both a developed and an undeveloped watershed in coastal South Carolina. Influent and effluent samples from several wastewater treatment plants (WWTPs) in the same area were also collected. Most Probable Numbers (MPNs) of fecal coliforms were determined for all samples. Escherichia coli isolates were analyzed for antibiotic resistance (AR) to 10 antibiotics. Then, AR indices (no. of resistant/total no. of antibiotics tested), were calculated for each isolate and site. Results indicated that MPNs from the WWTP samples were significantly higher than those from the developed watershed which were significantly higher than those from the undeveloped watershed (p<0.0001). The AR analyses suggested that there was a trend toward increased antibiotic resistance in samples for the urbanized Broad Creek (BC) watershed. In the Okatee River (OR), E. coli isolates from three sites (20%) showed resistance to a single antibiotic (penicillin) but in BC, isolates from seven sites (47%) were resistant to multiple antibiotics, and the predominant resistance pattern was chlortetracycline-oxytetracycline-tetracycline. Raw sewage isolates from most WWTPs contained E. coli that exhibited resistance to multiple antibiotics. Cluster analysis indicated that all resistant OR sites had antibiotic resistant isolates that matched AR patterns found in isolates from WWTPs. Similarly, six of the seven sites in BC had AR patterns that matched with resistance patterns in WWTPs. These results suggest that AR testing may be a useful tool for differentiating E. coli from human and wildlife sources. Further testing of bacterial isolates from known animal sources is necessary to better assess the utility of this approach.     

重组蛋白融合信号肽在大肠杆菌周质空间表达的研究进展

重组蛋白在大肠杆菌中表达时,往往面临着形成包涵体的问题,而重组蛋白若是分泌至周质空间则基本解决了这一问题,周质空间的周质蛋白不仅能帮助重组蛋白正确折叠还有利于二硫键的生成。信号肽是一段由15-30个氨基酸组成,被融合在重组蛋白N端的短肽,按照结构、功能的不同可以划分为N区、H区和C区,具有引导重组蛋白转运至细胞周质空间的作用。本文综述了信号肽的结构组成、作用机理和基本分泌途径,讨论了信号肽的高效转运和筛选方法,总结了在大肠杆菌中重组蛋白融合信号肽实现周质表达的新进展,并对未来高效信号肽选择方面的研究进行了探讨。     

A multiplex PCR procedure for the detection of six major virulence genes in Escherichia coli O157:H7

A multiplex PCR procedure that detects six major virulence genes, fliC, stx1, stx2, eae, rfbE, and hlyA, in Escherichia coli O157:H7 was developed. Analyses of the available sequences of the six major virulence genes and the published primers allowed us to develop the six-gene, multiplex PCR protocol that maintained the specificity of each primer pair. The resulting six bands for fliC, stx1, stx2, eae, rfbE, and hlyA were even and distinct with product sizes of 949, 655, 477, 375, 296, and 199 bp, respectively. The procedure was validated with a total of 221 E. coli strains that included 4 ATCC, 84 cattle, and 57 human E. coli O157:H7 strains as well as 76 non-O157 cattle and human E. coli strains. The results of all 221 strains were similar to the results generated by established multiplex PCR methods that involved two separate reactions to detect five virulence genes (stx1, stx2, eae, fliC, and hlyA). Specificity of the O antigen was indicated by amplification of only O157, and not O25, O26, O55, O78, O103, O111, O127, and O145 E. coli serotypes. Sensitivity tests showed that the procedure amplified genes from a fecal sample spiked with a minimum of 10⁴ CFU/g (10 cells/reaction) of E. coli O157. After a 6-h enrichment of E. coli O157-spiked samples, a sensitivity level of 10 CFU/g was achieved.     

Global/temporal gene expression analysis of Escherichia coli in the early stages of symbiotic relationship development with the cellular slime mold Dictyostelium discoideum

Escherichia coli and the cellular slime mold Dictyostelium discoideum form stable viscous symbiotic colonies in the laboratory. To examine changes in E. coli gene expression during establishment of this symbiotic relationship, cells of symbiotic co-cultures and monocultures at various time points were subjected to microarrays analysis. Genes changed significantly over time compared to the initial gene expression level were determined as characteristics of GO function categories. The categories that appeared significantly at the same sampling time points between the two cultures were also identified. Up-regulation of genes from several GO categories associated with polysaccharide synthesis, cell wall degradation, and iron acquisition as well as down-regulation of genes from GO categories associated with biosynthesis through starvation response were observed in co-cultures, indicating exchange of molecules between the two organisms. Up-regulation of genes from several GO categories associated with anaerobic respiration and flagella biosynthesis were also observed, indicating that the environment inside symbiotic colonies was similar to that in developed biofilms. Up-regulation of genes associated with energy-generating systems indicated that E. coli prolonged survival within the symbiotic colony. Thus, E. coli showed not only molecule exchange but also altered expression of various genes in symbiosis with D. discoideum.     

The role of lipopolysaccharide moieties in macrophage response to Escherichia coli

Lipopolysaccharide (LPS) is the main component of Gram-negative bacteria that - upon infection - activates the host immune system and is crucial in fighting pathogens as well as in the induction of sepsis. In the present study we addressed the question whether the key structural components of LPS equally take part in the activation of different macrophage immune responses. By genomic modifications of Escherichia coli MG1655, we constructed a series of strains harboring complete and truncated forms of LPS in their cell wall. These strains were exposed to RAW 264.7 macrophages, after which phagocytosis, fast release of pre-synthesized TNF and activation of NF-κB signal transduction pathway were quantified. According to our results the core and lipid A moieties are involved in immune recognition. The most ancient part, lipid A is crucial in evoking immediate TNF release and activation of NF-κB. The O-antigen inhibits phagocytosis, leading to immune evasion.     

Time-resolved fluorescence-based assay for rapid detection of Escherichia coli

Fast and simple detection of pathogens is of utmost importance in health care and the food industry. In this article, a novel technology for the detection of pathogenic bacteria is presented. The technology uses lytic-specific bacteriophages and a nonspecific interaction of cellular components with a luminescent lanthanide chelate. As a proof of principle, Escherichia coli-specific T4 bacteriophage was used to infect the bacteria, and the cell lysis was detected. In the absence of E. coli, luminescent Eu³⁺–chelate complex cannot be formed and low time-resolved luminescence signal is monitored. In the presence of E. coli, increased luminescence signal is observed as the cellular contents are leached to the surrounding medium. The luminescence signal is observed as a function of the number of bacteria in the sample. The homogeneous assay can detect living E. coli in bacterial cultures and simulated urine samples within 25 min with a detection limit of 1000 or 10,000 bacterial cells/ml in buffer or urine, respectively. The detection limit is at the clinically relevant level, which indicates that the method could also be applicable to clinical settings for fast detection of urine bacteria.     

Resistance of a recombinant Escherichia coli to dehydration

Dehydration of microorganisms, rendering them anhydrobiotic, is often an efficient method for the short and long term conservation of different strain-producers. However, some biotechnologically important recombinant bacterial strains are extremely sensitive to conventional treatment. We describe appropriate conditions during dehydration of the recombinant Escherichia coli strain HB 101 (GAPDH) that can result dry cells having a ∼88% viability on rehydration. The methods entails air-drying after addition of 100 mM trehalose to the cultivation medium or distilled water (for short term incubation).     

Enhanced resistance to peroxide stress in Escherichia coli grown outside their niche temperatures

Extended exposure of Escherichia coli to temperatures above and below their growth optimum led to significant changes in oxidant production and antioxidant defense. At 20 °C an increase in the intracellular H₂O₂ concentration and oxidized glutathione (GSSG) level was observed against a background of low levels of reduced glutathione (GSH) and decreased catalase and glutathione reductase (GOR) activities. The intracellular H₂O₂ and GSSG concentrations had minimal values at 30 and 37 °C, but rose again at 42 °C, suggesting that oxidative processes were intensified at high temperatures. An increase in temperature from 20 to 42 °C led to an elevation in the oxygen respiration rate and superoxide production; a 5-fold increase in the intracellular GSH concentration and in the GSH:GSSG ratio occurred simultaneously. Catalase HPI and GOR activities were elevated 4.4- and 1.5-fold, respectively. Prolonged exposure to sublethal temperatures facilitated an adaptation to subsequent oxidative stress produced by the addition of H₂O₂.     

An improved 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) reduction assay for evaluating the viability of Escherichia coli cells

To reduce the interference in MTT reduction assay, we developed an improved protocol for evaluating the viability of Escherichia coli cells by conducting MTT reduction in 1.5-mL centrifuge tubes and formazan dissolution in test tubes. Our study is helpful in developing protocols for measuring the viability of other gram-negative bacteria.     

A metabolomics-based method for studying the effect of yfcC gene in Escherichia coli on metabolism

Metabolomics is a potent tool to assist in identifying the function of unknown genes through analysis of metabolite changes in the context of varied genetic backgrounds. However, the availability of a universal unbiased profiling analysis is still a big challenge. In this study, we report an optimized metabolic profiling method based on gas chromatography–mass spectrometry for Escherichia coli. It was found that physiological saline at −80 °C could ensure satisfied metabolic quenching with less metabolite leakage. A solution of methanol/water (21:79, v/v) was proved to be efficient for intracellular metabolite extraction. This method was applied to investigate the metabolome difference among wild-type E. coli, its yfcC deletion, and overexpression mutants. Statistical and bioinformatic analysis of the metabolic profiling data indicated that the expression of yfcC potentially affected the metabolism of glyoxylate shunt. This finding was further validated by real-time quantitative polymerase chain reactions showing that expression of aceA and aceB, the key genes in glyoxylate shunt, was upregulated by yfcC. This study exemplifies the robustness of the proposed metabolic profiling analysis strategy and its potential roles in investigating unknown gene functions in view of metabolome difference.     

Development and application of a differential method for reliable metabolome analysis in Escherichia coli

Quantitative metabolomics of microbial cultures requires well-designed sampling and quenching procedures. We successfully developed and applied a differential method to obtain a reliable set of metabolome data for Escherichia coli K12 MG1655 grown in steady-state, aerobic, glucose-limited chemostat cultures. From a rigorous analysis of the commonly applied quenching procedure based on cold aqueous methanol, it was concluded that it was not applicable because of release of a major part of the metabolites from the cells. No positive effect of buffering or increasing the ionic strength of the quenching solution was observed. Application of a differential method in principle requires metabolite measurements in total broth and filtrate for each measurement. Different methods for sampling of culture filtrate were examined, and it was found that direct filtration without cooling of the sample was the most appropriate. Analysis of culture filtrates revealed that most of the central metabolites and amino acids were present in significant amounts outside the cells. Because the turnover time of the pools of extracellular metabolites is much larger than that of the intracellular pools, the differential method should also be applicable to short-term pulse response experiments without requiring measurement of metabolites in the supernatant during the dynamic period.     

Bacteriophage-based biosorbents coupled with bioluminescent ATP assay for rapid concentration and detection of Escherichia coli

Wild type T4 bacteriophage and recombinant T4 bacteriophages displaying biotin binding peptide (BCCP) and cellulose binding module (CBM) on their heads were immobilized on nano-aluminum fiber-based filter (Disruptor™), streptavidin magnetic beads and microcrystalline cellulose, respectively. Infectivity of the immobilized phages was investigated by monitoring the phage-mediated growth inhibition of bioluminescent E. coli B and cell lysis using bioluminescent ATP assay. The results showed that phage immobilization resulted in a partial loss of infectivity as compared with the free phage. Nevertheless, the use of a biosorbent based on T4 bacteriophage immobilized on Disruptor™ filter coupled with a bioluminescent ATP assay allowed simultaneous concentration and detection of as low as 6 × 10³ cfu/mL of E. coli in the sample within 2 h with high accuracy (CV = 1-5% in log scale). Excess of interfering microflora at levels 60-fold greater than the target organism did not affect the results when bacteriophage was immobilized on the filter prior to concentration of bacterial cells.     

The transport and mediation mechanisms of the common sugars in Escherichia coli

Escherichia coli can uptake and utilize many common natural sugars to form biomass or valuable target bio-products. Carbon catabolite repression (CCR) will occur and hamper the efficient production of bio-products if E. coli strains are cultivated in a mixture of sugars containing some preferred sugar, such as glucose. Understanding the transport and metabolism mechanisms of the common and inexpensive sugars in E. coli is important for further improving the efficiency of sugar bioconversion and for reducing industrial fermentation costs using the methods of metabolic engineering, synthetic biology and systems biology. In this review, the transport and mediation mechanisms of glucose, fructose, sucrose, xylose and arabinose are discussed and summarized, and the hierarchical utilization principles of these sugars are elucidated.     

Structural studies of the O-antigenic polysaccharides from the enteroaggregative Escherichia coli strain 94/D4 and the international type strain Escherichia coli O82

The structure of the O-antigen polysaccharides (PS) from the enteroaggregative Escherichia coli strain 94/D4 and the international type strain E. coli O82 have been determined. Component analysis and ¹H, ¹³C, and ³¹P NMR spectroscopy experiments were employed to elucidate the structure. Inter-residue correlations were determined by ¹H, ¹³C-heteronuclear multiple-bond correlation, and ¹H, ¹H-NOESY experiments. d-GroA as a substituent is linked via its O-2 in a phosphodiester-linkage to O-6 of the α-d-Glcp residue. The PS is composed of tetrasaccharide repeating units with the following structure:→4)-α-d-Glcp6-(P-2-d-GroA)-(1→4)-β-d-Galp-(1→4)-β-d-Glcp-(1→3)-β-d-GlcpNAc-(1→Cross-peaks of low intensity from an α-d-Glcp residue were present in the NMR spectra and spectral analysis indicates that they originate from the terminal residue of the polysaccharide. Consequently, the biological repeating unit has a 3-substituted N-acetyl-d-glucosamine residue at its reducing end. Enzyme immunoassay using specific anti-E. coli O82 rabbit sera showed identical reactivity to the LPS of the two strains, in agreement with the structural analysis of their O-antigen polysaccharides.