Development of a 16S rRNA gene-based prototype microarray for the detection of selected actinomycetes genera

Actinomycetes are known for their secondary metabolites, which have been successfully used as drugs in human and veterinary medicines. However, information on the distribution of this group of Gram-positive bacteria in diverse ecosystems and a comprehension of their activities in ecosystem processes are still scarce. We have developed a 16S rRNA-based taxonomic microarray that targets key actinomycetes at the genus level. In total, 113 actinomycete 16S rRNA probes, corresponding to 55 of the 202 described genera, were designed. The microarray accuracy was evaluated by comparing signal intensities with probe/target-weighted mismatch values and the Gibbs energy of the probe/target duplex formation by hybridizing 17 non-actinomycete and 29 actinomycete strains/clones with the probe set. The validation proved that the probe set was specific, with only 1.3% of false results. The incomplete coverage of actinomycetes by a genus-specific probe was caused by the limited number of 16S rRNA gene sequences in databases or insufficient 16S rRNA gene polymorphism. The microarray enabled discrimination between actinomycete communities from three forest soil samples collected at one site. Cloning and sequencing of 16S rRNA genes from one of the soil samples confirmed the microarray results. We propose that this newly constructed microarray will be a valuable tool for genus-level comparisons of actinomycete communities in various ecological conditions.     

Vanadium Requirements and Uptake Kinetics in the Dinitrogen-Fixing Bacterium Azotobacter vinelandii

Vanadium is a cofactor in the alternative V-nitrogenase that is expressed by some N₂-fixing bacteria when Mo is not available. We investigated the V requirements, the kinetics of V uptake, and the production of catechol compounds across a range of concentrations of vanadium in diazotrophic cultures of the soil bacterium Azotobacter vinelandii. In strain CA11.70, a mutant that expresses only the V-nitrogenase, V concentrations in the medium between 10⁻⁸ and 10⁻⁶ M sustain maximum growth rates; they are limiting below this range and toxic above. A. vinelandii excretes in its growth medium micromolar concentrations of the catechol siderophores azotochelin and protochelin, which bind the vanadate oxoanion. The production of catechols increases when V concentrations become toxic. Short-term uptake experiments with the radioactive isotope ⁴⁹V show that bacteria take up the V-catechol complexes through a regulated transport system(s), which shuts down at high V concentrations. The modulation of the excretion of catechols and of the uptake of the V-catechol complexes allows A. vinelandii to precisely manage its V homeostasis over a range of V concentrations, from limiting to toxic.     

Frequent,independent transfers of a catabolic gene from bacteria to contrasted filamentous eukaryotes

Even genetically distant prokaryotes can exchange genes between them, and these horizontal gene transfer events play a central role in adaptation and evolution. While this was long thought to be restricted to prokaryotes, certain eukaryotes have acquired genes of bacterial origin. However, gene acquisitions in eukaryotes are thought to be much less important in magnitude than in prokaryotes. Here, we describe the complex evolutionary history of a bacterial catabolic gene that has been transferred repeatedly from different bacterial phyla to stramenopiles and fungi. Indeed, phylogenomic analysis pointed to multiple acquisitions of the gene in these filamentous eukaryotes—as many as 15 different events for 65 microeukaryotes. Furthermore, once transferred, this gene acquired introns and was found expressed in mRNA databases for most recipients. Our results show that effective inter-domain transfers and subsequent adaptation of a prokaryotic gene in eukaryotic cells can happen at an unprecedented magnitude.     

Melon resistance to the aphid Aphis gossypii: behavioural analysis and chemical correlations with nitrogenous compounds

In the melon, the Vat (monogenic, dominant) resistance gene governs both an antixenotic reaction to the melon aphid Aphis gossypii Glover (Homoptera, Aphididae) and a resistance to non-persistent virus transmission, restricted to this vector species. We investigated the behavioural features and tissue localisation of the antixenosis resistance by the electrical penetration graph technique (EPG, DC system). We also compared the chemical composition in amino compounds and proteins of the phloem sap collected from two isogenic lines of melon (Cucumis melo L.), carrying the Vat gene or not. All behavioural and chemical data indicated that this resistance is constitutive. EPG analysis clearly showed that access to phloem, although delayed by alterations in pathway activities, was not impaired in terms of frequency of access or initiation of feeding. The most striking feature was, however, a very reduced duration of ingestion from phloem of resistant plants, making this compartment one of the tissues where the effects of the Vat gene are unambiguously expressed. This was confirmed by clear differential activity of phloem extracts in artificial no-choice bioassays. Chemical analyses have shown that phloem saps from the two isogenic lines were extremely similar in profiles of ninhydrin positive compounds, and contained a low total amount of free amino acids (less than 10 mM). Out of more than 40 distinguishable peaks in the chromatograms (protein and non-protein amino acids, as well as small peptides), only five differentiated the two genotypes. Two of them were increased in the resistant genotype: glutamic acid and a major unknown peak, probably a non-protein amino acid (different from pyrazolyl-alanine, a Cucumis-specific amino acid). The three others were depressed in resistant plants, and included the sulphur amino acid cystine and a peptide peak partly composed of the cysteine-containing peptide glutathione (reduced form). Sap collection also showed that phloem exudation rates, as well as total protein and glutathione levels, were depressed in phloem sap from resistant plants. Such data are all indicative of a modified phloem-sealing physiology, linked to sulfhydryl oxidation processes, in plants carrying the Vat gene. The originality of the mechanism of Vat resistance to aphids is discussed.     

Role of the host cell cycle in theAgrobacterium-mediated genetic transformation ofPetunia: Evidence of an S-phase control mechanism for T-DNA transfer

Chimeric -glucuronidase (GUS) gene expression in an efficientAgrobacterium-mediated transformation system utilising mesophyll cells ofPetunia hybrida synchronized with cell cycle phase-specific inhibitors (mimosine and colchicine) was used to show the absolute requirement of S-phase for transfer and/or integration of the transferred DNA (T-DNA). Flow-cytometric analysis of nuclear DNA content and immunohistological detection of bromodeoxyuridine (BrdUrd) incorporation showed that, prior to phytohormone treatment, most (98%) mesophyll cells were at GO-Gl-phase (quiescent phase) and no cell division was occurring. After 48 h and 72 h of phytohormone treatment, there was a rapid increase in S-G2-M-phase populations (> 75%) and a concomitant decrease (down to 24%) in G0–-G1-phase cells. Assays of GUS showed that maximum transformation (> 95% of explants) also occurred after this period. Our data showed that mimosine and colchicine blocked the mesophyll cells at late Gl-phase and M-phase, respectively. No transformation (= GUS expression) was observed in phytohormone-treated cells inhibited in late G1 by mimosine. However, after removal of mimosine, 82% of the explants were transformed, indicating the non-toxic and reversible effect of the inhibitor. On the other hand, a relatively high transformation frequency (65% of explants) was observed after blocking the cell cycle at M-phase with colchicine. However, only transient, but no stable, gene expression (= kanamycin-resistant callus formation) was observed in colchicine-treated M-phase-arrested cells. Similarly, endoreduplication of nuclear DNA, which occurred during the 48 h of phytohormone treatment in some mesophyll cells and cells located along the minor veins in the leaf explants, resulted in transient GUS expression only. These observations indicate a direct correlation between endoreduplication and transient GUS gene expression. Obviously, for stable GUS gene expression, cell division and proliferation are required, indicating that both DNA duplication (S-phase) and cell division (M-phase) are strongly related to stable transformation. We propose that the present system should facilitate further dissection of the process of T-DNA integration in the host genome and therefore should aid in developing new strategies for transformation of recalcitrant plants.Abbreviations BAP 6-benzylaminopurine - BM basal medium - BrdUrd bromodeoxyuridine - GUS -glucuronidase - Km^R kanamycin resistant - T-DNA transferred DNA