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The gene coding for xylose isomerase from the thermophilic bacterium Fervidobacterium gondwanense was cloned and overexpressed in Escherichia coli. The produced xylose isomerase (XylA), which closely resembles counterparts from Thermotoga maritima and T. neapolitana, was purified and characterized. It is optimally active at 70 degrees C, pH 7.3, with a specific activity of 15.0 U/mg for the interconversion of glucose to fructose. When compared with T. maritima XylA at 85 degrees C, a higher catalytic efficiency was observed. Divalent metal ions Co2+ and Mg2+ were found to enhance the thermostability.

Two conjugative plasmids (CPs) were isolated and characterized from the same ‘Sulfolobus islandicus’ strain, SOG2/4. The plasmids were separated from each other and transferred intoSulfolobus solfataricus. One has a high copy number and is not stable (pSOG1) whereas the other has a low copy number and is stably maintained (pSOG2). Plasmid pSOG2 is the firstSulfolobusCP found to have these characteristics. The genomes of both pSOG plasmids have been sequenced and were compared to each other and the availableSulfolobusCPs. Interestingly, apart from a very well-conserved core, 70 % of the pSOG1 and pSOG2 genomes is largely different and composed of a mixture of genes that often resemble counterparts in previously describedSulfolobusCPs. However, about 20 % of the predicted genes do not have known homologues, not even in other CPs. Unlike pSOG1, pSOG2 does not contain a gene for the highly conserved PlrA protein nor for obvious homologues of partitioning proteins. Unlike pNOB8 and pKEF9, both pSOG plasmids lack the so-called clustered regularly interspaced short palindrome repeats (CRISPRs). The sites of recombination between the two genomes can be explained by the presence of recombination motifs previously identified in otherSulfolobusCPs. Like otherSulfolobusCPs, the pSOG plasmids possess a gene encoding an integrase of the tyrosine recombinase family. This integrase probably mediates plasmid site-specific integration into the host chromosome at the highly conserved tRNAGluloci.

Paracoccus denitrificans strains with mutations in the genes encoding the cytochrome c 550 , c 552 , or c 1 and in combinations of these genes were constructed, and their growth characteristics were determined. Each mutant was able to grow heterotrophically with succinate as the carbon and free-energy source, although their specific growth rates and maximum cell numbers fell variably behind those of the wild type. Maximum cell numbers and rates of growth were also reduced when these strains were grown with methylamine as the sole free-energy source, with the triple cytochrome c mutant failing to grow on this substrate. Under anaerobic conditions in the presence of nitrate, none of the mutant strains lacking the cytochrome bc 1 complex reduced nitrite, which is cytotoxic and accumulated in the medium. The cytochrome c 550 -deficient mutant did denitrify provided copper was present. The cytochrome c 552 mutation had no apparent effect on the denitrifying potential of the mutant cells. The studies show that the cytochromes c have multiple tasks in electron transfer. The cytochrome bc 1 complex is the electron acceptor of the Q-pool and of amicyanin. It is also the electron donor to cytochromes c 550 and c 552 and to the cbb 3 -type oxidase. Cytochrome c 552 is an electron acceptor both of the cytochrome bc 1 complex and of amicyanin, as well as a dedicated electron donor to the aa 3 -type oxidase. Cytochrome c 550 can accept electrons from the cytochrome bc 1 complex and from amicyanin, whereas it is also the electron donor to both cytochrome c oxidases and to at least the nitrite reductase during denitrification. Deletion of the c -type cytochromes also affected the concentrations of remaining cytochromes c , suggesting that the organism is plastic in that it adjusts its infrastructure in response to signals derived from changed electron transfer routes.

The gene encoding a short-chain alcohol dehydrogenase, AdhA, has been identified in the hyperthermophilic archaeon Pyrococcus furiosus, as part of an operon that encodes two glycosyl hydrolases, the beta-glucosidase CelB and the endoglucanase LamA. The adhA gene was functionally expressed in Escherichia coli, and AdhA was subsequently purified to homogeneity. The quaternary structure of AdhA is a dimer of identical 26-kDa subunits. AdhA is an NADPH-dependent oxidoreductase that converts alcohols to the corresponding aldehydes/ketones and vice versa, with a rather broad substrate specificity. Maximal specific activities were observed with 2-pentanol (46 U x mg(-1)) and pyruvaldehyde (32 U x mg(-1)) in the oxidative and reductive reaction, respectively. AdhA has an optimal activity at 90 degrees C, at which temperature it has a half life of 22.5 h. The expression of the adhA gene in P. furiosus was demonstrated by activity measurements and immunoblot analysis of cell extracts. A role of this novel type of archaeal alcohol dehydrogenase in carbohydrate fermentation is discussed.

Pentameric ligand gated ion-channels, or Cys-loop receptors, mediate rapid chemical transmission of signals. This superfamily of allosteric transmembrane proteins includes the nicotinic acetylcholine (nAChR), serotonin 5-HT3, gamma-aminobutyric-acid (GABAA and GABAC) and glycine receptors. Biochemical and electrophysiological information on the prototypic nAChRs is abundant but structural data at atomic resolution have been missing. Here we present the crystal structure of molluscan acetylcholine-binding protein (AChBP), a structural and functional homologue of the amino-terminal ligand-binding domain of an nAChR alpha-subunit. In the AChBP homopentamer, the protomers have an immunoglobulin-like topology. Ligand-binding sites are located at each of five subunit interfaces and contain residues contributed by biochemically determined 'loops' A to F. The subunit interfaces are highly variable within the ion-channel family, whereas the conserved residues stabilize the protomer fold. This AChBP structure is relevant for the development of drugs against, for example, Alzheimer's disease and nicotine addiction.

An a-glucosidase activity (EC 3.2.1.20) isolated from Sulfolobus solfataricus strain MT-4 was characterised and found of interest at industrial level in the saccharification step of hydrolysis process of starch. The gene encoding for the enzyme was expressed in Escherichia coli BL21 (DE3). with a yield of 87.5 U/g of wet biomass. The recombinant cytosolic enzyme was purified to homogeneity with a rapid purification procedure employing only steps of selective and progressive thermal precipitations with a final yield of 75.4% and a purification of 14.5-fold. The properties of this thermophilic a-glucosidase were compared with those of the a-glucosidase of a commercial preparation from Aspergillus niger used in the starch processing.

The original strategy used in the Sulfolobus solfataricus genome project was to sequence non overlapping, or minimally overlapping, cosmid or lambda inserts without constructing a physical map. However, after only about two thirds of the genome sequence was completed, this approach became counter-productive because there was a high sequence bias in the cosmid and lambda libraries. Therefore, a new approach was devised for linking the sequenced regions which may be generally applicable. BAC libraries were constructed and terminal sequences of the clones were determined and used for both end mapping and PCR screening. The PCR approaches included a novel chromosome walking method termed "paired-PCR". 21 gaps were filled by BAC end sequence analyses and 6 gaps were filled by PCR including three large ones by paired-PCR. The complete map revealed that 0.9 Mb remained to be sequenced and 34 BAC clones were selected for walking over small gaps and preparing template libraries for larger ones. It is concluded that an optimal strategy for sequencing microorganism genomes involves construction of a high-resolution physical map by BAC end analyses, PCR screening and paired-PCR chromosome walking after about half the genome sequence has been accumulated.

Pyrococcus furiosus uses a modified Embden-Meyerhof pathway involving two ADP-dependent kinases. Using the N-terminal amino acid sequence of the previously purified ADP-dependent glucokinase, the corresponding gene as well as a related open reading frame were detected in the genome of P. furiosus. Both genes were successfully cloned and expressed in Escherichia coli, yielding highly thermoactive ADP-dependent glucokinase and phosphofructokinase. The deduced amino acid sequences of both kinases were 21.1% identical but did not reveal significant homology with those of other known sugar kinases. The ADP-dependent phosphofructokinase was purified and characterized. The oxygen-stable protein had a native molecular mass of approximately 180 kDa and was composed of four identical 52-kDa subunits. It had a specific activity of 88 units/mg at 50 degrees C and a pH optimum of 6.5. As phosphoryl group donor, ADP could be replaced by GDP, ATP, and GTP to a limited extent. The K(m) values for fructose 6-phosphate and ADP were 2.3 and 0.11 mM, respectively. The phosphofructokinase did not catalyze the reverse reaction, nor was it regulated by any of the known allosteric modulators of ATP-dependent phosphofructokinases. ATP and AMP were identified as competitive inhibitors of the phosphofructokinase, raising the K(m) for ADP to 0.34 and 0.41 mM, respectively.

The fermentative conversion of glucose in anaerobic hyperthermophilic Archaea is a variant of the classical Embden-Meyerhof pathway found in Bacteria and Eukarya. A major difference of the archaeal glycolytic pathway concerns the conversion of glyceraldehyde-3-phosphate. In the hyperthermophilic archaeon Pyrococcus furiosus, this reaction is catalyzed by an unique enzyme, glyceraldehyde-3-phosphate ferredoxin oxidoreductase (GAPOR). Here, we report the isolation, characterization, and transcriptional analysis of the GAPOR-encoding gene. GAPOR is related to a family of ferredoxin-dependent tungsten enzymes in (hyper)thermophilic Archaea and, in addition, to a hypothetical protein in Escherichia coli. Electron paramagnetic resonance analysis of the purified P. furiosus GAPOR protein confirms the anticipated involvement of tungsten in catalysis. During glycolysis in P. furiosus, GAPOR gene expression is induced, whereas the activity of glyceraldehyde-3-phosphate dehydrogenase is repressed. It is discussed that this unprecedented unidirectional reaction couple in the pyrococcal glycolysis and gluconeogenesis gives rise to a novel site of glycolytic regulation that might be widespread among Archaea.

We report here the first molecular characterization of an endo-beta-1,3-glucanase from an archaeon. Pyrococcus furiosus is a hyperthermophilic archaeon that is capable of saccharolytic growth. The isolated lamA gene encodes an extracellular enzyme that shares homology with both endo-beta-1,3- and endo-beta-1,3-1,4-glucanases of the glycosyl hydrolase family 16. After deletion of the N-terminal leader sequence, a lamA fragment encoding an active endo-beta-1,3-glucanase was overexpressed in Escherichia coli using the T7-expression system. The purified P. furiosus endoglucanase has highest hydrolytic activity on the beta-1,3-glucose polymer laminarin and has some hydrolytic activity on the beta-1,3-1,4 glucose polymers lichenan and barley beta-glucan. The enzyme is the most thermostable endo-beta-1,3-glucanase described up to now; it has optimal activity at 100-105 degrees C. In the predicted active site of glycosyl hydrolases of family 16 that show predominantly endo-beta-1,3-glucanase activity, an additional methionine residue is present. Deletion of this methionine did not change the substrate specificity of the endoglucanase, but it did cause a severe reduction in its catalytic activity, suggesting a structural role of this residue in constituting the active site. High performance liquid chromatography analysis showed in vitro hydrolysis of laminarin by the endo-beta-1,3-glucanase proceeds more efficiently in combination with an exo-beta-glycosidase from P. furiosus (CelB). This most probably reflects the physiological role of these enzymes: cooperation during growth of P. furiosus on beta-glucans.

The Paracoccus denitrificans fnrP gene encoding a homologue of the Escherichia coli FNR protein was localized upstream of the gene cluster that encodes the high-affinity cbb3-type oxidase. FnrP harbours the invariant cysteine residues that are supposed to be the ligands of the redox-sensitive [4Fe-4S] cluster in FNR. NNR, another FNR-like transcriptional regulator in P. denitrificans, does not. Analysis of FnrP and NNR single and double mutants revealed that the two regulators each exert exclusive control on the expression of a discrete set of target genes. In FnrP mutants, the expression of cytochrome c peroxidase was blocked, that of membrane-bound nitrate reductase and the cbb3-type oxidase was significantly reduced, whilst the activity of the bb3-type quinol oxidase was increased. The amounts of the nitrite and nitric oxide reductases in these FnrP mutants were the same as in the wild type. NNR mutants, on the other hand, were disturbed exclusively in the concentrations of nitrite reductase and nitric oxide reductase. An FnrP.NNR double mutant combined the phenotypes of the single mutant strains. In all three mutants, the concentrations and/or activities of the aa3-type oxidase, cytochrome C550, cytochrome C552, and nitrous oxide reductase equalled those in the wild type. As the FNR boxes in front of the FnrP- and NNR-regulated genes are highly similar to or even identical to each other, the absence of cross-talk between the regulation by FnrP and NNR implies that as yet unidentified factors are important in the control. It is proposed that the redox state of an intracellular redox couple other than the oxygen/water couple is one of the factors that modulates the activity of FnrP.

The taxonomy of Paracoccus denitrificans and related bacteria is discussed. Evidence is given which shows that the physiological differences between P. denitrificans and Thiosphaera pantotropha are less fundamental than previously thought. A proposal to consider a species P. pantotropha is mentioned. The properties of the denitrifying enzymes and the genes involved in their formation in P. denitrificans is discussed. The synthesis of the membrane-bound-nitrate reductase is regulated by FNR, that of the nitrite- and nitric oxide reductase by NNR. Evidence is given that FNR acts as a redox sensor rather than an oxygen sensor. The occurrence of aerobic denitrification and coupled heterotrophic nitrification-denitrification in the original strain of Thiosphaera pantotropha are explained by a limiting respiratory activity which activates FNR. Aerobic denitrification leads to a lower growth yield and an increase in mumax in batch culture when a limiting respiratory activity is assumed and when excess substrate is present. Coupled heterotrophic nitrification-denitrification gives a smaller increase in mumax and a more drastic reduction in yield. Both processes are thus advantageous to the organism. In a chemostat with limiting substrate these processes are disadvantageous. T. pantotropha has lost the ability for aerobic denitrification during extended cultivation. Possibly the substrate concentration was limiting during extended cultivation giving a selective advantage to variants which have lost these properties. The calculations predict that P. denitrificans should be able to grow chemolithotrophically with hydroxylamine.

The genes that encode the hc-type nitric-oxide reductase from Paracoccus denitrificans have been identified. They are part of a cluster of six genes (norCBQDEF) and are found near the gene cluster that encodes the cd1-type nitrite reductase, which was identified earlier [de Boer, A. P. N., Reijnders, W. N. M., Kuenen, J. G., Stouthamer, A. H. & van Spanning, R. J. M. (1994) Isolation, sequencing and mutational analysis of a gene cluster involved in nitrite reduction in Paracoccus denitrificans, Antonie Leeu wenhoek 66, 111-127]. norC and norB encode the cytochrome-c-containing subunit II and cytochrome b-containing subunit I of nitric-oxide reductase (NO reductase), respectively. norQ encodes a protein with an ATP-binding motif and has high similarity to NirQ from Pseudomonas stutzeri and Pseudomonas aeruginosa and CbbQ from Pseudomonas hydrogenothermophila. norE encodes a protein with five putative transmembrane alpha-helices and has similarity to CoxIII, the third subunit of the aa3-type cytochrome-c oxidases. norF encodes a small protein with two putative transmembrane alpha-helices. Mutagenesis of norC, norB, norQ and norD resulted in cells unable to grow anaerobically. Nitrite reductase and NO reductase (with succinate or ascorbate as substrates) and nitrous oxide reductase (with succinate as substrate) activities were not detected in these mutant strains. Nitrite extrusion was detected in the medium, indicating that nitrate reductase was active. The norQ and norD mutant strains retained about 16% and 23% of the wild-type level of NorC, respectively. The norE and norF mutant strains had specific growth rates and NorC contents similar to those of the wild-type strain, but had reduced NOR and NIR activities, indicating that their gene products are involved in regulation of enzyme activity. Mutant strains containing the norCBQDEF region on the broad-host-range vector pEG400 were able to grow anaerobically, although at a lower specific growth rate and with lower NOR activity compared with the wild-type strain.

Cytochrome c-550 of Thiobacillus versutus functions as an electron transfer protein in a chain of redox proteins that enables T. versutus to grow on methylamine. It is a single-heme protein of 134 residues, related to mitochondrial cytochrome c. Cytochrome c-550, as well as several other bacterial c2-type cytochromes, contain a C-terminal extension of 13-16 amino acids of unknown function, compared to mitochondrial cytochrome c. NMR experiments were performed to obtain structural and dynamic information on the protein in solution. For this purpose, T. versutus cytochrome c-550 was labeled with 15N and 13C using 13C-methanol grown Paracoccus denitrificans as a host for heterologous expression. NMR assignments were obtained for the 1H, 15N, and 13C nuclei in the backbone and the beta-positions of the protein and the secondary structure was determined. 15N-relaxation studies were performed to characterize the dynamic properties of the protein. The results indicate that the main part of T. versutus ferrocytochrome c-550 exists in solution as a rigid, well-ordered molecule with a secondary structure that is very similar to that of P. denitrificans cytochrome c-550, as observed in crystals. The C-terminal extension, however, is unstructured and highly mobile. The possible origin and function of the extension are discussed.

We present evidence that, in contrast to plasmids from other hyperthermophilic archaea, which are in the relaxed to positively supercoiled state, plasmid pGS5 (2.8 kb) from Archaeoglobus profundus is negatively supercoiled. This might be due to the presence of a gyrase introducing negative supercoils, since gyrase genes are present in the genome of its close relative A. fulgidus, and suggests that gyrase activity predominates over reverse gyrase whenever the two topoisomerases coexist in cells. Previous topological analyses of native plasmids from hyperthermophiles belonging to the two major phylogenetic lineages within the domain Archaea revealed a relaxed to positively supercoiled state of DNA (1, 2, 10). This was in contrast to the negatively supercoiled state of DNA in all mesophiles, including mesophilic archaea (1). The strains investigated in those studies were members of the orders Sulfolobales and Thermococcales, representing the two major archaeal kingdoms, Crenarchaeota and Euryarchaeota, respectively. All of them possessed reverse gyrase, a topoisomerase specific for hyperthermophiles that is able to introduce positive DNA supercoils (5). Therefore, reverse gyrase appeared responsible for this peculiar topological state. Accordingly, the idea that DNA was stabilized in organisms living at very high temperatures by an overall linking excess seemed to be supported (6). However, this hypothesis was called into question when plasmid pRQ7 from the hyperthermophilic bacterium Thermotoga maritima was found to be negatively supercoiled (7). In addition to reverse gyrase, T. maritima possesses DNA gyrase, a typical bacterial topoisomerase that introduces negative supercoils into DNA and is responsible for the negative supercoiling of pRQ7 (7). Two alternative possibilities could be then considered to explain the different plasmid topologies in hyperthermophiles. Either gyrase activity dominates whenever both reverse gyrase and gyrase are present in the cell, maintaining an overall DNA negative supercoiling, or, alternatively, the relaxed to positively supercoiled plasmid topology could be a taxonomic characteristic specific to hyperthermophilic archaea. The discovery of pGS5, a plasmid of 2,802 bp in the sulfate-reducing hyperthermophilic archaeon Archaeoglobus profundus (G. Erauso et al., unpublished data), allowed us to test which possibility was correct since the complete genome sequence of the closely related species Archaeoglobus fulgidus revealed the existence of genes encoding both gyrase and reverse gyrase (9). A. profundus strain AV18 was obtained from the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSM 5631) and cultivated under strict anaerobic conditions in a standard medium (Erauso et al., unpublished) consisting of a basal salt solution (containing, per liter, 20 g of NaCl ,3go f MgCl2 z 6H2 O, 4go f Na 2SO4, 0.5 g of KCl, 0.25 g of NH4Cl, 0.15 g of CaCl2 z 2H2O, and 0.14 g of K2HPO4 )p lus 1m l of a trace element solution (containing, per liter, 100 mg of Na2WO4 z 2H2O and 100 mg of NaSeO3 z 2H2O) buffered to pH 6.8 with 4go fpiperazine-N-N9-bis(2-ethanesulfonic acid) (PIPES) sodium salt and supplemented wit h1go fyeast extract (Difco) an d4go fsodium acetate. Resazurine (1 mg liter 21 ) was used as a redox indicator, and Na2S z 9H2O was

Introduction The enzymes which comprise the prokaryotic and eukaryotic respiratory chains are typically membrane-bound multi-subunit complexes. In mitochondria they tend to be more complex protein conglomerates than in bacteria. In both eubacteria and archaebacteria respiratory enzymes seem to contain only the functional cores of their eukaryotic sisters. In the simplest cases they may have only a single subunit but still retain at least a part of the relevant catalytic function [ 1,2]. The archaetype of this family is the mitochondrial cytochrome c oxidiase (1.9.3.1) or cytochrome aa3, which has, depending on the species, from seven to thirteen different subunits, contains two haem A groups and two redox-active copper ions, and functions as a redox-linked proton pump [3-51. Many bacteria have a similar cytochrome aa, but this has only three or four subunits [4, 61. Different types of terminal oxidases, many of which are known to have subunits homologous to those in the mitochondrial enzyme but which contain different prosthetic groups, are frequently found in bacteria [6-81. This short review will focus on these bacterial quinol and cytochrome c oxidases.

Publisher Summary This chapter presents that biocatalysis is gradually taking over from chemical catalysis in many industrial applications. Enzymes are environmentally friendly, biodegradable, efficient, and low cost in terms of resource requirements; they provide benefits compared with traditional chemical approaches in various industrial processes. In many instances, however, natural enzymes do not perform optimally in a particular unnatural process and can be unsuitable for large-scale industrial applications. Reflecting their participation in complex metabolic networks inside living cells, enzymes are often inhibited by their own substrates or products, either of which may severely limit the productivity of an industrial biocatalytic process. During natural evolution, enzymes are optimized and often highly specialized for certain biological functions within the context of a living organism. In contrast, biotechnology needs enzymes that have (1) a high activity over longer periods of time, (2) a high stability under harsh physical and chemical conditions, and (3) a high specificity and selectivity that does allow the enzyme to generate efficiently specific products that are not necessarily present in nature. The literature contains a large number of studies in which enzymes have been improved by rational or random approaches. It has been shown that both approaches have strengths, but also weaknesses. Rationale-based engineering requires a high-resolution three-dimensional (3D) model and insight into the structure–function relationships of the biocatalyst of interest. Laboratory evolution requires the ability to screen large libraries; when major adjustments are desired, the leap in sequence space may be too big to bridge.

The gene encoding a short-chain alcohol dehydrogenase, AdhA, has been identified in the hyperthermophilic archaeon Pyrococcus furiosus, as part of an operon that encodes two glycosyl hydrolases, the beta-glucosidase CelB and the endoglucanase LamA. The adhA gene was functionally expressed in Escherichia coli, and AdhA was subsequently purified to homogeneity. The quaternary structure of AdhA is a dimer of identical 26-kDa subunits. AdhA is an NADPH-dependent oxidoreductase that converts alcohols to the corresponding aldehydes/ketones and vice versa, with a rather broad substrate specificity. Maximal specific activities were observed with 2-pentanol (46 U x mg(-1)) and pyruvaldehyde (32 U x mg(-1)) in the oxidative and reductive reaction, respectively. AdhA has an optimal activity at 90 degrees C, at which temperature it has a half life of 22.5 h. The expression of the adhA gene in P. furiosus was demonstrated by activity measurements and immunoblot analysis of cell extracts. A role of this novel type of archaeal alcohol dehydrogenase in carbohydrate fermentation is discussed.

Publisher Summary One of the key enzymes of the hyperthermophilic archaeon Pyrococcus furiosus involved in growth on B-linked sugars is the inducible B-glucosidase (CelB). CelB from P. furiosus serves as a very suitable model glycosylhydrolase to study substrate specificity as well as adaptations of stability and activity to extreme temperatures. Stable production in E. coli and an efficient purification protocol allow for simple and rapid preparation of pure wildtype and mutant CelB enzymes. This chapter an presents an overview of the state of the art on this hyperthermostable enzyme, protocols for heterologous production and enzyme purification, and development and application of a directed evolution procedure that has resulted in the isolation and characterization of an active site mutant.

Publisher Summary Currently several serine proteases have been purified from hyperthermophiles with a variety of approaches and characterized at the enzyme and, in most cases also the gene level. Those that are genetically characterized all belong to the subtilisin-like family of serine proteases, also known as “subtilases.” Moreover, a variety of homologs of subtilases can be detected in the present databases of sequenced genomes of hyperthermophiles. The first of these serine proteases to be characterized at the enzyme and gene level has been pyrolysin from the hyperthermophilic archaeon Pyrococcus furiosus , which is the most thermostable protease to date and retains half of its activity after boiling for several hours. A characteristic feature of pyrolysin and other proteases is the fact that these enzymes are substrates of their proteolytic activity and degrade themselves in a process termed autoproteolysis. This has for a long time hampered the purification of pyrolysin and prevented its further characterization at the molecular level. This chapter discusses the procedures for the biochemical and genetic characterization of pyrolysin and other related thermostable serine proteases and the prediction of their properties by homology comparisons, database searches, and molecular modelling.

Publisher Summary ADP-dependent glucokinase and phosphofructokinase were demonstrated for the first time by Kengen et al . in the hyperthermophile Pyrococcus furiosus . This anaerobic archaeon grows optimally at 100° by fermenting sugar polymers and polypeptides to mainly acetate and alanine. The catabolism of sugars, such as starch, laminarin, maltose, or cellobiose, has been investigated in detail and has led to the discovery of novel type Embden–Meyerhof pathway, involving several enzymatic steps that are different from the classical ones. In addition to ADP-dependent kinases, the pathway involves a one-step, ferredoxin-dependent conversion of glyceraldehyde-3-phosphate to 3-phosphoglycerate instead of the conventional two-step, NAD-dependent and ATP-generating conversion. Furthermore, a remarkable AMP-dependent, ATP-generating pyruvate kinase has been described, which is different from the normal ADP-dependent pyruvate kinase. Moreover, the available P. furiosus genome sequence shows that for several other glycolytic enzymes, no homologous sequences can be identified, suggesting that these enzymes may be different from the classical ones as well. This chapter describes the procedures for assay and purification of both kinases and the cloning and expression of their genes.

The substrate specificity of the beta-glucosidase (CelB) from the hyperthermophilic archaeon Pyrococcus furiosus, a family 1 glycosyl hydrolase, has been studied at a molecular level. Following crystallization and X-ray diffraction of this enzyme, a 3.3 A resolution structural model has been obtained by molecular replacement. CelB shows a homo-tetramer configuration, with subunits having a typical (betaalpha)(8)-barrel fold. Its active site has been compared to the one of the previously determined 6-phospho-beta-glycosidase (LacG) from the mesophilic bacterium Lactococcus lactis. The overall design of the substrate binding pocket is very well conserved, with the exception of three residues that have been identified as a phosphate binding site in LacG. To verify the structural model and alter its substrate specificity, these three residues have been introduced at the corresponding positions in CelB (E417S, M424K, F426Y) in different combinations: single, double, and triple mutants. Characterization of the purified mutant CelB enzyme revealed that F426Y resulted in an increased affinity for galactosides, whereas M424K gave rise to a shifted pH optimum (from 5.0 to 6.0). Analysis of E417S revealed a 5-fold and a 3-fold increase of the efficiency of hydrolyzing o-nitrophenol-beta-D-galactopyranoside-6-phosphate, in the single and triple mutants, respectively. In contrast, their activity on nonphosphorylated sugars was largely reduced (30-300-fold). The residue at position E417 in CelB seems to be the determining factor for the difference in substrate specificity between the two types of family 1 glycosidases.

The hyperthermophilic archaea are a group of phylogenetically related microorganisms which by definition grow optimally at or above 80°C, with a maximal growth temperature of 90°C or higher (5). The majority of these are strictly anaerobic heterotrophs, most of which are obligately dependent upon the reduction of elemental sulfur (S0) to H2S. A limited number of facultative S0-reducing species are able to grow in the absence of S0 by means of an alternative fermentative-type metabolism. An example of this type of organism is Pyrococcus furiosus, which grows optimally at 100°C, with a temperature maximum of 105°C, by the fermentation of peptides and various carbohydrates, including starch, glycogen, β-glucans, cellobiose, and maltose (12). In addition, pyruvate can also be utilized as a carbon and energy source (9, 33). P. furiosus utilizes a modified Embden-Meyerhof pathway for the catabolism of sugars, which involves a pair of unprecedented ADP-dependent kinases (glucokinase and phosphofructokinase) and a unique glyceraldehyde-3-phosphate:ferredoxin oxidoreductase (18, 26, 35, 37). The main products produced during the fermentation of sugars include acetate, CO2, H2, and alanine (19). During growth on either peptides or carbohydrates, reduced ferredoxin is generated (1, 20). Regeneration of oxidized ferredoxin is assumed to be accomplished by three mechanisms: either by S0 reduction to H2S, by proton reduction to H2, or by the formation of alanine. The third alternative, formation of alanine, is found when P. furiosus is grown in the absence of S0. In addition to acetate, a significant amount of alanine is excreted into the medium (19, 33). The amount of alanine produced varies, with an increase in the H2 partial pressure resulting in an increase in the amount of alanine produced. The transamination of pyruvate with glutamate by the action of an alanine aminotransferase (AlaAT) was detected in cell extracts of P. furiosus (19). Furthermore, this activity was shown to be affected by both the partial pressure of hydrogen as well as the available carbon source, suggesting some form of regulation. Glutamate must be replenished through the action of the NADP-dependent glutamate dehydrogenase (GDH). However, there is some controversy concerning the exact role of GDH in the metabolism, since it has been proposed to serve an anabolic role (7, 27), as well as a catabolic role (32). Interestingly, the activity of the GDH reacted similarly to that of AlaAT under the same growth conditions, further suggesting some coordinated regulation of these enzymes (19). The necessary NADPH can be generated by the transfer of reducing equivalents from reduced ferredoxin to NADP+ by the ferredoxin:NADP oxidoreductase activity of the sulfide dehydrogenase. These initial findings suggest that P. furiosus is able to shift its metabolism in response to its environment and in particular the redox potential of the available terminal electron acceptor. In addition to P. furiosus, l-alanine production has been detected in the related archeaon Thermococcus profundus (21), as well as the hyperthermophilic bacteria belonging to the order of the Thermotogales (31). Pyrococcus and Thermococcus are considered to be one of the deepest branches in the domain of the Archaea, with Thermotogales being one of the deepest branches within the domain Bacteria. Based on this finding, it has been proposed that alanine production from sugar fermentation can be regarded as an ancestral metabolic characteristic (31). However, l-alanine production has also been reported during the fermentation of sugars under anaerobic conditions for the intestinal parasite Giardia lamblia (11) and a moderately thermophilic Clostridium species (28), suggesting that this pathway may be more common among the three domains than previously thought. Moreover, homoalanine fermentation was recently established by metabolic engineering in a lactic acid bacterium, indicating that this pathway may function as an electron sink in a wide range of organisms (16). Further analysis of this pathway may provide insight into not only the biology of these organisms, but also the evolution of fermentative metabolism.

The sequence of a 281-kbp contig from the crenarchaeote Sulfolobus solfataricus P2 was determined and analysed. Notable features in this region include 29 ribosomal protein genes, 12 tRNA genes (four of which contain archaeal-type introns), operons encoding enzymes of histidine biosynthesis, pyrimidine biosynthesis, and arginine biosynthesis, an ATPase operon, numerous genes for enzymes of lipopolysaccharide biosynthesis, and six insertion sequences. The content and organization of this contig are compared with sequences from crenarchaeotes, euryarchaeotes, bacteria, and eukaryotes. Key words: Archaea, Sulfolobus Genome Project, comparative genomics, bioinformatics.

ortho-Chlorophenol reductive dehalogenase of the halorespiring Gram-positive Desulfitobacterium dehalogenans was purified 90-fold to apparent homogeneity. The purified dehalogenase catalyzed the reductive removal of a halogen atom from the ortho position of 3-chloro-4-hydroxyphenylacetate, 2-chlorophenol, 2,3-dichlorophenol, 2,4-dichlorophenol, 2,6-dichlorophenol, pentachlorophenol, and 2-bromo-4-chlorophenol with reduced methyl viologen as electron donor. The dechlorination of 3-chloro-4-hydroxyphenylacetate was catalyzed by the enzyme at a Vmax of 28 units/mg protein and a Km of 20 microM. The pH and temperature optimum were 8.2 and 52 degrees C, respectively. EPR analysis indicated one [4Fe-4S] cluster (midpoint redox potential (Em) = -440 mV), one [3Fe-4S] cluster (Em = +70 mV), and one cobalamin per 48-kDa monomer. The Co(I)/Co(II) transition had an Em of -370 mV. Via a reversed genetic approach based on the N-terminal sequence, the corresponding gene was isolated from a D. dehalogenans genomic library, cloned, and sequenced. This revealed the presence of two closely linked genes: (i) cprA, encoding the o-chlorophenol reductive dehalogenase, which contains a twin-arginine type signal sequence that is processed in the purified enzyme; (ii) cprB, coding for an integral membrane protein that could act as a membrane anchor of the dehalogenase. This first biochemical and molecular characterization of a chlorophenol reductive dehalogenase has revealed structural resemblance with haloalkene reductive dehalogenases.

The role of an 18-residue ion-pair network, that is present in the glutamate dehydrogenase from the hyperthermophilic archaeon Pyrococcus furiosus, in conferring stability to other, less stable homologous enzymes, has been studied by introducing four new charged amino acid residues into the subunit interface of glutamate dehydrogenase from the hyperthermophilic bacterium Thermotoga maritima. These two GDHs are 55 % identical in amino acid sequence, differ greatly in thermo-activity and stability and derive from microbes with different phylogenetic positions. Amino acid substitutions were introduced as single mutations as well as in several combinations. Elucidation of the crystal structure of the quadruple mutant S128R/T158E/N117R/S160E T. maritima glutamate dehydrogenase showed that all anticipated ion-pairs are formed and that a 16-residue ion-pair network is present. Enlargement of existing networks by single amino acid substitutions unexpectedly resulted in a decrease in resistance towards thermal inactivation and thermal denaturation. However, combination of destabilizing single mutations in most cases restored stability, indicating the need for balanced charges at subunit interfaces and high cooperativity between the different members of the network. Combination of the three destabilizing mutations in triple mutant S128R/T158E/N117R resulted in an enzyme with a 30 minutes longer half-life of inactivation at 85 degrees C, a 3 degrees C higher temperature optimum for catalysis, and a 0.5 degrees C higher apparent melting temperature than that of wild-type glutamate dehydrogenase. These findings confirm the hypothesis that large ion-pair networks do indeed stabilize enzymes from hyperthermophilic organisms.

The taxonomy of Paracoccus denitrificans and related bacteria is discussed. Evidence is given which shows that the physiological differences between P. denitrificans and Thiosphaera pantotropha are less fundamental than previously thought. A proposal to consider a species P. pantotropha is mentioned. The properties of the denitrifying enzymes and the genes involved in their formation in P. denitrificans is discussed. The synthesis of the membrane-bound-nitrate reductase is regulated by FNR, that of the nitrite- and nitric oxide reductase by NNR. Evidence is given that FNR acts as a redox sensor rather than an oxygen sensor. The occurrence of aerobic denitrification and coupled heterotrophic nitrification-denitrification in the original strain of Thiosphaera pantotropha are explained by a limiting respiratory activity which activates FNR. Aerobic denitrification leads to a lower growth yield and an increase in mumax in batch culture when a limiting respiratory activity is assumed and when excess substrate is present. Coupled heterotrophic nitrification-denitrification gives a smaller increase in mumax and a more drastic reduction in yield. Both processes are thus advantageous to the organism. In a chemostat with limiting substrate these processes are disadvantageous. T. pantotropha has lost the ability for aerobic denitrification during extended cultivation. Possibly the substrate concentration was limiting during extended cultivation giving a selective advantage to variants which have lost these properties. The calculations predict that P. denitrificans should be able to grow chemolithotrophically with hydroxylamine.

Paracoccus denitrificans is a facultative anaerobic bacterium that has the capacity to adjust its metabolic infrastructure, quantitatively and/or qualitatively, to the prevailing growth condition. In this bacterium the relative activity of distinct catabolic pathways is subject to a hierarchical control. In the presence of oxygen the aerobic respiration, the most efficient way of electron transfer-linked phosphorylation, has priority. At high oxygen tensions P. denitrificans synthesizes an oxidase with a relatively low affinity for oxygen, whereas under oxygen limitation a high-affinity oxidase appears specifically induced. During anaerobiosis, the pathways with lower free energy-transducing efficiency are induced. In the presence of nitrate, the expression of a number of dehydrogenases ensures the continuation of oxidative phosphorylation via denitrification. After identification of the structural components that are involved in both the aerobic and the anaerobic respiratory networks of P. denitrificans, the intriguing next challenge is to get insight in its regulation. Two transcription regulators have recently been demonstrated to be involved in the expression of a number of aerobic and/or anaerobic respiratory complexes in P. denitrificans. Understanding of the regulation machinery is beginning to emerge and promises much excitement in discovery.

Cytochrome c oxidase contains a copper center, CuA, which is involved in electron transfer from cytochrome c to the oxygen-reducing active site. This center is distinct from types 1, 2, and 3 copper sites and related only to a purple copper center in nitrous oxide reductase. At present it is not clear whether this site is mononuclear or is comprised of two copper atoms in a mixed valence (Cu(I)-Cu(II)) configuration. Here we use a model of CuA, engineered into a structurally related but initially copperless protein, to study the structure of this copper center. The results from biochemical analysis, site-directed mutagenesis, and electrospray mass spectrometry support the binuclear model. Two cysteines, two histidines, and one methionine are the major ligands of two coppers. Substitution of these residues results in either a complete loss of color or dramatic changes in the absorbance spectrum. In contrast, substitution of the invariant glutamate residue, which is located between the copper-binding cysteines, leads to a minor perturbation of the optical spectrum.

Summary The ctaBCDEF genes coding for cytochrome c oxidase were found to reside adjacent to a regulatory gene ctaA at 127° on the Bacillus subtilis chromosome. The structural genes for subunits I and II, ctaD and ctaC, were deleted by gene-replacement using a phleomycin-resistance marker. The mutant was unable to oxidize N,N,N′,N′-tetramethyl-p-phenylene-diamine and oxidized cytochrome c at a significantly lower rate. Absorption spectra of the mutant and wild-type membranes confirmed the presence of two haem A-containing enzymes in B. subtilis. Another mutant, with a spontaneous deletion upstream from CtaC, was found to express neither of these enzymes. Radioactive haem-labelling was used to identify sub-unit ii, which contains a haem C, and cytochrome c-550 among the membrane-bound c-type cytochromes of B. subtilis.

The hydrogenase-catalyzed hydrogen production exhibited by the unicellular cyanobacterium Cyanothece 7822 during anoxic incubation in the dark is a result of the fermentative degradation of carbon reserves. Simultaneously with hydrogen production, evolution of carbon dioxide was detected, and excretion of ethanol, lactate, formate and acetate was demonstrated. The fermentation balance indicates that carbohydrates are fermented via a branched pathway, in which both the pentose phosphate pathway and glycolysis appear to be involved. It is proposed that the physiological function of hydrogen production is the introduction of protons as terminal electron acceptors. This removal of reducing equivalents might give rise to continuation of the pyruvate decarboxylation and consequently of the acetate formation, thereby increasing the efficiency of fermentative energy generation.

S beta gly and CelB are well-studied hyperthermophilic glycosyl hydrolases, isolated from the Archaea Sulfolobus solfataricus and Pyrococcus furiosus, respectively. Previous studies revealed that the two enzymes are phylogenetically related; they are very active and stable at high temperatures, and their overall three-dimensional structure is very well conserved. To acquire insight in the molecular determinants of thermostability and thermoactivity of these enzymes, we have performed a detailed comparison, under identical conditions, of enzymological and biochemical parameters of S beta gly and CelB, and we have probed the basis of their stability by perturbations induced by temperature, pH, ionic strength, and detergents. The major result of the present study is that, although the two enzymes are remarkably similar with respect to kinetic parameters, substrate specificity, and reaction mechanism, they are strikingly different in stability to the different physical or chemical perturbations induced. These results provide useful information for the design of further experiments aimed at understanding the structure-function relationships in these enzymes.

The cupredoxin fold, a Greek key beta-barrel, is a common structural motif in a family of small blue copper proteins and a subdomain in many multicopper oxidases. Here we show that a cupredoxin domain is present in subunit II of cytochrome c and quinol oxidase complexes. In the former complex this subunit is thought to bind a copper centre called CuA which is missing from the latter complex. We have expressed the C-terminal fragment of the membrane-bound CyoA subunit of the Escherichia coli cytochrome o quinol oxidase as a water-soluble protein. Two mutants have been designed into the CyoA fragment. The optical spectrum shows that one mutant is similar to blue copper proteins. The second mutant has an optical spectrum and redox potential like the purple copper site in nitrous oxide reductase (N2OR). This site is closely related to CuA, which is the copper centre typical of cytochrome c oxidase. The electron paramagnetic resonance (EPR) spectra of both this mutant and the entire cytochrome o complex, into which the CuA site has been introduced, are similar to the EPR spectra of the native CuA site in cytochrome oxidase. These results give the first experimental evidence that CuA is bound to the subunit II of cytochrome c oxidase and open a new way to study this peculiar copper site.

Regulation of gene expression in the domain Archaea , and specifically hyperthermophiles, has been poorly investigated so far. Biochemical experiments and genome sequencing have shown that, despite the prokaryotic cell and genome organization, basal transcriptional elements of members of the domain Archaea (i.e., TATA box-like sequences, RNA polymerase, and transcription factors TBP, TFIIB, and TFIIS) are of the eukaryotic type. However, open reading frames potentially coding for bacterium-type transcription regulation factors have been recognized in different archaeal strains. This finding raises the question of how bacterial and eukaryotic elements interact in regulating gene expression in Archaea . We have identified a gene coding for a bacterium-type transcription factor in the hyperthermophilic archaeon Sulfolobus solfataricus . The protein, named Lrs14, contains a potential helix-turn-helix motif and is related to the Lrp-AsnC family of regulators of gene expression in the class Bacteria . We show that Lrs14, expressed in Escherichia coli , is a highly thermostable DNA-binding protein. Bandshift and DNase I footprint analyses show that Lrs14 specifically binds to multiple sequences in its own promoter and that the region of binding overlaps the TATA box, suggesting that, like the E. coli Lrp, Lrs14 is autoregulated. We also show that the lrs14 transcript is accumulated in the late growth stages of S. solfataricus .

Cytochrome c oxidase contains a copper center, CuA, which is involved in electron transfer from cytochrome c to the oxygen-reducing active site. This center is distinct from types 1, 2, and 3 copper sites and related only to a purple copper center in nitrous oxide reductase. At present it is not clear whether this site is mononuclear or is comprised of two copper atoms in a mixed valence (Cu(I)-Cu(II)) configuration. Here we use a model of CuA, engineered into a structurally related but initially copperless protein, to study the structure of this copper center. The results from biochemical analysis, site-directed mutagenesis, and electrospray mass spectrometry support the binuclear model. Two cysteines, two histidines, and one methionine are the major ligands of two coppers. Substitution of these residues results in either a complete loss of color or dramatic changes in the absorbance spectrum. In contrast, substitution of the invariant glutamate residue, which is located between the copper-binding cysteines, leads to a minor perturbation of the optical spectrum.

CRISPR-Cas is an extraordinary prokaryotic adaptive immune system, divided into two classes that each contain three types and a wide variety of subtypes. Class 2 effector proteins are the most exploited because of their compact structures with multi-functional properties. Amongst the class 2 systems, the more recently discovered type V systems appears to be the most diverse type, with new mechanistic features still to be uncovered. The research described in this thesis focusses on the characterization and subsequent development of applications of type V CRISPR-Cas systems, more specifically types V-A and V-U1.Chapter 1 introduces CRISPR-Cas as a sophisticated prokaryotic adaptive immune system. CRISPR-Cas systems. Each CRISPR-Cas system has unique features, but all actively participate in adaptive immunity through a three-step mechanism: adaptation, expression and interference. Although all variants follow the same steps, the different CRISPR-Cas (sub)types are highly diverse with unique structural and functional features at all levels of the mechanism.After describing the underlying molecular mechanism of CRISPR-Cas, an overview is given in chapter 2 on the different genome editing applications. The main focus is on DNA-targeting class 2 effector proteins, such as Cas9, Cas12a and Cas12b. Apart from natural variants, engineered CRISPR-Cas nuclease variants that increase editing precision or regulate nuclease activity were presented as well. One of the biggest bottle necks of genome editing in eukaryotes is the delivery of a specific nuclease. Therefore, different approaches of nuclease/guide delivery were discussed. In addition, various host repair pathways were examined based on the type of DNA damage and the type of repair template available.In chapter 3, the first step of the CRISPR-Cas adaptive immunity, adaptation of the CRISPR memory, is studied in two type V systems, namely type V-A and V-B from Francisella novicida tularensis subsp. novicida U112 and Alicyclobacillus acidoterrestris ATCC 49025, respectively. The type V-A locus encodes Cas12a, Cas4, Cas1 and Cas2, whereas that of V-B encodes Cas12b, a Cas4/1 fusion protein and Cas2. It was found that in type V-A, only Cas1 and Cas2 are required for adaptation, and in type V-B, Cas4/1 and Cas2 are required for adaptation, but Cas4 activity is dispensable. Spacers acquired without a functional Cas4, appeared to target protospacers containing mostly non-conical PAMs. Thus, Cas4 activity is required for PAM selection and acquisition of suitable spacers in both type V-A and V-B. The role of Cas12a in the adaptation process has not been elucidated yet in this chapter, but will be addressed in future studies.Following adaptation, chapter 4 describes crRNA maturation in type V-A. Type V-A crRNA maturation is distinct to that found in type II, where Cas9 requires both a crRNA and a tracrRNA, and gets processed by endogenous RNaseIII after ribonucleoprotein complex formation. This study demonstrated that Cas12a does not require a tracrRNA nor RNase III for crRNA maturation. Instead Cas12a itself is able to process pre-crRNA into mature crRNA using a previously unknown RNase domain found in Cas12a. Having Cas12a able to process its own crRNA is greatly advantageous for genome editing applications and allows for simple simultaneous multi-gene (multiplex) editing using a single CRISPR-array. Using a single CRISPR-array containing four spacers, Cas12a was able to simultaneously edit up to four genes in mammalian cells (ex vivo) and up to three genes in mouse brain cells (in vivo).Apart from processing its own pre-crRNA, another distinct feature of Cas12a is the generation of staggered ends after cleavage of dsDNA. These staggered ends were exploited in chapter 5 to create in a novel genome editing approach in E. coli, termed “cut and paste. Cleavage by Cas12a generates double-stranded DNA breaks with 4-5 nt compatible staggered ends. These staggered ends can be repaired by ligation using T4 ligase. Using cut & paste, a genomic deletion in E. coli was successfully achieved, albeit with a relatively low editing efficiency.To further explore other type V systems, chapter 6 focusses on the characterization of a novel compact type V systems, type V-U1 from Mycolicibacterium mucogenicum CCH10. The type V-U1 CRISPR-Cas locus express a small effector protein MmuCas12u1. MmuCas12u1 is roughly half the size of Cas12a. Despite its small size, MmuCas12u1 seems to retain some functional features also found in Cas12a. Features such as processing its own pre-crRNA, targeting dsDNA and recognizing a 5’-TTN-3’ PAM. The RuvC domain of MmuCas12u1 does not cleave dsDNA, but instead is hypothesized to be involved dsDNA-activated transcriptional silencing. By leveraging this property, MmuCas12u1 has been used for single- and multiplex- transcriptional silencing in E. coli.Chapter 7 described how the fundamental knowledge gained on MmuCas12u1 is used to develop small Mmu base editors (MmuBE). MmuBEs are fusion proteins consisting of MmuCas12u1, cytidine deaminase and uracil glycosylase inhibitor, which is a tool for RNA-guided targeted nucleotide (C à T) substitution. Several MmuBE variants were constructed and characterized in E. coli. Most variants are relatively efficient, with a base editing window consisting of two regions, a PAM-proximal (2-5) and a PAM-distal (13-19) region, with the PAM-proximal region having more edits. In addition, a small-scale pilot experiment also demonstrated on-target base-editing by MmuBE in eukaryotic cells, namely in Saccharomyces cerevisiae. MmuBEs are currently the smallest base editors (genes ~2.8 kb) known, further expanding the current toolbox for prokaryotic base editing, and with great promise for eukaryotic base editing.

Host-pathogen interactions are among the most prevalent and evolutionary important interactions known today. The predation of prokaryotes by their viruses is happening on an especially large scale and had a major influence on the evolutionary history of prokaryotes. Since most viruses are lytic at some point in their life-cycle, there is a high selection pressure for prokaryotes to develop defense mechanisms. As described in Chapter 1, the CRISPR-Cas system is a relatively recently discovered defense system and is also the first adaptive defense system discovered in prokaryotes. CRISPR-Cas systems are widespread, occurring in the majority of archaea and also a considerable fraction of bacteria. This diversity is also reflected in the diversity of different types of CRISPR-Cas systems, currently being divided into 6 major types with a large number of subtypes. The type I-E system of Escherichia coli is a well-studied model system and of high relevance, since it is a major subtype of type I systems which make up around 50 % of all discovered CRISPR-Cas systems. CRISPR-Cas systems basically comprise the CRISPR array, made up of repeats and foreign derived spacers, and a set of cas genes. Immunity is commonly divided into three functional stages, adaptation, expression and interference. Adaptation is the acquisition of new spacers from the foreign nucleic acid and its incorporation into the CRISPR array. During expression, the CRISPR array is transcribed, processed and assembled with Cas proteins into CRISPR RNA (crRNA) guided ribonucleoprotein complexes (crRNP). Interference is the detection, binding and destruction of foreign nucleic acids by the crRNP and in type I systems the Cas3 nuclease. The type I-E system contains another function, called primed adaptation. Primed adaptation is a more rapid and efficient version of regular (naïve) adaptation. In addition to the adaptation machinery, primed adaptation also requires the interference machinery. Chapter 2 describes and compares a fundamental feature of most, if not all, CRISPR-Cas systems and also many other small RNA based systems. The mode of action of small RNAs relies on protein-assisted base pairing of the guide RNA with target mRNA or DNA to interfere with their transcription, translation or replication. Several unrelated classes of small non-coding RNAs have been identified including eukaryotic RNA silencing associated small RNAs, prokaryotic small regulatory RNAs and prokaryotic CRISPR (clustered regularly interspaced short palindromic repeats) RNAs. All three groups identify their target sequence by base pairing after finding it in a pool of millions of other nucleotide sequences in the cell. In this complicated target search process, a region of 6 to 12 nucleotides of the small RNA termed the ‘seed’ plays a critical role. The seed is often a structurally pre-ordered region that increases accessibility and lowers the energy barrier of RNA-DNA duplex formation. Furthermore, the length of the seed is optimally chosen to allow rapid probing and also rejection of potential target sites. The seed is a perfect example of parallel evolution, showing that nature comes up with the same strategy independently multiple times. Chapter 3 provides a description and protocol of the Electrophoretic Mobility Shift Assay (EMSA) and its use for studying crRNPs. EMSA is a straightforward and inexpensive method for the determination and quantification of protein–nucleic acid interactions. It relies on the different mobility of free and protein-bound nucleic acid in a gel matrix during electrophoresis. Nucleic acid affinities of crRNPs can be quantified by calculating the dissociation constant (Kd ). Protocols for two types of EMSA assays are described using the Cascade ribonucleoprotein complex from Escherichia coli as an example. One protocol uses plasmid DNA as substrate, while the other uses short linear oligonucleotides. Plasmids can be easily visualized with traditional DNA staining, while oligos have to be radioactively labelled using the 32Phosphate isotope. The EMSA method and these protocols are applied throughout the other chapters of this thesis. Chapter 4 focusses on the processes of interference and primed adaptation, specifically on their tolerance of mutations. Invaders can escape Type I-E CRISPR-Cas immunity in E. coli by making point mutations in the protospacer (especially in the seed) or its adjacent motif (PAM), but hosts quickly restore immunity by integrating new spacers in a positive feedback process termed priming. Here, we provide a systematic analysis of the constraints of both direct interference and subsequent priming in E. coli. We have defined a high-resolution genetic map of direct interference by Cascade and Cas3, which includes five positions of the protospacer at 6 nt intervals that readily tolerate mutations. Importantly, we show that priming is an extremely robust process capable of utilizing degenerate target regions with up to at least eleven mutations throughout the PAM and protospacer region. Priming is influenced by the number of mismatches, their position and is nucleotide dependent. Our findings imply that even out-dated spacers containing many mismatches can induce a rapid primed CRISPR response against diversified or related invaders, giving microbes an advantage in the co- evolutionary arms race with their invaders. In Chapter 5 we elucidate the mechanism of priming. Specifically, we determine how new spacers are produced and selected for integration into the CRISPR array during priming. We show that priming is directly dependent on interference. Rapid priming occurs when the rate of interference is high, delayed priming occurs when the rate of interference is low. Using in vitro assays and next generation sequencing, we show that Cas3 couples CRISPR interference to adaptation by producing DNA breakdown products that fuel the spacer integration process in a two-step, PAM-associated manner. The helicase-nuclease Cas3 pre-processes target DNA into fragments of about 30–100 nt enriched for thymine-stretches in their 3’ ends. By reconstituting the spacer integration process in vitro, we show that the Cas1-2 complex further processes these fragments and integrates them sequence- specifically into CRISPR repeats by coupling of a 3’ cytosine of the fragment. Our results highlight that the selection of PAM-compliant spacers during priming is enhanced by the combined sequence specificities of Cas3 and the Cas1-2 complex, leading to an increased propensity of integrating functional CTT-containing spacers. In Chapter 6 we look deeper into a nucleotide specific effect on priming that was discovered in Chapter 4. Immunity is based on the complementarity of host encoded spacer sequences with protospacers on the foreign genetic element. The efficiency of both direct interference and primed acquisition depends on the degree of complementarity between spacer and protospacer. Previous studies focused on the amount and positions of mutations, not the identity of the substituted nucleotide. In Chapter 4, we describe a nucleotide bias, showing a positive effect on priming of C substitutions and a negative effect on priming of G substitutions in the basepairing strand of the protospacer. Here we show that these substitutions rather directly influence the efficiency of interference and therefore indirectly influence the efficiency of interference dependent priming. We show that G substitutions have a profoundly negative effect on interference, while C substitutions are readily tolerated when in the same positions. Furthermore, we show that this effect is based on strongly decreased binding of the effector complex Cascade to G mutants, while C mutants only minimally affect binding. In Chapter 5 we showed a connection between the rate of interference and the time of occurrence of priming. Here, we also quantify the extent of priming and show that priming is very prevalent in a population that shows intermediate levels of interference, while high or low levels of interference lead to a lower prevalence of priming. Chapter 7 describes an attempt to make use of our knowledge about the Cascade complex and develop it into a genome editing tool. The development of genome editing tools has made major leaps in the last decade. Recently, RNA guided endonucleases (RGENs) such as Cas9 or Cpf1 have revolutionized genome editing. These RGENs are the hallmark proteins of class II CRISPR-Cas systems. Here, we have explored the possibility to develop a new genome editing tool that makes use of the Cascade complex from E. coli. This RNA guided protein complex is fused to a FokI nuclease domain to sequence specifically cleave DNA. We validate the tool in vitro using purified protein and two sets of guide RNAs, showing specific cleavage activity. The tool requires two target sites of 32 nt each at a distance of 30-40 nt and inward facing three nucleotide flexible PAM sequences. Cleavage occurs in the middle between the two binding sites and primarily creates 4 nt overhangs. Furthermore, we show that an additional RFP can be fused to FokI-Cascade, allowing visualization of the complex in target cells. Unfortunately, we were not able to successfully apply the tool in vivo in eukaryotic cells.