Shedding Light on the Bioluminescence “Paradox” Although Luminescence Provides Host Squids with Obvious Advantages, How Does It Beneﬁt Light-Producing Bacteria?

Shedding Light on the Bioluminescence “Paradox” Although luminescence provides host squids with obvious advantages, how does it benefit light-producing bacteria? Eric V. Stabb he fascinating biochemistry, genetics, have special significance for this bacterium. Bi- and cell density-dependent regula- oluminescence offers many such puzzles, and it tion of bacterial bioluminescence is unlikely a single solution will solve them all. T provoke a challenging question. Nonetheless, it is an exciting moment in bio- What good is it to bioluminescent luminescence research, with recent advances of- bacteria? fering the promise of answering the longstand- There may be no single answer to this seem- ing question, “how can bioluminescence ingly simple question. However, two recent ad- help bacteria?” Although researchers learned vances shed new light on the problem. First, nearly a century ago that luminescence reduces studies of the symbiosis between the biolumines- oxygen and that symbiotic bacteria inhabit the cent bacterium Vibrio fischeri and the Hawaiian light organs of squids, what was unimaginable bobtail squid bring an ecologically relevant until very recently is our ability to analyze the V. niche for a bioluminescent bacterium into focus fischeri genome sequence and to combine this under the discerning lens of controlled labora- knowledge with the ability to genetically manip- tory experimentation. Second, progress in under- ulate these bacteria and observe them under standing the genetics of V. fischeri, including controlled laboratory conditions in the ecologi- genomic sequencing of a squid symbiont, is en- cally relevant environment of a natural squid abling researchers to analyze how luminescence host. integrates into the physiology of this bacterial species and to test specific hypotheses about Biochemistry and Genetics of what advantage light production confers on the V. fischeri Bioluminescence bacteria. In bacteria such as V. fischeri, light is generated Understanding how bioluminescence aids V. by an enzyme, luciferase, that contains two pro- fischeri in a squid light organ will likely not be teins, designated LuxA and LuxB (Fig. 1). the final word on how bioluminescence benefits LuxAB sequentially binds FMNH2,O2, and an bacteria. Differences among bioluminescent aliphatic aldehyde (RCHO) that are converted bacteria intimate that this ancient system con- to an aliphatic acid, FMN, and water. In turn, fers varied selective advantages. For example, they are released from the enzyme with the con- “cryptically luminescent” Vibrio pathogens, comitant production of light (Fig. 1A). Addi- such as Vibrio salmonicida, produce luciferase, tional proteins, LuxC, LuxD, and LuxE, are the enzyme responsible for bioluminescence, but responsible for (re)generating the aldehyde, little or no aldehyde substrate, and may use while another protein, LuxG, shuttles reducing Eric V. Stabb is an luciferase to form “dark reaction” hydrogen power from NAD(P)H to FMN to (re)generate Assistant Professor peroxide as a host-damaging virulence factor. FMNH2. in the Department In contrast, some strains of Vibrio logei pro- In V. fischeri, the luxC, luxD, luxE, and luxG of Microbiology at duce an accessory Y1 protein that shifts their genes flank luxA and luxB. These genes are the University of emitted light to a yellow wavelength, which may cotranscribed with luxI, while luxR is adjacent Georgia, Athens. Volume 71, Number 5, 2005 / ASM News Y 223 FIGURE 1 ulated when intracellular AI concentra- tion exceeds a threshold. Thus, light is produced at high cell densities, such as A Biochemistry and physiology during growth in a squid light organ, Substratered Substrateox but not when planktonic cells are grow- ing at low densities. O e– transport 2 NADH NAD+ H+ LuxG Bioluminescence at First Analysis + H motive force H2O Appears To Be a Drag on Cell Energy FMNH2 FMN Despite having a good understanding of ATP Products LuxAB the biochemical and genetic mechan- FMN ics of bioluminescence, researchers re- main uncertain over what its selective LuxAB LuxAB -FMNH2 advantage is to bacteria. In particular, H O O 2 the apparent costs in energy to cells 2 from bioluminescence make its exis- tence appear paradoxical. Light In addition to the sizable biosyn- O - 2 LuxAB -FMNH2 thetic cost in producing the Lux pro- LuxE + LuxC RCHO RCOOH teins, generating light consumes both biochemical reducing power and oxy- + NADP NADPH + ATP gen, seemingly competing for substrates LuxD + AMP + PPi with aerobic respiration, which recovers O2- LuxAB -FMNH2 energy through electron transport (Fig. acyl-ACP 1A). Furthermore, energy stored as ATP RCHO is consumed in regenerating the aldehyde substrate (Fig. 1A). B Genetics and quorum sensing Indications that bioluminescence luxR luxI luxC luxD luxA luxB luxE luxG hinders cultured cells date at least as far back as E. Newton Harvey’s work at Princeton University in the early 1900s. Generate light Those findings were extended by J. Woodland (Woody) Hasting at Harvard OO University and his collaborators, who O showed that some relatively darker mu- N tants outgrew their brighter parents. Sim- H LuxR O LuxI ilarly, Paul Dunlap, now at the University AI-dependent Autoinducer (AI) of Michigan, characterized bright mu- transcriptional activator tants of V. fischeri that grew more slowly than do their relatively dim parent. V. fischeri bioluminescence. (A) Biochemistry and physiology. (B) Genetics and quorum In 1980, Ken Nealson and David Karl, sensing. currently at the University of Southern California and the University of Ha- waii, respectively, also found that cells to the other lux genes but not transcribed with expend appreciable energy for luminescence. them (Fig. 1B). Together LuxI and LuxR under- However, these two researchers did not find that lie a well-characterized quorum-sensing regula- bioluminescence slows growth rates. tory circuit, in which LuxI generates an autoin- These apparently inconsistent findings are dif- ducer (AI) that interacts with LuxR to stimulate ficult to interpret. Part of the explanation for lux transcription. The expression of lux is stim- the inconsistencies is that the best technology 224 Y ASM News / Volume 71, Number 5, 2005 FIGURE 2 Squids Provide a Means for Scrutinizing Bioluminescence under Controlled Conditions A major advance for bioluminescence research came when Margaret McFall- Ngai and Edward Ruby, who now are at the University of Wisconsin, Madi- son, brought the V. fischeri-E. scolopes symbiosis into the laboratory. Al- though several other hosts of bioluminescent bacteria cannot be bred in Symbiotic bioluminescence. Light organs of E. scolopes juveniles under white light (left captivity, E. scolopes is an exception. panel) or lit from within by bioluminescent V. fischeri symbionts (right panel). It is thought In a series of ecological studies, Ruby that adult E. scolopes use this ventrally directed luminescence to obscure their and his collaborators found that V. fis- silhouette. Solid bars are ϳ100 ␮m. cheri in Hawaii are specifically adapted to E. scolopes, and they are more abun- of the day required comparisons of noniso- dant in waters inhabited by the host. genic strains, analyses of undefined pleiotro- Later, Karen Visick at Loyola University in pic mutants, or induction of luminescence by Chicago, Ill., provided evidence that bacteria AI, which also regulates non-lux genes. Re- derive an advantage from bioluminescence in cently, however, Grzegorz Wegrzyn’s group at this symbiosis. Specifically, although a luxA mu- the University of Gdansk in Poland used de- tant colonizes E. scolopes, this mutant (unlike fined strains to show that luminescence indeed its parental strain) does not persist well. One slows the growth of bacteria in cultures. Spe- possibility is that this mutant is attenuated be- cifically, he finds that a luxA mutant of V. har- cause of detrimental effects from expressing the veyi outcompetes its isogenic parent. Simi- full set of LuxCDBEG proteins in the absence of larly, we find that a luxCDABEG deletion a functional LuxA. However, we tested an in- frame luxCDABEG deletion mutant and came mutant of V. fischeri outcompetes its isogenic up with a result equivalent to Visick’s, namely wild-type parent in mixed culture when AI is poor persistence of this mutant in the squid. present. Thus, at least under some conditions, Thus, although bioluminescence slows V. fisch- light production slows bacterial growth. eri growth in culture, it enhances the bacteri- Presumably, however, luminescence confers um’s colonization of the squid light organ. How? an advantage to bacteria in some settings. For instance, bioluminescent bacteria naturally inhabit and illuminate the light-emitting organs of animals such as the Hawaiian bobtailed How Bacteria May Benefit squid, Euprymna scolopes (Fig. 2). Some biol- from Being Luminescent ogists argue that, in such associations, “what’s good for the host is good for the Researchers have proposed several hypotheses symbiont.” In this specific case, E. scolopes to explain how bioluminescence could confer apparently uses V. fischeri’s luminescence to advantages to light-producing bacteria (see ta- elude its predators, suggesting that increased ble). Several of these ideas downgrade the im- host fitness offsets the cost of bioluminescence portance of luminescence itself, describing it as to the bacteria, because the host “pays” the little more than an eye-catching distraction. bacteria back with nutrients. Rather, some researchers argue that the impor- Even so, without other

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