Background and aim: A role for the intestinal microbial community (microbiota) in the onset and chronicity of Crohn’s disease (CD) is strongly suspected. However, investigation of such a complex ecosystem is difficult, even with culture independent molecular approaches. Methods: We used, for the first time, a comprehensive metagenomic approach to investigate the full range of intestinal microbial diversity. We used a fosmid vector to construct two libraries of genomic DNA isolated directly from faecal samples of six healthy donors and six patients with CD. Bacterial diversity was analysed by screening the two DNA libraries, each composed of 25 000 clones, for the 16S rRNA gene by DNA hybridisation. Results: Among 1190 selected clones, we identified 125 non-redundant ribotypes mainly represented by the phyla Bacteroidetes and Firmicutes. Among the Firmicutes, 43 distinct ribotypes were identified in the healthy microbiota, compared with only 13 in CD (p<0.025). Fluorescent in situ hybridisation directly targeting 16S rRNA in faecal samples analysed individually (n = 12) confirmed the significant reduction in the proportion of bacteria belonging to this phylum in CD patients (p<0.02). Conclusion: The metagenomic approach allowed us to detect a reduced complexity of the bacterial phylum Firmicutes as a signature of the faecal microbiota in patients with CD. It also indicated the presence of new bacterial species.

Background: The structure and function of human gut microbiota is currently inferred from metagenomic and metatranscriptomic analyses. Recovery of intact DNA and RNA is therefore a critical step in these studies. Here, we evaluated how different storage conditions of fecal samples affect the quality of extracted nucleic acids and the stability of their microbial communities.Results: We assessed the quality of genomic DNA and total RNA by microcapillary electrophoresis and analyzed the bacterial community structure by pyrosequencing the 16S rRNA gene. DNA and RNA started to fragment when samples were kept at room temperature for more than 24 h. The use of RNAse inhibitors diminished RNA degradation but this protection was not consistent among individuals. DNA and RNA degradation also occurred when frozen samples were defrosted for a short period (1 h) before nucleic acid extraction. The same conditions that affected DNA and RNA integrity also altered the relative abundance of most taxa in the bacterial community analysis. In this case, intra-individual variability of microbial diversity was larger than inter-individual one.Conclusions: Though this preliminary work explored a very limited number of parameters, the results suggest that storage conditions of fecal samples affect the integrity of DNA and RNA and the composition of their microbial community. For optimal preservation, stool samples should be kept at room temperature and brought at the laboratory within 24 h after collection or be stored immediately at −20°C in a home freezer and transported afterwards in a freezer pack to ensure that they do not defrost at any time. Mixing the samples with RNAse inhibitors outside the laboratory is not recommended since proper homogenization of the stool is difficult to monitor.

The mechanism of inhibition of eukaryotic DNA topoisomerase II [DNA topoisomerase (ATP-hydrolyzing), EC 5.99.1.3] by a member of the bisdioxopiperazine family of anticancer drugs, ICRF-193, was investigated by using purified yeast DNA topoisomerase II. In the absence of ATP, ICRF-193 has little effect on the binding of the enzyme to various forms of DNA. In the presence of ATP, the drug converts the enzyme to a form incapable of binding circular DNA. Incubation of a preformed circular DNA-enzyme complex with ICRF-193 and ATP converts the complex to a form stable in molar concentrations of salt. These results can be interpreted in terms of the ATP-modulated protein-clamp model of type II DNA topoisomerases [Roca, J. & Wang, J. C. (1992) Cell 71, 833-840]; ICRF-193 can bind to the closed-clamp form of the enzyme and prevents its conversion to the open-clamp form. This interpretation is further supported by the finding that whereas both ATP and the drug are needed to form the salt-stable circular DNA-enzyme complex, ATP is not needed for maintaining this complex; furthermore, a signature of the closed-clamp form of the enzyme, Staphylococcus aureus strain V8 endoproteinase cleavage site at Glu-680, is observed if the enzyme is incubated with both ATP and ICRF-193. Inhibition of interconversion between the open- and closed-clamp forms of type II DNA topoisomerases offers a new mechanism in the selection and design of therapeutics targeting this class of enzymes.

When linear double-stranded DNA is packed inside bacteriophage capsids, it becomes highly compacted. However, the phage is believed to be fully effective only if the DNA is not entangled. Nevertheless, when DNA is extracted from a tailless mutant of the P4 phage, DNA is found to be cyclic and knotted (probability of 0.95). The knot spectrum is very complex, and most of the knots have a large number of crossings. We quantified the frequency and crossing numbers of these knots and concluded that, for the P4 tailless mutant, at least half the knotted molecules are formed while the DNA is still inside the viral capsid rather than during extraction. To analyze the origin of the knots formed inside the capsid, we compared our experimental results to Monte Carlo simulations of random knotting of equilateral polygons in confined volumes. These simulations showed that confinement of closed chains to tightly restricted volumes results in high knotting probabilities and the formation of knots with large crossing numbers. We conclude that the formation of the knots inside the viral capsid is driven mainly by the effects of confinement.