Structural analysis and proteolytic activation of Enterococcus faecalis cytolysin, a novel lantibiotic

Clinical isolates of Enterococcus faecalis more commonly produce a cytolysin than do commensal isolates. Epidemiologic evidence and animal-model studies have established a role for the cytolysin in the pathogenesis of enterococcal disease. The cytolysin consists of two structural subunits, CylL_L and CylL_s, that are activated by a third component, CylA. Genetic and biochemical characterization of CylA indicate that it is a serine protease, and that activation putatively results from cleavage of one or both cytolysin subunits. Genetic evidence also suggests that the cytolysin subunits are related to the rapidly growing class of bacteriocins termed lantibiotics. However, unlike lantibiotics, the cytolysin is lytic for eukaryotic as well as prokaryotic cells, and it consists of two structural subunits. This report describes the purification and characterization of the cytolysin subunits and detection of lanthionine-type post-translational modifications within their structures. Furthermore, the cleavage specificity of the CylA activator is reported and it is shown that proteolytic activation of both subunits is essential for activity.     

Structural and functional differentiation of two clinally distributed glucosephosphate isomerase allelic isozymes from the teleost Fundulus heteroclitus

The teleost Fundulus heteroclitus (L.) possesses two loci, Gpi-A and Gpi-B, for the glycolytic enzyme, glucose-phosphate isomerase (GPI; D- glucose-6-phosphate ketol-isomerase; E.C. 5.3.1.9). The Gpi-B locus is polymorphic in Fundulus, with two common alleles, Gpi-Bb and Gpi-Bc, distributed in a clinal manner in populations along the east coast of North America. Since this clinal distribution is strongly correlated with a temperature gradient, we asked whether the GPI-B2 allozymes were functionally adapted to the thermal environment in which a given phenotype predominated. The two major GPI-B2 allozymes were purified to homogeneity and were characterized as to molecular weight, isoelectric pH, thermal denaturation, and kinetic parameters. Both GPI-Bb2 and GPI- Bc2 allozymes have molecular masses of 110 kD, and they have isoelectric pHs of 6.4 and 6.6, respectively. The GPI-Bb2 allozyme was more stable to thermal denaturation than was the GPI-Bc2 enzyme. Kinetic properties of the allelic isozymes were investigated both as a function of pH and as a function of temperature. At 25 degrees C, over the pH range considered, there were no significant differences between allozymes, either in Km for fructose-6-phosphate or in Ki for 6- phosphogluconate, but apparent Vmax values differed between pH 7.5 and pH 8.5. All steady-state kinetic parameters showed strong temperature dependence, but the allozymes differed only in the Ki for 6- phosphogluconate at temperatures greater than 30 degrees C. On the basis of the observed structural and functional differences alluded to above, the hypothesis that the major allelic isozymes of the Gpi-B locus were functionally equivalent was rejected. However, it is not yet known whether these structural and functional differences have any significance at higher levels of biological organization.      

Structure and properties of hemocyanins. XIV. Reassociation of Helix pomatia alpha-hemocyanin

Helix pomatia α-hemocyanin dissociates within minutes into $\text{1}\text{10}$-size and $\text{1}\text{20}$-size molecules, on increase of pH in the alkaline region.The rate and final state of reassociation of $\text{1}\text{10}$-size and $\text{1}\text{20}$-size molecules have been studied, by turbidity measurements and ultracentrifugation, on lowering of pH or on the addition of calcium ions.Reassociation of $\text{1}\text{10}\text{-size}$ molecules proceeds in two phases, with half-times in the order of minutes and one hour, respectively. The slow phase is linked to the disappearance of transitory intermediates, presumably dimers and tetramers of $\text{1}\text{10}$-size molecules.A hysteresis is observed in the final state of pH-dependent and Ca²⁺-dependent dissociation-reassociation.Reassociation of $\text{1}\text{20}$-size molecules depends on the initial (isomeric) state of these molecules. The F(fast sedimenting)-$\text{1}\text{20}$-size molecules reassociate almost completely to whole molecules, whereas the S(slow sedimenting)-form does not reassociate at all.     

Structural homology of lens crystallins. II. Homology expressed by correlation coefficients and hydropathy profiles

Bovine lens alpha A- and alpha B-crystallin polypeptides show extensive sequence homology with each other, but apparently none with beta Bp- and gamma 2-crystallin. Despite only 30% sequence homology, the latter two proteins are assumed to have a strong correspondence in tertiary structure, consisting of four structurally similar folding units of antiparallel beta-sheet. We have tested for internal structural repeats in all crystallins, and structural homology between crystallins, by comparing various physical properties of the amino acid residues, such as bulkiness and propensity to form beta-sheet and beta-turn structure. Two procedures used a combination of five physical parameters to calculate correlation coefficients. The 4-fold structural repeat in gamma 2-crystallin and the internal duplication in beta Bp-crystallin were readily detectable, as was also the strong structural homology between corresponding folding units in beta Bp- and gamma 2-crystallin. However, for alpha-crystallin polypeptides, no conclusive support was obtained for either a four-unit or a six-unit folding, the two models previously considered by us. The third procedure compared smoothened hydropathy plots, representing hydrophilic and hydrophobic regions along the polypeptide sequences. Hydropathy profiles were found to show strong correspondence, particularly between alpha B-crystallin and beta Bp-crystallin. These observations support a similar 4-fold folding pattern for all bovine crystallins. A possible role in subunit interactions of the N-terminal folding unit, which has hydrophobic surface characteristics in both alpha- and beta-crystallin polypeptides, is proposed.     

Primary structure and organization of the gene for a procaryotic, cell envelope-located serine proteinase

We have determined the complete nucleotide sequence of the gene for the cell envelope-located proteinase of Lactococcus lactis SK11. The gene contains a very AT-rich promoter region followed by the coding sequence of a protein of 1962 amino acids. Comparison of the NH2-terminal amino acid sequence of the mature proteinase and the expected primary translation product of the proteinase gene indicates that the enzyme is probably synthesized as a pre-pro-protein. This is confirmed by expression studies of the proteinase gene in Escherichia coli. The amino acid sequence of the proteinase shows significant homology to a number of serine proteinases of the subtilisin family. Compared with the related proteinase of L. lactis Wg2, the proteinase of L. lactis SK11 contains a 60-amino acids duplication and a total of 44-amino acid substitutions, some of which may account for the different cleavage specificity of both enzymes. Furthermore, a region was identified in the Lactococcus proteinase, which shows homology to the membrane-anchoring domains of a number of proteins from other Gram-positive bacteria.     

Echocardiographic assessment and percutaneous closure of multiple atrial septal defects

Percutaneous coronary intervention can be associated with distal embolization of thrombotic material causing myocardial necrosis and infarction. We discuss the role of intravascular imaging to guide the use of a distal protection device by describing the outcome of a young woman presenting with non-ST elevation myocardial infarction. Coronary angiography demonstrated an isolated minor stenosis in the proximal left anterior descending coronary artery with slight haziness beyond the lesion. Intravascular ultrasound confirmed an extensive thrombus overlying a bulky atherosclerotic plaque. A distal filter wire was therefore successfully used to reduce the risk of distal embolization. The use of intravascular ultrasound in patients presenting with acute coronary syndrome may reveal large thrombi that are difficult to image using conventional angiographic techniques. Intravascular ultrasound can therefore be used as a tool to select lesions requiring distal protection.     

Exclusion chromatography of concentrated hemoglobin solutions: Comparison of the self-association behavior of the oxy and deoxy forms of the α2β2 species

Theory pertaining to the interpretation of partition chromatography results obtained with self-associating protein systems studied at high total concentrations is extended to permit consideration of situations in which both monomeric and dimeric states partition. This development, which includes considerations of thermodynamic nonideality effects, permits a quantitative correlation of human oxyhemoglobin results reported previously and obtained in this work employing a different stationary matrix of controlled-pore glass beads. The two sets of results, obtained at pH 7.3 and 20°C- indicate that the α₂β₂ species of oxyhemoglobin self-associates. Two types of association pattern, discrete dimerization and an indefinite self-association, are examined. This is done for a realistic range of values for the radius, r, of the effective hard sphere appropriate to the calculation of the covolume of the α₂β₂ species in the assessment of the thermodynamic nonideality contribution. Assessed values of the isodesmic association constant range from 66 = 23 M ^?1(r = 2.84 nm) to 154 = 26 M^?1' (r = 3.13 nm). This mode of indefinite association is marginally favored over a dimerization when the larger value of r is considered, the two patterns becoming virtually indistinguishable for the lower value of r. Partition chromatography results are also presented for human deoxyhemoglobin up to a total concentration of 225 $\text{g}\text{I}$, and are analyzed in a similar fashion to show that the indefinite self-association pattern is favored, governed by an isodesmic constant in the range 91 = 9 M^?1(r = 2.84 nm) to 223 = 84 M^?1 (r = 3.13 nm). Comparison of the constants assessed for the oxy and deoxy systems permits discussion of the concept that oxygen binds preferentially to the α₂β₂ species of deoxyhemoglobin in comparison with its polymers.     

Structure and properties of hemocyanins. XIII. Dissociation of Helix pomatia alpha-hemocyanin at alkaline pH

Helix pomatia α-hemocyanin can dissociate stepwise into $\text{1}\text{2}$-size, $\text{1}\text{10}$-size and $\text{1}\text{20}$-size molecules. Both $\text{1}\text{10}$-size and $\text{1}\text{20}$-size molecules can occur in two isomeric forms, differing considerably in sedimentation behaviour.The effects of pH, ionic strength, temperature, Ca²⁺ concentration and oxygen pressure on these dissociation and isomerization steps were investigated systematically by sedimentation analysis.Dissociation and isomerization are favoured by increasing pH or temperature. Changes in ionic strength affect each step differently. Calcium ions are extremely effective in preventing dissociation and isomerization at low ionic strength, but this stabilizing ability diminishes at higher ionic strengths. Oxygen binding shifts the pH-dependent dissociation of whole into $\text{1}\text{2}$-size molecules to higher pH. Oxygen has no effect on the other dissociation steps. Intermolecular interactions appear to be predominantly electrostatic.     

Complete resequencing and reannotation of the Lactobacillus plantarum WCFS1 genome

There is growing interest in the beneficial effects of Lactobacillus plantarum on human health. The genome of L. plantarum WCFS1, first sequenced in 2001, was resequenced using Solexa technology. We identified 116 nucleotide corrections and improved function prediction for nearly 1,200 proteins, with a focus on metabolic functions and cell surface-associated proteins.     

Natural diversity and adaptive responses of Lactococcus lactis

Lactococcus lactis is the primary model organism for lactic acid bacteria (LAB) and is widely used in the production of fermented dairy products. In recent years there has been increasing interest in strains isolated from non-dairy environments, as these exhibit a high metabolic diversity and have unique flavour-forming activities. Recent progress has been made in understanding the natural diversity and adaptive responses of L. lactis from dairy and non-dairy origins. Genome sequencing and comparative genomics have also had an impact on understanding natural diversity within the species, and have provided new opportunities for industrial strain development.