Degradation of 2-chloroethanol by wild type and mutants of Pseudomonas putida US2

A strain of Pseudomonas putida was isolated that was able to degrade 2-chloroethanol. The degradation proceeded via 2-chloroacetaldehyde and chloroacetate to glycolate. In crude extracts the enzymes for this degradation pathway could be detected. All enzymes proved to be inducible. The dehalogenase that catalyzed the dehalogenation of chloroacetate to glycolate was further characterized. It consisted of a single polypeptide chain with a molecular mass of 28 kDa. After induction the dehalogenase was expressed at a high level. In a mutant resistant to high concentrations of 2-chloroethanol the dehalogenase was no longer expressed. The mechanism of resistance seemed to be due to the inability to convert chloroacetate and export of this compound out of the cell.Non-standard abbreviations CEO 2-chloroethanol - DCPIP 2,6-dichlorophenolindophenol - FPLC fast protein liquid chromatography - PAGE polyacrylamide gelelectrophoresis - PES phenazine ethosulfate - PMS phenazine methosulfate - PQQ pyrroloquinoline quinone     

Binding of 2'(3')-O-(2,4,6-trinitrophenyl)-adenosine 5'-diphosphate opens the pathway for protons through the chloroplast ATPase complex

The effect of 2'(3')-O-(2,4,6-trinitrophenyl)-adenosine 5'-diphosphate (TNP-ADP) on photophosphorylation and on the proton conductivity of the thylakoid membrane has been investigated. The results show that TNP-ADP is a potent competitive inhibitor of photophosphorylation (Ki = 1-2 microM). Moreover, in the absence of ADP and Pi, TNP-ADP accelerates basal electron transport of chloroplasts. Addition of ADP, which promotes release of the analogue from CF1, completely reverses this effect of TNP-ADP; likewise Pi alone reverses stimulation of electron transport by TNP-ADP. Dicyclohexylcarbodiimide treatment, which is known to close CF0 to H+, completely abolishes the effect of TNP-ADP. The measurements of the alkalization of the medium and the acidification of the thylakoid lumen following single turnover flashes showed that binding of TNP-ADP to CF1 increased membrane permeability for H+. Further results suggest that binding of TNP-ADP to the catalytic site of CF1 opens the CF0-CF1 complex for H+. Since ADP, as well as Pi alone, reverses the effect, it is concluded that TNP-ADP induces a conformation of the CF0-CF1 complex similar to the one triggered by simultaneous binding of ADP plus Pi. This may be achieved by interaction of the TNP residue with the Pi binding site. Thus it seems that the status of the catalytic site(s) in CF1 can be transmitted to the CF0 part to control proton flux through the ATPase complex in an economically reasonable way.     

Functional renaturation of receptor polypeptides eluted from SDS polyacrylamide gels

In order to gain further support for the concept that a homo-oligomeric protein-complex may be sufficient to form a functional ligand-activated ion channel and to explore additional possibilities for the reconstitution of channel activity, a single polypeptide band of the purified neuronal AChR from insects has been electroeluted from SDS-polyacrylamide gels, the SDS removed and the polypeptides incorporated into liposomes. Liposomes were fused into planar lipid bilayers which were subsequently analysed for channel activity. Fluctuations of cation-channels were detected after addition of agonists (carbamylcholine); channel activity was blocked by antagonists (d-tubocurarine). The channels formed by electroeluted polypeptides gave conductance values, as well as kinetic data, quite similar to channels formed by the native receptor protein. Sedimentation experiments using sucrose density gradient centrifugation revealed that a considerable portion of the electroeluted polypeptides assembled during the reconstitution process to form oligomeric complexes with a sedimentation coefficient of about 10 S; thus resembling the native receptor complex. Offprint requests to: W. Hanke     

Phylogeny of Greya (Lepidoptera: Prodoxidae), based on nucleotide sequence variation in mitochondrial cytochrome oxidase I and II: congruence with morphological data

The phylogeny of Greya Busck (Lepidoptera: Prodoxidae) was inferred from nucleotide sequence variation across a 765-bp region in the cytochrome oxidase I and II genes of the mitochondrial genome. Most parsimonious relationships of 25 haplotypes from 16 Greya species and two outgroup genera (Tetragma and Prodoxus) showed substantial congruence with the species relationships indicated by morphological variation. Differences between mitochondrial and morphological trees were found primarily in the positions of two species, G. variabilis and G. pectinifera, and in the branching order of the three major species groups in the genus. Conflicts between the data sets were examined by comparing levels of homoplasy in characters supporting alternative hypotheses. The phylogeny of Greya species suggests that host-plant association at the family level and larval feeding mode are conservative characters. Transition/transversion ratios estimated by reconstruction of nucleotide substitutions on the phylogeny had a range of 2.0-9.3, when different subsets of the phylogeny were used. The decline of this ratio with the increase in maximum sequence divergence among taxa indicates that transitions are masked by transversions along deeper internodes or long branches of the phylogeny. Among transitions, substitutions of A-->G and T-->C outnumbered their reciprocal substitutions by 2-6 times, presumably because of the approximately 4:1 (77%) A+T-bias in nucleotide base composition. Of all transversions, 73%-80% were A<-->T substitutions, 85% of which occurred at third positions of codons; these estimates did not decrease with an increase in maximum sequence divergence of taxa included in the analysis. The high frequency of A<-->T substitutions is either a reflection or an explanation of the 92% A+T bias at third codon positions.