Evaluation of PCR-DGGE methodology to monitor fungal communities on grapes.

AIMS: Some fungi present on the surface of grapes may have a negative effect on the quality of wine. The aim of this study was to evaluate PCR-denaturing gradient gel electrophoresis (PCR-DGGE), for the establishment of fungal community profiles from grapes, in order to monitor fungi potentially involved in wine defects. METHODS AND RESULTS: A fragment of the beta-tubulin gene was amplified from filamentous fungi and yeasts described from grapes and analysed using two different denaturing gradient gels to constitute a reference database. The use of beta-tubulin sequences instead of ITS rDNA in PCR-DGGE showed a progress in the discrimination of these fungal species but comigration problems were still observed. The technique was then applied on grape samples. The profiles counted up to 10 bands of which half corresponded to species which were not recorded in the reference database. CONCLUSION: PCR-DGGE represents a useful tool to compare environmental samples for the study of the dynamics of fungal communities, but comigrations represent a limit in its use to describe the species present. SIGNIFICANCE AND IMPACT OF THE STUDY: A better knowledge of the fungal diversity on grapes, particularly species responsible for wine defect, is necessary to develop accurate molecular detection tools.

Analysis of fungal diversity of grapes by application of temporal temperature gradient gel electrophoresis - potentialities and limits of the method.

AIMS: Fungi could be responsible for several problems in wines but the fungal ecosystem of grapes remains little known. The use of traditional methods does not allow to describe quickly this ecosystem. Therefore, we need to improve the knowledge about these fungi to prevent defects in wine. This study aims at evaluating the potentialities of the temporal temperature gradient gel electrophoresis (TTGE) method to describe the fungal ecosystem of grapes. METHODS AND RESULTS: The internal transcribed spacer (ITS) region was amplified and analysed using TTGE. A reference database of 56 fungal species was set up to evaluate the discrimination power of the method. The database was used for the direct identification of the fungal species present in complex samples. The sensitivity of the method is below 10(4) spores per ml. CONCLUSIONS: This method allows to describe the fungal diversity of grapes, but does not always allow to directly identify all fungal species, because of the taxonomic resolution of the ITS sequences. However, this identification strategy is less time consuming than traditional analysis by cloning and sequencing the bands. SIGNIFICANCE AND IMPACT OF THE STUDY: With this method, it will be possible to compare the fungal species present in different vineyards and to connect the presence of some fungi with particular defects in wine.

Filamentous bacterial population dominance in activated sludges subject to stresses.

Evolution of a filamentous bacterial population was monitored on four wastewater treatment pilot plants subject to stresses which have consisted of oxygen deficiencies and/or loading shocks. Fluorescent in situ hybridization (FISH) was used to perform filaments identification and quantification. Results obtained on the different pilot plants have led us to conclude that independently of the nature of the stresses, a single filamentous bacteria species was involved in the increase of the sludge volume index associated to the filamentous growth. In addition, when serial stresses were used, substitutions in dominant filamentous populations occurred: if another filament began to proliferate it caused the regression of the one which was formerly dominant.

Influence of transient substrate overloads on the proliferation of filamentous bacterial populations in an activated sludge pilot plant.

Using oligonucleotide probes directed at the rRNA of filamentous bacteria, this study looks at the influence of the components of transient substrate overloads on the growth of the dominant filamentous bacteria of activated sludge fed by a synthetic substrate. By dissociating the massive input of organic matter from the oxygen shortage that the latter generally induces, it is revealed that each of these factors applied alone, induces only transitory, small-scale growth of the filaments Nostocoida limicola, Haliscomenobacter hydrossis. Thiothrix and of type 021N. In contrast, combining them during a reconstituted transient substrate overload with an artificially created oxygen deficit, induces very fast growth of H. hydrossis which is responsible for establishing major proliferation. This massive proliferation was easily reduced by chlorination.

A possible role for L24 of Bacillus subtilis in nucleoid organization and segregation.

The condensation of DNA in bacterial nucleoids during cell cycle is a complex and dynamic process. Proteins displaying the physico-chemical properties of histones are known to contribute to this process. During a search for B. subtilis nucleoid associated proteins, HBsu and L24 were identified as the most abundant proteins in nucleoid containing fractions. Purified L24 binds and condenses DNA in vitro. In this paper we describe immunofluorescence studies that demonstrated that L24 is located at the poles of the nucleoids in exponentially growing cells. In contrast, the protein is dispersed in the cytoplasm during stationary phase. Moreover, overexpression of the rplX gene encoding L24 disrupts nucleoid segregation and positioning.

Phosphorylation of the lymphoid cell kinase p56lck is stimulated by micromolar concentrations of Zn2+.

In particulate fractions from LSTRA lymphoma cells, tyrosine phosphorylation of the lymphoid specific tyrosine kinase p56lck is elicited by Zn2+ in the absence of other divalent cations. Zn2+ alone also induces autophosphorylation of immunoprecipitated p56lck. The effect of Zn2+ is dose dependent; it is detected at concentrations of Zn2+ as low as 5 microM and reaches a maximum at 100 microM Zn2+. Among other divalent cations tested, Mn2+, and Co2+ to a lesser extent, were also effective. Zn2+ also stimulated p56lck phosphorylation in the presence of Mg2+ ions at physiological concentration, whereas orthovanadate had no effect. These results suggest that Zn2+ activates the autophosphorylation of p56lck; this fact could be related with the stimulating effect of Zn2+ in the activation of T lymphocytes.

Human skeletal muscle myosin light chains analyzed by immobilized pH gradients during ontogenesis: identification of new phosphorylatable isoforms of light chain 2.

Previous studies using two-dimensional gel electrophoresis have described adult and fetal isoforms of skeletal muscle myosin light chains (MLC). They have also revealed an embryo-specific light chain (LC1emb), apparently absent in most adult skeletal muscles. In order to characterize more thoroughly the MLC family, we have analyzed the MLCs from human skeletal muscle at different developmental stages using a two-dimensional electrophoresis technique with an immobilized pH gradient in the first dimension. The high resolution of this novel technique, resolving components which in isoelectric points are less than or equal to 0.01 pH, combined with sensitive silver staining, has allowed us to identify four phosphorylatable isoforms of MLC2: two slow-myosin light chains (MLC2Sa and b), two fast myosin light chains (MLC2Fa and b), and their phosphorylated counterparts: MLC2SaP and bP, MLC2FaP and bP. The following major modifications during development were observed: (i) The embryonic LC (LC1emb) persists up to at least 26 weeks of fetal life. (ii) The polymorphism of LC2 is already evident at 10 weeks of development but only the nonphosphorylated forms of LC2S and LC2F seem to be present. The LC2Fa form is predominant. As early as 26 weeks of fetal life, the 4 phosphorylated forms are detected. In the adult, LC2Fb is a minor component. (iii) LC3F (fast) is already expressed at an early embryonic stage (10 weeks).

2-D analysis of human skeletal muscle myosin light chains with immobilized pH gradients.

Myosin light chains (LC) are a low molecular mass fraction non-covalently bound to the heavy chains. They are present in the myosin molecules and exhibit various degrees of polymorphism among the different species. By utilizing a highly-resolving 2-D technique, in narrow immobilized pH gradients, we have compared the LC forms of skeletal muscle in human and rabbit. Our findings: (1) both forms, LC1 and LC3, migrate in the two species with rather similar electrophoretic constants (both in terms of pI and Mr); (2) the LC2 forms of rabbit and humans exhibit the same Mr but quite different pI values, the rabbit forms being more acidic; (3) the chain LC2Sb is resolved into two spots in both rabbit and humans. In the former, the two bands have equal intensity, while in the latter the high pI component is clearly the most abundant.

Nebulin seen in DMD males including one patient with a large DNA deletion encompassing the DMD gene.

The presence of nebulin in a muscle specimen from a patient with Duchenne muscular dystrophy (DMD) due to a large deletion precludes the possibility that this protein is the DMD gene product.