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.     

"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.     

The lux genes in Photobacterium leiognathi are closely linked with genes corresponding in sequence to riboflavin synthesis genes.

Three open reading frames (ORFs) have been found in the region downstream of the luxG gene in the Photobacterium leiognathi lux operon. These genes (ORF I, II, and III) are not only closely linked to the lux operon and transcribed in the same direction but also show the same organization and code for proteins homologous in sequence to the gene products of ribB, ribA, and ribH of Bacillus subtilis, respectively. The Photobacterium leiognathi gene (ORF II) corresponding to ribA was expressed in Escherichia coli in the bacteriophage T7 promoter-RNA polymerase system and a 40 kDa 35S-labeled polypeptide has been detected on SDS-PAGE. Expression of DNA extending from luxBEG to ORF II inserted between a strong promoter and a reporter gene and transferred by conjugation into Vibrio harveyi did not affect the expression of the reporter gene. The results provide evidence that neither promoter nor terminator sites were present in the DNA between the luxG and ORF II indicating that these genes might be part of the lux operon.     

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.     

Nucleotide sequence, expression, and properties of luciferase coded by lux genes from a terrestrial bacterium

The lux genes required for expression of luminescence have been cloned from a terrestrial bacterium, Xenorhabdus luminescens, and the nucleotide sequences of the luxA and luxB genes coding for the alpha and beta subunits of luciferase determined. The lux gene organization was closely related to that of marine bacteria from the Vibrio genus with the luxD gene being located immediately upstream and the luxE downstream of the luciferase genes, luxAB. A high degree of homology (85% identity) was found between the amino acid sequences of the alpha subunits of X. luminescens luciferase and the luciferase from a marine bacterium, Vibrio harveyi, whereas the beta subunits of the two luciferases had only 60% identity in amino acid sequence. The similarity in the sequences of the alpha subunits of the two luciferases was also reflected in the substrate specificities and turnover rates with different fatty aldehydes supporting the proposal that the alpha subunit almost exclusively controls these properties. The luciferase from X. luminescens was shown to have a remarkably high thermal stability being stable at 45 degrees C (t 1/2 greater than 3 h) whereas V. harveyi luciferase was rapidly inactivated at this temperature (t 1/2 = 5 min). These results indicate that the X. luminescens lux system may be the bacterial bioluminescent system of choice for application in coupled luminescent assays and expression of lux genes in eukaryotic systems at higher temperatures.     

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.     

Cloning and expression of the Photobacterium phosphoreum luminescence system demonstrates a unique lux gene organization

The organization of the lux structural genes (A-E) in Photobacterium phosphoreum has been determined and a new gene designated as luxF discovered. The P. phosphoreum luminescence system was cloned into Escherichia coli using a pBR322 vector and identified by cross-hybridization with Vibrio fischeri lux DNA. The lux genes were located by specific expression of P. phosphoreum DNA fragments in the T7-phage polymerase/promoter system in E. coli and identification of the labeled polypeptide products. The luxA and luxB gene products (luciferase subunits) were shown to catalyze light emission in the presence of FMNH2, O2, and aldehyde. The luxC, luxD, and luxE gene products (fatty acid reductase subunits) responsible for aldehyde biosynthesis could be specifically acylated with 3H-labeled fatty acids. The order of the lux genes in P. phosphoreum was found to be luxCDABFE with luxF coding for a new polypeptide of 26 kDa. The presence of a new gene in the P. phosphoreum luminescence system between luxB and luxE as compared to the organization of the lux structural gene in V. fischeri and Vibrio harveyi (luxCDABE) demonstrates that the luminescent systems in the marine bacteria have significantly diverged. The discovery of the luxF gene provides the basis for elucidating the role of its gene product in the expression of luminescence 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.     

Lateral transfer of the lux gene cluster

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.     

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.     

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.     

Nucleotide sequence of the LuxC gene and the upstream DNA from the bioluminescent system of Vibrio harveyi.

The nucleotide sequence of the luxC gene (1431 bp) and the upstream DNA (1049 bp) of the luminescent bacterium Vibrio harveyi has been determined. The luxC gene can be translated into a polypeptide of 55 kDa in excellent agreement with the molecular mass of the reductase polypeptide required for synthesis of the aldehyde substrate for the bioluminescent reaction. Analyses of codon usage showed a high frequency (1.9%) of the isoleucine codon, AUA, in the luxC gene compared to that found in Escherichia coli genes (0.2%) and its absence in the luxA, B and D genes. The low G/C content of the luxC gene and upstream DNA (38-39%) compared to that found in the other lux genes of V. harveyi (45%) was primarily due to a stretch of 500 nucleotides with only a 24% G/C content, extending from 200 bp inside lux C to 300 bp upstream. Moreover, an open reading frame did not extend for more than 48 codons between the luxC gene and 600 bp upstream at which point a gene transcribed in the opposite direction started. As the lux system in the luminescent bacterium, V. fischeri, contains a regulatory gene immediately upstream of luxC transcribed in the same direction, these results show that the organization and regulation of the lux genes have diverged in different luminescent bacteria.