Gioacchino Micheli

Thermoregulation of Shigella and Escherichia coli EIEC pathogenicity. A temperature-dependent structural transition of DNA modulates accessibility of virF promoter to transcriptional repressor H-NS

The expression of plasmid‐borne virF of Shigella encoding a transcriptional regulator of the AraC family, is required to initiate a cascade of events resulting in activation of several operons encoding invasion functions. H‐NS, one of the main nucleoid‐associated proteins, controls the temperature‐dependent expression of the virulence genes by repressing the in vivo transcription of virF only below a critical temperature (∼32°C). This temperature‐dependent transcriptional regulation has been reproduced in vitro and the targets of H‐NS on the virF promoter were identified as two sites centred around −250 and −1 separated by an intrinsic DNA curvature. H‐NS bound cooperatively to these two sites below 32°C, but not at 37°C. DNA supercoiling within the virF promoter region did not influence H‐NS binding but was necessary for the H‐NS‐mediated transcriptional repression. Electrophoretic analysis between 4 and 60°C showed that the virF promoter fragment, comprising the two H‐NS sites, undergoes a specific and temperature‐dependent conformational transition at ∼32°C. Our results suggest that this modification of the DNA target may modulate a cooperative interaction between H‐NS molecules bound at two distant sites in the virF promoter region and thus represents the physical basis for the H‐NS‐dependent thermoregulation of virulence gene expression.

The virF promoter in Shigella: more than just a curved DNA stretch

In the human enteropathogen Shigella transcription of virF, the primary regulator of the invasion functions, is strictly temperature‐dependent and is antagonistically mediated by H‐NS and FIS, which bind to specific sites on the virF promoter. Here we report on the relevance of DNA geometry to the themoregulation of virF and demonstrate that the virF promoter hosts a major DNA bend halfway between two H‐NS sites. The bent region has been mutagenized in vitro to mimic temperature‐induced changes of DNA curvature. Functional analysis of curvature mutants and of promoter constructs in which the two H‐NS sites are phased‐out by a half–helix turn reveals that modifying the spatial relationships between these sites severely affects the interaction of H‐NS with the virF promoter, as well as its in vivo and in vitro temperature‐dependent activity. The role of promoter curvature as thermosensor is also compatible with the present observation that, with increasing temperature, the virF bending centre moves downstream at a rate having its maximum around the transition temperature, abruptly unmasking a binding site for the transcriptional activator FIS.

A role for H-NS in the regulation of the virF gene of Shigella and enteroinvasive Escherichia coli.

We have investigated the role of H-NS, one of the major components of the bacterial nucleoid, in the expression of the virF gene present on the large virulence plasmid of Shigella and enteroinvasive Escherichia coli in response to different environmental conditions. VirF is an AraC-like protein which activates at least two promoters, virB and virG, both repressed by H-NS. Band shift experiments reveal that the affinity of H-NS for the virF and virB promoters is comparable, while the affinity for the virG promoter is higher. Polyacrylamide gel electrophoresis of three DNA fragments containing the virF, the virB and the VirG promoters demonstrates, in agreement with computer predictions, that they have an intrinsically curved structure, confirming the preference of H-NS for bent DNA. In vivo transcriptional analysis of virF mRNA shows that H-NS negatively controls the expression of virF at 30 degrees C. The expression of a virF-lacZ translational fusion in E.coli wild type and in an hns-defective derivative grown at 30 degrees or 37 degrees C and at pH 6.0 or 7.0 indicates that, in the absence of H-NS, virF expression becomes insensitive to temperature and to limited pH changes. Our results strongly suggest that H-NS controls virF expression by binding to the virF promoter and by repressing its expression at low temperature and at low pH.

Fingerprinting and sequence homology of plasmids from different virulent strains of Agrobacterium rhizogenes.

Abstract Several wild-type virulent strains of Agrobacterium rhizogenes, belonging to both biotypes 1 and 2, were analyzed for plasmid content. All the strains were found to harbor at least one large plasmid, of a size comparable to that of A. tumefaciens Ti plasmids as determined by electron microscopy. The plasmids were characterized by restriction endonuclease finger-printing and compared for sequence homology by the Southern blot-hybridization technique. Despite the diversity of the restriction cleavage patterns, the plasmids of the various strains share rather extensive sequence homology. All of the biotype 1 organisms analyzed were shown to harbor one common plasmid.

Polyamines: emerging players in bacteria-host interactions.

Polyamines are small polycationic molecules found in almost all cells and associated with a wide variety of physiological processes. In recent years it has become increasingly clear that, in addition to core physiological functions, polyamines play a crucial role in bacterial pathogenesis. Considerable evidence has built up that bacteria have evolved mechanisms to turn these molecules to their own advantage and a novel standpoint to look at host-bacterium interactions emerges from the interplay among polyamines, host cells and infecting bacteria. In this review, we highlight how human bacterial pathogens have developed their own resourceful strategies to exploit polyamines or manipulate polyamine-related processes to optimize their fitness within the host. Besides contributing to a better understanding of the complex relationship between a pathogen and its host, acquisitions in this field have a significant potential towards the development of novel antibacterial therapeutic approaches.

Histone-like proteins and the Shigella invasivity regulon.

The contribution of histone-like proteins to the transcriptional regulation of virulence gene networks is a common feature among pathogenic bacteria. In this article we review current knowledge about the regulative role of major histone-like proteins in the silencing/activation of the invasivity phenotype of Shigella, the etiological agent of bacillary dissentery.

Chromosome length and DNA loop size during early embryonic development of Xenopus laevis

The looped organization of the eukaryotic genome mediated by a skeletal framework of non-histone proteins is conserved throughout the cell cycle. The radial loop/scaffold model envisages that the higher order architecture of metaphase chromosomes relies on an axial structure around which looped DNA domains are radially arranged through stable attachment sites. In this light we investigated the relationship between the looped organization and overall morphology of chromosomes. In developing Xenopus laevis embryos at gastrulation, the bulk of the loops associated with histone-depleted nuclei exhibit a significant size increase, as visualized by fluorescence microscopy of the fully extended DNA halo surrounding high salt treated, ethidium bromide stained nuclei. This implies a reduction in the number of looped domains anchored to the supporting nucleoskeletal structure. The cytological analysis of metaphase plates from acetic acid fixed whole embryos, carried out in the absence of drugs inducing chromosome condensation, reveals a progressive thickening and shortening of metaphase chromosomes during development. We interpret these findings as a strong indication that the size and number of DNA loops influence the thickness and length of the chromosomes, respectively. The quantitative analysis of chromosome length distributions at different developmental stages suggests that the shortening is timed differently in different embryonic cells.

Shedding of genes that interfere with the pathogenic lifestyle: The Shigella model

Pathoadaptive mutations are evolutionary events leading to the silencing of specific anti-virulence loci. This reshapes the core genome of a novel pathogen, adapts it to the host and boosts its harmful potential. A paradigmatic case is the emergence of Shigella, the causative agent of bacillary dysentery, from its innocuous Escherichia coli ancestor. Here we summarize current views on how pathoadaptation has allowed Shigella to progressively increase its virulence. In this context, modification of the polyamine pattern emerges as a crucial step towards full expression of the virulence program in Shigella.

The looped domain organization of the nucleoid in histone-like protein defective Escherichia coli strains**

We have investigated the major Escherichia coli histone-like proteins (H-NS, HU, FIS, and IHF) as putative factors involved in the maintenance of the overall DNA looped arrangement of the bacterial nucleoid. The long-range architecture of the chromosome has been studied by means of an assay based on in vivo genomic fragmentation mediated by endogenous DNA gyrase in the presence of oxolinic acid. The fragmentation products were analysed by CHEF electrophoresis. The results indicate that in vivo a large fraction of the bacterial chromatin constitutes an adequate substrate for the enzyme. DNA fragments released upon oxo-treatment span a size range from about 1000 kb to a limit-size of about 50 kb. The latter value is in excellent agreement with the average size reported for bacterial chromosomal domains. The DNA gyrase-mediated fragmentation does not appear to be significantly altered in strains depleted in histone-like proteins as compared to an E. coli wild type strain. This suggests that these proteins may not represent critical determinants for the maintenance of the supercoiled loop organisation of the E. coli chromosome.

The Salmonella wien virulence plasmid pZM3 carries Tn1935, a multiresistance transposon containing a composite IS1936-kanamycin resistance element.

Tn1935, a 23.5-kb transposon mediating resistance to ampicillin, kanamycin, mercury, spectinomycin, and sulfonamide was isolated from pZM3, an IncFIme virulence plasmid from Salmonella wien. Tn1935 possesses the entire sequence of Tn21 and contains two additional DNA segments of 0.95 and 2.7 kb carrying the ampicillin and kanamycin resistance genes, respectively. The latter is part of a composite element since it is flanked by two IS15-like insertion sequences (IS1936) in direct orientation. IS1936 is about 800 bp long and is closely related to IS15 delta, IS26, IS46, IS140, and IS176. Functional analysis of IS1936-mediated cointegrates shows that both insertion sequences are active and able to form cointegrates at the same frequency. Resolution of the cointegrates requires the presence of the host Rec system. The presence of the composite IS1936-element within Tn1935 supports the hypothesis that multidrug resistance transposons evolved by insertion of antibiotic determinants which are themselves transposable.