Expression of a Highly Toxic Protein, Bax, in Escherichia coli by Attachment of a Leader Peptide Derived from the GroES Cochaperone

Expression of the human apoptosis modulator protein Bax in Escherichia coli is highly toxic, resulting in cell lysis at very low concentrations (Asoh, S., et al., J. Biol. Chem. 273, 11384–11391, 1998). Attempts to express a truncated form of murine Bax in the periplasm by using an expression vector that attached the OmpA signal sequence to the protein failed to alleviate this toxicity. In contrast, attachment of a peptide based on a portion of the E. coli cochaperone GroES reduced Bax's toxicity significantly and allowed good expression. The peptide, which was attached to the N-terminus, included the amino acid sequence of the mobile loop of GroES that has been demonstrated to interact with the chaperonin, GroEL. Under normal growth conditions, expression of this construct was still toxic, but generated a small amount of detectable recombinant Bax. However, when cells were grown in the presence of 2% ethanol, which stimulated overproduction of the molecular chaperones GroEL and DnaK, toxicity was reduced and good overexpression occurred. Two-dimensional gel electrophoresis analysis showed that approximately 15-fold more GroES-loop-Bax was produced under these conditions than under standard conditions and that GroEL and DnaK were elevated approximately 3-fold.     

B-chromosome condensation inPhleum pollen grains

Pollen grains were investigated in 11 specimens ofPhleum Boehmeri Wib. [P. phleoides (L.) Karst.] differing in the number of B-chromosomes (2n=14+0–3B). In some uninucleate pollen grains there were seen large chromocentres, not observed in other tissues. They resembled meiotically and mitotically condensed Bs both in size and shape. They occurred only in plants with 2 or 3 Bs. In the plants with 2 Bs, 2.5% pollen grains had a nucleus with 1 chromocentre; in the plants with 3 Bs, 41.4% of uninucleate pollen grains had 1 chromocentre and 0.2% of grains had 2 chromocentres. Comparison of the frequency of Bs in the metaphase of the first pollen mitosis and that of chromocentres in the nuclei of the uninucleate pollen grains was carried out. The author suggests that in pollen-grain nuclei one B chromosome per genome remains decondensed, and every next one (if it occurs) forms a chromocentre.     

Crystallographic studies on E. coli Trp aporepressor

Two crystal forms of Trp aporepressor, an inactive, unliganded form of Trp repressor have been obtained which are suitable for high resolution X-ray diffraction analysis. Trp aporepressor crystallizes in two forms: orthorhombic, P 2(1)2(1)2 and tetragonal P 4(1) (or P 4(3] which diffract to 1.8 A and 2.4 A, respectively. The orthorhombic crystals contain one monomer in the asymmetric unit, therefore the twofold axis relates two subunits of the dimer as in the case of the previously described Trp repressor (R. Schevitz et al., 1985, Nature, 317, 782-786) and Trp pseudorepressor (C. Lawson & P. B. Sigler, 1988, Nature, 333, 869-871). The tetragonal crystals have two dimers in the asymmetric unit and are nearly isomorphous with the tetragonal crystals of Trp repressor and Trp pseudorepressor grown under similar conditions but in the presence of an activator or inhibitor, respectively.     

An integrated approach to the ligand binding specificity of Neisseria meningitidis M1 alanine aminopeptidase by fluorogenic substrate profiling,inhibitory studies and molecular modeling

Neisseria meningitides is a gram-negative diplococcus bacterium and is the main causative agent of meningitis and other meningococcal diseases. Alanine aminopeptidase from N. meningitides (NmAPN) belongs to the family of metallo-exopeptidase enzymes, which catalyze the removal of amino acids from the N-terminus of peptides and proteins, and are found among all the kingdoms of life. NmAPN is suggested to be mostly responsible for proteolysis and nutrition delivery, similar to the orthologs from other bacteria.     

Crystal structure of YfeU protein from Haemophilus influenzae: a predicted etherase involved in peptidoglycan recycling

The crystal structure of the H. influenzae YfeU protein, was determined at 1.90 Å resolution using multi-wavelength anomalous diffraction. YfeU belongs to a very large conserved family of proteins found mainly in bacteria but also in archaea and eukaryota. The protein is a homolog of eukaryotic glucokinase regulator and is predicted to be a sugar phosphate isomerase or aminotransferase. Here we describe the structure of YfeU and discuss the possible function as an etherase possibly involved in peptidoglycan recycling.     

The structure of pyogenecin immunity protein, a novel bacteriocin-like immunity protein from Streptococcus pyogenes

Background  

Many Gram-positive lactic acid bacteria (LAB) produce anti-bacterial peptides and small proteins called bacteriocins, which enable them to compete against other bacteria in the environment. These peptides fall structurally into three different classes, I, II, III, with class IIa being pediocin-like single entities and class IIb being two-peptide bacteriocins. Self-protective cognate immunity proteins are usually co-transcribed with these toxins. Several examples of cognates for IIa have already been solved structurally. Streptococcus pyogenes, closely related to LAB, is one of the most common human pathogens, so knowledge of how it competes against other LAB species is likely to prove invaluable.