A pH-dependent shift of redox cofactor specificity in a benzyl alcohol dehydrogenase of aromatoleum aromaticum EbN1.

We characterise a reversible bacterial zinc-containing benzyl alcohol dehydrogenase (BaDH) accepting either NAD+ or NADP+ as a redox cofactor. Remarkably, its redox cofactor specificity is pH-dependent with the phosphorylated cofactors favored at lower and the dephospho-forms at higher pH. BaDH also shows different steady-state kinetic behavior with the two cofactor forms. From a structural model, the pH-dependent shift may affect the charge of a histidine in the 2'-phosphate-binding pocket of the redox cofactor binding site. The enzyme is phylogenetically affiliated to a new subbranch of the Zn-containing alcohol dehydrogenases, which share this conserved residue. BaDH appears to have some specificity for its substrate, but also turns over many substituted benzyl alcohol and benzaldehyde variants, as well as compounds containing a conjugated C=C double bond with the aldehyde carbonyl group. However, compounds with an sp3-hybridised C next to the alcohol/aldehyde group are not or only weakly turned over. The enzyme appears to contain a Zn in its catalytic site and a mixture of Zn and Fe in its structural metal-binding site. Moreover, we demonstrate the use of BaDH in an enzyme cascade reaction with an acid-reducing tungsten enzyme to reduce benzoate to benzyl alcohol. KEY POINTS: •Zn-containing BaDH has activity with either NAD + or NADP+ at different pH optima. •BaDH converts a broad range of substrates. •BaDH is used in a cascade reaction for the reduction of benzoate to benzyl alcohol.

Modeling the Initiation Phase of the Catalytic Cycle in the Glycyl-Radical Enzyme Benzylsuccinate Synthase.

The reaction of benzylsuccinate synthase, the radical-based addition of toluene to a fumarate cosubstrate, is initiated by hydrogen transfer from a conserved cysteine to the nearby glycyl radical in the active center of the enzyme. In this study, we analyze this step by comprehensive computer modeling, predicting (i) the influence of bound substrates or products, (ii) the energy profiles of forward- and backward hydrogen-transfer reactions, (iii) their kinetic constants and potential mechanisms, (iv) enantiospecificity differences, and (v) kinetic isotope effects. Moreover, we support several of the computational predictions experimentally, providing evidence for the predicted H/D-exchange reactions into the product and at the glycyl radical site. Our data indicate that the hydrogen transfer reactions between the active site glycyl and cysteine are principally reversible, but their rates differ strongly depending on their stereochemical orientation, transfer of protium or deuterium, and the presence or absence of substrates or products in the active site. This is particularly evident for the isotope exchange of the remaining protium atom of the glycyl radical to deuterium, which appears dependent on substrate or product binding, explaining why the exchange is observed in some, but not all, glycyl-radical enzymes.

Bacteria at Work - Experimental and Theoretical Studies Reveal the Catalytic Mechanism of Ectoine Synthase.

Ectoine synthase (EctC) catalyses the ultimate step of ectoine biosynthesis, a kosmotropic compound produced as compatible solute by many bacteria and some archaea or eukaryotes. EctC is an Fe2+-dependent homodimeric cytoplasmic protein. Using Mössbauer spectroscopy, molecular dynamics simulations and QM/MM calculations, we determined the most likely coordination number and geometry of the Fe2+ ion and proposed a mechanism of the EctC-catalysed reaction. Most notably, we show that apart from the three amino acids binding to the iron ion (Glu57, Tyr84 and His92), one water molecule and one hydroxide ion are required as additional ligands for the reaction to occur. They fill the first coordination sphere of the Fe2+-cofactor and act as critical proton donors and acceptors during the cyclization reaction.

A Synthetic Pathway for the Production of Benzylsuccinate in Escherichia coli.

(R)-Benzylsuccinate is generated in anaerobic toluene degradation by the radical addition of toluene to fumarate and further degraded to benzoyl-CoA by a ß-oxidation pathway. Using metabolic modules for benzoate transport and activation to benzoyl-CoA and the enzymes of benzylsuccinate ß-oxidation, we established an artificial pathway for benzylsuccinate production in Escherichia coli, which is based on its degradation pathway running in reverse. Benzoate is supplied to the medium but needs to be converted to benzoyl-CoA by an uptake transporter and a benzoate-CoA ligase or CoA-transferase. In contrast, the second substrate succinate is endogenously produced from glucose under anaerobic conditions, and the constructed pathway includes a succinyl-CoA:benzylsuccinate CoA-transferase that activates it to the CoA-thioester. We present first evidence for the feasibility of this pathway and explore product yields under different growth conditions. Compared to aerobic cultures, the product yield increased more than 1000-fold in anaerobic glucose-fermenting cultures and showed further improvement under fumarate-respiring conditions. An important bottleneck to overcome appears to be product excretion, based on much higher recorded intracellular concentrations of benzylsuccinate, compared to those excreted. While no export system is known for benzylsuccinate, we observed an increased product yield after adding an unspecific mechanosensitive channel to the constructed pathway.

A bacterial tungsten-containing aldehyde oxidoreductase forms an enzymatic decorated protein nanowire.

Aldehyde oxidoreductases (AORs) are tungsten enzymes catalyzing the oxidation of many different aldehydes to the corresponding carboxylic acids. In contrast to other known AORs, the enzyme from the denitrifying betaproteobacterium Aromatoleum aromaticum (AORAa) consists of three different subunits (AorABC) and uses nicotinamide adenine dinucleotide (NAD) as an electron acceptor. Here, we reveal that the enzyme forms filaments of repeating AorAB protomers that are capped by a single NAD-binding AorC subunit, based on solving its structure via cryo-electron microscopy. The polyferredoxin-like subunit AorA oligomerizes to an electron-conducting nanowire that is decorated with enzymatically active and W-cofactor (W-co) containing AorB subunits. Our structure further reveals the binding mode of the native substrate benzoate in the AorB active site. This, together with quantum mechanics:molecular mechanics (QM:MM)-based modeling for the coordination of the W-co, enables formulation of a hypothetical catalytic mechanism that paves the way to further engineering for applications in synthetic biology and biotechnology.

Electrocatalytic Aldehyde Oxidation by a Tungsten Dependent Aldehyde Oxidoreductase from Aromatoleum Aromaticum.

In contrast to their molybdenum dependent relatives, tungsten enzymes operate at significantly lower redox potentials, and in some cases they can carry out reversible redox transformations of their substrates and products. Still, the electrochemical properties of W enzymes have received much less attention than their Mo relatives. Herein we analyse the tungsten enzyme aldehyde oxidoreductase (AOR) from the mesophilic bacterium Aromatoleum aromaticum which has been immobilised on a glassy carbon working electrode. This generates a functional system that electrochemically oxidises a wide variety of aromatic and aliphatic aldehydes in the presence of the electron transfer mediators benzyl viologen, methylene blue or dichlorophenol indophenol. Simulation of the cyclic voltammetry has enabled a thorough kinetic analysis of the system, which reveals that methylene blue acts as a two-electron acceptor. In contrast, the other two mediators act as single electron oxidants. The different electrochemical driving forces imparted by these mediators also lead to significantly different outer sphere electron transfer rates with AOR. This work shows that electrocatalytic aldehyde oxidation can be achieved at a low applied electrochemical potential leading to an extremely energy efficient catalytic process.

IncP-type plasmids carrying genes for antibiotic resistance or for aromatic compound degradation are prevalent in sequenced Aromatoleum and Thauera strains.

Self-transferable plasmids of the incompatibility group P-1 (IncP-1) are considered important carriers of genes for antibiotic resistance and other adaptive functions. In the laboratory, these plasmids have a broad host range; however, little is known about their in situ host profile. In this study, we discovered that Thauera aromatica K172T , a facultative denitrifying microorganism capable of degrading various aromatic compounds, contains a plasmid highly similar to the IncP-1 ε archetype pKJK5. The plasmid harbours multiple antibiotic resistance genes and is maintained in strain K172T for at least 1000 generations without selection pressure from antibiotics. In a subsequent search, we found additional nine IncP-type plasmids in a total of 40 sequenced genomes of the closely related genera Aromatoleum and Thauera. Six of these plasmids form a novel IncP-1 subgroup designated θ, four of which carry genes for anaerobic or aerobic degradation of aromatic compounds. Pentanucleotide sequence analyses (k-mer profiling) indicated that Aromatoleum spp. and Thauera spp. are among the most suitable hosts for the θ plasmids. Our results highlight the importance of IncP-1 plasmids for the genetic adaptation of these common facultative denitrifying bacteria and provide novel insights into the in situ host profile of these plasmids.

Structure of the membrane-bound formate hydrogenlyase complex from Escherichia coli.

The prototypical hydrogen-producing enzyme, the membrane-bound formate hydrogenlyase (FHL) complex from Escherichia coli, links formate oxidation at a molybdopterin-containing formate dehydrogenase to proton reduction at a [NiFe] hydrogenase. It is of intense interest due to its ability to efficiently produce H2 during fermentation, its reversibility, allowing H2-dependent CO2 reduction, and its evolutionary link to respiratory complex I. FHL has been studied for over a century, but its atomic structure remains unknown. Here we report cryo-EM structures of FHL in its aerobically and anaerobically isolated forms at resolutions reaching 2.6 Å. This includes well-resolved density for conserved loops linking the soluble and membrane arms believed to be essential in coupling enzymatic turnover to ion translocation across the membrane in the complex I superfamily. We evaluate possible structural determinants of the bias toward hydrogen production over its oxidation and describe an unpredicted metal-binding site near the interface of FdhF and HycF subunits that may play a role in redox-dependent regulation of FdhF interaction with the complex.

Tungsten Enzyme Using Hydrogen as an Electron Donor to Reduce Carboxylic Acids and NAD.

Tungsten-dependent aldehyde oxidoreductases (AORs) catalyze the oxidation of aldehydes to acids and are the only known enzymes reducing non-activated acids using electron donors with low redox potentials. We report here that AOR from Aromatoleum aromaticum (AOR Aa ) catalyzes the reduction of organic acids not only with low-potential Eu(II) or Ti(III) complexes but also with H2 as an electron donor. Additionally, AOR Aa catalyzes the H2-dependent reduction of NAD+ or benzyl viologen. The rate of H2-dependent NAD+ reduction equals to 10% of that of aldehyde oxidation, representing the highest H2 turnover rate observed among the Mo/W enzymes. As AOR Aa simultaneously catalyzes the reduction of acids and NAD+, we designed a cascade reaction utilizing a NAD(P)H-dependent alcohol dehydrogenase to reduce organic acids to the corresponding alcohols with H2 as the only reductant. The newly discovered W-hydrogenase side activity of AOR Aa may find applications in either NADH recycling or conversion of carboxylic acids to more useful biochemicals.

Finis tolueni: a new type of thiolase with an integrated Zn-finger subunit catalyzes the final step of anaerobic toluene metabolism.

Anaerobic toluene degradation involves ß-oxidation of the first intermediate (R)-2-benzylsuccinate to succinyl-CoA and benzoyl-CoA. Here, we characterize the last enzyme of this pathway, (S)-2-benzoylsuccinyl-CoA thiolase (BbsAB). Although benzoylsuccinyl-CoA is not available for enzyme assays, the recombinantly produced enzymes from two different species showed the reverse activity, benzoylsuccinyl-CoA formation from benzoyl-CoA and succinyl-CoA. Activity depended on the presence of both subunits, the thiolase family member BbsB and the Zn-finger protein BbsA, which is affiliated to the DUF35 family of unknown function. We determined the structure of BbsAB from Geobacter metallireducens with and without bound CoA at 1.7 and 2.0 Å resolution, respectively. CoA binding into the well-known thiolase cavity triggers an induced-fit movement of the highly disordered covering loop, resulting in its rigidification by forming multiple interactions to the outstretched CoA moiety. This event is coupled with an 8 Å movement of an adjacent hairpin loop of BbsB and the C-terminal domain of BbsA. Thereby, CoA is placed into a catalytically productive conformation, and a putative second CoA binding site involving BbsA and the partner BbsB' subunit is simultaneously formed that also reaches the active center. Therefore, while maintaining the standard thioester-dependent Claisen-type mechanism, BbsAB represents a new type of thiolase.

Characterisation of the redox centers of ethylbenzene dehydrogenase.

Ethylbenzene dehydrogenase (EbDH), the initial enzyme of anaerobic ethylbenzene degradation from the beta-proteobacterium Aromatoleum aromaticum, is a soluble periplasmic molybdenum enzyme consisting of three subunits. It contains a Mo-bis-molybdopterin guanine dinucleotide (Mo-bis-MGD) cofactor and an 4Fe-4S cluster (FS0) in the α-subunit, three 4Fe-4S clusters (FS1 to FS3) and a 3Fe-4S cluster (FS4) in the ß-subunit and a heme b cofactor in the γ-subunit. Ethylbenzene is hydroxylated by a water molecule in an oxygen-independent manner at the Mo-bis-MGD cofactor, which is reduced from the MoVI to the MoIV state in two subsequent one-electron steps. The electrons are then transferred via the Fe-S clusters to the heme b cofactor. In this report, we determine the midpoint redox potentials of the Mo-bis-MGD cofactor and FS1-FS4 by EPR spectroscopy, and that of the heme b cofactor by electrochemically induced redox difference spectroscopy. We obtained relatively high values of > 250 mV both for the MoVI-MoV redox couple and the heme b cofactor, whereas FS2 is only reduced at a very low redox potential, causing magnetic coupling with the neighboring FS1 and FS3. We compare the results with the data on related enzymes and interpret their significance for the function of EbDH.

Inactive pseudoenzyme subunits in heterotetrameric BbsCD, a novel short-chain alcohol dehydrogenase involved in anaerobic toluene degradation.

Anaerobic toluene degradation proceeds by fumarate addition to produce (R)-benzylsuccinate as first intermediate, which is further degraded via ß-oxidation by five enzymes encoded in the conserved bbs operon. This study characterizes two enzymes of this pathway, (E)-benzylidenesuccinyl-CoA hydratase (BbsH), and (S,R)-2-(α-hydroxybenzyl)succinyl-CoA dehydrogenase (BbsCD) from Thauera aromatica. BbsH, a member of the enoyl-CoA hydratase family, converts (E)-benzylidenesuccinyl-CoA to 2-(α-hydroxybenzyl)succinyl-CoA and was subsequently used in a coupled enzyme assay with BbsCD, which belongs to the short-chain dehydrogenases/reductase (SDR) family. The BbsCD crystal structure shows a C2-symmetric heterotetramer consisting of BbsC2 and BbsD2 dimers. BbsD subunits are catalytically active and capable of binding NAD+ and substrate, whereas BbsC subunits represent built-in pseudoenzyme moieties lacking all motifs of the SDR family required for substrate binding or catalysis. Molecular modeling studies predict that the active site of BbsD is specific for conversion of the (S,R)-diastereomer of 2-(α-hydroxybenzyl)succinyl-CoA to (S)-2-benzoylsuccinyl-CoA by hydride transfer to the re-face of nicotinamide adenine dinucleotide (NAD)+ . Furthermore, BbsC subunits are not engaged in substrate binding and merely serve as scaffold for the BbsD dimer. BbsCD represents a novel clade of related enzymes within the SDR family, which adopt a heterotetrameric architecture and catalyze the ß-oxidation of aromatic succinate adducts.

Benzylmalonyl-CoA dehydrogenase, an enzyme involved in bacterial auxin degradation.

A novel acyl-CoA dehydrogenase involved in degradation of the auxin indoleacetate by Aromatoleum aromaticum was identified as a decarboxylating benzylmalonyl-CoA dehydrogenase (IaaF). It is encoded within the iaa operon coding for enzymes of indoleacetate catabolism. Using enzymatically produced benzylmalonyl-CoA, the reaction was characterized as simultaneous oxidation and decarboxylation of benzylmalonyl-CoA to cinnamoyl-CoA and CO2. Oxygen served as electron acceptor and was reduced to H2O2, whereas electron transfer flavoprotein or artificial dyes serving as electron acceptors for other acyl-CoA dehydrogenases were not used. The enzyme is homotetrameric, contains an FAD cofactor and is enantiospecific in benzylmalonyl-CoA turnover. It shows high catalytic efficiency and strong substrate inhibition with benzylmalonyl-CoA, but otherwise accepts only a few medium-chain alkylmalonyl-CoA compounds as alternative substrates with low activities. Its reactivity of oxidizing 2-carboxyacyl-CoA with simultaneous decarboxylation is unprecedented and indicates a modified reaction mechanism for acyl-CoA dehydrogenases, where elimination of the 2-carboxy group replaces proton abstraction from C2.

Structural and Functional Characterization of an Electron Transfer Flavoprotein Involved in Toluene Degradation in Strictly Anaerobic Bacteria.

(R)-Benzylsuccinate is the characteristic initial intermediate of anaerobic toluene metabolism, which is formed by a radical-type addition of toluene to fumarate. Its further degradation proceeds by activation to the coenzyme A (CoA)-thioester and ß-oxidation involving a specific (R)-2-benzylsuccinyl-CoA dehydrogenase (BbsG) affiliated with the family of acyl-CoA dehydrogenases. In this report, we present the biochemical properties of electron transfer flavoproteins (ETFs) from the strictly anaerobic toluene-degrading species Geobacter metallireducens and Desulfobacula toluolica and the facultatively anaerobic bacterium Aromatoleum aromaticum We determined the X-ray structure of the ETF paralogue involved in toluene metabolism of G. metallireducens, revealing strong overall similarities to previously characterized ETF variants but significantly different structural properties in the hinge regions mediating conformational changes. We also show that all strictly anaerobic toluene degraders utilize one of multiple genome-encoded related ETF paralogues, which constitute a distinct clade of similar sequences in the ETF family, for ß-oxidation of benzylsuccinate. In contrast, facultatively anaerobic toluene degraders contain only one ETF species, which is utilized in all ß-oxidation pathways. Our phylogenetic analysis of the known sequences of the ETF family suggests that at least 36 different clades can be differentiated, which are defined either by the taxonomic group of the respective host species (e.g., clade P for Proteobacteria) or by functional specialization (e.g., clade T for anaerobic toluene degradation).IMPORTANCE This study documents the involvement of ETF in anaerobic toluene metabolism as the physiological electron acceptor for benzylsuccinyl-CoA dehydrogenase. While toluene-degrading denitrifying proteobacteria use a common ETF species, which is also used for other ß-oxidation pathways, obligately anaerobic sulfate- or ferric-iron-reducing bacteria use specialized ETF paralogues for toluene degradation. Based on the structure and sequence conservation of these ETFs, they form a new clade that is only remotely related to the previously characterized members of the ETF family. An exhaustive analysis of the available sequences indicated that the protein family consists of several closely related clades of proven or potential electron-bifurcating ETF species and many deeply branching nonbifurcating clades, which either follow the host phylogeny or are affiliated according to functional criteria.

Two Different Quinohemoprotein Amine Dehydrogenases Initiate Anaerobic Degradation of Aromatic Amines in Aromatoleum aromaticum EbN1.

Aromatic amines like 2-phenylethylamine (2-PEA) and benzylamine (BAm) have been identified as novel growth substrates of the betaproteobacterium Aromatoleum aromaticum EbN1, which degrades a wide variety of aromatic compounds in the absence of oxygen under denitrifying growth conditions. The catabolic pathway of these amines was identified, starting with their oxidative deamination to the corresponding aldehydes, which are then further degraded via the enzymes of the phenylalanine or benzyl alcohol metabolic pathways. Two different periplasmic quinohemoprotein amine dehydrogenases involved in 2-PEA or BAm metabolism were identified and characterized. Both enzymes consist of three subunits, contain two heme c cofactors in their α-subunits, and exhibit extensive processing of their γ-subunits, generating four intramolecular thioether bonds and a cysteine tryptophylquinone (CTQ) cofactor. One of the enzymes was present in cells grown with 2-PEA or other substrates, showed an α2ß2γ2 composition, and had a rather broad substrate spectrum, which included 2-PEA, BAm, tyramine, and 1-butylamine. In contrast, the other enzyme was specifically induced in BAm-grown cells, showing an αßγ composition and activity only with BAm and 2-PEA. Since the former enzyme showed the highest catalytic efficiency with 2-PEA and the latter with BAm, they were designated 2-PEADH and benzylamine dehydrogenase (BAmDH). The catalytic properties and inhibition patterns of 2-PEADH and BAmDH showed considerable differences and were compared to previously characterized quinohemoproteins of the same enzyme family.IMPORTANCE The known substrate spectrum of A. aromaticum EbN1 is expanded toward aromatic amines, which are metabolized as sole substrates coupled to denitrification. The characterization of the two quinohemoprotein isoenzymes involved in degrading either 2-PEA or BAm expands the knowledge of this enzyme family and establishes for the first time that the necessary maturation of their quinoid CTQ cofactors does not require the presence of molecular oxygen. Moreover, the study revealed a highly interesting regulatory phenomenon, suggesting that growth with BAm leads to a complete replacement of 2-PEADH by BAmDH, which has considerably different catalytic and inhibition properties.

Characterization of an Aldehyde Oxidoreductase From the Mesophilic Bacterium Aromatoleum aromaticum EbN1, a Member of a New Subfamily of Tungsten-Containing Enzymes.

The biochemical properties of a new tungsten-containing aldehyde oxidoreductase from the mesophilic betaproteobacterium Aromatoleum aromaticum EbN1 (AOR Aa ) are presented in this study. The enzyme was purified from phenylalanine-grown cells of an overexpressing mutant lacking the gene for an aldehyde dehydrogenase normally involved in anaerobic phenylalanine degradation. AOR Aa catalyzes the oxidation of a broad variety of aldehydes to the respective acids with either viologen dyes or NAD+ as electron acceptors. In contrast to previously known AORs, AOR Aa is a heterohexameric protein consisting of three different subunits, a large subunit containing the W-cofactor and an Fe-S cluster, a small subunit containing four Fe-S clusters, and a medium subunit containing an FAD cofactor. The presence of the expected cofactors have been confirmed by elemental analysis and spectrophotometric methods. AOR Aa has a pH optimum of 8.0, a temperature optimum of 40°C and is completely inactive at 50°C. Compared to archaeal AORs, AOR Aa is remarkably resistant against exposure to air, exhibiting a half-life time of 1 h as purified enzyme and being completely unaffected in cell extracts. Kinetic parameters of AOR Aa have been obtained for the oxidation of one aliphatic and two aromatic aldehydes, resulting in about twofold higher k cat values with benzyl viologen than with NAD+ as electron acceptor. Finally, we obtained evidence that AOR Aa is also catalyzing the reverse reaction, reduction of benzoate to benzaldehyde, albeit at very low rates and under conditions strongly favoring acid reduction, e.g., low pH and using Ti(III) citrate as electron donor of very low redox potential. AOR Aa appears to be a prototype of a new subfamily of bacterial AOR-like tungsten-enzymes, which differ from the previously known archaeal AORs mostly by their multi-subunit composition, their low sensitivity against oxygen, and the ability to use NAD+ as electron acceptor.

Illuminating the catalytic core of ectoine synthase through structural and biochemical analysis.

Ectoine synthase (EctC) is the signature enzyme for the production of ectoine, a compatible solute and chemical chaperone widely synthesized by bacteria as a cellular defense against the detrimental effects of osmotic stress. EctC catalyzes the last step in ectoine synthesis through cyclo-condensation of the EctA-formed substrate N-gamma-acetyl-L-2,4-diaminobutyric acid via a water elimination reaction. We have biochemically and structurally characterized the EctC enzyme from the thermo-tolerant bacterium Paenibacillus lautus (Pl). EctC is a member of the cupin superfamily and forms dimers, both in solution and in crystals. We obtained high-resolution crystal structures of the (Pl)EctC protein in forms that contain (i) the catalytically important iron, (ii) iron and the substrate N-gamma-acetyl-L-2,4-diaminobutyric acid, and (iii) iron and the enzyme reaction product ectoine. These crystal structures lay the framework for a proposal for the EctC-mediated water-elimination reaction mechanism. Residues involved in coordinating the metal, the substrate, or the product within the active site of ectoine synthase are highly conserved among a large group of EctC-type proteins. Collectively, the biochemical, mutational, and structural data reported here yielded detailed insight into the structure-function relationship of the (Pl)EctC enzyme and are relevant for a deeper understanding of the ectoine synthase family as a whole.

Type IV CRISPR RNA processing and effector complex formation in Aromatoleum aromaticum.

Type IV CRISPR-Cas modules belong to class 1 prokaryotic adaptive immune systems, which are defined by the presence of multisubunit effector complexes. They usually lack the known Cas proteins involved in adaptation and target cleavage, and their function has not been experimentally addressed. To investigate RNA and protein components of this CRISPR-Cas type, we located a complete type IV cas gene locus and an adjacent CRISPR array on a megaplasmid of Aromatoleum aromaticum EbN1, which contains an additional type I-C system on its chromosome. RNA sequencing analyses verified CRISPR RNA (crRNA) production and maturation for both systems. Type IV crRNAs were shown to harbour unusually short 7 nucleotide 5'-repeat tags and stable 3' hairpin structures. A unique Cas6 variant (Csf5) was identified that generates crRNAs that are specifically incorporated into type IV CRISPR-ribonucleoprotein (crRNP) complexes. Structures of RNA-bound Csf5 were obtained. Recombinant production and purification of the type IV Cas proteins, together with electron microscopy, revealed that Csf2 acts as a helical backbone for type IV crRNPs that include Csf5, Csf3 and a large subunit (Csf1). Mass spectrometry analyses identified protein-protein and protein-RNA contact sites. These results highlight evolutionary connections between type IV and type I CRISPR-Cas systems and demonstrate that type IV CRISPR-Cas systems employ crRNA-guided effector complexes.

Compatible Solute Synthesis and Import by the Moderate Halophile Spiribacter salinus: Physiology and Genomics.

Members of the genus Spiribacter are found worldwide and are abundant in ecosystems possessing intermediate salinities between seawater and saturated salt concentrations. Spiribacter salinus M19-40 is the type species of this genus and its first cultivated representative. In the habitats of S. salinus M19-40, high salinity is a key determinant for growth and we therefore focused on the cellular adjustment strategy to this persistent environmental challenge. We coupled these experimental studies to the in silico mining of the genome sequence of this moderate halophile with respect to systems allowing this bacterium to control its potassium and sodium pools, and its ability to import and synthesize compatible solutes. S. salinus M19-40 produces enhanced levels of the compatible solute ectoine, both under optimal and growth-challenging salt concentrations, but the genes encoding the corresponding biosynthetic enzymes are not organized in a canonical ectABC operon. Instead, they are scrambled (ectAC; ectB) and are physically separated from each other on the S. salinus M19-40 genome. Genomes of many phylogenetically related bacteria also exhibit a non-canonical organization of the ect genes. S. salinus M19-40 also synthesizes trehalose, but this compatible solute seems to make only a minor contribution to the cytoplasmic solute pool under osmotic stress conditions. However, its cellular levels increase substantially in stationary phase cells grown under optimal salt concentrations. In silico genome mining revealed that S. salinus M19-40 possesses different types of uptake systems for compatible solutes. Among the set of compatible solutes tested in an osmostress protection growth assay, glycine betaine and arsenobetaine were the most effective. Transport studies with radiolabeled glycine betaine showed that S. salinus M19-40 increases the pool size of this osmolyte in a fashion that is sensitively tied to the prevalent salinity of the growth medium. It was amassed in salt-stressed cells in unmodified form and suppressed the synthesis of ectoine. In conclusion, the data presented here allow us to derive a genome-scale picture of the cellular adjustment strategy of a species that represents an environmentally abundant group of ecophysiologically important halophilic microorganisms.