Molecular Microbiology (1992) 6(4), 443-449
MicroReview Evolutionary origins of bacterial bioluminescence Dennis J. O'Kane^ and Douglas C. Prasher'*
Bioluminescence
'Department of Biochemistry, University of Georgia, Athens, Georgia 30602, USA. ^Department of Biology. Woods Hole Oceanographic Institute. Woods Hole, Massachusetts 02543, USA,
Bacterial bioluminescence involves the enzymatic oxidation of a long-chain aliphatic aldehyde and reduced flavin mononucleotide (FMNH2) by luciferase. The enzymatic mechanism is believed to be similar to cyclohexanone oxygenase (see McCapra, 1990). With luciferase, the excess free energy from this oxidation is liberated as light. In several species of bacteria, the excess free energy of the reaction is transferred to antennae proteins which subsequently emit with a wavelength distribution distinct from luciferase. Bacterial bioluminescenoe has been reviewed numerous times (Nealson and Hastings, 1979; Zeigler and Baldwin, 1981; Hastings, 1983; Lee, 1985; Meighen, 1988; 1991; Lee e/a/., 1991 a) and no attempt is made herein to review the field comprehensively. Several genes are involved in bacterial bioluminescence. With the exceptions of an oxidoreductase that reduces flavin mononucleotide (FMN), and an antenna protein described below, the genes are localized in lux operons (Fig. 1). The lux genes are contained in two operons, termed regulons, in Vibrio fischeri (Baldwin et ai, 1989). At present the structural organization of the Photobacterium operons is less completely established. The right-hand operon encodes six to nine structural genes, depending on the species, and is transcribed as a polycistronic mRNA (Miyamoto et ai, 1985). Three of these genes {luxC, luxD, and luxE] encode proteins involved in synthesis of aldehyde, while luxA and luxB, respectively, encode the a- and p-subunits of luciferase. In Photobacterium phosphoreum and Photobacterium leiognathi. luxE (fatty acid synthetase) is separated from luxB by another gene which has been variously designated /uxF(Mancini etai. 1988; 1989; Soly etai, 1988), /uxW(Baldwin etai. 1989), or luxG (Illarinov et ai, 1990). This gene encodes an unusual flavoprotein, variously referred to as non-fluorescent flavoprotein (NFP) (Visser et ai. 1983; O'Kane 3t ai, 1987), FP390 (Kasai etai, 1987), and green flavoprotein (Raibekas, 1991). This gene is absent from Vibrio species (Mancini et ai, 1989). A left-hand operon is present in V. fischeri and contains the luxR gene which encodes a regulatory protein sensitive to an autoinducer synthesized by the luxl gene product. The functions of the luxG and luxH gene products have not been described (Swartzman etai, 1990; Meighen, 1991). Two additional structural genes of interest encode
Summary In bacteria, most genes required for the bioluminescence phenotype are contained in lux operons. Sequence alignments of several lux gene products show the existence of at least two groups of paraiogous products. The a- and p-subunits of bacterial luciferase and the non-fluorescent flavoprotein are paralogous, and two antennae proteins (lumazine protein and yeiiow fluorescence protein) are paralogous with riboflavin synthetase. Modeis describing the evolution of these paralogous proteins are suggested, as well as a postulate for the identity of the gene encoding a protobioluminescent luciferase. Introduction A variety of evolutionariiy distinct iuciferases cataiyse biolurTiinescence, the iight emission that accompanies the oxidation of organic compounds (luciferins). Seliger (1975) has postuiated tinat bioiuminescence evolved foliowing the evolution of visuai photo receptors, in this model, protobioluminescent structural genes encode enzymes capable of oxidizing luciferins and emitting light with a low quantum efficiency. Providing that this bioluminescence capacity served a function which increased the fitness of the organism, the efficiency of light emission could be increased by natural selection following mutational events. Seliger (1987) postulated that the protobioluminescent progenitor of bacterial luciferase was a flavin-dependent, aldehyde oxidizing enzyme which emitted light with a low quantum efficiency, in this review, we extend Seliger's hypotheses by describing evolutionary scenarios, incorporating gene duplications and deletion events as common themes, to explain the origins of several structural genes which encode extant proteins responsible for bacterial bioluminescence. Received 1 October, 1991;revisedandaccepted5Nov8mber, 1991. *For correspondence. Tel. (508) 457 2000; Fax (508) 457 2195.
444
D. J. O'KaneandD. C. Prasher
p. phosphoreum
Fig. 1. Organization of the /uxoperons. Arrowheads indicate the directions of transcription.
antennae proteins that modify the colour of the bioluminescence emission (Baldwin etai., 1990; Prasher et ai., 1990). First, /uxV encodes the yellow fluorescence protein (YFP) in V. fischeri{Ba\d\N\u etal., 1990); its physical location within the genome has not been reported. Second, luxL, which encodes the other antenna protein, named lumazine protein (LumP), is located upstream and transcribed in the opposite direction from luxC in P. phosphoreum (Prasher ef al., 1990). This review focuses on the evolution of tuxA. luxB, luxF, luxL, and luxY. Sufficient data are not available to hypothesize about the origins of luxC, luxD, luxE, tuxG, and tuxi-l.
Bacterial lucrferase Available information supports the hypothesis that progenitor luciferase was encoded by a single gene, as opposed to two different genes like those encoding extant luciferase. The homology of the two subunits in all extant tuciferases examined has been demonstrated (Baldwin et ai,, 1979; Xi etai, 1991). Alignment of the a- and p-subunits shows the likely occurrence of a deletion event during the evolution of the |i-subunit (Fig. 2). A model consistent with these results suggests that the p-subunit evolved from the a-subunit following gene duplication (Murata and Lee, 1987; Xi etai., 1991; Baldwin eta!., 1979; Meighen, 1991). Even though the subunits are homologous, they have different properties. Different mechanistic and kinetic properties have been attributed to the two subunits (Meighen and Bartlet, 1980). as well as different susceptibility to chemical modification (Meighen et al., 1971; see Zeigler and Baldwin, 1981; Meighen, 1988). The a-subunit has a molecular size of 40000-45000 while the psubunit has a molecular size of 35 0 0 0 ^ 0 000 depending upon the species of bacterium (Meighen, 1988). Numerous attempts have been made to assign the various enzymatic functions required for bioluminescence emission to one or the other subunit of luciferase. For example, a single catalytic flavin-binding site is proposed from kinetic
data (Becvar and Hastings, 1975). On the basis of subunit recombination studies (Meighen etal., 1971; Meighen and Bartlet, 1980) and protein sequence alignment studies (Murata and Lee, 1987), this binding site has been suggested to reside on the a-subunit. However, Vervocrt etal. (1986) demonstrated two [^^C]-NMR (nuclear magnetic resonance) signals from isotopically labelled FMN bound to luciferase from P. phosphoreum and from Vibrio harveyi. suggesting that there might be two distinct flavinbinding sites. Could an ancestral form, consisting solely of the a-subunit, be the protobioluminescent progenitor postulated by Seliger (1987)? Waddle and Baldwin (1991) found weak bioluminescence activity (c. 10^-fold less than the wildtype ap-luciferase) in partially purified extracts of Escherichia co//separately containing recombinant forms of either the a-subunit or the p-subunit of luciferase. These data suggest that both luciferase subunits have flavin-binding sites and can interact with aldehyde. Thus, we propose that the protobioluminescent progenitor of luciferase was a protein very similar to the a-subunit of luciferase. Support for this proposal could be obtained from studies with non-bioluminescent bacteria which have iux genes (Palmer and Colwell, 1991) or proteins that cross-react antigenically with luciferase antibody (Kou and Makemson, 1988). What new property might the p-subunit have contributed to the bioluminescent phenotype that was not demonstrated solely by the a-subunit of luciferase? One property seems to be a promotion of temperature stability. Escher etai. (1989) proposed that V, rtarvey/luciferase evolved by gene duplication which resulted originally in the formation of a fused gene {iuxAB). A fusion protein produced following site-directed mutagenesis was more temperature-sensitive than luciferase (Escher ef al., 1989; 1991). Such a fusion protein would be problematic for V. harveyi, which is prevalent in coastal and estuarine waters that can reach 31 °C (O'Brien and Sizemore, 1979). Escher ef ai. (1989) postulated that increased thermostability was gained by mutations, resulting in the formation of the separate iuxA and iuxB genes. More recently, Escher etai. (1992) have demonstrated that the amino acid sequence of the p-subunit strongly affects the thermostability of V. harveyi luciferase.
Non-fluorescent flavoprotein luxE. which encodes NFP, is a second example of a gene duplication event in the /uxoperons (Baldwin etai., 1989; Mancini etal., 1988; 1989; Soly etai., 1988; Illarinov ef al,. 1990). Presumably the luxFgene has a more recent evolutionary origin than the other lux genes since it is found only in Photobacterium. NFP is a flavoprotein that contains a non-covalently bound, unusual flavin (Visser ef
Bacterial bioluminescence
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MicroReview Evolutionary origins of bacterial bioluminescence Dennis J. O'Kane^ and Douglas C. Prasher'*
Bioluminescence
'Department of Biochemistry, University of Georgia, Athens, Georgia 30602, USA. ^Department of Biology. Woods Hole Oceanographic Institute. Woods Hole, Massachusetts 02543, USA,
Bacterial bioluminescence involves the enzymatic oxidation of a long-chain aliphatic aldehyde and reduced flavin mononucleotide (FMNH2) by luciferase. The enzymatic mechanism is believed to be similar to cyclohexanone oxygenase (see McCapra, 1990). With luciferase, the excess free energy from this oxidation is liberated as light. In several species of bacteria, the excess free energy of the reaction is transferred to antennae proteins which subsequently emit with a wavelength distribution distinct from luciferase. Bacterial bioluminescenoe has been reviewed numerous times (Nealson and Hastings, 1979; Zeigler and Baldwin, 1981; Hastings, 1983; Lee, 1985; Meighen, 1988; 1991; Lee e/a/., 1991 a) and no attempt is made herein to review the field comprehensively. Several genes are involved in bacterial bioluminescence. With the exceptions of an oxidoreductase that reduces flavin mononucleotide (FMN), and an antenna protein described below, the genes are localized in lux operons (Fig. 1). The lux genes are contained in two operons, termed regulons, in Vibrio fischeri (Baldwin et ai, 1989). At present the structural organization of the Photobacterium operons is less completely established. The right-hand operon encodes six to nine structural genes, depending on the species, and is transcribed as a polycistronic mRNA (Miyamoto et ai, 1985). Three of these genes {luxC, luxD, and luxE] encode proteins involved in synthesis of aldehyde, while luxA and luxB, respectively, encode the a- and p-subunits of luciferase. In Photobacterium phosphoreum and Photobacterium leiognathi. luxE (fatty acid synthetase) is separated from luxB by another gene which has been variously designated /uxF(Mancini etai. 1988; 1989; Soly etai, 1988), /uxW(Baldwin etai. 1989), or luxG (Illarinov et ai, 1990). This gene encodes an unusual flavoprotein, variously referred to as non-fluorescent flavoprotein (NFP) (Visser et ai. 1983; O'Kane 3t ai, 1987), FP390 (Kasai etai, 1987), and green flavoprotein (Raibekas, 1991). This gene is absent from Vibrio species (Mancini et ai, 1989). A left-hand operon is present in V. fischeri and contains the luxR gene which encodes a regulatory protein sensitive to an autoinducer synthesized by the luxl gene product. The functions of the luxG and luxH gene products have not been described (Swartzman etai, 1990; Meighen, 1991). Two additional structural genes of interest encode
Summary In bacteria, most genes required for the bioluminescence phenotype are contained in lux operons. Sequence alignments of several lux gene products show the existence of at least two groups of paraiogous products. The a- and p-subunits of bacterial luciferase and the non-fluorescent flavoprotein are paralogous, and two antennae proteins (lumazine protein and yeiiow fluorescence protein) are paralogous with riboflavin synthetase. Modeis describing the evolution of these paralogous proteins are suggested, as well as a postulate for the identity of the gene encoding a protobioluminescent luciferase. Introduction A variety of evolutionariiy distinct iuciferases cataiyse biolurTiinescence, the iight emission that accompanies the oxidation of organic compounds (luciferins). Seliger (1975) has postuiated tinat bioiuminescence evolved foliowing the evolution of visuai photo receptors, in this model, protobioluminescent structural genes encode enzymes capable of oxidizing luciferins and emitting light with a low quantum efficiency. Providing that this bioluminescence capacity served a function which increased the fitness of the organism, the efficiency of light emission could be increased by natural selection following mutational events. Seliger (1987) postulated that the protobioluminescent progenitor of bacterial luciferase was a flavin-dependent, aldehyde oxidizing enzyme which emitted light with a low quantum efficiency, in this review, we extend Seliger's hypotheses by describing evolutionary scenarios, incorporating gene duplications and deletion events as common themes, to explain the origins of several structural genes which encode extant proteins responsible for bacterial bioluminescence. Received 1 October, 1991;revisedandaccepted5Nov8mber, 1991. *For correspondence. Tel. (508) 457 2000; Fax (508) 457 2195.
444
D. J. O'KaneandD. C. Prasher
p. phosphoreum
Fig. 1. Organization of the /uxoperons. Arrowheads indicate the directions of transcription.
antennae proteins that modify the colour of the bioluminescence emission (Baldwin etai., 1990; Prasher et ai., 1990). First, /uxV encodes the yellow fluorescence protein (YFP) in V. fischeri{Ba\d\N\u etal., 1990); its physical location within the genome has not been reported. Second, luxL, which encodes the other antenna protein, named lumazine protein (LumP), is located upstream and transcribed in the opposite direction from luxC in P. phosphoreum (Prasher ef al., 1990). This review focuses on the evolution of tuxA. luxB, luxF, luxL, and luxY. Sufficient data are not available to hypothesize about the origins of luxC, luxD, luxE, tuxG, and tuxi-l.
Bacterial lucrferase Available information supports the hypothesis that progenitor luciferase was encoded by a single gene, as opposed to two different genes like those encoding extant luciferase. The homology of the two subunits in all extant tuciferases examined has been demonstrated (Baldwin et ai,, 1979; Xi etai, 1991). Alignment of the a- and p-subunits shows the likely occurrence of a deletion event during the evolution of the |i-subunit (Fig. 2). A model consistent with these results suggests that the p-subunit evolved from the a-subunit following gene duplication (Murata and Lee, 1987; Xi etai., 1991; Baldwin eta!., 1979; Meighen, 1991). Even though the subunits are homologous, they have different properties. Different mechanistic and kinetic properties have been attributed to the two subunits (Meighen and Bartlet, 1980). as well as different susceptibility to chemical modification (Meighen et al., 1971; see Zeigler and Baldwin, 1981; Meighen, 1988). The a-subunit has a molecular size of 40000-45000 while the psubunit has a molecular size of 35 0 0 0 ^ 0 000 depending upon the species of bacterium (Meighen, 1988). Numerous attempts have been made to assign the various enzymatic functions required for bioluminescence emission to one or the other subunit of luciferase. For example, a single catalytic flavin-binding site is proposed from kinetic
data (Becvar and Hastings, 1975). On the basis of subunit recombination studies (Meighen etal., 1971; Meighen and Bartlet, 1980) and protein sequence alignment studies (Murata and Lee, 1987), this binding site has been suggested to reside on the a-subunit. However, Vervocrt etal. (1986) demonstrated two [^^C]-NMR (nuclear magnetic resonance) signals from isotopically labelled FMN bound to luciferase from P. phosphoreum and from Vibrio harveyi. suggesting that there might be two distinct flavinbinding sites. Could an ancestral form, consisting solely of the a-subunit, be the protobioluminescent progenitor postulated by Seliger (1987)? Waddle and Baldwin (1991) found weak bioluminescence activity (c. 10^-fold less than the wildtype ap-luciferase) in partially purified extracts of Escherichia co//separately containing recombinant forms of either the a-subunit or the p-subunit of luciferase. These data suggest that both luciferase subunits have flavin-binding sites and can interact with aldehyde. Thus, we propose that the protobioluminescent progenitor of luciferase was a protein very similar to the a-subunit of luciferase. Support for this proposal could be obtained from studies with non-bioluminescent bacteria which have iux genes (Palmer and Colwell, 1991) or proteins that cross-react antigenically with luciferase antibody (Kou and Makemson, 1988). What new property might the p-subunit have contributed to the bioluminescent phenotype that was not demonstrated solely by the a-subunit of luciferase? One property seems to be a promotion of temperature stability. Escher etai. (1989) proposed that V, rtarvey/luciferase evolved by gene duplication which resulted originally in the formation of a fused gene {iuxAB). A fusion protein produced following site-directed mutagenesis was more temperature-sensitive than luciferase (Escher ef al., 1989; 1991). Such a fusion protein would be problematic for V. harveyi, which is prevalent in coastal and estuarine waters that can reach 31 °C (O'Brien and Sizemore, 1979). Escher ef ai. (1989) postulated that increased thermostability was gained by mutations, resulting in the formation of the separate iuxA and iuxB genes. More recently, Escher etai. (1992) have demonstrated that the amino acid sequence of the p-subunit strongly affects the thermostability of V. harveyi luciferase.
Non-fluorescent flavoprotein luxE. which encodes NFP, is a second example of a gene duplication event in the /uxoperons (Baldwin etai., 1989; Mancini etal., 1988; 1989; Soly etai., 1988; Illarinov ef al,. 1990). Presumably the luxFgene has a more recent evolutionary origin than the other lux genes since it is found only in Photobacterium. NFP is a flavoprotein that contains a non-covalently bound, unusual flavin (Visser ef
Bacterial bioluminescence
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