The SpoIIE phosphatase, the sporulation septum and the establishment of forespore-specific transcription in Bacillus subtilis: a reassessment

Making a spore in Bacillus subtilis requires the formation of two cells, the forespore and the mother cell, which follow dissimilar patterns of gene expression. Cell specificity is first established in the forespore under the control of the sigma F factor, which is itself activated through the action of the SpoIIE serine phosphatase, an enzyme targeted to the septum between the two cells. Deletion of the 10 transmembrane segments of the SpoIIE protein leads to random distribution of SpoIIE in the cytoplasm. Activation of sigma F is slightly delayed and less efficient than in wild type, but it remains restricted to the forespore in a large proportion of cells and the bacteria sporulate with 30% efficiency. Overexpression of the complete SpoIIE protein in a divIC mutant leads to significant sigma F activity, indicating that the septum requirement for activating sigma F can be bypassed. In contradiction to current models, we propose that genetic asymmetry is not created by unequal distribution of SpoIIE within the sporangium, but by exclusion of an inhibitor of SpoIIE from the forespore. This putative inhibitor would be a cytoplasmic molecule that interacts with SpoIIE and shuts off its phosphatase activity until it disappears specifically from the forespore.     

Immunoassay of 7-hydroxysteroids: 2. Radioimmunoassay of 7alpha-hydroxy-dehydroepiandrosterone

High sensitivity radioimmunoassay of 3beta, 7alpha-dihydroxy-5-androsten-17-one (7alpha-OH-DHEA) has been developed and evaluated. The method is based on polyclonal rabbit antisera raised against 19-O-(carboxymethyl)oxime bovine serum albumin conjugate and bridge- and position homologous [(125)I]iodotyrosine methyl ester as a tracer. Sensitivity of the assay amounted to 3.12 fmol (0.95 pg)/tube, precision as a mean intra- and interassay coefficient of variation was 7.1 and 10.6%, respectively, and the average recovery of the analyte added to steroid-free serum was 110%. Out of the steroids occurring in human serum which may interfere with the assay, the only important cross-reactants were dehydroepiandrosterone and 3beta, 7beta-dihydroxy-5-androsten-17-one (7beta-OH-DHEA) with cross-reactivities of 1.95 and 1.16%, respectively. The levels of free (unconjugated) 7alpha-OH-DHEA have been determined in 29 sera from healthy volunteers (23 females and 6 males), and from 48 patients (43 females and 5 males) in which dehydroepiandrosterone and its sulfate (DHEA/S) had been measured for various endocrinopathies. The levels in healthy subjects ranged from 0.21 to 6.57 (mean 2.33+/-1.50) nM, while those of the patients from 0 to 5. 99 (mean 1.46+/-1.52) nM. The levels of 7alpha-OH-DHEA in patients significantly correlated with those of DHEA and its sulfate.     

Root sprouting in Rumex acetosella under different nutrient levels

Growth of Rumex acetosella, a root sprouting plant, was studied in a pot experiment. Each plant of R. acetosella consisted of two ramets which were interconnected by a root about 9 cm long. One of the ramets was placed in a compartment with nutrient-rich soil, the other with nutrient-poor soil. The root connection between the ramets either remained intact or was severed at the nutrient interface after planting. Growth of new roots was prevented at the nutrient interface.The presence of a connection between the ramets did not affect biomass or shoot production in either soil compartment, indicating a poor integration of the interconnected plant systems. In the nutrient-rich environment, two to four times more shoots and biomass were produced than in the low nutrient regime. A large proportion of buds initiated on roots remained dormant, forming a bud bank. When the number of shoots or buds was expressed per g of root dry weight or per m of root length, the nutrient response was no longer evident or, in a few cases, a significant effect in the opposite direction was obtained. These results show that the greater production of buds and shoots in the nutrient-rich environment reflected an allometric relationship between root biomass and the number of buds and shoots initiated on the roots.     

Ecological Succession of a Relict Central European Peat Bog and Variability of Its Insect Biodiversity

The isolated habitat of the ervené Blato bog (South Bohemia, Czech Republic) and its relict insect fauna have been the subject of long-term monitoring. The species composition and abundance of Lepidoptera (light traps) and Coleoptera (pitfall traps) were monitored for 4 years (1994–1997) simultaneously on two sites – in the edaphic climax pine forest and in wetland successional habitats. The method of statistical evaluation by RDA and CCA ordination, representing the habitat preference of species of Coleoptera (Carabidae only) and Lepidoptera (all nocturnal phototactic taxa) between the edaphic climax forest and succession stages, was used. All categories of the peatland taxa (tyrphobiontic, tyrphophilous and tyrphoneutral species) were analysed. Ten highly stenotopic tyrphobiontic species and 23 tyrphophilous species of Lepidoptera (out of 487) were most characteristic of the bog habitat. Only two tyrphophilous carabid species (out of 20) were characteristic of the bog. The most important relict species (tyrphobionts) of Lepidoptera are most diverse and abundant within the successional habitats and in the open wet forest. The relict fauna of the closed climax pine forest is much less diverse and composed mostly of abundant tyrphophilous and tyrphoneutral forest species. Preservation or restoration of sufficiently constant hydrological conditions, which prevents formation of the closed forest, is the basic management for habitat conservation of all relict tyrphobiontic species of the ervené Blato bog and similar peat land habitat islands. The peat bog is a unified complex system of specific diverse and relict taxa. The most specific taxa are tyrphobiontic Lepidoptera, but a number of other vulnerable tyrphophilous and tyrphoneutral insects are associated with the peat bog as well.     

Alterations in surface hydrophobicity ofAcinetobacter baumannii induced by meropenem

Six strains ofAcinetobacter baumannii out of eleven strains tested revealed a strong hydrophobic character. This was demonstrated by adherence of bacteria to xylene in the range of 90–94%. Changes in surface hydrophobicity of these strains were studied after treatment with meropenem at subinhibitory concentrations (sub-MICs) (1/4, 1/8, 1/16 or 1/32 of the MICs). All strains showed a reduced adherence to xylene after the action of meropenem at 1/4 or 1/16 of the MICs. Hydrophobicity of the treated bacteria was decreased to 1.3–70% (1/16 of the MICs) or to 12–86% (1/4 of the MICs), depending on the strain. A decrease in surface hydrophobicity of three strains was also observed after their exposure to meropenem at 1/8 of the MICs (to 18–71% of the control values). Meropenem at 1/32 of the MICs practically did not affect bacterial hydrophobic properties, with the exception of one strain.     

Conversion of glutamate to glutamine by permeabilizedCorynebacterium glutamicum

Glutamate was converted to glutamine byCorynebacterium glutamicum permeabilized by ethanol (10%). ATP was supplied to the reaction by driedSaccharomyces cerevisiae or regenerated by acetyl kinase. High glutamine synthetase activity inC. glutamicum was induced by cultivation of the microorganism in media containing glutamate as the sole source of nitrogen.     

Matrilin-2, a large, oligomeric matrix protein, is expressed by a great variety of cells and forms fibrillar networks

Matrilin-2 is a member of the protein superfamily with von Willebrand factor type A-like modules. Mouse matrilin-2 cDNA fragments were expressed in 293-EBNA cells, and the protein was purified, characterized, and used to immunize rabbits. The affinity-purified antiserum detects matrilin-2 in dense and loose connective tissue structures, subepithelial connective tissue of the skin and digestive tract, specialized cartilages, and blood vessel walls. In situ hybridization of 35S-labeled riboprobes localizes the matrilin-2 mRNA to fibroblasts of dermis, tendon, ligaments, perichondrium, and periosteum; connective tissue elements in the heart; smooth muscle cells; and epithelia and loose connective tissue cells of the alimentary canal and respiratory tract. RNA blot hybridization and immunoblotting revealed both matrilin-2 mRNA and protein in cultures of a variety of cell types, confirming the tissue distribution. Alternative splicing affects a module unique for matrilin-2 in all of the above RNA sources. SDS-polyacrylamide gel electrophoresis and electron microscopy reveals matrilin-2 from tissue extracts and cell line cultures as a mixture of mono-, di-, tri-, and tetramers. Matrilin-2 is substituted with N-linked oligosaccharides but not with glycosaminoglycans. Because of other, yet unidentified, cell-type dependent posttranslational modifications, the monomer is heterogeneous in size. Immunofluorescence showed that matrilin-2 functions by forming an extracellular, filamentous network.     

Phosphorylated aspartate in the structure of a response regulator protein

Phosphorylation of aspartic acid residues is the hallmark of two- component signal transduction systems that orchestrate the adaptive responses of micro-organisms to changes in their surroundings. Two-component systems consist of a sensor kinase that interprets environmental signals and a response regulator that activates the appropriate physiological response. Although structures of response regulators are known, little is understood about their activated phosphorylated forms, due to the intrinsic instability of the acid phosphate linkage. Here, we report the phosphorylated structure of the receiver/phosphoacceptor domain of Spo0A, the master regulator of sporulation, from Bacillus stearothermophilus. The phosphoryl group is covalently bonded to the invariant aspartate 55, and co-ordinated to a nearby divalent metal cation, with both species fulfilling their electrostatic potential through interactions with solvent water molecules, the protein main chain, and with side-chains of amino acid residues strongly conserved across the response regulator family. This is the first direct visualisation of a phosphoryl group covalently linked to an aspartic acid residue in any protein, with implications for signalling within the response regulator family.     

Effect of killer toxin K1 on yeast membrane potential reported by the diS-C3(3) probe reflects strain-and physiological state-dependent variations

The rate and extent of uptake of the fluorescent probe diS-C₃(3) reporting on membrane potential inS. cerevisiae is affected by the strain under study, cell-growth phase, starvation and by the concentration of glucose both in the growth medium and in the monitored cell suspension under non-growth conditions. Killer toxin K1 brings about changes in membrane potential. In all types of cells tested,viz. in glucose-supplied stationary or exponential cells of the killer-sensitive strain S6/1 or a conventional strain RXII, or in glucose-free exponential cells of both strains, both active and heat-inactivated toxin slow down the potential-dependent uptake of diS-C₃(3) into the cells. This may reflect “clogging” of pores in the cell wall that hinders, but does not prevent, probe passage to the plasma membrane and its equilibration. The clogging effect of heat-inactivated toxin is stronger than that exerted by active toxin. In susceptible cells,i.e. in exponential-phase glucose-supplied cells of the sensitive strain S6/1, this phase of probe uptake retardation is followed by an irreversible red shift in probe fluorescence maximumλ _max indicating damage to membrane integrity and cell permeabilization. A similar fast red shift inλ _max signifying lethal cell damage was found in heat-killed or nystatin-treated cells.     

Novel organization of genes involved in prophage excision identified in the temperate lactococcal bacteriophage TP901-1

In this work, the phage-encoded proteins involved in site-specific excision of the prophage genome of the temperate lactococcal bacteriophage TP901-1 were identified. The phage integrase is required for the process, and a low but significant frequency of excision is observed when the integrase is the only phage protein present. However, 100% excision is observed when the phage protein Orf7 is provided as well as the integrase. Thus, Orf7 is the TP901-1 excisionase, and it is the first excisionase identified that is used during excisive recombination catalyzed by an integrase belonging to the family of extended resolvases. Orf7 is a basic protein of 64 amino acids, and the corresponding gene (orf7) is the third gene in the early lytic operon. This location of an excisionase gene of a temperate bacteriophage has never been described before. The experiments are based on in vivo excision of specifically designed excision vectors carrying the TP901-1 attP site which are integrated into attB on the chromosome of Lactococcus lactis. Excision of the vectors was investigated in the presence of different TP901-1 genes. In order to detect very low frequencies of excision, a method for positive selection of loss of genetic material based upon the upp gene (encoding uracil phosphoribosyltransferase) was designed, since upp mutants are resistant to fluorouracil. By using this system, frequencies of excision on the order of 10(-5) per cell could easily be measured. The described selection principle may be of general use for many organisms and also for types of deletion events other than excision.