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).     

Purification of endo-(1→3)-β-D-glucanases lysing yeast cell walls from Rhizoctonia solani

Three forms of $\text{endo}\text{-(1→3)-β-}\text{g}\text{-glucanases}$ lysing yeast cell walls from Rhizoctonia solani were separated by precipitation with ammonium sulfate and by successive chromatographies on CM Bio-Gel A and Bio-Gel P-60 or P-30, and were finally purified by substrate affinity chromatography on short-chain pachyman-AH-Sepharose CL 6B column. Each preparation was found to be homogeneous on gel filtration and by electrophoresis on acrylamide gel with sodium dodecyl sulfate. They exhibit high activity against insoluble pachyman, but only restricted activity against soluble short-chain pachyman. In the affinity chromatography, three enzymes were found to be strongly absorbed on the column, so that they could be easily eluted with substrate solution using biospecific counter-ligand. It was thus revealed that covalent binding of such a soluble glucan to aminohexyl-Sepharose provides a useful carrier for separation of $\text{endo}\text{-(1→3)-β-}\text{D}\text{-glucanases}$ lysing yeast cell walls.     

Expression and purification of a recombinant LL-37 from Escherichia coli

Human cathelicidin-derived LL-37 is a 37-residue cationic, amphipathic α-helical peptide. It is an active component of mammalian innate immunity. LL-37 has several biological functions including a broad spectrum of antimicrobial activities and LPS-neutralizing activity. In order to determine the high-resolution three-dimensional structure of LL-37 using NMR spectroscopy, it is important to obtain the peptide with isotopic labels such as ¹⁵N, ¹³C and/or ²H. Since it is less expensive to obtain such a peptide biologically, in this study, we report for the first time a method to express in E. coli and purify LL-37 using Glutathione S-transferase (GST) fusion system. LL-37 gene was inserted into vector pGEX-4T3 and expressed as a GST-LL-37 fusion protein in BL21(DE3) strain. The recombinant GST-LL-37 protein was purified with a yield of 8 mg/l by affinity chromatography and analyzed its biochemical and spectroscopic properties. Factor Xa was used to cleave a 4.5-kDa LL-37 from the GST-LL-37 fusion protein and the peptide was purified using a reverse-phase HPLC on a Vydac C₁₈ column with a final yield of 0.3 mg/l. The protein purified using reverse-phase HPLC was confirmed to be LL-37 by the analyses of Western blot and MALDI-TOF-Mass spectrometry. E. coli cells harboring the expression vector pGEX-4T3-LL-37 were grown in the presence of the ¹⁵N-labeled M9 minimal medium and culture conditions were optimized to obtain uniform ¹⁵N enrichment in the constitutively expressed LL-37 peptide. These results suggest that our production method will be useful in obtaining a large quantity of recombinant LL-37 peptide for NMR studies.     

Screening of recombinant Escherichia coli using activation of green fluorescent protein as an indicator

A novel cloning vector that can be used to identify recombinant Escherichia coli colonies by activation of the green fluorescent protein gene (GFP) was constructed. Screening using the vector does not require special reagents. The recombinant plasmid activates GFP, and the rate of false-positive results is low.     

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.     

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.     

IbpA the small heat shock protein from Escherichia coli forms fibrils in the absence of its cochaperone IbpB

Small heat shock proteins (sHsps) associate with aggregated proteins, changing their physical properties in such a way that chaperone mediated disaggregation becomes much more efficient. In Escherichia coli two small Hsps, IbpA and IbpB, exist. They are 48% identical at the amino acid level, yet their roles in stabilisation of protein aggregates are quite distinct. Here we analysed the biochemical properties of IbpA. We found that IbpA assembles into protofilaments which in turn form mature fibrils. Such fibrils are atypical for sHsps. Interaction of IbpA with either its cochaperone IbpB or an aggregated substrate blocks IbpA fibril formation.

Structured summary

MINT-7876715: ibpA (uniprotkb:P0C054) and ibpA (uniprotkb:P0C054) bind (MI:0407) by molecular sieving (MI:0071)MINT-7888427: ibpB (uniprotkb:P0C058) and ibpB (uniprotkb:P0C058) bind (MI:0407) by molecular sieving (MI:0071)MINT-7888448: ibpA (uniprotkb:P0C054) and ibpA (uniprotkb:P0C054) bind (MI:0407) by electron microscopy (MI:0040)MINT-7888434: ibpB (uniprotkb:P0C058) and ibpB (uniprotkb:P0C058) bind (MI:0407) by electron microscopy (MI:0040)MINT-7888459: ibpA (uniprotkb:P0C054) and ibpA (uniprotkb:P0C054) bind (MI:0407) by fluorescence microscopy (MI:0416)     

Crystal Structure of Metal-Dependent Allantoinase from Escherichia coli

Allantoinase acts as a key enzyme for the biogenesis and degradation of ureides by catalyzing the conversion of (S)-allantoin into allantoate, the final step in the ureide pathway. Despite limited sequence similarity, biochemical studies of the enzyme suggested that allantoinase belongs to the amidohydrolase family. In this study, the crystal structure of allantoinase from Escherichia coli was determined at 2.1 Å resolution. The enzyme consists of a homotetramer in which each monomer contains two domains: a pseudo-triosephosphate-isomerase barrel and a β-sheet. Analogous to other enzymes in the amidohydrolase family, allantoinase retains a binuclear metal center in the active site, embedded within the barrel fold. Structural analyses demonstrated that the metal ions in the active site ligate one hydroxide and six residues that are conserved among allantoinases from other organisms. Functional analyses showed that the presence of zinc in the metal center is essential for catalysis and enantioselectivity of substrate. Both the metal center and active site residues Asn94 and Ser317 play crucial roles in dictating enzyme activity. These structural and functional features are distinctively different from those of the metal-independent allantoinase, which was very recently identified.     

Characterisation of Escherichia coli cell surface by isoelectric equilibrium analysis

A characterisation of the lipopolysaccharide (outermost) layer of Escherichia coli cells has been made by isoelectric equilibrium analysis. Unmodified E. coli cells show a surface isoelectric point (pI) of 5.6. Cells treated with ethyleneimine in order to esterify the carboxyl groups are isoelectric at pH 8.55. When amino groups are blocked the bacterial surface has a pI of 3.85. An analysis of these results suggests that the ionisable groups occurring in the isoelectric zone i.e. the zone amenable to investigation by the isoelectric equilibrium method are: carboxyl groups and amino groups of polysaccharide and protein components. The carboxyl groups have a pK between 3.2 and 4.5 and the amino groups have a pK of 7.5. ε-Amino groups, phenolic hydroxyl groups and guanidyl groups do not occur, and phosphate and amino groups of the phospholipid complex are not detected. The number of thiol groups in the isoelectric zone has been determined using 6,6′-dithiodinicotinic acid. The number of anionogenic and cationogenic groups has been determined. From the density of the negative charges on the surface it is estimated that the isoelectric zone might extend up to 60 Å below the cell surface. The data discussed in this paper relate to the outermost layer of the bacterial cell wall composed of lipopolysaccharide-phospholipid-protein complex. Since reactive groups of the phosphilipid component of the complex have not been detected in the isoelectric zone, it is suggested that the arrangement of lipopolysaccharide phospholipid protein complex is such that the phospholipids are located at a depth of more than 60 Å from the bacterial surface.     

Studies on the damage to Escherichia coli cell membrane caused by different rates of freeze-thawing

Freeze-thawing of Escherichia coli cells caused a release of cell membrane components such as protein, phospholipids and lipopolysaccharides. A greater amount of release and a lesser extent of cell survival were seen in slow freeze-thawing than in rapid freeze-thawing. Several dehydrogenases in the cells were also freed. The mode of release was also dependent on the rate of freeze-thawing.The materials released by slow freeze-thawing were found to be mostly composed of outer membrane components, whereas the materials released by rapid freeze-thawing contained cytoplasmic as well as outer membrane components. The chemical composition of these fragments differed significantly from that of the original membranes. The relative content of cytoplasmic membrane-bound enzymes in these fragments also differed from that of the cytoplasmic membrane.The fragmentation was assumed to have resulted mainly from the crystallization of external water. In slow freeze-thawing, it was considered that the phase separation of the membrane phospholipid bilayer increased the possibility of outer membrane fragmentation. Rapid freeze-thawing caused cytoplasmic membrane damage to the cells as well as to the outer membrane. In rapid freeze-thawing, the effect of phase separation appeared to be small because of rapid passage through the transition temperatures.The presence of 10% glycerol completely inhibited the release of cellular materials and enzymes. Cell survival was maintained at a high level in the glycerol-treated samples whether freeze-thawed slowly or rapidly.     

Immobilization of bioluminescent Escherichia coli cells using natural and artificial fibers treated with polyethyleneimine

Biosensors based on whole-cell bioluminescence have the potential to become a cost-effective alternative to conventional detection methods upon validation of target selectivity and sensitivity. However, quantitative analysis of bioluminescence is greatly hindered due to lack of control over the total number of cells in a suspending culture. In this study, the effect of surface properties of genetically engineered luminous E. coli cells and fibrous matrices on the immobilization capacity and effectiveness under various environmental conditions were characterized. Four different fibers, including cotton, polyester, viscose rayon, and silk, were investigated. Although cell adhesion was observed on untreated viscose and cotton fibers, viscose fiber pretreated with 0.667% polyethyleneimine (PEI) was found capable of immobilizing the most viable E. coli DPD2234 cells, followed by viscose treated with 0.33% and 1% PEI. The cells immobilized on PEI-treated viscose remained viable and yielded 20% or more bioluminescence signals immediately upon contact with the inducer up to 72 h without feeding nutrients to the cells, suggesting that viscose treated with 0.667% PEI could provide a stable immobilization mechanism for bioluminescent E. coli cells with long sensing period, quick response time, and good signal reproducibility.     

The role of the L2 loop in the regulation and maintaining the proteolytic activity of HtrA (DegP) protein from Escherichia coli

The aim of this study was to characterize the role of particular elements of the regulatory loop L2 in the activation process and maintaining the proteolytic activity of HtrA (DegP) from Escherichia coli. We measured the effects of various mutations introduced to the L2 loop’s region (residues 228-238) on the stability of HtrA molecule and its proteolytic activity. We demonstrated that most mutations affected the activity of HtrA. In the case of the following substitutions: L229N, N235I, I238N, the proteolytic activity was undetectable. Thus, the majority of interactions mediated by the studied amino-acid residues seem to play important role in maintaining the active conformation. Formation of contacts between the apical parts (residues 231-234) of the L2 loops within the HtrA trimer, in particular the residues D232, was shown to play a crucial role in the activation process of HtrA. Stabilization of these intermolecular interactions by substitution of D232 with valine caused a stimulation of proteolytic activity whereas deletion of this region abolished the activity. Since the pathogenic E. coli strains require active HtrA for virulence, the apical part of L2 is of particular interest in terms of structure-based drug design for treatment E. coli infections.     

Coupling effects of distal loops on structural stability and enzymatic activity of Escherichia coli dihydrofolate reductase revealed by deletion mutants

Residues distal from the active site in dihydrofolate reductase (DHFR) have regulatory roles in catalytic reaction and also folding stability. The couplings of the distal residues to the ones in the active site have been analyzed using site-directed mutants. To expand our understanding of the structural and functional influences of distal residue mutation, we explored the structural stability and enzymatic activity of deletion mutants. Deletion has greater structural and dynamical impacts on the corresponding part than site-directed mutation does. Thus, deletion amplifies the effects caused by distal mutations, which should make the mutual couplings among the distant residues more apparent. We focused on residues 52, 67, 121, and 145 in the four distinct loops of DHFR. All the single-residue deletion mutants showed marked reduction in stability, except for Δ52 in an αC–βC loop. Double deletion mutants showed that the loop αC–βC has nonadditive couplings with the βF–βG and βG–βH loops regarding stability. Single deletion to the loops αC–βC or βC–βD resulted in considerable activity reduction, demonstrating that the loops couple to the residues near the active site. The four loops were shown to be functionally interdependent from the double deletion experiments.     

Reductive amination by recombinant Escherichia coli: Whole cell biotransformation of 2-keto-3-methylvalerate to l-isoleucine

A whole cell biotransformation system for reductive amination has been studied in recombinant Escherichia coli cells. Reductive amination of 2-keto-3-methylvalerate to l-isoleucine by a two-enzyme-cascade was achieved by overproduction of endogenous l-alanine dependent transaminase AvtA and heterologous l-alanine dehydrogenase from Bacillus subtilis in recombinant E. coli. Up to 100 mM l-isoleucine were produced from 100 mM 2-keto-3-methylvalerate and 100 mM ammonium sulfate. Regeneration of NADH as cofactor in the whole cell system was driven by glucose catabolism. The effects of defined gene deletions in the central carbon metabolism on biotransformation were tested. Strains lacking the NuoG subunit of NADH:ubiquinone oxidoreductase (complex I) or aceA encoding the glyoxylate cycle enzyme isocitrate lyase exhibited increased biotransformation rates.     

Synthesis of a tetrasaccharide related to the repeating unit of the O-antigen from Escherichia coli K-12

Synthesis of a tetrasaccharide related to the repeating unit of the O-antigen from Escherichia coli K-12 is reported in the form of its octyl glycoside. Syntheses of the 1,2-cis glycosidic linkages have been accomplished by using NIS in conjunction with H₂SO₄-silica, and it was found to be stereoselective and productive. The synthesized tetrasaccharide will be utilized as the substrate for galactofuranosyltransferase, WbbI.     

Identification in various chlorate-resistant mutants of a protein involved in the activation of nitrate reductase in the soluble fraction of a chlA mutant of Escherichia coli K-12

We report some properties of Protein PA which has been isolated from the soluble fraction of a chlB mutant after anaerobic growth in the presence of KNO₃. This protein has been identified by its capacity to reactivate nitrate reductase present in the soluble fraction of a chlA mutant by the complementation process. The presence of active Protein PA in the chlB mutant is independent of the presence of oxygen or of nitrate during growth. In contrast, the addition of sodium tungstate to the growth medium leads to the formation of inactive Protein PA which is not able to activate nitrate reductase in the chlA-soluble extract by complementation. Inactive Protein PA has been quantitated immunologically. The partial purification of Protein PA has been achieved from various chlorate-resistant mutants (chlA?chlG). The establishment of particular complementation systems comprising the soluble extracts of chlA or chlB mutants and partially purified Protein PA from soluble fractions of different chlorate-resistant mutants, has allowed the quantitative estimation of this protein. The analysis by ‘rocket immunoelectrophoresis’ using an antiserum specific for Protein PA has shown that inactive Protein PA is present in approximately equivalent amounts in the chlA, chlE, chlG and chlD mutants     

Structural alteration of Escherichia coli Hsp31 by thermal unfolding increases chaperone activity

Escherichia coli Hsp31, encoded by hchA, is a heat-inducible molecular chaperone. We found that Hsp31 undergoes a conformational change via temperature-induced unfolding, generating a high molecular weight (HMW) form with enhanced chaperone activity. Although it has previously been reported that some subunits of the Hsp31 crystal structure show structural heterogeneity with increased hydrophobic surfaces, Hsp31 basically forms a dimer. We found that a C-terminal deletion (CΔ19) of Hsp31 exhibited structurally and functionally similar characteristics to that of the HMW form. Both the CΔ19 and HMW forms achieved a structure with considerably more β-sheets and less α-helices than the native dimeric form, exposing a portion of its hydrophobic surfaces. The structural alterations were determined from its spectral changes in circular dichroism, intrinsic fluorescence of tryptophan residues, and fluorescence of bis-ANS binding to a hydrophobic surface. Interestingly, during thermal transition, the dimeric Hsp31 undergoes a conformational change to the HMW species via the CΔ19 structure, as monitored with near-UV CD spectrum, implying that the CΔ19 resembles an intermediate state between the dimer and the HMW form. From these results, we propose that Hsp31 transforms itself into a fully functional chaperone by altering its tertiary and quaternary structures.     

alr0882 encoding a hypothetical protein of Anabaena PCC7120 protects Escherichia coli from nutrient starvation and abiotic stresses

This study is the first to demonstrate cloning of alr0882, a hypothetical protein gene of Anabaena PCC7120, its heterologous expression in Escherichia coli strain LN29MG1655 (?uspA::Kan) and functional complementation of abiotic stress tolerance of E. coli UspA. The recombinant vector pGEX-5X-2-alr0882 was used to transform ?uspA E. coli strain. The IPTG induced expression of a 56.6 kDa GST fusion protein was visualized on SDS–PAGE and attested by immunoblotting. E. coli ?uspA strain harboring pGEX-5X-2-alr0882 when grown under carbon, nitrogen, phosphorus and sulphur limitation and abiotic stresses e.g. nalidixic acid, cycloserine, CdCl₂, H₂O₂, UV-B, phenazine methosulphate (PMS), dinitrophenol (DNP), NaCl, heat, carbofuron and CuCl₂ demonstrated about 22.6–51.6% increase in growth over the cells transformed with empty vector. Expression of alr0882 gene in mutant E. coli as measured by semi-quantitative RT-PCR at different time points under selected treatments reaffirmed its role in tolerance against stresses employed in this study. Thus the results of this study vividly demonstrated that the novel protein alr0882, although appreciably different from the known UspA of E. coli, offers tolerance to abiotic stresses hence holds potential for the development of transgenic cyanobacteria.     

Quantitative in vitro assay to evaluate the capability of yeast cell wall fractions from Trichosporon mycotoxinivorans to selectively bind gram negative pathogens

Yeast cell wall fractions have been proposed to bind enteropathogenic bacteria. The aim of this study was to develop a quantitative assay by measuring the optical density as growth parameter of adhering bacteria. The exponential growth phase of adhering bacteria was determined by optical density reading and compared with the colony count (CFU/mL). A linear regression was compiled and the bacterial number bound to the yeast cell wall product could be determined. Further focus was the investigation of a yeast cell wall from strain Trichosporon mycotoxinivorans (MTV) for its ability to bind gram negative Salmonella, E. coli and Campylobacter strains and gram positive probiotic bacteria of the genera lactobacilli and bifidobacteria as well as gram positive Clostridium perfringens quantitatively. The gram negative probiotic strain E. coli Nissle 1917 was also investigated. Seven out of 10 S. Typhimurium and S. Enteritidis strains adhered to the cell wall product with an amount between 10³ and 10⁴ CFU/10 μg. Four out of 7 E. coli strains showed an average binding capability (10² CFU/10 µg) whereas 4 × 10³E. coli F4 cells bound per 10 μg yeast cell wall. E. coli 0149 K91, E. coli 0147 K89, C. jejuni and C. perfringens as well the genera lactobacilli and bifidobacteria did not bind to the yeast cell wall. E. coli Nissle 1917 was bound with 2 × 10² CFU/10 μg. These results demonstrate that cell wall from MTV can be used to differentially bind E. coli spp. and Salmonella spp. up to 8 × 10⁴ CFU/10 μg. Thus certain yeast cell walls may prevent enteric infections caused by selective bacteria. This methodical approach would be an accurate tool in the feed industry for quality control of yeast cell wall products.