Propionate and butyrate dependent bacterial sulfate reduction at extremely haloalkaline conditions and description of Desulfobotulus alkaliphilus sp. nov.

Evidence on the utilization of simple fatty acids by sulfate-reducing bacteria (SRB) at extremely haloalkaline conditions are practically absent, except for a single case of syntrophy by Desulfonatronum on acetate. Our experiments with sediments from soda lakes of Kulunda Steppe (Altai, Russia) showed sulfide production with sulfate as electron acceptor and propionate and butyrate (but not acetate) as an electron donor at a pH 10–10.5 and a salinity 70–180 g l^?1. With propionate as substrate, a highly enriched sulfidogenic culture was obtained in which the main component was identified as a novel representative of the family Syntrophobacteraceae. With butyrate as substrate, a pure SRB culture was isolated which oxidized butyrate and some higher fatty acids incompletely to acetate. The strain represents the first haloalkaliphilic representative of the family Desulfobacteraceae and is described as Desulfobotulus alkaliphilus sp. nov.     

Studies of formation of bivalent copper complexes with native and denatured DNA

The formation of Cu2+ complexes with native and denatured DNA is studied by the methods of differential UV spectroscopy, CD spectroscopy, and viscometry. On ion binding to the bases of native DNA the latter transforms into a new conformation. This transition is accompanied with a sharp increase in UV absorption and a decrease in the intrinsic viscosity though the high degree of helicity persists. Possible sites of Cu2+ ion binding on DNA of various conformations are found along with corresponding constants of complex formation.     

Transferrin endocytosis and iron uptake in developing myogenic cells in culture: effects of microtubular and metabolic inhibitors, sulphydryl reagents and lysosomotrophic agents

The experiments described in this study were designed to investigate receptor-mediated endocytosis of transferrin and its role in iron uptake by cultured chick presumptive myoblasts (dividing and non-dividing) and myotubes. The effects of a variety of inhibitors on the internalization of transferrin and iron were investigated and three main effects were found: (i) sulphydryl reagents and microtubular inhibitors reduced the rate of transferrin and iron internalization to similar degrees, (ii) metabolic inhibitors reduced the rate of iron uptake more than that of transferrin endocytosis, and (iii) lysosomotrophic agents almost completely abolished iron accumulation by the cells without any effect on the rate of transferrin internalization. The results suggest that metabolic energy is required not only for the endocytosis of transferrin but also for subsequent steps in the iron uptake process, and that iron release from transferrin occurs in acidified endosomes. Overall, these experiments show that all or virtually all of the iron taken up by developing muscle cells from transferrin occurs as a consequence of receptor-mediated endocytosis of the protein.     

Demonstration of an intact complex of epidermal growth factor and its receptor in the endosomes of A-431 cells

The compartmentalization of the epidermal growth factor (EGF) receptors in A-431 cells was studied using centrifugation of the microsomal fraction of these cells in continuous Percoll gradient. The existence of an intact (non-degraded) EGF receptor in plasma membrane and endosome fraction was demonstrated by electrophoretic analysis of in vitro phosphorylated Percoll fractions. No phosphorylated receptor was revealed in lysosomal fraction by this method. The existence of non dissociated EGF-receptor complexes in intracellular compartments 30 minutes after the start of internalization was proven using a synthesized photoreactive labeled EGF derivative (125I-EGF-SANAH). The removing of pH gradient in organellar membranes by 10 mkM of monensin did not affect dissociation from its receptor. The data obtained proved the existence of non-dissociated and non-degraded EGF-receptor complexes in the endosomal compartment of A-431 cells.     

A universal instinct for designing thermoregulated promotors in gram-positive bacteria]

The construction of plasmid pVKH300, which is useful for modifying any promoter into the thermoregulated form in B. subtilis cells, is presented. The main features of the plasmid are the presence of effectively expressed in B. subtilis lambda C1857 gene and recognition site of BglII restriction enzyme between OR2 and OR3 lambda phage operator sites. Promoterless alpha-amylase gene of B. amyloliquefaciens is used as a reporter gene for promoter cloning into BglII site of pVKH300. Examples of promoter-containing DNA fragments cloning with pVKH300 as vector are presented. It was found that the best regulated promoter, in a plasmid named pVKH332, was cloned in such a way that the distance between central nucleotides of OR2 and OR3 is equal to integer number of DNA helix turns (84 b.p. in the case).     

Molecular genetic studies of the DNA promotor regions in Bacillus thuringiensis subsp. galleriae 69-6. I. Structural and functional analysis

Three recombinant plasmids pPBT9, pPBT10 and pPBT74 carrying promoter-containing regions of DNA of Bacillus thuringiensis which are responsible for the expression of the promoterless tet gene, were studied. In the in vitro experiments, it had been shown that these promoter-active HindIII fragments of bacillar DNA contained RNA polymerase binding sites. The AluI subfragments that specifically bind to Escherichia coli RNA polymerase promote the tet gene expression, similar to the whole HindIII fragments. Sequence analysis revealed that the approximately 220 base pair AluI subfragment of the bacillar insertion of the pPBT10 plasmid contained sites typical for "-10" and "-35" homology regions of promoters specific for sigma 55-RNA polymerase from Bac. subtilis. The 1.45 kb HindII bacillar fragment of the plasmid pPBT9 had three AluI subfragments that bind to E. coli RNA polymerase. Only approximately 400 base pair AluI subfragment among these restored the tet gene expression in vivo. Bireplicon pBP plasmids were constructed that promoted the expression of the enterobacterial antibiotic resistance gene under the control of Bac. thuringiensis promoters in Bac. subtilis cells.     

Structure of the Bacillus brevis metalloprotease gene

Primary structure of DNA fragment of 2355 b.p., encoding metalloprotease gene of Bac. brevis, had been determined. Open reading frame for a protein with size of 528 amino acid residues was found in this sequence. The encoding protein is homologous to metalloproteases of Bac. stearothermophilus, Bac. cereus, Bac. subtilis and Bac. amyloliquefaciens. The structure of Bac. brevis metalloprotease gene reveals that this enzyme is synthesised as pre-pro-protease with signal peptide and pro-region, which are cut during its synthesis. The proposed size of mature protease is 304 amino acid residues. The residues, essential for catalysis, binding of Zn ion and Ca ions were found on the basis of Bacilli metalloproteases structures comparison.