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We describe identification and functional characterization of ISEcll, a new insertion sequence that is widespread in enteroinvasive E. coli (EIEC), in which it is always present on the virulence plasmid (pINV) and very frequently also present on the chromosome. ISEcll is flanked by subterminal 13-bp inverted repeats (IRs) and is bounded by 3-bp terminal sequences, and it transposes with target specificity without generating duplication of the target site. ISEcll is characterized by an atypical transposase containing the DEDD motif of the Piv/MooV family of DNA recombinases, and it is closely related to the ISllll family. Transposition occurs by formation of minicircles through joining of the abutted ends and results in assembly of a junction promoter ([P.sub.juncC]) containing a--10 box in the interstitial sequence and a -35 box upstream of the right IR. A natural variant of ISEcll (ISEcllp), found on EIEC pINV plasmids, contains a perfect duplication of the outermost 39 bp of the right end. Upon circularization, ISEcllp forms a junction promoter ([P.sub.juncP]) which, despite carrying--10 and--35 boxes identical to those of [P.sub.juncC] exhibits 30-fold-greater strength in vivo. The discovery of only one starting point in primer extension experiments rules out the possibility that there are alternative promoter sites within the 39-bp duplication. Analysis of in vitro-generated transcripts confirmed that at limiting RNA polymerase concentrations, the activity of [P.sub.juncP] is 20-fold higher than the activity of [P.sub.juncC]. These observations suggest that the 39-bp duplication might host cis-acting elements that facilitate the binding of RNA polymerase to the promoter.

Shigella spp , the etiological agents of bacillary dysentery in humans, have evolved an intricate regulatory strategy to ensure fine-tuned expression of virulence genes in response to environmental stimuli. A key component in this regulation is VirF, an AraC-like transcription factor, which at the host temperature (37°C) triggers, directly or indirectly, the expression of > 30 virulence genes important for invasion of the intestinal epithelium. Previous work identified two different forms of VirF with distinct functions: VirF30 activates virulence gene expression, while VirF21 appears to negatively regulate virF itself. Moreover, VirF21 originates from either differential translation of the virF mRNA or from a shorter leaderless mRNA (llmRNA). Here we report that both expression of the virF21 llmRNA and the VirF21:VirF30 protein ratio are higher at 30°C than at 37°C, suggesting a possible involvement of VirF21 in minimizing virulence gene expression outside the host (30°C). Ectopic elevation of VirF21 levels at 37°C indeed suppresses Shigella ´s ability to infect epithelial cells. Finally, we find that the VirF21 C-terminal portion, predicted to contain a Helix-Turn-Helix motif (HTH2), is required for the functionality of this negative virulence regulator. [ABSTRACT FROM AUTHOR]

Virulence gene expression in enteroinvasive Escherichia coli requires all three virulence regulatory genes: virB, virF and hns. The integration of the virulence plasmid pINV produces a noninvasive strain by repressing virB transcription, despite virF and hns remaining unaffected. H-NS in the pINV-strain binds more stably to virB, preventing virF activation but an increase in virF gene dosage, for autonomously replicating pINV, overcomes H-NS' negative regulatory effect on virB transcription.

Abstract Background All bacterial genomes contain repetitive sequences which are members of specific DNA families. Such repeats may occur as single units, or found clustered in multiple copies in a head-to-tail configuration at specific loci. The number of clustered units per locus is a strain-defining parameter. Assessing the length variability of clusters of repeats is a versatile typing methodology known as multilocus variable number of tandem repeat analysis (MLVA). Results Stenotrophomonas maltophilia is an environmental bacterium increasingly involved in nosocomial infections and resistant to most antibiotics. The availability of the whole DNA sequence of the S. maltophilia strain K279a allowed us to set up fast and accurate PCR-based diagnostic protocols based on the measurement of length variations of loci carrying a variable number of short palindromic repeats marking the S. maltophilia genome. On the basis of the amplimers size, it was possible to deduce the number of repeats present at 12 different loci in a collection of S. maltophilia isolates, and therefore label each of them with a digit. PCR-negative regions were labelled 0. Co-amplification of two pairs of loci provided a 4-digit code sufficient for immediate subtyping. By increasing the number of loci analyzed, it should be possible to assign a more specific digit profile to isolates. In general, MLVA data match genotyping data obtained by PFGE (pulsed-field gel electrophoresis). However, some isolates exhibiting the same PCR profiles at all loci display distinct PFGE patterns. Conclusion The utilization of the present protocol allows to type several S. maltophilia isolates in hours. The results are immediately interpretable without the need for sophisticated softwares. The data can be easily reproducible, and compared among different laboratories.

A real-time in vitro transcription model system based on a specific fluorescence molecular beacon was used to quantitatively analyze the role of sequence-mediated DNA curvature in thermoregulated gene expression. The DNA curvature-mediated regulation of gene expression identified as a possible strategy to fine-tune control possibilities might represent one of the most primitive forms of gene regulation.

The genetic context of tetracycline resistance determinants in these natural strains, tet(M), is characterized at the molecular level. The gene is associated with the broad-host, conjugative transposon Tn916, and the novel coupling sequence has conferred low conjugation frequency in mating experiments with the recipient strain JH2-2 of Enterococcus faecalis.