Strain JM101 Research Articles

Pathway engineering for efficient biosynthesis of violaxanthin in Escherichia coli.

Carotenoids are naturally synthesized in some species of bacteria, archaea, and fungi (including yeasts) as well as all photosynthetic organisms. Escherichia coli has been the most popular bacterial host for the heterologous production of a variety of carotenoids, including even xanthophylls unique to photosynthetic eukaryotes such as lutein, antheraxanthin, and violaxanthin. However, conversion efficiency of these epoxy-xanthophylls (antheraxanthin and violaxanthin) from zeaxanthin remained substantially low. We here examined several factors affecting their productivity in E. coli. Two sorts of plasmids were introduced into the bacterial host, i.e., a plasmid to produce zeaxanthin due to the presence of the Pantoea ananatis crtE, crtB, crtI, crtY, and crtZ genes in addition to the Haematococcus pluvialis IDI gene, and one containing each of zeaxanthin epoxidase (ZEP) genes originated from nine photosynthetic eukaryotes. It was consequently found that paprika (Capsicum annuum) ZEP (CaZEP) showed the highest conversion activity. Next, using the CaZEP gene, we performed optimization experiments in relation to E. coli strains as the production hosts, expression vectors, and ribosome-binding site (RBS) sequences. As a result, the highest productivity of violaxanthin (231 μg/g dry weight) was observed, when the pUC18 vector was used with CaZEP preceded by a RBS sequence of score 5000 in strain JM101(DE3).

Salmonella Utilizes D-Glucosaminate via a Mannose Family Phosphotransferase System Permease and Associated Enzymes

Salmonella enterica is a globally significant bacterial food-borne pathogen that utilizes a variety of carbon sources. We report here that Salmonella enterica subsp. enterica serovar Typhimurium (S. Typhimurium) uses d-glucosaminate (2-amino-2-deoxy-d-gluconic acid) as a carbon and nitrogen source via a previously uncharacterized mannose family phosphotransferase system (PTS) permease, and we designate the genes encoding the permease dgaABCD (d-glucosaminate PTS permease components EIIA, EIIB, EIIC, and EIID). Two other genes in the dga operon (dgaE and dgaF) were required for wild-type growth of S. Typhimurium with d-glucosaminate. Transcription of dgaABCDEF was dependent on RpoN (σ(54)) and an RpoN-dependent activator gene we designate dgaR. Introduction of a plasmid bearing dgaABCDEF under the control of the lac promoter into Escherichia coli strains DH5α, BL21, and JM101 allowed these strains to grow on minimal medium containing d-glucosaminate as the sole carbon and nitrogen source. Biochemical and genetic data support a catabolic pathway in which d-glucosaminate, as it is transported across the cell membrane, is phosphorylated at the C-6 position by DgaABCD. DgaE converts the resulting d-glucosaminate-6-phosphate to 2-keto-3-deoxygluconate 6-phosphate (KDGP), which is subsequently cleaved by the aldolase DgaF to form glyceraldehyde-3-phosphate and pyruvate. DgaF catalyzes the same reaction as that catalyzed by Eda, a KDGP aldolase in the Entner-Doudoroff pathway, and the two enzymes can substitute for each other in their respective pathways. Examination of the Integrated Microbial Genomes database revealed that orthologs of the dga genes are largely restricted to certain enteric bacteria and a few species in the phylum Firmicutes.

A method for DNA-based detection of E. coli O157:H7 in a proteinous background using piezoelectric-excited cantilever sensors

We show for the first time the detection of ~700 E. coli O157:H7 (EC) in both buffer and proteinous sample (beef wash) using the virulent gene stx2 as a sensing molecule immobilized on piezoelectric-excited millimeter cantilever (PEMC) sensors without a sample preparation step. Genomic DNA of EC suspended in buffer or beef wash was extracted using a 30-minute procedure, or using a commercial extraction kit, and then was exposed to the sensor immobilized with a 19-mer probe that is complement to stx2 gene. As hybridization occurred, the resonant frequency of the cantilever sensor decreased due to the increased attached mass indicating the presence of stx2 gene in the sample. Wild strain JM101 subjected to the same preparation and procedure did not induce a hybridization response, nor did the genomic extract of EC with a bare sensor. PEMC sensor responses to diluted genomic extracts indicate that a much lower concentration of 700 cells is detectable. In order to compare the results with antibody-based detection, samples with EC at 2500 cells per mL were exposed to antibody-immobilized PEMC sensors which gave reproducible responses confirming previous experiments with such samples.

Metabolic flux ratio analysis and multi-objective optimization revealed a globally conserved and coordinated metabolic response of E. coli to paraquat-induced oxidative stress

The ability of a microorganism to adapt to changes in the environment, such as in nutrient or oxygen availability, is essential for its competitive fitness and survival. The cellular objective and the strategy of the metabolic response to an extreme environment are therefore of tremendous interest and, thus, have been increasingly explored. However, the cellular objective of the complex regulatory structure of the metabolic changes has not yet been fully elucidated and more details regarding the quantitative behaviour of the metabolic flux redistribution are required to understand the systems-wide biological significance of this response. In this study, the intracellular metabolic flux ratios involved in the central carbon metabolism were determined by fractional (13)C-labeling and metabolic flux ratio analysis (MetaFoR) of the wild-type E. coli strain JM101 at an oxidative environment in a chemostat. We observed a significant increase in the flux through phosphoenolpyruvate carboxykinase (PEPCK), phosphoenolpyruvate carboxylase (PEPC), malic enzyme (MEZ) and serine hydroxymethyltransferase (SHMT). We applied an ε-constraint based multi-objective optimization to investigate the trade-off relationships between the biomass yield and the generation of reductive power using the in silico iJR904 genome-scale model of E. coli K-12. The theoretical metabolic redistribution supports that the trans-hydrogenase pathway should not play a direct role in the defence mounted by E. coli against oxidative stress. The agreement between the measured ratio and the theoretical redistribution established the significance of NADPH synthesis as the goal of the metabolic reprogramming that occurs in response to oxidative stress. Our work presents a framework that combines metabolic flux ratio analysis and multi-objective optimization to investigate the metabolic trade-offs that occur under varied environmental conditions. Our results led to the proposal that the metabolic response of E. coli to paraquat-induced oxidative stress is globally conserved and coordinated.

New insights into Escherichia coli metabolism: carbon scavenging, acetate metabolism and carbon recycling responses during growth on glycerol

BackgroundGlycerol has enhanced its biotechnological importance since it is a byproduct of biodiesel synthesis. A study of Escherichia coli physiology during growth on glycerol was performed combining transcriptional-proteomic analysis as well as kinetic and stoichiometric evaluations in the strain JM101 and certain derivatives with important inactivated genes.ResultsTranscriptional and proteomic analysis of metabolic central genes of strain JM101 growing on glycerol, revealed important changes not only in the synthesis of MglB, LamB and MalE proteins, but also in the overexpression of carbon scavenging genes: lamB, malE, mglB, mglC, galP and glk and some members of the RpoS regulon (pfkA, pfkB, fbaA, fbaB, pgi, poxB, acs, actP and acnA). Inactivation of rpoS had an important effect on stoichiometric parameters and growth adaptation on glycerol. The observed overexpression of poxB, pta, acs genes, glyoxylate shunt genes (aceA, aceB, glcB and glcC) and actP, suggested a possible carbon flux deviation into the PoxB, Acs and glyoxylate shunt. In this scenario acetate synthesized from pyruvate with PoxB was apparently reutilized via Acs and the glyoxylate shunt enzymes. In agreement, no acetate was detected when growing on glycerol, this strain was also capable of glycerol and acetate coutilization when growing in mineral media and derivatives carrying inactivated poxB or pckA genes, accumulated acetate. Tryptophanase A (TnaA) was synthesized at high levels and indole was produced by this enzyme, in strain JM101 growing on glycerol. Additionally, in the isogenic derivative with the inactivated tnaA gene, no indole was detected and acetate and lactate were accumulated. A high efficiency aromatic compounds production capability was detected in JM101 carrying pJLBaroGfbrtktA, when growing on glycerol, as compared to glucose.ConclusionsThe overexpression of several carbon scavenging, acetate metabolism genes and the absence of acetate accumulation occurred in JM101 cultures growing on glycerol. To explain these results it is proposed that in addition to the glycolytic metabolism, a gluconeogenic carbon recycling process that involves acetate is occurring simultaneously in this strain when growing on glycerol. Carbon flux from glycerol can be efficiently redirected in JM101 strain into the aromatic pathway using appropriate tools.

Detection of single nucleotide polymorphisms in the conserved ESTs regions of Gossypium arboreum

Cellulase is a group of enzymes (endoglucanase, exoglucanase and beta-glucosidase) required for cellulosic feedstock hydrolysis during bioethanol production. The use of recombinant cellulase is a strategy to reduce the enzyme cost. In this context, the present work describes the construction of a cellulase expression vector (pEglABglA), which allowed constitutive co-expression of endoglucanase A (EglA) from an endophytic Bacillus pumilus and the hyperthermophilic β-glucosidase A (BglA) from Fervidobacterium sp. in Escherichia coli . When compared to the non-modified strain DH5α, the recombinant Escherichia coli DH5α (pEglABglA) reduced fivefold the viscosity of the carboxymethylcellulose medium (CMC-M). Also, it presented almost 30-fold increase in reducing sugar released from CMC-M, enabling the recombinant strain to grow using CMC as the sole carbon and energy source. When cultivated in rich media, specific growth rates of recombinant E. coli strains BL21, JM101 and Top10 were higher than those of DH5α and DH10B strains. The constructed plasmid (pEglABglA) can be used as backbone for further cellulase gene addition, which may enhance even more E. coli cellulolytic capacity and growth rate.

Transcription Analysis of Central Metabolism Genes in Escherichia coli. Possible Roles of σ38 in Their Expression, as a Response to Carbon Limitation

The phosphoenolpyruvate: carbohydrate transferase system (PTS) transports glucose in Escherichia coli. Previous work demonstrated that strains lacking PTS, such as PB11, grow slow on glucose. PB11 has a reduced expression of glycolytic, and upregulates poxB and acs genes as compared to the parental strain JM101, when growing on glucose. The products of the latter genes are involved in the production of AcetylCoA. Inactivation of rpoS that codes for the RNA polymerase σ38 subunit, reduces further (50%) growth of PB11, indicating that σ38 plays a central role in the expression of central metabolism genes in slowly growing cells. In fact, transcription levels of glycolytic genes is reduced in strain PB11rpoS − as compared to PB11. In this report we studied the role of σ70 and σ38 in the expression of the complete glycolytic pathway and poxB and acs genes in certain PTS− strains and their rpoS − derivatives. We determined the transcription start sites (TSSs) and the corresponding promoters, in strains JM101, PB11, its derivative PB12 that recovered its growth capacity, and in their rpoS− derivatives, by 5′RACE and pyrosequencing. In all these genes the presence of sequences resembling σ38 recognition sites allowed the proposition that they could be transcribed by both sigma factors, from overlapping putative promoters that initiate transcription at the same site. Fourteen new TSSs were identified in seventeen genes. Besides, more than 30 putative promoters were proposed and we confirmed ten previously reported. In vitro transcription experiments support the functionality of putative dual promoters. Alternatives that could also explain lower transcription levels of the rpoS − derivatives are discussed. We propose that the presence if real, of both σ70 and σ38 dependent promoters in all glycolytic genes and operons could allow a differential transcription of these central metabolism genes by both sigma subunits as an adaptation response to carbon limitation.

Production and characterization of poly-(3-hydroxybutyrate) from recombinant Escherichia coli grown on cheap renewable carbon substrates

Although the biopolymer poly-(3-hydroxybutyrate), P[3HB], presents physicochemical properties that make it an alternative material to conventional plastics, its biotechnological production is quite expensive. As carbon substrates contribute greatly to P[3HB] production cost, the utilization of a cheaper carbon substrate and less demanding micro-organisms should decrease its cost. In the present study a 2(3) factorial experimental design was applied, aiming to evaluate the effects of using hydrolysed corn starch (HCS) and soybean oil (SBO) as carbon substrates, and cheese whey (CW) supplementation in the mineral medium (MM) on the responses, cell dried weigh (DCW), percentage P[3HB] and mass P[3HB] by recombinant Escherichia coli strains JM101 and DH10B, containing the P[3HB] synthase genes from Cupriavidus necator (ex-Ralstonia eutropha). The analysis of effects indicated that the substrates and the supplement and their interactions had positive effect on CDW. Statistically generated equations showed that, at the highest concentrations of HCS, SO and CW, theoretically it should be possible to produce about 2 g L(- 1) DCW, accumulating 50% P[3HB], in both strains. To complement this study, the strain that presented the best results was cultivated in MM added to HCS, SBO and CW (in best composition observed) and complex medium (CM) to compare the obtained P[3HB] in terms of physicochemical parameters. The obtained results showed that the P[3HB] production in MM (1.29 g L( -1)) was approximately 20% lower than in CM (1.63 g L(- 1)); however, this difference can be compensated by the lower cost of the MM achieved by the use of cheap renewable carbon sources. Moreover, using differential scanning calorimetry and thermogravimetry analyses, it was observed that the polymer produced in MM was the one which presented physicochemical properties (Tg and Tf) that were more similar to those found in the literature for P[3HB].

New Insights into the Role of Sigma Factor RpoS as Revealed in Escherichia coli Strains Lacking the Phosphoenolpyruvate:Carbohydrate Phosphotransferase System

It has been demonstrated that about 10% of the Escherichia coli genes are under direct or indirect control of RpoS. Therefore, Weber et al. [2005] proposed that this sigma subunit should be considered a second vegetative sigma factor under non-optimal growth conditions. In this report we demonstrate that in the phosphoenolpyruvate:carbohydrate phosphotransferase system-deficient (PTS^–) derivatives, PB11 and PB12 of strain JM101 that permanently grow slowly on glucose, the inactivation of rpoS resulted in decreased growth rates of 50 and 10%, respectively. Real-time PCR (RT-PCR) analysis confirmed the important role of this sigma factor in the PTS^– strains and allowed the identification of 19 genes including almost all the glycolytic genes, not previously reported, to be at least partially dependent on RpoS. The transcription level of gpp, spoT, ppa and ndk whose products are involved in ppGpp metabolism was upregulated in strain PB12 as compared to the parental strains PB11 and JM101. In the PTS^– strains, at least three of these genes (gpp, spoT and ppa) were mainly or partially regulated by RpoS which is known to require ppGpp for activation, while only gpp was highly RpoS-dependent in the parental PTS⁺ strain JM101. The role of RpoS in the transcription of these genes was analyzed and evidence that the expression of this group of genes could be regulated by a common factor in addition to RpoS was discussed.

Pigment Binding, Fluorescence Properties, and Oligomerization Behavior of Lhca5, a Novel Light-harvesting Protein

A new potential light-harvesting protein, named Lhca5, was recently detected in higher plants. Because of the low amount of Lhca5 in thylakoid membranes, the isolation of a native Lhca5 pigment-protein complex has not been achieved to date. Therefore, we used in vitro reconstitution to analyze whether Lhca5 binds pigments and is actually an additional light-harvesting protein. By this approach we could demonstrate that Lhca5 binds pigments in a unique stoichiometry. Analyses of pigment requirements for light-harvesting complex formation by Lhca5 revealed that chlorophyll b is the only indispensable pigment. Fluorescence measurements showed that ligated chlorophylls and carotenoids are arranged in a way that allows directed energy transfer within the light-harvesting complex. Reconstitutions of Lhca5 together with other Lhca proteins resulted in the formation of heterodimers with Lhca1. This result demonstrates that Lhca5 is indeed a protein belonging to the light-harvesting antenna of photosystem I. The properties of Lhca5 are compared with those of previously characterized Lhca proteins, and the consequences of an additional Lhca protein for the composition of the light-harvesting antenna of photosystem I are discussed in view of the recently published photosystem I structure of the pea.

Role of Pyruvate Oxidase in Escherichia coli Strains Lacking the Phosphoenolpyruvate:Carbohydrate Phosphotransferase System

We report a study to determine the role of pyruvate oxidase among Escherichia coli isogenic strains with active and inactive phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS). Strain PB11, displaying a specific growth rate (µ) in glucose minimal medium of 0.1 h^–1 is a ptsHI, crr operon deletion derivative of wild-type JM101 (displaying a µ of 0.70 h^–1). Strain PB12 is a spontaneous mutant obtained from PB11 after selection for its capacity to grow on glucose with a µ of 0.40 h^–1. In minimal medium cultures supplemented with glucose plus acetate, strain JM101 displayed preferential consumption of glucose, whereas strains PB11 and PB12 did not display glucose catabolic repression of acetate consumption. Inactivation of poxB caused a severe reduction in growth rate in strain PB11 when grown in the fermentor with medium containing glucose or glucose plus acetate, whereas under the same conditions poxB^–derivative strains of JM101 and PB12 were not affected. Relative transcript levels for 29 genes related to poxB transcriptional regulation and central metabolism were determined using RT-PCR. This analysis revealed 2-fold lower transcript levels for genes encoding subunits of the pyruvate dehydrogenase complex (Pdh) in strain PB11 and 4- to 6-fold higher transcript levels for poxB in strains PB11 and PB12, when compared to JM101. In addition, in the PTS^– strains, upregulation of the poxB transcription factors rpoS, soxS and marA, was detected. The results presented here strongly suggest that AcCoA is mainly synthesized from acetate produced by pyruvate oxidase in strain PB11, whereas in strains JM101 and PB12, AcCoA is synthesized preferentially from pyruvate by Pdh.

Cloning and Expression of a Prokaryotic Enzyme, Arginine Deiminase, from a Primitive Eukaryote Giardia intestinalis

Arginine deiminase (EC 3.5.3.6) catalyzes the irreversible catabolism of arginine to citrulline in the arginine dihydrolase pathway. This pathway has been regarded as restricted to prokaryotic organisms but is an important source of energy to the primitive protozoan Giardia intestinalis. In this paper we report the cloning and expression of the arginine deiminase gene from this parasite. Degenerate oligonucleotides based on amino acid sequences of tryptic peptides from the purified protein were used to amplify a portion of the arginine deiminase gene. This was then used as a probe to screen HindIII and PstI "mini" libraries to obtain two overlapping clones that contained the arginine deiminase gene. The open reading frame encoded 581 amino acids including all of the tryptic peptides that were sequenced and corresponded to a molecular mass of 67 kDa. Northern blot analysis identified a single 1.8-kilobase transcript in both trophozoites and encysting cells. Arginine deiminase was successfully expressed in Escherichia coli and purified to homogeneity. The recombinant protein was found to have characteristics comparable with those of the native enzyme.

Enhancement of extracellular production of a Cellulomonas fimi exoglucanase in Escherichia coli by the reduction of promoter strength

The enzymatic approach to the treatment of cellulosic wastes depends on the availability of a cost-effective means for the production of cellulases. We have engineered an excretion construct, tacIQpar8cex, to investigate the extracellular production of a Cellulomonas fimi exoglucanase (Exg) in Escherichia coli. The overall yield of Exg expressed by the culture of JM101 (tacIQpar8cex) was 2–11 times higher than that obtained using other systems. Over 20% of the activity was detected in the medium. When the culture was induced with IPTG, the overall production of Exg dropped dramatically. The lower yield was found to be caused by both rapid cell death and plasmid curing. A derivative of tacIQpar8cex containing the weaker lacUV5 promoter, designated lacUV5par8cex, was constructed to enhance excretion of Exg from strain JM101. Even with IPTG induction, the JM101 (lacUV5par8cex) culture was found to show a high level of cell viability and plasmid stability as well as the ability to provide efficient expression and excretion of Exg. Upon IPTG induction for 12 h, the activity and specific activity of the excreted Exg obtained from the lacUV5par8cex construct were 143 U ml −1 and 793 U mg −1 protein, respectively, which are 2–5 times higher than that detected from the tacIQpar8cex construct and from the best construct expressing the same gene reported previously.

Scanning Alanine Mutagenesis and De-peptidization of a Candida albicans Myristoyl-CoA:ProteinN-Myristoyltransferase Octapeptide Substrate Reveals Three Elements Critical for Molecular Recognition

Candida albicans produces a single myristoyl-CoA:protein N-myristoyltransferase (Nmt) that is essential for its viability. An ADP-ribosylation factor (Arf) is included among the few cellular protein substrates of this enzyme. An octapeptide (GLYASKLS-NH2) derived from a N-terminal Arf sequence was used as the starting point to identify elements critical for recognition by the acyltransferases's peptide-binding site. In vitro kinetic studies, employing purified Nmt and a panel of peptides with single Ala substitutions at each position of GLYASKLS-NH2, established that its Gly1, Ser5, and Lys6 residues play predominant roles in binding. ALYASKLS-NH2 was found to be an inhibitor competitive for peptide (Ki = 15.3 +/- 6.4 microM) and noncompetitive for myristoyl-CoA (Ki = 31.2 +/- 0.7 microM). A survey of 26 derivatives of this inhibitor, representing (i) a complete alanine scan, (ii) progressive C-terminal truncations, and (iii) manipulation of the physical-chemical properties of its residues 1, 5, and 6, confirmed the important stereochemical requirements for the N-terminal amine, the beta-hydroxyl of Ser5, and the epsilon-amino group of Lys6. Remarkably, replacement of the the N-terminal tetrapeptide of ALYASKLS-NH2 with an 11-aminoundecanoyl group produced a competitive inhibitor, 11-aminoundecanoyl-SKLS-NH2, that was 38-fold more potent (Ki = 0.40 +/- 0.03 microM) than the starting octapeptide. Removing the primary amine (undecanoyl-SKLS-NH2), or replacing it with a methyl group (dodecanoyl-SKLS-NH2), resulted in 26- and 34-fold increases in IC50, confirming the important contribution of the amine to recognition. Removal of LeuSer from the C terminus (11-aminoundecanoyl-SK-NH2) yielded a competitive dipeptide inhibitor with a Ki (11.7 +/- 0.4 microM) equivalent to that of the starting octapeptide, ALYASKLS-NH2. Substitution of Ser with homoserine, cis-4-hydroxyproline, or tyrosine reduces potency by 3-70-fold, emphasizing the requirement for proper presentation of the hydroxyl group in the dipeptide inhibitor. Substituting D- for L-Lys decreases its inhibitory activity >100-fold, while deletion of the epsilon-amino group (Nle) or masking its charge (epsilon-N-acetyl-lysine) produces 4-7-fold attenuations. L-His, but not its D-isomer, can fully substitute for L-Lys, producing a competitive dipeptide inhibitor with similar potency (Ki = 11.9 +/- 1.0 microM). 11-Aminoundecanoyl-SK-NH2 and 11-aminoundecanoyl-SH-NH2 establish that a simple alkyl backbone can maintain an appropriate distance between three elements critical for recognition by the fungal enzyme's peptide-binding site: a simple omega-terminal amino group, a beta-hydroxyl, and an epsilon-amino group or an imidazole. These compounds contain one peptide bond and two chiral centers, suggesting that it may be feasible to incorporate these elements of recognition, or functionally equivalent mimics, into a fully de-peptidized Nmt inhibitor.

The Role of the Variable Region in Tet Repressor for Inducibility by Tetracycline

A set of deletions and substitutions to alanine was introduced into the loop separating helices alpha8 and alpha9 of Tn10 Tet repressor (TetR). This region appears as an unstructured loop in the crystal structure of the TetR(D).([Mg-tc]+)2 complex and is the only internal segment of variable length in an alignment of Tet repressors from seven different resistance determinants. In vivo analysis of 10 mutants shows that this loop is important for inducibility by tetracycline (tc), whereas DNA binding is not or only marginally affected. All deletions have an induction-deficient TetRS phenotype, but the corresponding substitutions do not or only slightly affect inducibility. The purified mutant TetR proteins have a reduced affinity for tc in vitro that correlates with their lack of inducibility. The association rate of [Mg-tc]+ to the TetR mutants is enhanced. Since none of the mutated residues contacts tc directly in the crystal structure, we propose that the length of the loop is important for the structural transition between a closed, tc binding and an open, operator binding conformation of TetR. We propose that the deletions in the loop shift the equilibrium between both forms toward the open, operator binding conformation.

1] Generation and characterization of cellular retinoic acid-binding proteins from Escherichia coli expression systems

This chapter describes the generation and characterization of cellular retinoic acid-binding proteins from Escherichia coli ( E. Coli ) expression systems. The cellular retinoic acid-binding proteins (CRABP)-II is expressed in the protease-deficient E. coli strain BL21(DE3) ( lon - ompT - ). This prevents proteolysis of the CRABP-II, which has been observed in other E. coli strains such as JM101. CRABP-I has been successfully expressed in E. coli strains BL21(DE3) and JM101. Significant expression can be obtained in a variety of media. The enhanced solubilization of CRABP expressed in a supplemented, M9-based medium. The expression and purification of CRABP can be evaluated by denaturing SDS–polyacrylamide gel electrophoresis (SDS–PAGE) analysis on a 15% (w/v) polyacrylamide gel followed by Coomassie staining. Bacterial samples for SDS–PAGE analysis should be processed quickly because insults to the bacteria may artifactually induce the recA promoter. Both isoforms of CRABP exhibit an absorption peak at 280 nm typical of tryptophan-containing proteins. Analysis of purified CRABP by sequential Edman degradation can be a measure of protein purity, and should reproduce the expected sequence.

Expression of Extracellular Ligand-Binding Domain of Murine Guanylate Cyclase/Atrial Natriuretic Factor Receptor cDNA in Escherichia coli

The membrane-bound form of guanylate cyclase/atrial natriuretic factor receptor (GC/ANF-R) is a 135 kDa transmembrane glycoprotein which binds ANF with high affinity. We have expressed the extracellular ligand-binding domain of murine guanylate cyclase ANF-R (GC/ANFR-LBD) cDNA in Escherichia coli. The cDNA encoding the extracellular ANF-binding domain (nucleotide positions covering from 432-1755 base pair) of GC/ANF-R was amplified by polymerase chain reaction, cloned into BamHI site of pGEX-3X prokaryotic expression vector and was transfected into E. coli, strain JM101. After isopropyl-β-D-thiogalactopyranoside (IPTG) induction of bacterial cells, the GC/ANFR-LBD was expressed as the glutathione-S-transferase (GST) fusion protein, yielding a molecular mass of 70 kDa. The expressed fusion protein was characterized for binding affinity to both full length and truncated ANF molecules. After expression in E. coli, the binding 125I-ANF to the extracellular region of GC/ANF-R was similar and corresponded to the pharmacological class of native receptor protein. The 70 kDa fusion product was purified as a predominant single protein band by glutathione-affinity chromatography. These findings establish that E. coli may be utilized as an effective heterologous model system to delineate the structure-function analysis of guanylate cyclase-coupled ANF receptor molecules.

Biomass, Nutrient and Product Models for Nutrient-limited Batch Growth of Recombinant E. Coli

Both the Monod and the logistic models for single-nutrient-limited growth and two models for two-nutrient-limited growth were were fitted to data from the growth of recombinant E. coli as well as host strains, under various conditions of nutrient concentration and nutrient feeding in complex medium. The biomass data for the growth of strain JM101 in L-broth plus glycerol as the energy source fits best to a simple Monod model, whereas with acetate as an energy source, it fits best to a logistic model. The data from the growth of E. coli HB101 in L-broth with initial glucose is best described by a model in which the growth rate is limited by glucose and the acetate derived from it. The biomass data for the growth of strain JM101 in L-broth plus glucose feeding fits best to a model with two noncomplementary, sequential nutrients.

A novel technique for the measurement of disruption in high‐pressure homogenization: Studies on E. coli containing recombinant inclusion bodies

The high-pressure homogenization of Escherichia coli, strain JM101, containing inclusion bodies of recombinant porcine somatotropin was investigated. A novel technique employing an analytical disc centrifuge was used to monitor the disruption. This a direct technique which measures cell disintegration rather than soluble protein release. The technique is particularly suited to measurements where the disruption approaches 100%. The disk centrifuge provides a size distribution of the homogenate, and furnishes evidence for the preferential disruption of larger cells. For E. coli containing inclusion bodies, and increase in the cell feed concentration from 145 g/L (wet weight) to 330 g/L resulted is poorer homogenization. Poorer disruption was also obtained by lowering the feed temperature from 20 degrees C to 5 degrees C. Only slight variations in performance were obtained by increasing the feed pH from 7.5 to 9.0 or by storing the feed at 4 degrees C for 24 h prior to disruption. Comparison with uninduced E. coli strain JM101, showed that the disruption obtained is higher for bacteria containing a recombinant inclusion body.

Variations in the activity of glutathione reductase and the cellular glutathione content in relation to sensitivity to methylviologen in Escherichia coli

To study the function of glutathione reductase and glutathione in Escherichia coli the coding sequence of the bacterial glutathione reductase gene ( gor gene) was cloned into the vector pBR322, and the gor gene was expressed under the control of the promoter of the tetracycline-resistance gene ( tet gene) in different Escherichia coli strains. Cells of the gor-mutant strain SG5 containing the vector pBR322 (SG5:pBR322) had no detectable glutathione reductase activity and a significantly lower total glutathione (GSH + GSSG) content relative to control cells of the strain JM101 (JM101: pBR322). The gor mutant cells were less sensitive to inhibition by methylviologen (as defined by changes in growth) than cells of the strain JM101. Elevated levels of both glutathione reductase activity and the total glutathione content (GSH + GSSG) were found when the gor gene was expressed in cells of the gor-mutant strain SG5 (SG5:pJIK1). Thus the activity of glutathione reductase is essential in order to maintain a high glutathione content. Furthermore, cells of the strain SG5: pJIK1 showed an increased sensitivity to methylviologen compared to cells of the gor mutant containing the vector pBR322 alone without the cloned gor gene insert (SG5:pBR322). In all experiments, the glutathione pool (GSH + GSSG) of bacterial cells was 90% reduced. In methylviologen-sensitive sodB mutant cells lacking iron superoxide dismutase activity (QC773:pBR322) overexpression of the cloned gor gene resulted in an elevated level of glutathione reductase activity which partially protected sodB mutant cells (QC773:pJIK1) against methylviologen toxicity. In sodB mutant cells expressing the gor gene (QC773:pJIK1) protection by glutathione reductase was, however, less effective than protection provided by expression of the iron superoxide dismutase gene ( sodB gene) in these mutant cells (QC773:pJIK2). In sodA mutant cells lacking manganese superoxide dismutase activity but expressing the cloned gor gene (QC772:pJIK1) increased cellular glutathione reductase activity did not provide protection against methylviologen.