Characterization of growth and metabolism of the haloalkaliphile Natronomonas pharaonis

Natronomonas pharaonis is an archaeon adapted to two extreme conditions: high salt concentration and alkaline pH. It has become one of the model organisms for the study of extremophilic life. Here, we present a genome-scale, manually curated metabolic reconstruction for the microorganism. The reconstruction itself represents a knowledge base of the haloalkaliphile's metabolism and, as such, would greatly assist further investigations on archaeal pathways. In addition, we experimentally determined several parameters relevant to growth, including a characterization of the biomass composition and a quantification of carbon and oxygen consumption. Using the metabolic reconstruction and the experimental data, we formulated a constraints-based model which we used to analyze the behavior of the archaeon when grown on a single carbon source. Results of the analysis include the finding that Natronomonas pharaonis, when grown aerobically on acetate, uses a carbon to oxygen consumption ratio that is theoretically near-optimal with respect to growth and energy production. This supports the hypothesis that, under simple conditions, the microorganism optimizes its metabolism with respect to the two objectives. We also found that the archaeon has a very low carbon efficiency of only about 35%. This inefficiency is probably due to a very low P/O ratio as well as to the other difficulties posed by its extreme environment

Metabolism of halophilic archaea

In spite of their common hypersaline environment, halophilic archaea are surprisingly different in their nutritional demands and metabolic pathways. The metabolic diversity of halophilic archaea was investigated at the genomic level through systematic metabolic reconstruction and comparative analysis of four completely sequenced species: Halobacterium salinarum, Haloarcula marismortui, Haloquadratum walsbyi, and the haloalkaliphile Natronomonas pharaonis. The comparative study reveals different sets of enzyme genes amongst halophilic archaea, e.g. in glycerol degradation, pentose metabolism, and folate synthesis. The carefully assessed metabolic data represent a reliable resource for future system biology approaches as it also links to current experimental data on (halo)archaea from the literature

Computational Genome and Pathway Analysis of Halophilic Archaea

Halophilic archaea inhabit hypersaline environments and share common physiological features such as acidic protein machineries in order to adapt to high internal salt concentrations as well as electron transport chains for oxidative respiration. Surprisingly, nutritional demands were found to differ considerably amongst haloarchaeal species, though, and in this project several complete genomes of halophilic archaea were analysed to predict their metabolic capabilities. Comparative analysis of gene equipments showed that haloarchaea adopted several strategies to utilize abundant cell material available in brines such as the acquisition of catabolic enzymes, secretion of hydrolytic enzymes, and elimination of biosynthesis gene clusters. For example, metabolic genes of the well-studied Halobacterium salinarum were found to be consistent with the known degradation of glycerol and amino acids. Further, the complex requirement of H. salinarum for various amino acids and vitamins in comparison with other halophiles was explained by the lack of several genes and gene clusters, e.g. for the biosynthesis of methionine, lysine, and thiamine. Nitrogen metabolism varied also among halophilic archaea, and the haloalkaliphile Natronomonas pharaonis was predicted to apply several modes of N-assimilation to cope with severe ammonium deficiencies in its highly alkaline habitat. This species was experimentally shown to possess a functional respiratory chain, but comparative analysis with several archaea suggests a yet unknown complex III analogue in N. pharaonis. Respiratory chains of halophilic and other respiratory archaea were found to share similar genes for pre-quinone electron transfer steps but show great diversity in post-quinone electron transfer steps indicating adaptation to changing environmental conditions in extreme habitats. Finally, secretomes of halophilic and non-halophilic archaea were predicted proposing that haloarchaea secretion proteins are predominantly exported via the twin-arginine pathway and commonly exhibit a lipobox motif for N-terminal lipid anchoring. In N. pharaonis, lipoboxcontaining proteins were most frequent suggesting that lipid anchoring might prevent protein extraction under alkaline conditions. By contrast, non-halophilic archaea seem to prefer the general secretion pathway for protein translocation and to retain only few secretion proteins by N-terminal lipid anchors. Membrane attachment was preferentially observed for interacting components of ABC transporters and respiratory chains and might further occur via postulated C-terminal anchors in archaea. Within this project, the complete genome of the newly sequenced N. pharaonis was analysed with focus on curation of automatically generated data in order to retrieve reliable gene prediction and protein function assignment results as a basis for additional studies. Through the development of a post-processing routine and expert validation as well as by integration of proteomics data, a highly reliable gene set was created for N. pharaonis which was subsequently used to assess various microbial gene finders. This showed that all automatic gene tools predicted a rather correct gene set for the GC-rich N. pharaonis genome but produced insufficient results in respect to their start codon assignments. Available proteomics results for N. pharaonis and H. salinarum were further analysed for posttranslational modifications, and N-terminal peptides of haloarchaeal proteins were found to be commonly processed by N-terminal methionine cleavage and to some extent further modified by N-acetylation. For general function assignment of predicted N. pharaonis proteins and for enzyme assignment in H. salinarum, similarity-based searches, genecontext methods such as neighbourhood analysis but also manual curation were applied in order to reduce the number of hypothetical proteins and to avoid cross-species transfer of misassigned functions. This permitted to reliably reconstruct the metabolism of H. salinarum and N. pharaonis. Generated metabolic data were stored in a newly developed metabolic database that also integrates experimental data retrieved from the literature. The pathway data can be assessed as coloured KEGG maps and were combined with data resulting from transcriptomics and proteomics techniques. In future, expert-curated reaction entries of the created metabolic database will be a valuable source for the design of metabolic experiments and will deliver a reliable input for metabolic models of halophilic archaea

Genome-Scale Metabolic Modeling of Archaea Lends Insight into Diversity of Metabolic Function

Decades of biochemical, bioinformatic, and sequencing data are currently being systematically compiled into genome-scale metabolic reconstructions (GEMs). Such reconstructions are knowledge-bases useful for engineering, modeling, and comparative analysis. Here we review the fifteen GEMs of archaeal species that have been constructed to date. They represent primarily members of the Euryarchaeota with three-quarters comprising representative of methanogens. Unlike other reviews on GEMs, we specially focus on archaea. We briefly review the GEM construction process and the genealogy of the archaeal models. The major insights gained during the construction of these models are then reviewed with specific focus on novel metabolic pathway predictions and growth characteristics. Metabolic pathway usage is discussed in the context of the composition of each organism’s biomass and their specific energy and growth requirements. We show how the metabolic models can be used to study the evolution of metabolism in archaea. Conservation of particular metabolic pathways can be studied by comparing reactions using the genes associated with their enzymes. This demonstrates the utility of GEMs to evolutionary studies, far beyond their original purpose of metabolic modeling; however, much needs to be done before archaeal models are as extensively complete as those for bacteria

Characterization of the DNA methyltransferase M.NmaphiCh1I and further characterization of a transformation system of haloalkaliphilic Archaea

Diese Arbeit beschreibt den Versuch der Charakterisierung der M.NmaphiCh1 und der weiteren Charakterisierung eines entwickelten Transformationssystems in haloalkaliphilien Archaea. Der Virus aus dem das Methyltransferasegen isoliert wurde ist ein temperenter Phage, dessen einzig bekannter Wirt Nab. magadii ist. Die optimalen Lebensbedingungen dieses Archaeons sind hohe Salzkonzentrationen und extrem hohe pH Werte. Es wurden drei Konstrukte die entweder das ganze Methyltransferasegen oder Teile davon enthalten durch PCR angefertigt und in verschiedene Vektoren kloniert, nämlich pro-5, pQE32 und pRSET-C. Die drei Vektoren, die entweder das gesamte Mtase-Gen oder Teile davon enthielten, wurden benutzt, um die Transformationseffiezienz in Nab. magadii L13 zu prüfen. Allerdings mußte dafür zuerst die Promotorsequenz vom ORF34 davor kloniert weden, da bis dato der vermeintliche Initiationsstartpunkt der Mtase nicht bekannt war. Die pQE32 Konstrukte wurden verwendet um in vivo die Aktivität auf ihre Existenz zu prüfen. Zuletzt wurde anhand der pRSET-C Vektoren, die ebenfalls die Mtasefragmente enthielten die Bindung des Proteins an verschiedene unmethylierte DNA Sequenzen getestet. Dabei hat sich z.B. herausgestellt, dass für die erfolgreiche Bindung Chelat bindende Substanzen wie EDTA notwendig sind. Der zweite Teil dieser Arbeit hat sich mit dem Transformationssystem und dessen weitere Charakterisierung beschäftigt. Zu Beginn wurde versucht heraus zu finden welches Enzym eine bessere Spheroplastenbildung hervorruft, Proteinase K oder Pronase E. Dabei hat sich heraus gestellt, dass durch den Gebrauch von Proteinase K mehr Spheroplasten gebildet werden, nur im Falle von Nmn. pharaonis kann auf den Gebrauch von Pronase E nicht verzichtet werden. Verschiedene Plasmide sogar mit unterschiedlichen Antibiotikaresistenzen wurden versucht zu transformieren. Diese Versuche zeigten dass es möglich ist Mevinolin als Selektionsmarker zu verwenden. Die optimalen Wachstumsbedingungen waren in diesem Fall 7.5 µg/ml Mevinolin. Anhand dieser Ergebnisse wurden folgende Vektoren erfolgreich in Nab. magadii L13 transformiert: pRo-5, pRo-4, pro-4/Mev, pNB102 und zwei neu konstruierte, pNov-1/101 und pRo-5/Bop. Obwohl pNov-1/101 den gleichen Replikationsursprung wie pNB102 enthält wurde es effiezienter von den Zellen aufgenommen als pNB102. Plasmide wie pMDS11 und pMDS24 konnten leider nicht transformiert werden. Auch andere Organismen, nämlich Nmn. pharaonis wurden auch bezüglich ihrer Transformationsfähigkeit untersucht. Die oben genannten Plasmide wurden auch hier erfolgreich transformiert, allerdings mit einer geringeren Ausbeute. Zusätzlich wurden auch die Vektoren pMG100, pMG200 und pMG300 in die Testreihe miteinbezogen, deren Transformanten mit Southern blot Analysen bestätigt wurden.This thesis describes the analysis for the characterization of the M.NmaphiCh1 and further characterization of a transformation system in haloalkaliphilic Archaea. The virus from which the methyltransferase gene was isolated is a temerate one and its only known host is the archaeon Nab. magadii, whose optimal living conditions are in a high alkaine environment with high pH values. Three constructs including the whole or parts of the mtase gene were performed by PCR and cloning into different plasmids like pRo-5, pQE32 and pRSET-C. pRo-5 plasmids including the different fragments of the Mtase gene were used for transformation experiments in Nab. magadii L13 to test the efficiencv. Therefore, it was needed to clone the promotorsequence of ORF34 infront of the fragments because till this thesis it putative initiation sequence was not known. The pQE32 constructs were used for in vivo assays to determine the activity of the gene. Binding assays with different unmethylated DNA substrates were performed with the pRSET-C constructs, which result in the assumption that a chelat binding agent like EDTA is required for binding of the protein. The second part of this study is the further characterization of the established trans-formation system in Archaea. Therefore it was tested what kind of enzyme is better for forming spheroplasts, proteinase K or pronase E. It became apparent that proteinase K is the more efficient enzyme. Only in case of Nmn. pharaonis the pronase E was needed to successed in transformation. Different plasmids even with an other antibiotic resistance were tried to transform, which lead to the possibility to use mevinolin as a marker gene. Growth experiments and a few transformations showed that the optimal concentration of mevinolin is 7.5 µl/ml. With these data plasmids like pRo-5, pRo-4, pro-4/Mev, pNB102 and two constructed ones pNov-1/101 and pRo-5/Bop were successfully transformed into Nab. magadii L13. pNov-1/101 which containes the same origin of replication as pNB102 was anyhow more efficient. Plasmids like pMDS11 and pMDS24 were not transformable. Other organisms were also tested namely Nmn. pharaonis. The same plasmids were indeed with a lower efficiency but successfully transformed and additionally the con-structs pMG100, pMG200 and pMG300 which were proven by Southern blot analysis

Halophiles and Their Biomolecules: Recent Advances and Future Applications in Biomedicine

The organisms thriving under extreme conditions better than any other organism living on Earth, fascinate by their hostile growing parameters, physiological features, and their production of valuable bioactive metabolites. This is the case of microorganisms (bacteria, archaea, and fungi) that grow optimally at high salinities and are able to produce biomolecules of pharmaceutical interest for therapeutic applications. As along as the microbiota is being approached by massive sequencing, novel insights are revealing the environmental conditions on which the compounds are produced in the microbial community without more stress than sharing the same substratum with their peers, the salt. In this review are reported the molecules described and produced by halophilic microorganisms with a spectrum of action in vitro: antimicrobial and anticancer. The action mechanisms of these molecules, the urgent need to introduce alternative lead compounds and the current aspects on the exploitation and its limitations are discussed.España, MINECO CGL2017-83385-