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Significant advances have been made in the characterization of luciferases and other lux-specific proteins as well as the lux genes from a number of different species of marine and terrestrial luminescent bacteria. A common lux gene organization (luxCDAB..E) modulated by the presence of specific genes involved in regulation and flavin binding and metabolism (luxF-I,L,R,Y) has been found with the luciferase genes (luxAB) flanked by the genes involved in synthesis of its fatty aldehyde substrate (luxCDE). For many species, light intensity per cell is highly dependent on cellular growth resulting in a spectacular autoinduction of luminescence at high cell density. Consequently, the bacterial lux system is of particular interest as it can serve as an excellent model for more general signal transduction systems involved in developmental processes, intercellular communication, and even symbioses. Identification of the lux autoinducers and regulatory proteins of Vibrio harveyi and Vibrio fischeri has provided the biochemical and genetic basis for dissection of the luminescent system. Isolation of the lux genes and the ability to transfer these genes into prokaryotic and eukaryotic organisms have greatly expanded the scope and potential uses of bacterial bioluminescence as a safe, rapid, and sensitive sensor for a wide variety of compounds and metabolic processes.

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...

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The crystal structure of a myristoyl acyl carrier protein specific thioesterase (C14ACP-TE) from a bioluminescent bacterium, Vibrio harveyi, was solved by multiple isomorphous replacement methods and refined to an R factor of 22% at 2.1-A resolution. This is the first elucidation of a three-dimensional structure of a thioesterase. The overall tertiary architecture of the enzyme resembles closely the consensus fold of the rapidly expanding superfamily of alpha/beta hydrolases, although there is no detectable homology with any of its members at the amino acid sequence level. Particularly striking similarity exists between the C14ACP-TE structure and that of haloalkane dehalogenase from Xanthobacter autotrophicus. Contrary to the conclusions of earlier studies [Ferri, S. R., & Meighen, E. A. (1991) J. Biol. Chem. 266, 12852-12857] which implicated Ser77 in catalysis, the crystal structure of C14ACP-TE reveals a lipase-like catalytic triad made up of Ser114, His241, and Asp211. Surprisingly, the gamma-turn with Ser114 in a strained secondary conformation (phi = 53 degrees, psi = -127 degrees), characteristic of the so-called nucleophilic elbow, does not conform to the frequently invoked lipase/esterase consensus sequence (Gly-X-Ser-X-Gly), as the positions of both glycines are occupied by larger amino acids. Site-directed mutagenesis and radioactive labeling support the catalytic function of Ser114. Crystallographic analysis of the Ser77-->Gly mutant at 2.5-A resolution revealed no structural changes; in both cases the loop containing the residue in position 77 is disordered.(ABSTRACT TRUNCATED AT 250 WORDS)

The cloning and expression of the lux genes from different luminescent bacteria including marine and terrestrial species have led to significant advances in our knowledge of the molecular biology of bacterial bioluminescence. All lux operons have a common gene organization of luxCDAB(F)E, with luxAB coding for luciferase and luxCDE coding for the fatty acid reductase complex responsible for synthesizing fatty aldehydes for the luminescence reaction, whereas significant differences exist in their sequences and properties as well as in the presence of other lux genes (I, R, F, G, and H). Recognition of the regulatory genes as well as diffusible metabolites that control the growth-dependent induction of luminescence (autoinducers) in some species has advanced our understanding of this unique regulatory mechanism in which the autoinducers appear to serve as sensors of the chemical or nutritional environment. The lux genes have now been transferred into a variety of different organisms to generate new luminescent species. Naturally dark bacteria containing the luxCDABE and luxAB genes, respectively, are luminescent or emit light on addition of aldehyde. Fusion of the luxAB genes has also allowed the expression of luciferase under a single promoter in eukaryotic systems. The ability to express the lux genes in a variety of prokaryotic and eukaryotic organisms and the ease and sensitivity of the luminescence assay demonstrate the considerable potential of the widespread application of the lux genes as reporters of gene expression and metabolic function.