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.     

The O-antigen of Salmonella enterica O13 and its relation to the O-antigen of Escherichia coli O127

The following structure of the O-polysaccharide (O-antigen) of Salmonella enterica O13 was established by chemical analyses along with 2D ¹H and ¹³C NMR spectroscopy:→2)-α-l-Fucp-(1→2)-β-d-Galp-(1→3)-α-d-GalpNAc-(1→3)-α-d-GlcpNAc-(1→The O-antigen of S. enterica O13 was found to be closely related to that of Escherichia coli O127, which differs only in the presence of a GalNAc residue in place of the GlcNAc residue and O-acetylation. The location of the O-acetyl groups in the E. coli O127 polysaccharide was determined. The structures of the O-polysaccharides studied are in agreement with the DNA sequence of the O-antigen gene clusters of S. enterica O13 and E. coli O127 reported earlier.     

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.     

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.     

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).     

Four products from Escherichia coli pseudogenes increase hydrogen production

Pseudogenes are considered to be nonfunctional genes that lack a physiological role. By screening 3985 Escherichia coli mutants using chemochromic membranes, we found four pseudogenes involved in hydrogen metabolism. Knockouts of pseudogenes ydfW and ypdJ had a defective hydrogen phenotype on glucose and formate, respectively. Also, the knockout of pseudogene yqiG formed hydrogen from formate but not from glucose. For the yqiG mutant, 100% hydrogen recovery was obtained by the complementation of YqiG via a plasmid. The knockout of pseudogene ylcE showed hydrogen deficiency in minimal media which suggested that the role of YlcE is associated with cell growth. Hence, the products of these four pseudogenes play an important physiological role in hydrogen production in E. coli.     

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.     

The quaternary structure of pyruvate kinase type 1 from Escherichia coli at low nanomolar concentrations

Pyruvate kinase (PK) is the key control point of glycolysis—the biochemical pathway central to energy metabolism and the production of precursors used in biosynthesis. PK type 1 from Escherichia coli (Ec-PK1) is activated by both fructose-1,6-bisphosphate (FBP) and its substrate, phosphoenol pyruvate (PEP). To date, it has not been possible to determine whether the enzyme is tetrameric at the low concentrations (i.e. low nM range) used to study the steady-state kinetics, or assess whether its allosteric effectors alter the oligomeric state of the enzyme at these concentrations. Employing the new technique of analytical ultracentrifugation with fluorescence detection we have, for the first time, shown that the K_D^4–2 for Ec-PK1 is in the subnanomolar range, well below the concentrations used in kinetic studies. In addition, we show that, unlike some other PK isoenzymes, the modulation of oligomeric state by the allosteric effectors FBP and PEP does not occur at a concentration of 10 nM or above.     

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.     

Comparison of surface proteomes of enterotoxigenic (ETEC) and commensal Escherichia coli strains

Pathogenesis of enterotoxigenic Escherichia coli (ETEC) infections involves colonization of the small intestine mediated by cell-surface fimbriae (CS) or colonization fimbriae antigens (CFA). However, protection against reinfection of ETEC is also conferred by somatic antigens rather than by virulence factors. To discover ETEC specific somatic antigens, the surface proteome of the ETEC H10406 strain was compared with that of non-pathogenic E. coli K12 strains. In this study, we were using stable isotope labelling with amino acids in cell culture (SILAC) technology for the labelling and relative quantification of surface proteins in order to identify polypeptides that are specifically present on ETEC strains. Outer membrane proteins were isolated, separated by gel electrophoresis, and identified by mass spectrometry. Twenty-three differentially expressed cell-surface polypeptides of ETEC were identified and evaluated by bioinformatics for protein vaccine candidates. The combination of being surface-exposed and present differentially makes these polypeptides highly suitable as targets for antibodies and thus for use in passive or active immunisation/vaccination.     

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.     

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.     

Enhanced production of recombinant streptokinase in Escherichia coli using fed-batch culture

Fed-batch culture strategy is often used for increasing production of heterologous recombinant proteins in Escherichia coli. This study was initiated to investigate the effects of dissolved oxygen concentration (DOC), complex nitrogen sources and pH control agents on cell growth and intracellular expression of streptokinase (SK) in recombinant E. coli BL21(DE3). Increase in DOC set point from 30% to 50% did not affect SK expression in batch culture where as similar increase in fed-batch cultivation led to a significant improvement in SK expression (from 188 to 720 mg l⁻¹). This increase in SK could be correlated with increase in plasmid segregational stability. Supplementation of production medium with yeast extract and tryptone and replacement of liquid ammonia with NaOH as pH control agent further enhanced SK expression without affecting cell growth. Overall, SK concentration of 1120 mg l⁻¹ representing 14-fold increase in SK production on process scale-up from flask to bioreactor scale fed-batch culture is the highest reported concentration of SK to date.     

Synthesis of the pentasaccharide repeating unit of Escherichia coli O128 antigen

A pentasaccharide, 4-methoxyphenyl 2-acetamido-2-deoxy-β-d-galactopyranosyl-(1→4)-α-d-galactopyranosyl-(1→3)-2-acetamido-2-deoxy-β-d-galactopyranosyl-(1→6)-[α-l-fucopyranosyl-(1→2)]-β-d-galactopyranoside (1), representing the repeating unit of Escherichia coli O128 antigen, was successfully prepared in 23% overall yield via a convergent ‘2+3’ glycosylation strategy.     

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.     

The C-terminal residues of the 2b protein of Cucumber mosaic virus are important for efficient expression in Escherichia coli and DNA-binding

The RNA silencing suppressor 2b protein of Cucumber mosaic virus (CMV) is difficult to produce in Escherichia coli. We compared two CMV 2b proteins that differ in their toxicity against E. coli and found that the acidic amino acid residues in the C-terminal significantly affected the toxicity and expression level of the protein in E. coli. In addition, in a DNA-binding assay, 2b had the ability to bind to DNA, and this ability was affected by the charge on the C-terminal residues of 2b. We concluded that the C-terminal residues were important for 2b’s DNA-binding ability, which may partly explain the toxicity of the protein.     

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 site for the allosteric activator GTP of Escherichia coli UMP kinase

UMP kinase (UMPK), a key bacterial pyrimidine nucleotide biosynthesis enzyme, is UTP-inhibited and GTP-activated. We delineate the GTP site of Escherichia coli UMPK by alanine mutagenesis of R92, H96, R103, W119 or R130, abolishing GTP activation; of S124 and R127, decreasing affinity for GTP; and of N111 and D115, with little detrimental effect. We exclude the correspondence with the modulatory ATP site of Bacillus anthracis UMPK, confirming the functionality of the GTP site found by Evrin. Mutants R92A, H96A and R127A are constitutively activated, suggesting key roles of these residues in allosteric signal transduction and of positive charge neutralization in triggering activation. No mutation hampered UTP inhibition, excluding overlapping of the UTP and GTP sites.     

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.