SciCombinator

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Concept: Bioluminescence

198

We report the discovery of light organs (photophores) adjacent to the dorsal defensive spines of a small deep-sea lanternshark (Etmopterus spinax). Using a visual modeling based on in vivo luminescence recordings we show that this unusual light display would be detectable by the shark’s potential predators from several meters away. We also demonstrate that the luminescence from the spine-associated photophores (SAPs) can be seen through the mineralized spines, which are partially translucent. These results suggest that the SAPs function, either by mimicking the spines' shape or by shining through them, as a unique visual deterrent for predators. This conspicuous dorsal warning display is a surprising complement to the ventral luminous camouflage (counterillumination) of the shark.

Concepts: In vivo, Bioluminescence, Deep sea fish, Fish anatomy, Etmopterus, Velvet belly lantern shark, Etmopteridae, Display

185

Beetle luciferases are thought to have evolved from fatty acyl-CoA synthetases present in all insects. Both classes of enzymes activate fatty acids with ATP to form acyl-adenylate intermediates, but only luciferases can activate and oxidize d-luciferin to emit light. Here we show that the Drosophila fatty acyl-CoA synthetase CG6178, which cannot use d-luciferin as a substrate, is able to catalyze light emission from the synthetic luciferin analog CycLuc2. Bioluminescence can be detected from the purified protein, live Drosophila Schneider 2 cells, and from mammalian cells transfected with CG6178. Thus, the nonluminescent fruit fly possesses an inherent capacity for bioluminescence that is only revealed upon treatment with a xenobiotic molecule. This result expands the scope of bioluminescence and demonstrates that the introduction of a new substrate can unmask latent enzymatic activity that differs significantly from an enzyme’s normal function without requiring mutation.

Concepts: Protein, Metabolism, Adenosine triphosphate, Enzyme, Insect, Catalysis, Bioluminescence, Luciferase

183

Bioluminescence methodologies have been extraordinarily useful due to their high sensitivity, broad dynamic range, and operational simplicity. These capabilities have been realized largely through incremental adaptations of native enzymes and substrates, originating from luminous organisms of diverse evolutionary lineages. We engineered both an enzyme and substrate in combination to create a novel bioluminescence system capable of more efficient light emission with superior biochemical and physical characteristics. Using a small luciferase subunit (19 kDa) from the deep sea shrimp Oplophorus gracilirostris, we have improved luminescence expression in mammalian cells ∼2.5 million-fold by merging optimization of protein structure with development of a novel imidazopyrazinone substrate (furimazine). The new luciferase, NanoLuc, produces glow-type luminescence (signal half-life >2 h) with a specific activity ∼150-fold greater than that of either firefly (Photinus pyralis) or Renilla luciferases similarly configured for glow-type assays. In mammalian cells, NanoLuc shows no evidence of post-translational modifications or subcellular partitioning. The enzyme exhibits high physical stability, retaining activity with incubation up to 55 °C or in culture medium for >15 h at 37 °C. As a genetic reporter, NanoLuc may be configured for high sensitivity or for response dynamics by appending a degradation sequence to reduce intracellular accumulation. Appending a signal sequence allows NanoLuc to be exported to the culture medium, where reporter expression can be measured without cell lysis. Fusion onto other proteins allows luminescent assays of their metabolism or localization within cells. Reporter quantitation is achievable even at very low expression levels to facilitate more reliable coupling with endogenous cellular processes.

Concepts: Protein, Protein structure, Bacteria, Enzyme, Cell biology, Firefly, Bioluminescence, Luciferase

174

Quorum sensing (QS) is a bacterial cell-cell communication process that relies on the production and detection of extracellular signal molecules called autoinducers. QS allows bacteria to perform collective activities. Vibrio cholerae, a pathogen that causes an acute disease, uses QS to repress virulence factor production and biofilm formation. Thus, molecules that activate QS in V. cholerae have the potential to control pathogenicity in this globally important bacterium. Using a whole-cell high-throughput screen, we identified eleven molecules that activate V. cholerae QS: eight molecules are receptor agonists and three molecules are antagonists of LuxO, the central NtrC-type response regulator that controls the global V. cholerae QS cascade. The LuxO inhibitors act by an uncompetitive mechanism by binding to the pre-formed LuxO-ATP complex to inhibit ATP hydrolysis. Genetic analyses suggest that the inhibitors bind in close proximity to the Walker B motif. The inhibitors display broad-spectrum capability in activation of QS in Vibrio species that employ LuxO. To the best of our knowledge, these are the first molecules identified that inhibit the ATPase activity of a NtrC-type response regulator. Our discovery supports the idea that exploiting pro-QS molecules is a promising strategy for the development of novel anti-infectives.

Concepts: Bacteria, Microbiology, Receptor antagonist, Biofilm, Vibrio cholerae, Bioluminescence, Vibrio

168

Quorum sensing is a process of chemical communication that bacteria use to monitor cell density and coordinate cooperative behaviors. Quorum sensing relies on extracellular signal molecules and cognate receptor pairs. While a single quorum-sensing system is sufficient to probe cell density, bacteria frequently use multiple quorum-sensing systems to regulate the same cooperative behaviors. The potential benefits of these redundant network structures are not clear. Here, we combine modeling and experimental analyses of the Bacillus subtilis and Vibrio harveyi quorum-sensing networks to show that accumulation of multiple quorum-sensing systems may be driven by a facultative cheating mechanism. We demonstrate that a strain that has acquired an additional quorum-sensing system can exploit its ancestor that possesses one fewer system, but nonetheless, resume full cooperation with its kin when it is fixed in the population. We identify the molecular network design criteria required for this advantage. Our results suggest that increased complexity in bacterial social signaling circuits can evolve without providing an adaptive advantage in a clonal population.

Concepts: Bacteria, Microbiology, Pseudomonas aeruginosa, Biofilm, Bacillus, Quorum sensing, Bioluminescence, Vibrio harveyi

152

Fireflies have drawn considerable attention for thousands of years due to their highly efficient bioluminescence, which is important for fundamental research and photonic applications. However, there are few reports on the reflector layer (RL) of firefly lantern, which contributes to the bright luminescence. Here we presented the detailed microstructure of the RL consisting of random hollow granules, which had high reflectance in the range from 450 nm to 800 nm. Inspired by the firefly lantern, artificial films with high reflectance in the visible region were fabricated using hollow silica microparticles mimicking the structure of the RL. Additionally, the bioinspired structures provided an efficient RL for the chemiluminescence system and could substantially enhance the initial chemiluminescence intensity. The work not only provides new insight into the bright bioluminescence of fireflies, but also is importance for the design of photonic materials for theranostics, detection, and imaging.

Concepts: Optics, Light, Structure, Luminescence, Firefly, Bioluminescence, Phengodidae, Reflector

137

The sensitivity of bioluminescence imaging in animals is primarily dependent on the amount of photons emitted by the luciferase enzyme at wavelengths greater than 620 nm where tissue penetration is high. This area of work has been dominated by firefly luciferase and its substrate, D-luciferin, due to the system’s peak emission (~ 600 nm), high signal to noise ratio, and generally favorable biodistribution of D-luciferin in mice. Here we report on the development of a codon optimized mutant of click beetle red luciferase that produces substantially more light output than firefly luciferase when the two enzymes are compared in transplanted cells within the skin of black fur mice or in deep brain. The mutant enzyme utilizes two new naphthyl-luciferin substrates to produce near infrared emission (730 nm and 743 nm). The stable luminescence signal and near infrared emission enable unprecedented sensitivity and accuracy for performing deep tissue multispectral tomography in mice.

Concepts: Enzyme, Enzyme substrate, Light, Electromagnetic radiation, Firefly, Signal-to-noise ratio, Bioluminescence, Luciferase

50

Gelatinous organisms apparently play a central role in deep pelagic ecosystems, but lack of observational methodologies has restricted information on their behaviour. We made acoustic records of diel migrating jellyfish Periphylla periphylla forming small, ephemeral groups at the upper fringe of an acoustic scattering layer consisting of krill. Groups of P. periphylla were also documented photographically using a remotely operated vehicle (ROV). Although the adaptive value of group formation remains speculative, we clearly demonstrate the ability of these jellyfishes to locate and team up with each other.

Concepts: Psychology, Animal, Sociology, Jellyfish, Bioluminescence, Pelagic zone, Remotely operated underwater vehicle

42

Bioluminescence is a fascinating phenomenon occurring in numerous animal taxa in the ocean. The reef dwelling splitfin flashlight fish (Anomalops katoptron) can be found in large schools during moonless nights in the shallow water of coral reefs and in the open surrounding water. Anomalops katoptron produce striking blink patterns with symbiotic bacteria in their sub-ocular light organs. We examined the blink frequency in A. katoptron under various laboratory conditions. During the night A. katoptron swims in schools roughly parallel to their conspecifics and display high blink frequencies of approximately 90 blinks/minute with equal on and off times. However, when planktonic prey was detected in the experimental tank, the open time increased compared to open times in the absence of prey and the frequency decreased to 20% compared to blink frequency at night in the absence of planktonic prey. During the day when the school is in a cave in the reef tank the blink frequency decreases to approximately 9 blinks/minute with increasing off-times of the light organ. Surprisingly the non-luminescent A. katoptron with non-functional light organs displayed the same blink frequencies and light organ open/closed times during the night and day as their luminescent conspecifics. In the presence of plankton non-luminescent specimens showed no change in the blink frequency and open/closed times compared to luminescent A. katoptron. Our experiments performed in a coral reef tank show that A. katoptron use bioluminescent illumination to detect planktonic prey and that the blink frequency of A. katoptron light organs follow an exogenous control by the ambient light.

Concepts: Algae, Symbiosis, Coral, Coral reef, Wavelength, Ocean, Bioluminescence, Deep sea fish

37

Vampire squid (Vampyroteuthis infernalis) are considered phylogenetic relics with cephalopod features of both octopods and squids. They lack feeding tentacles, but in addition to their eight arms, they have two retractile filaments, the exact functions of which have puzzled scientists for years. We present the results of investigations on the feeding ecology and behaviour of Vampyroteuthis, which include extensive in situ, deep-sea video recordings from MBARI’s remotely operated vehicles (ROVs), laboratory feeding experiments, diet studies and morphological examinations of the retractile filaments, the arm suckers and cirri. Vampire squid were found to feed on detrital matter of various sizes, from small particles to larger marine aggregates. Ingested items included the remains of gelatinous zooplankton, discarded larvacean houses, crustacean remains, diatoms and faecal pellets. Both ROV observations and laboratory experiments led to the conclusion that vampire squid use their retractile filaments for the capture of food, supporting the hypothesis that the filaments are homologous to cephalopod arms. Vampyroteuthis' feeding behaviour is unlike any other cephalopod, and reveals a unique adaptation that allows these animals to spend most of their life at depths where oxygen concentrations are very low, but where predators are few and typical cephalopod food is scarce.

Concepts: Bioluminescence, Cuttlefish, SQUID, Octopodiformes, Vampire squid, Cephalopod, Cephalopods, Muscular hydrostat