Opposite roles of domains 2 + 3 of Escherichia coli EF-Tu and Bacillus stearothermophilus EF-Tu in the regulation of EF-Tu GTPase activity

The effect of noncatalytic domains 2 + 3 on the intrinsic activity and thermostability of the EF-Tu GTPase center was evaluated in experiments with isolated domains 1 and six chimeric variants of mesophilic Escherichia coli (Ec) and thermophilic Bacillus stearothermophilus (Bst) EF-Tus. The isolated catalytic domains 1 of both EF-Tus displayed similar GTPase activities at their optimal temperatures. However, noncatalytic domains 2 + 3 of the EF-Tus influenced the GTPase activity of domains 1 differently, depending on the domain origin. Ecdomains 2 + 3 suppressed the GTPase activity of the Ecdomain 1, whereas those of BstEF-Tu stimulated the Bstdomain 1 GTPase. Domain 1 and domains 2 + 3 of both EF-Tus positively cooperated to heat-stabilize their GTPase centers to attain optimal activity at a temperature close to the optimal growth temperature of either organism. This can be explained by a stabilization effect of domains 2 + 3 on α-helical regions of the G-domain as revealed by CD spectroscopy.     

Secretion of β-lactamase by Escherichia coli in vivo and in vitro: effect of cerulenin

The effect of cerulenin on the production of -lactamase and other periplasmic proteins was studied in Escherichia coli IA199 carrying plasmid pBR322. Cerulenin (10 to 25 g/ml) had almost no effect on the growth rate of E. coli but it decreased the amount of -lactamase and other periplamic proteins in shock fluid. Higher amounts of the antibiotic (40 to 100 g/ml)decreased turbidity and almost completely prevented synthesis of -lactamase and other periplasmic proteins. Cerulenin decreased incorporation of l-[³⁵S]methionine into membranes during growth as well. Spheroplasts secreted -lactamase into the external medium, but during a 3-h incubation in the presence of cerulenin (25 g/ml) this secretion was prevented by more than 90%. -Lactamase was secreted into the isolated membrane vesicles from E. coli IA199. However, only 5% of the total amount of pre--lactamase was secreted and processed by the membranes in vitro. Cerulenin did not prevent processing in vitro but the membranes prepared from the cells grown in the presence of cerulenin (25 g/ml) did not catalyze processing of pre--lactamase at all. Membrane preparations from Bacillus subtilis did not process pre--lactamase either in the absence or in the presence of cerulenin.     

Redefining Escherichia coli σ(70) promoter elements: -15 motif as a complement of the -10 motif

Classical elements of σ(70) bacterial promoters include the -35 element ((-35)TTGACA(-30)), the -10 element ((-12)TATAAT(-7)), and the extended -10 element ((-15)TG(-14)). Although the -35 element, the extended -10 element, and the upstream-most base in the -10 element ((-12)T) interact with σ(70) in double-stranded DNA (dsDNA) form, the downstream bases in the -10 motif ((-11)ATAAT(-7)) are responsible for σ(70)-single-stranded DNA (ssDNA) interactions. In order to directly reflect this correspondence, an extension of the extended -10 element to a so-called -15 element ((-15)TGnT(-12)) has been recently proposed. I investigated here the sequence specificity of the proposed -15 element and its relationship to other promoter elements. I found a previously undetected significant conservation of (-13)G and a high degeneracy at (-15)T. I therefore defined the -15 element as a degenerate motif, which, together with the conserved stretch of sequence between -15 and -12, allows treating this element analogously to -35 and -10 elements. Furthermore, the strength of the -15 element inversely correlates with the strengths of the -35 element and -10 element, whereas no such complementation between other promoter elements was found. Despite the direct involvement of -15 element in σ(70)-dsDNA interactions, I found a significantly stronger tendency of this element to complement weak -10 elements that are involved in σ(70)-ssDNA interactions. This finding is in contrast to the established view, according to which the -15 element provides a sufficient number of σ(70)-dsDNA interactions, and suggests that the main parameter determining a functional promoter is the overall promoter strength.     

Construction and expression of sTRAIL–melittin combining enhanced anticancer activity with antibacterial activity in Escherichia coli

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), as an anticancer protein with tumor-selective apoptotic activity, has been examined for use in clinical application. Melittin, an antibacterial peptide isolated from the bee Apis mellifera, has shown strong cytotoxicity to both tumor and normal cells. To ameliorate the cytotoxicity of melittin on cells and enhance the activity of TRAIL on cancer cells, we constructed a novel fusion protein, sTRAIL–melittin, containing a small ubiquitin-related modifier (SUMO) tag and expressed this fusion protein in Escherichia coli. Data showed that expression of the soluble fusion protein with the SUMO tag was approximately 85 % of total target protein which was much higher than that without the SUMO tag (approximately 10 %); sTRAIL–melittin was easily purified using Ni-NTA affinity chromatography and the tag was removed easily using SUMO-specific protease. To assay anticancer activity and side effects, methyl thiazolyl tetrazolium, hemolytic, and apoptosis assays were employed. Results demonstrated that sTRAIL–melittin had cytotoxic and apoptotic activity in K562 leukemia cells and HepG2 liver carcinoma cells, while it had only a minimal effect on erythrocytes and normal HEK293 cells. This indicates that the cytotoxicity of sTRAIL–melittin in normal cells was low and the anticancer activity of the fusion protein in tumor cells was significantly enhanced compared with sTRAIL (P?<?0.01). Furthermore, we found that sTRAIL–melittin also showed antibacterial activity to Staphylococcus aureus due to the presence of the melittin domain. Therefore, TRAIL fused with an antibacterial peptide may be a promising novel TRAIL-based anticancer treatment strategy.     

Cloning and expression in Escherichia coli of Clostridium thermocellum DNA encoding β-glucosidase activity

Sau3A fragments of Clostridium thermocellum (NCIB 10682) DNA were ligated into the BamHI site of pBR322 and expressed in a Lac⁻mutant of Escherichia coli HB101. Five clones expressing β-glucosidase activity were shown by restriction enzyme analysis to contain a common 4.4 kbp fragment of inserted DNA. Hybridization of recombinant plasmids with chromosomal DNA ratified the physical maps of the inserted DNA and was further used to confirm that the 4.4 kbp fragment was common to all five clones. Enzyme activity, comprising cellobiase and aryl-β-glucosidase, was similar with respect to substrate specificity for each of the five clones, and was expressed independently of the orientation of the cloned DNA. A differential effect of temperature on activity of the cellobiase and aryl-β-glucosidase activities was observed but in other respects, the properties of the cloned β-glucosidase corresponded to those of the single β-glucosidase previously described for C. thermocellum.     

An “energy-auxotroph” Escherichia coli provides an in vivo platform for assessing NADH regeneration systems

An efficient in vivo regeneration of the primary cellular resources NADH and ATP is vital for optimizing the production of value-added chemicals and enabling the activity of synthetic pathways. Currently, such regeneration routes are tested and characterized mainly in vitro before being introduced into the cell. However, in vitro measurements could be misleading as they do not reflect enzyme activity under physiological conditions. Here, we construct an in vivo platform to test and compare NADH regeneration systems. By deleting dihydrolipoyl dehydrogenase in Escherichia coli, we abolish the activity of pyruvate dehydrogenase and 2-ketoglutarate dehydrogenase. When cultivated on acetate, the resulting strain is auxotrophic to NADH and ATP: acetate can be assimilated via the glyoxylate shunt but cannot be oxidized to provide the cell with reducing power and energy. This strain can, therefore, serve to select for and test different NADH regeneration routes. We exemplify this by comparing several NAD-dependent formate dehydrogenases and methanol dehydrogenases. We identify the most efficient enzyme variants under in vivo conditions and pinpoint optimal feedstock concentrations that maximize NADH biosynthesis while avoiding cellular toxicity. Our strain thus provides a useful platform for comparing and optimizing enzymatic systems for cofactor regeneration under physiological conditions.     

Escherichia coli β-galactosidase as an in vitro and in vivo reporter enzyme and stable transfection marker in the intracellular protozoan parasite Toxoplasma gondii

We have developed several protocols for the use of β-galactosidase (βGal) from Escherichia coli as a reporter enzyme in transfection studies of Toxoplasma gondii (Tg) and as a readily screenable marker for stable transformation. Three Tg expression vectors with different promoters driving lacZ were constructed and shown in transient transfections to differ in their relative expression levels. Using a fluorescent βGal substrate, it was possible to detect enzymatic activity with as little as 50 ng of transfected lacZ-containing plasmid DNA. When stably transformed intracellular parasites were cultivated in microtiter plates in the presence of the color substrate, chlorophenol red-β-D-galactopyranoside (CPRG), the signal from as few as 400 Tg could be readily detected by eye. Using serial dilutions of transfected parasite cultures in the presence of CPRG, we were able to clone stably expressing βGal-positive Tg without the need for another selectable marker. Such lacZ transgenics could also be visualized histochemically in the tissue of infected mice. Thus, the application of βGal to studies on Tg provides not only a much needed second reporter for transient transfection, it also comprises a safe and sensitive marker for the generation and analysis of stably transfected parasites     

Immobilization of Escherichia coli containing ω-transaminase activity in LentiKats®

Whole Escherichia coli cells overexpressing ω‐transaminase (ω‐TA) and immobilized cells entrapped in LentiKats® were used as biocatalysts in the asymmetric synthesis of the aromatic chiral amines 1‐phenylethylamine (PEA) and 3‐amino‐1‐phenylbutane (APB). Whole cells were permeabilized with different concentrations of cetrimonium bromide (CTAB) and ethanol; the best results were obtained with CTAB 0.1% which resulted in an increase in reaction rate by 40% compared to the whole cells. The synthesis of PEA was carried out using isopropyl amine (IPA) and L ‐alanine (Ala) as amino donors. Using whole cell biocatalysis, the reaction with IPA was one order of magnitude faster than with Ala. No reaction was detected when permeabilized E. coli cells containing ω‐TA were employed using Ala as the amino donor. Additionally, the synthesis of APB from 4‐phenyl‐2‐butanone and IPA was studied. Whole and permeabilized cells containing ω‐TA and their immobilized LentiKats® counterparts showed similar initial reactions rates and yields in the reaction systems, indicating 100% of immobilization efficiency (observed activity/activity immobilized) and absence of diffusional limitations (due to the immobilization). Immobilization of whole and permeabilized cells containing ω‐TA in LentiKats® allowed improved stability as the biocatalyst was shown to be efficiently reused for five reaction cycles, retaining around 80% of original activity. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012     

Engineering in vivo instability of firefly luciferase and Escherichia coli β-glucuronidase in higher plants using recognition elements from the ubiquitin pathway

The ubiquitin pathway targets proteins for degradation through the post-translational covalent attachment of the 76 amino acid protein ubiquitin to -amino lysyl groups on substrate proteins. Two instability determinants recognized by the ubiquitin pathway in Saccharomyces cerevisiae have been identified. One is described by the N-end rule and requires specific destabilizing residues at the substrate protein N-termini along with a proximal lysyl residue for ubiquitin conjugation. The second is a linear uncleavable N-terminal ubiquitin moiety. The ability of these two determinants to function in higher plants was investigated in tobacco protoplast transient transfection assays using DNA encoding variants of well characterized reporter enzymes as substrates: firefly luciferase that is localized to peroxisomes (pxLUC), a cytosolic version of LUC (cLUC), and Escherichia coli -glucuronidase (GUS). cLUC with phenylalanine encoded at its mature N-terminus was 10-fold less abundant than cLUC with methionine at its mature N-terminus. GUS with phenylalanine encoded at its mature N-terminus was 3-fold less abundant than GUS with methionine at its mature N-terminus. The presence of a uncleavable N-terminal ubiquitin fusion resulted in 50-fold lower protein accumulation of cLUC, but had no effect on GUS. Both instability determinants had a much larger effect on cLUC than on pxLUC, suggesting that these degradation signals are either unrecognized or poorly recognized in the peroxisomes.     

Formation and Stability of the Bacteriophage λ Replication Complexes in UV-Irradiated Escherichia coli

Bacteriophage λ replication complex, containing the phage-encoded O initiator protein protected from proteases by other elements of this complex, is a stable structure that can be inherited by one of the two daughter λ DNA copies after a replication round in Escherichia coli. In normal growth conditions in bacteria bearing a plasmid derived from bacteriophage λ, such a complex may be stable for many cell generations. However, it was found that this stable structure is disassembled under certain conditions, namely, after heat shock. Therefore, we asked whether other environmental stresses may cause disassembly of the λ replication complex. We found that UV irradiation of the host cells prevented formation of the stable λ replication complex (though not preventing phage replication), while the same UV doses did not affect the stability of the replication complex assembled prior to the irradiation. These results indicate that the stable λ replication complex, although sensitive to heat shock, is resistant to some other environmental stresses and that formation of at least two types of λ replication complexes is possible. Both stable and unstable λ replication complexes are functional because replication of λ DNA under conditions preventing formation of the stable complex proceeds efficiently. Received: 18 January 2000 / Accepted: 2 March 2000     

DNA duplex stability as discriminative characteristic for Escherichia coli σ54- and σ28- dependent promoter sequences

The advent of modern high-throughput sequencing has made it possible to generate vast quantities of genomic sequence data. However, the processing of this volume of information, including prediction of gene-coding and regulatory sequences remains an important bottleneck in bioinformatics research. In this work, we integrated DNA duplex stability into the repertoire of a Neural Network (NN) capable of predicting promoter regions with augmented accuracy, specificity and sensitivity. We took our method beyond a simplistic analysis based on a single sigma subunit of RNA polymerase, incorporating the six main sigma-subunits of Escherichia coli. This methodology employed successfully re-discovered known promoter sequences recognized by E. coli RNA polymerase subunits σ²⁴, σ²⁸, σ³², σ³⁸, σ⁵⁴ and σ⁷⁰, with highlighted accuracies for σ²⁸- and σ⁵⁴- dependent promoter sequences (values obtained were 80% and 78.8%, respectively). Furthermore, the discrimination of promoters according to the σ factor made it possible to extract functional commonalities for the genes expressed by each type of promoter. The DNA duplex stability rises as a distinctive feature which improves the recognition and classification of σ²⁸- and σ⁵⁴- dependent promoter sequences. The findings presented in this report underscore the usefulness of including DNA biophysical parameters into NN learning algorithms to increase accuracy, specificity and sensitivity in promoter beyond what is accomplished based on sequence alone.