The lux genes of the luminous bacterial symbiont, Photobacterium leiognathi, of the ponyfish. Nucleotide sequence, difference in gene organization, and high expression in mutant Escherichia coli

The lux genes required for light expression in the luminescent bacterium Photobacterium leiognathi (ATCC 25521) have been cloned and expressed in Escherichia coli and their organization and nucleotide sequence determined. Transformation of a recombinant 9.5-kbp chromosomal DNA fragment of P. leiognathi into an E. coli mutant (43R) gave luminescent colonies that were as bright as those of the parental strain. Moreover, expression of the lux genes in the mutant E. coli was strong enough so that not only were high levels of luciferase detected in crude extracts, but the fatty-acid reductase activity responsible for synthesis of the aldehyde substrate for the luminescent reaction could readily be measured. Determination of the 7.3-kbp nucleotide sequence of P. leiognathi DNA, including the genes for luciferase (luxAB) and fatty-acid reductase (luxCDE) as well as a new lux gene (luxG) found recently in luminescent Vibrio species, showed that the order of the lux genes was luxCDABEG. Moreover, luxF, a gene homologous to luxB and located between luxB and luxE in Photobacterium but not Vibrio strains, was absent. In spite of this different lux gene organization, an intergenic stem-loop structure between luxB and luxE was discovered to be highly conserved in other Photobacterium species after luxF.     

Riboflavin synthesis genes are linked with the lux operon of Photobacterium phosphoreum.

Four genes immediately downstream of luxG in the Photobacterium phosphoreum lux operon (ribEBHA) have been sequenced and shown to be involved in riboflavin synthesis. Sequence analyses and complementation of Escherichia coli riboflavin auxotrophs showed that the gene products of ribB and ribA are 3,4-dihydroxy-2-butanone 4-phosphate (DHBP) synthetase and GTP cyclohydrolase II, respectively. By expression of P. phosphoreum ribE in E. coli using the bacteriophage T7 promoter-RNA polymerase system, ribE was shown to code for riboflavin synthetase, which catalyzes the conversion of lumazine to riboflavin. Increased thermal stability of RibE on expression with RibH indicated that ribH coded for lumazine synthetase. The organization of the rib genes in P. phosphoreum is quite distinct, with ribB and ribA being linked but separated by ribH, whereas in E. coli, they are unlinked and in Bacillus subtilis, RibB and RibA functions are coded by a single gene.     

A new Vibrio fischeri lux gene precedes a bidirectional termination site for the lux operon.

The DNA downstream of the lux structural genes in the Vibrio fischeri lux operon has been sequenced and a new lux gene (luxG) has been identified. A hairpin loop that begins with a poly(A) region and ends with a poly(T) region and thus can function as a bidirectional termination site for luxG and a convergent gene is located immediately downstream of luxG. 3' S1 nuclease mapping has demonstrated that the luxG mRNA was induced in a cell-density-dependent fashion consistent with it being part of the lux system and that the lux mRNA terminated immediately after the hairpin loop. The mRNA coded by an open reading frame convergent to luxG on the complementary strand was also shown by S1 nuclease mapping to overlap the lux mRNA for at least 20 nucleotides before termination. Expression of DNA containing the hairpin loop, placed between a strong promoter and a reporter gene and transferred by conjugation into luminescent bacteria, demonstrated the very high efficiency of termination by this hairpin loop oriented in either direction. These results also demonstrate that the organization of the genes at the 3' ends of the lux operons of V. fischeri and V. harveyi has clearly diverged.     

Delineation of the transcriptional boundaries of the lux operon of Vibrio harveyi demonstrates the presence of two new lux genes

The 5' and 3' ends of the lux mRNA of Vibrio harveyi, which extends over 8 kilobases, have been mapped, and two new genes, luxG and luxH, were identified at the 3' end of the lux operon. Both S1 nuclease and primer extension mapping demonstrated that the start site for the lux mRNA was 26 bases before the initiation codon of the first gene, luxC. The promoter region contained a typical -10 but not a recognizable -35 consensus sequence. By using S1 nuclease mapping the mRNA was found to be induced in a cell density- and arginine-dependent manner. The DNA downstream of the five known V. harveyi lux genes, luxCDABE, was sequenced and found to contain coding regions for two new genes, designated luxG and luxH, followed by a classical rho-independent termination signal for RNA polymerase. luxG codes for a protein of 233 amino acids with a molecular weight of 26,108, and luxH codes for a protein of 230 amino acids with a molecular weight of 25,326. The termination signal is active in vivo as demonstrated by 3' S1 nuclease mapping, confirming that the two genes are part of the V. harveyi lux operon. Comparison of the luxG amino acid sequence with coding regions immediately downstream from luxE in other luminescent bacteria has demonstrated that this gene may be a common component of the luminescent systems in different marine bacteria.     

Phylogenetic analysis of the incidence of lux gene horizontal transfer in Vibrionaceae

Horizontal gene transfer (HGT) is thought to occur frequently in bacteria in nature and to play an important role in bacterial evolution, contributing to the formation of new species. To gain insight into the frequency of HGT in Vibrionaceae and its possible impact on speciation, we assessed the incidence of interspecies transfer of the lux genes (luxCDABEG), which encode proteins involved in luminescence, a distinctive phenotype. Three hundred three luminous strains, most of which were recently isolated from nature and which represent 11 Aliivibrio, Photobacterium, and Vibrio species, were screened for incongruence of phylogenies based on a representative housekeeping gene (gyrB or pyrH) and a representative lux gene (luxA). Strains exhibiting incongruence were then subjected to detailed phylogenetic analysis of horizontal transfer by using multiple housekeeping genes (gyrB, recA, and pyrH) and multiple lux genes (luxCDABEG). In nearly all cases, housekeeping gene and lux gene phylogenies were congruent, and there was no instance in which the lux genes of one luminous species had replaced the lux genes of another luminous species. Therefore, the lux genes are predominantly vertically inherited in Vibrionaceae. The few exceptions to this pattern of congruence were as follows: (i) the lux genes of the only known luminous strain of Vibrio vulnificus, VVL1 (ATCC 43382), were evolutionarily closely related to the lux genes of Vibrio harveyi; (ii) the lux genes of two luminous strains of Vibrio chagasii, 21N-12 and SB-52, were closely related to those of V. harveyi and Vibrio splendidus, respectively; (iii) the lux genes of a luminous strain of Photobacterium damselae, BT-6, were closely related to the lux genes of the lux-rib(2) operon of Photobacterium leiognathi; and (iv) a strain of the luminous bacterium Photobacterium mandapamensis was found to be merodiploid for the lux genes, and the second set of lux genes was closely related to the lux genes of the lux-rib(2) operon of P. leiognathi. In none of these cases of apparent HGT, however, did acquisition of the lux genes correlate with phylogenetic divergence of the recipient strain from other members of its species. The results indicate that horizontal transfer of the lux genes in nature is rare and that horizontal acquisition of the lux genes apparently has not contributed to speciation in recipient taxa.     

Natural merodiploidy of the lux-rib operon of Photobacterium leiognathi from coastal waters of Honshu, Japan

Sequence analysis of the bacterial luminescence (lux) genes has proven effective in helping resolve evolutionary relationships among luminous bacteria. Phylogenetic analysis using lux genes, however, is based on the assumptions that the lux genes are present as single copies on the bacterial chromosome and are vertically inherited. We report here that certain strains of Photobacterium leiognathi carry multiple phylogenetically distinct copies of the entire operon that codes for luminescence and riboflavin synthesis genes, luxCDABEG-ribEBHA. Merodiploid lux-rib strains of P. leiognathi were detected during sequence analysis of luxA. To define the gene content, organization, and sequence of each lux-rib operon, we constructed a fosmid library of genomic DNA from a representative merodiploid strain, lnuch.13.1. Sequence analysis of fosmid clones and genomic analysis of lnuch.13.1 defined two complete, physically separate, and apparently functional operons, designated lux-rib1 and lux-rib2. P. leiognathi strains lelon.2.1 and lnuch.21.1 were also found to carry lux-rib1 and lux-rib2, whereas ATCC 25521T apparently carries only lux-rib1. In lnuch.13.1, lelon.2.1, lnuch.21.1, and ATCC 25521T, lux-rib1 is flanked upstream by lumQ and putA and downstream by a gene for a hypothetical multidrug efflux pump. In contrast, transposase genes flank lux-rib2 of lnuch.13.1, and the chromosomal location of lux-rib2 apparently differs in lnuch.13.1, lelon.2.1, and lnuch.21.1. Phylogenetic analysis demonstrated that lux-rib1 and lux-rib2 are more closely related to each other than either one is to the lux and rib genes of other bacterial species, which rules out interspecies lateral gene transfer as the origin of lux-rib2 in P. leiognathi; lux-rib2 apparently arose within a previously unsampled or extinct P. leiognathi lineage. Analysis of 170 additional strains of P. leiognathi, for a total of 174 strains examined from coastal waters of Japan, Taiwan, the Philippine Islands, and Thailand, identified 106 strains that carry only a single lux-rib operon and 68 that carry multiple lux-rib operons. Strains bearing a single lux-rib operon were obtained throughout the geographic sampling range, whereas lux-rib merodiploid strains were found only in coastal waters of central Honshu. This is the first report of merodiploidy of lux or rib genes in a luminous bacterium and the first indication that a natural merodiploid state in bacteria can correlate with geography.     

Expression of Photobacterium leiognathi bioluminescence system genes in Escherichia coli

Expression of Photobacterium leiognathi bioluminescence genes under the control of lac, tac, tet promoters in Escherichia coli cells has been studied. The position of the genes for aliphatic aldehyde biosynthesis and for the synthesis of luciferase subunits was identified. The plasmid pBRPL1 has been constructed containing the system of bioluminescence genes devoid of promoter following the polylinker DNA fragment. The plasmid can be used for selection of promoter containing DNA sequences as well as for studying the promoters regulation in process of Escherichia coli cells growth.     

Transcriptional regulation of lux genes transferred into Vibrio harveyi

Past work has shown that transformed Escherichia coli is not a suitable vehicle for studying the expression and regulation of the cloned luminescence (lux) genes of Vibrio harveyi. Therefore, we have used a conjugative system to transfer lux genes cloned into E. coli back into V. harveyi, where they can be studied in the parental organism. To do this, lux DNA was inserted into a broad-spectrum vector, pKT230, cloned in E. coli, and then mobilized into V. harveyi by mating aided by the conjugative plasmid pRK2013, also contained in E. coli. Transfer of the wild-type luxD gene into the V. harveyi M17 mutant by this means resulted in complementation of the luxD mutation and full restoration of luminescence in the mutant; expression of transferase activity was induced if DNA upstream of luxC preceded the luxD gene on the plasmid, indicating the presence of a strong inducible promoter. To extend the usefulness of the transfer system, the gene for chloramphenicol acetyltransferase was inserted into the pKT230 vector as a reporter. The promoter upstream of luxC was verified to be cell density regulated and, in addition, glucose repressible. It is suggested that this promoter may be the primary autoregulated promoter of the V. harveyi luminescence system. Strong termination signals on both DNA strands were recognized and are located downstream from luxE at a point complementary to the longest mRNA from the lux operon. Structural lux genes transferred back into V. harveyi under control of the luxC promoter are expressed at very high levels in V. harveyi as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis: the gene transfer system is thus useful for expression of proteins as well as for studying the regulation of lux genes in their native environment.     

A biosensor for environmental genotoxin screening based on an SOS lux assay in recombinant Escherichia coli cells.

A genetically controlled luminescent bacterial reporter assay, the SOS lux test, was developed for rapid detection of environmental genotoxins. The bioassay is based on the recombinant plasmid pPLS-1, which was constructed as a derivative of pBR322, carrying the promoterless luxCDABFE genes of Photobacterium leiognathi downstream of a truncated cda gene from ColD with a strong SOS promoter. E. coli recA+ strains containing this construction are inducible to high levels of light production in the presence of substances or agents that cause damage to the DNA of the cells. The light signal, reflecting the SOS-inducing potency, is recorded from the growing culture within 1 s, and the test results are available within 1 to 2 h. Induction of bioluminescence was demonstrated by treatment of E. coli C600(pPLS-1) with 6 genotoxic chemicals (mitomycin C, N-methyl-N'-nitro-N-nitrosoguanidine, nalidixic acid, dimethylsulfate, hydrogen peroxide, and formaldehyde) and with UV and gamma radiation. A clear dose-response relationship was established for all eight genotoxins. The sensitivity of the SOS lux test is similar to that of other bioassays for genotoxicity or mutagenicity, such as the SOS chromotest, umu test, and Ames mutatest. These results indicate that the SOS lux test is potentially useful for the in situ and continuous detection of genotoxins.     

Cloning and expression of genes of the luminescence system in Photobacterium leiognathi

The genes of Photobacterium leiognathi luminescence system were cloned in plasmid pUC18. Escherichia coli cells harboring a recombinant plasmid pPHL1 are luminescent. pPHL1 contains luciferase genes and genes responsible for aldehyde biosynthesis. The luminescence of Escherichia coli is subject to autoinductor regulation similar to the one existing in luminescent bacteria. The 2.7 kb fragment of Photobacterium leiognathi DNA containing the genes for alpha- and beta-luciferase subunits were cloned in pUC19.     

"Quorum sensing" regulation of lux gene expression and the structure of lux operon in marine bacteria Alivibrio logei

A group of luminescent strains of marine bacteria Alivibrio logei has been isolated (basins of the Okhotsk, White and Bering Seas). Strains A. logei were shown to be psycrophiic bacteria with an optimal growth temperature of approximately 15 degrees C. Biolumiscent characteristics of strains were studied, and the expression of lux genes was shown to be regulated by the "quorum sensing" system. The A. logei lux operon was cloned in Escherichia coli cells and the structure of this operon and its nucleotide sequence were determined. The structure of A. logei lux operon differs markedly from that in the closely related species of luminescent marine bacteria A. fischeri. In the structure of the A. logei lux operon, the the luxI gene is absent in front of luxC, and a fragment containing luxR2-luxI genes is located immediately after luxG gene. Luminescent psycrophiic marine bacteria of A. logei are assumed to be widely distributed in cold waters of northern seas.     

Multiple repetitive elements and organization of the lux operons of luminescent terrestrial bacteria.

The complete nucleotide sequences of the luxA to luxE genes, as well as the flanking regions, were determined for the lux operons of two Xenorhabdus luminescens strains isolated from insects and humans. The nucleotide sequences of the corresponding lux genes (luxCDABE) were 85 to 90% identical but completely diverged 350 bp upstream of the first lux gene (luxC) and immediately downstream of the last lux gene (luxE). These results show that the luxG gene found immediately downstream of luxE in luminescent marine bacteria is missing at this location in terrestrial bacteria and raise the possibility that the lux operons are at different positions in the genomes of the X. luminescens strains. Four enteric repetitive intergenic consensus (ERIC) or intergenic repetitive unit (IRU) sequences of 126 bp were identified in the 7.7-kbp DNA fragment from the X.luminescens strain isolated from humans, providing the first example of multiple ERIC structures in the same operon including two ERIC structures at the same site. Only a single ERIC structure between luxB and luxE is present in the 7-kbp lux DNA from insects. Analysis of the genomic DNAs from five X. luminescens strains or isolates by polymerase chain reaction has demonstrated that an ERIC structure is between luxB and luxE in all of the strains, whereas only the strains isolated from humans had an ERIC structure between luxD and luxA. The results indicate that there has been insertion and/or deletion of multiple 126-bp repetitive elements in the lux operons of X.luminescens during evolution.     

Conditions that influence bacterial luminescence in the presence of blood serum

Conditions that influence the luminescence of natural and recombinant luminescent bacteria in the presence of blood serum were studied. In general, blood serum quenched the luminescence of the marine Photobacterium phosphoreum and the recombinant Escherichia coli strains harboring the luminescent system genes of Photobacterium leiognathi, but enhanced the luminescence of the soil bacterium Photorhabdus luminescens Zm1 and the recombinant E. coli strain harboring the lux operon of P. luminescens Zm1. The quenching effect of blood serum increased with its concentration and the time and temperature of incubation. The components of blood serum that determine the degree and specificity of its action on bacterial luminescence were identified.     

Heterogeneity of the populations of marine luminescent bacteria Photobacterium leiognathi under different conditions of cultivation

Manifestation of pleiotropic effects in the isogenic variants of luminescent bacteria Photobacterium leiognathi 54 was investigated. The decrease or increase of the expression level of bioluminescence was caused by changes in lux operon regulation. The dynamics of the bioluminescence of dark and dim variants did not differ from the dynamics of the initial luminescent variant, but dependence of the level of luminescence intensity on the exogenous autoinductor of the lux operon was revealed. The investigated variants of P. leiognathi 54 inherited fairly stable morphological characteristics, colony architectonics, level of luminescence, and activity of some enzymes; variants with reduced bioluminescence formed colonies of the S type. Stable bright variants with S- and R-type colonies appeared both in the initial strain population and in the dark variant population, but with smaller frequency. Populations of the bright variant with R-type colonies were most heterogeneous; this can be determined by the lack of glucose repression of the bioluminescence in contrast to other investigated variants of P. leiognathi.     

Nucleotide sequence of genes for alpha- and beta-subunits of luciferase from Photobacterium leiognathi

Nucleotide sequence of the Photobacterium leiognathi DNA containing genes of alpha and beta subunits of luciferase has been determined. We also deduced amino acid sequence and molecular mass of luciferase and localized luciferase genes in the sequenced DNA fragment.     

Limited geographic distribution of certain strains of the bioluminescent symbiont Photobacterium leiognathi

Photobacterium leiognathi is a facultative bioluminescent symbiont of marine animals. Strains of P.?leiognathi that are merodiploid for the luminescence genes (lux-rib operon) have been previously obtained only from Japan. In contrast, strains bearing a single lux-rib operon have been obtained from all the areas sampled in Japan and the western Pacific. In this study, we tested whether distribution of merodiploid P.?leiognathi is limited by physical barriers in the environment, or because fish in the western Pacific preferentially form symbiosis with bacteria bearing a single lux-rib operon. We collected light organ symbionts from Secutor indicius, a fish species that is typically found in the western Pacific and has only recently expanded its geographic range to Japan. We found that all S.?indicius specimens collected from Japan formed symbiosis only with single lux-rib operon-bearing strains, although fish from other species collected from the same geographic area frequently contained merodiploid strains. This result shows that S.?indicius were preferentially colonized by bacteria bearing a single lux-rib operon and suggests that the limited geographic distribution of merodiploid P.?leiognathi can be attributed to preferential colonization of fish species found in the western Pacific by strains bearing only a single lux-rib operon.     

LuxA gene of light organ symbionts of the bioluminescent fish Acropoma japonicum (Acropomatidae) and Siphamia versicolor (Apogonidae) forms a lineage closely related to that of Photobacterium leiognathi ssp. mandapamensis

A molecular phylogenetic analysis of luxA gene sequences of light organ symbionts of the fish Acropoma japonicum (Acropomatidae) and Siphamia versicolor (Apogonidae) revealed that the sequences were related to those of Photobacterium leiognathi ssp. mandapamensis, which is not known to occur as a light organ symbiont among bioluminescent P. leiognathi clades. The presence of another lux gene element, luxF, coding for nonfluorescent protein, provided additional support for the identity of the light organ symbionts of the fish. Cladogenesis of the light organ symbiont P. leiognathi may be influenced by the radiation of host fishes.