No description available.

There is a need for a sensitive, specific, rapid and cost-effective assay that can be used as an early warning signal of contamination of aquatic ecosystems. The purpose of this work was to develop a sensitive stress-specific microbial bioreporter responsive to pro-oxidants. Furthermore, the bioreporter was designed to be applicable in environments possibly affected by metal processing activities. An E.coli bioreporter was developed containing a plasmid with the katG promoter sequence as the sensing sequence and with mCherry as the reporter protein. The bioreporter responded to metal pro-oxidants (Cd, As, Zn, Pb, Ag and Ag nanoparticles). A new assay growth-medium was developed and contributed to improve the sensitivity of our assay that has the best detection limit to inorganic pro-oxidants compared to other oxidative-stress sensitive bioreporters in the literature. The bioreporter detected pro-oxidants in environmental samples. The assay has a reasonable sensitivity, however, it still lacks sensitivity to detect pro-oxidants at concentrations lower than those shown to be toxic to many aquatic species. Within-lab reproducibility and robustness were determined to be acceptable. For stress-specific bioreporters to be incorporated in regulative legislations and industrial monitoring programs there is a need to improve the sensitivity of these assays, they need to be calibrated with other relevant pro-oxidants, inter-lab reproducibility needs to be established and robustness to environmental samples needs to be further tested. To further validate the sensitivity and ecotoxicological relevance of the bioreporter as a relevant predictive tool, stress-specific bioreporter assays need to be performed in parallel with traditional ecotoxicological assays using contaminated environmental samples.

In the nature, lots of organism apply different kinds of lights such as flourscence or luminoscence for some purposes such as defence or hunting. Firefly luciferase and Bacterial luciferase are the most famous ones which have been used to design Biosensors or Bioreporters in recent decades. Their applications are so extensive from detecting pollutions in the environment to medical and treatment usages. To design Copper Bioreporter, copper resistance promoter from COP operon which plays an important role in Pseudomonas syringae and pGL3 plasmid which has luciferase gene were utilized. To achieve that target, sequences of promoter were synthesized and inserted to pCR2.1 vector, then suitable primers with considering restriction sites were designed to get high concentration of DNA. After digestion of pGL3 and interested gene by Nhe I and Sac I enzymes, ligation was performed, and then recombinat plasmids were transferred to E. coli BL-21 as a host cell. Finallay, luciferase assay of designed bioreporter was performed by Luminometer in presence of different concentration of CuSO4. The result was maginificant that confirmed design of Copper Bioreporter.

Alkaline phosphatase (ALP) is a natural milk enzyme. It has been used as reporter for process controls in food industry. Since ALP denatures at pasteurization temperature (at 63&deg / C or 72&deg / ) its detection in milk confirms the unproper pasteurization. There are different detection procedures such as colorimetric, fluorometric methods and immunoassays for ALP in milk. However, they are time consuming processes and require specific instruments and qualified staff. In this study, new, semiquantitative, disposable, cheap and practical paper bioreporter have been developed for ALP detection. In optimization studies, 1mg/mL p-NPP in 0.1 M glycine buffer at pH 9.5 and 0.5 mg/mL bromocresol green in 1.0 M Tris-HCl buffer at pH 9.5 were determined as optimum for ALP bioreporter as a result of visual inspection and green color intensity analyses.The effects of samples temperature and pH of on the response of bioreporter were tested. Milk samples at pH 5.0, 5.5, 6.0 and 6.5 and milks stored at 37&deg / C, room temperature and 4&deg / C did not affect the response of bioreporter. Also the response of bioreporter against milk samples from different animals (cattle, sheep and goat) and cow&rsquo / s milk from different location in Turkey were evaluated. The appropriate responses were observed by bioreporter. Whatman filter papers, cotton and bandage were used as support materials to construct bioreporter and Whatman filter papers were selected as the most applicaple support material. Finally, stability tests were carried out at 4&deg / C and room temperature and 40 days at 4&deg / C was determined as shelf life of bioreporter.

No description available.

Whole cell biosensors have been extensively used for monitoring toxicity and contamination of compounds in environmental biology and microbial ecology. However, their application in the pharmaceutical and cosmetics industries for preservative efficacy testing (PET) has been limited. According to several pharmacopoeias, preservatives should be tested for microbial activity using traditional viable count techniques; the use of whole cell microbial biosensors potentially provides an alternative, fast, and efficient method. The aim of the study was to construct and develop whole cell microbial biosensors with Pseudomonas aeruginosa ATCC 9027. Constitutive promoters: PlysS, Pspc, Ptat, Plpp and PldcC and the lux-cassette were inserted into plasmid pME4510 and transformed into P. aeruginosa ATCC 9027 cells to produce bioluminescent strains. Plasmids were found to be maintained stably (~50 copies per cell) throughout the growth and death cycle. The novel bioluminescent strains were validated in accordance with the pharmacopoeia using bioluminescence detection and quantification followed by comparison with the traditional plate counting method. The bioluminescent method was found to be accurate, precise and equivalent at a range of 103 – 107 CFU/mL, as compared with plate counting. Recovery of bacterial cells was quantified using bioluminescence; this method proved to be accurate with percentage recoveries between 70-130% for all bioluminescent strains. The method was also more precise (relative standard deviation less than 15%) than the traditional plate counting method or the ATP bioluminescent method. Therefore, the bioluminescent constructs passed/exceeded pharmacopoeial specified criteria for range, limit of detection, accuracy, precision and equivalence. Physiology of the validated bioluminescent strains was studied by assessing the growth and death patterns using constitutive gene expression linked with bacterial replication. Promoter strengths were evaluated at various stages of the growth and death pattern and related to promoter sequences. PlysS, Ptat and Plpp were relatively strong promoters whilst PldcC and Pspc were relatively weak promoters. Relative promoter strength decreased in the order of Plpp>Ptat>PlysS>PldcC>Pspc during the exponential phase whilst Ptat was stronger than Plpp during the stationary phase of growth. Plpp had its highest level of expression during the exponential phase, while Ptat had relatively stable lux expression during the stationary phase. Correlations between relative bioluminescence and CFU at 24 hours were greater than 0.9 indicating a strong relationship for all bioluminescent strains. Reduction in correlation coefficients to approximately 0.6 between relative bioluminescence and CFU and between relative fluorescence and CFU beyond 24 hours indicated that a certain proportion of cells were viable but non-culturable. Tat-pME-lux showed steady bioluminescence compared to CFU count (R>0.9) throughout 28 days of growth. Equivalence analysis showed no significant difference between the bioluminescence and plate count method throughout 28 days of growth for all five bioluminescent strains. Applicability of these novel bioluminescent strains was evaluated for preservative efficacy tests (PET) using bacterial replication and bioluminescence as a measure of constitutive gene expression. PET using benzalkonium chloride and benzyl alcohol showed no significant difference between the bioluminescent method and the plate count method. Good correlations between bioluminescence, CFU count and fluorescence were obtained for benzalkonium chloride (BKC) concentrations (R>0.9) between 0.0003% and 0.0025% against strains lysR25, lppR4 and tatH5. Similarly, good correlations (R>0.9) between the three parameters were obtained for benzyl alcohol (BA) concentrations between 0.125% and 2% against strains lysR25, lppR4 and tatH5. The bioluminescent method and traditional plate counting method were equivalent for concentrations of BKC (0.0003 - 0.02%) and BA (0.25 - 2%) during preservative efficacy tests. These bioluminescent constructs therefore are good candidates for selection for preservative efficacy testing. The bioluminescent method and traditional plate counting method were also found to be equivalent for construct tatH5 at a concentration of 0.125% BA. PET testing with BKC and BA showed that tatH5-pMElux (R>0.9) had consistently high correlation coefficients between CFU and relative bioluminescence. Together with the results from growth and death kinetics, where tatH5 showed the greatest constitutive expression, it can be concluded that P. aeruginosa ATCC 9027 tatH5-pMElux is the best construct for testing various antimicrobial agents. This study has shown that according to the pharmacopoeial requirements, the bioluminescent method is more accurate, precise and equivalent to the traditional plate counting method and therefore can be utilised instead of the traditional plate counting method for the purpose of preservative efficacy testing.

Chloromethane is the most abundant halocarbon in the environment, and responsible for substantial ozone destruction in the stratosphere. Sources and sinks of chloromethane are still poorly constrained. Although synthesized and used industrially, chloromethane is mainly produced naturally, with major emissions from vegetation and especially the phyllosphere, i.e. the aerial parts of plants. Some phyllosphere epiphytes are methylotrophic bacteria which can use single carbon compounds such as methanol and chloromethane as the sole source of carbon and energy for growth. Most chloromethane-degrading strains isolated so far utilize the cmu pathway for growth with chloromethane which was characterized by the team. The main objective of this work was to investigate whether epiphytes may act as filters for plant emissions of chloromethane, by using a laboratory bipartite system consisting of the model plant Arabidopsis thaliana, known to produce chloromethane mainly by way of the HOL1 gene, and the reference chloromethane-degrading bacterial strain Methylobacterium extorquens CM4, possessing the cmu pathway and of known genome sequence. Three A. thaliana Col-0 variants with different levels of expression of HOL1, i.e. the wild-type strain, its homozygous HOL1 knockout mutant hol1 and an HOL1-OX HOL1 overexpressor, were selected using PCR and qRT-PCR. Chloromethane-degrading strains were isolated from the A. thaliana phyllosphere, and shown to contain the cmu pathway. A plasmid-based bacterial bioreporter for chloromethane was constructed which exploits the promoter region of the conserved chloromethane dehalogenase gene cmuA of strain CM4. It yields rapid, highly sensitive, specific and methyl halide concentration-dependent fluorescence. Application of the bioreporter to the three A. thaliana variants differing in expression of HOL1 investigated in this work suggested that they indeed synthesize different levels of chloromethane. Analysis by qPCR and qRT-PCR of metagenomic DNA from the leaf surface of these variants showed that the relative proportion and expression of cmuA in this environment paralleled HOL1 gene expression. Taken together, the results obtained indicate that even minor amounts of chloromethane produced by A. thaliana in the face of large emissions of methanol may provide a selective advantage for chloromethane-degrading methylotrophic bacteria in the phyllosphere environment. This suggests that chloromethane-degrading epiphytes may indeed act as filters for emissions of chloromethane from plants. Further experiments are envisaged to further assess the adaptation mechanisms of chloromethane-degrading bacteria in the phyllosphere, building upon the comparative genomic analysis of chloromethane-degrading strains which was also performed in this work, and on the preliminary investigations using high-throughput sequencing that were initiated.

Le chlorométhane est le plus abondant des composés organo-halogénés dans l’atmosphère et il est impliqué dans la destruction de l’ozone dans la stratosphère. Les sources et les puits de chlorométhane restent mal évalués. Bien que synthétisé et utilisé de manière industrielle, il est principalement produit naturellement, avec comme sources majeures les émissions provenant des végétaux et plus particulièrement de la phyllosphère, qui correspond aux parties aériennes des plantes. Certaines bactéries épiphytes de la phyllosphère sont des méthylotrophes capables d’utiliser des composés organiques sans liaison carbone-carbone comme le méthanol et le chlorométhane comme unique source de carbone et d’énergie pour leur croissance. La plupart des bactéries chlorométhane-dégradantes isolées jusqu’à présent utilisent une voie métabolique pour leur croissance sur chlorométhane appelée voie cmu (pour chloromethane utilisation), caractérisée par l’équipe. L’objectif principal de cette thèse a été de déterminer si des bactéries de la phyllosphère peuvent jouer le rôle de filtre pour l’émission de chlorométhane par les plantes. Dans ce but, un modèle de laboratoire a été mis en place, constitué de la plante Arabidopsis thaliana connue pour produire du chlorométhane par une réaction impliquant le gène HOL1, et la bactérie Methylobacterium extorquens CM4, souche de référence pour l’étude du métabolisme de dégradation du chlorométhane, qui possède la voie cmu et dont le génome complet a été séquencé et analysé. Des variants d’A. thaliana avec différents niveaux d’expression du gène HOL1 (le type sauvage, le mutant homozygote « knock-out » hol1 et un variant HOL1-OX avec surexpression) ont été sélectionnés par PCR et qPCR. Des souches bactériennes chlorométhane-dégradantes ont été isolées à partir de la phyllosphère d’A. thaliana, dont il a été montré qu’elles possèdent la voie cmu. Un bio-rapporteur bactérien pour le chlorométhane a été construit à l’aide d’un plasmide exploitant la région promotrice du gène conservé de la déshalogénase (cmuA) de la souche M. extorquens CM4. Il présente une réponse fluorescente rapide, sensible, et spécifique aux méthyl-halogénés de manière concentration-dépendante. L’application du bio-rapporteur aux trois variants d’A. thaliana étudiés suggère des niveaux d’émissions de chlorométhane différents. L’analyse, par qPCR et qRT-PCR, de l’ADN métagénomique extrait de la surface des feuilles a montré une corrélation entre la proportion relative de bactéries portant le gène cmuA et l’exprimant dans cet environnement, et l’expression du gène HOL1. Ces résultats indiquent qu’une production de chlorométhane, même très modeste par rapport aux fortes émissions de méthanol par A. thaliana, confère un avantage sélectif pour les bactéries épiphytes chlorométhane-dégradantes. Ces dernières pourraient ainsi bien jouer un rôle de filtre pour les émissions de chlorométhane de la phyllosphère vers l’atmosphère. En perspective, de nouvelles expériences complémentaires, basées sur l’analyse par génomique comparative des souches chlorométhane-dégradantes également effectuée dans le cadre du projet et sur une analyse par séquençage à haut-débit initiée dans ce travail, sont proposées pour améliorer la compréhension des mécanismes d’adaptation des bactéries chlorométhane-dégradantes dans la phyllosphère. / Chloromethane is the most abundant halocarbon in the environment, and responsible for substantial ozone destruction in the stratosphere. Sources and sinks of chloromethane are still poorly constrained. Although synthesized and used industrially, chloromethane is mainly produced naturally, with major emissions from vegetation and especially the phyllosphere, i.e. the aerial parts of plants. Some phyllosphere epiphytes are methylotrophic bacteria which can use single carbon compounds such as methanol and chloromethane as the sole source of carbon and energy for growth. Most chloromethane-degrading strains isolated so far utilize the cmu pathway for growth with chloromethane which was characterized by the team. The main objective of this work was to investigate whether epiphytes may act as filters for plant emissions of chloromethane, by using a laboratory bipartite system consisting of the model plant Arabidopsis thaliana, known to produce chloromethane mainly by way of the HOL1 gene, and the reference chloromethane-degrading bacterial strain Methylobacterium extorquens CM4, possessing the cmu pathway and of known genome sequence. Three A. thaliana Col-0 variants with different levels of expression of HOL1, i.e. the wild-type strain, its homozygous HOL1 knockout mutant hol1 and an HOL1-OX HOL1 overexpressor, were selected using PCR and qRT-PCR. Chloromethane-degrading strains were isolated from the A. thaliana phyllosphere, and shown to contain the cmu pathway. A plasmid-based bacterial bioreporter for chloromethane was constructed which exploits the promoter region of the conserved chloromethane dehalogenase gene cmuA of strain CM4. It yields rapid, highly sensitive, specific and methyl halide concentration-dependent fluorescence. Application of the bioreporter to the three A. thaliana variants differing in expression of HOL1 investigated in this work suggested that they indeed synthesize different levels of chloromethane. Analysis by qPCR and qRT-PCR of metagenomic DNA from the leaf surface of these variants showed that the relative proportion and expression of cmuA in this environment paralleled HOL1 gene expression. Taken together, the results obtained indicate that even minor amounts of chloromethane produced by A. thaliana in the face of large emissions of methanol may provide a selective advantage for chloromethane-degrading methylotrophic bacteria in the phyllosphere environment. This suggests that chloromethane-degrading epiphytes may indeed act as filters for emissions of chloromethane from plants. Further experiments are envisaged to further assess the adaptation mechanisms of chloromethane-degrading bacteria in the phyllosphere, building upon the comparative genomic analysis of chloromethane-degrading strains which was also performed in this work, and on the preliminary investigations using high-throughput sequencing that were initiated.