Mutagenic scan of the H-N-H motif of colicin E9: implications for the mechanistic enzymology of colicins,homing enzymes and apoptotic endonucleases

Colicin E9 is a microbial toxin that kills bacteria through random degradation of chromosomal DNA. Within the active site of the cytotoxic endonuclease domain of colicin E9 (the E9 DNase) is a 32 amino acid motif found in the H-N-H group of homing endonucleases. Crystal structures of the E9 DNase have implicated several conserved residues of the H-N-H motif in the mechanism of DNA hydrolysis. We have used mutagenesis to test the involvement of these key residues in colicin toxicity, metal ion binding and catalysis. Our data show, for the first time, that the H-N-H motif is the site of DNA binding and that Mg²⁺-dependent cleavage of double-stranded DNA is responsible for bacterial cell death. We demonstrate that more active site residues are required for catalysis in the presence of Mg²⁺ ions than transition metals, consistent with the recent hypothesis that the E9 DNase hydrolyses DNA by two distinct, cation-dependent catalytic mechanisms. The roles of individual amino acids within the H-N-H motif are discussed in the context of the available structural information on this and related DNases and we address the possible mechanistic similarities between caspase-activated DNases, responsible for the degradation of chromatin in eukaryotic apoptosis, and H-N-H DNases.     

Differences in Cold Inactivation of Phosphoenolpyruvate Carboxylase among C4 Species: The Effect of pH and of Enzyme Concentration

Among various C4 plants we found a wide range in the level of inactivation of phosphoenolpyruvate carboxylase (PEPC) at low temperature (0 °C). The activity of the 2-fold diluted enzyme in crude leaf extracts after 60 min incubation (compared to zero time incubation) at pH 7.5, remained above 87 % at low temperatures for the species Setaria verticillata, Portulaca oleracea, and Saccharum officinarum, and between 11 and 17 % in the species Cynodon dactylon and Atriplex halimus. The enzyme exhibited intermediate levels of inactivation (42 to 58 %) for the species Amaranthus sp., Zea mays, Salsola kali, and Digitaria sanguinalis. The enzyme activity for S. verticillata was unaffected between pH 5.7 and 8.4 during incubation at room and low temperatures. Under similar conditions, the activity of the enzyme from C. dactylon was stable between pH 5.7 and 7.0 and decreased at pH above 7.0, but for Z. mays it was enhanced between pH 5.7 and 6.8 and decreased at pH above 7.0.     

Mutation-Directed Chemical Cross-Linking of Human Immunodeficiency Virus Type 1 gp41 Oligomers

The human immunodeficiency virus type 1 transmembrane protein gp41 oligomer anchors the attachment protein, gp120, to the viral envelope and mediates viral envelope-cell membrane fusion following gp120-CD4 receptor-chemokine coreceptor binding. We have used mutation-directed chemical cross-linking with bis(sulfosuccinimidyl)suberate (BS³) to investigate the architecture of the gp41 oligomer. Treatment of gp41 with BS³ generates a ladder of four bands on sodium dodecyl sulfate-polyacrylamide gels, corresponding to monomers, dimers, trimers, and tetramers. By systematically replacing gp41 lysines with arginine and determining the mutant gp41 cross-linking pattern, we observed that gp41 N termini are cross-linked. Lysine 678, which is close to the transmembrane sequence, was readily cross-linked to Lys-678 on other monomers within the oligomeric structure. This arrangement appears to be facilitated by the close packing of membrane-anchoring sequences, since the efficiency of assembly of heterooligomers between wild-type and mutant Env proteins is improved more than twofold if the mutant contains the membrane-anchoring sequence. We also detected close contacts between Lys-596 and Lys-612 in the disulfide-bonded loop/glycan cluster of one monomer and lysines in the N-terminal amphipathic α-helical oligomerization domain (Lys-569 and Lys-583) and C-terminal α-helical sequence (Lys-650 and Lys-660) of adjacent monomers. Precursor-processing efficiency, gp120-gp41 association, soluble recombinant CD4-induced shedding of gp120 from cell surface gp41, and acquisition of gp41 ectodomain conformational antibody epitopes were unaffected by the substitutions. However, the syncytium-forming function was most dependent on the conserved Lys-569 in the N-terminal α-helix. These results indicate that gp160-derived gp41 expressed in mammalian cells is a tetramer and provide information about the juxtaposition of gp41 structural elements within the oligomer.     

Novel cystic fibrosis mutation associated with mild disease in Cypriot patients

Cyprus is an island in the eastern Mediter-ranean basin inhabited by people of Caucasian extraction, mostly Greek-Cypriots. The most common inherited disease among Caucasians is cystic fibrosis (CF). Although no careful scientific study had ever been done the impression was that CF was extremely rare among the Greek-Cypriots, with an incidence estimated at around 130,000. About 2 years ago, we introduced molecular diagnostic methodology in an effort to assist clinicians in safer diagnosis of patients presenting with atypical CF symptomatology, and also for testing the hypothesis that mutations that cause milder phenotypes might be responsible for misdiagnosis or for missing entirely some cases of CF. Initial screening for F508 revealed that it is indeed rare in the general population. Further screening of suspected CF patients revealed a novel mutation that converted leucine at position 346 to proline (L346P) in two unrelated families. The second CF mutation was F508 and 1677delTA in the two families respectively, both reportedly associated with severe phenotypes. Yet our patients did not present with typical CF pictures possibly because of the dominant nature of this novel mild mutation in exon 7. Symptoms included failure to thrive, chest infections and electrolyte disturbances. These findings raise the possibility that Cyprus might have been spared very severe CF phenotypes but not cystic fibrosis transmembrane conductance regulator (CFTR) mutations.     

Structural and immunological characterization of a biosurfactant produced by Bacillus licheniformis JF-2.

Bacillus licheniformis JF-2 produces a very active biosurfactant under both aerobic and anaerobic conditions. We purified the surface-active compound to homogeneity by reverse-phase C18 high-performance liquid chromatography and showed that it is a lipopeptide with a molecular weight of 1,035. Amino acid analysis, fast atom mass and infrared spectroscopy, and, finally, 1H, 13C, and two-dimensional nuclear magnetic resonance demonstrated that the biosurfactant consists of a heterogeneous C15 fatty acid tail linked to a peptide moiety very similar to that of surfactin, a lipopeptide produced by Bacillus subtilis. Polyclonal antibodies were raised against surfactin and shown to exhibit identical reactivity towards purified JF-2 lipopeptide in competition enzyme-linked immunosorbent assays, thus providing further evidence for the structural similarity of these two compounds. Under optimal conditions, the B. licheniformis JF-2 biosurfactant exhibits a critical micelle concentration of 10 mg/liter and reduces the interfacial tension against decane to 6 x 10(-3) dyne/cm, which is one of the lowest interfacial tensions ever reported for a microbial surfactant.     

Demonstration of efficient trichloroethylene biodegradation in a hollow‐fiber membrane bioreactor

Rapid cometabolism of trichloroethylene (TCE) by pure cultures of Methylosinus trichosporium OB3b PP358 was demonstrated in a two‐stage hollow‐fiber membrane bioreactor over the course of 3 weeks. PP358 was grown in a continuous‐flow chemostat and circulated through the shell of a hollow‐fiber membrane module (HFMM), while TCE contaminated water (160 to 1450 μg/L) was pumped through the fiber lumen (fiber interior). In parallel‐flow HFMM biological experiments, 82% to 89% of the influent TCE was removed from the lumen (5.1‐min residence time) with 99% of the transferred TCE undergoing biodegradation. Biological experiments in a larger capacity baffled radial‐flow HFMM resulted in 66% to 99% TCE transferred and 93% to 96% TCE biodegradation at lumen residence times of between 1.5 and 3.7 min. Biodegradation was maintained throughout the experiments at pseudo‐first‐order biodegradation rate constants of 0.41 to 2.8 L/mg TSS/day. Best‐fit computer modeling of the baffled radial‐flow biological process estimated mass transfer coefficients as large as 2.7 × 10⁻² cm/min. The computer model was also shown to simulate the experimental results quite well. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 62: 681–692, 1999.     

Rate controls on the oxidation of heaps of pyritic material imposed by upper temperature limits on the bacterially catalysed process

Abstract: Oxidation of a fraction of the pyrite in gold-bearing pyrite ores before leaching often increases gold recovery rates. There are a number of techniques availablc, one of which is dump oxidation. Commercial viability of such an operation requires a net oxidation of 50% or more in 1 year. This is possible in a dump provided convection is established quickly. Since convection requires temperatures in the dump to be well above ambient, a question arises as to the constraints that might be imposed by the upper temperature limit ( T _kill) of the microorganisms which catalyse the oxidation of the pyrite. Our objective is to investigate, by way of a computer model, the importance of T _kill as a parameter effecting the oxidation rate and the fraction of pyrite oxidised after 1 year in dumps where the goal is to oxidise about 50% of the pyrite in a year or less.     

Phase transitions in crystals of racemic long chain 2-amino alcohols

Various techniques, namely differential scanning calorimetry, optical microscopy, dielectric and Raman spectroscopy, all covering a wide range of temperatures, were used to study the thermodynamically stable phases and molecular mobility of crystals of long chain 2-amino alcohols. The results showed that two different crystal forms are present in each sample. The temperature behaviour of the phases is studied in details.     

A biocatalyst for the removal of sulfite from alcoholic beverages

The presence of sulfites in alcoholic beverages, particularly in wines, can cause allergic responses with symptoms ranging from mild gastrointestinal problems to life threatening anaphylactic shock in a substantial portion of the population. We have developed a simple and inexpensive biocatalytic method that employs wheatgrass (Triticum aestivum) chloroplasts for the efficient oxidation of sulfites in wines to innocuous sulfates. A sufficiently high rate of sulfite oxidation was obtained in the presence of ethanol at concentrations commonly found in most wines. Crude chloroplast preparations at a concentration as low as 5 mg/mL were capable of reducing sulfite in commercial white wines from 150 ppm to under 7.5 ppm within 3 hours. A 93% removal of sulfite in commercial red wines was observed with 1 mg/mL chloroplasts within 45 min. Optimal sulfite removal efficiency was observed at pH 8.5 and was promoted by illumination, indicating the participation of light-induced photosynthetic electron transport processes in sulfite oxidation. Overall, this work indicates that biocatalytic oxidation using wheatgrass chloroplasts can be employed to remove sulfites from beverages prior to consumption.