A reverse genetic approach for generating gene replacement mutants in<Emphasis Type="Italic"> Ustilago maydis</Emphasis>

We describe a versatile strategy for generating gene replacement mutants in the phytopathogenic fungus Ustilago maydis. The system includes the choice of 32 different insertion cassettes for genetic engineering purposes, such as gene disruption and more sophisticated insertions of reporter genes, heterologous promoters or combinations of the two. PCR-amplified flanking sequences needed for homologous recombination are ligated to the respective insertion cassettes via Sfi I sites. As proof of principle we generated two replacement mutants in which the endogenous promoter of the pheromone gene mfa1 drives expression of the Green Fluorescent Protein gene (gfp). Simultaneously, expression of the mfa1 ORF is controlled either by the carbon source-regulated crg1 promoter or the nitrogen source-regulated nar1 promoter. In both cases gfp expression was pheromone-inducible and pheromone expression was only detected when the heterologous promoters were active.Communicated by G. JürgensThe first two authors contributed equally to this work     

Genetically modified Streptococcus mutans for the prevention of dental caries

There are many examples of positive and negative interactions between different species of bacteria inhabiting the same ecosystem. This observation provides the basis for a novel approach to preventing microbial diseases called replacement therapy. In this approach, a harmless effector strain is permanently implanted in the host's microflora. Once established, the presence of the effector strain prevents the colonization or outgrowth of a particular pathogen. In the case of dental caries, replacement therapy has involved construction of an effector strain called BCS3-L1, which was derived from a clinical Streptococcus mutans isolate. Recombinant DNA technology was used to delete the gene encoding lactate dehydrogenase in BCS3-L1 making it entirely deficient in lactic acid production. This effector strain was also designed to produce elevated amounts of a novel peptide antibiotic called mutacin 1140 that gives it a strong selective advantage over most other strains of S. mutans. In laboratory and rodent model studies, BCS3-L1 was found to be genetically stable and to produce no apparent deleterious side effects during prolonged colonization. BCS3-L1 was significantly less cariogenic than wild-type S. mutansin gnotobiotic rats, and it did not contribute at all to the cariogenic potential of the indigenous flora of conventional Sprague-Dawley rats. And, its strong colonization properties indicated that a single application of the BCS3-L1 effector strain to human subjects should result in its permanent implantation and displacement over time of indigenous, disease-causing S. mutans strains. Thus, BCS3-L1 replacement therapy for the prevention of dental caries is an example of biofilm engineering that offers the potential for a highly efficient, cost effective augmentation of conventional prevention strategies. It is hoped that the eventual success of replacement therapy for the prevention of dental caries will stimulate the use of this approach in the prevention of other bacterial diseases.     

The fenestrin antigen in submembrane skeleton of the ciliate Tetrahymena thermophila is proposed as a marker of cell polarity during cell division and in oral replacement

Tetrahymena thermophila cells have two types of polarized morphogenesis: divisional morphogenesis and oral reorganization (OR). The aim of this research is the analysis of cortical patterns of immunostaining during cell division and in OR using previously characterized antibodies against fenestrin and epiplasm B proteins. During cell division, the anarchic field of basal body proliferation of the new developing oral apparatus (AF) showed concomitant strong binding of the fenestrin antigen and withdrawal of a signal of the epiplasm B antigen. At a specific stage, the fenestrin antigen also appeared as a character of the anterior cortex pole, with a co-localized decrease in the detected epiplasm B antigen. The fenestrin antigen also showed a polarity of duplicating basal bodies in ciliary rows. Indirect immunofluorescence and immunogold labeling experiments were performed in the absence and presence of an inhibitor of activity of serine/threonine kinases, 6-dimethylaminopurine (6-DMAP) as an inducer of the oral replacement process. In the presence of 6-DMAP, one class of cells started OR, and some others were trapped and affected in cell division. Both types of cells showed an instability of oral structures and formed enlarged primordial oral fields. These anarchic fields (AFs) bind the fenestrin antigen, with disappearance of epiplasmic antigen staining. Only one protein (about 64 kDa) is detected in western blots by the anti-fenestrin antibody and it accumulated in 6-DMAP-treated cells that are involved in uncompleted morphogenetic activity. At a defined stage of oral development, both during cell division and in OR, the fenestrin antigen served as a marker of polarity of the cell of the anterior pole character.     

A method for allelic replacement in Francisella tularensis

A vector for mutagenesis of Francisella tularensis was constructed based on the pUC19 plasmid. By inserting the sacB gene of Bacillus subtilis, oriT of plasmid RP4, and a chloramphenicol resistance gene of Shigella flexneri, a vector, pPV, was obtained that allowed specific mutagenesis. A protocol was developed that allowed introduction of the vector into the live vaccine strain, LVS, of F. tularensis by conjugation. As a proof of principle, we aimed to develop a specific mutant defective in expression of a 23-kDa protein (iglC) that we previously have shown to be prominently upregulated during intracellular growth of F. tularensis. A plasmid designated pPV-DeltaiglC was developed that contained only the regions flanking the encoding gene, iglC. By a double crossover event, the chromosomal iglC gene was deleted. However, the resulting strain, denoted DeltaiglC1, still had an intact iglC gene. Southern blot analysis verified that LVS harbors two copies for the iglC gene. The mutagenesis was therefore repeated and a mutant defective in both iglC alleles, designated DeltaiglC1+2, was obtained. The DeltaiglC1+2 strain, in contrast to DeltaiglC1, was shown to display impaired intracellular macrophage growth and to be attenuated for virulence in mice. The developed genetic system has the potential to provide a tool to elucidate virulence mechanisms of F. tularensis and the specific F. tularensis mutant illustrates the critical role of the 23-kDa protein, iglC, for the virulence of F. tularensis LVS.     

Effect of iron supplementation on oxidative stress and antioxidant status in iron-deficiency anemia

This study was designed to measure the effect of iron supplementation on antioxidant status in iron-deficient anemia, including the time for hemoglobin normalization and at the time of filling of iron body stores. The extent of plasma lipid peroxidation was evaluated by measuring the levels of malondialdehyde and glutathione peroxidase (GSH-Px), and the activities of superoxide dismutase (SOD) and catalase in 63 patients with iron-deficiency anemia before and after 6 wk of iron supplementation and at the time when body iron stores are saturated. After 6 wk of iron supplementation, a significant decrease of oxidative stress was observed in the treated subjects relative to controls (p<0.05). No significant differences existed between treated patients at 6 wk and at the end of the study. The erythrocyte levels of catalase, SOD, and GSH-Px were significantly lower in treated patients relative to controls (p<0.05). These levels increased after 6 wk of supplementation (p<0.05) and showed no significant differences with those at the end of the study.     

Decrease of plasma taurine in Gaucher disease and its sustained correction during enzyme replacement therapy

Summary. Gaucher disease is caused by an autosomal-recessive deficiency of glucocerebrosidase. Cells of monocytic/macrophagic origin accumulate glucosylceramide. This leads to hepatosplenomegaly, bone destruction, thrombocytopenia and anemia. Enzyme replacement therapy (ERT) with macrophage-targeted glucocerebrosidase leads to normalization of these parameters. The way of macrophage activation in Gaucher disease is not known. Recently, the osmolytes taurine, betaine and inositol were identified as important regulators of macrophage function in liver. Therefore, the role of plasma taurine in Gaucher disease as a primarily macrophage-derived disease was studied. Fasting plasma levels were measured from blood samples of healthy control subjects (n = 29, m : f = 11 : 18, mean age 37 ± 3 years), from un-treated Gaucher patients (n = 16, m : f = 7 : 9, mean age 44 ± 4 years) and those treated for 37 ± 2 months (n = 54, m : f = 19 : 35, mean age 47 ± 2 years). Amino acid analysis was carried out in a BioChrom amino acid analyzer. In the untreated patients, plasma taurine was 45 ± 3 μM, as compared to the controls with a plasma taurine of 63 ± 4 μM (p < 0.01). The aver-age increase of plasma taurine during the first year of ERT was 18 ± 8 μM (n = 10). Patients treated for an average of 37 months (range 1–9 years of ERT) had a plasma taurine of 65 ± 4 μM (n = 54), which was not different from the controls. It is concluded that Gaucher patients show decreased plasma taurine levels and that therapy of Gaucher disease might correct this. It has to be established, whether decreased taurine availability is a cofactor of the permanent activation of glucosylceramide-storing monocytes/macrophages in this disease. Received January 25, 2000/Accepted January 31, 2000     

Response to orphaning in two Neotropical termites: shape Armitermes euamignathus and shape Embiratermes festivellus

In this paper we examine the potential of the termites Armitermes euamignathus Silvestri: 1901 and Embiratermes festivellus (Silvestri, 1901) (Isoptera, Termitidae, Nasutitermitinae) to produce neotenics experimentally. Three nests of the mound-building termite A. euamignathus, from the Brazilian cerrado, had their primary queens removed in August 1994. After 12 months, only one mound survived; it had a normal appearance. In this healthy, orphaned colony we found the primary king, six physogastric nymphoid female replacement reproductives, two ergatoid female replacement reproductives, 46 nymphs, several presoldiers, soldiers, workers, larvae and many eggs. These data show that neotenics in A. euamignathus may originate from both workers and nymphs, but nymphoids are produced in larger numbers. The biometric study of nymphs and nymphoids suggests that these brachypterous neotenics were derived from third instar nymphs after a single moult or from four instar nymphs after a reduction of wing bud length. A piece of an E. festivellus nest with some third instar nymphs, soldiers and workers was kept under laboratory conditions. After 12 months, the whole experimental subcolony was examined and appeared to contain two pigmented nymphoid females, two pigmented nymphoid males, only one larva, seven nymphs of the same instar, 148 workers, five soldiers and many eggs. These results also indicate the capacity of the termite E. festivellus to produce nymphoid neotenics. These neotenic females were laying eggs, but they were not physogastric after a year, unlike some nymphoids of the same species collected from natural colonies.     

Mutagenesis of histidine 26 demonstrates the importance of loop-loop and loop-protein interactions for the function of iso-1-cytochrome c.

In yeast iso-1-cytochrome c, the side chain of histidine 26 (His26) attaches omega loop A to the main body of the protein by forming a hydrogen bond to the backbone atom carbonyl of glutamic acid 44. The His26 side chain also forms a stabilizing intra-loop interaction through a hydrogen bond to the backbone amide of asparagine 31. To investigate the importance of loop-protein attachment and intra-loop interactions to the structure and function of this protein, a series of site-directed and random-directed mutations were produced at His26. Yeast strains expressing these variant proteins were analyzed for their ability to grow on non-fermentable carbon sources and for their intracellular production of cytochrome c. While the data show that mutations at His26 lead to slightly decreased intracellular amounts of cytochrome c, the level of cytochrome c function is decreased more. The data suggest that cytochrome c reductase binding is affected more than cytochrome c oxidase or lactate dehydrogenase binding. We propose that mutations at this residue increase loop mobility, which, in turn, decreases the protein's ability to bind redox partners.     

Tooth replacement rates in early chondrichthyans: a qualitative approach

The continuous replacement of teeth throughout their lifetime is a common characteristic of most chondrichthyans. This process was already present in the earliest representatives of the group. It has been well established that different species of extant sharks show rapid tooth replacement rates; however, some authors have suggested that in early chondrichthyans this rate might have been much slower. Here we present a qualitative approach to analyse tooth replacement rates in the Early Devonian shark Leonodus carlsi , the earliest tooth-bearing shark known to date. For this, we have examined 1,103 isolated teeth from Celtiberia, Spain. Our study provides strong evidences of an extremely slow dental replacement in this primitive chondrichthyan based on three independents analyses: (1) statistical analysis of the wear degree, demonstrating that teeth remain functional for a long period of time; (2) analysis of both the histological and the morphological features of the teeth cusps suggests that this chondrichthyan used a maturation process that optimizes its function, thus worn teeth show an efficient working shape that implies their teeth remained functional for a long time after being modelled by use; and (3) estimations of size increments between teeth (Δs) of the same dental family for some recent sharks whose rates of replacement were known prove that Δs is inversely proportional to the rate of replacement ( R ² = 0.8327). The estimated values of tooth replacement rates obtained from Δs for L. carlsi and for some Late Devonian cladoselachian sharks are significatively slower than those observed in current sharks.