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.     

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.     

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

Manifestation of pleiotropic effects in the isogenic variants of the luminescent bacterium 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 autoinducer 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 inherited variants of P. leiognathi.     

Purification of lumazine proteins from Photobacterium leiognathi and Photobacterium phosphoreum: bioluminescence properties

Bright strains of the marine bioluminescent bacterium Photobacterium leiognathi produce a "lumazine protein" in amounts comparable to that previously found in Photobacterium phosphoreum. New protocols are developed for the purification to homogeneity of the proteins from both species in yields up to 60%. In dimmer strains the amounts of lumazine protein in extracts are less, and also there is an accompanying shift of the bioluminescence spectral maximum to longer wavelength, 492 nm. Both types of lumazine proteins have identical fluorescence spectra, with maxima at 475 nm, so it is suggested that, whereas lumazine protein is the major emitter in bright strains, there is a second emitter also present with a fluorescence maximum at longer wavelength. The two species of lumazine protein have the same 276 nm/visible absorbance ratio, 2.2, but differ in visible maxima: P. phosphoreum, 417 nm; P. leiognathi, 420 nm. For the latter the bound lumazine has epsilon 420 = 10 100 M-1 cm-1, practically the same as in free solution. The two lumazine proteins also differ quantitatively in their effect on the in vitro bioluminescence reaction, i.e., at blue shifting the bioluminescence spectrum or altering the kinetics. The P. phosphoreum lumazine protein is more effective with its homologous luciferase or with P. leiognathi luciferase than is the lumazine protein from P. leiognathi. These differences may have an electrostatic origin.     

The primary structure of iron-superoxide dismutase from Photobacterium leiognathi

The complete amino acid sequence of iron-superoxide dismutase from Photobacterium leiognathi was determined. The sequence was deduced following characterization of the peptides obtained from tryptic, chymotryptic, and Staphylococcus aureus V-8 protease digests of the apoprotein. The amino acid sequence listed below is made up of 193 residues. It is the first complete sequence to be determined for an iron-superoxide dismutase. The iron-superoxide dismutase shows the same order of homology with the manganese-superoxide dismutases as these enzymes show among themselves. No homology was observed with the copper/zinc-containing class of superoxide dismutases. Ala-Phe-Glu-Leu-Pro-Ala-Leu-Pro-Phe-Ala-Met-Asn-Ala-Leu-Glu-Pro-His-Ile- Ser-Gln-Glu-Thr-Leu-Glu-Tyr-His-Tyr-Gly-Lys-His-His-Asn-Thr-Tyr-Val-Val- Lys-Leu-Asn-Gly-Leu-Val-Glu-Gly-Thr-Glu-Leu-Ala-Glu-Lys-Ser-Leu-Glu-Glu- Ile-Ile-Lys-Thr-Ser-Thr-Gly-Gly-Val-Phe-Asn-Asn-Ala-Ala-Gln-Val-Trp-Asn- His-Thr-Phe-Tyr-Trp-Asn-Cys-Leu-Ala-Pro-Asn-Ala-Gly-Gly-Glu-Pro-Thr-Gly- Glu-Val-Ala-Ala-Ala-Ile-Glu-Lys-Ala-Phe-Gly-Ser-Phe-Ala-Glu-Phe-Lys-Ala- Lys-Phe-Thr-Asp-Ser-Ala-Ile-Asn-Asn-Phe-Gly-Ser-Ser-Trp-Thr-Trp-Leu-Val- Lys-Asn-Ala-Asn-Gly-Ser-Leu-Ala-Ile-Val-Asn-Thr-Ser-Asn-Ala-Gly-Cys-Pro- Ile-Thr-Glu-Glu-Gly-Val-Thr-Pro-Leu-Leu-Thr-Val-Asp-Leu-Trp-Glu-His-Ala- Tyr-Tyr-Ile-Asp-Tyr-Arg-Asn-Leu-Arg-Pro-Ser-Tyr-Met-Asp-Gly-Phe-Trp-Ala- Leu-Val-Asn-Trp-Asp-Phe-Val-Ser-Lys-Asn-Leu-Ala-Ala.     

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.     

一株与鳆发光杆菌最相似细菌的研究

对从海鲜食品中分离到的1株少见细菌M1进行系统分类鉴定。采用常规方法[1]进行分离培养,以形态学特征、培养特性、生理生化特征以及分子生物学等方法对其进行分类鉴定。结果可见,该菌株为革兰氏阴性杆状细菌,16SrDNA核甘酸序列测定与鳆发光杆菌最为相似,生理生化特征与发光杆菌属、弧菌属相近,但都不完全相符,而与鳆发光杆菌相似性最高。     

Chemical characterization of lumazine protein from Photobacterium leiognathi: comparison with lumazine protein from Photobacterium phosphoreum

The properties of lumazine proteins purified from the marine bioluminescent bacteria Photobacterium phosphoreum, a psychrophile, and Photobacterium leiognathi, a relatively thermophilic species, are compared. An accurate 1:1 stoichiometry of binding of the ligand 6,7-dimethyl-8-ribityllumazine to each lumazine protein is established by back-titration of the apoprotein with the authentic ligand, using both fluorescence and absorption measurements. Neither protein contains metal cofactors, organic phosphorus, or carbohydrate. Both proteins are anionic and hydrophilic. They each contain a single Trp residue and have blocked amino terminals but otherwise differ in amino acid composition and other properties (P. phosphoreum and P. leiognathi, respectively): Met (internal), 1, 2; Cys, 2, 1; Arg, 4, 7; pI, 4.78 and 4.83, 4.38 and 4.45; Mr, 19 750, 21 300. In the P. phosphoreum protein both Cys residues are accessible, but in the P. leiognathi protein the single Cys is "buried". Modification of this buried Cys and at least one Cys in the P. phosphoreum protein prevents binding of the ligand. The UV and visible absorption spectra of both lumazine proteins denatured in 6 M guanidine hydrochloride can be accurately modeled by using the number of equivalents of the lumazine derivative and blocked aromatic amino acid model compounds determined by chemical and spectrophotometric analyses for Trp, Tyr, and Phe.     

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     

Comparative restriction analysis of chromosomal DNA of strains of Photobacterium leiognathi

Chromosomal DNA in 5 hereditary variants occurring in Photobacterium leiognathi population was subjected to restriction analysis. The variants differed in the levels and regulation of luminescence and colony morphology. Agarose electrophoresis of DNA fragments isolated after exposure to Hind II, Bam HI, Bgl I and Pst I restriction endonucleases revealed respectively 38, 28, 35 and 29 fragments equally distributed by their molecular weights. Electrophoregrams of the 5 strains were absolutely identical. After exposure of DNA of all the strains to PVu II, Xho II, Sal GI and Eco RI restriction endonucleases there were detected no fragments. The pleoiotropic genetic variation in these strains was not associated with large deletions or amplification of chromosomal DNA regions.     

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.     

Comparative biochemistry of the cell envelopes of Photobacterium leiognathi and Escherichia coli

Photobacterium leiognathi closely resembles Escherichia coli with respect to cell lysis by lysozyme, and the fractionation of outer and cytoplasmic membranes. The two organisms differ in their phospholipid contents and, more significantly, in outer membrane protein compositions.     

Osmotic control of luminescence and growth in Photobacterium leiognathi from ponyfish light organs

Osmolarity was found to control the luminescence and growth of Photobacterium leiognathi strain LN-1a isolated from the light organ of the ponyfish Leiognathus nuchalis (family Leiognathidae). Low osmolarity (ca. 400 mOsm) stimulated luminescence per cell 80 to 100-fold to a level (ca. 2.0×10⁴ quanta·s^-1·cell^-1) equal to that of bacteria taken directly from the light organ and increased the level of luciferase per cell 8 to 10-fold compared to high osmolarity (ca. 800 mOsm). Conversely, high osmolarity stimulated oxygen uptake and growth rate 2 to 4-fold compared to low osmolarity. Of 21 additional tested strains of P. leiognathi from light organs of 9 other ponyfish species, all responded similarly. Low osmolarity may be a host control factor that functions to stimulate the luminescence and restrict the growth of ponyfish light organ bacteria in situ.     

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.     

The effect of Hg2+ on the bioluminescence of Photobacterium leiognathi

Photobacteria were used as test objects for rapid monitoring of ecotoxicants. Specific inhibitory effects of Hg²⁺ on bioluminescence and cell growth as well as the lux gene expression of Photobacterium leiognathi were studied. The 3‐(4,5‐Dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide reduction assay was used to evaluate cellular proliferation and mortality. The luminescent inhibition effect on P. leiognathi cells was found to increase as cellular mortality increased; y = 0.744x ‐ 4.8916, R² = 0.9794. However, this trend was not observed in cell growth processes. Quantitation of lux mRNAs by semi‐quantitative RT‐qPCR indicated that increases and decreases in luciferase mRNA integral level coincided with changes in luminescence intensity (R² = 0.93). Addition of Hg²⁺ changed luminescence but without concomitant changes in extractable luciferase activity. Nevertheless, the presence of Hg²⁺ changed lux gene expression. This phenomenon requires further research. Copyright © 2012 John Wiley & Sons, Ltd.     

Interaction of aromatic compounds with Photobacterium leiognathi luciferase: fluorescence anisotropy study

The time‐resolved and steady‐state fluorescence techniques were employed to elucidate possible interactions of four aromatic compounds (anthracene, POPOP, MSB and 1,4‐naphthalendiol) with bacterial luciferase. Fluorescence spectra and fluorescence anisotropy decays of these compounds were studied in ethanol, water–ethanol solutions and in the presence of bacterial luciferase. Shifts of fluorescent spectra and differences in rotational correlation times are interpreted in terms of weak (hydrophobic) interactions of the molecules with the enzyme. These interactions suggest the feasibility of intermolecular energy transfer by an exchange resonance mechanism with a collision‐interaction radius as a way of excitation of these compounds in the reaction catalysed by bacterial luciferase. Copyright © 2003 John Wiley & Sons, Ltd.     

Crystallographic characterization of a Cu,Zn superoxide dismutase from Photobacterium leiognathi

Crystals of a copper-zinc superoxide dismutase from Photobacterium leiognathi, a luminescent marine bacterium that is the species-specific symbiont of the ponyfish, have been obtained from 2-methyl-2,4-pentanediol solutions. The space group was determined using screenless small-angle precession photographs, and was confirmed by analyzing area detector diffraction data with the XENGEN programs for indexing and refinement. The crystals are monoclinic, space group C2 (a = 126.4 A, b = 87.0 A, c = 44.4 A, beta = 92.8 A), and have two 32,000 Mr dimers per asymmetric unit. The crystals diffract to at least 2.7 A resolution, are resistant to radiation damage, and are suitable for determination of the structure by X-ray diffraction.