Int J Food Microbiol, 1995 Aug, 26(3), 319 - 33 Qualitative and quantitative characterization of spoilage bacteria from packed fish; Dalgaard P; The large cells recently suggested to be responsible for spoilage of packed cod, have been identified as Photobacterium phosphoreum . The spoilage activity of these cells, of Shewanella putrefaciens and of other microorganisms isolated form spoiled packed cod has been studied . Both qualitative and quantitative tests were used for characterization of the microbial spoilage activity . The importance of the different groups of microorganisms was evaluated by comparison of microbial spoilage activity determined in model substrates and in product experiments . The yield factor for production of trimethylamine (YTMA/CFU) and the cell concentration determined at the time of off-odour detection were used as quantitative measurements of microbial spoilage activity . On average cells of P . phosphoreum produced 30 times more TMA than cells of S . putrefaciens, YTMA/CFU of the two organisms were 10(-8.0) mg-N TMA/cfu and 10(-9.5) mg-N TMA/cfu, respectively . With these yield factors the level of TMA found in spoiled packed cod (30 mg-N TMA/100g) corresponds to about 10(7) cfu/g of P . phosphoreum and to 10(8)-10(9) cfu/g of S . putrefaciens . 10(7) cfu/g of P . phosphoreum were actually found in spoiled packed cod suggesting this organism could be responsible for spoilage . High cell concentrations of more than 10(8) cfu/g of S . putrefaciens were required for production of detectable off-odours and is was concluded that this organism is without importance for spoilage of packed cod. Int J Food Microbiol, 1995 Aug, 26(3), 305 - 17 Modelling of microbial activity and prediction of shelf life for packed fresh fish; Dalgaard P; Prediction of shelf life based on growth of specific spoilage organisms (SSO) in model substrates was studied . The effect of CO2 on the growth kinetics for Photobacterium phosphoreum and Shewanella putrefaciens was quantified and modelled . Results showed that microbial spoilage of packed cod stored with various concentrations of CO2 was accurately predicted from the effect of CO2 on P . phosphoreum grown in model substrates . The short shelf life extensions previously reported for packed cod therefore can be explained by the high CO2 resistance of this Gram negative organism . S . putrefaciens was very sensitive to CO2 and growth rates could not be related to the shelf life of packed cod . Growth curves without lag phases were found for all concentrations of CO2 and for both the microorganisms studied . For the fitting of these growth curves the log-transformed Logistic models were selected after comparison with the 'modified Gompertz' models and with the model of Baranyi et al . (1993) . The effect of CO2 on mu max was well described by a 2 parameter square root model . Validation of kinetic models by comparison of shelf life predictions with shelf life determined by sensory evaluations in product experiments was preferred for comparison of microbial growth rates determined in product and model system experiments . Kinetic modelling was found to be valuable for both evaluation and prediction of microbial fish spoilage and an iterative approach for development of kinetic shelf life models was suggested. Gene, 1995 Jul 28, 160(2), 303 - 4 Isolation of cDNAs encoding GTP cyclohydrolase II from Arabidopsis thaliana; Kobayashi M et al.; A GTP cyclohydrolase II-encoding gene from Arabidopsis thaliana was isolated through functional complementation of a mutant of Escherichia coli, BSV18, deficient in this protein . The derived amino-acid sequence constitutes a polypeptide of 27 kDa and shows 37-58% identity with previously published sequences of Escherichia coli, Bacillus subtilis, Photobacterium leiognathi and P . phosphoreum. Ecotoxicol Environ Saf, 1995 Jul, 31(2), 99 - 103 Microbial bioassays to assess the toxicity of solid-associated contaminants; Ronnpagel K et al.; Due to the effects that sediment or soil matrices have on the bioavailability of compounds, it has been difficult to screen toxicity of solid-associated contaminants . The majority of microbial assays for testing toxicity of soils and sediments have been performed on water or solvent extracts . These procedures lead to a fractionation of the toxicity, which may underestimate or overestimate exposure routes and consequently potential adverse environmental effects . Recently, a solid-phase Microtox assay which eliminates the need for soil extracts and utilizes whole sediments or soils has been developed . This report describes a toxicity testing procedure using the inhibition of dehydrogenase enzyme activity of Bacillus cereus as test parameter . Studies with soil samples and a synthetic sediment spiked with organic contaminants and copper indicate the higher sensitivity of both solid-phase bioassays compared to water extract testing . A comparison of the results demonstrates that the B . cereus contact test is more sensitive for copper than the Photobacterium phosphoreum solid-phase test. Chemosphere, 1995 Jul, 31(1), 2521 - 8 Quantitative structure-activity relationships as a tool to assess the comparative toxicity of organic chemicals; Dearden JC et al.; Quantitative structure-activity relationships of toxicity are discussed as a means of assessing the value of the Microtox test which uses the light-emitting bacterium Vibrio fisheri (Photobacterium phosphoreum) as a replacement for toxicity testing in higher species . The Microtox test is found to be a good surrogate for testing in fish, for compounds acting by the narcosis mechanism . However, for reactive chemicals the Microtox test significantly underestimates the potential hazard . It should not therefore be used in isolation for such chemicals, but rather as part of a battery of tests. Mol Microbiol, 1995 Jul, 17(2), 345 - 56 Characterisation of the yenI/yenR locus from Yersinia enterocolitica mediating the synthesis of two N-acylhomoserine lactone signal molecules; Throup JP et al.; Yersinia enterocolitica produces compounds capable of transcriptionally activating the Photobacterium fischeri bioluminescence (lux) operon . Using high-performance liquid chromatography, high resolution tandem mass spectrometry in conjunction with chemical synthesis, two signal molecules were identified and shown to be N-hexanoyl-L-homoserine lactone (HHL) and N-(3-oxohexanoyl)-L-homoserine lactone (OHHL) . A gene (yenI) was isolated from Y . enterocolitica and demonstrated to direct the synthesis of both HHL and OHHL . DNA sequence analysis revealed an open reading frame (ORF) of 642 bp encoding a protein (YenI) of 24.6 kDa with approximately 20% identity to the LuxI family of proteins . Northern blot analysis of yenI expression indicated yenI is transcribed as a single gene and 5' transcript mapping of yenI identified a transcriptional start site 89 bp upstream of the ORF . DNA sequence analysis of the region downstream of yenI located a second ORF, termed yenR, with significant homology to the LuxR family of transcriptional activators . An insertion mutation of yenI abolishes HHL and OHHL production, indicating its central role in N-acylhomoserine lactone synthesis in Y . enterocolitica . Transcriptional analysis using a chromosomal yenI::luxAB fusion has demonstrated that yenI is not subject to autoinduction but is expressed constitutively . Whilst production of the Yop proteins in the wild type and in yenI mutants is indistinguishable, two-dimensional SDS-PAGE analysis of total cell proteins indicated that a number of proteins lack the yenI mutant. Biochem Biophys Res Commun, 1995 Jun 26, 211(3), 774 - 9 The yellow bioluminescence bacterium, Vibrio fischeri Y1, contains a bioluminescence active riboflavin protein in addition to the yellow fluorescence FMN protein; Petushkov VN et al.; The yellow bioluminescence Y1 strain of Vibrio fischeri can produce a 22 kDa protein with either FMN or riboflavin as a bound fluorophore . Both forms are active for shifting the bioluminescence spectral maximum . The fluorescence spectral distribution of the two proteins differs slightly and the in vivo emission appears to be an equal mixture of the two . The bioluminescence activity of the riboflavin Y1 protein contrasts with the inactivity of the related Photobacterium type. J Mol Biol, 1995 May 26, 249(1), 195 - 214 Structural refinement of the non-fluorescent flavoprotein from Photobacterium leiognathi at 1.60 A resolution; Moore SA et al.; The crystallographically-determined structure of the non-fluorescent flavoprotein (NFP) from Photobacterium leiognathi, a homolog of the bacterial luciferase subunits, has been refined to a conventional R-factor {formula: see text} of 0.175 using synchrotron data between 10.0 and 1.60 A resolution . The molecular structure is a homodimer of beta/alpha domains, the monomer having structural similarities to (beta alpha)8 barrel proteins . However, one beta-strand and three alpha-helices of a typical (beta alpha)8 domain are not present in the NFP structure . The refined structure of NFP consists of the 228 amino acid polypeptide, 191 water molecules, a sulfate ion, and two flavin mononucleotides (FMNs) each with a covalently-attached myristate (C14 fatty acid) . Both flavin adducts are well-ordered and have exceptional electron density for both the FMN and the myristate moieties . Each flavin mononucleotide-myristate adduct is characterized by a stereospecific linkage (the S enantiomer) between C-6 of the flavin isoalloxazine ring and the C-3' atom of the fatty acyl chain . The stereospecific nature of this flavin-fatty acid linkage suggests that it is the result of an enzyme-catalyzed reaction, most likely the bioluminescence reaction itself . The myristate chains are buried from solvent in hydrophobic pockets in the interior of the protein . Four amino acid side-chains of the NFP polypeptide have been modeled with alternate conformations . Five of the protein's seven alpha-helices have classical C-capping boxes . NFP is dimeric and many of the extensive contacts at the dimer interface are mediated by hydrogen-bonded water molecules as well as by hydrophobic interactions . One of the myristate acyl chains sits between NFP monomers and contributes a significant portion of the hydrophobic interactions at the NFP dimer interface. Biochem Biophys Res Commun, 1995 May 25, 210(3), 938 - 47 Nucleotide sequence and functional analysis of regulatory region of the lumP and the lux operon from Photobacterium leiognathi; Lin JW et al.; The lumP gene is linked to the lux operon, but runs in the opposite direction in Photobacterium leiognathi PL741 . The gene order of the lumP and the lux operon is < -lumP-R & R-luxC-luxD-luxA-luxB-luxN-luxE- > (R & R: regulatory region) . The nucleotide sequence of the regulatory region (827-bp) between the lumP and the lux operon was determined . Sequence analysis illustrates that the regulatory region includes two divergent promoter systems, PR-promoter system for the lux operon (R-operon) and PL-promoter system for the lumP or lum operon (L-operon) . Functional analysis of the regulatory region shows that the PR- and PL-promoter systems both are able to lead the gene expression . The deletion experiment result elicits that the PR- and PL-promoter are coordinatively and negatively regulated; the PR- and PL-promoter might be competing for recognition by RNA polymerase to initiate transcription . The fact of the LumP responsible for the spectral blue shift in P . leiognathi implied that the lumP gene closedly linked to the lux operon is for coordinative regulation with the lux operon . In addition, the glucose repression on the PR-promoter system shows that the expression of the lux operon is regulated by cAMP-CRP induction in E . coli. J Biolumin Chemilumin, 1995 May-Jun, 10(3), 157 - 67 Formation of active bacterial luciferase between interspecific subunits in vivo; Almashanu S et al.; Interspecific complementation between luxAs and luxBs from Vibrio harveyi, Vibrio fischeri, Photobacterium leiognathi and Xenorhabdus luminescens was examined in vivo . The individual genes from these species were cloned on different compatible plasmids or amplified by PCR and brought together to yield cis combinations without extraneous DNA . The beta subunits from V . harveyi and X . luminescens form active enzyme only with alpha subunits from one of these species . All other combinations yield active enzymes . The lack of activity of the V . harveyi and X . luminescens beta subunits with the alpha subunits from V . fischeri and P . leiognathi results from a lack of association . This was shown by in vivo competition in which these beta subunits were overproduced in comparison with the beta and alpha of V . fischeri . No reduction in light was found . Overall, the in vivo results parallel those found in vitro using isolated denatured subunits and renaturation by removal of the denaturant. Ecotoxicol Environ Saf, 1995 Apr, 30(3), 221 - 51 Review of whole-organism bioassays: soil, freshwater sediment, and freshwater assessment in Canada; Keddy CJ et al.; Whole organism bioassays for the assessment of soil, freshwater sediment, and freshwater quality were evaluated for their application in the assessment and remediation of contaminated sites in Canada under the National Contaminated Sites Remediation Program . Using 3 essential and 12 desirable methodological criteria, bioassays were categorized as currently usable, prototype, or under development . Based on further considerations related to bioassay application, a battery of usable screening and definitive tests was recommended (with suggestions for augmentation) for each medium . Of the 18 bioassays reviewed for soil quality assessment, 6 were usable, 5 were prototypes, and 7 were under development . Battery screening and definitive tests included 14-day Eisenia andrei survival, 120-hr lettuce and radish seedling emergence, and 72-hr Selenastrum capricornutum growth inhibition . Augmentation with the following bioassays was recommended: soil/freshwater bacterial growth, arthropod reproduction, earthworm reproduction, and reproduction of other soil-dependent organisms . Of the 9 bioassays reviewed for freshwater sediment quality assessment, 1 was usable, 2 were prototypes, and 6 were under development . Three bioassays in the latter two groups were considered usable with the imminent completion of research underway . Screening tests selected included 10-day Chironomus tentans survival, 10-day Hyalella azteca survival, 10-day Hexagenia spp . survival, and 72-hr S . capricornutum growth inhibition . Definitive tests included screening tests, substituting 28-day H . azteca sexual maturation for 10-day survival . Augmentation with the following bioassays was recommended: sediment/freshwater bacterial test, 28-day Tubifex tubifex reproduction, and rooted aquatic plant growth . Of the 25 bioassays considered for freshwater quality assessment, 8 were usable, 7 were prototypes, and 10 were under development . Screening tests selected included 72-hr S . capricornutum growth inhibition; 48-hr Daphnia sp . survival, and 5- and 15-min Photobacterium phosphoreum bioluminescence . Definitive tests included first screening test, 7-day Ceriodaphnia dubia, 7-day fathead minnow larval survival, or 96-hr rainbow trout survival . Augmentation with the following bioassays was recommended: Brachionus calyciflorus 24-hr survival, 48-hr reproduction; freshwater bacterial growth; and aquatic vascular plant growth. Biochemistry, 1995 Mar 14, 34(10), 3300 - 9 Properties of recombinant fluorescent proteins from Photobacterium leiognathi and their interaction with luciferase intermediates; Petushkov VN et al.; Ligand binding and luciferase interaction properties of the recombinant protein corresponding to the lumazine protein gene (EMBL X56534) of Photobacterium leiognathi have been determined by fluorescence dynamics, circular dichroism, gel filtration, and SDS-PAGE . Scatchard analysis of a fluorescence titration shows that the apoprotein possess one binding site, and at 30 degrees C the KdS (microM) are as follows: 6,7-dimethyl-8-ribityllumazine, 0.26; riboflavin, 0.53; and much more weakly bound FMN, 30 . All holoproteins are highly fluorescent and have absorption spectra distinct from each other and from the free ligands . The longest wavelength absorption maxima are, respectively (nm, 2 degrees C), 420, 463, and 458 . Ligand binding produces no change in the far-UV circular dichroism; all have mean residual ellipticity at 210 nm of -6500 deg cm2 dmol-1, the same as the native protein . However, in the bioluminescence reaction only the lumazine holoprotein shows a bioluminescence effect . Fluorescence emission anisotropy decay was used to establish that none of these holoproteins complexed with native luciferase and that the lumazine protein alone formed a 1:1 complex with the luciferase hydroxyflavin fluorescent transient and the luciferase peroxyflavin intermediates, revealed by a dominant channel of anisotropy loss, with rotational correlation time of 2.5 ns, and attributed to excitation transfer from the luciferase flavin donor to the acceptor, the lumazine ligand . The complex stability was sufficient to allow its isolation by FPLC gel filtration and verification by SDS-PAGE . These methods also confirmed the absence of interaction of the holoflavoproteins. J Biochem (Tokyo), 1995 Mar, 117(3), 575 - 8 Crystal structure determination of a flavoprotein FP390 from a luminescent bacterium, Photobacterium phosphoreum; Kita A et al.; The three-dimensional structure of a flavoprotein, FP390, purified from a luminescent bacterium, Photobacterium phosphoreum, has been determined at 3 A resolution by X-ray crystallography . Crystallographic refinements of the structural model have led to an R-factor of 0.24 for the intensity data between 6 to 3 A resolution collected with synchrotron radiation . It was found that a homodimer of the FP390 molecules related by a non-crystallographic 2-fold axis is comprised in the asymmetric unit . Two homodimers are arranged around a crystallographic 2-fold axis to form a tetrameric assembly . The monomer molecule of FP390, to which two molecules of the flavin cofactor (Q-flavin) are bound, consists of a seven-stranded parallel beta-sheet which forms a half of the beta-barrel structure and seven alpha-helices which surround one side of the beta-barrel . We suggest that the reason why the Q-flavin sample prepared from FP390 is always a mixture of two components is connected with the fact that the monomer molecules has two flavin binding sites, at the dimer interface and at the molecular surface. J Bacteriol, 1995 Feb, 177(4), 1008 - 16 ompH gene expression is regulated by multiple environmental cues in addition to high pressure in the deep-sea bacterium Photobacterium species strain SS9; Bartlett DH et al.; Photobacterium species strain SS9 is a moderately barophilic (pressure-loving) deep-sea bacterial species which induces the expression of the ompH gene in response to elevated pressure . Here we demonstrate that at 1 atm (1 atm = 1.01325 x 10(5) Pa), ompH expression increases with cell density in 2216 marine medium batch culture and is subject to catabolite repression and the OmpH synthesis is inducible by energy (carbon) starvation . Regulatory mutants which are impaired in ompH gene expression at high pressure are also impaired in cell density regulation of ompH gene expression, indicating that the two inducing conditions overlap in their signal transduction pathways . The same promoter was activated by high cell density at 1 atm of pressure as well as during low-cell-density growth at 272 atm . Catabolite repression of ompH gene expression was induced by a variety of carbon sources, and this repression could be partially reversed in most cases by the addition of cyclic AMP (cAMP) . Surprisingly, glucose repression of ompH transcription occurred only at 1 atm, not at 272 atm, despite the fact that catabolite repression was operational in SS9 under both conditions . It is suggested that ompH expression is cAMP and catabolite repressor protein dependent at 1 atm but becomes cAMP and perhaps catabolite repressor protein independent at 272 atm . Possible mechanisms of ompH gene activation are discussed. FEMS Microbiol Lett, 1995 Jan 1, 125(1), 101 - 5 A 26-kDa outer membrane protein, OmpK, common to Vibrio species is the receptor for a broad-host-range vibriophage, KVP40; Inoue T et al.; KVP40 is a broad-host-range vibriophage forming plaques on strains of at least eight Vibrio and one Photobacterium species . A spontaneous KVP40-resistant mutant, R4000, derived from Vibrio parahaemolyticus 1010 lacked a 26-kDa outer membrane protein designated OmpK . KVP40 was inactivated by outer membrane and OmpK prepared from 1010, but not by outer membrane from R4000 . These results strongly suggest that OmpK is the receptor for KVP40 . Immunoblotting analyses using an anti-OmpK rabbit serum revealed that OmpK or its homologs of molecular masses 25-29 kDa were distributed widely among Vibrio and Photobacterium strains including those naturally resistant to KVP40. J Biolumin Chemilumin, 1995 Jan-Feb, 10(1), 21 - 7 Interactions between aldehyde derivatives and the aldehyde binding site of bacterial luciferase; Jockers R et al.; The interaction of triazine aldehydes with the aldehyde binding site of bacterial luciferases was investigated using a series of triazine aldehydes with different aldehyde chain length, and substituents on the s-triazine ring . Substrate activity was determined using luciferase from Photobacterium fischeri and Vibrio harveyi in a dithionite-based luciferases assay . The chain length optimum was determined for two triazine aldehyde classes to be C-10 and C-11, respectively . Only the substrate activity of 10-(4-chloro-6-methylthio-s-triazine-2-yl)aminodecanal (5) was as high as n-decanal, the reference aldehyde . All other triazine derivatives reduced light emission, probably by hindered binding of the substrates . The degree of activity reduction correlated with the volume of the triazine ring moiety . The triazine moiety volume of compound 5 was estimated to be 200 x 10(-30) m3 . Triazine aldehydes which showed reduced light emission had an estimated volume of 228 x 10(-30) m3 or greater . All triazine aldehydes showed approximately 10-fold lower activities for Vibrio harveyi than for Photobacterium fischeri luciferase . Substrate specificity was the same for both luciferases . A schematic superposition of quinone aldehydes and triazine aldehydes which showed substrate activities equivalent to n-decanal, indicated potential interaction sites of aldehyde substrates with the aldehyde binding site of bacterial luciferases . The in vivo relevance of the results is discussed. Biochim Biophys Acta, 1994 Nov 11, 1201(2), 251 - 8 Expression and properties of the recombinant lumazine (riboflavin) protein from Photobacterium leiognathi; Illarionov B et al.; Photobacterium leiognathi lumazine protein has been expressed in Escherichia coli in high yield, 30 mg/l . The cloned gene was one previously reported by Illarionov (EMBL X56534), that had a similar sequence and was located in the same position as the lumazine protein gene in P . phosphoreum . This gene was placed downstream of the T7 gene 10 promoter of the plasmid pT7-7 . When the E . coli are grown at 37 degrees C the protein accumulates in inclusion bodies but solubilization can be achieved in 6 M urea . By a simple procedure of dialysis in the presence of riboflavin and centrifugation, without any chromatography, the recombinant holoprotein is purified to 95% homogeneity . The spectral properties of this recombinant riboflavin protein are the same as those of a fluorescent riboflavin-bound protein produced by many strains of P . leiognathi . The absorption spectrum has the same maxima, 276, 386, 464 nm, the circular dichroism is also the same, and both absorption spectrum and CD are the same as that of apo-lumazine protein having riboflavin bound . The riboflavin on the recombinant can be easily replaced by 6,7-dimethyl-8-ribityllumazine . The absorption and fluorescence spectra, fluorescence yield, and bioluminescence properties of this rebound protein identify it as authentic lumazine protein. Protein Sci, 1994 Nov, 3(11), 1914 - 26 Common structural features of the luxF protein and the subunits of bacterial luciferase: evidence for a (beta alpha)8 fold in luciferase; Moore SA et al.; The amino acid sequence identity and potential structural similarity between the subunits of bacterial luciferase and the recently determined structure of the luxF molecule are examined . The unique beta/alpha barrel fold found in luxF appears to be conserved in part in the luciferase subunits . From secondary structural predictions of both luciferase subunits, and from structural comparisons between the protein product of the luxF gene, NFP, and glycolate oxidase, we propose that it is feasible for both luciferase subunits to adopt a (beta alpha)8 barrel fold with at least 2 excursions from the (beta alpha)8 topology . Amino acids conserved between NFP and the luciferase subunits cluster together in 3 distinct "pockets" of NFP, which are located at hydrophobic interfaces between the beta-strands and alpha-helices . Several tight turns joining the C-termini of beta-strands and the N-termini of alpha-helices are found as key components of these conserved regions . Helix start and end points are easily demarcated in the luciferase subunit protein sequences; the N-cap residues are the most strongly conserved structural features . A partial model of the luciferase beta subunit from Photobacterium leiognathi has been built based on our crystallographically determined structure of luxF at 1.6 A resolution. Ecotoxicol Environ Saf, 1994 Nov, 29(2), 200 - 13 Mechanism-based comparisons of acute toxicities elicited by industrial organic chemicals in procaryotic and eucaryotic systems; Jaworska JS et al.; Comparisons of toxicities elicited by nonpolar and polar narcotics, weak acid uncouplers of oxidative phosphorylation, and bioreactive chemicals between the eucaryotic systems Pimephales promelas and Tetrahymena pyriformis and the procaryotic systems Escherichia coli and Photobacterium phosphoreum were performed . Each chemical had been a priori assigned a mechanism/mode of action based on the results from previous studies with eucaryotic systems . Hydrophobicity-dependent QSARs for nonpolar narcosis for both the E . coli and the P . phosphoreum endpoints was developed . However, due to the lack of a significant relationship between P . phosphoreum toxicity and log Kow, such a QSAR for polar narcosis was developed only for the E . coli endpoint . Except for 4-nitroaniline (the only chemical in the examined group that required activation to become the Michael receptor), all chemicals containing reactive substructures revealed excess toxicity over polar narcosis QSAR for E . coli endpoints . Moreover, chloroacidic acid and ethyl chloroacetate in this system also appear to be bioreactive . The only mechanism that seemed to not exist in the procaryotic system was uncoupling of oxidative phosphorylation . Chemicals from this group, except 2,4-dinitroaniline, did not exhibit excess toxicity over polar narcosis QSAR . This was thought to be explained by the lack of mitochondria in procaryotes, the target site of uncoupling agents in eucaryotes . In addition, evaluation of toxicities of halogen-substituted short-chain carboxylic alcohols indicated that their mechanisms vary, depending upon the type of substitution and the system. Eur J Biochem, 1994 Aug 1, 223(3), 1007 - 17 The lumazine synthase/riboflavin synthase complex of Bacillus subtilis . X-ray structure analysis of hollow reconstituted beta-subunit capsids; Ladenstein R et al.; The lumazine synthase/riboflavin synthase complex of Bacillus subtilis consists of an icosahedral capsid of 60 beta subunits enclosing a triplet of alpha subunits . An X-ray structure of 0.32 nm resolution has been obtained for the icosahedral capsid of the native alpha 3 beta 60 complex {Ladenstein, R., Schneider, M., Huber, R., Bartunik, H . D., Wilson, K., Schott, K . & Bacher, A . (1988) J . Mol . Biol . 203, 1045-1070} . beta subunits were isolated after denaturation of the alpha 3 beta 60 complex and were subsequently reconstituted in a ligand-driven reaction yielding artifactual, hollow beta 60 capsids with icosahedral symmetry . Hexagonal crystals (space group P6(3)22) of the reconstituted capsids diffracted X-rays to a resolution of 0.32 nm . Crystallographic intensity data were obtained using synchrotron radiation . Freeze-etched electron-microscopic images and rotation function calculations showed that the hexagonal crystal forms of the artifactual beta 60 capsids and the native alpha 3 beta 60 complex are isomorphous . Orientation and translation parameters of the beta-subunit model were refined by XPLOR rigid-body refinement . The electron-density map was improved by cyclic icosahedral averaging and phase extension from 0.5-0.32 nm resolution . The beta-subunit structure was partially refined by energy minimization and crystallographic refinement (XPLOR) assuming strict icosahedral symmetry (final R factor 30.9% for data at 0.8-0.32 nm resolution) . The topology and chain folding of the beta subunits in the artifactual beta 60 capsid are similar to the native alpha 3 beta 60 enzyme . Structural features of the substrate-binding site and the binding of the substrate-analogous ligand 5-nitro-6-ribitylamino-2,4(1H,3H)-pyrimidinedione are discussed . Ligand binding occurs at the pentamer interfaces and includes van der Waals' interactions and hydrogen bonding . The binding pocket shows a hydrophobic region which accomodates the pyrimidinedione ring and a hydrophilic region to which the ribityl side chain binds . Most amino acid residues involved in the active site are conserved as shown by sequence comparisons with the putative lumazine-synthase genes of Escherichia coli and Photobacterium leiognathi . In the final electron-density map, a residual density feature was tentatively assigned to a bound phosphate ion which mimics the binding of the second substrate, 3,4-dihydroxy-2-butanone 4-phosphate . This putative phosphate-binding site involves a highly conserved amino acid sequence containing three basic residues. Ecotoxicol Environ Saf, 1994 Aug, 28(3), 317 - 28 The environmental risks of industrial waste disposal: an experimental approach including acute and chronic toxicity studies; Lambolez L et al.; The toxicity of 15 leachates of various solid industrial wastes accepted in an engineered landfill has been studied . A cost-effective battery of tests allowing evaluation of acute and chronic toxicity, as well as genotoxicity, and investigations on different trophic levels in the aquatic environment has been used . Acute toxicity was tested on bacteria (Microtox assay with Photobacterium phosphoreum) and microcrustaceans (Daphnia magna immobilization assay) . A growth inhibition test of microalgae was carried out on Raphidocelis subcapitata . A 28-day chronic test with Daphnia magna was used to detect effects on reproduction . Genotoxicity was evaluated by means of the Ames test conducted on the crude aqueous phase and also on the concentrated fractions of water-extractable micropollutants (liquid-liquid and freeze-dried extracts) . Chemical analyses of leachates were carried out simultaneously . The toxicity varied greatly between the different wastes . Toxic effects were observed in the short and/or in the long term . Four samples were potentially genotoxic . In most cases, toxicity registered could not be correlated with results of the chemical analyses . This study demonstrates the usefulness of associating a toxicological monitoring with chemical analyses in waste management. Chemosphere, 1994 Jul, 29(2), 371 - 90 Ecotox-evaluation strategy for soil bioremediation exemplified for a PAH-contaminated site; Hund K et al.; During a bioremediation of a PAH-contaminated site chemical and biological analyses were carried out . The biological investigations included ecotoxicological analyses in the aqueous extract, (Pseudomonas putida, Photobacterium phosphoreum, daphnids, algae, fish) and analyses in the soil with introduced organisms (plants, earthworms) and natural soil organisms (nematodes, microorganisms) . In all test systems a correspondence between decreasing toxicity and degradation of the easily biodegradable PAHs was found . From investigations with aqueous extracts therefore not only conclusions on potential risks for groundwater can be drawn, but these tests also seem to allow risk assessments for soil inhabitants . Furthermore with these tests comprising dilution series the extent of toxicity for soil organisms can be quantified more precisely than with terrestrial investigations . Of all aquatic test systems a constant remaining toxicity was found only in the Microtox text . The test with Daphnia magna indicated the intermediate formation of organism specific toxic metabolites . Therefore useful information may be obtained with biological analyses which complement chemical analyses . For an extensive assessment of a contaminated site a test battery is advisable. Biochemistry, 1994 Jun 21, 33(24), 7634 - 40 (Trifluoromethyl)lumazine derivatives as 19F NMR probes for lumazine protein; Scheuring J et al.; Lumazine protein acts as an electronic excited state transducer in bioluminescence of Photobacterium species . The protein binds 6,7-dimethyl-8-(D-ribityl)lumazine (1) which serves as the fluorophore . This compound also serves as a biosynthetic precursor of riboflavin and is the substrate of the enzyme riboflavin synthase . This enzyme and lumazine protein show considerable sequence homology . The interaction of lumazine apoprotein with several trifluoromethyl analogs of 6,7-dimethyl-8-ribityllumazine was investigated by 19F NMR spectroscopy . Upon binding to the protein, the 19F NMR resonances of the ligand shift to lower field with broadened line widths to around 30 Hz . By comparison, all ligands studied show more complex NMR spectra when bound to riboflavin synthase . Only one position 7 epimer (designated epimer A) of 6,7-bis(trifluoromethyl)-7-hydroxy-8-(D-ribityl)lumazine binds to lumazine apoprotein and riboflavin synthase . The apoprotein can also bind lumazine derivatives with a quarternary C-7 . It is suggested that the binding site of lumazine protein corresponds to the donor binding site of riboflavin synthase. Biomed Environ Sci, 1994 Jun, 7(2), 101 - 8 UV-A coexposure enhances the toxicity of aromatic hydrocarbons, munitions, and metals to Photobacterium phosphoreum; Arfsten DP et al.; Johnson et al . (1993) showed that coexposure to UV-A between 300-400 nm enhanced the toxicity of nitrotoluenes to Photobacterium phosphoreum, a marine bioluminescent bacteria used in the Microtox test (Microbics Inc.) . This paper reports that UV-A photoenhanced the toxicity of polynuclear aromatic hydrocarbons, other types of organic compounds, and some transition metals to P . phosphoreum . Coexposure to 400 muw/cm2 for 15 min increased the toxicity of psoralen, alpha-terthienyl, anthracene, acridine, fluoranthene, TNT, Cu2-, As3-, Ni2, and Cd2+ . Phenanthrene was photoenhanced after 30 min coexposure at 400 muw/cm2-, and Mn2+ at 800 muw/cm2 after 15 min . Naphthalene was not enhanced at 800 muw/cm2 for 30 min. J Bacteriol, 1994 Apr, 176(7), 2100 - 4 Riboflavin synthesis genes are linked with the lux operon of Photobacterium phosphoreum; Lee CY et al.; 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. J Biochem (Tokyo), 1994 Apr, 115(4), 670 - 4 Preparation of P-flavin-bound and P-flavin-free luciferase and P-flavin-bound beta-subunit of luciferase from Photobacterium phosphoreum; Kasai S; P-flavin-bound luciferase, P-flavin-free luciferase, and P-flavin-bound beta-subunit of luciferase were prepared from Photobacterium phosphoreum using hydrophobic interaction chromatography after conventional purification using DEAE-cellulose chromatography and gel-filtration . The P-flavin-bound luciferase preparation contained about 20% P-flavin-free luciferase not removable by the present procedure . Since the specific activity of the P-flavin-bound luciferase preparation was about 20% of that of the P-flavin-free luciferase, it was concluded that the P-flavin-bound luciferase is an enzyme-product complex and has no more luciferase activity . Unlike the absorption spectrum of FP390 or other flavoproteins, that of P-flavin-bound luciferase preparation has a high absorption peak around 370 nm and resembles the spectrum synthesized by superposing the P-flavin-free luciferase spectrum on the P-flavin-bound beta-subunit spectrum: the P-flavin-bound beta-subunit spectrum is similar to that of FP390, while that of P-flavin-free luciferase has an absorption peak around 370 nm but practically no peak around 450 nm . In addition, P-flavin-free luciferase exhibits a weak but distinct NADH-FMN oxidoreductase activity . These results suggest that a prosthetic group, which absorbs around 370 nm, binds to the luciferase and that this compound is required to yield P-flavin; and they support the hypothesis that the physiological function of bacterial luciferase is to produce P-flavin . Furthermore, the presence of P-flavin-bound beta-subunit of the luciferase in the cell extract supports the hypothesis that physiological function of the lux operon is the biosynthesis of FP390 including its prosthetic group. J Biol Chem, 1994 Mar 4, 269(9), 6683 - 8 An essential histidine residue required for fatty acylation and acyl transfer by myristoyltransferase from luminescent bacteria; Ferri SR et al.; The lux-specific acyltransferases are serine esterases responsible for preferential diversion of myristic acid from fatty acid biosynthesis to the luminescent system . In contrast to other acyltransferases, an acylated enzyme intermediate can readily be detected making it ideal for the study of the mechanism of acyl transfer . Although the transferase readily cleaves acyl carrier protein and acyl-CoA, an alternate more rapid and convenient assay involving the cleavage of p-nitrophenyl acyl esters was developed and applied in these studies . The cleavage of the oxyesters by the transferase was shown to have a similar dependence on fatty acid chain length and organic solvents as the cleavage of thioesters . Using this assay, it could be demonstrated that the Photobacterium phosphoreum transferase was inactivated at pH 6 with diethyl pyrocarbonate at a rate (73 M-1 s-1, 10 degrees C) even faster than that reported for other enzymes with reactive histidyl residues at their active site . Spectral changes during chemical modification as well as restoration of activity by neutral hydroxylamine showed that the loss of activity was associated with modification of a single histidine residue . Replacement of the four histidine residues, conserved in all lux-specific acyltransferases, by asparagine demonstrated that cleavage of both thioesters and oxyesters by the P . phosphoreum acyltransferase as well as acylation of the enzyme was blocked on mutation of His-244 but not the other three conserved histidines (His-12, -52, and -75) . These results suggest that the histidine residue near the carboxyl terminus (His-244) may be part of a catalytic triad essential for cleavage of acyl esters and transfer of the acyl group to the enzyme. Food Chem Toxicol, 1994 Feb, 32(2), 173 - 87 Human acute toxicity prediction of the first 50 MEIC chemicals by a battery of ecotoxicological tests and physicochemical properties; Calleja MC et al.; Five acute bioassays consisting of three cyst-based tests (with Artemia salina, Streptocephalus proboscideus and Brachionus calyciflorus), the Daphnia magna test and the bacterial luminescence inhibition test (Photobacterium phosphoreum) are used to determine the acute toxicity of the 50 priority chemicals of the Multicentre Evaluation of In Vitro Cytotoxicity (MEIC) programme . These tests and five physiocochemical properties (n-octanol-water partition coefficient, molecular weight, melting point, boiling point and density) are evaluated either singly or in combination to predict human acute toxicity . Acute toxicity in human is expressed both as oral lethal doses (HLD) and as lethal concentrations (HLC) derived from clinical cases . A comparison has also been made between the individual tests and the conventional rodent tests, as well as between rodent tests and the batteries resulting from partial least squares (PLS), with regard to their predictive power for acute toxicity in humans . Results from univariate regression show that the predictive potential of bioassays (both ecotoxicological and rodent tests) is generally superior to that of individual physicochemical properties for HLD . For HLC prediction, however, no consistent trend could be discerned that indicated whether bioassays are better estimators than physicochemical parameters . Generally, the batteries resulting from PLS regression seem to be more predictive than rodent tests or any of the individual tests . Prediction of HLD appears to be dependent on the phylogeny of the test species: cructaceans, for example, appear to be more important components in the test battery than rotifers and bacteria . For HLC prediction, one anostracan and one cladoceran crustacean are considered to be important . When considering both ecotoxicological tests and physicochemical properties, the battery based on the molecular weight and the cladoceran crustacean predicts HLC substantially better than any other combination. Curr Opin Biotechnol, 1994 Feb, 5(1), 54 - 9 Immobilized cells used for detection and analysis; Karube I et al.; Many kinds of biosensor have now been developed and utilized for various types of analysis including clinical, medical and environmental monitoring, industrial process control, as well as many other applications . The microbial biosensor has advantages, such as longer lifetime and lower cost, over other types of biosensor . Recently, photobacteria and recombinant bacteria have been employed in biosensors both for determining biochemical oxygen demand and for detecting heavy metals and other toxic compounds. Ecotoxicol Environ Saf, 1994 Feb, 27(1), 23 - 33 Phototoxicology . 2 . Near-ultraviolet light enhancement of Microtox assays of trinitrotoluene and aminodinitrotoluenes; Johnson LR et al.; Coexposure of 2,4,6-trinitrotoluene (TNT), 2-amino-4,6-dinitrotoluene (2A), or 4-amino-2,6-dinitrotoluene (4A) to near-ultraviolet (nuv) light (lambda max-354 nm) significantly enhanced their toxicity toward Photobacterium phosphoreum (Microtox bioassay) during 30 min but not 15 min . Based on the slopes of the dose-response lines, the nuv coexposure and dark toxic mechanisms of action for TNT, 2A, and 4A appeared to be similar . nuv coexposure of binary mixtures significantly enhanced (supraadditivity) the toxicity of these compounds to P . phosphoreum . Under normal laboratory lighting, the toxicity of TNT + 2A and 2A + 4A mixtures were supraadditive but the toxicity of TNT + 4A mixtures could be explained by simple addition . Supporting these conclusions, the response curves of alpha-terthienyl, a compound known not to require nuv for toxicity, were similar in the dark and with nuv coexposure . In contrast, angelicin and psoralen, compounds known to require nuv coexposure to damage DNA, gave response curves having different slopes in the dark and with nuv coexposure . The nuv coexposure Microtox assay was able to detect and quantify phototoxicity in psoralen, angelicin, alpha-terthienyl, anthracene, TNT, and aminodinitrotoluenes. Biochem Biophys Res Commun, 1994 Jan 14, 198(1), 40 - 4 An upper limit for the effect of 60 Hz magnetic fields on bioluminescence from the photobacterium Vibrio fischeri; Greenbaum MP; Bioluminescence from Vibrio fischeri was measured in the presence and absence of 60 Hz magnetic fields . The peak value of the field was approximately 1.3 mT, a value approximately 13 times the Earth's background static field and comparable to the AC field near heavy-duty electrical equipment such as generators . The objective of this work was a search for causality between the applied magnetic field and a basic biological function at the biochemical, membrane or cellular level based on the direct linkage of bioluminescence to many of the cells mandatory functions such as enzyme (luciferase) activity, electron transport, proton translocation, iron uptake, oxidative metabolism, and cellular communication via the autoinducer N-{3-oxohexanoyl} homoserine lactone . A variation in the activity of any one of these functions will cause a change in bioluminescence . The key result of this work is that, for a signal to noise ratio of 1:1, an effect, if present at all, must be less than 1% of the baseline level of continuously monitored bioluminescence. Arch Microbiol, 1994, 162(5), 323 - 8 Genetic characterization of ompH mutants in the deep-sea bacterium Photobacterium sp . strain SS9; Bartlett D et al.; OmpH is an outer membrane protein produced by the deep-sea bacterium Photobacterium species strain SS9 in response to elevated hydrostatic pressure . In order to facilitate studies of the function of this protein, a series of OmpH+ and OmpH- strains were obtained from SS9 by Tn5 gene replacement mutagenesis . A previously isolated ompH::lacZ strain and a derivative of this strain harboring a plasmid expressing the wild-type ompH gene were also utilized . The acridine mutagen ICR 191 preferentially inhibited the growth of OmpH+ over OmpH- cells . Indeed, OmpH+ cultures treated with the mutagen rapidly accumulated mutants producing reduced levels of OmpH . In addition, OmpH+ cells took up the peptide Met-Leu-Phe approximately 15 times more rapidly than OmpH- cells . The results are consistent with the hypothesis that OmpH functions as a relatively large, nonspecific diffusion channel. Biophys Chem, 1993 Dec, 48(2), 149 - 58 Lumazine protein and the excitation mechanism in bacterial bioluminescence; Lee J; The spectral properties of lumazine protein and mixtures with the intermediates of the bacterial luciferase reaction, are reviewed . Measurements of fluorescence dynamics in particular have been employed with the aim of elucidating the mechanism by which lumazine protein functions in the bioluminescence of the bacteria of the type Photobacterium . The reaction of bacterial luciferase with its substrates produces bioluminescence emission with a spectral maximum at 496 nm . This spectrum is the same as the fluorescence of a luciferase flavin intermediate in the reaction, called the Fluorescent Transient . When lumazine protein is also present in the reaction; however, the bioluminescence emission now corresponds to the fluorescence of lumazine protein, which has a maximum at 475 nm . From measurements of the decay of fluorescence anisotropy of lumazine protein alone and in mixtures with the luciferase fluorescent transient, it is shown that a protein-protein complex is formed and that there is rapid energy transfer between the flavin on the luciferase and the lumazine derivative bound to its protein . An approximate calculation estimates the rate of this energy transfer to be faster than 10(9) s-1, and this would account for the efficient transfer of excitation from the flavin on the associated luciferase in the mixed protein bioluminescence reaction. J Bacteriol, 1993 Dec, 175(23), 7533 - 40 Use of a reporter gene to follow high-pressure signal transduction in the deep-sea bacterium Photobacterium sp . strain SS9; Chi E et al.; Photobacterium sp . strain SS9 is a deep-sea bacterium which modulates the abundances of several outer membrane proteins as a function of hydrostatic pressure . These proteins include the product of the previously cloned ompH gene (D . H . Bartlett, M . Wright, A . A . Yayanos, and M . Silverman . Nature (London) 342:572-574, 1989) . Subsequent to conjugal plasmid delivery it was possible to cross an ompH::lacZ transcriptional fusion into the genome of SS9, replacing the wild-type ompH gene, generating strain EC10 . EC10 is not impaired in growth at high pressure, indicating that under the growth conditions employed, OmpH is not required for baroadaptation . beta-Galactosidase production in EC10 is induced by high pressure to approximately the same extent that OmpH production is in the parental strain, SS9 . Therefore, OmpH abundance appears to be primarily regulated at the transcriptional level . EC10 was used for the isolation of ompH regulatory mutants . Derivatives of EC10 which produce reduced levels of beta-galactosidase at both low and high pressure and which appeared to possess mutations outside the ompH::lacZ locus were obtained . All of these regulatory mutants displayed alterations in the high-pressure repression of a second outer membrane protein, designated OmpL, and two of the mutants were also deficient in the high-pressure induction of a third outer membrane protein, designated OmpI . The most dramatic phenotype was present in mutant EC1002, whose growth was extremely barosensitive . EC1002 is the first pressure-sensitive mutant ever isolated . Prolonged incubation of EC1002 at high pressure led to the accumulation of cells with wild-type growth characteristics at high pressure . These cells are suggested to possess suppressor mutations, as they remain deficient in beta-galactosidase production and maintain their high-pressure-adapted phenotype for many generations in the absence of high-pressure selection. J Biolumin Chemilumin, 1993 Nov-Dec, 8(6), 293 - 9 Growth and luminescence of luminous bacteria promoted by agents of microbial origin; Rodicheva EK et al.; The examination of four species of luminous bacteria Photobacterium leiognathi, Photobacterium phosphoreum, Vibrio fischeri and Vibrio harveyi has enabled us to reveal some nutrient medium components effecting growth, luminescence intensity and luciferase synthesis . These agents are nucleic components (nucleotides, nucleotides and amine bases), amino acids and vitamins, which are part of hydrolysates from the biomass of various lithotrophic microorganisms, hydrogen-oxidizing, iron-oxidizing and carboxydobacteria . The effect of promoting agents essentially alters the physiological state and ultrastructure of the cells of luminous bacteria and increases luciferase biosynthesis two- to three-fold compared to a control. Int J Food Microbiol, 1993 Sep, 19(4), 283 - 94 Spoilage and shelf-life of cod fillets packed in vacuum or modified atmospheres; Dalgaard P et al.; Microbial growth, sensory and chemical changes and composition of gas atmosphere were studied in vacuum packed (VP) and modified atmosphere packed (MAP) cod fillets stored at 0 degree C . Contrary to previous studies, coccobacilli and pleomorphic Gram-negative microorganisms (2-4 by 2-5 microns) and not Shewanella putrefaciens were found most likely to be the main spoilage organisms . These microorganisms, which may be Photobacterium phosphoreum, can explain the short shelf-life extension of VP and MAP fish products compared to meat products . It is suggested that they may inhibit the typical H2S-producing fish spoilage bacteria, S . putrefaciens, as the maximum concentration of H2S-producing bacteria found in MAP fish products is very low . Compared to VP, a shelf-life extension of 6-7 days was obtained with 48% CO2 in MAP . However, with pure CO2 the shelf life was only extended by 2-3 days . Poor texture and high drip loss indicated that the shelf life of these fillets was limited by chemical reactions and not only by microbial activity. Nature, 1993 May 13, 363(6425), 154 - 6 Bioluminescent symbionts of flashlight fishes and deep-sea anglerfishes form unique lineages related to the genus Vibrio; Haygood MG et al.; Bioluminescent symbioses range from facultative associations to highly adapted, apparently obligate ones . The family Anomalopidae (flashlight fishes) encompasses five genera of tropical reef fishes that have large suborbital light organs . The suborder Ceratioidei (deep-sea anglerfishes) contains 11 families . In nine of these, females have a bioluminescent lure that contains bacterial symbionts . In all other fish light-organ symbioses (occurring in 10 families in 5 orders), the symbionts belong to three Photobacterium species; nonsymbiotic luminous bacteria are Vibrio species . The bacteria are extracellular and tightly packed in tubules that communicate with the exterior, releasing bacteria into the gut of the host or the surrounding sea water . The released bacteria are usually cultivable and can contribute to planktonic populations . Although anomalopids release bacteria and ceratioids have pores that would allow release, the fate of these bacteria is unknown and they cannot be cultured by standard isolation techniques . We report here phylogenetic analysis of 16S ribosomal RNA gene sequences from light organs that show that anomalopid and ceratioid symbionts are not known luminous bacteria, but are new groups related to Vibrio spp . They are characterized by host specificity, deep divergence between symbionts from different genera (anomalopids) or families (ceratioids) and, possibly, parallel divergence of hosts and symbionts. EMBO J, 1993 May, 12(5), 1767 - 74 Crystal structure of a flavoprotein related to the subunits of bacterial luciferase; Moore SA et al.; The molecular structure of the luxF protein from the bioluminescent bacterium Photobacterium leiognathi has been determined by X-ray diffraction techniques and refined to a conventional R-factor of 17.8% at 2.3 A resolution . The 228 amino acid polypeptide exists as a symmetrical homodimer and 33% of the monomer's solvent-accessible surface area is buried upon dimerization . The monomer displays a novel fold that contains a central seven-stranded beta-barrel . The solvent-exposed surface of the monomer is covered by seven alpha-helices, whereas the dimer interface is primarily a flat surface composed of beta-strands . The protein monomer binds two molecules of flavin mononucleotide, each of which has C6 of the flavin isoalloxazine moiety covalently attached to the C3' carbon atom of myristic acid . Both myristyl groups of these adducts are buried within the hydrophobic core of the protein . One of the cofactors contributes to interactions at the dimer interface . The luxF protein displays considerable amino acid sequence homology with both alpha- and beta-subunits of bacterial luciferase, especially the beta-subunit . Conserved amino acid residues shared between luxF and the luciferase subunits cluster predominantly in two distinct regions of the luxF protein molecule . These homologous regions in the luciferase subunits probably share a three-dimensional fold similar to that of the luxF protein. Gene, 1993 Apr 15, 126(1), 155 - 6 Sequence of the luxD gene encoding acyltransferase of the lux operon from Photobacterium leiognathi; Chao YF et al.; The nucleotide sequence of luxD (EMBL accession No . X65611), encoding acyltransferase (ACT), of the lux operon from Photobacterium leiognathi PL741 was determined, and the amino acid (aa) sequence was deduced . ACT is a component of the fatty acid reductase complex, which is responsible for converting fatty acid to aldehyde that serves as the substrate in the luciferase-catalyzed bioluminescent reactions . The protein has a calculated M(r) of 34,384 and comprises 305 aa residues . Alignment and comparison of the ACT of P . leiognathi with that of Vibrio fischeri ATCC7744, V . harveyi B392 and Xenorhabdus luminescens Hm shows that there is 66%, 59% and 61% aa identity, respectively. Gene, 1993 Apr 15, 126(1), 153 - 4 The lumazine protein-encoding gene in Photobacterium leiognathi is linked to the lux operon; Lin JW et al.; The nucleotide (nt) sequence of the lumP (EMBL accession No . X65612) gene of Photobacterium leiognathi PL741 was determined and the amino acid (aa) sequence deduced . The encoded aa sequence of lumP was identified as that of the lumazine protein (LumP) by homology with that of Photobacterium phosphoreum (56%) . This small protein has a calculated M(r) of 19,997 and comprises 186 aa residues . Biochemical studies suggested that LumP is the protein which, when combined with luciferase, is responsible for the bioluminescent spectrum shift from blue-green light (490-505 nm) to blue (470 nm) in P . leiognathi . The nt sequence of the flanking region showed that lumP is linked to the lux operon but runs in the opposite direction . The gene order of the lumP and lux operon is as follows: <--lumP-R&R-luxC-luxD-luxA-luxB-luxN-lu xE-->; the R&R regulatory region sequence included two promoter systems, PR for the lux operon and PL for the lumP or the lum operon. Biochem Biophys Res Commun, 1993 Feb 26, 191(1), 314 - 8 Nucleotide sequence of the luxC gene encoding fatty acid reductase of the lux operon from Photobacterium leiognathi; Lin JW et al.; The nucleotide sequence of the luxC gene (EMBL Accession No . 65156) encoding fatty acid reductase (FAR) of the lux operon from Photobacterium leiognathi PL741 was determined and the encoded amino acid sequence deduced . The fatty acid reductase is a component of the fatty acid reductase complex . The complex is responsible for converting fatty acid to aldehyde which serves as the substrate in the luciferase-catalyzed bioluminescent reaction . The protein comprises 478 amino acid residues and has a calculated M(r) of 53,858 . Alignment and comparison of the fatty acid reductase of P . leiognathi with that of Vibrio harveyi B392 and Vibrio fischeri ATCC 7744 shows that there is 70% and 59% amino acid residues identity, respectively. J Biolumin Chemilumin, 1993 Jan-Feb, 8(1), 39 - 48 Separation of pH, dilution, ionic strength and chemical matrix effects for biological monitoring of urines with the Microtox test using nicotine, cotinine and reference urines; Chou CC et al.; The aim was to investigate the factors influencing light emission from Photobacterium phosphoreum in the Microtox test to interpret bioassay results for urine . Four reference urines were assessed as reference materials for the bioassay . Nicotine and cotinine were investigated as urinary markers for tobacco exposure . The optimum luminescence conditions were: 1.85%-3.25% NaCl, 0.33-0.58 mol/L ionic strength, and pH 5.8-6.7 . Low pH values and high concentration of toxic trace metals were important factors in this study . Unexpected toxicity for a Standard Reference Material was attributed to zinc contamination . Nicotine and cotinine together exhibited antagonistic effects in 2% saline but this could not be observed in the urines because of substantial urine toxicity . Thus practical urinary biological monitoring with the Microtox test necessitates excretion of metabolites and compounds that are much more toxic than the urine components . Also, separation of the effects of physical factors like pH, ionic strength and dilution is essential before chemical toxicity effects can be assigned . This is the first report of Microtox EC50 values for nicotine and cotinine . The results have application to environmental samples since analyses are often uncontrolled relative to pH, ionic strength and dilution. Crit Rev Microbiol, 1993, 19(4), 191 - 216 Light organ symbioses in fishes; Haygood MG; Most bioluminescent fishes are self-luminescent, but a substantial minority of bioluminescent teleosts produce light that is due to symbiotic luminous bacteria housed in elaborate light organs . The majority of symbiotically bioluminescent fishes (ten families in five orders) harbors common free-living species of marine luminous bacteria: Photobacterium phosphoreum, P . leiognathi, and P . fischeri (= Vibrio fischeri) . Others, associated with the beryciform family Anomalopidae and nine families in the lophiiform suborder Ceratioidei, have apparently obligate symbionts that have recently been identified by small subunit (16S) rRNA analysis as new groups within the genus Vibrio . This article summarizes what is currently known about relationships between light organ symbionts and their hosts, including characteristics of light organ environments, physiology of light organ symbionts, and the evolution of light organ symbionts and their associations. J Biomater Sci Polym Ed, 1993, 5(1-2), 37 - 48 Covalent immobilization of microorganisms in polymeric hydrogels; Valuev LI et al.; A method of covalent immobilization of microorganisms (marine luminescent bacteria and yeast) in polymeric hydrogels is described . It is shown that cell immobilization leads to the creation of materials having properties of both synthetic polymers and physiologically active systems . Application of systems containing covalent immobilized yeast and photobacteria in biotechnological and other processes is proposed. Eur J Biochem, 1992 Dec 15, 210(3), 711 - 9 Fluorescence study of the ligand stereospecificity for binding to lumazine protein; Lee J et al.; 6,7-Dimethyllumazine derivatives, substituted at the 8-position with aldityls or monohydroxyalkyl groups, have been examined for their binding ability to lumazine apo-protein from two strains of Photobacterium phosphoreum using fluorescence dynamics techniques . On the protein the lumazine has a nearly monoexponential decay of fluorescence with lifetime 13.8 ns (20 degrees C) . In free solution the lifetime is 9.6 ns . The concentration of free and bound lumazine in an equilibrium mixture can be recovered readily by analysis of the fluorescence decay . Only the aldityl derivatives D-xylityl and 3'-deoxy-D-ribityl, having stereoconfigurations at the 2' and 4' positions identical to the natural ligand, 8-(1'-D-ribityl), show comparable dissociation constants (0.3 microM, 20 degrees C, pH 7.0) . D-Erythrityl and L-arabityl have dissociation constants of 1-2 microM . All other ligands show no interaction at all or have dissociation constants in the range 6-80 microM, which can still be determined semi-quantitatively using the fluorescence decay technique . In the case of these very weakly bound ligands, unambiguous detection of bound ligand can be shown by a long correlation time (23 ns, 2 degrees C) for the fluorescence anisotropy decay . Examination of the bound D-xylityl compound's fluorescence anisotropy decay at high time resolution (< 100 ps) shows rigid association, i.e . no mobility independent of the macromolecule . All bound ligands appear to be similarly positioned in the binding site . The influence of the stereoconfiguration at the 8-position found for lumazine protein parallels that previously observed for the enzyme riboflavin synthase, where the lumazines are substrates or inhibitors . This is consistent with the finding of significant sequence similarity between these proteins . The binding rigidity may have implications for the mechanism of the enzyme. Appl Environ Microbiol, 1992 Nov, 58(11), 3694 - 700 Development of monoclonal antibodies that identify Vibrio species commonly isolated from infections of humans, fish, and shellfish; Chen D et al.; Monoclonal antibodies (MAbs) against Vibrio species that infect humans, fish, and shellfish were developed for application in rapid identifications . The pathogens included Vibrio alginolyticus, V . anguillarum, V . carchariae, V . cholerae, V . damsela, V . furnissii, V . harveyi, V . ordalii, V . parahaemolyticus, and V . vulnificus . Three types of MAbs were selected . The first important group included MAbs that reacted with only a single species . A second group comprised a number of MAbs that reacted with two, taxonomically closely related Vibrio species . For example, of 22 MAbs raised against V . alginolyticus, 6 recognized a 52-kDa flagellar H antigen common to both V . alginolyticus and V . parahaemolyticus; V . anguillarum and V . ordalii also shared antigens . A third group included three genus-specific MAbs that reacted with almost all Vibrio species but did not react with other members of the family Vibrionaceae (e.g., members of the Aeromonas, Photobacterium, and Plesiomonas genera) or a wide range of gram-negative bacteria representing many genera . This last group indicated the possible existence of an antigenic determinant common to Vibrio species . Two of these three genus-specific MAbs reacted with heat-stable antigenic determinants of Vibrio species as well as lipopolysaccharide extracted from Vibrio species . The use of the MAbs in blind tests and diagnosis of clinical isolates indicated that three different types of bacteria, viz., live, formalin-fixed, and sodium azide-killed bacteria, were detected consistently . Overall, it was found that the genus-specific MAbs were very useful for rapidly identifying vibrios in the screening of acute infections, while the species-specific MAbs and others were useful for completing the diagnosis. Biochem Biophys Res Commun, 1992 Jul 31, 186(2), 690 - 7 The lux genes in Photobacterium leiognathi are closely linked with genes corresponding in sequence to riboflavin synthesis genes; Lee CY et al.; 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. FEMS Microbiol Lett, 1992 Jul 1, 73(1-2), 191 - 4 Evidence for the existence of a restriction-modification system common to several species of the family Vibrionaceae; Matsuzaki S et al.; A broad-host-range vibriophage KVP40 originally isolated on Vibrio parahaemolyticus 1010 was restricted and modified by strains of at least five Vibrio and one Photobacterium species . 1010 was a non-restricting host . An anti-restriction mutant KVP40 aar1 was isolated after propagating the phage on a restricting host, V . anguillarum VIB36 . KVP40 aar1 grown on either 1010 or VIB36, as well as the parental phage grown on VIB36, showed much higher efficiencies of plating on all the restricting hosts as compared with the parental phage grown on 1010, indicating that these restricting hosts probably share a common restriction-modification system active in vivo on KVP40. Ecotoxicol Environ Saf, 1992 Jun, 23(3), 355 - 63 Microtox EC50 values for drinking water by-products produced by ozonolysis; Chou CC et al.; The aim was to determine the Microtox EC50 values of some aliphatic aldehydes and carboxylic acids of normal chain length with 1-14 carbon atoms since these compounds have been detected as ozonolysis by-products in drinking water . The aqueous EC50 values decreased with increasing chain length except for formaldehyde and for the C1-C7 acids . At chain lengths above C7, where methanolic saline solutions were utilized to promote solubility, the aldehydes were more toxic than their corresponding carboxylic acids . Below a chain length of C7, the reverse situation applied . More precise and sensitive EC50 values were observed at 15 and 25 min than at 5 min . Both C14 aldehyde and acid as well as palmitoleic acid showed luminescence in methanolic saline solutions . The C14 compounds are known natural substrates for bacterial luciferase, and the present findings confirm this for Photobacterium phosphoreum . The concentrations of the aldehyde and/or carboxylic acid ozonolysis by-products reported in drinking water could not be detected by the Microtox test . This is the first report of Microtox EC50 values for these carboxylic acids and for aldehydes of chain lengths greater than C4. J Mol Biol, 1992 Mar 20, 224(2), 523 - 6 Crystallization of Photobacterium leiognathi non-fluorescent flavoprotein, an unusual flavoprotein with limited sequence identity to bacterial luciferase; Moore SA et al.; 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. Ecotoxicol Environ Saf, 1992 Feb, 23(1), 46 - 63 Integrated assessment of contaminated sediments in the lower Fox River and Green Bay, Wisconsin; Ankley GT et al.; Samples of sediment and biota were collected from sites in the lower Fox River and southern Green Bay to determine existing or potential impacts of sediment-associated contaminants on different ecosystem components of this Great Lakes area of concern . Evaluation of benthos revealed a relatively depauperate community, particularly at the lower Fox River sites . Sediment pore water and bulk sediments from several lower Fox River sites were toxic to a number of test species including Pimephales promelas, Ceriodaphnia dubia, Hexagenia limbata, Selenastrum capricornutum, and Photobacterium phosphorum . An important component of the observed toxicity appeared to be due to ammonia . Evaluation of three bullhead (Ictalurus) species from the lower Fox River revealed an absence of preneoplastic or neoplastic liver lesions, and the Salmonella typhimurium bioassay indicated relatively little mutagenicity in sediment extracts . Apparent adverse reproductive effects were noted in two species of birds nesting along the lower Fox River and on a confined disposal facility for sediments near the mouth of the river, and there were measurable concentrations of potentially toxic 2,3,7,8-substituted polychlorinated dibenzo-p-dioxins (PCDDs) and dibenzofurans (PCDFs), and planar polychlorinated biphenyls (PCBs) both in the birds and in sediments from several of the study sites . Based on toxic equivalency factors and the results of an in vitro bioassay with H4IIE rat hepatoma cells, it appeared that the majority of potential toxicity of the PCB/PCDF/PCDD mixture in biota from the lower Fox River/Green Bay system was due to the planar PCBs . The results of these studies are discussed in terms of an integrated assessment focused on providing data for remedial action planning. Microbiol Immunol, 1992, 36(1), 93 - 7 A broad-host-range vibriophage, KVP40, isolated from sea water; Matsuzaki S et al.; A broad-host-range vibriophage, KVP40, was isolated from sea water by using Vibrio parahaemolyticus 1010 (EB101) as the indicator host . The host range of KVP40 extended over at least 8 Vibrio and 1 Photobacterium species . KVP40 was a large tailed phage containing double-stranded DNA and belonged to Ackermann's morphotype A2 . KVP40 DNA was cleaved by 11 different type II restriction endonucleases including EcoRI and HindIII, but not by 17 other enzymes including BamHI, KpnI and SalI. J Toxicol Clin Toxicol, 1992, 30(4), 585 - 96 The effects of histamine administered in fish samples to healthy volunteers; van Gelderen CE et al.; The effects of histamine administered in samples of fish to eight healthy volunteers (4 females and 4 males), aged 21-30 years, were studied . The subjects were given 0, 45 and 90 mg of histamine that had been metabolized from histidine by photobacteria in the fish and 90 mg of histamine added to fresh fish, for breakfast . The subjects were observed during 6 h after breakfast . Special attention was paid to clinical symptoms, blood pressure and ECG . The pH of the gastric contents was recorded continuously from 5 min before to 6 h after the meal . Blood samples to measure the histamine concentration were taken at intervals during 24 h after breakfast . Two of the subjects showed effects (facial flushing, headache) that could be attributed to the ingestion of histamine . No significant changes were observed in the blood pressure and ECG . The pH of the gastric fluids did not decrease significantly . The histamine concentration in plasma correlated closely with the histamine dose ingested (p < 0.001, r = 0.996) . The Cmax of the dose of 90 mg did not differ statistically significant from the Cmax of the dose of 90 mg histamine added to unspoiled fish. Chin J Biotechnol, 1992, 8(4), 269 - 76 Partition coefficient of luciferase from photobacteria in PEG/salt two aqueous phase system; Sun W et al.; The relationship between the logarithmic partition coefficient (K) of luciferase of photobacteria and PEG MW in the PEG/salt two aqueous phase system was shown to be a linear function . The hydrophilic PEG of lower MW facilitated the partition of luciferase into the top PEG-riching phase and gave a higher K value, while PEG of higher MW, its hydrophobic characteristic, made more enzyme partition into the bottom salt-riching phase and lower K value was obtained . In the PEG/trivalent salt system, such as phosphate and citrate, there was a turning-point on the linear relation between the log K and the PEG MW, but which never appeared if a divalent salt such as sulfate, succinate or tartrate was used in the system . When the system was composed of homogeneous PEG and ammonium sulfate, the K value was increased with the increment of the salt concentration, but after the salt concentration had reached at certain level, the K value was uninfluenced . When two kinds of PEG with different MW were used in this system a minimal K value appeared at certain concentration of ammonium sulfate, and the K value was raised when the salt concentration was either increased or decreased . Neither the proportion of the two kinds of PEG nor their total concentration used in the system showed any effect on the above patterns, although the K value may give some corresponding changes . (ABSTRACT TRUNCATED AT 250 WORDS) Schriftenr Ver Wasser Boden Lufthyg, 1992, 89, 591 - 624 {The luminescent bacteria test for clean water legislation}; Krebs F; Luminescent bacteria (Photobacterium phosphoreum) may be used, on the one hand, for classical toxicity tests based on the inhibition of respiration or growth and, on the other hand, their luminescence itself may become the test criterion . Such a luminescence inhibition test is commonly called a luminescent bacteria test . It may be conducted with fresh bacteria or with conserved ones . The luminescence parameter is, just like the parameter oxygen consumption, a summative parameter for undisturbed metabolic processes . Dilution series are prepared to determine the concentration level at which no inhibition of the luminous intensity is detectable in comparison with reference samples . This paper highlights the necessity of standardizing the test methods (the influence of toxic substances depends on test duration and temperature) and describes the standardized procedure established by the DIN-Arbeitskreis "Leuchtbakterientest" (Working Group of the German Institute for Standardization for the luminescent bacteria test) using freeze-dried, liquid-dried, and fresh bacteria (DIN 38,412, part 34) . Results of wastewater analyses are presented and the wide range of applications is described . The choice of biotests for applications under the Federal Water Resources Management Act (Wasserhaushaltsgesetz--WHG) and the Wastewater Charges Act (Abwasserabgabengesetz--AbwAG) is discussed and the reasons for the selection of the luminescent bacteria test are explained . Within the framework of a test battery of bioassays, this test shall be used according to section 7a WHG both for evaluating the technological level of wastewater treatment and for routine monitoring of wastewater discharges . In March 1992, this test (DIN 38,412, part 34, edition March 1991) became part of the general administrative regulations for wastewater (Rahmen-Abwasser-Verwaltungsvorschrift-RAV). Sci Total Environ, 1991 Dec, 109-110, 499 - 514 Regression and cluster analysis of the acute toxicity of 267 chemicals to six species of biota and the octanol/water partition coefficient; Kaiser KL et al.; The acute toxicities of 267 compounds to six aquatic and one terrestrial species were investigated with correlation, principal component and cluster analysis techniques for relationships with each other and with the compounds' octanol/water partition coefficient . Selection of the investigated chemicals was based on the availability of at least three of the following measured parameters: acute (24-h to 96-h) lethal concentrations (LC50) to the fish fathead minnow (Pimephales promelas), the fish goldorfe (Leuciscus idus melanotus), the zooplankter Daphnia magna, the ciliate Tetrahymena pyriformis, and the algae Scenedesmus quadricauda; the (30-min) inhibitory concentrations (EC50) to the luminescent marine bacterium Photobacterium phosphoreum (the Microtox test); the acute oral dose (LD50) for the common Norway rat and the octanol/water partition coefficient (log P or log Kow) . The results indicate highly significant correlations between the fathead minnow, goldorfe and Daphnia LC50 and the Photobacterium EC50 concentrations . The cluster and principal components analyses did not detect any clearly defined groups of compounds . The toxicities were also highly collinear with the octanol/water partition coefficients for all species except the rat, where two relationships are indicated, with the division at log P = 2.00. Sci Total Environ, 1991 Dec, 109-110, 431 - 9 QSAR studies of comparative toxicity in aquatic organisms; Cronin MT et al.; This study investigated the relationships between the toxicities of common organic pollutants to the fathead minnow (Pimephales promelas), to Daphnia magna, to Tetrahymena pyriformis and in the Microtox test, which uses the luminescent bacterium Photobacterium phosphoreum . The toxicity data were compiled from the literature, with the exception of 40 experimentally determined Microtox data . Encouraging correlations are seen, indicating significant relationships between fish toxicities and those to lower organisms . When the toxicities of individual chemical classes are studied, further improvement is often seen in the correlations . Analysis of significant outliers from the inter-species relationships has led to the suggestion that the fathead minnow may be more susceptible to chemicals that are metabolised to reactive intermediates (such as the aldehydes) . The fish may, however, be less susceptible to other chemical classes such as ketones and alcohols. J Biochem (Tokyo), 1991 Nov, 110(5), 748 - 50 Crystallization and preliminary X-ray diffraction studies of a flavoprotein, FP390, from a luminescent bacterium, Photobacterium phosphoreum; Kita A et al.; A flavoprotein, FP390, obtained from a luminescent bacterium, Photobacterium phosphoreum, in the purification of luciferase has been crystallized by the vapor-diffusion procedure . Crystals obtained from polyethylene glycol 4000 solutions, whose X-ray photographs show powder diffraction patterns, were unsuitable for further crystallographic work . However, tetragonal crystals grown from potassium phosphate solution well diffracted X-rays beyond 3 A resolution . The space group of this crystal is P4(1)22 or P4(3)22 with unit-cell dimensions of a = b = 76.8 and c = 241 A . Assuming two or three molecules in an asymmetric unit, the value for the crystal volume per unit molecular mass, Vm, is calculated as 3.3 or 2.2 A3/Da, respectively . A total of 13,555 independent reflections for the native crystal was collected up to 3 A resolution using a Weissenberg camera attached to the synchrotron radiation source, the merging R factor being 0.077 for 79,335 measurements. Eur J Biochem, 1991 Oct 1, 201(1), 161 - 7 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; Lee CY et al.; 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. Int J Syst Bacteriol, 1991 Oct, 41(4), 529 - 34 Evaluation of the genus Listonella and reassignment of Listonella damsela (Love et al.) MacDonell and Colwell to the genus Photobacterium as Photobacterium damsela comb . nov . with an emended description; Smith SK et al.; The genus Listonella, which was recently described on the basis of 5S rRNA sequence data, was found to be of dubious value on the basis of the results of a comparison of a number of taxonomic studies involving members of the Vibrionaceae . The available data suggest that 5S rRNA sequences may be of limited taxonomic use at the intra- and intergeneric levels, at least for apparently recently evolved groups, such as the Vibrionaceae . In this light, we assessed the generic assignment of the species Listonella damsela . Phenotypic characterization of 12 strains of bacteria assigned to L . damsela, including type strain ATCC 33539, revealed a strong resemblance to members of the genus Photobacterium . All of the strains conformed to major characteristics common to all known Photobacterium species . The characteristics of these organisms included the absence of a flagellar sheath and accumulation of poly-beta-hydroxybutyrate during growth on glucose coupled with the inability to utilize DL-beta-hydroxybutyrate as a sole carbon source . On the basis of the phenotypic data, we propose that L . damsela should be reassigned to the genus Photobacterium as Photobacterium damsela comb . nov. Biochemistry, 1991 Jul 16, 30(28), 6825 - 35 Electronic excitation transfer in the complex of lumazine protein with bacterial bioluminescence intermediates; Lee J et al.; Fluorescence dynamics measurements have been made on the bioluminescence reaction intermediates using Photobacterium leiognathi, Vibrio fischeri, and Vibrio harveyi luciferases, both alone and in mixtures with Photobacterium phosphoreum lumazine protein . Each luciferase produces a "fluorescent transient" intermediate on reaction with the bioluminescence substrates, FMNH2, tetradecanal, and O2, and all have a fluorescence quantum yield about 0.3, with a predominant lifetime around 10 ns . The P . leiognathi luciferase fluorescent transient has a rotational correlation time of 79 ns at 2 degrees C, as expected for the rotational diffusion of a 77-kDa macromolecule . In the presence of lumazine protein however a faster correlation time of about 3 ns predominates . This rapid channel of anisotropy loss is attributed to energy transfer from the flavin intermediate bound on the luciferase to the lumazine ligand, reflects the presence of protein-protein complexation, and is greatest in the case of P . leiognathi, but not at all for V . fischeri . This fact is consistent with the strong influence of lumazine protein on the bioluminescence reaction of P . leiognathi, and not at all with V . fischeri . The rate of energy transfer is of order 10(9) s-1, much greater than the 10(8) s-1 fluorescence rate of the donor . Thus the bioluminescence excitation of lumazine protein could occur by a similar photophysical mechanism of interprotein energy transfer from a chemically excited fluorescent transient donor to the lumazine acceptor. J Biol Chem, 1991 Jul 15, 266(20), 12852 - 7 A lux-specific myristoyl transferase in luminescent bacteria related to eukaryotic serine esterases; Ferri SR et al.; The diversion of fatty acids from fatty acid biosynthesis into the luminescent system is catalyzed by a lux-specific acyltransferase that catalyzes the cleavage of fatty acyl-acyl carrier protein (ACP) . Analysis of the substrate specificities for fatty acyl-ACPs of the transferases from divergent luminescent bacteria, Photobacterium phosphoreum and Vibrio harveyi, has demonstrated that myristoyl-ACP is cleaved at the highest rate . Inhibition by phenylmethanesulfonyl fluoride as well as resistance of the acylated enzyme intermediate to cleavage by hydroxylamine showed that the transferase is a serine esterase . Moreover, activity was dependent on a basic residue with a pKa of 6.3 implicating a histidine residue as part of a charge relay system found in serine esterases . The nucleotide sequence of the P . phosphoreum luxD gene coding for the transferase was determined resulting in the identification of the active site motif for serine esterases, G-X-S-X-G . Replacement of the serine residue at the center of this motif by threonine, alanine, or glycine blocked the transferase acyl-ACP cleavage activity, its ability to be acylated, and complementation of a transferase defective mutant on transconjugation with the luxD gene . The sequence and location of the serine as well as a histidine residue in the lux-specific transferases were found to be similar to those involved in the charge relay system in vertebrate thioesterases . Combined with the similar kinetic properties, these results support a common metabolic role for both enzymes in the diversion of fatty acids from the fatty acid biosynthetic pathway. J Biolumin Chemilumin, 1991 Jul-Sep, 6(3), 169 - 76 Green flavoprotein from P . leiognathi: purification, characterization and identification as the product of the lux G(N) gene; Raibekas AA; A green flavoprotein (GFP) was isolated and purified to homogeneity from Photobacterium leiognathi, strain 208 . GFP is a homodimer of molecular weight 54,000 and contains two molecules of an unusual flavin per molecule of protein . Various biochemical characteristics including isoelectric point, trypsin and chymotrypsin degradation, SDS and temperature influence on subunit dissociation and the dissociation of the flavin chromophore, were investigated . The sequence of 23 N-terminal amino acids was determined and found to be concurrent with the N-terminal amino acid sequence encoded by the lux G(N) gene of P . leiognathi . This fact suggests that GFP is a structural component of the Photobacterium luminescence system. Sci Total Environ, 1991 May 15, 104(3), 229 - 37 The acute toxicity of pulse-dosed, para-substituted phenols to larval American flagfish (Jordanella floridae): a comparison with toxicity to photoluminescent bacteria and predicted toxicity using log Kow; Holdway DA et al.; The acute toxicity of nine para-substituted phenols was determined using a pulse-exposure testing protocol and 8-day-old larval American flagfish (Jordanella floridae) . Relative tolerance was assessed by determining the 2-h pulse exposure concentration causing 20 and 50% mortality (PE LC20 and PE LC50) over the subsequent 94 h . Four bioassays were run for each phenol and yielded the following mean PE LC20 values (mg 1(-1)) in descending order of toxicity: p-aminophenol, 0.06; hydroquinone, 0.13; phenol, 0.70; p-nitrophenol, 0.81; p-cyanophenol, 3.0; p-chlorophenol, 3.3; p-hydroxyacetophenone, 4.2; p-hydroxybenzyl alcohol, 6.4; and p-hydroxybenzoic acid, 170 . These toxicities did not correlate significantly with either previously reported toxicity values for the photoluminescent bacteria Photobacterium phosphoreum, or with the log octanol-water partition coefficient . For some of the compounds, however, sensitivities were quite close to previously reported rainbow trout chronic no-observed-effect concentrations based on continuous exposure . Caution is urged with respect to applying "low-level" biota techniques or simple quantitative structure-activity correlations such as Kow when attempting to predict the toxicity of specific chemicals to fish. J Mol Biol, 1991 May 5, 219(1), 69 - 77 Identification of the acyl transfer site of fatty acyl-protein synthetase from bioluminescent bacteria; Soly RR et al.; Fatty acid activation, transfer, and reduction by the fatty acid reductase multienzyme complex from Photobacterium phosphoreum to generate fatty aldehydes for the luminescence reaction is regulated by the interaction of the synthetase and reductase subunits of this complex . Identification of the specific site involved in covalent transfer of the fatty acyl group between the sites of activation and reduction on the synthetase and reductase subunits, respectively, is a critical step in understanding how subunit interactions modulate the flow of fatty acyl groups through the fatty acid reductase complex . To accomplish this goal, the nucleotide sequence of the luxE gene coding for the acyl-protein synthetase subunit (373 amino acid residues) was determined and the conserved cysteinyl residues implicated in fatty acyl transfer identified . Using site-specific mutagenesis, each of the five conserved cysteine residues was converted to a serine residue, the mutated synthetases expressed in Escherichia coli, and the properties of the mutant proteins examined . On complementation of four of the mutants with the reductase subunit, the synthetase subunit was acylated and the acyl group could be reversibly transferred between the reductase and synthetase subunits, and fatty acid reductase activity was fully regenerated . As well, sensitivity of the acylated synthetases to hydroxylamine cleavage (under denaturation conditions to remove any conformational effects on reactivity) was retained, showing that a cysteine and not a serine residue was still acylated . However, substitution of a cysteine residue only ten amino acid residues from the carboxyl terminal (C364S) prevented acylation of the synthetase and regeneration of fatty acid reductase activity . Moreover, this mutant protein preserved its ability to activate fatty acid to fatty acyl-AMP but could not accept the acyl group from the reductase subunit, demonstrating that the C364S synthetase had retained its conformation and specifically lost the fatty acylation site . These results provide evidence that the flow of fatty acyl groups in the fatty acid reductase complex is modulated by interaction of the reductase subunit with a cysteine residue very close to the carboxyl terminal of the synthetase, which in turn acts as a flexible arm to transfer acyl groups between the sites of activation and reduction. Appl Environ Microbiol, 1991 May, 57(5), 1319 - 24 Development of species-specific hybridization probes for marine luminous bacteria by using in vitro DNA amplification; Wimpee CF et al.; By using two highly conserved region of the luxA gene as primers, polymerase chain reaction amplification methods were used to prepare species-specific probes against the luciferase gene from four major groups of marine luminous bacteria . Laboratory studies with test strains indicated that three of the four probes cross-reacted with themselves and with one or more of the other species at low stringencies but were specific for members of their own species at high stringencies . The fourth probe, generated from Vibrio harveyi DNA, cross-reacted with DNAs from two closely related species, V . orientalis and V . vulnificus . When nonluminous cultures were tested with the species-specific probes, no false-positive results were observed, even at low stringencies . Two field isolates were correctly identified as Photobacterium phosphoreum by using the species-specific hybridization probes at high stringency . A mixed probe (four different hybridization probes) used at low stringency gave positive results with all of the luminous bacteria tested, including the terrestrial species, Xenorhabdus luminescens, and the taxonomically distinct marine bacterial species Shewanella hanedai; minimal cross-hybridization with these species was seen at higher stringencies. Biochem Biophys Res Commun, 1991 Apr 15, 176(1), 541 - 8 Structure and properties of luciferase from Photobacterium phosphoreum; Ferri SR et al.; The nucleotide sequences of the luxA and luxB genes coding for the alpha and beta subunits, respectively, of luciferase from Photobacterium phosphoreum have been determined . The predicted amino acid sequences of the alpha and beta subunits were shown to be significantly different from other bacterial luciferases with 62 to 88% identity with the alpha subunits and 47 to 71% identity with the beta subunits of other species . Expression of the different luciferases appear to correlate with the number of modulator codons . Kinetic properties of P . phosphoreum luciferase were shown to reflect the bacterium's natural cold temperature habitat. Proc Natl Acad Sci U S A, 1991 Feb 15, 88(4), 1100 - 4 Borrowed proteins in bacterial bioluminescence; O'Kane DJ et al.; A library of Photobacterium phosphoreum DNA was screened in lambda 2001 for the lumazine protein gene, using two degenerate 17-mer oligonucleotide probes that were deduced from a partial protein primary sequence . The lumazine protein gene was localized to a 3.4-kilobase BamHI/EcoRI fragment in one clone . The fragment contained an open reading frame, encoding a 189-residue protein, that had a predicted amino acid sequence that concurred with the partial sequence determined for lumazine protein . Considerable sequence similarity was detected between lumazine protein, the yellow fluorescence protein from Vibrio fischeri, and the alpha subunit of riboflavin synthetase (EC 2.5.1.9) . A highly conserved sequence in lumazine protein corresponds to the proposed lumazine binding sites in the alpha subunit of riboflavin synthetase . Several secondary structure programs predict the conformation of this site in lumazine protein to be a beta-sheet . A minimal model with three interactions between the ligand and this beta-sheet structure is proposed, which is consistent with the results of NMR and ligand binding studies. Can J Microbiol, 1991 Feb, 37(2), 148 - 53 Further study on polyamine compositions in Vibrionaceae; Yamamoto S et al.; It has previously been reported that norspermidine, one of the unusual polyamines, is present in Vibrio species . To expand this observation, the cellular polyamine compositions of additional species and strains in the family Vibrionaceae (Vibrio, Photobacterium, Listonella, and Shewanella) as well as Aeromonas species and Plesiomonas shigelloides, which have been proposed to be excluded from Vibrionacea, were determined by using gas-liquid chromatography . Some Vibrio species previously reported were reexamined under the same conditions, and their results are included in this report . Norspermidine was detected as a major triamine in 23 of 24 Vibrio species, all of 4 Listonella species, and 3 of 5 Photobacterium species . Vibrio costicola, Photobacterium fischeri, and Photobacterium phosphoreum contained no norspermidine . Listonella species were indistinguishable from Vibrio species in their polyamine profiles . However, Schewanella putrefaciens ATCC 8071, formerly allocated in the genus Alteromonas, contained no norspermidine, and its polyamine profile was similar to those of four Aeromonas species, in which putrescine was exclusively found . Plesiomonas shigelloides was very similar to Escherichia coli in that putrescine and spermidine were predominant polyamines . Our data indicate that the occurrence of norspermidine may be very helpful as a generic marker in identification and classification of Vibrio and Listonella species . A gas-liquid chromatographic method with a nitrogen-selective detector was presented for rapid and sensitive detection of cellular norspermidine. Free Radic Res Commun, 1991, 12-13 Pt 1, 437 - 41 Induction of superoxide dismutases in Photobacterium leiognathi; Kobayashi H et al.; We investigated the induction of Cu,Zn-SOD (bacteriocuprein) and Fe-SOD in Photobacterium leiognathi DK-A1 which was isolated from the light organ of the squid, Droteuthis kensaki . The induction of superoxide dismutases depended on the addition of paraquat to the medium . Induction of SOD by paraquat was attributed mostly to the bacteriocuprein by measuring of the activities of both SODs by using densitometry of isoelectrofocusing gel . When paraquat was added to the culture at various times in the early log phase of growth, the most efficient induction of the SODs, which was measured at the time of harvesting the cells (17 hours after inoculation), was observed when paraquat was added at 60 min after the inoculation . Catalase was not significantly induced by the addition of paraquat or increasing of oxygen concentration . We developed an assay of SOD by modification of a cytochrome c-xanthine oxidase method using a computer equipped absorption spectrophotometer. Free Radic Res Commun, 1991, 12-13 Pt 1, 363 - 70 The rate of Cu,Zn superoxide dismutase evolution; Kwiatowski J et al.; The rate of amino acid replacement in Cu,Zn SOD greatly departs from the expectations of the molecular clock . We examine 27 Cu,Zn SOD sequences available and conclude that: (1) the SOD enzymes from different mammal families differ from each other by roughly the same number of replacements, which is consistent with a simultaneous mammalian radiation; (2) over the most recent 60 million years (MY) the rate of SOD evolution is fairly high (15 aa/100 aa/100 MYR) and may be considered constant; (3) the rate of accumulation of amino acid replacements since the divergence of fungi, plants and animals to the present is inconstant along different branches of the evolutionary tree; moreover it steadily decreases with time, to the same extent in all lineages; (4) some comparisons exhibit divergences that are in any case greater than expected from a Poisson process on the assumption of a molecular clock; (5) plant chloroplast enzymes display fewer differences from each other than cytoplasmic ones; (6) bacteriocuprein (from Photobacterium leiognathi), fluke and human extracellular SOD are all three extremely remotely related to one another and to the SOD of other eukaryotes . The process of consistent decline of the rate of evolution of Cu, Zn SOD can be described by a number of mathematical functions . We explore simple models that assume constant rates and might be applicable to other proteins or genes that apparently evolve at disparate rates. Free Radic Res Commun, 1991, 12-13 Pt 1, 349 - 61 Evolutionary aspects of superoxide dismutase: the copper/zinc enzyme; Bannister WH et al.; Copper/zinc superoxide dismutase is typically an enzyme of eukaryotes . The presence of the enzyme in the ponyfish symbiont Photobacterium leiognathi and some free living bacteria does not have an immediate explanation . Amino acid sequence alignment of 19 Cu/Zn superoxide dismutases shows 21 invariant residues in key positions related to maintenance of the beta-barrel fold, the active site structure including the electrostatic channel loop, and dimer contacts . Nineteen other residues are invariant in 18 of the 19 sequences . Thirteen of these nearly invariant residues show substitutions in Photobacterium Cu/Zn superoxide dismutase . Copper/zinc superoxide dismutase from the trematode Schistosoma mansoni shows an N-terminal sub-domain with a hydrophobic leader peptide, as in human extracellular superoxide dismutase which is a Cu/Zn enzyme . The latter also has a C-terminal sub-domain with preponderance of hydrophilic and positively charged residues . The amino acid sequence of this superoxide dismutase between the N-terminal and C-terminal regions shares many features of cytosolic Cu/Zn superoxide dismutase, including 20 of the 21 invariant residues found in 19 Cu/Zn enzymes, suggesting a similar type of beta-barrel fold and active site structure for the extracellular enzyme. J Biolumin Chemilumin, 1991 Jan-Mar, 6(1), 13 - 8 Analysis of synchronous photon emissions from the bacterium Photobacterium phosphoreum during colony formation from a single cell; Watanabe H et al.; Light emission from Photobacterium phosphoreum was analysed during cell growth on an agar plate from a single cell to colony formation . Temporal analysis of image intensified light was set so that a quadratic window covered a single cell . Intensity of light emission from a single cell through colony formation showed an initial decrease, a prolonged lag phase, and then a rapid increase . These responses on an agar plate were similar to those from liquid cultures . The image analysis showed repeated bursts of light emission in the phases when light was increasing and decreasing . Statistical analysis of light emission also emphasized the presence of bursts of light emission, suggesting the metabolic synchronism of luciferase reactions in either a single cell or suggesting the metabolic synchronism of luciferase reactions in either a single cell or synchronously divided cells . The repetitive bursts of light occurred in a single cell and continued during the growth phase in which the cell population and the light emission was increasing . In a single cell, however, periodicity of light emission was not defined directly from fast Fourier transformation, although it was indicated on oscillation of mean level of fluctuated light emission, at initial phase of culture on agar plate. Braz J Med Biol Res, 1991, 24(6), 573 - 82 Evaluation of the genotoxic activity and acute toxicity of Euphorbia splendens latex, a molluscicide for the control of schistosomiasis; Schall VT et al.; 1 . The latex of Euphorbia splendens var . hislopii has a molluscicidal action at low concentration (LD90 less than 1.5 ppm or 1.5 micrograms/ml) against the vector snails of schistosomiasis . 2 . In the present study, the latex in natura or after lyophilization was submitted to the Ames test and the chromotest to evaluate genotoxicity, to the Microtox System to determine acute toxicity, and to the Chinese hamster ovary cell assay (CHO) to measure cytotoxicity . 3 . The latex had no mutagenic activity in the presence or absence of S9 toward the TA98 and TA100 strains of Salmonella typhimurium (Ames test) at concentrations up to 200 microliters/plate (in natura) and of 200 micrograms/plate (lyophilized) . The lyophilized latex had no genotoxic activity (Chromotest) and no acute toxic effect on Photobacterium phosphoreum at concentrations up to 445 micrograms/ml, whereas the sample in natura had a toxic effect with an EC50 of 148,000 microliters/l (or ppm) . In the CHO/cytotoxicity assay, the lyophilized latex had no cytotoxic effect in quantities up to 200 micrograms . 4 . The latex was found to have no acute toxicity or mutagenic activity at the concentrations of 10 to 12 micrograms/ml (or ppm) that are being proposed for molluscicidal use in the field. Comp Biochem Physiol C, 1991, 100(1-2), 129 - 32 Investigations of phagocytosis concerning the immunological defence mechanism of Mytilus edulis using a sublethal luminescent bacterial assay (Photobacterium phosphoreum); Hansen PD et al.; 1 . A simple method for the determination of phagocytosis activity using mussel hemocytes by measuring the bioluminescence is presented . 2 . The immunological defence activity based on phagocytosis is measured and quantified by a luminescent bacterial assay with Photobacterium phosphoreum . 3 . The measuring system allows us to establish the stress of the immunological defence mechanism of organisms exposed to chemicals and polluted rivers or sewage . Results with reference substances and the phagocytosis indices of exposed mussels from Norwegian aquaculture plants compared to those of mussels from the German Wadden Sea are given as examples. J Acoust Soc Am, 1990 Dec, 88(6), 2527 - 32 Bacterial luminescence: a new tool for investigating the effects of acoustic energy and cavitation; McInnes C et al.; An assay utilizing luminescent bacteria, Photobacterium phosphoreum, was adapted to assess the antibacterial effects of acoustic energy . Acoustic pressures up to 67 kPa in the 100- to 800-Hz frequency range were applied to bacteria freely suspended in a liquid medium . Bacterial luminescence decreased after sonication, thus showing sensitivity to the effects of acoustic energy . This decreased luminescence was linearly related to exposure duration, appeared independent of acoustic frequency in this range, and was significantly heightened by the presence of cavitation . High-frequency components of the acoustic emission were recorded from the sonicated fluid, and it was found that the decrease in luminescence due to sonication was directly related to the logarithm of the acoustic emission . Viability studies on exposed bacteria indicated a diminution of luminescence without bacterial death . The potential use of luminescent bacteria in assessing the biological effects of acoustic energy-generating systems is discussed. Biochem Cell Biol, 1990 Jul-Aug, 68(7-8), 1045 - 51 A soluble fatty acyl-acyl carrier protein synthetase from the bioluminescent bacterium Vibrio harveyi; Byers DM et al.; An enzyme catalyzing the ligation of long chain fatty acids to bacterial acyl carrier protein (ACP) has been detected and partially characterized in cell extracts of the bioluminescent bacterium Vibrio harveyi . Acyl-ACP synthetase activity (optimal pH 7.5-8.0) required millimolar concentrations of ATP and Mg2+ and was slightly activated by Ca2+, but was inhibited at high ionic strength and by Triton X-100 . ACP from either Escherichia coli (apparent Km = 20 microM) or V . harveyi was used as a substrate . Of the {14C}fatty acids tested as substrates (8-18 carbons), a preference for fatty acids less than or equal to 14 carbons in length was observed . Vibrio harveyi acyl-ACP synthetase appears to be a soluble hydrophilic enzyme on the basis of subcellular fractionation and Triton X-114 phase partition assay . The enzyme was not coinduced with luciferase activity or light emission in vivo during the late exponential growth phase in liquid culture . Acyl-ACP synthetase activity was also detected in extracts from the luminescent bacterium Vibrio fischeri, but not Photobacterium phosphoreum . The cytosolic nature and enzymatic properties of V . harveyi acyl-ACP synthetase indicate that it may have a different physiological role than the membrane-bound activity of E . coli, which has been implicated in phosphatidylethanolamine turnover . Acyl-ACP synthetase activity in V . harveyi could be involved in the intracellular activation and elongation of exogenous fatty acids that occurs in this species or in the reactivation of free myristic acid generated by luciferase. J Biolumin Chemilumin, 1990 Jul-Sep, 5(3), 187 - 92 Affinity purification of bacterial luciferase and NAD(P)H:FMN oxidoreductases by FMN-sepharose for analytical applications; Lavi JT et al.; A modified purification method for bacterial luciferases and NAD(P)H:FMN oxidoreductases is described which uses FMN-Sepharose alone or coupled to DEAE ion exchange chromatography for the simultaneous purification of luciferase and the various oxidoreductases from Vibrio harveyi, a bright mutant of Vibrio harveyi, Vibrio fischeri, and Photobacterium phosphoreum . This purification method is compared with DEAE-Sepharose Cl 6B fractionations from these organisms . Both methods allow the separation of oxidoreductases specific for either NADH or NADPH . The use of FMN-Sepharose coupled to DEAE-Sepharose fractionation allows the isolation of highly purified enzymes . Lacking interfering factors, these are very suitable for various analytical applications based on bacterial bioluminescence enzymes . The partially purified enzymes from the affinity column have higher specific activities than those obtained using DEAE-Sepharose. Biochim Biophys Acta, 1990 Jun 26, 1017(3), 229 - 34 Inhibition of bioluminescence in Photobacterium phosphoreum by sulfamethizole and its stimulation by thymine; Watanabe H et al.; In bioluminescent bacteria very few agents have been reported that can selectively inhibit the luminescence . In sensitivity tests with Photobacterium phosphoreum, using 55 different antibiotics, it was found that sulfamethizole, an inhibitor of dihydropteroate synthetase and the formation of folic acid, inhibited bioluminescence more than growth . Likewise, in mutants requiring thymine for growth, the luminescence per cell was much less in a medium low in thymine . In neither case could the decreased specific luminescence be attributed to a decrease in the cellular level of luciferase or aldehyde factor; the involvement of additional but unidentified factors in the regulation of in vivo bioluminescence is postulated. J Bacteriol, 1990 Jun, 172(6), 2901 - 10 Copper-zinc superoxide dismutase of Caulobacter crescentus: cloning, sequencing, and mapping of the gene and periplasmic location of the enzyme; Steinman HM et al.; Although widely found in the cytoplasm of eucaryotes, the copper-zinc form of superoxide dismutase (CuZnSOD) has been identified in only a small number of bacterial species . One species is the freshwater bacterium Caulobacter crescentus, which also contains an SOD with iron as the metal cofactor (FeSOD) . To investigate the function of this CuZnSOD and its structural relationship to the eucaryotic CuZnSODs, the gene encoding CuZnSOD (sodC) of C . crescentus CB15 was cloned and sequenced . By hybridization to pulsed-field electrophoresis gels, sodC was mapped near cysE in the C . crescentus chromosome . Through analysis of spheroplasts, the two SODs of C . crescentus were shown to be differently localized, Cu