BackgroundTo date, only few compounds targeting the AI-2 based quorum sensing (QS) system are known. In the present study, we screened cinnamaldehyde and substituted cinnamaldehydes for their ability to interfere with AI-2 based QS. The mechanism of QS inhibition was elucidated by measuring the effect on bioluminescence in several Vibrio harveyi mutants. We also studied in vitro the ability of these compounds to interfere with biofilm formation, stress response and virulence of Vibrio spp. The compounds were also evaluated in an in vivo assay measuring the reduction of Vibrio harveyi virulence towards Artemia shrimp.ResultsOur results indicate that cinnamaldehyde and several substituted derivatives interfere with AI-2 based QS without inhibiting bacterial growth. The active compounds neither interfered with the bioluminescence system as such, nor with the production of AI-2. Study of the effect in various mutants suggested that the target protein is LuxR. Mobility shift assays revealed a decreased DNA-binding ability of LuxR. The compounds were further shown to (i) inhibit biofilm formation in several Vibrio spp., (ii) result in a reduced ability to survive starvation and antibiotic treatment, (iii) reduce pigment and protease production in Vibrio anguillarum and (iv) protect gnotobiotic Artemia shrimp against virulent Vibrio harveyi BB120.ConclusionCinnamaldehyde and cinnamaldehyde derivatives interfere with AI-2 based QS in various Vibrio spp. by decreasing the DNA-binding ability of LuxR. The use of these compounds resulted in several marked phenotypic changes, including reduced virulence and increased susceptibility to stress. Since inhibitors of AI-2 based quorum sensing are rare, and considering the role of AI-2 in several processes these compounds may be useful leads towards antipathogenic drugs.

Summary This study aimed at getting a deeper insight in the molecular mechanism by which the natural furanone (5Z)‐4‐bromo‐5‐(bromomethylene)‐3‐butyl‐2(5H)‐furanone disrupts quorum sensing in Vibrio harveyi. Bioluminescence experiments with signal molecule receptor double mutants revealed that the furanone blocks all three channels of the V. harveyi quorum sensing system. In further experiments using mutants with mutations in the quorum sensing signal transduction pathway, the compound was found to block quorum sensing‐regulated bioluminescence by interacting with a component located downstream of the Hfq protein. Furthermore, reverse transcriptase real‐time polymerase chain reaction with specific primers showed that there was no effect of the furanone on luxRVh mRNA levels in wild‐type V. harveyi cells. In contrast, mobility shift assays showed that in the presence of the furanone, significantly lower levels of the LuxRVh response regulator protein were able to bind to its target promoter sequences in wild‐type V. harveyi. Finally, tests with purified LuxRVh protein also showed less shifts with furanone‐treated LuxRVh, whereas the LuxRVh concentration was found not to be altered by the furanone (as determined by SDS‐PAGE). Therefore, our data indicate that the furanone blocks quorum sensing in V. harveyi by rendering the quorum sensing master regulator protein LuxRVh unable to bind to the promoter sequences of quorum sensing‐regulated genes.

SummaryVibrio fischeri is the bacterial symbiont within the light-emitting organ of the sepiolid squid Euprymna scolopes. Upon colonizing juvenile squids, bacterial symbionts grow on host-supplied nutrients, while providing a bioluminescence that the host uses during its nocturnal activities. Mutant bacterial strains that are unable to emit light have been shown to be defective in normal colonization. A 606 bp open reading frame was cloned from V. fischeri that encoded a protein, which we named LitR, that had about 60% identity to four related regulator proteins: Vibrio cholerae HapR, Vibrio harveyi LuxR, Vibrio parahaemolyticus OpaR and Vibrio vulnificus SmcR. When grown in culture, cells of V. fischeri strain PMF8, in which litR was insertionally inactivated, were delayed in the onset of luminescence induction and emitted only about 20% as much light per cell as its parent. Protein-binding studies suggested that LitR enhances quorum sensing by regulating the transcription of the luxR gene. Interestingly, when competed against its parent in mixed inocula, PMF8 became the predominant symbiont present in 83% of light organs. Thus, the litR mutation appears to represent a novel class of mutations in which the loss of a regulatory gene function enhances the bacterium's competence in initiating a benign infection. required for successful colonization and/or the initiation of host development . Preliminary evidence has suggested that V. fischeri produces an extracellular proteolytic activity similar to that exhibited by the Vibrio cholerae Hap (Finkelstein et al., 1983), Vibrio vulnificus Vvp (Nishina et al., 1992) or Vibrio anguillarum EmpA (Garcia et al., 1997) proteins. This activity might allow symbiosis-competent cells of V. fischeri to (i) move through the mucous barrier outside the light organ pores (Nyholm et al., 2000) and/or (ii) gain access to hostderived peptides in the crypts (Graf and Ruby, 1998). The expression of all three of these other Vibrio spp. proteases has been shown to be dependent on TetR family regulator proteins [i.e. HapR (Jobling and Holmes, 1997); SmcR (McDougald et al., 2001;Shao and Hor, 2001); and VanT (Milton et al., 1999) respectively]. In addition, two other homologues have been described: OpaR, which controls colony opacity in Vibrio parahaemolyticus (McCarter, 1998), and LuxR, which is required for luminescence in Vibrio harveyi (Showalter et al., 1990). To date, there have been no reports of a homologous regulatory protein in V. fischeri. To understand better the control of both protease activity and luminescence in V. fischeri, and to examine how these activities might be modulated in the symbiosis, we searched for a gene that might encode a member of this family of regulators.We report here the discovery in V. fischeri of litR, a gene that encodes a protein with high sequence identity to the other TetR family transcriptional regulators present in Vibrio spp. Its product, designated LitR, not only has functional characteristics that are like those reported for some of the o...

Spatial chromatin organization is emerging as an important mechanism to regulate the expression of genes. However, very little is known about genome architecture at high-resolution in vivo. Here, we mapped the three-dimensional organization of the human Hox clusters with chromosome conformation capture (3C) technology. We show that computational modeling of 3C data sets can identify candidate regulatory proteins of chromatin architecture and gene expression. Hox genes encode evolutionarily conserved master regulators of development which strict control has fascinated biologists for over 25 years. Proper transcriptional silencing is key to Hox function since premature expression can lead to developmental defects or human disease. We now show that the HoxA cluster is organized into multiple chromatin loops that are dependent on transcription activity. Long-range contacts were found in all four silent clusters but looping patterns were specific to each cluster. In contrast to the Drosophila homeotic bithorax complex (BX-C), we found that Polycomb proteins are only modestly required for human cluster looping and silencing. However, computational three-dimensional Hox cluster modeling identified the insulator-binding protein CTCF as a likely candidate mediating DNA loops in all clusters. Our data suggest that Hox cluster looping may represent an evolutionarily conserved structural mechanism of transcription regulation.

Biochemical characterization of the purified bile salt hydrolase (BSH) from Bifidobacterium bifidum ATCC 11863 revealed some distinct characteristics not observed in other species of Bifidobacterium. The bsh gene was cloned from B. bifidum, and the DNA flanking the bsh gene was sequenced. Comparison of the deduced amino acid sequence of the cloned gene with previously known sequences revealed high homology with BSH enzymes from several microorganisms and penicillin V amidase (PVA) of Bacillus sphaericus. The proposed active sites of PVA were highly conserved, including that of the Cys-1 residue. The importance of the SH group in the N-terminal cysteine was confirmed by substitution of Cys with chemically and structurally similar residues, Ser or Thr, both of which resulted in an inactive enzyme. The transcriptional start point of the bsh gene has been determined by primer extension analysis. Unlike Bifidobacterium longum bsh, B. bifidum bsh was transcribed as a monocistronic unit, which was confirmed by Northern blot analysis. PCR amplification with the type-specific primer set revealed the high level of sequence homology in their bsh genes within the species of B. bifidum.