Structural and functional analysis of aa3-type and cbb3-type cytochrome c oxidases of Paracoccus denitrificans reveals significant differences in proton-pump design

In Paracoccusdenitrificans the aa₃-type cytochrome c oxidase and the bb₃-type quinol oxidase have previously been characterized in detail, both biochemically and genetically. Here we report on the isolation of a genomic locus that harbours the gene cluster ccoNOQP, and demonstrate that it encodes an alternative cbb₃-type cytochrome c oxidase. This oxidase has previously been shown to be specifically induced at low oxygen tensions, suggesting that its expression is controlled by an oxygen-sensing mechanism. This view is corroborated by the observation that the ccoNOQP gene cluster is preceded by a gene that encodes an FNR homologue and that its promoter region contains an FNR-binding motif. Biochemical and physiological analyses of a set of oxidase mutants revealed that, at least under the conditions tested, cytochromes aa₃, bb₃. and cbb₃ make up the complete set of terminal oxidases in P. denitrificans. Proton-translocation measurements of these oxidase mutants indicate that all three oxidase types have the capacity to pump protons. Previously, however, we have reported decreased H⁺/e coupling efficiencies of the cbb₃-type     

Siderophore production byBradyrhizobium spp. strains nodulating groundnut

EighteenBradyrhizobium spp. strains, fourRhizobium spp. strains and oneAzorhizobium caulinodans strain were grown under Fe limitation and assayed for siderophore production. It was further assessed if Fe accumulation in two groundnut cultivars was influenced by inoculant strain or nitrate fertilisation. Growth ofBradyrhizobium spp. strains nodulating groundnut was slow with mean generation times from 11–24 h. All strains, except MAR 967, showed a reduced growth rate when deprived of Fe; none of the strains showed starvation at 1 M Fe. In the CAS (chrome azurol S)-agar assay, all strains, which formed colonies, produced siderophores as visualised by orange halos around the colonies on blue plates.Bradyrhizobium strains produced much smaller halos than the referenceRhizobium meliloti strain. In the CAS-supernatant assay, all strains, except MAR 967, gave positive responses (measured as absorbance at 630 nm) when supernatants of Fe-depleted cultures were assayed with CAS-indicator complex in comparison with Fe-supplemented cultures. Responses of all fourRhizobium spp. strains were large, while responses of allBradyrhizobium strains, exceptB. japonicum MAR 1491 (USDA 110), were small and mostly insignificant. A small response, i.e. a low Fe-scavenging ability, implies either the production of small quantities of siderophores or the production of low affinity siderophores. Among theBradyrhizobium strains, MAR 1574 and MAR 1587 gave the largest responses taken over the two assays. Fe accumulation in groundnut cultivar Falcon was seven times larger than in cultivar Natal Common. No correlation was found between the quantity of nodule tissue and Fe accumulation, making it unlikely that bacteroids are involved in Fe acquisition by groundnuts. Nitrate-fertilised plants accumulated significantly more Fe, suggesting involvement of nitrate reductase in Fe assimilation in groundnut. The two most successful Fe-scavengingBradyrhizobium spp. strains were also the most effective in nodulating groundnut, the reverse also being true. Strain MAR 967, with the lowest Fe requirement, produced the largest nodule dry weight. These data indicate that improved Fe scavenging properties and/or reduced Fe requirement improve rhizospheric growth and with that nodulation effectiveness.     

The complete karyotype of Aspergilius niger: the use of introduced electrophoretic mobility variation of chromosomes for gene assignment studies

A method is described for unambiguous assignment of cloned genes to Aspergillus niger chromosomes by CHEF gel electrophoresis and Southern analysis. All of the eight linkage groups (LGs), with the exception of LG VII, have previously been assigned to specific chromosomal bands in the electrophoretic karyotype of A. niger. Using a LG VII-specific probe (nicB gene of A. niger) we have shown that LG VII corresponds to a chromosome of about 4.1 Mb. Furthermore, genetic localization of three unassigned genes (glaA, agIA and pepA) in strains in which these genes had been replaced by a selectable marker gene led to a revised karyotype for the chromosomes corresponding to LGs VIII and VI. The revised electrophoretic karyotype reveals only 5 distinct bands. The presence of three pairs of equally sized chromosomes precluded assignment of genes to one specific chromosome in the wild-type strain. However, unambiguous chromosome assignment of cloned genes using CHEF-Southern analysis was demonstrated using a set of A. niger strains with introduced chromosomal size variation. The availability of these tester strains obviates the need to isolate or construct mutant. strains for the purpose of chromosome assignment.     

Seasonal analysis of the tropane alkaloid ecgonine methyl ester and the occurrence of other highly-valued tropanes in the South African <Emphasis Type="Italic">Erythroxylum</Emphasis> trees

The coca family (Erythroxylaceae) consists of trees and shrubs sub-divided into four genera: Aneulophus, Nectaropetalum, Pinacopodium, and Erythroxylum, which include species with highly valuable medicinal compounds. E. delagoense, E. emarginatum, and E. pictum are endemic to southern Africa and have great pharmaceutical potential based on their traditional uses. Previous studies have shown certain inconsistencies in terms of the presence or absence of tropane alkaloids in these species, resulting in a need for further research and clarification. Therefore, the aim of this study was to determine the seasonal variation of the immediate biosynthetic precursor of cocaine, the tropane alkaloid, ecgonine methyl ester in the three South African Erythroxylum species by means of gas chromatography–mass spectrometry, as well as to conduct a phytochemical screening for observing the presence of other potential compounds and tropane alkaloids. We found significant differences in tropane concentrations from the seasonal variation study, explaining the discrepancies in previous reports on its presence/absence in these species. Furthermore, we report for the first time on the occurrence of selected highly valuable tropane alkaloids in E. emarginatum currently used in ‘blockbuster medicine’.     

New data on the use of stone tools by chimpanzees in Guinea and Liberia

Two types of use of “hammers” for cracking nuts by wild-living chimpanzees have been distinguished: (1) Relatively small stones are used by the chimpanzee community at Bossou in Guinea to crack the nuts of oil palms growing on abandoned farmland, while no nuts of wild tree species are cracked. (2) Larger hammer stones (and, at some sites, wooden clubs) are used in a more sophisticated manner to crack the nuts of wild trees, but not of oil palms, in an area ranging from south-east Sierra Leone through Liberia to the south-west of the Ivory Coast. The first author (1986) has proposed that Type I has been copied by the chimpanzees, under pressure of food shortage, from the local human population. New data now indicate that, at Bossou, while habitat deterioration has continued, the number of hammer and anvil stones per utilized oil palm tree has approximately tripled in the last six years. The quantity of food obtained from oil palm nut kernels, however, amounts to only a few percent of the total diet. For the rest these apes depend to a large extent on many other agricultural products cultivated at Bossou which they are allowed freely to consume, including even cassava (manioc) roots and sweet potatoes dug by them from the ground. Some factors determining the chosen size of hammers were analyzed. Two abnormal hammers were found whose wear suggested a tentative, human-like manner of use. No evidence has been found to indicate the use of stone tools by chimpanzees in the adjoining chimpanzee-inhabited areas around the range of the Bossou community. Type II stone tool use was found, however, in a primary forest on a mountain≈13 km west of Bossou. This is especially intriguing because the site is separated by a wide belt of drier rain forest from the belt of very humid rain forest in the south where all the other known Type II sites are located. More research on the geographical distribution of the use of stone tools by chimpanzees and on the underlying ecological factors is recommended.     

A comparison between aerobic growth of Bacillus licheniformis in continuous culture and partial-recycling fermentor,with contributions to the discussion on maintenance energy demand

Energy costs of biomass synthesis are relatively higher at low than at high specific growth rates () because of an increased protein content of the cell and increased costs of protein synthesis as such at low values. A comparison of aerobic, glucose limited cultures of Bacillus licheniformis in a chemostat and in a partial-recycling fermentor indicated that pulse-wise nutrient addition increased the maintenance energy demand (m). In the chemostat experiments, we also found a striking deviation from linearity between substrate consumption and , with large implications for the maintenance coefficient. The deviation is mainly due to a large shift in metabolic carbon flows at specific growth rates between 50 and 100% of _max. At those growth rates, uncoupled growth occurs, presumably as a necessary condition for faster growth, since uncoupling results in a faster energysupply for biosynthetic purposes.The maintenance coefficient as determined by chemostat studies should be regarded as a compounds parameter, constituted of maintenance energy demands like ppGpp accumulation, variable costs of mRNA and protein accumulation, kinetic proofreading etc. and influenced by fermentor operation parameters like the substrate addition rate; moreover, both constancy of m and a linear relation between m and appear quite unlikely.     

Quantitative analysis of cellular differentiation during early embryogenesis ofPlatynereis dumerilii

Summary As in many spiralian embryos with unequal cleavage, cleavage inPlatynereis follows an invariant pattern. Preceding each cleavage the cytoplasm is reorganized, allowing the spiral cleavage mode to produce cells with different cytoplasmic composition. The fertilized egg undergoes a dramatic ooplasmic segregation after the completion of the cortical reaction. As a consequence, a plug of clear cytoplasm becomes located at the animal pole. Once the four quadrants of the embryo have been established, the cleavage sequence of the D quadrant differs clearly from that of the other three quadrants. The results presented here suggest that differential distribution of the clear cytoplasm governs this sequence. The first quartet of micromeres, which will form the ectoderm and the cerebral ganglia of the head, is clearly bilaterally symmetrical from the onset of the third cleavage. Dorsoventral polarity and bilateral symmetry in the ectoderm of the trunk is expressed most markedly by the dorsal location of the large 2d cell, whose rapid proliferation is bilaterally symmetrical with respect to the median plane. As a result of this proliferation it comes to fill most of the posttrochal region (ectoderm, three pairs of anlagen for the setal sacs, and the ventral plate which forms the nerve cord). The other micromeres contribute only a minor portion of the ventral ectoderm and are involved in the formation of the stomodaeum. The mesentoblast, 4d, i.e. the stem cell of the primary mesoderm, forms at the sixth cleavage, also in a position on the dorsal mid-line. The daughter cells, which arise from 4d by strictly bilaterally symmetrical cleavage, form the mesodermal germ bands, which lie beneath the ectoderm. The trochoblasts are formed by asynchronously cleaving founder cells, but further cleavages in these cells are synchronous. This suggests that cell-cell interaction is involved in the development of this alleged mosaic embryo.     

Hydrogen oxidation and nitrogen fixation in rhizobia,with special attention focused on strain ORS 571

In this survey we describe the influence of hydrogen oxidation on the physiology ofRhizobium ORS 571. The presence of hydrogen is required for the synthesis of hydrogenase. Carbon substrates do not repress the synthesis of hydrogenase. The respiratory system contains cytrochromes of theb- andc-type. Cytochromea ₆₀₀ is present after growth at high oxygen tensions. The nature of the terminal oxidases functioning at low oxygen tensions has not been established yet. → H⁺/O values with endogenous substrates are between 6 and 7. The results show the presence of two phosphorylation sites: site 1 (ATP/2e=1.0) and site 2(ATP/2e=1.33). By measuring molar growth yields it has been demonstrated that carbon-limited, nitrogen-fixing cultures obtain additional ATP from hydrogen oxidation, and that site 2 of oxidative phosphorylation is passed during hydrogen oxidation. A method is described to calculate ATP/N₂ values (the total amount of ATP used by nitrogenase during the fixation of 1 mol N₂) and H₂/N₂ ratios (mol hydrogen formed per mol N₂ fixed) in aerobic organisms. ForRhizobium ORS 571 the ATP/N₂ value is about 40 and the H₂/N₂ ratio is between 5 and 7.5. Cells obtained from oxygen-limited nitrogen-fixing cultures contain 30–40% poly-β-hydroxybutyrate, which explains the high molar growth yields found. Hydrogen has not been detected in the effluent gas of these cultures, which may point to reoxidation of the hydrogen formed at nitrogen fixation. Calculations show that the effect of hydrogen reoxidation on the efficiency of nitrogen fixation (g N fixed × mol⁻¹ substrate converted) is not very large and that the actual H₂/N₂ ratio is of much more importance. After addition of hydrogen to succinate-limited, ammonia-assimilating cultures, an initial increase of the Y_succinate value (g dry wt × mol⁻¹ succinate) is followed by a gradual decrease. This is accompanied by a large decrease of the value, and an increased permeability of the cytoplasmic membrane to protons. The results may be explained by a transition of the culture from an energy-limited state to a carbon-limited state.     

Thermal Preference of Juvenile Dover Sole (Solea solea) in Relation to Thermal Acclimation and Optimal Growth Temperature

Dover sole (Solea solea) is an obligate ectotherm with a natural thermal habitat ranging from approximately 5 to 27°C. Thermal optima for growth lie in the range of 20 to 25°C. More precise information on thermal optima for growth is needed for cost-effective Dover sole aquaculture. The main objective of this study was to determine the optimal growth temperature of juvenile Dover sole (Solea solea) and in addition to test the hypothesis that the final preferendum equals the optimal growth temperature. Temperature preference was measured in a circular preference chamber for Dover sole acclimated to 18, 22 and 28°C. Optimal growth temperature was measured by rearing Dover sole at 19, 22, 25 and 28°C. The optimal growth temperature resulting from this growth experiment was 22.7°C for Dover sole with a size between 30 to 50 g. The temperature preferred by juvenile Dover sole increases with acclimation temperature and exceeds the optimal temperature for growth. A final preferendum could not be detected. Although a confounding effect of behavioural fever on temperature preference could not be entirely excluded, thermal preference and thermal optima for physiological processes seem to be unrelated in Dover sole.