Subunit Positioning and Stator Filament Stiffness in Regulation and Power Transmission in the V1 Motor of the Manduca sexta V-ATPase

The vacuolar H⁺-ATPase (V-ATPase) is an ATP-driven proton pump essential to the function of eukaryotic cells. Its cytoplasmic V₁ domain is an ATPase, normally coupled to membrane-bound proton pump V_o via a rotary mechanism. How these asymmetric motors are coupled remains poorly understood. Low energy status can trigger release of V₁ from the membrane and curtail ATP hydrolysis. To investigate the molecular basis for these processes, we have carried out cryo-electron microscopy three-dimensional reconstruction of deactivated V₁ from Manduca sexta. In the resulting model, three peripheral stalks that are parts of the mechanical stator of the V-ATPase are clearly resolved as unsupported filaments in the same conformations as in the holoenzyme. They are likely therefore to have inherent stiffness consistent with a role as flexible rods in buffering elastic power transmission between the domains of the V-ATPase. Inactivated V₁ adopted a homogeneous resting state with one open active site adjacent to the stator filament normally linked to the H subunit. Although present at 1:1 stoichiometry with V₁, both recombinant subunit C reconstituted with V₁ and its endogenous subunit H were poorly resolved in three-dimensional reconstructions, suggesting structural heterogeneity in the region at the base of V₁ that could indicate positional variability. If the position of H can vary, existing mechanistic models of deactivation in which it binds to and locks the axle of the V-ATPase rotary motor would need to be re-evaluated.     

Isozyme Variation among Biological Species in the Gibberella fujikuroi Species Complex (Fusarium Section Liseola)

Isozyme phenotypes were determined for 101 strains of Gibberella fujikuroi and 2 strains of Gibberella nygamai that represent seven biological species (mating populations) isolated from a variety of plant hosts in dispersed geographic locations. Fourteen enzymes were resolved in one or more of three buffer systems. Two of the enzymes, arylesterase and acid phosphatase, were polymorphic within two or more biological species and are suitable for intraspecific studies of population variation. Six enzymes, alcohol dehydrogenase, aspartate aminotransferase, glucose-6-phosphate dehydrogenase, mannitol dehydrogenase, phosphoglucomutase, and phosphogluconate dehydrogenase, were monomorphic in all of the isolates examined. The remaining six enzymes, fumarase, glucose phosphate isomerase, glutamate dehydrogenase (NADP), isocitrate dehydrogenase (NADP), malate dehydrogenase, and triose-phosphate isomerase, could potentially be used to distinguish the different biological species. Mating populations C and D are the most similar, since the mating population C isolates examined had the same isozyme phenotype as did a subset of the isolates in mating population D. Mating population E is the least similar to the other taxa examined. Unique isozyme phenotypes are present but are composed of banding patterns shared among the biological species. This finding supports the hypothesis that these biological species, with the possible exception of mating populations C and D, are reproductively isolated from one another and that no significant gene flow is occurring between them. Isozyme analysis is a useful method to distinguish these closely related biological species. Examination of isozyme phenotypes is more rapid than the present technique, which is based on sexual crosses; can be applied to strains that are not sexually fertile; and is more sensitive than traditional morphological characters, which cannot distinguish more than three or four morphological groups among the seven biological species. While emphasizing the discreteness of the mating populations as biological entities, our isozyme data also reaffirm the close genetic relationship among these groups.     

Importance of Photobacterium phosphoreum in relation to spoilage of modified atmosphere-packed fish products

Occurrence and growth of Photobacterium phosphoreum were studied in 20 experiments with fresh fish from Denmark, Iceland and Greece. The organism was detected in all marine fish species but not in fish from fresh water. Growth of P. phosphoreum to high levels (>10⁷ cfu g⁻¹) was observed in most products and the organism is likely to be of importance for spoilage of several modified atmosphere-packed (MAP) marine fish species when stored at chill temperatures. Some microbiological methods recommended for control of fish products by national and international authorities are inappropriate for detection of psychrotolerant and heat-labile micro-organisms like P. phosphoreum . These methods have been used in many previous studies of MAP fish and this could explain why, contrary to the findings in the present study, P. phosphoreum in general was not detected previously in spoiled MAP fish.     

Deoxyribonucleic acid reassociation and interspecies relationships of the genusChlorella (Chlorophyceae)

Phylogenetic relationships within the genusChlorella were studied by means of DNA/DNA hybridization under both optimal and relaxed reassociation conditions as well as by determination of the thermal stability of hybrid DNA duplexes. The results indicate a relationship betweenC. fusca var.fusca, C. fusca var.rubescens, C. fusca var.vacuolata, and the genusScenedesmus. In addition, the strains endosymbiotic withParamecium bursaria seem to be related with theC. vulgaris/sorokiniana group. The relations between most other species, however, could not be sufficiently resolved by the above methods. This implies considerable phylogenetic divergency within the genusChlorella.     

Distribution of Clostridium botulinum.

The distribution of Clostridium botulinum in the natural environments of Denmark, The Faroe Islands, Iceland, Greenland, and Bangladesh was examined. A total of 684 samples were tested. Type E was found in 90% of samples from the aquatic environment of Denmark, including sediments from young artificial lakes, and in 86% of samples from the marine environment of Greenland. Type E was not found in Danish cultivated soil and woodlands, including cultivated soil from reclaimed sea beds, but type B was frequently demonstrated in these environments. C. botulinum types A, B, or E were found in 2.6% of samples from the environments of the Faroe Islands and Iceland, whereas types C or D were demonstrated in 42% of samples from Bangladesh. The incidence of type E in aquatic sediments was not related to general industrial pollution or a high content of rotting vegetation. Fish or a rich aquatic fauna, on the other hand, appeared to contribute to a high incidence of type E. Based on these findings, it is suggested that type E is a true aquatic organism, because this environment offers the best conditions for survival of the spore in nature. It is further suggested that its presence in aquatic bottom deposits is based on sedimentation after proliferation in the carrion of the aquatic fauna and dissemination by water currents and migrating fish.     

Role of T-region borders in Agrobacterium host range

The limited host range AB3 strain of Agrobacterium tumefaciens induces tumors by transferring two T-regions, TA and TB. TA is a deleted version of the well-known biotype I octopine TL-region that lacks the iaa and ipt genes, but carries an intact oncogene, gene 6b, and typical left and right border sequences. TB carries two iaa genes that together code for the synthesis of indoleacetic acid. Gene 6b and the iaa gene act synergistically when transferred in a coinoculation experiment. The TA-region of the limited host range isolate Ag57 is related to the TA-region of AB3, but differs from it at several positions. The most significant difference is the absence of the right border region. In spite of this, Ag57 and the exconjugant strain C58C9(pTiAg57) induce normal tumors on Nicotiana rustica and Vitis vinifera. Various experiments indicate that gene 6b of the Ag57 TA-region is active and transferred in spite of the absence of the right border. On N. tabacum, C58C9(pTiAg57) is nononcogenic but becomes oncogenic when the pTiAg57 TA-region is restored by the right TA border sequence of pTiAB3. Thus, the right TA border sequence of the biotype III limited host range strains is required for tumor induction on some hosts, but not on others.