Lysis protein T of bacteriophage T4

Summary Lysis protein T of phage T4 is required to allow the phage's lysozyme to reach the murein layer of the cell envelope and cause lysis. Using fusions of the cloned gene t with that of the Escherichia coli alkaline phosphatase or a fragment of the gene for the outer membrane protein OmpA, it was possible to identify T as an integral protein of the plasma membrane. The protein was present in the membrane as a homooligomer and was active at very low cellular concentrations. Expression of the cloned gene t was lethal without causing gross leakiness of the membrane. The functional equivalent of T in phage is protein S. An amber mutant of gene S can be complemented by gene t, although neither protein R of (the functional equivalent of T4 lysozyme) nor S possess any sequence similarity with their T4 counterparts. The murein-degrading enzymes (including that of phage P22) have in common a relatively small size (molecular masses of ca. 18 000) and a rather basic nature not exhibited by other E. coli cystosolic proteins. The results suggest that T acts as a pore that is specific for this type of enzyme.     

Expression and purification of antimicrobial peptide adenoregulin with C-amidated terminus in Escherichia coli

Adenoregulin is a 33 amino acid antimicrobial peptide isolated from the skin of the arboreal frog Phyllomedusa bicolor. Natural adenoregulin is synthesized with an amidated valine residue at C-terminus and shows lethal effects against filamentous fungi, as well as a broad spectrum of pathogenic microorganisms. A synthetic gene for adenoregulin (ADR) with an additional amino acid glutamine at C-terminus was cloned into pET32a vector to allow expression of ADR as a Trx fusion protein in Escherichia coli BL21(DE3). The resulting expression level of the fusion protein could reach up to 20% of the total cell proteins. The fusion protein could be purified effectively by Ni2+-chelating chromatography. Released from the fusion protein by enterokinase cleavage and purified to homogeneity, the recombinant ADR displayed antimicrobial activity similar to that of the synthetic ADR reported earlier. Comparing the antimicrobial activities of the recombinant adenoregulin with C-amidated terminus to that without an amidated C-terminus, we found that the amide of glutamine at C-terminus of ADR improved its potency on certain microorganisms such as Tritirachium album and Saccharomyces cerevisiae.     

Mechanism of 2-aminopurine-stimulated mutagenesis in Escherichia coli

2-Aminopurine (2AP), a base analog, causes both transition and frameshift mutations in Escherichia coli. The analog is thought to cause mutations by two mechanisms: directly, by mispairing with cytosine, and indirectly, by saturation of mismatch repair (MMR). The goal of this work was to measure the relative contribution of these two mechanisms to the occurrence of transition mutations. Our data suggest that, in contrast to 2-aminopurine-stimulated frameshift mutations, the majority of transition mutations are a direct effect of base mispairing.     

Effect of chitosan derivatives on the reproduction of coliphages T2 and T7

The effect of chitosan derivatives with different degrees of polymerization and deamination, as well as of chitosan 6-O-sulfate and chitosanN-succinate-6-O-sulfate, on the reproduction of coliphages T2 and T7 inEscherichia coli and on the growth of this bacterium was studied. Chitosan derivatives decreased the yield of coliphages and exhibited antibacterial activity. The efficiency of inhibition of viral infection and the antibacterial activity of chitosan were found to be dependent on the degree of its polymerization. At the same time, there was no correlation between the degree of chitosan deamination and the extent of inhibition of viral infection. Anionic chitosan derivatives virtually did not possess antiviral or antibacterial activity. It is assumed that chitosan blocks some stages of phage reproduction. The decrease in the phage-producing ability ofE. coli may also be due to the antibacterial effect of chitosan.     

Isolation and genetic characterization of new uvsW alleles of bacteriophage T4

Summary TheuvsW gene of bacteriophage T4 is required for wild-type levels of recombination, for normal survival and mutagenesis after UV irradiation, and for wild-type resistance to hydroxyurea. Additionally,uvsW mutations restore the arrested DNA synthesis caused by mutations in any of several genes that block secondary initiation (recombination-primed replication, the major mode of initiation at late times), but only partially restore the reduced burst size. AuvsW deletion mutation was constructed to establish the null-allele phenotype, which is similar but not identical to the phenotype of the canonicaluvsW mutation, and to demonstrate convincingly that theuvsW gene is non-essential (althoughuvsW mutations severely compromise phage production). In an attempt to uncouple the diverse effects ofuvsW mutations, temperature-sensitiveuvsWts mutants were isolated. Recombination and replication effects were partially uncoupled in these mutants, suggesting distinct and separable roles foruvsW in the two processes. Furthermore, the restoration of DNA synthesis but not recombination in the double mutantsuvsW uvsX anduvsW uvsY prompts the hypothesis that the restored DNA synthesis is not recombinationally initiated.     

The DD-carboxypeptidase activity encoded by pbp4B is not essential for the cell growth of Escherichia coli

The gene (pbp4B) encoding a putative DD-carboxypeptidase has been deleted in Escherichia coli and it is shown to be not essential for cell division. Disruption of the gene in a genetic background where all putative activities of DD-carboxypeptidases and/or DD-endopeptidases had been eliminated indicates that these activities are not required for cell growth in enterobacteria. The penicillin-binding capacity and a low DD-carboxypeptidase activity of PBP4B are demonstrated. Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

Expression, purification, and renaturation of bone morphogenetic protein-2 from Escherichia coli

Homodimeric bone morphogenetic protein-2 (BMP-2) is a member of the transforming growth factor beta superfamily that has been used for bone grafting. We were interested in exploring the functions of BMP-2 in other disease areas and focused on expressing and purifying active BMP-2 proteins. We have developed a new approach which involves using FoldIt refolding buffer to refold BMP-2 followed by a heparin affinity column to separate correctly folded dimer from monomer. A high yield of 29.4 mg BMP-2 dimer per gram cell wet weight was achieved. The purified BMP-2 dimer was shown to possess the same level of activity as BMP-2 from CHO cells as tested by the induction of alkaline phosphatase activity in C2C12 cells. This approach has potential application in refolding and purifying other homodimeric proteins.     

Ethylene formation by cell-free extracts of Escherichia coli

The pathway leading to the formation of ethylene as a secondary metabolite from methionine by Escherichia coli strain B SPAO has been investigated. Methionine was converted to 2-oxo-4-methylthiobutyric acid (KMBA) by a soluble transaminase enzyme. 2-Hydroxy-4-methylthiobutyric acid (HMBA) was also a product, but is probably not an intermediate in the ethylene-forming pathway. KMBA was converted to ethylene, methanethiol and probably carbon dioxide by a soluble enzyme system requiring the presence of NAD(P)H, Fe³⁺ chelated to EDTA, and oxygen. In the absence of added NAD(P)H, ethylene formation by cell-free extracts from KMBA was stimulated by glucose. The transaminase enzyme may allow the amino group to be salvaged from methionine as a source of nitrogen for growth. As in the plant system, ethylene produced by E. coli was derived from the C-3 and C-4 atoms of methionine, but the pathway of formation was different. It seems possible that ethylene production by bacteria might generally occur via the route seen in E. coli.Abbreviations EDTA ethylenediaminetetraacetic acid - HMBA 2-hydroxy-4-methylthiobutyric acid (methionine hydroxy analogue) - HSS high speed supernatant - KMBA 2-oxo-4-methylthiobutyric acid - PCS phase combining system     

High-level expression of bovine rotavirus VP4 protein in Escherichia coli using a hepatitis B core antigen vector

Six regions of the VP4 protein of bovine rotavirus strain UKtc were expressed using hepatitis B core antigen (HBcAg) as a carrier. Following induction by IPTG, the six fusion proteins, AHBcAg through FHBcAg, were expressed in Escherichia coli to a level of 20–37% of total cellular protein. Soluble fusion proteins in a particulate form were partially purified with yields ranging from 0.5–6.4 mg l^–1 of culture.     

Synthesis of recombinant human interleukin-2 via controlled feed of lactose-complex media in fed-batch cultures of Escherichia coli BL21(DE3)[pT7-G3IL2]

Using lactose as an inducer, recombinant human interleukin-2 (rhIL-2) was synthesized with an N-terminus fusion partner, G3 (three tandem-arranged glucagon peptides) in fed-batch cultures at high cell concentration (60–90 g l^–1) of Escherichia coli BL21(DE3) [pT7-G3IL2]. With batch additions of lactose (4 × 13.5 g), the fusion rhIL-2 was synthesized up to 9.3 g l^–1. However, if all the lactose (54 g) was added at once to the culture, synthesized fusion rhIL-2 decreased to 5.4 g l^–1 with a decreased cell growth rate. A statistical optimization of the production medium containing glucose, yeast extract, and lactose led to fusion rhIL-2 being produced at > 9 g l^–1.     

Expression of antimicrobial peptide LH multimers in <Emphasis Type="Italic">Escherichia coli</Emphasis> C43(DE3)

The tandem repeats of LFB15(W4,10)-HP(4-16) (LH) gene were cloned into vector pET32a(+) for recombinant expression in Escherichia coli. The E. coli C43(DE3) was successfully used as the expression host to avoid the cell death during induction in E. coli BL21(DE3). Fusion LH dimer was expressed as inclusion body at a portion of 35% of total cell protein and could be well purified by Ni²⁺-chelating chromatography. The recombinant LH was released by the cleavage of 50% formic acid, and its yield reached 11.3 mg/l with purity of 95%. The MIC₅₀ of 3.6 and 1.9 μM of recombinant LH against E. coli CMCC 44102 and Bacillus subtilis ATCC 6633 were determined, respectively. The results demonstrated that expression of tandem LH gene in E. coli C43(DE3) and formic acid cleavage would provide a potent efficient platform for the production of interested peptides. Zi-gang Tian and Tian-tang Dong contributed equally to this paper.     

A newly discovered tRNA(1Asp) gene (aspV) of Escherichia coli K12

Summary We report a new tRNA ₁ ^Asp gene near the dnaQ gene, which is located at 5 min on the Escherichia coli linkage map. We named it aspV. The sequence corresponding to the mature tRNA is identical with that of the two previously identified tRNA ₁ ^Asp genes (aspT and aspU), but there is no homology in the sequences of their 3-and 5-flanking regions.Abbreviations kb kilo base pair(s) - rrn ribosomal RNA     

Relation of potassium uptake to proton transport and activity of hydrogenases in Escherichia coli grown at low pH

Escherichia coli grown under anaerobic conditions in acidic medium (pH 5.5) upon hyperosmotic stress accumulates potassium ions mainly through the Kup system, the functioning of which is associated with proton efflux decrease. It was shown that H⁺ secretion but not glucose-induced K⁺ uptake was inhibited by N,N′-dicyclohexylcarbodiimide (DCC). The inhibitory effect of DCC on the H⁺ efflux was stronger in the trkA mutant with defective potassium transport. The K⁺ and H⁺ fluxes depended on the extent of hyperosmotic stress in the absence or presence of DCC. The decrease in external oxidation/reduction potential and H₂ liberation insensitive to DCC were recorded. It was found that the atpD mutant with nonfunctional F₀F₁-ATPase produced a substantial amount of H₂, while in the hyc mutant (but not the hyf mutant defective in hydrogenases 3 (Hyd-3) and 4 (Hyd-4)) the H₂ production was significantly suppressed. At the same time, the rate of K⁺ uptake was markedly lower in hyfR and hyfB-R but not in hycE or hyfA-B mutants; H⁺ transport was lowered and sensitive to DCC in hyf but not in hyc mutants. The results point to the relationship of K⁺ uptake with the Hyd-4 activity. Novel options of the expression of some hyf genes in E. coli grown at pH 5.5 are proposed. It is possible that the hyfB-R genes expressed under acidic conditions or their gene products interact with the gene coding for the Kup protein or directly with the Kup system.     

Evolutionary relationship between Enterobacteriaceae: comparison of the ATP synthases (F1F0) of Escherichia coli and Klebsiella pneumoniae

The ATP synthase complex of Klebsiella pneumoniae (KF₁F₀) has been purified and characterized. SDS-gel electrophoresis of the purified F₁F₀ complexes revealed an identical subunit pattern for E. coli (EF₁F₀) and K. pneumoniae. Antibodies raised against EF₁ complex and purified EF₀ subunits recognized the corresponding polypeptides of EF₁F₀ and KF₁F₀ in immunoblot analysis. Protease digestion of the individual subunits generated an identical cleavage pattern for subunits , , , , a, and c of both enzymes. Only for subunit different cleavage products were obtained. The isolated subunit c of both organisms showed only a slight deviation in the amino acid composition. These data suggest that extensive homologies exist in primary and secondary structure of both ATP synthase complexes reflecting a close phylogenetic relationship between the two enterobacteric tribes.Abbreviations ACMA 9-amino-6-chloro-2-methoxyacridine - DCCD N,N-dicyclohexylcarbodiimide - FITC fluorescein isothiocyanate - SDS sodium dodecyl sulfate - TTFB 4,5,6,7-tetrachloro-2-trifluoromethylbenzimidazole     

Photo-catalytic preparation of silver-coated TiO2 particles for antibacterial applications

Colloidal silver has been known to have unique antimicrobial activity that may be useful in the construction of antibacterial materials (self-cleaning materials) to aid in the fight against bacteria-related infections. In this study, silver-coated TiO₂ (Ag/TiO₂) particles prepared through the photo-reduction of Ag⁺ were investigated as an antibacterial agent against Escherichia coli and Staphylococcus aureus. The deposition of Ag onto the surface was confirmed with SEM and EDS analysis of the post-reaction particles. It was also determined that the initial concentration of Ag⁺ in solution played a significant role in the effective size of the post-irradiation particles. The antibacterial effectiveness of the Ag/TiO₂ was evaluated through the determination of the minimum inhibitory concentration (MIC) of AgTiO₂ for each species of bacteria. The MIC values for the Ag/TiO₂, on both E. coli and S. aureus, were much lower than the MIC values for Ag metal, and quite comparable to the MIC values for AgNO₃. A disc diffusion/antibiotic sensitivity test was also performed using the Ag/TiO₂ particles and the results compared with the results obtained for Ag metal, AgNO₃ and common antibacterial agents; tetracycline, chloramphenicol, erythromycin, and neomycin. The zone of inhibition diameters for the Ag/TiO₂ particles were found to be comparable with those of the other antimicrobial agents.     

Methylated cytosine at Dcm (CC T A GG) sites in Escherichia coli: Possible function and evolutionary implications

The frequency and distribution of methylated cytosine (5-MeC) at CC _T ^A GG (Dcm sites) in 49 E. coli DNA loci (207,530 bp) were determined. Principal observations of this analysis were: (1) Dcm frequency was higher than expected from random occurrence but lower than calculated with Markov chain analysis; (2) CCTGG sites were found more frequently in coding than in noncoding regions, while the opposite was true for CCAGG sites; (3) Dcm site distribution does not exhibit any identifiably regular pattern on the chromosome; (4) Dcm sites at oriC are probably not important for accurate initiation of DNA replication; (5) 5-MeC in codons was more frequently found in first than in second and third positions; (6) there are probably few genes in which the mutation rate is determined mainly by DNA methylation. It is proposed that the function of Dcm methylase is to protect chromosomal DNA from restriction-enzyme EcoRII. The Dcm methylation contribution to determine frequency of oligonucleotides, mutation rate, and recombination level, and thus evolution of the E. coli genome, could be interpreted as a consequence of the acquisition of this methylation.Correspondence to: M.C. Gómez-Eichelmann     

Mutation of Thr445 and Ile500 of initiation factor 2 G-domain affects Escherichia coli growth rate at low temperature

The Escherichia coli protein synthesis initiation factor IF2 is a member of the large family of G-proteins. Along with translational elongation factors EF-Tu and EF-G and translational release factor RF-3, IF2 belongs to the subgroup of G-proteins that are part of the prokaryotic translational apparatus. The roles of IF2 and EF-Tu are similar: both promote binding of an aminoacyl-tRNA to the ribosome and hydrolyze GTP. In order to investigate the differences and similarities between EF-Tu and IF2 we have created point mutations in the G-domain of IF2, Thr445 to Cys, Ile500 to Cys, and the double mutation. Threonine 445 (X1), which corresponds to cysteine 81 in EF-Tu, is well conserved in the DX1X2GH consensus sequence that has been proposed to interact with GTP. The NKXD motif, in which X is isoleucine 500 in IF2, corresponds to cysteine 137 in EF-Tu, and is responsible for the binding of the guanine ring. The recombinant mutant proteins were expressed and tested in vivo for their ability to sustain growth of an Escherichia coli strain lacking the chromosomal copy of the infB gene coding for IF2. All mutated proteins resulted in cell viability when grown at 42 degrees C or 37 degrees C. However, Thr445 to Cys mutant showed a significant decrease in the growth rate at 25 degrees C. The mutant proteins were overexpressed and purified. As observed in vivo, a reduced activity at low temperature was measured when carrying out in vitro ribosome dependent GTPase and stimulation of ribosomal fMet-tRNAfMet binding.     

The impact of electrochemical disinfection on Escherichia coli and Legionella pneumophila in tap water

In order to reduce the risks of Legionnaires' disease, caused by the bacterium Legionella pneumophila, disinfection of tap water systems contaminated with this bacterium is a necessity. This study investigates if electrochemical disinfection is able to eliminate such contamination. Hereto, water spiked with bacteria (10(4)CFU Escherichia coli or L. pneumophila/ml) was passed through an electrolysis cell (direct effect) or bacteria were added to tap water after passage through such disinfection unit (residual effect). The spiked tap water was completely disinfected, during passage through the electrolysis cell, even when only a residual free oxidant concentration of 0.07 mg/l is left (L. pneumophila). The residual effect leads to a complete eradication of cultivable E. coli, if after reaction time at least a free oxidant concentration of 0.08 mg/l is still present. Similar conditions reduce substantially L. pneumophila, but a complete killing is not realised.