On the mechanism of cytochrome oxidation in bacterial photosynthesis Quantum tunnelling effects revisited

We propose that the unique temperature dependence of the Chance-De Vault cytochrome oxidation reaction in Chromatium is not due to a transition from low-temperature nuclear tunnelling to a high-temperature activated electron transfer (ET), but rather originates from two parallel ET processes from two distinct low-potential cytochromes to the bacteriochlorophyll dimer cation. These involve a slow activationless process, which dominates at low temperatures (T 120 K) and an activated process, which is practically exclusive at high temperatures. This conjecture provides plausible nuclear and electronic coupling terms and structural data for the two cytochrome oxidation reactions.     

Mitochondrial DNA polymorphisms in subterranean mole-rats of the Spalax ehrenbergi superspecies in Israel, and its peripheral isolates

Patterns of mitochondrial DNA (mtDNA) variation were examined in 133 mole-rats constituting all four chromosomal species (2n = 52, 2n = 54, 2n = 58, and 2n = 60) of the Spalax ehrenbergi superspecies in Israel, as well as the peripheral isolates of 2n = 60. In the main range of the complex, a total of 28 mtDNA haplotypes were found in 64 mole-rats, with most haplotypes being unique to either a single chromosomal species or population. mtDNA divergence increased from low to high diploid number in a north-to-south direction in Israel. Overall levels of mtDNA diversity were unexpectedly the highest in the 2n = 60, the youngest species of the complex. The mtDNA haplotypes can be separated into two major groups, 2n = 52-54 and 2n = 58-60, and a phylogenetic analysis for each group revealed evidence of a few haplotypes not sorted by diploid number. The overall patterns of mtDNA divergence seen within and among the four chromosomal species are consistent with the parapatric mode of speciation as suggested from previous studies of allozyme and DNA hybridization. In a separate data set the patterns of mtDNA variation were examined across the main geographic range and across peripheral semi-isolates and isolates of the 2n = 60 chromosomal species. Fifteen haplotypes were found in 69 mole-rats. High levels of mtDNA diversity characterized the main range, semi-isolated, and even some desert isolated populations. The peripheral isolates contain much mtDNA diversity, including novel haplotypes.      

Expression and properties of arginyl-tRNA synthetase from jack bean (Canavalia ensiformis)

The coding region for arginyl-tRNA synthetase from jack bean (Canavalia ensiformis) has been sequenced and cloned into the bacterial expression vector pET32a. Transformation of BL21 cells and induction with IPTG results in the high level expression of the protein fused N-terminally with thioredoxin and bearing a His-tag. A substantial proportion of the enzyme is recovered in the soluble fraction of the cell lysate (10 mg per litre cell culture) and can be isolated with metal-affinity technology. The thioredoxin component and the His-tag portion of the fused protein could be removed with thrombin, resulting in a homogeneous product retaining an N-terminal extension of 3.2 kDa compared to the native arginyl-tRNA synthetase. Both full-length fusion and thrombin-treated products proved to be active in aminoacylation, with similar kinetic parameters.     

Redox-controlled,in vivo and in vitro phosphorylation of the α subunit of the light-harvesting complex I in Rhodobacter capsulatus

Labelling of Rhodobacter capsulatus cells with (³²P)Pi in a phototrophic culture results in phosphorylation of a membrane-bound polypeptide identified as the subunit of the LHI antenna complex of the photosynthetic apparatus. Phosphorylation of the same polypeptide was also observed by incubation of chromatophores with (³²P)ATP or under conditions of photophosphorylation with ADP and (³²P)Pi. The identity of the phosphorylated LHI- subunit was demonstrated by N-terminal protein sequencing of the phosphorylated polypeptide and by failure of labelling in LHI-defective mutants. Pre-aeration of the samples or addition of the oxidant potassium ferrcyanide stimulated the kinase activity whereas the presence of soluble cytoplasmic proteins impaired phosphorylation in an in vitro assay. No effect resulted from addition of reductants to the assay medium. The results indicate the presence of a membrane-bound protein kinase in R. capsulatus that phosphorylates the subunit of the LHI antenna complex under redox control.Abbreviations Pi inorganic phosphate - SDS-PAGE sodium dodecyl-sulfate polyacrylamide gel electrophoresis     

Dual chaperone role of the C-terminal propeptide in folding and oligomerization of the pore-forming toxin aerolysin

Throughout evolution, one of the most ancient forms of aggression between cells or organisms has been the production of proteins or peptides affecting the permeability of the target cell membrane. This class of virulence factors includes the largest family of bacterial toxins, the pore-forming toxins (PFTs). PFTs are bistable structures that can exist in a soluble and a transmembrane state. It is unclear what drives biosynthetic folding towards the soluble state, a requirement that is essential to protect the PFT-producing cell. Here we have investigated the folding of aerolysin, produced by the human pathogen Aeromonas hydrophila, and more specifically the role of the C-terminal propeptide (CTP). By combining the predictive power of computational techniques with experimental validation using both structural and functional approaches, we show that the CTP prevents aggregation during biosynthetic folding. We identified specific residues that mediate binding of the CTP to the toxin. We show that the CTP is crucial for the control of the aerolysin activity, since it protects individual subunits from aggregation within the bacterium and later controls assembly of the quaternary pore-forming complex at the surface of the target host cell. The CTP is the first example of a C-terminal chain-linked chaperone with dual function.