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.     

Isolation of the lux genes from Photobacterium leiognathi and expression in Escherichia coli

Genes necessary for luminescence (lux genes) in the marine bacterium Photobacterium leiognathi, strain PL721, were isolated and expressed in Escherichia coli. A 15-kb fragment obtained from a partial digestion of PL721 DNA with HindIII was cloned into the plasmid pACYC184, resulting in the hybrid plasmid pSD721. When pSD721 was transformed into E. coli ED8654, the resulting transformants were luminous with no additions to the cells, indicating that it contained the structural genes coding for the alpha and beta subunits of luciferase (luxA and luxB), and for components involved in aldehyde biosynthesis. Hybridization analysis with luxA and luxB 32P probes confirmed the location of these two genes on the 15-kb insert. When pSD721 was transformed into four different strains of E. coli, luminescence expression varied widely in amount and in pattern. In some strains, luminescence developed like an autoinducible system, and at maximum induction was very bright, even with no addition of aldehyde, while in others, luminescence was 100-fold less, and no induction was seen. In no case was luminescence affected by shifts in temperature, osmolarity, or iron concentration. These results indicate that, while the complete lux regulon is apparently contained on the 15-kb cloned fragment, the regulation of the lux regulon in pSD721 is subject to host controls by E. coli, controls which vary widely among different E. coli strains.     

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.     

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.     

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.     

Crystallization of Photobacterium leiognathi non-fluorescent flavoprotein, an unusual flavoprotein with limited sequence identity to bacterial luciferase.

Single crystals of the non-fluorescent flavoprotein (NFP) purified from Photobacterium leiognathi strain S1 have been grown from ammonium sulphate solutions using the hanging drop vapour diffusion technique. The crystals grow as thin (0.06 mm) plates and belong to the orthorhombic space group C222(1): a = 57.06(3) A, b = 92.41(6) A, c = 99.52(6) A. There is one NFP monomer per asymmetric unit and crystals diffract to 2.2 A spacings on film. A complete native data set to 2.5 A resolution has been collected on a San Diego Multiwire Detector system at the University of Alberta and a heavy-atom derivative search is presently in progress.     

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.     

Control of luciferase synthesis in a newly isolated strain of Photobacterium leiognathi

In previous studies with luminous bacteria of all different species it has been reported that the synthesis of luciferase is autoinducible: during growth at low cell densities synthesis is effectively repressed while after induction, at higher cell densities, the rate of synthesis of enzyme is up to five times the growth rate. In this paper we report on newly isolated strains of Photobacterium leiognathi which show continued luciferase synthesis irrespective of the cell density. The specific synthesis rate may nevertheless differ from the rate of growth and depends on the luciferase content of the inoculated cells. A ratio of 1 was established for cells having a maximum luciferase content varying to a ratio of about 2 for cells that contained only 1% of the maximum.Abbreviation BSA bovine serum albumin     

Strain variation in bacteriocuprein superoxide dismutase from symbiotic Photobacterium leiognathi.

Photobacterium leiognathi ATCC 25521 (the type strain and light-organ symbiont of ponyfish) is one of the few bacteria that produces a copper-zinc superoxide dismutase, termed bacteriocuprein. We enzymologically and immunologically characterized the bacteriocuprein superoxide dismutases in sonicates from the type strain and nine additional strains of P. leiognathi, each isolated from the light organ of a separate ponyfish specimen, representing seven ponyfish species. The results indicate considerable strain variation. (i) The level of bacteriocuprein enzymatic activity varied greatly among strains from different species of ponyfish. In four of the nine strains, activity was low or undetectable, while in five strains it was comparable to that in the type strain. (ii) The bacteriocuprein in one strain had a specific activity much lower than that of the type strain, and in another strain, no bacteriocuprein activity and no cross-reactive polypeptide were detectable. (iii) A new electrophoretic variant, which migrated slower than that of strains from fish captured in Thailand and Japan, was identified in strains from fish captured in the Philippine Islands. (iv) Enzymological and immunological differences were observed in bacteriocupreins of strains from male and female specimens of the same ponyfish species, for the two species in which specimens of both sexes were examined. These observations raise the possibility that specific variations in the bacteriocupreins of P. leiognathi might be characteristic of the species, geographical source, or sex of the ponyfish host. Thus, the data indicate that the possibility of strain variation should be considered when other species are screened for bacteriocupreins.     

Spectroscopic investigations of the single tryptophan residue and of riboflavin and 7-oxolumazine bound to lumazine apoprotein from Photobacterium leiognathi

Spectroscopic techniques have been applied to investigate the conformation, local structure, and dynamic properties of the apoprotein of the lumazine protein from Photobacterium leiognathi and the holoprotein reconstituted with either the natural ligand 6,7-dimethyl-8-ribityllumazine or the closely related analogues riboflavin and 6-methyl-7-oxo-8-ribityllumazine (7-oxolumazine). The analogues are bound similarly to the natural prosthetic group. They exhibit similar shifts on binding in their absorption and fluorescence spectra, single-exponential fluorescence decays, and no independent motion from the protein as evident from a long-lived anisotropy decay (single-exponential phi = 10 ns, 20 degrees C) and high initial anisotropy. Steady-state anisotropy measurements result in similar KD's (40 nM, 20 degrees C, 50 mM inorganic phosphate) for all ligands. Circular dichroism in the far-UV region (190-250 nm) indicates no change in secondary structure on binding to the apoprotein. In the spectral region of 250-310 nm relatively large changes occur, indicating changes in the environment of the tyrosine and tryptophan residues. The single tryptophan residue shows a three-exponential decay of its fluorescence in both the apoprotein and the holoprotein. Radiationless energy transfer also occurs from the tryptophan to the bound ligand, especially evident with 7-oxolumazine. We have designed a new method for evaluation of the rate constant of energy transfer by measuring the (picosecond) rise time of the acceptor fluorescence. The anisotropy decay of the tryptophan residue shows two correlation times, a short one (phi approximately equal to 0.4 ns) representing rapid but restriced oscillation of this residue and a longer one (phi 2 = 5-7 ns, 20 degrees C) representing the motion of a larger segment of the protein.     

The alpha-A-crystallin and cystathionine beta-synthase genes are physically very closely linked in proximal mouse chromosome 17

Murine genes homologous to those contributing to the Down syndrome (DS) phenotype in man are currently of interest because of their potential for providing animal models for the study of specific DS symptoms. Most of the genes mapping to human chromosome 21q22, where the DS genes are concentrated, are related to sequences located on mouse chromosome 16. Others, however, are known to map to mouse chromosome 10, and two genes, cystathionine beta-synthase (Cbs) and alpha-A-crystallin (Crya-1), have been localized to the proximal portion of mouse chromosome 17. In this paper, we show that the two genes mapping to human chromosome 21q22 and mouse chromosome 17 are very tightly linked in mouse, being separated by at least 70 kb, but not more than 130 kb. The very close physical linkage of mouse Cbs and Crya-1, combined with data that localize homologs of the closely flanking markers H2k and Pim-1 to human chromosome 6, suggests that the human 21q22/mouse chromosome 17 conserved segment is of a very limited total physical size and is likely to contain a relatively small number of genes.     

The Myb and Ahi-1 genes are physically very closely linked on mouse Chromosome 10

Ahi-1 has previously been identified as a common helper provirus integration site on mouse Chromosome (Chr) 10 in 16% of Abelson pre-B-cell lymphomas and shown to be closely linked to the Myb protooncogene. By using long-range restriction mapping, we have mapped the Myb and Ahi-1 regions within a 120-kbp DNA fragment. The Ahi-1 region is located approximately 35 kbp downstream of the Myb gene. A further comfirmation of this finding was obtained by screening a mouse YAC library. The three positive clones obtained contained both the Myb and Ahi-1 gene sequences. To test whether provirus integration in the Ahi-1 region enhances the expression of Myb by a cis-acting mechanism, we have also examined Myb gene expression in A-MuLV-induced pre-B-lymphomas. Our data have revealed that there is no clear evidence for such activation in the tumors we have tested, indicating that provirus insertion in the Ahi-1 region is activating a novel gene, apparently involved in tumor formation.