Gene_locus Report for: phopo-luxd

Six representatives of a luminous bacterium commonly found in association with deep, cold-dwelling marine fishes were isolated from the light organs and skin of different fish species. These bacteria were Gram-negative, catalase-positive, and weakly oxidase-positive or oxidase-negative. Morphologically, cells of these strains were coccoid or coccoid-rods, occurring singly or in pairs, and motile by means of polar flagellation. After growth on seawater-based agar medium at 22 degrees C for 18 h, colonies were small, round and white, with an intense cerulean blue luminescence. Analysis of 16S rRNA gene sequence similarity placed these bacteria in the genus Photobacterium. Phylogenetic analysis based on seven housekeeping gene sequences (16S rRNA gene, gapA, gyrB, pyrH, recA, rpoA and rpoD), seven gene sequences of the lux operon (luxC, luxD, luxA, luxB, luxF, luxE and luxG) and four gene sequences of the rib operon (ribE, ribB, ribH and ribA), resolved the six strains as members of the genus Photobacterium and as a clade distinct from other species of Photobacterium. These strains were most closely related to Photobacterium phosphoreum and Photobacterium iliopiscarium. DNA-DNA hybridization values between the designated type strain, Photobacterium kishitanii pjapo.1.1(T), and P. phosphoreum LMG 4233(T), P. iliopiscarium LMG 19543(T) and Photobacterium indicum LMG 22857(T) were 51, 43 and 19 %, respectively. In AFLP analysis, the six strains clustered together, forming a group distinct from other analysed species. The fatty acid C(17 : 0) cyclo was present in these bacteria, but not in P. phosphoreum, P. iliopiscarium or P. indicum. A combination of biochemical tests (arginine dihydrolase and lysine decarboxylase) differentiates these strains from P. phosphoreum and P. indicum. The DNA G+C content of P. kishitanii pjapo.1.1(T) is 40.2 %, and the genome size is approximately 4.2 Mbp, in the form of two circular chromosomes. These strains represent a novel species, for which the name Photobacterium kishitanii sp. nov. is proposed. The type strain, pjapo.1.1(T) (=ATCC BAA-1194(T)=LMG 23890(T)), is a luminous symbiont isolated from the light organ of the deep-water fish Physiculus japonicus.

The lux operon is an uncommon gene cluster. To find the pathway through which the operon has been transferred, we sequenced the operon and both flanking regions in four typical luminous species. In Vibrio cholerae NCIMB 41, a five-gene cluster, most genes of which were highly similar to orthologues present in Gram-positive bacteria, along with the lux operon, is inserted between VC1560 and VC1563, on chromosome 1. Because this entire five-gene cluster is present in Photorhabdus luminescens TT01, about 1.5 Mbp upstream of the operon, we deduced that the operon and the gene cluster were transferred from V. cholerae to an ancestor of Pr. luminescens. Because in both V. fischeri and Shewanella hanedai, luxR and luxI were found just upstream of the operon, we concluded that the operon was transferred from either species to the other. Because most of the genes flanking the operon were highly similar to orthologues present on chromosome 2 of vibrios, we speculated that the operon of most species is located on this chromosome. The undigested genomic DNAs of five luminous species were analysed by pulsed-field gel electrophoresis and Southern hybridization. In all the species except V. cholerae, the operons are located on chromosome 2.

The diversion of fatty acids from fatty acid biosynthesis into the luminescent system is catalyzed by a lux-specific acyltransferase that catalyzes the cleavage of fatty acyl-acyl carrier protein (ACP). Analysis of the substrate specificities for fatty acyl-ACPs of the transferases from divergent luminescent bacteria, Photobacterium phosphoreum and Vibrio harveyi, has demonstrated that myristoyl-ACP is cleaved at the highest rate. Inhibition by phenylmethanesulfonyl fluoride as well as resistance of the acylated enzyme intermediate to cleavage by hydroxylamine showed that the transferase is a serine esterase. Moreover, activity was dependent on a basic residue with a pKa of 6.3 implicating a histidine residue as part of a charge relay system found in serine esterases. The nucleotide sequence of the P. phosphoreum luxD gene coding for the transferase was determined resulting in the identification of the active site motif for serine esterases, G-X-S-X-G. Replacement of the serine residue at the center of this motif by threonine, alanine, or glycine blocked the transferase acyl-ACP cleavage activity, its ability to be acylated, and complementation of a transferase defective mutant on transconjugation with the luxD gene. The sequence and location of the serine as well as a histidine residue in the lux-specific transferases were found to be similar to those involved in the charge relay system in vertebrate thioesterases. Combined with the similar kinetic properties, these results support a common metabolic role for both enzymes in the diversion of fatty acids from the fatty acid biosynthetic pathway.

Six representatives of a luminous bacterium commonly found in association with deep, cold-dwelling marine fishes were isolated from the light organs and skin of different fish species. These bacteria were Gram-negative, catalase-positive, and weakly oxidase-positive or oxidase-negative. Morphologically, cells of these strains were coccoid or coccoid-rods, occurring singly or in pairs, and motile by means of polar flagellation. After growth on seawater-based agar medium at 22 degrees C for 18 h, colonies were small, round and white, with an intense cerulean blue luminescence. Analysis of 16S rRNA gene sequence similarity placed these bacteria in the genus Photobacterium. Phylogenetic analysis based on seven housekeeping gene sequences (16S rRNA gene, gapA, gyrB, pyrH, recA, rpoA and rpoD), seven gene sequences of the lux operon (luxC, luxD, luxA, luxB, luxF, luxE and luxG) and four gene sequences of the rib operon (ribE, ribB, ribH and ribA), resolved the six strains as members of the genus Photobacterium and as a clade distinct from other species of Photobacterium. These strains were most closely related to Photobacterium phosphoreum and Photobacterium iliopiscarium. DNA-DNA hybridization values between the designated type strain, Photobacterium kishitanii pjapo.1.1(T), and P. phosphoreum LMG 4233(T), P. iliopiscarium LMG 19543(T) and Photobacterium indicum LMG 22857(T) were 51, 43 and 19 %, respectively. In AFLP analysis, the six strains clustered together, forming a group distinct from other analysed species. The fatty acid C(17 : 0) cyclo was present in these bacteria, but not in P. phosphoreum, P. iliopiscarium or P. indicum. A combination of biochemical tests (arginine dihydrolase and lysine decarboxylase) differentiates these strains from P. phosphoreum and P. indicum. The DNA G+C content of P. kishitanii pjapo.1.1(T) is 40.2 %, and the genome size is approximately 4.2 Mbp, in the form of two circular chromosomes. These strains represent a novel species, for which the name Photobacterium kishitanii sp. nov. is proposed. The type strain, pjapo.1.1(T) (=ATCC BAA-1194(T)=LMG 23890(T)), is a luminous symbiont isolated from the light organ of the deep-water fish Physiculus japonicus.

"Photobacterium mandapamensis" (proposed name) and Photobacterium leiognathi are closely related, phenotypically similar marine bacteria that form bioluminescent symbioses with marine animals. Despite their similarity, however, these bacteria can be distinguished phylogenetically by sequence divergence of their luminescence genes, luxCDAB(F)E, by the presence (P. mandapamensis) or the absence (P. leiognathi) of luxF and, as shown here, by the sequence divergence of genes involved in the synthesis of riboflavin, ribBHA. To gain insight into the possibility that P. mandapamensis and P. leiognathi are ecologically distinct, we used these phylogenetic criteria to determine the incidence of P. mandapamensis as a bioluminescent symbiont of marine animals. Five fish species, Acropoma japonicum (Perciformes, Acropomatidae), Photopectoralis panayensis and Photopectoralis bindus (Perciformes, Leiognathidae), Siphamia versicolor (Perciformes, Apogonidae), and Gadella jordani (Gadiformes, Moridae), were found to harbor P. mandapamensis in their light organs. Specimens of A. japonicus, P. panayensis, and P. bindus harbored P. mandapamensis and P. leiognathi together as cosymbionts of the same light organ. Regardless of cosymbiosis, P. mandapamensis was the predominant symbiont of A. japonicum, and it was the apparently exclusive symbiont of S. versicolor and G. jordani. In contrast, P. leiognathi was found to be the predominant symbiont of P. panayensis and P. bindus, and it appears to be the exclusive symbiont of other leiognathid fishes and a loliginid squid. A phylogenetic test for cospeciation revealed no evidence of codivergence between P. mandapamensis and its host fishes, indicating that coevolution apparently is not the basis for this bacterium's host preferences. These results, which are the first report of bacterial cosymbiosis in fish light organs and the first demonstration that P. leiognathi is not the exclusive light organ symbiont of leiognathid fishes, demonstrate that the host species ranges of P. mandapamensis and P. leiognathi are substantially distinct. The host range difference underscores possible differences in the environmental distributions and physiologies of these two bacterial species.

The lux operon is an uncommon gene cluster. To find the pathway through which the operon has been transferred, we sequenced the operon and both flanking regions in four typical luminous species. In Vibrio cholerae NCIMB 41, a five-gene cluster, most genes of which were highly similar to orthologues present in Gram-positive bacteria, along with the lux operon, is inserted between VC1560 and VC1563, on chromosome 1. Because this entire five-gene cluster is present in Photorhabdus luminescens TT01, about 1.5 Mbp upstream of the operon, we deduced that the operon and the gene cluster were transferred from V. cholerae to an ancestor of Pr. luminescens. Because in both V. fischeri and Shewanella hanedai, luxR and luxI were found just upstream of the operon, we concluded that the operon was transferred from either species to the other. Because most of the genes flanking the operon were highly similar to orthologues present on chromosome 2 of vibrios, we speculated that the operon of most species is located on this chromosome. The undigested genomic DNAs of five luminous species were analysed by pulsed-field gel electrophoresis and Southern hybridization. In all the species except V. cholerae, the operons are located on chromosome 2.

Substantial ambiguity exists regarding the phylogenetic status of facultatively psychrophilic luminous bacteria identified as Photobacterium phosphoreum, a species thought to be widely distributed in the world's oceans and believed to be the specific bioluminescent light-organ symbiont of several deep-sea fishes. Members of the P. phosphoreum species group include luminous and non-luminous strains identified phenotypically from a variety of different habitats as well as phylogenetically defined lineages that appear to be evolutionarily distinct. To resolve this ambiguity and to begin developing a meaningful knowledge of the geographic distributions, habitats and symbiotic relationships of bacteria in the P. phosphoreum species group, we carried out a multilocus, fine-scale phylogenetic analysis based on sequences of the 16S rRNA, gyrB and luxABFE genes of many newly isolated luminous strains from symbiotic and saprophytic habitats, together with previously isolated luminous and non-luminous strains identified as P. phosphoreum from these and other habitats. Parsimony analysis unambiguously resolved three evolutionarily distinct clades, phosphoreum, iliopiscarium and kishitanii. The tight phylogenetic clustering within these clades and the distinct separation between them indicates they are different species, P. phosphoreum, Photobacterium iliopiscarium and the newly recognized 'Photobacterium kishitanii'. Previously reported non-luminous strains, which had been identified phenotypically as P. phosphoreum, resolved unambiguously as P. iliopiscarium, and all examined deep-sea fishes (specimens of families Chlorophthalmidae, Macrouridae, Moridae, Trachichthyidae and Acropomatidae) were found to harbour 'P. kishitanii', not P. phosphoreum, in their light organs. This resolution revealed also that 'P. kishitanii' is cosmopolitan in its geographic distribution. Furthermore, the lack of phylogenetic variation within 'P. kishitanii' indicates that this facultatively symbiotic bacterium is not cospeciating with its phylogenetically divergent host fishes. The results of this fine-scale phylogenetic analysis support the emerging view that bacterial species names should designate singular historical entities, i.e. discrete lineages diagnosed by a significant divergence of shared derived nucleotide characters.

The luminous marine bacterium Photobacterium mandapamensis was synonymized several years ago with Photobacterium leiognathi based on a high degree of phenotypic and genetic similarity. To test the possibility that P. leiognathi as now formulated, however, actually contains two distinct bacterial groups reflecting the earlier identification of P. mandapamensis and P. leiognathi as separate species, we compared P. leiognathi strains isolated from light-organ symbiosis with leiognathid fishes (i.e., ATCC 25521(T), ATCC 25587, lequu.1.1 and lleuc.1.1) with strains from seawater originally described as P. mandapamensis and later synonymized as P. leiognathi (i.e., ATCC 27561(T) and ATCC 33981) and certain strains initially identified as P. leiognathi (i.e., PL-721, PL-741, 554). Analysis of the 16S rRNA and gyrB genes did not resolve distinct clades, affirming a close relationship among these strains. However, strains ATCC 27561(T), ATCC 33981, PL-721, PL-741 and 554 were found to bear a luxF gene in the lux operon ( luxABFE), whereas ATCC 25521(T), ATCC 25587, lequu.1.1 and lleuc.1.1 lack this gene ( luxABE). Phylogenetic analysis of the luxAB(F)E region confirmed this distinction. Furthermore, ATCC 27561(T), ATCC 33981, PL-721, PL-741 and 554 all produced a higher level of luminescence on high-salt medium, as previously described for PL-721, whereas ATCC 25521(T), ATCC 25587, lequu.1.1 and lleuc.1.1 all produced a higher level of luminescence on low-salt medium, a characteristic of P. leiognathi from leiognathid fish light organs. These results demonstrate that P. leiognathi contains two evolutionarily and phenotypically distinct clades, P. leiognathi subsp. leiognathi (strains ATCC 25521(T), ATCC 25587, lequu.1.1 and lleuc.1.1), and P. leiognathi subsp. mandapamensis (strains ATCC 27561(T), ATCC 33981, PL-721, PL-741 and 554).

The crystal structure of a myristoyl acyl carrier protein specific thioesterase (C14ACP-TE) from a bioluminescent bacterium, Vibrio harveyi, was solved by multiple isomorphous replacement methods and refined to an R factor of 22% at 2.1-A resolution. This is the first elucidation of a three-dimensional structure of a thioesterase. The overall tertiary architecture of the enzyme resembles closely the consensus fold of the rapidly expanding superfamily of alpha/beta hydrolases, although there is no detectable homology with any of its members at the amino acid sequence level. Particularly striking similarity exists between the C14ACP-TE structure and that of haloalkane dehalogenase from Xanthobacter autotrophicus. Contrary to the conclusions of earlier studies [Ferri, S. R., & Meighen, E. A. (1991) J. Biol. Chem. 266, 12852-12857] which implicated Ser77 in catalysis, the crystal structure of C14ACP-TE reveals a lipase-like catalytic triad made up of Ser114, His241, and Asp211. Surprisingly, the gamma-turn with Ser114 in a strained secondary conformation (phi = 53 degrees, psi = -127 degrees), characteristic of the so-called nucleophilic elbow, does not conform to the frequently invoked lipase/esterase consensus sequence (Gly-X-Ser-X-Gly), as the positions of both glycines are occupied by larger amino acids. Site-directed mutagenesis and radioactive labeling support the catalytic function of Ser114. Crystallographic analysis of the Ser77-->Gly mutant at 2.5-A resolution revealed no structural changes; in both cases the loop containing the residue in position 77 is disordered.

The diversion of fatty acids from fatty acid biosynthesis into the luminescent system is catalyzed by a lux-specific acyltransferase that catalyzes the cleavage of fatty acyl-acyl carrier protein (ACP). Analysis of the substrate specificities for fatty acyl-ACPs of the transferases from divergent luminescent bacteria, Photobacterium phosphoreum and Vibrio harveyi, has demonstrated that myristoyl-ACP is cleaved at the highest rate. Inhibition by phenylmethanesulfonyl fluoride as well as resistance of the acylated enzyme intermediate to cleavage by hydroxylamine showed that the transferase is a serine esterase. Moreover, activity was dependent on a basic residue with a pKa of 6.3 implicating a histidine residue as part of a charge relay system found in serine esterases. The nucleotide sequence of the P. phosphoreum luxD gene coding for the transferase was determined resulting in the identification of the active site motif for serine esterases, G-X-S-X-G. Replacement of the serine residue at the center of this motif by threonine, alanine, or glycine blocked the transferase acyl-ACP cleavage activity, its ability to be acylated, and complementation of a transferase defective mutant on transconjugation with the luxD gene. The sequence and location of the serine as well as a histidine residue in the lux-specific transferases were found to be similar to those involved in the charge relay system in vertebrate thioesterases. Combined with the similar kinetic properties, these results support a common metabolic role for both enzymes in the diversion of fatty acids from the fatty acid biosynthetic pathway.