Dipeptidyl peptidase IV (DPP-IV) is a serine protease, which executes its proteolytic activity by cleaving X-Pro dipeptides from the N-termini of its substrates. Furthermore, DPP-IV exhibits many biological functions independent of its enzymatic aktivity. Previous studies in our laboratory proved increased expression of DPP-IV in high-grade astrocytic tumours. To evaluate the enzymatic and non-enzymatic functions of DPP-IV in a glioma model, clones of asctrocytic cell line U373MG transfected by enzymatically inactive, mutated DPP-IV (mutDPP-IV) and enzymatically active, wild type DPP-IV (wtDPP-IV), were prepared. Enzymatically inactive mutDPP-IV was prepared using point mutation the active site serine residue. Cells U373MG were transfected using a doxycycline inducible Tet-On® system. For further analysis of the transgenic forms of DPP-IV, methods were used for verification of protein expression, enzymatic activity and subcellular localization. Doxycycline induced U373MG mutDPP-IV and U373MG wtDPP-IV cells, expressing mutated and wild type DPP-IV, respectivelly, exhibited increased expression of transgenic DPP-IV in a concentration and time dependent manner. Doxycycline induced U373MG wtDPP-IV cells exhibited both increased expression and enzymatic activity of DPP-IV. In contrast, DPP-IV enzymatic...

Two common strategies used to identify specific genes that influence aging in Drosophila melanogaster are overexpression screens and candidate gene approaches. Both of these strategies rely on gene expression systems. A very popular gene expression system in Drosophila is the bipartite UAS/GAL4 system, where binding of GAL4 to a UAS sequence can direct the expression of a UAS-linked transgene in a pattern determined by GAL4. Although the UAS/GAL4 system allows for spatial regulation of transgene expression, it does not allow researchers to control when transgene expression will occur. This is an important consideration since aging research is primarily interested in identifying genes that influence aging during adulthood, therefore requiring that transgene expression be effectively blocked during pre-adult stages. Both the Gene-Switch and the Tet-Off/GAL80 systems are attempts to establish temporal control over GAL4 activity. The Gene-Switch system is based on a modified form of GAL4 whose transcriptional activity can be controlled through the antiprogestin molecule RU486. The Tet-Off/GAL80 system, where expression of GAL80 (a negative regulator of GAL4) is under the control of a tetracycline sensitive expression system, allows regulation of GAL4 activity through the antibiotic tetracycline. Characterization of these systems reveals that although neither system can completely repress leaky transgene expression, the Tet-Off/GAL80 system is much better at preventing unwanted transgene expression at most stages of the fly life cycle. Furthermore, comparison of muscle specific GAL4 and Gene-Switch strains revealed that upon treatment with their respective inducers, the Tet-Off/GAL80 system allows for GAL4 activity in the muscles, while the Gene-Switch system results in GAL4 activity in other tissues in addition to the muscles. In other characterized Gene-Switch strains, GAL4 activity is achieved only in a subset of the cells of the targeted tissue, suggesting that the Gene-Switch system may be ill-suited for aging studies. These findings, along with the fact that the Tet-Off/GAL80 but not the Gene-Switch system is compatible with the hundreds of characterized GAL4 lines presently available which allows transgene expression to be targeted to most tissues, indicate that the Tet-Off/GAL80 system is the best-suited for aging studies in Drosophila at present. / Thesis (Ph.D, Biology) -- Queen's University, 2008-12-22 17:13:42.089

Efficient gene transfer has been achieved in several animal models using different vector systems, leading to stable transgene expression. The tight control of this expression is now an important outcome for the field of gene therapy. Such regulation is likely to be required for therapeutic applications and in some instances for safety reasons. For this purpose, several regulatable systems depending on small molecules have been developed. Among these, the tetracycline and the rapamycin dependent systems have been largely used. However, if long-term regulation of the transgene has been obtained in small animal models using these inducible systems, when translational studies were initiated in larger animals, the development of an immune response against proteins involved in transgene regulation were often observed. Such immune response was especially documented when using the TetOn tetracycline regulatable system in nonhuman primates (NHP). Humoral and destructive cellular immune responses against the transactivator involved in this regulation system were documented in a large majority of NHP leading to the complete loss of the transgene regulation and expression. This review will describe the immune responses observed in these different model systems applied for transgene regulation. Focus will be finally given on future directions in which such immune responses might be surmounted, enabling long-term transgene regulation in future clinical developments of gene transfer.

Murine polyomavirus is an important member of Polyomaviridae family offering potential applications in gene therapy and immunotherapy. Viral mutant analysis is crucial for study of the virus, however, commonly used methods of its production are laborious and give low yields. This thesis involves development of the new experimental system that can produce intact viral genome from recombinant plasmid in vivo using Cre/loxP-mediated recombination. One loxP site is unavoidably introduced into newly generated viral genome during recombination. Two variants of production plasmids generating wild type viral genome with incorporation of loxP between the poly(A) signal sites of early and late genes or into the intronic region of early genes were prepared. LoxP insertion between the poly(A) signal sites has a dramatic effect on viral gene expression and leads to complete loss of virus infectivity. Conversely, the infectious virus was obtained from the viral genome containing loxP site in the early intronic region. To ensure expression of Cre recombinase I also prepared stably transfected cell lines which can simplify the virus production. This thesis shows that newly designed system gives satisfactory yield of the virus, solves restrictions connected with commonly used methods and can be used for low infectious viral...

Les plasmides pSci de Spiroplasma citri GII3 possèdent une structure mosaïque avec de nombreuses régions conservées. Des dérivés du plasmide pSci2 ont été produits par délétions successives et leur capacité de réplication a été évaluée. Le plus petit réplicon obtenu ne contient plus qu’une CDS (pE) et ses régions flanquantes. L’inactivation dans un vecteur navette du gène pE est suffisante pour abolir la réplication de ce plasmide dans S. citri. Des dérivés des pSci ont été introduits efficacement dans S. kunkelii et S. phoeniceum, deux spiroplasmes phytopathogènes pour lesquels aucun outil génétique n’était disponible jusqu’à présent. La stabilité des dérivés des pSci a également été évaluée par leur capacité à persister en l’absence de pression de sélection. L’instabilité ségrégationnelle des plasmides dans lesquels soj a été délété ou inactivé indique que la protéine de partition Soj/ParA est essentielle au maintien des plasmides pSci. L’incompatibilité sélective entre un plasmide pSci et ses dérivés a été exploitée pour produire une collection de souches possédant des profils plasmidiques différents. L’analyse des phénotypes d’acquisition et de transmission de plusieurs de ces mutants suggère que la CDS traG portée par le pSci6 est essentielle à la transmission de S. citri GII3. En revanche, les plasmides pSci1-5, codant les protéines adhésine-like ScARPs, ne sont indispensables ni à l’acquisition ni à la transmission. Dans le cadre de ce travail, plusieurs outils génétiques ont été adaptés aux mollicutes. Le promoteur Pxyl/tetO2 a été utilisé pour contrôler l’expression du gène de la spiraline chez S. citri et M. agalactiae. Enfin, un système de linéarisation de plasmide in vivo basé sur l’expression de l’endonucléase I-SceI a été utilisé pour l’élimination de plasmides pSci chez S. citri. / Plasmids pSci from Spiroplasma citri GII3 display a mosaic gene organization with highly conserved regions. Through successive deletions, various pSci2 derivatives were constructed and assessed for their ability to replicate. The smallest functional replicon consists of one single CDS (pE) and its flanking, intergenic regions. Furthermore, shuttle (S. citri/E. coli) plasmids, in which the pE gene was disrupted, failed to replicate in S. citri, suggesting that PE is the replication protein. S. citri plasmids were efficiently introduced into S. kunkelii and S. phoeniceum, two plant pathogenic spiroplasmas, the transformation of which had never been described before. Studying stability of various pSci-derived plasmids in the absence of selection pressure strongly suggests the occurrence of an active partition system involving the soj-like gene. Selective incompatibility between a given pSci plasmid and its derivatives was used to remove plasmids from the wild-type strain. As a result, a collection of S. citri GII3 mutants differing in their plasmid contents was produced. Experimental transmission of these mutants through injection to or ingestion by the leafhopper vector will provide new insights into the role of plasmid encoded determinants in the biology of S. citri. First data indicated that pSci6 traG is required for insect transmission of S. citri GII3. In contrast, pSci1-5, encoding adhesin-like proteins, are not essential for both transmission and acquisition. In the frame of this work, new genetic tools have been adapted for use in mollicutes. The tetracycline inducible promoter, Pxyl/tetO2, was used to control spiraline gene in S. citri and M. agalactiae. Also, an in vivo linearization system based on the expression of the I-SceI-endonuclease was used to remove pSci plasmids from S. citri.

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[ES] La Biología Sintética de Plantas es un campo en rápida expansión, con un gran potencial para potenciar la producción de compuestos en plantas. Las plantas pueden actuar como biofactorías sostenibles y económicas para la producción de moléculas de alto valor; sin embargo, la producción continua a menudo genera problemas de toxicidad para la propia planta y bajos rendimientos. Un objetivo clave en este ámbito es lograr un control preciso e inducible sobre la expresión génica mediante circuitos genéticos sintéticos que incluyen sensores y procesadores modulares. Esta tesis se centra en el desarrollo de un sensor de cobre y procesadores basados en geminivirus, diseñados como componentes modulares para circuitos sintéticos en Nicotiana benthamiana, una planta modelo con gran potencial para transformaciones génicas tanto transitorias como estables. El primer capítulo de esta tesis presenta un sistema de expresión génica inducible por cobre, diseñado para permitir una regulación precisa dependiente de sulfato de cobre (CuSO4). Este sistema incluye un elemento sensor compuesto por un factor de transcripción que se une al cobre, CUP2, fusionado al dominio de activación Gal4 y un promotor sintético con sitios de unión al cobre (CBS) ubicado antes de un promotor mínimo. El sensor de cobre se utilizó exitosamente para controlar un activador transcripcional programable basado en CRISPR/dCas9 (dCasEV2.1) en N. benthamiana. Este sensor, combinado con el procesador dCasEV2.1 (CS/dCasEV2.1), confirió un control robusto de la expresión génica, con mínima actividad basal y fuerte activación en respuesta a cobre, ofreciendo una herramienta robusta para la activación específica de genes endógenos. Este sistema tiene un gran potencial para la regulación dirigida de genes en Biología Sintética de Plantas, especialmente en aplicaciones que requieren activación bajo demanda y baja expresión de fondo. El segundo capítulo se centra en el desarrollo de un circuito de amplificación génica regulado por cobre, denominado CuBe, en referencia al ion cobre (Cu²⁺) y al virus del enanismo amarillo del frijol (BeYDV). CuBe combina el sensor de cobre con un vector replicativo derivado específicamente del geminivirus BeYDV y sus correspondientes proteínas asociadas a replicación (Rep/RepA). Esta combinación permite la activación inducida por cobre del replicón y la posterior expresión de proteínas recombinantes en N. benthamiana. Diseñado para lograr una baja expresión basal y alta inducibilidad, CuBe facilita la producción controlada de proteínas de interés farmacéutico, como anticuerpos. Se generaron líneas transgénicas estables con CuBe, confirmando su funcionalidad a largo plazo y robustez con métodos alternativos de aplicación de cobre, como en post-cosecha e hidroponía. Este sistema destaca el potencial de los circuitos regulados por cobre para aplicaciones escalables de Agricultura Molecular en condiciones controladas. El tercer capítulo amplía el uso de sistemas basados en geminivirus investigando y optimizando varios vectores geminivirales (derivados de BeYDV, TYLCV y BCTV) para aplicaciones en biotecnología vegetal. Utilizando un sistema reportero de bioluminiscencia autosostenida, este estudio caracteriza la dinámica y los niveles de expresión basal de diferentes configuraciones de vectores, evaluando su eficacia para la expresión transitoria de una enzima recombinante ligada a la bioluminiscencia. Estos análisis proporcionaron información sobre los puntos fuertes y las limitaciones de cada vector para aplicaciones específicas, como la expresión de múltiples genes, lo que enriquece el conjunto de herramientas geminivirales disponibles para la Biología Sintética de Plantas. / [CA] La Biologia Sintètica de Plantes és un camp en ràpida expansió, amb un gran potencial per a potenciar la producció de compostos en plantes. Les plantes poden actuar com biofactories sostenibles i econòmiques per a la producció de molècules d'alt valor; no obstant això, la producció contínua sovint genera problemes de toxicitat per a la pròpia planta i baixos rendiments. Un objectiu clau en aquest àmbit és aconseguir un control precís i induïble sobre l'expressió gènica mitjançant circuits genètics sintètics que inclouen sensors i processadors modulars. Aquesta tesi se centra en el desenvolupament d'un sensor de coure i processadors basats en geminivirus, dissenyats com a components modulars per a circuits sintètics en Nicotiana benthamiana, una planta model amb gran potencial per a transformacions gèniques tant transitòries com estables. El primer capítol d'aquesta tesi presenta un sistema d'expressió gènica induïble per coure, dissenyat per a permetre una regulació precisa dependent de sulfat de coure (CuSO4). Aquest sistema inclou un element sensor compost per un factor de transcripció que s'uneix al coure, CUP2, fusionat al domini d'activació Gal4 i un promotor sintètic amb llocs d'unió al coure (CBS) situat abans d'un promotor mínim. El sensor de coure es va utilitzar amb èxit per a controlar un activador transcripcional programable basat en CRISPR/dCas9 (dCasEV2.1) en N. benthamiana. Aquest sensor, combinat amb el processador dCasEV2.1 (CS/dCasEV2.1), va conferir un control robust de l'expressió gènica, amb mínima activitat basal i forta activació en resposta a coure, oferint una eina robusta per a l'activació específica de gens endògens. Aquest sistema té un gran potencial per a la regulació dirigida de gens en Biologia Sintètica de Plantes, especialment en aplicacions que requereixen activació sota demanda i baixa expressió de fons. El segon capítol se centra en el desenvolupament d'un circuit d'amplificació gènica regulat per coure, denominat CuBe, en referència a l'ió coure (Cu²¿) i al virus del nanisme groc del fesol (BeYDV). CuBe combina el sensor de coure amb un vector replicatiu derivat específicament del geminivirus BeYDV i les seues corresponents proteïnes associades a replicació (Rep/RepA). Aquesta combinació permet l'activació induïda per coure del replicon i la posterior expressió de proteïnes recombinants en N. benthamiana. Dissenyat per a aconseguir una baixa expressió basal i alta inducibilitat, CuBe facilita la producció controlada de proteïnes d'interés farmacèutic, com a anticossos. Es van generar línies transgèniques estables amb CuBe, confirmant la seua funcionalitat a llarg termini i robustesa amb mètodes alternatius d'aplicació de coure, com en post-collita i hidroponia. Aquest sistema destaca el potencial dels circuits regulats per coure per a aplicacions escalables d'Agricultura Molecular en condicions controlades. El tercer capítol amplia l'ús de sistemes basats en geminivirus investigant i optimitzant diversos vectors geminivirals (derivats de BeYDV, TYLCV i BCTV) per a aplicacions en biotecnologia vegetal. Utilitzant un sistema reporter de bioluminescència autosostinguda, aquest estudi caracteritza la dinàmica i els nivells d'expressió basal de diferents configuracions de vectors, avaluant la seua eficàcia per a l'expressió transitòria d'un enzim recombinant lligat a la bioluminescència. Aquestes anàlisis van proporcionar informació sobre els punts forts i les limitacions de cada vector per a aplicacions específiques, com l'expressió de múltiples gens, la qual cosa enriqueix el conjunt d¿eines geminivirals disponibles per a la Biologia Sintètica de Plantes. / [EN] Plant Synthetic Biology is a rapidly advancing field with significant potential to enhance plant-based biomanufacturing. Plants can serve as sustainable and cost-effective biofactories for producing valuable compounds, yet sustained production often leads to issues like toxicity for the plant itself and low yield. A major goal in plant biomanufacturing is to enable precise and inducible control over gene expression through the use of synthetic gene circuits containing modular sensor and processor elements. This thesis focuses on the development of a copper sensor and geminivirus-based processors, designed as modular components for synthetic gene circuits in Nicotiana benthamiana, a model plant with strong potential for transient and stable gene transformation. The first chapter of this thesis presents a copper-inducible gene expression system designed to achieve precise, copper sulfate (CuSO4)-dependent regulation of transcriptional activation. This system comprises a sensor element consisting of the copper-binding transcription factor CUP2 fused to the Gal4 activation domain, and a synthetic promoter containing copper-binding sites (CBS) positioned upstream of a minimal promoter. The copper sensor was successfully applied to control the activation of a CRISPR/dCas9-based programmable transcriptional activator (dCasEV2.1) in N. benthamiana. The copper sensor, combined with the dCasEV2.1 processor (CS/dCasEV2.1), conferred robust control of gene expression, with minimal basal activity and strong activation in response to copper sulfate, providing a reliable tool for fine-tuned induction of endogenous genes. This system offers significant potential for targeted gene regulation in Plant Synthetic Biology, especially for applications requiring on-demand activation with minimal background expression. The second chapter focuses on the development of a copper-regulated gene amplification circuit called CuBe, which takes its name from the chemical symbol for copper (Cu²¿) and the initials of the bean yellow dwarf virus (BeYDV). In this system, the copper sensor is combined with a replicative vector and the associated replication proteins (Rep/RepA), both derived from BeYDV. This integration enables copper-inducible activation of the replicon, allowing for controlled expression of recombinant proteins in N. benthamiana. Designed to achieve low basal expression and high inducibility, CuBe facilitates controlled expression of recombinant proteins with pharmaceutical relevance, such as antibodies. Stable transgenic lines carrying CuBe were generated, confirming its long-term functionality and robustness with alternative copper application methods, including post-harvest and hydroponic. The CuBe system highlights the potential of copper-inducible circuits for scalable Molecular Farming applications in controlled conditions. The third chapter expands on geminivirus-based systems by investigating and optimizing various geminiviral vectors (derived from BeYDV, TYLCV, and BCTV) for plant biotechnology applications. Using a self-sustained bioluminescence-based reporter system, this study characterizes the dynamics and basal expression levels of different vector configuration, comparing their efficacy for the transient expression of a recombinant enzyme linked to bioluminescence. These analyses provided insights into the strengths and limitations of each vector for specific applications, such as multi-gene expression, thereby enhancing the geminiviral toolbox available for Plant Synthetic Biology. In conclusion, this thesis contributes novel tools for Plant Synthetic Biology, with significant implications for Molecular Farming and plant-based biocomputing. By integrating inducible systems with optimized vector design, this research provides a foundation for future advancements in synthetic gene circuits tailored for plant biotechnology applications that demand tightly regulated and customizable gene expression. / Esta tesis doctoral ha sido financiada mediante la Subvención para la contratación de personal investigador de carácter predoctoral (ACIF/2020/309) de la Generalitat Valenciana. / García Pérez, E. (2025). Development of a Copper Sensor and Geminivirus-Based Processors for Engineering Synthetic Gene Circuits in Plants [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/214743

Les cellules souches embryonnaires (cellules ES) sont pluripotentes et ont donc le potentiel de se différencier en cellules des trois feuillets embryonnaires, ainsi qu’en cellules de la lignée germinale. Ces propriétés en font un modèle pour l’étude des mécanismes de prolifération et de différenciation. Le facteur de transcription Lmx1b est impliqué dans la maintenance du phénotype différencié des neurones dopaminergiques mésencéphaliques. Et il a aussi été montré comme un facteur clef dans la différenciation et la maintenance des neurones sérotoninergiques du rhombencéphale générés dans les noyaux du Raphé. Dans ce travail, nous nous sommes intéressés aux capacités de Lmx1b d’influencer la différenciation des cellules ES de souris en neurones sérotoninergiques. La première stratégie adoptée a résulté en une expression ectopique stable de Lmx1b dans les cellules ES et leurs dérivés. Le niveau d’expression de Lmx1b a fortement influencé les capacités de différenciation neuronale des cellules. Puis, l’analyse de marqueurs de différenciation spécifiques a montré une augmentation de l’expression des marqueurs sérotoninergiques, au contraire des marqueurs dopaminergiques ou de neurones moteur. La seconde stratégie a consisté en une surexpression inductible de Lmx1b dans les précurseurs neuraux dérivés de cellules ES pour mimer l’expression physiologique de Lmx1b. Après induction, Lmx1b était bien exprimé dans les cellules durant toutes les étapes de différenciation neuronale. L’activation de l’expression de Lmx1b au stade des colonies neuroépithéliales a aussi résulté en une amélioration de la différenciation sérotoninergique. Les résultats de ce travail soulignent les capacités de Lmx1b à diriger la différenciation des précurseurs neuraux dérivés de cellules ES vers la voie sérotoninergique in vitro. / Pluripotent Embryonic Stem Cells (ESC) have the potential to develop into cells of the three germ layers and of the germ line. Therefore, they are used as a model to study the proliferation and differentiation mechanisms. The LIM homeodomain transcription factor Lmx1b is involved in the maintenance of the differentiated phenotype of midbrain dopaminergic neurons. And it has been also demonstrated to be a key factor in differentiation and maintenance of hindbrain serotonergic neurons generated in the Raphe Nuclei. Here, we explored the capacity of Lmx1b to direct differentiation of mouse ESC (mESC) into serotonergic neurons. In the first approach, stable ectopic expression of Lmx1b was achieved. First, the level of Lmx1b expression was found to strongly influence the capacity of mESC to accomplish neuronal differentiation. Then, analysis of lineage-specific differentiation markers showed an increase in serotonergic markers’ expression by contrast to dopaminergic or motor neurons markers. In the second approach, Lmx1b was over-expressed in mESC-derived neural precursors by an inducible system in order to mimic the physiological onset of Lmx1b expression. After induction, Lmx1b was found to be stably expressed throughout neuronal differentiation. Activation of Lmx1b expression in neuroepithelial colonies resulted in enhancement of serotonergic differentiation, consistently with the stable system results. The results of this work highlight the capacity of Lmx1b to promote the shift of mESC-derived neural precursors toward a serotonergic fate in vitro.

The high affinity, ATP-dependent K+ uptake system KdpFABC of Halobacterium salinarum, is highly induced under K+ limitation. In contrast to the well-characterized Kdp system in Escherichia coli, in which the kdpFABC genes are transcriptionally regulated by the sensor kinase/response regulator system KdpD/KdpE, transcriptional regulation of the kdp genes in H. salinarum was unknown due to the absence of halobacterial homologues of KdpD/KdpE. Furthermore, the physiological relevance of the KdpFABC K+ uptake system of H. salinarum was puzzling, since hypersaline habitats usually comprise K+ concentrations which do not induce kdp expression. In order to analyze the regulation of kdp gene expression, it was essential to gain information about the transcriptional unit(s) involved. Northern blotting, primer extension analysis and real-time RT-PCR revealed the presence of a polycistronic leaderless kdpFABCQ transcript with a putative kdp terminator or at least a potential mRNA processing site downstream of kdpQ. Furthermore, promoter truncation studies verified the so far only predicted basal transcription elements together with an upstream-located operator sequence. Since deletions of this putative operator sequence did not lead to a constitutive expression, a further component has to be involved in the regulation of the kdpFABCQ genes. However, truncation and scanning mutagenesis analyses of the kdp promoter as well as translational fusions of a halophilic beta-galactosidase to the kdp promoter excluded an additional regulatory element up- or downstream of the basal transcription elements and in the kdp-coding region. These results lead to speculations of multiple basal transcription factors to be involved. Furthermore, an inducible expression vector (shuttle vector) was constructed based on the promoter of the kdpFABCQ operon due to its, K+-sensitive features. Inducible expression systems are yet not available for H. salinarum. The resulting, replicating vector pKIX is functional and enables a K+-dependent expression from the kdp promoter with rather high induction ratios of 50-fold. Expression levels could further be improved by plasmid- and additional chromosomally encoded kdpQ and mutations generated in the kdp promoter. Since transcript levels from pKIX were found to be independent of differential target genes, the general application of pKIX as an inducible expression system is strongly supported and pKIX could, thus, be made accessible to the scientific community. To decipher the physiological relevance of the halobacterial Kdp system, H. salinarum was encountered to desiccation stress and salt crystal (halite) entombment. Halite crystals grown under non-inducing K+ concentrations with entombed strains of H. salinarum and H. salinarum deleted in the kdpFABCQ genes revealed a significantly reduced survival rate of the deletion strain upon recultivation. Additionally, a kdpFABCQ-inducing desiccation stress could already be determined on agar plates under non-limiting K+ concentrations. Furthermore, the cell morphology of H. salinarum entrapped in halite crystals resembled that of H. salinarum grown under K+-limiting conditions. Therefore, the Kdp system promotes survival of H. salinarum under desiccation stress. Furthermore, the Kdp system could be identified as at least one of the systems important for long-term survival of H. salinarum in halite.