Occurrence of P-flavin binding protein in Vibrio fischeri and properties of the protein.

In previous studies involving Photobacterium species we proposed that (i) P-flavin is the product of luciferase, (ii) the physiological function of the lux operon is not to produce light but to produce FP(390) (luxF protein), including its prosthetic group, P-flavin, and (iii) FP(390) reactivates oxidatively inactivated cobalamin-dependent methionine synthase similar to flavodoxin but at relatively high ionic strength. It seems difficult to extend this idea to all luminous bacteria because the luxF gene is not present in the lux operon in Vibrio or Xenorhabdus. But we predicted that a luciferase fragment which binds P-flavin should function like FP(390) in these species. In this study, we isolated P-flavin binding protein from Vibrio fischeri ATCC 7744. The obtained protein was a modified luciferase as expected, in which the beta-subunit was intact but about 25 amino acid residues at the C-terminus of the alpha-subunit were deleted and the prosthetic group was the fully reduced P-flavin. These results strongly support that the physiological function of the lux operon is as described above even in luminous bacteria other than Photobacterium species. We propose that chromophore B reported by Tu and Hastings [Tu, S.-C. and Hastings, J.W. (1975) Biochemistry 14, 1975-1980] is the reduced P-flavin.     

Mutational analysis of the Vibrio fischeri LuxI polypeptide: critical regions of an autoinducer synthase.

Synthesis of the Vibrio fischeri autoinducer, a signal involved in the cell density-dependent activation of bioluminescence, is directed by the luxI gene product. The LuxI protein catalyzes the synthesis of N-acyl-homoserine lactones from S-adenosylmethionine and acylated-acyl carrier protein. We have gained an appreciation of the LuxI regions and amino acid residues involved in autoinducer synthesis by isolating and analyzing mutations generated by random and site-specific mutagenesis of luxI. By random mutagenesis we isolated 13 different single amino acid substitutions in the LuxI polypeptide. Eleven of these substitutions resulted in no detectable autoinducer synthase activity, while the remaining two amino acid substitutions resulted in reduced but detectable activity. The substitutions that resulted in no detectable autoinducer synthase activity mapped to two small regions of LuxI. In Escherichia coli, wild-type luxI showed dominance over all of the mutations. Because autoinducer synthesis has been proposed to involve formation of a covalent bond between an acyl group and an active-site cysteine, we constructed site-directed mutations that altered each of the three cysteine residues in LuxI. All of the cysteine mutants retained substantial activity as an autoinducer synthase in E. coli. Based on the analysis of random mutations we propose a model in which there are two critical regions of LuxI, at least one of which is an intimate part of an active site, and based on the analysis of site-directed mutations we conclude that an active-site cysteine is not essential for autoinducer synthase activity.     

Critical regions of the Vibrio fischeri luxR protein defined by mutational analysis.

Expression of Vibrio fischeri luminescence genes requires an inducer, termed autoinducer, and a positive regulatory element, the luxR gene product. A plasmid containing a tac promoter-controlled luxR was mutagenized in vitro with hydroxylamine, and luxR mutant plasmids were identified by their inability to complement a luxR deletion mutation in trans. Sixteen luxR mutant plasmids were obtained, ten of which encoded full-length but inactive luxR gene products as demonstrated by a Western immunoblot analysis. The effects of 1 of the 10 mutations could be overcome by the addition of autoinducer at a high concentration. The mutations in each of the 10 mutant plasmids that directed the synthesis of an inactive LuxR protein were identified by DNA sequencing. Of the 10 proteins encoded by the mutant luxR plasmids, 9 differed from the normally active LuxR in only a single amino acid residue. The amino acid residue substitutions in the proteins encoded by the nine mutant luxR genes clustered in two regions. One region around the middle of the polypeptide encoded by luxR was hypothesized to represent an autoinducer-binding domain, and the other region towards the carboxy terminus of the gene product was hypothesized to constitute a lux operator DNA-binding domain or a lux operator DNA recognition domain.     

Expression of luciferases from Vibrio harveyi and Vibrio fischeri in filamentous cyanobacteria.

Shuttle vectors that had previously been shown to replicate both in Escherichia coli and in strains of Anabaena spp. were used to transfer the lux genes from Vibrio harveyi and Vibrio fischeri into Anabaena spp. The level of expression of luciferase in the cyanobacteria (up to 7,000 quanta cell-1 s-1) makes these genes good candidates for use as promoter probes during the differentiation of certain cells in a filament into heterocysts.     

Magnesium promotes flagellation of Vibrio fischeri

The bacterium Vibrio fischeri requires bacterial motility to initiate colonization of the Hawaiian squid Euprymna scolopes. Once colonized, however, the bacterial population becomes largely unflagellated. To understand environmental influences on V. fischeri motility, we investigated migration of this organism in tryptone-based soft agar media supplemented with different salts. We found that optimal migration required divalent cations and, in particular, Mg2+. At concentrations naturally present in seawater, Mg2+ improved migration without altering the growth rate of the cells. Transmission electron microscopy and Western blot experiments suggested that Mg2+ addition enhanced flagellation, at least in part through an effect on the steady-state levels of flagellin protein.