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.     

Manipulating ribulose bisphosphate carboxylase/oxygenase in the chloroplasts of higher plants

Transgenic manipulation of the photosynthetic CO2-fixing enzyme, ribulose bisphosphate carboxylase/oxygenase (Rubisco) in higher plants provides a very specific means of testing theories about photosynthesis and its regulation. It also encourages prospects for radically improving the efficiencies with which photosynthesis and plants use the basic resources of light, water, and nutrients. Manipulation was once limited to variation of the leaf's total content of Rubisco by transforming the nucleus with antisense genes directed at the small subunit. More recently, technology for transforming the small genome of the plastid of tobacco has enabled much more precise manipulation and replacement of the plastome-encoded large subunit. Engineered changes in Rubisco's properties in vivo are reflected as profound changes in the photosynthetic gas-exchange properties of the leaves and the growth requirements of the plants. Unpredictable expression of plastid transgenes and assembly requirements of some foreign Rubiscos that are not satisfied in higher-plant plastids provide challenges for future research.     

The deletion of Ffh in Escherichia coli induces adverse physiological and morphological changes

The Escherichia coli Ffh protein is homologous to the SRP54 subunit of the eukaryotic signal recognition particle (SRP) that is involved in targeting and translocation of membrane proteins. The functions of Ffh in E. coli were investigated using the mutant with the Ffh deficiency. The mutant showed lower growth rate at 30°C and rapidly lost viability at the non-permissive temperature of 42°C. In addition, the amount of the total membrane proteins decreased sharply in the mutant. The mutant cells cultured at either 30 or 42°C appeared to have an elongated shape as compared to the wild type cells. Transmission electron microscopy revealed that the membrane layer of the mutant cells was thinner than that of the wild type cells. The article is published in the original.     

Preparation and characterization of truncated human lipopolysaccharide-binding protein in Escherichia coli

Lipopolysaccharide (LPS) is a component of the outer membrane of Gram-negative bacteria, and is the causative agent of endotoxin shock. LPS induces signal transduction in immune cells when it is recognized by the cell surface complex of toll-like receptor 4 (TLR4) and MD-2. The complex recognizes the lipid A structure in LPS, which is buried in the membrane of the outer envelope. To present the Lipid A structure to the TLR4/MD-2, processing of LPS by LPS-binding protein (LBP) and CD14 is required. In previous studies, we expressed recombinant proteins of human MD-2 and CD14 as fusion proteins with thioredoxin in Escherichia coli, and demonstrated their specific binding abilities to LPS. In this study, we prepared a recombinant fusion protein containing 212 amino terminal residues of human LBP (HLB212) by using the same expression system. The recombinant protein expressed in E. coli was purified as a complex form with host LPS. The binding was not affected by high concentrations of salt, but was prevented by low concentrations of various detergents. Both rough-type LPS lacking the O antigen and smooth-type LPS with the antigen bound to HLBP212. Therefore, oligosaccharide repeats appeared to be unnecessary for the binding. A nonpathogenic penta-acylated LPS also bound to HLBP212, but the binding was weaker than that of the wild type. The hydrophobic interaction between the LBP and acyl chains of lipid A appears to be important for the binding. The recombinant proteins of LPS-binding molecules would be useful for analyzing the defense mechanism against infections.     

An essential gene of Escherichia coli that has sequence similarity to a chloroplast gene of unknown function

Summary The dedB gene of Escherichia coli has sequence similarity to the zfpA gene of the chloroplast chromosome. The functions of dedB and zfpA are unknown. We constructed derivatives of temperature-sensitive polA strains into whose chromosomes a plasmid containing the disrupted dedB gene was integrated by homologous recombination. These strains contained normal and disrupted dedB genes in their chromosomes. We then selected plasmid-segregated strains and found no cells containing the disrupted dedB gene, indicating that disruption of the dedB gene was lethal in polA strains of E. coli.     

Alkali-tolerant high-activity catalase from a thermophilic bacterium and its overexpression in Escherichia coli

We have purified an alkali-tolerant catalase from the thermophilic bacterium Metallosphaera hakonensis. The catalase gene, which encodes 303 amino acids and has a calculated molecular mass of 33 kDa, including its putative signal peptide encoding sequence, was cloned. The deduced amino acid sequence exhibited a region-specific homology with the sequences of manganese catalases from thermophilic bacteria such as Thermus thermophilus and Thermus brockianus. When this gene was overexpressed in Escherichia coli, proteins of the expected size (33 kDa) were overproduced in the inactive form. We made several attempts to obtain active forms of or to activate these overproduced proteins. Upon their induction into E. coli, a 100-fold increase in the catalase activity was detected when high-concentration manganese was used as the medium. The catalase activity of the purified enzyme was optimal at a pH of 10.0. The alkali-tolerant property of this catalase makes it a promising enzyme in biotechnological applications such as H(2)O(2)-detoxifying systems.     

Pole cap formation in Escherichia coli following induction of the maltose-binding protein

After induction with maltose, 30–40% of the total protein in the osmotic shock fluid consist of maltose-binding protein while the induction ratio (maltose versus glycerol grown cells) for the amount of binding protein synthesized as well as for maltose transport is in the order of 10. Induction of maltose transport does not occur during all times of the cell cycle, but only shortly before cell division. Electronmicroscopic analysis of cells grown logarithmically on glycerol or maltose revealed in the latter the formation of large pole caps. These pole caps arise from an enlargement of the periplasmic space. Small cells contain one pole cap, large cells contain two. Pulse label studies with strain BUG-6, a mutant that is temperature sensitive for cell division reveal the following: Growth at the non-permissive temperature prevents maltose-binding protein synthesis and formation of new transport capacity.After shifting to the permissive temperature the cells regain both functions. Simultaneously, the newly formed cells exhibit pole caps.We conclude that the induction of maltose-binding protein is responsible for the formation of pole caps. In addition, beside the presence of inducer, cell cycle events occuring during division are necessary for the synthesis of maltose-binding protein.Non Standard Abbreviations GLPT periplasmic protein, related to transport of glycerolphosphate in Escherichia coli (Silhavy et al., 1976b)     

Subcellular location and expression level of a chimeric protein consisting of the maize waxy transit peptide and the beta-glucuronidase of Escherichia coli in transgenic potato plants

Summary The transit peptide of the maize waxy protein (a nuclear-encoded amyloplast protein of the maize endosperm) was studied with respect to its role in subcellular protein targeting in transgenic potato plants. TP30, a chimeric precursor protein consisting of the waxy transit peptide and an additional 34 amino acids of the mature waxy protein fused to the -glucuronidase of Escherichia coli, was expressed in potato plants under the control of the 35S promoter of cauliflower mosaic virus. This fusion protein is imported not only into amyloplasts, the natural target organelles in the maize plant, but also into chloroplasts. In contrast, Gus, the -glucuronidase alone, which was also expressed in parallel experiments in transgenic potato plants is always found in the cytosol of the plant cells. As a consequence of the different subcellular locations of TP30 and Gus, we observed differences in the expression rates of the respective proteins in leaf cells, resulting in higher steady state levels of TP30 compared to Gus. In tuber cells, no correlation between intracellular location and expression of the proteins was found.     

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.     

Signal recognition particle mediated protein targeting in Escherichia coli

The signal recognition particle (SRP) is a conserved ribonucleoprotein complex that binds to targeting sequences in nascent secretory and membrane proteins. The SRP guides these proteins to the cytoplasmic membrane in prokaryotes and the endoplasmic reticulum membrane in eukaryotes via an interaction with its cognate receptor. The E. coli SRP is relatively small and is currently used as a model for fundamental and applied studies on translation-linked protein targeting. In this review recent advances in our understanding of the structure and function of the E. coli SRP and its receptor are discussed. In particular, the interplay between the SRP pathway and other targeting routes, the role of guanine nucleotides in cycling of the SRP and the substrate specificity of the SRP are highlighted     

Cloning, overexpression, purification, and characterization of O-acetylserine sulfhydrylase-B from Escherichia coli

O-Acetylserine sulfhydrylase-B (OASS-B, EC 2.5.1.47) is one of the two isozymes produced by Escherichia coli that catalyze the synthesis of L-cysteine from O-acetyl-L-serine and sulfide. The cysM gene encoding OASS-B was cloned and the enzyme was overexpressed in E. coli using pUC19 with a lacUV5 promoter. The enzyme was purified to homogeneity, as evidenced by SDS-PAGE. Approximately 300 mg of purified OASS-B was obtained from 1600 mL of culture broth with a purification yield of 60% or higher. The purified OASS-B was characterized and its properties compared with OASS-A. OASS-B did not form a complex with E. coli serine acetyltransferase (SAT, EC 2.3.1.30) and showed a wide range of substrate specificity in nonproteinaceous amino acid synthesis.     

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.     

On the precision and accuracy achieved by Escherichia coli cells at fission about their middle

Length and width of each of the prospective siblings of constricted Escherichia coli cells from different strains and culture conditions were measured from electron micrographs. The data were statistically analyzed to investigate how equally the length and volume of one cell was divided into two. The analysis showed that, for all cultures, bipartition is unbiased or very nearly so, i.e. sibling cells were on the average equally long and large. The precision of bipartition attained by the cells was usually high; it was related to the average cell shape (length/width): slender E. coli cells divided into two less precisely than squat cells. Absolute size, growth rate and strain specificity affected the precision of bipartition only indirectly, i.e. in as much as they influenced cell shape.     

Cloning and expression of rapeseed procruciferin in Escherichia coli and crystallization of the purified recombinant protein

Two rapeseed cruciferin cDNAs (cru2/3a and cru2/3b) were cloned and sequenced. A comparison of their DNA and protein sequences with other cruciferins, indicated cru2/3b to be a novel clone and, among them, an inherent and highly conserved sequence of twelve amino acids was identified. Procruciferin 2/3a and 2/3b were expressed in Eschericha coli, and procruciferin 2/3a was obtained in a soluble form. The expressed procruciferin 2/3a has a trimeric structure and formed crystals although the quality was not good, suggesting that this expression system is useful for protein engineering of procruciferin 2/3a.     

Engineering the Escherichia coli outer membrane protein OmpC for metal bioadsorption

The outer membrane protein, OmpC, from Escherichia coli was used to display metal-binding poly-histidine peptides on the surface of this bacterium. SDS-PAGE analysis of outer membrane protein preparations confirmed the expression of the metal-binding epitopes inserted in position 162 of the mature OmpC protein. Display of these epitopes was confirmed by epifluorescence microscopy of cells bound to Ni²⁺-NTA-agarose beads and metal adsorption experiments. The cells harboring one or two copies of the metal binding epitope were able to adsorb 3 to 6 times more Zn²⁺ (13.8 mol g^–1 cell), Fe³⁺ (35.3 mol g^–1 cell), and Ni²⁺ (9.9 mol g^–1 cell) metallic ions than control cells expressing the wild-type OmpC.     

Initiation of DNA replication in Escherichia coli after overproduction of the DnaA protein

Summary Flow cytometry was used to study initiation of DNA replication in Escherichia coli K12 after induced expression of a plasmid-borne dnaA ⁺ gene. When the dnaA gene was induced from either the plac or the pL promoter initiation was stimulated, as evidenced by an increase in the number of origins and in DNA content per mass unit. During prolonged growth under inducing conditions the origin and DNA content per mass unit were stabilized at levels significantly higher than those found before induction or in similarly treated control cells. The largest increase was observed when using the stronger promoter pL compared to plac. Synchrony of initiation was reasonably well maintained with elevated DnaA protein concentrations, indicating that simultaneous initiation of all origins was still preferred under these conditions. A reduced rate of replication fork movement was found in the presence of rifampin when the DnaA protein was overproduced. We conclude that increased synthesis levels or increased concentrations of the DnaA protein stimulate initiation of DNA replication. The data suggest that the DnaA protein may be the limiting factor for initiation under normal physiological conditions.     

Toroidal nucleoids in Escherichia coli exposed to chloramphenicol

The DNA of growing cells of Escherichia coli occurs in one or a few lobular bodies known as nucleoids. Upon exposure to chloramphenicol, the nucleoids assume compact, rounded forms ("cm-nucleoids") that have been described as ring- or sphere-shaped. Multiple views of single cells or spheroplasts, however, support a different, curved toroid shape for cm-nucleoids. The multiple views were obtained either by DNA fluorescence imaging as the cells or spheroplasts reoriented in liquid medium or by optical sectioning using phase-contrast or fluorescence imaging of immobilized cells. The curved toroid shape is consistent with electron microscope images of thin sections of chloramphenicol-treated cells. The relationship of this structure to active and inactive nucleoids and to the smaller toroidal forms made by in vitro DNA condensation is discussed.     

Construction of Escherichia coli dnaK-deletion mutant infected by lambdaDE3 for overexpression and purification of recombinant GrpE proteins

Escherichia coli is widely employed to produce recombinant proteins because this microorganism is simple to manipulate, inexpensive to culture, and of short duration to produce a recombinant protein. However, contamination of molecular chaperone DnaK during purification of the recombinant protein is sometimes a problem, since DnaK sometimes has a negative effect on subsequent experiments. Previously, several efforts have been done to remove the DnaK contaminants by several sequential chromatography or washing with some expensive chemicals such as ATP. Here, we developed a simple and inexpensive method to express and purify recombinant proteins based on an E. coli dnaK-deletion mutant. The E. coli ΔdnaK52 mutant was infected by λDE3 phage to overexpress desired recombinant proteins under the control of T7 promoter. Using this host cell, recombinant hexa histidine-tag fused GrpE, which is well known as a co-chaperone for DnaK and to strongly interact with DnaK, was overexpressed and purified by one-step nickel affinity chromatography. As a result, highly purified recombinant GrpE was obtained without washing with ATP. The purified recombinant GrpE showed a folded secondary structure and a dimeric structure as previous findings. In vitro ATPase activity assay and luciferase-refolding activity assay demonstrated that the recombinant GrpE worked together with DnaK. Thus, this developed method would be rapid and useful for expression and purification of recombinant proteins which is difficult to remove DnaK contaminants.