Codon optimization of bacterial luciferase (lux) for expression in mammalian cells

Expression of the bacterial luciferase (lux) system in mammalian cells would culminate in a new generation of bioreporters for in vivo monitoring and diagnostics technology. Past efforts to express bacterial luciferase in mammalian cells have resulted in only modest gains due in part to low overall expression of the bacterial genes. To optimize expression, we have designed and synthesized codon-optimized versions of the luxA and luxB genes from Photorhabdus luminsecens. To evaluate these genes in vivo, stable HEK293 cell lines were created harboring wild type luxA and luxB (WTA/WTB), codon-optimized luxA and wild type luxB (COA/WTB), and codon-optimized versions of both luxA and luxB genes (COA/COB). Although mRNA levels within these clones remained approximately equal, LuxA protein levels increased significantly after codon optimization. On average, bioluminescence levels were increased by more than six-fold [5×10⁵ vs 2.9×10⁶ relative light units (RLU)/mg total protein] with the codon-optimized luxA and wild type luxB. Bioluminescence was further enhanced upon expression of both optimized genes (2.7×10⁷ RLU/mg total protein). These results show promise toward the potential development of an autonomous light generating lux reporter system in mammalian cells     

Stimulation of DNA repair as an evolutionary drive for bacterial luminescence

It was demonstrated recently that luminescence of a free‐living marine bacterium, Vibrio harveyi, stimulates DNA repair, most probably by activation of the photoreactivation process. Here, we ask whether the stimulation of DNA repair could be an evolutionary drive that ensured maintenance and development of early bacterial luminescent systems. To test this hypothesis, we cultivated V. harveyi lux⁺ bacteria and luxA mutants in mixed cultures. Initial cultures were mixed to obtain a culture consisting of roughly 50% lux⁺ cells and 50% luxA mutants. Then bacteria were cultivated for several days and ratio of luminescent to dark bacteria was measured. Under these conditions, luxA mutants became highly predominant within a few days of cultivation. This indicates that, without a selective pressure, the luminescence is a disadvantage for bacteria, perhaps due to consumption of significant portion of cell energy. However, when the same experiments were repeated but cultures were irradiated with low UV doses, luminescent bacteria started to predominate shortly after the irradiation. Therefore, we conclude that stimulation of photoreactivation may be an evolutionary drive for bacterial bioluminescence. Copyright © 2003 John Wiley & Sons, Ltd.     

Identification of Vibrio splendidus as a Member of the Planktonic Luminous Bacteria from the Persian Gulf and Kuwait Region with luxA Probes

Hybridization probes specific for the luxA genes of four groups of luminous bacteria were used to screen luminous isolates obtained from the Persian Gulf, near Al Khiran, Kuwait Nine of these isolates were identified as Vibrio harveyi, a commonly encountered planktonic isolate, while three others showed no hybridization to any of the four probes (V. harveyi, Vibrio fischeri, Photobacterium phosphoreum, or Photobacterium leiognathi) under high-stringency conditions. Polymerase chain reaction amplification was used to prepare a luxA probe against one of these isolates, K-1, and this probe was screened under high-stringency conditions against a collection of DNAs from luminous bacteria; it was found to hybridize specifically to the DNA of the species Vibrio splendidus. A probe prepared against the type strain of V. splendidus (ATCC 33369) was tested against the collection of luminous bacterial DNA preparations and against the Kuwait isolates and was found to hybridize only against the type strain and the three unidentified Kuwait isolates. Extensive taxonomic analysis by standard methods confirmed the identification of the 13 isolates.     

Enteric luminous microflora of the pond-cultured milk fishChanos chanos (Forskal)

Qualitative and quantitative investigations were made on the luminous bacteria associated with the gut of pond cultured milk fishChanos chanos. Significant differences in luminous bacterial numbers were found between gut and pond water and between gut and pond sediment, but not between pond water and sediment. No significant variation in luminous bacterial population among the gut regions was observed. The quantity of ingesta in the fish gut does not appear to influence the biomass of luminous bacteria.Vibrio harveyi andV. fischeri were the 2 most commonly encountered species, and of the 2 luminous species,V. harveyi was predominant.     

Growth and luminescence of luminous bacteria promoted by agents of microbial origin

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, nucleosides and amine bases), amino acids and vitamins, which are part of hydrolysates from the biomass of various lithotrophic microorganisms, hydrogen-oxidizing, ironoxidizing 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.     

Symbiotic association of Photobacterium fischeri with the marine luminous fish Monocentris japonica; a model of symbiosis based on bacterial studies.

Isolation of bacteria from the luminous organ of the fish Monocentris japonica has revealed that the organ contains a pure culture of luminous bacteria. For the four fish examined, all contained Photobacterium fischeri as their luminous bacterial symbiont. This is the first time that P. fischeri has been identified in a symbiotic association. A representative isolate (MJl) of the light organ population was selected for in vivo studies of its luminous system. Several physiological features suggest adaptation for symbiotic existence. First, MJl has been shown to produce and respond to an inducer of luciferase that could accumulate in the light organ. Secondly, the specific activity of light production was seen to be maximal under low, growth-limiting concentrations of oxygen. Thirdly, unlike another luminous species (Beneckea harveyi), synthesis of the light production system of these bacteria is not catabolite repressed by glucose--a possible source of nutrition in the light organ. Fourthly, when grown aerobically on glucose these bacteria excrete pyruvic acid into the medium. This production of pyruvate is a major process, accounting for 30-40% of the glucose utilized and may serve as a form of regulatory and nutritional communication with the host.     

Phylogenetic analysis of host–symbiont specificity and codivergence in bioluminescent symbioses

Several groups of marine fishes and squids form mutualistic bioluminescent symbioses with luminous bacteria. The dependence of the animal on its symbiont for light production, the animal's specialized anatomical adaptations for harboring bacteria and controlling light emission, and the host family bacterial species specificity characteristic of these associations suggest that bioluminescent symbioses are tightly coupled associations that might involve coevolutionary interactions. Consistent with this possibility, evidence of parallel cladogenesis has been reported for squid–bacterial associations. However, genetic adaptations in the bacteria necessary for and specific to symbiosis have not been identified, and unlike obligate endosymbiotic associations in which the bacteria are transferred vertically, bacterially bioluminescent hosts acquire their light‐organ symbionts from the environment with each new host generation. These contrasting observations led us to test the hypotheses of species specificity and codivergence in bioluminescent symbioses, using an extensive sampling of naturally formed associations. Thirty‐five species of fish in seven teleost families (Chlorophthalmidae, Macrouridae, Moridae, Trachichthyidae, Monocentridae, Acropomatidae, Leiognathidae) and their light‐organ bacteria were examined. Phylogenetic analysis of a taxonomically broad sampling of associations was based on mitochondrial 16S rRNA and cytochrome oxidase I gene sequences for the fish and on recA, gyrB and luxA sequences for bacteria isolated from the light organs of these specimens. In a fine‐scale test focused on Leiognathidae, phylogenetic analysis was based also on histone H3 subunit and 28S rRNA gene sequences for the fish and on gyrB, luxA, luxB, luxF and luxE sequences for the bacteria. Deep divergences were revealed among the fishes, and clear resolution was obtained between clades of the bacteria. In several associations, bacterial species identities contradicted strict host family bacterial species specificity. Furthermore, the fish and bacterial phylogenies exhibited no meaningful topological congruence; evolutionary divergence of host fishes was not matched by a similar pattern of diversification in the symbiotic bacteria. Re‐analysis of data reported for squids and their luminous bacteria also revealed no convincing evidence of codivergence. These results refute the hypothesis of strict host family bacterial species specificity and the hypothesis of codivergence in bioluminescent symbioses. © The Willi Hennig Society 2007.     

The Effects of Regulatory Proteins RcsA and RcsB on the Expression of the Vibrio fischeri lux Operon in Escherichia coli

A study was made of the effect of RcsA and RcsB on the Vibrio fischeri lux expression in Escherichia coli. RcsA suppressed the LuxR activity and thereby inhibited expression of the lux genes coding for luciferase and reductase. In osmotic shock, RcsA–RcsB activated lux expression and, consequently, the bioluminescence of E. coli cells in the early log phase.     

The uncultured luminous symbiont of Anomalops katoptron (Beryciformes: Anomalopidae) represents a new bacterial genus

Flashlight fishes (Beryciformes: Anomalopidae) harbor luminous symbiotic bacteria in subocular light organs and use the bacterial light for predator avoidance, feeding, and communication. Despite many attempts anomalopid symbionts have not been brought into laboratory culture, which has restricted progress in understanding their phylogenetic relationships with other luminous bacteria, identification of the genes of their luminescence system, as well as the nature of their symbiotic interactions with their fish hosts. To begin addressing these issues, we used culture-independent analysis of the bacteria symbiotic with the anomalopid fish, Anomalops katoptron, to characterize the phylogeny of the bacteria and to identify the genes of their luminescence system including those involved in the regulation of luminescence. Analysis of the 16S rRNA, atpA, gapA, gyrB, pyrH, recA, rpoA, and topA genes resolved the A. katoptron symbionts as a clade nested within and deeply divergent from other members of Vibrionaceae. The bacterial luminescence (lux) genes were identified as a contiguous set (luxCDABEG), as found for the lux operons of other luminous bacteria. Phylogenetic analysis based on the lux genes confirmed the housekeeping gene phylogenetic placement. Furthermore, genes flanking the lux operon in the A. katoptron symbionts differed from those flanking lux operons of other genera of luminous bacteria. We therefore propose the candidate name Candidatus Photodesmus (Greek: photo = light, desmus = servant) katoptron for the species of bacteria symbiotic with A. katoptron. Results of a preliminary genomic analysis for genes regulating luminescence in other bacteria identified only a Vibrio harveyi-type luxR gene. These results suggest that expression of the luminescence system might be continuous in P. katoptron.     

Genomic signatures of obligate host dependence in the luminous bacterial symbiont of a vertebrate

The majority of bacteria engaged in bioluminescent symbiosis are environmentally acquired and facultatively symbiotic. A few enigmatic bioluminescent symbionts have not been successfully cultured, which has led to speculation that they may be obligately dependent on their hosts. Here, we report the draft genome of the uncultured luminous symbiont of an anomalopid flashlight fish, ‘Candidatus Photodesmus katoptron’. The genome of the anomalopid symbiont is reduced by 80% compared with close relatives and lacks almost all genes necessary for amino acid synthesis and for metabolism of energy sources other than glucose, supporting obligate dependence on the host for growth. ‘Candidatus Photodesmus katoptron’ is the first described obligate mutualistic symbiont of a vertebrate. Unlike most other obligate mutualists, the anomalopid symbiont genome has retained complete pathways for chemotaxis and motility as well as most genes involved in cell wall production, consistent with the hypothesis that these bacteria may be transmitted environmentally during an extra‐host phase.     

Comparison of the spectral emission of lux recombinant and bioluminescent marine bacteria

The purpose of the present paper was to study the influence of bacteria harbouring the luciferase‐encoding Vibrio harveyi luxAB genes upon the spectral emission during growth in batch‐culture conditions. In vivo bioluminescence spectra were compared from several bioluminescent strains, either naturally luminescent (Vibrio fischeri and Vibrio harveyi) or in recombinant strains (two Gram‐negative Escherichia coli::luxAB strains and a Gram‐positive Bacillus subtilis::luxAB strain). Spectral emission was recorded from 400 nm to 750 nm using a highly sensitive spectrometer initially devoted to Raman scattering. Two peaks were clearly identified, one at 491–500 nm (± 5 nm) and a second peak at 585–595 (± 5 nm) with the Raman CCD. The former peak was the only one detected with traditional spectrometers with a photomultiplier detector commonly used for spectral emission measurement, due to their lack of sensitivity and low resolution in the 550–650 nm window. When spectra were compared between all the studied bacteria, no difference was observed between natural or recombinant cells, between Gram‐positive and Gram‐negative strains, and growth conditions and growth medium were not found to modify the spectrum of light emission. Copyright © 2003 John Wiley & Sons, Ltd.     

Role of luminous bacteria in chitin degradation in the intestine of fish

Summary Chitinolytic activity of three luminous bacterial species viz.,Vibrio harveyi, V. fischeri andPhotobacterium leiognathi, was estimated. The stomach of the fishTachysurus arius which harbours luminous bacteria also possesses chitinase activity. The magnitude of non-bacterial chitinase activity appeared to exceed that of the bacterial origin as evidenced by the enzyme activity detected in fish tested with antibiotics. In fishes which lack an indigenous chitinase, the microbial chitinase is likely to play an important role in chitin digestion.

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Role des bactéries lumineuses dans la dégradation de la chitine dans l'intestin de poisson

Résumé On a estimé l'activité chitinolytique de trois espèces de bactéries lumineuses, notammentVibrio harveyi, V. fischeri andPhoto bacterium leiognathi. L'estomac du poisson Tachysurus arius qui héberge les bactéries lumineuses possède aussi l'activité chitinasique. L'activité chitinasique non-bactérienne dépasse celle d'origine bactérienne ainsi qu'il en résulte de l'activité enzymatique chez les poissons traités aux antibiotiques. Chez les poissons déficients en activité chitinasique indigène, la chitinase microbienne joue probablement un rôle important dans la digestion de la chitine.

     

Vibrio harveyi Associated with Aglaophenia octodonta (Hydrozoa, Cnidaria)

A previously unknown association between a luminous bacterium, Vibrio harveyi, and a benthic hydrozoan, Aglaophenia octodonta, is described. Aglaophenia hydrocladia showed a clear fluorescence in the folds along the hydrocaulus and at the base of the hydrotheca, suggesting the presence of luminous bacteria. This hypothesis was confirmed by isolation of luminous bacteria from Aglaophenia homogenates. Phenotypic characterization of bacterial isolates was performed by several morphological, biochemical, and cultural tests, completed with 16S rDNA sequence analysis. All the isolates were referred to a single species: V. harveyi. The association between V. harveyi and A. octodonta has epidemiological as well as ecological significance. Therefore, A. octodonta may function as habitat “islands” providing a unique set of environmental conditions for luminous bacteria colonization, quite different from those already recorded from the plankton for other Vibrio species.     

Stratified bacterial community in the bladder of the medicinal leech, Hirudo verbana

Most animals harbour symbiotic microorganisms inside their body, where intimate interactions occur between the partners. The medicinal leech, Hirudo verbana, possesses 17 pairs of excretory bladders that harbour a large number of intracellular and extracellular symbiotic bacteria. In this study, we characterized the bladder symbionts using molecular phylogenetic analyses, transmission electron microscopy (TEM) and fluorescence in situ hybridization (FISH). Restriction fragment length polymorphism (RFLP) and sequence analyses of 16S rRNA gene clone libraries suggested that six bacterial species co‐colonize the leech bladders. Phylogenetic analyses revealed that these species belong to the α‐Proteobacteria (Ochrobactrum symbiont), β‐Proteobacteria (Beta‐1 and Beta‐2 symbionts), δ‐Proteobacteria (Bdellovibrio symbiont) and Bacteroidetes (Niabella and Sphingobacterium symbionts). Species‐specific PCR detection and FISH confirmed the localization of the symbiotic bacteria in the bladders. The Ochrobactrum, Beta‐1, Bdellovibrio and Sphingobacterium symbionts were consistently detected in 13 leeches from two populations, while infection rate of the other symbionts ranged between 20% and 100% in the two leech populations. Transmission electron microscopy observations of the bladders revealed epithelial cells harbouring a number of intracellular bacilli and an additional type of extracellular, rod‐shaped bacteria in the luminal region. Fluorescence in situ hybridization with group‐specific oligonucleotide probes revealed the spatial organization of the bacterial species in the bladder: the Ochrobactrum symbiont was located intracellularly inside epithelial cells; the Bacteroidetes were localized close to the epithelium in the lumen of the bladder; and the Bacteroidetes layer was covered with dense β‐proteobacterial cells. These results clearly demonstrate that a simple but organized microbial community exists in the bladder of the medicinal leech.     

Localisation of luciferase in luminous marine bacteria by gold immunocytochemical labelling

Abstract Bioluminescent bacteria are able to produce light by an oxidative reaction involving the enzyme luciferase. The biochemistry of the reaction has been well documented. It has been postulated from the reaction pathway that the enzyme luciferase is membrane bound. Two species of luminous bacteria, Vibrio fischeri and V. harveyi , were isolated from sea water, fixed and embedded in resin for transmission electron microscopy. A commercially available V. fischeri bacterial luciferase was used to immunise rats for production of an antibody against luciferase. This antibody was coupled with a protein A gold probe and used to immunolabel the sections of bacteria. It was shown that the luciferase was associated with the inner membrane, and to a lesser extent present in the cytoplasm. Our results confirm that the luciferase is predominantly membrane bound.     

Evidence of luminous bacterial symbionts in the light organs of myctophid and stomiiform fishes

The myctophids and stomiiforms represent two common groups of luminous fishes, but the source of luminescence in these animals has remained undetermined. In this study, labeled luciferase gene fragments from luminous marine bacteria were used to probe DNA isolated from specific fish tissues. A positive signal was obtained from skin DNA in all luminous fishes examined, whereas muscle DNA gave a weaker signal and brain DNA was negative. This observation is consistent with luminous bacteria acting as the light source in myctophids and stomiiforms and argues against the genes necessary for luminescence residing on the fish chromosomes. To confirm the location of this signal, a bacterial probe was hybridized in situ to sections of a stomiiform. A strong signal was generated directly over specific regions of the fish light organs, whereas no signal was found over other internal or epidermal tissues of the fish. Taken together, these data provide the first indication that luminous bacterial symbionts exist in myctophids and stomiiforms and that these symbionts account for luminescence in these fishes.