Sensitive In Situ Monitoring of a Recombinant Bioluminescent Yersinia enterocolitica Reporter Mutant in Real Time on Camembert Cheese

Bioluminescent mutants of Yersinia enterocolitica were generated by transposon mutagenesis using a promoterless, complete lux operon (luxCDABE) derived from Photorhabdus luminescens, and their production of light in the cheese environment was monitored. Mutant B94, which had the lux cassette inserted into an open reading frame of unknown function was used for direct monitoring of Y. enterocolitica cells on cheeses stored at 10°C by quantifying bioluminescence using a photon-counting, intensified charge-coupled device camera. The detection limit on cheese was 200 CFU/cm². Bioluminescence of the reporter mutant was significantly regulated by its environment (NaCl, temperature, and cheese), as well as by growth phase, via the promoter the lux operon had acquired upon transposition. At low temperatures, mutant B94 did not exhibit the often-reported decrease of photon emission in older cells. It was not necessary to include either antibiotics or aldehyde in the food matrix in order to gain quantitative, reproducible bioluminescence data. As far as we know, this is the first time a pathogen has been monitored in situ, in real time, in a “real-product” status, and at a low temperature.     

Evaluation of luciferase reporter bacteriophage A511::luxAB for detection of Listeria monocytogenes in contaminated foods.

A511::luxAB is a recombinant derivative of a broad-host-range bacteriophage specific for the genus Listeria, transducing bacterial bioluminescence into infected cells. In this study, we have evaluated its use for rapid and easy testing of contaminated foods and environmental samples for the presence of viable Listeria cells, in comparison to the standard plating procedure. With a short preenrichment step of 20 h, the system was capable of detecting very low initial contamination rates in several foods artificially contaminated with Listeria monocytogenes Scott A cells. In ricotta cheese, chocolate pudding, and cabbage, less than one cell per g of food could be clearly identified by comparing the light emission of phage-infected samples to that of controls without lux phage. In foods having a large and complex microbial background flora, such as minced meat and soft cheese, at least 10 cells per g were necessary to produce a positive bioluminescence signal. Of 348 potentially contaminated natural food and environmental samples, 55 were found to be Listeria positive by the lux phage method. The standard plating procedure detected 57 positive samples. Some differences were observed with respect to the individual samples, i.e., the lux phage procedure detected more positive samples among the dairy products and environmental samples, whereas the plating procedure revealed more contaminated meat and poultry samples. Overall, both methods performed similarly, i.e., were equally sensitive. However, the minimum time required for detection of Listeria with the luciferase phage assay was 24 h, which is much shorter than the 4 days needed by the standard plating method. Furthermore, a most probable number technique with three parallels, based on the use of A511::luxAB for differentiation of positive and negative tubes, is described. The method enables rapid enumeration of low levels of Listeria cells in several foods tested, against the background of a competing microflora.     

"Quorum sensing" regulation of lux gene expression and the structure of lux operon in marine bacteria Alivibrio logei

A group of luminescent strains of marine bacteria Alivibrio logei has been isolated (basins of the Okhotsk, White and Bering Seas). Strains A. logei were shown to be psycrophiic bacteria with an optimal growth temperature of approximately 15 degrees C. Biolumiscent characteristics of strains were studied, and the expression of lux genes was shown to be regulated by the "quorum sensing" system. The A. logei lux operon was cloned in Escherichia coli cells and the structure of this operon and its nucleotide sequence were determined. The structure of A. logei lux operon differs markedly from that in the closely related species of luminescent marine bacteria A. fischeri. In the structure of the A. logei lux operon, the the luxI gene is absent in front of luxC, and a fragment containing luxR2-luxI genes is located immediately after luxG gene. Luminescent psycrophiic marine bacteria of A. logei are assumed to be widely distributed in cold waters of northern seas.     

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

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

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.     

Construction of a bioluminescence reporter plasmid for Francisella tularensis

A Francisella tularensis shuttle vector that constitutively expresses the Photorhabdus luminescens lux operon in type A and type B strains of F. tularensis was constructed. The bioluminescence reporter plasmid was introduced into the live vaccine strain of F. tularensis and used to follow F. tularensis growth in a murine intranasal challenge model in real-time by bioluminescence imaging. The results show that the new bioluminescence reporter plasmid represents a useful tool for tularemia research that is suitable for following F. tularensis growth in both in vitro and in vivo model systems.     

Enhancement of the multi-channel continuous monitoring system through the use of Xenorhabdus luminescens lux fusions

The enhancement of the multi-channel continuous toxicity monitoring system developed previously was studied. To achieve better and more stable results from the system, the use of thermo-lux fusion strains that express the luxCDABE genes from Xenorhabdus luminescens was evaluated. A total of six recombinant Escherichia coli strains with the promoters from three oxidative-stress responsive genes, i.e. the katG, sodA and pqi-5 genes, fused to either the lux genes from Vibrio fischeri or X. luminescens were characterized and their responses to different chemicals compared. It was found that the basal level bioluminescence (BL) from the thermo-lux fusion strains was always higher while that of the V. fischeri lux strains were always near or below the lower limit of detection of the system. For example, the katG::V. fischeri lux strain, DPD2511, gave no discernible response due to its low level expression while a fusion of the katG promoter with the X. luminescens lux operon was clearly responsive and capable of detecting hydrogen peroxide down to about 1 ppm. The use of the thermo-lux strains found them to be as sensitive as the V. fischeri lux strains while providing a brighter, more stable basal level bioluminescence, making the analysis and monitoring of water-borne toxicity more reliable.