The natural furanone (5<i>Z</i>)-4-bromo-5-(bromomethylene)-3-butyl-2(5<i>H</i>)-furanone disrupts quorum sensing-regulated gene expression in <i>Vibrio harveyi</i> by decreasing the DNA-binding activity of the transcriptional regulator protein luxR

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

Enhanced sensitivity and responses to viologens from a whole-cell bacterial bioreporter treated with branched polyethyleneimines

Aims: Evaluate the use of polyethyleneimines (PEIs) as membrane permeabilizers to improve the responses and sensitivity of a bacterial bioreporter strain to viologens. Methods and Results: The responses from E. coli str. EBS, i.e., E. coli BW25113 carrying plasmid pSDS, when exposed to five different viologens were characterized, as were the toxicities of seven different PEIS, including two linear and five branched species. Based on these results, benzyl viologen led to the greatest responses, and 0??8-kDa branched PEI (BPEI) was the least toxic of the PEIs tested and, therefore, both were selected for the subsequent tests. The bioluminescence and relative responses from E. coli str. EBS exposed to various concentrations of 0??8 kDa BPEI identified 400 mg l&amp;#8722;1 as the optimal concentration. Using this concentration, tests were performed with all five of the viologens. Conclusions: The responses from E. coli str. EBS to the viologens were improved, with the maximum relative bioluminescence values increasing between 5??6 and 16??5-fold. The minimum detectable levels for four of the viologens were likewise improved 2- to 4-fold. Significance and Impact of Study: Improving bacterial membrane permeability in a controlled manner using BPEIs can improve biosensing of toxic compounds, as well as be used in biofuel and bioenergy applications where membrane permeability to a solute is important