Transduction-like gene transfer in the methanogen Methanococcus voltae. (49/6255)

Strain PS of Methanococcus voltae (a methanogenic, anaerobic archaebacterium) was shown to generate spontaneously 4.4-kbp chromosomal DNA fragments that are fully protected from DNase and that, upon contact with a cell, transform it genetically. This activity, here called VTA (voltae transfer agent), affects all markers tested: three different auxotrophies (histidine, purine, and cobalamin) and resistance to BES (2-bromoethanesulfonate, an inhibitor of methanogenesis). VTA was most effectively prepared by culture filtration. This process disrupted a fraction of the M. voltae cells (which have only an S-layer covering their cytoplasmic membrane). VTA was rapidly inactivated upon storage. VTA particles were present in cultures at concentrations of approximately two per cell. Gene transfer activity varied from a minimum of 2 x 10(-5) (BES resistance) to a maximum of 10(-3) (histidine independence) per donor cell. Very little VTA was found free in culture supernatants. The phenomenon is functionally similar to generalized transduction, but there is no evidence, for the time being, of intrinsically viral (i.e., containing a complete viral genome) particles. Consideration of VTA DNA size makes the existence of such viral particles unlikely. If they exist, they must be relatively few in number;perhaps they differ from VTA particles in size and other properties and thus escaped detection. Digestion of VTA DNA with the AluI restriction enzyme suggests that it is a random sample of the bacterial DNA, except for a 0.9-kbp sequence which is amplified relative to the rest of the bacterial chromosome. A VTA-sized DNA fraction was demonstrated in a few other isolates of M. voltae.  (+info)

In vivo processing and antibiotic activity of microcin B17 analogs with varying ring content and altered bisheterocyclic sites. (50/6255)

BACKGROUND: The Escherichia coli peptide antibiotic microcin B17 (MccB17) contains four oxazole and four thiazole rings, and inhibits DNA gyrase. The role of individual and tandem pairs of heterocycles in bioactivity has not been determined previously. RESULTS: The two tandem 4,2-bisheterocycles in MccB17 were varied by expression of MccB17 or mutants containing altered sequences at Gly39-Ser40-Cys41 or Gly54-Cys55-Ser56. A mixture of five-nine-ring MccB17 isoforms were separated and quantitated for antibiotic potency. Mutagenesis of the thiazole-oxazole pair significantly affected antibiotic activity compared with the upstream oxazole-thiazole, which might stabilize partially cyclized intermediates against proteolysis. CONCLUSIONS: Enzymatic heterocyclization in native MccB17 occurs distributively. Antibiotic activity correlates with the number of rings and is differentially sensitive to both the location and the identity of the 4,2-tandem heterocycle pairs in MccB17. Such tandem heterocycles might be useful pharmacophores in combinatorial libraries.  (+info)

Methanobacterium thermoformicicum thymine DNA mismatch glycosylase: conversion of an N-glycosylase to an AP lyase. (51/6255)

The thymine DNA mismatch glycosylase from Methanobacterium thermoformicicum, a member of the endonuclease III family of repair proteins, excises the pyrimidine base from T-G and U-G mismatches. Unlike endonuclease III, it does not cleave the phosphodiester backbone by a beta-elimination reaction. This cleavage event has been attributed to a nucleophilic attack by the conserved Lys120 of endonuclease III on the aldehyde group at C1' of the deoxyribose and subsequent Schiff base formation. The inability of TDG to perform this beta-elimination event appears to be due to the presence of a tyrosine residue at the position equivalent to Lys120 in endonuclease III. The purpose of this work was to investigate the requirements for AP lyase activity. We replaced Tyr126 in TDG with a lysine residue to determine if this replacement would yield an enzyme with an associated AP lyase activity capable of removing a mismatched pyrimidine. We observed that this replacement abolishes the glycosylase activity of TDG but does not affect substrate recognition. It does, however, convert the enzyme into an AP lyase. Chemical trapping assays show that this cleavage proceeds through a Schiff base intermediate and suggest that the amino acid at position 126 interacts with C1' on the deoxyribose sugar.  (+info)

Volatile anaesthetics and the atmosphere: atmospheric lifetimes and atmospheric effects of halothane, enflurane, isoflurane, desflurane and sevoflurane. (52/6255)

The atmospheric lifetimes of the halogenated anaesthetics halothane, enflurane, isoflurane, desflurane and sevoflurane with respect to reaction with the hydroxyl radical (OH.) and UV photolysis have been determined from observations of OH. reaction kinetics and UV absorption spectra. Rate coefficients for the reaction with OH radicals for all halogenated anaesthetics investigated ranged from 0.44 to 2.7 x 10(-14) cm3 molec-1 s-1. Halothane, enflurane and isoflurane showed distinct UV absorption in the range 200-350 nm. In contrast, no absorption in this wavelength range was detected for desflurane or sevoflurane. The total atmospheric lifetimes, as derived from both OH. reactivity and photolysis, were 4.0-21.4 yr. It has been calculated that up to 20% of anaesthetics enter the stratosphere. As a result of chlorine and bromine content, the ozone depletion potential (ODP) relative to chlorofluorocarbon CFC-11 varies between 0 and 1.56, leading to a contribution to the total ozone depletion in the stratosphere of approximately 1% for halothane and 0.02% for enflurane and isoflurane. Estimates of the greenhouse warming potential (GWP) relative to CFC-12 yield values of 0.02-0.14, resulting in a relative contribution to global warming of all volatile anaesthetics of approximately 0.03%. The stratospheric impact of halothane, isoflurane and enflurane and their influence on ozone depletion is of increasing importance because of decreasing chlorofluorocarbons globally. However, the influence of volatile anaesthetics on greenhouse warming is small.  (+info)

Investigation on the detergent role in the function of secondary quinone in bacterial reaction centers. (53/6255)

In this paper are reported studies on the detergent role in isolated reaction centers (RC) from Rhodobacter sphaeroides, over a large range of lauryldimethylamino-N-oxide (LDAO) concentrations, in influencing the thermodynamics of the quinone exchange reaction as well as the protein aggregation. The occurrence of the quinone exchange reaction between the QB-binding site (where QB is the second quinone molecule of two in the RC) and the ubiquinone 0 dissolved in the different environments (water, LDAO micelles and detergent phase of the protein-detergent complex) has also been analyzed. Measurements carried out in QB-depleted RC to which exogenous quinone has been added show that the relative amplitudes of the slow and fast phase of the recombination reaction depend on this parameter. The overall amount of the restored QB-functionality is affected by the concentration of the LDAO in solution. Interpolation of the titration curves with a quadratic function obtained by simple considerations allowed the binding constant of UQ0 to the QB-binding site to be calculated. From the fitting procedure, the distribution of the quinone in the different environments present in solution was evaluated, indicating that the exchange reaction can take place only between the QB-site and the detergent phase. The dependence of the quinone pool size upon the volume of the phase in which the interacting quinone is solubilized is also discussed. The increasing difficulty in saturating the QB-pocket above the LDAO critical micellar concentration is finally related to the association of protein-detergent complexes to form large protein clusters.  (+info)

Phosphatoquinones A and B, novel tyrosine phosphatase inhibitors produced by Streptomyces sp. (54/6255)

Phosphatoquinones A and B were isolated from the cultured broth of Streptomyces sp. TA-0363 and their structures were elucidated by spectroscopic analyses. Phosphatoquinones A and B inhibited the protein tyrosine phosphatase activity prepared from human Ball-1 cells with IC50 of 28 microM and 2.9 microM, respectively.  (+info)

Purification and characterization of 2-deoxy-scyllo-inosose synthase derived from Bacillus circulans. A crucial carbocyclization enzyme in the biosynthesis of 2-deoxystreptamine-containing aminoglycoside antibiotics. (55/6255)

The biosynthesis of 2-deoxystreptamine, the central aglycon of a major group of clinically important aminoglycoside antibiotics, commences with the initial carbocycle formation step from D-glucose-6-phosphate to 2-deoxy-scyllo-inosose. This crucial step is known to be catalyzed by 2-deoxy-scyllo-inosose synthase, which has not yet been characterized so far. Reported in this paper is the first purification of 2-deoxy-scyllo-inosose synthase from butirosin-producing Bacillus circulans SANK 72073 to electrophoretic homogeneity. The enzyme was isolated as a heterodimeric protein comprising from a 23 kDa- and a 42 kDa polypeptide chains. The Km of the enzyme for D-glucose-6-phosphate was estimated to be 9.0 x 10(-4) M and that for NAD+ 1.7 x 10(-4) M, kcat for D-glucose-6-phosphate being 7.3 x 10(-2) s(-1). The presence of Co2+ was essential for the enzyme activity, but Zn2+ was totally inhibitory. While the reaction mechanisms are quite similar, 2-deoxy-scyllo-inosose synthase appears to be distinct from dehydroquinate synthase in the shikimate pathway, with respect to the quaternary structure, metal ion requirement, and the kinetic parameters.  (+info)

Pseudo-native motifs in the noncovalent heme-apocytochrome c complex. Evidence from antibody binding studies by enzyme-linked immunosorbent assay and microcalorimetry. (56/6255)

When beef heart apocytochrome c is unfolded, it folds upon noncovalent heme binding (Dumont, M. E., Corin, A. F., and Campbell, G.A. (1994) Biochemistry, 33, 7368-7378). Here, the conformation of the heme-apocytochrome noncovalent complex is compared with that of holocytochrome c. A purification method was designed for obtaining in large amounts apocytochrome c that was shown by amino acid analysis and mass spectroscopy to be chemically intact. The apoprotein and its noncovalent complex were characterized by absorption, fluorescence, circular dichroism, and sedimentation velocity, confirming previous reports. Sedimentation-diffusion equilibrium showed that the apoprotein and its noncovalent complex with heme were monomeric. Surprisingly, whereas apocytochrome c was quite soluble, the noncovalent complex slowly formed heavy aggregates, thus precluding experiments at the concentrations needed for structural studies. Two monoclonal antibodies that bind strongly to distinct antigenic sites on native holocytochrome were used to probe the noncovalent complex conformation. For both antibodies, the affinity for the noncovalent complex was only about 5-10-fold smaller than that for native holocytochrome c, and about 50-100-fold larger than that for apocytochrome c. These results indicate that the noncovalent complex, although not entirely native, carries some pseudo-native structural motifs.  (+info)