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

Manifestation of pleiotropic effects in the isogenic variants of the luminescent bacterium Photobacterium leiognathi 54 was investigated. The decrease or increase of the expression level of bioluminescence was caused by changes in lux operon regulation. The dynamics of the bioluminescence of dark and dim variants did not differ from the dynamics of the initial luminescent variant, but dependence of the level of luminescence intensity on the exogenous autoinducer of the lux operon was revealed. The investigated variants of P. leiognathi 54 inherited fairly stable morphological characteristics, colony architectonics, level of luminescence, and activity of some enzymes; variants with reduced bioluminescence formed colonies of the S type. Stable bright variants with S-and R-type colonies appeared both in the initial strain population and in the dark variant population, but with smaller frequency. Populations of the bright variant with R-type colonies were most heterogeneous; this can be determined by the lack of glucose repression of the bioluminescence in contrast to other investigated inherited variants of P. leiognathi.     

Quantitative criteria for the estimation of the effectiveness of bioluminescence expression in natural and transgenic luminescent bacteria

Computation coefficients for estimating the effectiveness of bioluminescence expression in natural luminescent bacteria P. leiognathi 54 and transgenic strain E. coli Z905/pPHL7 bearing lux-operon in multicopy plasmid are suggested, and their use on the molecular, cell, and population levels was considered. It was shown that, on the population level, all transgenic variants got the better of natural variants of P. leiognathi 54 irrespective of the type of lux-operon regulation. On the cell level, in the bright and dim variants of the transgenic strain, the effectiveness of bioluminescence expression increases by several orders. On the level of one lux-operon, the effectiveness of expression of the bright variant of transgenic strain is substantially higher than in the natural bright variant; in dim variants, the efficiency values are similar, and the effectiveness of bioluminescence expression in the dark variant of E. coli Z905-2 /pPHL7 is by two orders lower than that in the dark variant of P. leiognathi 54.     

Quantitative criteria for estimating the effectiveness of bioluminescence expression in natural and transgenic luminescent bacteria

Computational coefficients for estimating the effectiveness of bioluminescence expression in natural luminescent bacteria Photobacterium leiognathi 54 and transgenic strain E. coli Z905/pPHL7 bearing lux-operon in a multicopy plasmid are suggested and their use at molecular, cell, and population levels was considered. It was shown that at the population level, all transgenic variants have an advantage over natural variants of P. leiognathi 54 irrespective of the type of lux-operon regulation. At the cell level, the effectiveness of bioluminescence expression in the bright and dim variants of the transgenic strain increased by several orders. At the level of one lux-operon, the effectiveness of expression in the bright variant of the transgenic strain is substantially higher than in the natural bright variant; in dim variants, the efficiency values are similar; the effectiveness of bioluminescence expression in the dark variant of E. coli Z905-2/pPHL7 is by two orders of magnitude lower than in the dark variant of P. leiognathi 54.     

Effect of salts on luminescence of natural and recombinant luminescent bacterial biosensors

Effect of cations K+, Na+, Mg2+, and Ca2+ and anions SO4(2-), HCO3(-), and CO3(2-) on the luminescence intensity of the marine luminescent bacterium Photobacterium phorphoreum (Microbiosensor B-17 677f) and the recombinant strain Escherichia coli with cloned lux operon of P. leiognathi (Ekolyum-9). It is found that small concentrations of chlorides and sulfates of the cations studied had a concentration-dependent stimulatory effect on bacterial bioluminescence; as the concentration of agents increased, activation was succeeded by quenching. The strength of the inhibitory effect, which is characterized by EC50, decreased in the series Ca2+ > Na+ > Mg2+ > K+. Carbonates and hydrocarbonates had a pronounced inhibitory effect on the bioluminescence intensity, determined by an increase in pH. We showed that some types of highly mineralized water with a high hydrocarbonate content have a marked inhibitory effect on the luminescence intensity of microbial luminescent biosensors, mimicking the effect of chemical pollutants.     

The lux genes of the luminous bacterial symbiont, Photobacterium leiognathi, of the ponyfish. Nucleotide sequence, difference in gene organization, and high expression in mutant Escherichia coli

The lux genes required for light expression in the luminescent bacterium Photobacterium leiognathi (ATCC 25521) have been cloned and expressed in Escherichia coli and their organization and nucleotide sequence determined. Transformation of a recombinant 9.5-kbp chromosomal DNA fragment of P. leiognathi into an E. coli mutant (43R) gave luminescent colonies that were as bright as those of the parental strain. Moreover, expression of the lux genes in the mutant E. coli was strong enough so that not only were high levels of luciferase detected in crude extracts, but the fatty-acid reductase activity responsible for synthesis of the aldehyde substrate for the luminescent reaction could readily be measured. Determination of the 7.3-kbp nucleotide sequence of P. leiognathi DNA, including the genes for luciferase (luxAB) and fatty-acid reductase (luxCDE) as well as a new lux gene (luxG) found recently in luminescent Vibrio species, showed that the order of the lux genes was luxCDABEG. Moreover, luxF, a gene homologous to luxB and located between luxB and luxE in Photobacterium but not Vibrio strains, was absent. In spite of this different lux gene organization, an intergenic stem-loop structure between luxB and luxE was discovered to be highly conserved in other Photobacterium species after luxF.     

Conditions that influence bacterial luminescence in the presence of blood serum

Conditions that influence the luminescence of natural and recombinant luminescent bacteria in the presence of blood serum were studied. In general, blood serum quenched the luminescence of the marine Photobacterium phosphoreum and the recombinant Escherichia coli strains harboring the luminescent system genes of Photobacterium leiognathi, but enhanced the luminescence of the soil bacterium Photorhabdus luminescens Zm1 and the recombinant E. coli strain harboring the lux operon of P. luminescens Zm1. The quenching effect of blood serum increased with its concentration and the time and temperature of incubation. The components of blood serum that determine the degree and specificity of its action on bacterial luminescence were identified.     

Natural merodiploidy of the lux-rib operon of Photobacterium leiognathi from coastal waters of Honshu, Japan

Sequence analysis of the bacterial luminescence (lux) genes has proven effective in helping resolve evolutionary relationships among luminous bacteria. Phylogenetic analysis using lux genes, however, is based on the assumptions that the lux genes are present as single copies on the bacterial chromosome and are vertically inherited. We report here that certain strains of Photobacterium leiognathi carry multiple phylogenetically distinct copies of the entire operon that codes for luminescence and riboflavin synthesis genes, luxCDABEG-ribEBHA. Merodiploid lux-rib strains of P. leiognathi were detected during sequence analysis of luxA. To define the gene content, organization, and sequence of each lux-rib operon, we constructed a fosmid library of genomic DNA from a representative merodiploid strain, lnuch.13.1. Sequence analysis of fosmid clones and genomic analysis of lnuch.13.1 defined two complete, physically separate, and apparently functional operons, designated lux-rib1 and lux-rib2. P. leiognathi strains lelon.2.1 and lnuch.21.1 were also found to carry lux-rib1 and lux-rib2, whereas ATCC 25521T apparently carries only lux-rib1. In lnuch.13.1, lelon.2.1, lnuch.21.1, and ATCC 25521T, lux-rib1 is flanked upstream by lumQ and putA and downstream by a gene for a hypothetical multidrug efflux pump. In contrast, transposase genes flank lux-rib2 of lnuch.13.1, and the chromosomal location of lux-rib2 apparently differs in lnuch.13.1, lelon.2.1, and lnuch.21.1. Phylogenetic analysis demonstrated that lux-rib1 and lux-rib2 are more closely related to each other than either one is to the lux and rib genes of other bacterial species, which rules out interspecies lateral gene transfer as the origin of lux-rib2 in P. leiognathi; lux-rib2 apparently arose within a previously unsampled or extinct P. leiognathi lineage. Analysis of 170 additional strains of P. leiognathi, for a total of 174 strains examined from coastal waters of Japan, Taiwan, the Philippine Islands, and Thailand, identified 106 strains that carry only a single lux-rib operon and 68 that carry multiple lux-rib operons. Strains bearing a single lux-rib operon were obtained throughout the geographic sampling range, whereas lux-rib merodiploid strains were found only in coastal waters of central Honshu. This is the first report of merodiploidy of lux or rib genes in a luminous bacterium and the first indication that a natural merodiploid state in bacteria can correlate with geography.     

Effects of luxCDABEG induction in Vibrio fischeri: enhancement of symbiotic colonization and conditional attenuation of growth in culture

Production of bioluminescence theoretically represents a cost, energetic or otherwise, that could slow Vibrio fischeri growth; however, bioluminescence is also thought to enable full symbiotic colonization of the Euprymna scolopes light organ by V. fischeri. Previous tests of these models have proven inconclusive, partly because they compared nonisogenic strains, or undefined and/or pleiotropic mutants. To test the influence of the bioluminescence-producing lux operon on growth and symbiotic competence, we generated dark luxCDABEG mutants in strains MJ1 and ES114 without disrupting the luxR-luxI regulatory circuit. The MJ1 luxCDABEG mutant out-competed its visibly luminescent parent approximately 26% per generation in a carbon-limited chemostat. Similarly, induction of luminescence in the otherwise dim ES114 strain slowed growth relative to DeltaluxCDABEG mutants. Some culture conditions yielded no detectable effect of luminescence on growth, indicating that luminescence is not always growth limiting; however, luminescence was never found to confer an advantage in culture. In contrast to this conditional disadvantage of lux expression, ES114 achieved approximately fourfold higher populations than its luxCDABEG mutant in the light organ of E. scolopes. These results demonstrate that induction of luxCDABEG can slow V. fischeri growth under certain culture conditions and is a positive symbiotic colonization factor.     

Biotests for mineral waters with natural and recombinant luminescent microorganisms

We have developed methods of biotesting mineral waters involving use of natural or recombinant luminescent strains with elimination of the effect of salt concentration and pH. To overcome the adverse effect of high salt concentrations, disguising the action of chemical pollutants, a special method of mineral water sample preparation is proposed. In this method, the marine luminescent bacterium Photobacterium phosphoreum (Microbiosensor B17 677f) is used as a test object. Samples to be analyzed are supplemented with NaCl depending on their natural salt concentration to adjust it to 3 g/l. Another approach, more universal and efficient, involves pH adjustment in the samples to 7.5. This value is suitable for application of both Microbiosensor B17 677f and the recombinant Escherichia coli strain harboring the cloned lux operon of P. leiognathi (Ecolum 9). It has been shown that this treatment, retaining the natural luminescence level of the bacterial biosensors, allows bioluminescent detection of exogenous pollutants added to the samples, including benzene and Cr(VI).     

Molecular biology of bacterial bioluminescence.

The cloning and expression of the lux genes from different luminescent bacteria including marine and terrestrial species have led to significant advances in our knowledge of the molecular biology of bacterial bioluminescence. All lux operons have a common gene organization of luxCDAB(F)E, with luxAB coding for luciferase and luxCDE coding for the fatty acid reductase complex responsible for synthesizing fatty aldehydes for the luminescence reaction, whereas significant differences exist in their sequences and properties as well as in the presence of other lux genes (I, R, F, G, and H). Recognition of the regulatory genes as well as diffusible metabolites that control the growth-dependent induction of luminescence (autoinducers) in some species has advanced our understanding of this unique regulatory mechanism in which the autoinducers appear to serve as sensors of the chemical or nutritional environment. The lux genes have now been transferred into a variety of different organisms to generate new luminescent species. Naturally dark bacteria containing the luxCDABE and luxAB genes, respectively, are luminescent or emit light on addition of aldehyde. Fusion of the luxAB genes has also allowed the expression of luciferase under a single promoter in eukaryotic systems. The ability to express the lux genes in a variety of prokaryotic and eukaryotic organisms and the ease and sensitivity of the luminescence assay demonstrate the considerable potential of the widespread application of the lux genes as reporters of gene expression and metabolic function.     

Intracellular redox equilibrium and growth phase affect the performance of luciferase-based biosensors

Light emission from the bacterial luciferase operon has been variously exploited during last two decades. The use of convenient inducible promoters has granted significant degrees of specificity to whole cell-based assays for high-throughput screening and environmental monitoring. Nevertheless, unexplained unspecific responses have been repeatedly reported. Here, we show that the impairment of the intracellular biochemical equilibrium interferes with the luminescence produced by Escherichia coli and Staphylococcus aureus strains carrying the lux operon under constitutive or inducible control. Compounds as trimethoprim and methotrexate, by indirectly inducing NADPH accumulation, enhance light emission. Conversely, molecules driving the cell toward an oxidized state, as dimethyl sulfoxide, inhibit luminescence. These findings fit into the accepted biochemical pathway for bioluminescence, where NADPH and reducing equivalents are necessary for the production of luciferase substrates, although they do not directly take part into the light-emitting reaction. Moreover, we investigated the influence of induction timing upon the bioluminescence response from inducible reporter systems and demonstrated a correlation between the emitted light and the growth phase at which induction is performed. Our results provide explanations for some unspecific responses recorded so far in whole cell-based luminescent biosensors and emphasize the intrinsic limitations of this kind of reporting system.     

A homoserine lactone autoinducer regulates virulence of an insect-pathogenic bacterium, Xenorhabdus nematophilus (Enterobacteriaceae).

N-beta-Hydroxybutanoyl homoserine lactone (HBHL), the autoinducer of the luminescent system of Vibrio harveyi, has been identified as the first small compound to restore virulence to avirulent mutants of Xenorhabdus nematophilus. HBHL stimulated the level of lipase activity excreted by avirulent X. nematophilus and lowered the phenoloxidase activity in the hemolymph of insects infected with X. nematophilus, parameters that are both associated with insect pathogenesis. Moreover, mortality of the insects infected with avirulent X. nematophilus was restored upon injection with HBHL. Chloroform extraction of medium conditioned with wild-type but not avirulent X. nematophilus led to the isolation of a compound with the same chromatographic mobility as HBHL as well as the ability to stimulate the luminescence of a dim autoinducer-dependent mutant of V. harveyi. Transfer of the V. harveyi lux operon into avirulent and wild-type X. nematophilus generated dim and bright luminescent strains, respectively, which responded to HBHL and an agonist and antagonist in a manner analogous to their effects on the luminescence of dim autoinducer-deficient and bright wild-type strains of V. harveyi, indicating that similar HBHL-dependent regulatory systems exist in these two bacterial species.     

Cloning and expression of the lux-operon of Photorhabdus luminescens, strain Zm1: nucleotide sequence of luxAB genes and basic properties of luciferase

A chromosomal fragment of bacteria Photorhabdus luminescence Zm1, which contains the lux operon, was cloned into the vector pUC18. The hybrid clone containing plasmid pXen7 with the EcoRI fragment approximately 7-kb was shown to manifest a high level of bioluminescence. By subcloning and restriction analysis of the EcoRI fragment, the location of luxCDABE genes relative to restriction sites was determined. The nucleotide sequence of the DNA fragment containing the luxA and luxB genes encoding alpha- and beta-subunits of luciferase was determined. A comparison with the nucleotide sequences of luxAB genes in Hm and Hw strains of Ph. luminescence revealed 94.5 and 89.7% homology, respectively. The enterobacterial repetitive intergenic sequence (ERIC) of 126 bp typical for Hw strains was identified in the spacer between the luxD and luxA genes. The lux operon of Zm1 is assumed to emerge through recombination between Hm and Hw strains. Luciferase of Ph. luminescence was shown to possess a high thermal stability: its activity decreased by a factor of 10 at 44 degrees C for 30 min, whereas luciferases of marine bacteria Vibrio fischeri and Vibrio harveyi were inactivated by one order of magnitude at 44 degrees C for 1 and 6 min, respectively. The lux genes of Ph. luminescence are suggested for use in gene engineering and biotechnology.     

Phylogenetic analysis of the incidence of lux gene horizontal transfer in Vibrionaceae

Horizontal gene transfer (HGT) is thought to occur frequently in bacteria in nature and to play an important role in bacterial evolution, contributing to the formation of new species. To gain insight into the frequency of HGT in Vibrionaceae and its possible impact on speciation, we assessed the incidence of interspecies transfer of the lux genes (luxCDABEG), which encode proteins involved in luminescence, a distinctive phenotype. Three hundred three luminous strains, most of which were recently isolated from nature and which represent 11 Aliivibrio, Photobacterium, and Vibrio species, were screened for incongruence of phylogenies based on a representative housekeeping gene (gyrB or pyrH) and a representative lux gene (luxA). Strains exhibiting incongruence were then subjected to detailed phylogenetic analysis of horizontal transfer by using multiple housekeeping genes (gyrB, recA, and pyrH) and multiple lux genes (luxCDABEG). In nearly all cases, housekeeping gene and lux gene phylogenies were congruent, and there was no instance in which the lux genes of one luminous species had replaced the lux genes of another luminous species. Therefore, the lux genes are predominantly vertically inherited in Vibrionaceae. The few exceptions to this pattern of congruence were as follows: (i) the lux genes of the only known luminous strain of Vibrio vulnificus, VVL1 (ATCC 43382), were evolutionarily closely related to the lux genes of Vibrio harveyi; (ii) the lux genes of two luminous strains of Vibrio chagasii, 21N-12 and SB-52, were closely related to those of V. harveyi and Vibrio splendidus, respectively; (iii) the lux genes of a luminous strain of Photobacterium damselae, BT-6, were closely related to the lux genes of the lux-rib(2) operon of Photobacterium leiognathi; and (iv) a strain of the luminous bacterium Photobacterium mandapamensis was found to be merodiploid for the lux genes, and the second set of lux genes was closely related to the lux genes of the lux-rib(2) operon of P. leiognathi. In none of these cases of apparent HGT, however, did acquisition of the lux genes correlate with phylogenetic divergence of the recipient strain from other members of its species. The results indicate that horizontal transfer of the lux genes in nature is rare and that horizontal acquisition of the lux genes apparently has not contributed to speciation in recipient taxa.     

Limited geographic distribution of certain strains of the bioluminescent symbiont Photobacterium leiognathi

Photobacterium leiognathi is a facultative bioluminescent symbiont of marine animals. Strains of P.?leiognathi that are merodiploid for the luminescence genes (lux-rib operon) have been previously obtained only from Japan. In contrast, strains bearing a single lux-rib operon have been obtained from all the areas sampled in Japan and the western Pacific. In this study, we tested whether distribution of merodiploid P.?leiognathi is limited by physical barriers in the environment, or because fish in the western Pacific preferentially form symbiosis with bacteria bearing a single lux-rib operon. We collected light organ symbionts from Secutor indicius, a fish species that is typically found in the western Pacific and has only recently expanded its geographic range to Japan. We found that all S.?indicius specimens collected from Japan formed symbiosis only with single lux-rib operon-bearing strains, although fish from other species collected from the same geographic area frequently contained merodiploid strains. This result shows that S.?indicius were preferentially colonized by bacteria bearing a single lux-rib operon and suggests that the limited geographic distribution of merodiploid P.?leiognathi can be attributed to preferential colonization of fish species found in the western Pacific by strains bearing only a single lux-rib operon.     

A new rapid and sensitive bioluminescence assay for antibiotics that inhibit protein synthesis

N aveh , A., P otasman , I., B assan , H. & U litzur , S. 1984. A new rapid and sensitive bioluminescence assay for antibiotics that inhibit protein synthesis. Journal of Applied Bacteriology 56 , 457–463.
A new sensitive, rapid and simple bioluminescence assay for antibiotics inhibiting protein synthesis is described. In this assay the ability of the tested antibiotic to inhibit the de novo synthesis of the enzymes participating in the bacterial luminescence system is determined by means of a dark variant of a luminous bacterium that undergoes prompt induction of the luminescence system with certain DNA-intercalating agents. Upon induction, the in vivo luminescence of the dark variant is increased more than 50-fold within 30 min. Antibiotics that block the de novo synthesis of protein limit the development of luminescence at a level that was found to be a function of the antibiotic concentration. The minimum detectable concentration of antibiotics in the bioluminescence test, after 45–60 min of incubation, was 0.1 μg ml for streptomycin, gentamicin, kanamycin, lincomycin and chlorampheni-col and 0.3 μg/ml for neomycin, clindamycin and spectinomycin. The new bioluminescence test has been used to assay these antibiotics in serum.     

Cloning and expression of genes of the luminescence system in Photobacterium leiognathi

The genes of Photobacterium leiognathi luminescence system were cloned in plasmid pUC18. Escherichia coli cells harboring a recombinant plasmid pPHL1 are luminescent. pPHL1 contains luciferase genes and genes responsible for aldehyde biosynthesis. The luminescence of Escherichia coli is subject to autoinductor regulation similar to the one existing in luminescent bacteria. The 2.7 kb fragment of Photobacterium leiognathi DNA containing the genes for alpha- and beta-luciferase subunits were cloned in pUC19.     

Induction of light emission by luminescent bacteria treated with UV light and chemical mutagens

Intensity of light emission by luminescent bacteria in response to UV irradiation and chemical mutagens was tested. We demonstrated that luminescence of six strains of marine bacteria (belonging to four species: Photobacterium leiognathi, P. phosphoreum, Vibrio fischeri and V. harveyi) is significantly increased by UV irradiation relatively shortly after dilution of cultures. Such a stimulation of luminescence was abolished in cells treated with chloramphenicol 15 min before UV irradiation, indicating that effective gene expression is necessary for UV-mediated induction of light emission. These results suggest that stimulation of luminescence in UV-irradiated bacterial cells may operate independently of the quorum sensing regulation. A significant induction of luminescence was also observed upon treatment of diluted cultures of all investigated strains with chemical mutagens: sodium azide (SA), 2-methoxy-6-chloro-9-(3-(2-chloroethyl)aminopropylamino)acridine x 2HCl (ICR-191), 4-nitro-o-phenylenediamine (NPD), 4-nitroquinolone-N-oxide (NQNO), 2-aminofluorene (2-AF), and benzo[alpha]pyrene. These results support the proposal that genes involved in bioluminescence belong to the SOS regulon. The use of bacterial luminescence systems in assays for detection of mutagenic compounds is discussed in the light of this proposal.