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.

Random mutagenesis of bacterial luciferase: critical role of Glu175 in the control of luminescence decay.

Bacterial luciferases (LuxAB) can be readily classed as slow or fast decay luciferases based on their rates of luminescence decay in a single turnover assay. Luciferases from Vibrio harveyi and Xenorhabdus (Photorhabdus) luminescens have slow decay rates, and those from the Photobacterium genus, such as Photobacterium fisheri, P. phosphoreum and P. leiognathi, have rapid decay rates. By substitution of a 67-amino-acid stretch of P. phosphoreum LuxA in the central region of the LuxA subunit, the 'slow' X. luminescens luciferase was converted into a chimaeric luciferase with a significantly more rapid decay rate [Valkova, Szittner and Meighen (1999) Biochemistry 38, 13820-13828]. To understand better the role of specific residues in the classification of luciferases as slow and fast decay, we have conducted random mutagenesis on this region. One of the mutants generated by a single mutation on LuxA at position 175 [E175G (Glu175-->Gly)] resulted in the 'slow decay' X. luminescens luciferase being converted into a luciferase with a significantly more rapid decay rate. These results indicate the importance of Glu175 in LuxA as a critical residue for differentiating between 'slow' and 'fast' luciferases and show that this distinction is primarily due to differences in aldehyde affinity and in the decomposition of the luciferase-flavin-oxygen intermediate.

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.

Changes in the kinetics and emission spectrum on mutation of the chromophore-binding platform in Vibrio harveyi luciferase.

The recently proposed model for the bacteria luciferase-flavin mononucleotide complex identifies a number of critical intermolecular interactions that define a binding platform for the isoalloxazine ring of flavin [Lin, L. Y., Sulea, T., Szittner, R., Vassilyev, V., Purisima, E. O., and Meighen, E. A. (2001) Protein Sci. 10, 1563-1571]. A key interaction involving van der Waals contact between the isopropyl side chain of alphaVal173 and the 7,8-dimethyl benzene plane of the isoalloxazine chromophore represents an important target to test the validity of the proposed model. Here, structure-function analysis of luciferase variants carrying single point mutations at position alpha173 have verified the functional layout of the active site architecture and implicated this site directly in flavin binding. Moreover, a decrease in the stability of the enzyme-bound C4a-hydroperoxyflavin intermediate in the mutants could account for changes in saturation with the fatty aldehyde substrate. A predicted red-shift on mutation of position alpha173 to increase its polarity confirmed that alphaVal173 was an integral component of the chromophore-binding microenvironment. Introduction of mutations in residues that contact the pyrimidine plane of the isoalloxazine chromophore (alphaA75G/C106V) into the alphaV173A, alphaV173C, alphaV173T, and alphaV173S mutants led to the retention of high levels of enzyme activity (10-40% of wild type) and further red-shifted the emission spectra in the triple mutants. The additivity of the mutation-induced red-shifts in the emission wavelength spectrum provides the basis toward engineering luciferase variants that emit different light colors with the proposed flavin-luciferase model complex as a design reference.

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.

Implications of the reactive thiol and the proximal non-proline cis-peptide bond in the Structure and function of Vibrio harveyi luciferase.

The role of a highly reactive cysteine residue, Cys106, in Vibrio harveyi luciferase in modulating the substrate-enzyme interactions and in turn affecting the enzyme activity has been extensively investigated over the past three decades. Replacing Cys106 with valine dramatically hinders the ability of luciferase to stabilize the C4a-hydroperoxyflavin intermediate [Abu-Soud, H. M., Clark, A. C., Francisco, W. A., Baldwin, T. O., and Raushel, F. M. (1993) J. Biol. Chem. 268, 7699-7706] and consume aldehyde substrate [Xi, L., Cho, K.-W., Herndon, M. E., and Tu, S.-C. (1990) J. Biol. Chem. 265, 4200-4203], therefore markedly decreasing enzyme activity. On the basis of the structure-activity relationship of flavin analogues and the location of the phosphate binding site of flavin mononucleotide (FMN) coupled with molecular modeling, the functional part of the isoalloxazine ring of FMN, the thiol side chain of Cys106, the methyl group of Ala75, and the unique non-prolyl cis-peptide bond between Ala74 and Ala75 were found to be closely packed [Lin, L. Y., Sulea, T., Szittner, R., Vassilyev, V., Purisima, E. O., and Meighen, E. A. (2001) Protein Sci. 10, 1563-1571]. Here, by mutating Ala75 to Gly, we restored key wild-type properties to the C106V mutant, in particular, high enzyme activity and a stable C4a-hydroperoxyflavin intermediate, demonstrating that the primary reason for the dark phenotype of the C106V mutant was the unfavorable steric interaction between Val106 and Ala75 side chains, which could in turn disturb the cis-oriented amide linkage of Ala74 and Ala75. Moreover, significant red shifts in light emission of 3-10 nm were measured for luciferases carrying Val106 with the spectrum of the double mutant C106V/A75G now red shifted to that of Photobacterium phosphoreum luciferase, which also has Val and Gly at positions 106 and 75, respectively. These results strengthen the validity of the binding geometry of the modeled flavin with the re-face of the pyrimidine end of the isoalloxazine ring next to Cys106 and implicate the Ala74-Ala75 cis-peptide as a key component in the bioluminescence reaction.

Cloning, sequencing, and functional studies of the rpoS gene from Vibrio harveyi.

The Vibrio harveyi rpoS gene which encodes an alternative sigma factor (sigma(s) or sigma(38)), has been cloned and characterized. The predicted protein sequence is closely related to RpoS proteins in other bacteria with up to 86% sequence identity. A rpoS null mutant of V. harveyi was constructed and the phenotype studied. Comparison of the properties of the V. harveyi wild type and rpoS deletion mutant showed that rpoS affected the ability of the cells to survive only under specific types of environmental stresses. The rpoS null mutant had a lower survival rate compared to the wild type parental strain at high concentrations of ethanol and in the stationary phase. In contrast to other bacteria, deletion of rpoS in V. harveyi did not affect the resistance of the cells to high osmolarity or hydrogen peroxide, suggesting the existence of alternative systems in V. harveyi responsible for resistance to these stresses. RpoS appears not to be involved in the control of luminescence in V. harveyi even though it is implicated in regulation of other acyl-homoserine dependent quorum sensing systems.

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.