The natural furanone (5Z)-4-bromo-5-(bromomethylene)-3-butyl-2(5H)-furanone disrupts quorum sensing-regulated gene expression in Vibrio harveyi 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 luxR(Vh) 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 LuxR(Vh) response regulator protein were able to bind to its target promoter sequences in wild-type V. harveyi. Finally, tests with purified LuxR(Vh) protein also showed less shifts with furanone-treated LuxR(Vh), whereas the LuxR(Vh) 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 LuxR(Vh) unable to bind to the promoter sequences of quorum sensing-regulated genes.

Involvement of LuxR, a quorum sensing regulator in Vibrio harveyi, in the promotion of metabolic genes: argA, purM, lysE and rluA.

Quorum sensing, involving signal transduction via the two-component response regulator LuxO to its downstream target LuxR, controls luminescence in the marine bacterium Vibrio harveyi. LuxR is a DNA binding protein that acts as both activator of the lux operon and repressor of its own gene. In order to determine if any other genes are affected by quorum sensing in V. harveyi, an assay for luxR-dependent promotion was devised using a genomic library maintained in a novel luxAB (luciferase) reporter. Screening in Escherichia coli DH-21 (lacI(sq)) entailed the addition of a second plasmid containing luxR under plac control. Four out of 5000 colonies showed luminescence stimulation upon IPTG induction of luxR. The four luxR-dependent promoters were upstream of argA, purM, lysE, and rluA, genes involved in arginine and purine biosyntheses, amino acid efflux, and pseudouridine synthesis, respectively. Based on analysis of luxR-dependent promoters, particularly that of argA, we describe a LuxR binding site, and implicate the coordination of LuxR with ArgR.

Cloning and characterization of the bile salt hydrolase genes (bsh) from Bifidobacterium bifidum strains.

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.

LuxO controls luxR expression in Vibrio harveyi: evidence for a common regulatory mechanism in Vibrio.

Quorum-sensing control of luminescence in Vibrio harveyi, which involves an indirect autoinducer-mediated phosphorelay signal transduction system, contrasts with the prototypical quorum-sensing system of Vibrio fischeri, in which the autoinducer and the transcriptional activator LuxR directly activate lux operon expression. In V. harveyi, a regulator not homologous to V. fischeri LuxR and also designated LuxR (LuxRvh), binds specifically to the lux operon promoter region and activates the expression of luminescence. A direct connection has not been identified previously between V. harveyi LuxRvh and the autoinducer-mediated phosphorelay system. Here, we demonstrate by mobility shift assays and measurement of luxRvh mRNA levels with luxO+ and luxO- cells that the central response regulator of the V. harveyi phosphorelay system (LuxO) represses the level of LuxRvh. Expression of a luxRvh-bearing plasmid strongly stimulated luminescence of a luxO- mutant but had no effect on luminescence of wild-type luxO+ cells, indicating tight regulation of luxRvh by LuxO. Furthermore, luxO null mutants of V. fischeri MJ-1 and two autoinducer mutants, MJ-211 (luxI-) and MJ-215 (luxI-ainS-), emitted more light and exhibited more elevated levels of litR, a newly identified V. harveyi luxRvh homologue, than their luxO+ counterparts. These results suggest that activity of the autoinducer-mediated phosphorelay system is coupled to LuxRvh/LitR control of luminescence through LuxO in V. harveyi and V. fischeri. The presence of homologues of V. harveyi LuxRvh, LuxO and other phosphorelay system proteins in various Vibrio species and the control of LuxRvh and its homologues by LuxO identified here in V. harveyi and V. fischeri and recently in Vibrio cholerae suggest that the luxO-luxRvh couple is a central feature of this quorum-sensing system in members of the genus Vibrio.

MetR and CRP bind to the Vibrio harveyi lux promoters and regulate luminescence.

The induction of luminescence in Vibrio harveyi at the later stages of growth is controlled by a quorum-sensing mechanism in addition to nutritional signals. However, the mechanism of transmission of these signals directly to the lux promoters is unknown and only one regulatory protein, LuxR, has been shown to bind directly to lux promoter DNA. In this report, we have cloned and sequenced two genes, crp and metR, coding for the nutritional regulators, CRP (cAMP receptor protein) and MetR (a LysR homologue), involved in catabolite repression and methionine biosynthesis respectively. The metR gene was cloned based on a general strategy to detect lux DNA-binding proteins expressed from a genomic library, whereas the crp gene was cloned based on its complementation of an Escherichia coli crp mutant. Both CRP and MetR were shown to bind to lux promoter DNA, with CRP being dependent on the presence of cAMP. Expression studies indicated that the two regulators had opposite effects on luminescence: CRP was an activator and MetR a repressor. Disruption of crp decreased luminescence by about 1,000-fold showing that CRP is a major activator of luminescence the same as LuxR, whereas disruption of MetR resulted in activation of luminescence over 10-fold, confirming its function as a repressor. Comparison of the levels of the autoinducers involved in quorum sensing excreted by V. harveyi, and the crp and metR mutants, showed that autoinducer production was not significantly different, thus indicating that the nutritional signals do not affect luminescence by changing the levels of the signals required for quorum sensing. Indeed, the large effects of these nutritional sensors show that luminescence is controlled by multiple signals related to the environment and the cell density which must be integrated at the molecular level to control expression at the lux promoters.

LitR, a new transcriptional activator in Vibrio fischeri, regulates luminescence and symbiotic light organ colonization.

Vibrio 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.

Generation and properties of a luminescent insect pathogen Xenorhabdus nematophilus (Enterobacteriaceae).

Studies on the interaction of the insect pathogenic bacterium, Xenorhabdus nematophilus (Enterobacteriaceae), with its nematode and insect hosts would be greatly assisted if a luminescent phenotype were generated that would allow the detection of viable bacteria in vivo without the necessity for disruption of the cellular interactions. The plasmid, pMGM221, containing the luminescence gene (luxCDABE) of Vibrio harveyi was introduced into different strains (DD136 and 19061) and phases (one and two) of X. nematophilus by triparental mating. For reproducible and efficient conjugation, it was necessary to use older cultures (96-160 h) in the stationary phase of X. nematophilus for mating with relatively small differences (<2-fold) in transconjugant yield for the different strains and phases of X. nematophilus. All transconjugants emitted high levels of light with optimum bioluminescence at 27 degrees C in Luria broth at pH 8.0 containing 20 g/L NaCl; pH, osmolarity, and temperature conditions were similar to those encountered by the bacteria in the hemolymph of the larvae of Galleria mellonella. Plasmids were detected in the transconjugants after 6 months of subculturing the bacteria without antibiotic selection. Aside from light emission, luminescent transconjugants had the same physiological properties as the nonluminescent parental strains, including identical rates of growth, production of exoenzymes, removal from and subsequent emergence into the insect's hemolymph, bacterial-induced hemocyte damage, suppression of prophenoloxidase activation, and the ability to kill G. mellonella larvae. Light-emitting larvae could readily be detected by eye in a dark room, and all bacteria reisolated from dead larvae were luminescent. These properties validate the use of luminescent X. nematophilus not only as a means of following bacterial host interactions, but also as a potential agent to follow the infection and death of the insect population.