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.     

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.     

A pulse-radiolysis study of the catalytic mechanism of the iron-containing superoxide dismutase from Photobacterium leiognathi.

The mechanism of the enzymic reaction of an iron-containing superoxide dismutase purified from the marine bacterium Photobacterium leiognathi was studied by using pulse radiolysis. Measurements of activity were done with two different preparations of enzyme containing either 1.6 or 1.15 g-atom of iron/mol. In both cases, identical values of the second-order rate constant for reaction between superoxide dismutase and the superoxide ion in the pH range 6.2-9.0 (k=5.5 X 10(8) M-1-S-1 at pH 8.0) were found. As with the bovine erythrocuprein, there was no evidence for substrate saturation. The effects of reducing agents (H2O2, sodium ascorbate or CO2 radicals) on the visible and the electron-paramagnetic-resonance spectra of the superoxide dismutase containing 1.6 g-atom of ferric iron/mol indicate that this enzyme contains two different types of iron. Turnover experiments demonstrate that only that fraction of the ferric iron that is reduced by H2O2 is involved in the catalysis, being alternately oxidized and reduced by O2; both the oxidation and the reduction steps have a rate constant equal to that measured under turnover conditions. These results are interpreted by assuming that the superoxide dismutase isolated from the organism contains 1 g-atom of catalytic iron/mol and a variable amount of non-catalytic iron. This interpretation is discused in relation to the stoicheiometry reported for iron-containing superoxide dismutases prepared from several other organisms.     

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.     

Evidence of stable monomeric species in the unfolding of Cu,Zn superoxide dismutase from Photobacterium leiognathi.

The equilibrium unfolding process of Photobacterium leiognathi Cu,Zn superoxide dismutase has been quantitatively monitored through circular dichroism (CD) and fluorescence spectroscopy, upon increasing the guanidinium hydrochloride concentration. The study has been undertaken for both the holo- and the copper-free derivative to work out the role of copper in protein stability. In both cases the unfolding was reversible. The denaturation curve derived from CD and fluorescence spectroscopy was not coincident, suggesting that the denaturation process occurs through a three-state model with formation of an intermediate monomeric species. The occurrence of an intermediate species has been unambiguously demonstrated following CD and steady-state fluorescence spectra of the enzyme at various concentrations in presence of a fixed amounts of guanidinium hydrochloride.     

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.     

Amino acid sequence of iron-superoxide dismutase from Pseudomonas ovalis

The amino acid sequence of iron-superoxide dismutase from Pseudomonas ovalis was deduced by the analyses of peptides derived from limited hydrolysis of the aminoethylated or pyridylethylated apoprotein with trypsin, Staphylococcus aureus V8 protease, and dilute acid hydrolysis. The polypeptide chain contains 195 amino acid residues and has a calculated Mr of 21,421. The sequence is highly homologous (65% identity) to the recently published sequence of the iron-superoxide dismutase from Photobacterium leiognathi. It is also homologous to the known sequences of the manganese-superoxide dismutase by sharing 33-53% identical residues. Alignment of the superoxide dismutase sequences and the available structural information from X-ray crystallography suggest that the ligands to the iron in the P. ovalis superoxide dismutase are His-26, His-74, Asp-156 and His-160, which align with the ligands to the manganese in the Thermus thermophilus manganese-superoxide dismutase. The sequence information of the P. ovalis dismutase will facilitate refinement of the X-ray crystallographic data that are now available at 2.9 A resolution.     

Genomic polymorphism in symbiotic populations of Photobacterium leiognathi

Photobacterium leiognathi forms a bioluminescent symbiosis with leiognathid fishes, colonizing the internal light organ of the fish and providing its host with light used in bioluminescence displays. Strains symbiotic with different species of the fish exhibit substantial phenotypic differences in symbiosis and in culture, including differences in 2-D PAGE protein patterns and profiles of indigenous plasmids. To determine if such differences might reflect a genetically based symbiont-strain/host-species specificity, we profiled the genomes of P. leiognathi strains from leiognathid fishes using PFGE. Individual strains from 10 species of leiognathid fishes exhibited substantial genomic polymorphism, with no obvious similarity among strains; these strains were nonetheless identified as P. leiognathi by 16S rDNA sequence analysis. Profiling of multiple strains from individual host specimens revealed an oligoclonal structure to the symbiont populations; typically one or two genomotypes dominated each population. However, analysis of multiple strains from multiple specimens of the same host species, to determine if the same strain types consistently colonize a host species, demonstrated substantial heterogeneity, with the same genomotype only rarely observed among the symbiont populations of different specimens of the same host species. Colonization of the leiognathid light organ to initiate the symbiosis therefore is likely to be oliogoclonal, and specificity of the P. leiognathi/leiognathid fish symbiosis apparently is maintained at the bacterial species level rather than at the level of individual, genomotypically defined strain types.     

Inactivation of Pseudomonas iron-superoxide dismutase by hydrogen peroxide

Pseudomonas Fe-superoxide dismutase (superoxide:superoxide oxidoreductase, EC 1.15.1.1) is inactivated by hydrogen peroxide by a mechanism which exhibits saturation kinetics. The pseudo-first-order rate constant of the inactivation increased with increasing pH, with an inflection point around pH 8.5. Two parameters of the inactivation were measured in the pH range 7.8 to 9.0; the total H2O2 concentration at which the enzyme is half-saturated (K inact) was found to be independent of pH (30 mM) and the maximum rate constant for inactivation (k max) increased progressively with increasing pH, from 3.3 min-1 at pH 7.8 to 21 min-1 at pH 9.0. This evidence suggests the presence of an ionization group (pKa approximately 8.5) which does not participate in the binding of H2O2 but which affects the maximum inactivation rate of the enzyme. The loss of dismutase activity of the Fe-superoxide dismutase is accompanied by a modification of 1.6, 1.1 and 0.9 residues of tryptophan, histidine and cysteine, respectively. Since the amino acid residues of the Cr-substituted enzyme, which has no enzymatic activity, were not modified by H2O2, the active iron of the enzyme is essential for the modification of the amino acid residues.     

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。     

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.     

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.     

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.