Bright Mutants ofVibrio fischeriES114 Reveal Conditions and Regulators That Control Bioluminescence and Expression of theluxOperon

Abstract

Vibrio fischeri ES114, an isolate from the Euprymna scolopes light organ, produces little bioluminescence in culture but is ϳ1,000-fold brighter when colonizing the host. Cell-density-dependent regulation alone cannot explain this phenomenon, because cells within colonies on solid medium are much dimmer than symbiotic cells despite their similar cell densities. To better understand this low luminescence in culture, we screened ϳ20,000 mini-Tn5 mutants of ES114 for increased luminescence and identified 28 independent "luminescence-up" mutants with insertions in 14 loci. Mutations affecting the Pst phosphate uptake system led to the discovery that luminescence is upregulated under low-phosphate conditions by PhoB, and we also found that ainS, which encodes an autoinducer synthase, mediates repression of luminescence during growth on plates. Other novel luminescence-up mutants had insertions in acnB, topA, tfoY, phoQ, guaB, and two specific tRNA genes. Two loci, hns and lonA, were previously described as repressors of bioluminescence in transgenic Escherichia coli carrying the light-generating lux genes, and mutations in arcA and arcB were consistent with our report that Arc represses lux. Our results reveal a complex regulatory web governing luminescence and show how certain environmental conditions are integrated into regulation of the pheromone-dependent lux system.Vibrio fischeri is a valuable model for examining bioluminescence, pheromone signaling, and symbiotic bacteria-animal interactions. Studies of V. fischeri's mutualistic interactions have gained momentum since the discovery that this bacterium's light organ symbiosis with the Hawaiian bobtail squid, Euprymna scolopes, can be reconstituted in the laboratory (54,70,83). Moreover, the bioluminescence induced by V. fischeri in the host light organ is a pheromone-mediated behavior, making this an attractive system for examining environmental influences on bacterial pheromone signaling in a natural infection. Largely because of interest in this symbiosis, strain ES114, which was isolated from the E. scolopes light organ, has become the experimental strain of choice for many studies of V. fischeri.The genetic basis of bioluminescence in V. fischeri ES114 is fundamentally similar to that of other characterized V. fischeri strains (31, 32). The lux genes responsible for bioluminescence, luxABCDE and -G, are found together with the regulatory genes luxR and luxI and are arranged with luxR divergently transcribed from the luxICDABEG operon, as shown in Fig. 1 (24, 25, 56). Light is generated when luciferase, comprised of LuxA and LuxB, binds to FMNH 2 , O 2 , and an aliphatic aldehyde, and then converts these substrates to FMN, water, and an aliphatic acid (35, 76). LuxC, LuxD, LuxE, and LuxG (re)generate luciferase's aldehyde and FMNH 2 substrates (12, 64).The remaining genes in this cluster, luxI and luxR, underlie a pheromone-mediated regulatory mechanism often referred to as quorum sensing. LuxI generates the membrane-permeable autoinducer pheromone N-3-oxoh...

Regulation of Bioluminescence in Photobacterium leiognathi Strain KNH6

“…To perform an unbiased search for gene products involved in regulation of luminescence in KNH6, a transposon-based mutagenesis screen was used. Approximately 12,000 mutant colonies were screened on agar plates for reduced luminescence, a number that theoretically falls short of a comprehensive screen of nonessential genes (26,27). However, multiple independent mutations were identified in several loci (Table 2), indicating diminishing returns for further screening efforts.…”

Monitoring of Vibrio harveyi quorum sensing activity in real time during infection of brine shrimp larvae

“…This also suggests an integration of a host cue into the quorum sensing system (either at the level of the small regulatory RNAs or at the level of luxR mRNA). Integration of a host cue into the QS system has also been documented before for Pseudomonas aeruginosa quinolone signalling during growth in sputum of cystic fibrosis patients (Brown et al, 2008) and in Vibrio fischeri during symbiosis with the bobtail squid, where low phosphate has been identified as one of the host cues (Lyell et al, 2010).…”

Substrate Specificity and Function of the Pheromone Receptor AinR in Vibrio fischeri ES114

“…The litR::ermR mutant JB18 is phenotypically indistinguishable from the litR::ermR mutant JB19 (32) and was constructed in the same way except that the ermR cassette was in the opposite orientation. To construct the ⌬ainSR luxI mutant JHK001, plasmid pVCW2A6 (17) bearing luxI with the frame-shifting 4-bp insertion allele originally on pHV200I Ϫ (37) was exchanged into the ⌬ainSR strain NL55 (38). The ⌬ainS allele in pNL62 was exchanged into VCW2G7 (luxI) to generate strain NL63.…”