Human metallothionein-II is synthesized as a stable membrane-localized fusion protein in Escherichia coli

A synthetic gene encoding human metallothionein-II (HMT) was cloned into the specially constructed high-copy-number expression vector, pUA7, and expressed in Escherichia coli. The plasmid construct includes the promoter/operator and regulatory sequences of the Salmonella typhimurium ara operon and part of the 5'-coding and all of the 3'-noncoding regions of the E. coli lpp. Upon induction with arabinose, the resulting Lpp::HMT fusion protein was produced 75,000-fold over uninduced cells, with a relatively stable mRNA (T1/2 of 8.3 min) and a completely stable protein. In addition, over 95% of the final fusion protein was localized in the outer membrane and was capable of binding heavy metals (especially cadmium) in vitro. Cells producing Lpp::HMT bioaccumulated heavy metals (e.g., cadmium) 66-fold over nonproducing cells.     

The oxygen affinity of haemoglobin Tak, a variant with an elongated beta chain.

The oxygen affinity was investigated of purified Hb Tak, a human haemoglobin variant with elongated beta-chains. A very low P50 value was found which was not influenced by the addition of 2,3 diphosphoglycerate. The n value was 1, indicating non-cooperativity. The oxygen equilibrium curve of the whole blood haemolysate containing Hbs A and Tak was close to that of Hb A at the top of the curve, while the bottom of the curve greatly deviated from the latter, indicative of small if any interaction between Hb A and Tak during oxygenation.     

Physical properties of microbial polythioesters: characterization of poly(3-mercaptoalkanoates) synthesized by engineered Escherichia coli

Physical properties of chiral poly(thioesters), PTEs, prepared by engineered Escherichia coli, were examined by GPC, 13C CP/MAS solid-state NMR, X-ray diffraction, and thermal analysis. Microbial homopolymers of PTEs, poly(3-mercaptopropionate), PMP, and poly(3-mercaptovalerate), PMV, showed different solubility characteristics compared to poly(hydroxyalkanoates), PHAs. Generally, PTEs required higher temperatures for dissolution. Poly(3-mercaptobutyrate), PMB, and PMV dissolve in chloroform, and the molecular weight values were revealed by GPC as 176,000 and 165,000, respectively. The density values for PMP and PMB were 1.42 and 1.27 g/cm3, respectively. These values are similar to those for oxygen analogues. The NMR spectra for PTEs showed that carbonyl carbons are greatly shifted downfield by the sulfur atoms in the chain backbone compared to the PHA family. X-ray powder diffraction data indicated that PTEs are crystalline materials, but they do not crystallize as well as in the PHA family. The melting point, Tm, for PMP was 170 degrees C, which is about 100 degrees C higher than the equivalent oxygen analogue, poly(3-hydroxypropionate), PHP, and almost the same as that of bacterial poly(3-hydroxybutyrate), PHB. According to thermal analysis, only the PMP sample had enhanced heat stability, e.g., the decomposition temperature for PMP was 277 degrees C at 5% weight loss, whereas the values for PHP and PHB were 233 and 260 degrees C at the same weight loss, respectively.     

Spectral properties of phosphopyridoxyl-Lys-7(-41)-ribonuclease A.

We have studied the spectral properties of RNAase A containing a phosphopyridoxyl residue at the epsilon-NH2 group of Lys-7 or Lys-14. The overall conformations of the native and modified enzymes were shown to be rather similar. All three proteins have similar circular dichroism spectra within the 220-300-nm region, and similar thermal transition temperatures. All the changes in the RNAase A molecule modified are located in close proximity to the alkylated lysine residue. The phosphopyridoxyl group of (P-Pxy)-epsilon-Lys-41-RNAase A is situated directly at the enzyme active site and is 25% butied in the protein globule. The P-pyridoxyl group of (P-Pxy)-epsilon-Lys-7-RNAase A was shown to be located in the vicinity of the active site and to be more exposed to the solvent. In the pyridoxyl phosphate absorption band, optical activity is induced in both proteins. Study of the pH dependence of the changes occurring in the circular dichroism and absorption spectra has shown that in the modified proteins, the pyridoxyl phosphate chromophore is rather sensitive to the ionic state of the surrounding medium and serves as a "reporter" group when the relationship between structure and function of the RNAase A active site is being investigated.     

Location of a gene (ssrA) for a small, stable RNA (10Sa RNA) in the Escherichia coli chromosome.

The gene for 10Sa RNA, which is a major small, stable RNA in Escherichia coli, is a unique gene in the E. coli chromosome. The 10Sa RNA gene (ssrA) has been located between 2,760 and 2,761 kilobases on the E. coli genome.     

Examination of newly synthesized DNA in Escherichia coli.

Summary The short (0-20S) Okazaki fragments seen upon pulse-labeling E. coli (thy ⁺, deo ⁺) with ³H-thymidine are actually composed of three types of DNA fragments: (1) true replication intermediates, (2) post-replication repair fragments (such as those which arise subsequent to the removal of misincorporated uracil), and (3) chromosomal fragments. Our experiments show that the number of pulse-labeled fragments decreases slightly with the introduction of the ung-1 mutation into E. coli K-12 (dut ⁺, thyl ⁺, polA ⁺), and that there are fewer fragments found in E. coli B/r than in E. coli K-12 ung-1. This suggests that while some fraction of pulse-labeled fragments may be due to repair, this fraction can vary among different strains; moreover, the majority of fragments appear to be replication intermediates. Selfhybridization (reannealing) of pulse-labeled fragments from E. coli B/r show that these fragments are asymmetric with respect to the strand origin and with respect to their size: the smaller (3-8S) fragments come from only one of the parental strands, whereas the larger (13-20S) fragments are synthesized equally from both parental strands. We interpret our results to mean that replication can be discontinuous on both strands but asymmetric with respect to both the size of the fragments and the size of the discontinuous region on the two strands.     

Two mutations in recombinant Hb beta F41(C7)Y, K82 (EF6)D show additive effects in decreasing oxygen affinity.

Based on the properties of two low oxygen affinity mutated hemoglobins (Hb), we have engineered a double mutant Hb (rHb beta YD) in which the beta F41Y substitution is associated with K82D. Functional studies have shown that the Hb alpha 2 beta 2(C7)F41Y exhibits a decreased oxygen affinity relative to Hb A, without a significantly increased autooxidation rate. The oxygen affinity of the natural mutant beta K82D (Hb Providence-Asp) is decreased due to the replacement of two positive charges by two negative ones at the main DPG-binding site. The functional properties of both single mutants are interesting in the view of obtaining an Hb-based blood substitute, which requires: (1) cooperative oxygen binding with an overall affinity near 30 mm Hg at half saturation, at 37 degrees C, and in the absence of 2,3 diphosphoglycerate (DPG), and (2) a slow rate of autooxidation in order to limit metHb formation. It was expected that the two mutations were at a sufficient distance (20 A) that their respective effects could combine to form low oxygen affinity tetramers. The double mutant does display additive effects resulting in a fourfold decrease in oxygen affinity; it can insure, in the absence of DPG, an oxygen delivery to the tissues similar to that of a red cell suspension in vivo at 37 degrees C. Nevertheless, the rate of autooxidation, 3.5-fold larger than that of Hb A, remains a problem.     

The isolated C-terminal (F2) fragment of the Escherichia coli tryptophan synthase beta 2-subunit folds into a stable, organized nonnative conformation

Proteolysis of the beta 2-subunit of Escherichia coli tryptophan synthase by the endoproteinase Glu C from Staphylococcus aureus V8 yields a peptide, F2, corresponding to the C-terminal 101 residues of the beta-chain. The conformation and stability of isolated F2 in phosphate buffer at pH 7.8 (where native beta 2 is stable) have been investigated. Circular dichroism spectra in the far-UV showed the presence of large amounts of secondary structure (19% alpha-helices, 34% extended beta-structures). Circular dichroism spectra in the near-UV and sedimentation velocity studies indicated an open globular structure with the aromatic side chains in a symmetric (or disordered) environment. NMR spectra and rates of amide proton exchange showed that F2 fluctuates rapidly between several conformations. The thermal denaturation of F2 observed by the loss of far-UV circular dichroism with increasing temperature appeared noncooperative, and indicates a high thermal stability (Tm = 70 degrees C). Differential scanning microcalorimetry confirmed the absence of cooperativity and indicated a very low value for the calorimetric enthalpy of denaturation (delta H = 17 kJ/mol). All these properties were compatible with a molten globule. However, the low sedimentation coefficient of F2 suggested a very hydrated and/or expanded structure, and the secondary structure content of isolated F2 (see above) differed widely from that reported in the literature for F2 within the context of native beta 2 (49% alpha-helices and 13% extended beta-structures). Thus, neither the secondary nor the tertiary structure of isolated F2 resembled those of native F2. In this respect, isolated F2 is not a "molten globule".(ABSTRACT TRUNCATED AT 250 WORDS)     

A new haemoglobin variant: haemoglobin Anantharaj alpha 11 (A9) lysine replaced by glutamic acid.

Four heterozygotes for a fast alpha-chain variant in a Thai family were detected on starch gel electrophoresis during a survey study on iron deficiency anaemia in a rural area not far from Bangkok. They were healthy and had normal haematological profiles except for the presence of around 44% abnormal pigment, quantitated by cellulose acetate electrophoresis. The structural characterization of the variant by globin chain separation, peptide mapping, and amino acid analyses of the abnormal peptides indicated that lysine residue 11 (A9) of alpha-chain was replaced by glutamic acid. This mutation has not been previously described and it is proposed that it be called Haemoglobin Anantharaj.     

Recognition properties of the beta subunit of Escherichia coli ribonucleic acid polymerase.

Changes in the phage protein patterns obtained by gel electrophoresis of extracts from phage S13 and phiX174 infection of rifampin-resistant hosts suggest that the beta subunit of ribonucleic acid polymerase of Escherichia coli has a function in the recognition of promoter or terminator sites or both. The altered protein patterns also provide information on the location of some ribonucleic acid polymerase recognition signals in S13 deoxyribonucleic acid. There is a promoter site before gene A, which lies either in gene H or between H and A. There is evidence for a promotor between genes C and D or in gene C. There is either a terminator or a promoter somewhere between the end of gene D and the beginning of gene F.     

Membrane structures in stable L-forms of Escherichia coli.

Microtubular and lamellate membrane structures were observed at the log phase of growth in the stable L-forms of Escherichia coli cultured in the absence of penicillin.     

Prevalence of verotoxin-producing Escherichia coli (VTEC) and E. coli O157:H7 in French pork

AIMS: To determination the prevalence of VTEC in pork products and the surrounding environment of the pork plant (slaughterhouse and cutting plant), and characterization of the VTEC strains isolated (virulence genes and serotype). METHODS AND RESULTS: Among the 2146 carcass and pork samples and 876 environmental samples (swabs of surfaces or materials), 328 (15%) and 170 (19%) were PCR-positive for stx genes respectively. VTEC strains were recovered from positive samples by colony hybridization or immunoconcentration, serotyped and genetically characterized. Strains of E. coli O157:H7 were not isolated from 3 uidA-positive samples detected by PCR. The VTEC isolates did not harbour eae, ehx and uidA genes. CONCLUSIONS: Pigs and pork meat may contain VTEC strains but characterization of the strains based on virulence factors showed that the potential danger of pork meat appears to be low since although all strains harboured a stx gene, they did not have other virulence genes. SIGNIFICANCE OF THE STUDY: General hygiene measures appear to be sufficient and specific hygiene measures for VTEC are not necessary at this time. The porcine VTEC strains isolated in our study probably do not present a hazard.     

The toxicity of recombinant proteins in Escherichia coli: a comparison of overexpression in BL21(DE3), C41(DE3), and C43(DE3)

Two mutant strains of Escherichia coli BL21(DE3), called C41(DE3) and C43(DE3) and originally described by Miroux and Walker, are frequently used to overcome the toxicity associated with overexpressing recombinant proteins using the bacteriophage T7 RNA polymerase expression system. Even when the toxicity of the plasmids is so high that it prevents transformation in the strain BL21(DE3), the toxic proteins can often be expressed successfully in C41(DE3) and/or C43(DE3). In this work, using a range of plasmids coding for several types of proteins, we investigated in BL21(DE3), C41(DE3), and C43(DE3) their ability to undergo transformation and to express. While transformation was always possible in C41(DE3) and C43(DE3), we could not obtain transformants in BL21(DE3) for 62% of the expression vectors tested. Moreover, after induction, the expression of heterologous proteins in both mutant strains is generally better than in BL21(DE3). In this study, we also enhanced the stability of plasmids in culture during the expression of proteins by adding the par locus from the plasmid pSC101 to the vector backbone. The stability of a subset of the plasmids (measured 3 h after induction) was determined in C41(DE3) and C43(DE3) and varies from 62 to 92% for C43(DE3) and from 10 to 90% for C41(DE3). This study demonstrates the usefulness of these strains C41(DE3) and C43(DE3) in solving the problem of plasmid instability during the expression of toxic recombinant proteins.     

The gene for a small stable RNA (10Sa RNA) of Escherichia coli

A gene that codes for a small stable RNA (362 nucleotides) has been sequenced. It is a monocistronic gene, with its own promoter and terminator. It produces a precursor that is about 100 nucleotides longer than the mature RNA with all the extra nucleotides at the 3' end. The gene contains an open reading frame that corresponds to a small protein 25 amino acids long.     

Assembly of an in vitro synthesized Escherichia coli outer membrane porin into its stable trimeric configuration

The folding of in vitro synthesized outer membrane protein PhoE of Escherichia coli was studied in immunoprecipitation experiments with monoclonal antibodies which recognize cell surface-exposed conformational epitopes. The signal sequence appears to interfere with the formation of these conformational epitopes, since a mutant PhoE protein which lacks the majority of the signal peptide could be precipitated four times better than the wild type precursor. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the immunoprecipitated PhoE protein revealed that part of the immunoprecipitated PhoE was present as a heat-modifiable form of the protein which migrated faster in the gels than the completely denatured protein. This form of the protein probably represents a folded monomer which might be an intermediate in the assembly of the protein. Outer membrane vesicles were required to induce the formation of small amounts of heat-stable trimers, the functional form of the protein in vivo.