Root-secreted nucleosides: signaling chemoattractants of rhizosphere bacteria.

The rhizosphere is a complex ecosystem, consisting of a narrow soil zone influenced by plant roots and inhabited by soil-borne microorganisms. Plants actively shape the rhizosphere microbiome through root exudates. Some metabolites are signaling molecules specifically functioning as chemoattractants rather than nutrients. These elusive signaling molecules have been sought for several decades, and yet little progress has been made. Root-secreted nucleosides and deoxynucleosides were detected in exudates of various plants by targeted ultra-performance liquid chromatography-mass spectrometry/mass spectrometry. Rhizobacteria were isolated from the roots of Helianthemum sessiliflorum carrying the mycorrhizal desert truffle Terfezia boudieri. Chemotaxis was determined by a glass capillary assay or plate assays on semisolid agar and through a soil plate assay. Nucleosides were identified in root exudates of plants that inhabit diverse ecological niches. Nucleosides induced positive chemotaxis in plant beneficial bacteria Bacillus pumilus, Bacillus subtilis, Pseudomonas turukhanskensis spp., Serratia marcescens, and the pathogenic rhizobacterium Xanthomonas campestris and E coli. In a soil plate assay, nucleosides diffused to substantial distances and evoked chemotaxis under conditions as close as possible to natural environments. This study implies that root-secreted nucleosides are involved in the assembly of the rhizosphere bacterial community by inducing chemotaxis toward plant roots. In animals, nucleoside secretion known as "purinergic signaling" is involved in communication between cells, physiological processes, diseases, phagocytic cell migration, and bacterial activity. The coliform bacterium E. coli that inhabits the lower intestine of warm-blooded organisms also attracted to nucleosides, implying that nucleosides may serve as a common signal for bacterial species inhabiting distinct habitats. Taken together, all these may indicate that chemotaxis signaling by nucleosides is a conserved universal mechanism that encompasses living kingdoms and environments and should be given further attention in plant rhizosphere microbiome research.

Blocking an epitope of misfolded SOD1 ameliorates disease phenotype in a model of amyotrophic lateral sclerosis.

The current strategies to mitigate the toxicity of misfolded superoxide dismutase 1 (SOD1) in familial amyotrophic lateral sclerosis via blocking SOD1 expression in the CNS are indiscriminative for misfolded and intact proteins, and as such, entail a risk of depriving CNS cells of their essential antioxidant potential. As an alternative approach to neutralize misfolded and spare unaffected SOD1 species, we developed scFv-SE21 antibody that blocks the ß6/ß7 loop epitope exposed exclusively in misfolded SOD1. The ß6/ß7 loop epitope has previously been proposed to initiate amyloid-like aggregation of misfolded SOD1 and mediate its prion-like activity. The adeno-associated virus-mediated expression of scFv-SE21 in the CNS of hSOD1G37R mice rescued spinal motor neurons, reduced the accumulation of misfolded SOD1, decreased gliosis and thus delayed disease onset and extended survival by 90 days. The results provide evidence for the role of the exposed ß6/ß7 loop epitope in the mechanism of neurotoxic gain-of-function of misfolded SOD1 and open avenues for the development of mechanism-based anti-SOD1 therapeutics, whose selective targeting of misfolded SOD1 species may entail a reduced risk of collateral oxidative damage to the CNS.

The Microbiome Structure of the Symbiosis between the Desert Truffle Terfezia boudieri and Its Host Plant Helianthemum sessiliflorum.

The desert truffle Terfezia boudieri is an ascomycete fungus that forms ect-endomycorrhiza in the roots of plants belonging to Cistaceae. The fungus forms hypogeous edible fruit bodies, appreciated as gourmet food. Truffles and host plants are colonized by various microbes, which may contribute to their development. However, the diversity and composition of the bacterial community under field conditions in the Negev desert are still unknown. The overall goal of this research was to identify the rhizosphere microbial community supporting the establishment of a symbiotic association between T. boudieri and Helianthemum sessiliflorum. The bacterial community was characterized by fruiting bodies, mycorrhized roots, and rhizosphere soil. Based on next-generation sequencing meta-analyses of the 16S rRNA gene, we discovered diverse bacterial communities of fruit bodies that differed from those found in the roots and rhizosphere. Families of Proteobacteria, Planctomycetes, and Actinobacteria were present in all four samples. Alpha diversity analysis revealed that the rhizosphere and roots contain significantly higher bacterial species numbers compared to the fruit. Additionally, ANOSIM and PCoA provided a comparative analysis of the bacterial taxa associated with fruiting bodies, roots, and rhizosphere. The core microbiome described consists of groups whose biological role triggers important traits supporting plant growth and fruit body development.

Exposure of ß6/ß7-Loop in Zn/Cu Superoxide Dismutase (SOD1) Is Coupled to Metal Loss and Is Transiently Reversible During Misfolding.

Upon losing its structural integrity (misfolding), SOD1 acquires neurotoxic properties to become a pathogenic protein in ALS, a neurodegenerative disease targeting motor neurons; understanding the mechanism of misfolding may enable new treatment strategies for ALS. Here, we reported a monoclonal antibody, SE21, targeting the ß6/ß7-loop region of SOD1. The exposure of this region is coupled to metal loss and is entirely reversible during the early stages of misfolding. By using SE21 mAb, we demonstrated that, in apo-SOD1 incubated under the misfolding-promoting conditions, the reversible phase, during which SOD1 is capable of restoring its nativelike conformation in the presence of metals, is followed by an irreversible structural transition, autocatalytic in nature, which takes place prior to the onset of SOD1 aggregation and results in the formation of atypical apo-SOD1 that is unable to bind metals. The reversible phase defines a window of opportunity for pharmacological intervention using metal mimetics that stabilize SOD1 structure in its nativelike conformation to attenuate the spreading of the misfolding signal and disease progression by preventing the exposure of pathogenic SOD1 epitopes. Phenotypically similar apo-SOD1 species with impaired metal binding properties may also be produced via oxidation of Cys111, underscoring the diversity of SOD1 misfolding pathways.

Agrobacterium tumefaciens-Mediated Genetic Transformation of the Ect-endomycorrhizal Fungus Terfezia boudieri.

Mycorrhizal desert truffles such as Terfezia boudieri, Tirmania nivea, and Terfezia claveryi, form mycorrhizal associations with plants of the Cistaceae family. These valued truffles are still collected from the wild and not cultivated under intensive farming due to the lack of basic knowledge about their biology at all levels. Recently, several genomes of desert truffles have been decoded, enabling researchers to attempt genetic manipulations to enable cultivation. To execute such manipulations, the development of molecular tools for genes transformation into truffles is needed. We developed an Agrobacterium tumefaciens-mediated genetic transformation system in T. boudieri. This system was optimized for the developmental stage of the mycelia explants, bacterial optical density, infection and co-cultivation durations, and concentrations of the selection antibiotics. The pFPL-Rh plasmid harboring hph gene conferring hygromycin resistance as a selection marker and the red fluorescent protein gene were used as visual reporters. The optimal conditions were incubation with 200 µM of acetosyringone, attaining a bacterial optical density of 0.3 OD600; transfer time of 45 min; and co-cultivation for 3 days. This is the first report on a transformation system for T. boudieri, and the proposed protocol can be adapted for the transformation of other important desert truffles as well as ectomycorrhizal species.

Primate differential redoxome (PDR) - A paradigm for understanding neurodegenerative diseases.

Despite different phenotypic manifestations, mounting evidence points to similarities in the molecular basis of major neurodegenerative diseases (ND). CNS has evolved to be robust against hazard of ROS, a common perturbation aerobic organisms are confronted with. The trade-off of robustness is system's fragility against rare and unexpected perturbations. Identifying the points of CNS fragility is key for understanding etiology of ND. We postulated that the 'primate differential redoxome' (PDR), an assembly of proteins that contain cysteine residues present only in the primate orthologues of mammals, is likely to associate with an added level of regulatory functionalities that enhanced CNS robustness against ROS and facilitated evolution. The PDR contains multiple deterministic and susceptibility factors of major ND, which cluster to form coordinated redox networks regulating various cellular processes. The PDR analysis revealed a potential CNS fragility point, which appears to associates with a non-redundant PINK1-PRKN-SQSTM1(p62) axis coordinating protein homeostasis and mitophagy.

Cu/Zn-superoxide dismutase and wild-type like fALS SOD1 mutants produce cytotoxic quantities of H2O2 via cysteine-dependent redox short-circuit.

The Cu/Zn-superoxide dismutase (SOD1) is a ubiquitous enzyme that catalyzes the dismutation of superoxide radicals to oxygen and hydrogen peroxide. In addition to this principal reaction, the enzyme is known to catalyze, with various efficiencies, several redox side-reactions using alternative substrates, including biological thiols, all involving the catalytic copper in the enzyme's active-site, which is relatively surface exposed. The accessibility and reactivity of the catalytic copper is known to increase upon SOD1 misfolding, structural alterations caused by a mutation or environmental stresses. These competing side-reactions can lead to the formation of particularly toxic ROS, which have been proposed to contribute to oxidative damage in amyotrophic lateral sclerosis (ALS), a neurodegenerative disease that affects motor neurons. Here, we demonstrated that metal-saturated SOD1WT (holo-SOD1WT) and a familial ALS (fALS) catalytically active SOD1 mutant, SOD1G93A, are capable, under defined metabolic circumstances, to generate cytotoxic quantities of H2O2 through cysteine (CSH)/glutathione (GSH) redox short-circuit. Such activity may drain GSH stores, therefore discharging cellular antioxidant potential. By analyzing the distribution of thiol compounds throughout the CNS, the location of potential hot-spots of ROS production can be deduced. These hot-spots may constitute the origin of oxidative damage to neurons in ALS.

Characterization of Morphology, Volatile Profiles, and Molecular Markers in Edible Desert Truffles from the Negev Desert.

Desert truffles are mycorrhizal, hypogeous fungi considered a delicacy. On the basis of morphological characters, we identified three desert truffle species that grow in the same habitat in the Negev desert. These include Picoa lefebvrei (Pat.), Tirmania nivea (Desf.) Trappe, and Terfezia boudieri (Chatain), all associated with Helianthemum sessiliflorum. Their taxonomy was confirmed by PCR-RFLP. The main volatiles of fruit bodies of T. boudieri and T. nivea were 1-octen-3-ol and hexanal; however, volatiles of the latter species further included branched-chain amino acid derivatives such as 2-methylbutanal and 3-methylbutanal, phenylalanine derivatives such as benzaldehyde and benzenacetaldehyde, and methionine derivatives such as methional and dimethyl disulfide. The least aromatic truffle, P. lefebvrei, contained low levels of 1-octen-3-ol as the main volatile. Axenic mycelia cultures of T. boudieri displayed a simpler volatile profile compared to its fruit bodies. This work highlights differences in the volatile profiles of desert truffles and could hence be of interest for selecting and cultivating genotypes with the most likable aroma.

Glyoxylate carboligase: a unique thiamin diphosphate-dependent enzyme that can cycle between the 4'-aminopyrimidinium and 1',4'-iminopyrimidine tautomeric forms in the absence of the conserved glutamate.

Glyoxylate carboligase (GCL) is a thiamin diphosphate (ThDP)-dependent enzyme, which catalyzes the decarboxylation of glyoxylate and ligation to a second molecule of glyoxylate to form tartronate semialdehyde (TSA). This enzyme is unique among ThDP enzymes in that it lacks a conserved glutamate near the N1' atom of ThDP (replaced by Val51) or any other potential acid-base side chains near ThDP. The V51D substitution shifts the pH optimum to 6.0-6.2 (pK(a) of 6.2) for TSA formation from pH 7.0-7.7 in wild-type GCL. This pK(a) is similar to the pK(a) of 6.1 for the 1',4'-iminopyrimidine (IP)-4'-aminopyrimidinium (APH(+)) protonic equilibrium, suggesting that the same groups control both ThDP protonation and TSA formation. The key covalent ThDP-bound intermediates were identified on V51D GCL by a combination of steady-state and stopped-flow circular dichroism methods, yielding rate constants for their formation and decomposition. It was demonstrated that active center variants with substitution at I393 could synthesize (S)-acetolactate from pyruvate solely, and acetylglycolate derived from pyruvate as the acetyl donor and glyoxylate as the acceptor, implying that this substitutent favored pyruvate as the donor in carboligase reactions. Consistent with these observations, the I393A GLC variants could stabilize the predecarboxylation intermediate analogues derived from acetylphosphinate, propionylphosphinate, and methyl acetylphosphonate in their IP tautomeric forms notwithstanding the absence of the conserved glutamate. The role of the residue at the position occupied typically by the conserved Glu controls the pH dependence of kinetic parameters, while the entire reaction sequence could be catalyzed by ThDP itself, once the APH(+) form is accessible.

Many of the functional differences between acetohydroxyacid synthase (AHAS) isozyme I and other AHASs are a result of the rapid formation and breakdown of the covalent acetolactate-thiamin diphosphate adduct in AHAS I.

Acetohydroxy acid synthase (AHAS; EC 2.2.1.6) is a thiamin diphosphate (ThDP)-dependent decarboxylase-ligase that catalyzes the first common step in the biosynthesis of branched-chain amino acids. In the first stage of the reaction, pyruvate is decarboxylated and the reactive intermediate hydroxyethyl-ThDP carbanion/enamine is formed. In the second stage, the intermediate is ligated to another 2-ketoacid to form either acetolactate or acetohydroxybutyrate. AHAS isozyme I from Escherichia coli is unique among the AHAS isozymes in that it is not specific for 2-ketobutyrate (2-KB) over pyruvate as an acceptor substrate. It also appears to have a different mechanism for inhibition by valine than does AHAS III from E. coli. An investigation of this enzyme by directed mutagenesis and knowledge of detailed kinetics using the rapid mixing-quench NMR method or stopped-flow spectroscopy, as well as the use of alternative substrates, suggests that two residues determine most of the unique properties of AHAS I. Gln480 and Met476 in AHAS I replace the Trp and Leu residues conserved in other AHASs and lead to accelerated ligation and product release steps. This difference in kinetics accounts for the unique specificity, reversibility and allosteric response of AHAS I. The rate of decarboxylation of the initially formed 2-lactyl-ThDP intermediate is, in some AHAS I mutants, different for the alternative acceptors pyruvate and 2-KB, putting into question whether AHAS operates via a pure ping-pong mechanism. This finding might be compatible with a concerted mechanism (i.e. the formation of a ternary donor-acceptor:enzyme complex followed by covalent, ThDP-promoted catalysis with concerted decarboxylation-carboligation). It might alternatively be explained by an allosteric interaction between the multiple catalytic sites in AHAS.

Allosteric regulation in Acetohydroxyacid Synthases (AHASs)--different structures and kinetic behavior in isozymes in the same organisms.

Acetohydroxyacid Synthases (AHASs) have separate small regulatory subunits which specifically activate the catalytic subunits with which they are associated. The binding sites for the inhibitory amino acid(s) (valine or leucine) are in the interface between two ACT (small ligand binding) domains, and are apparently found in all AHAS regulatory subunits. However, the structures and the kinetic mechanisms of the different enzymes are very heterogeneous. Among the three isozymes encoded in the enterobacteria, the regulatory patterns are different, and their different responses to the inhibitory end product valine can be rationalized, at least in part, on the basis of the regulatory subunit structures and differences in catalytic mechanisms. The regulatory subunits in "typical" single AHASs found in other bacteria are similar to that of Escherichia coli isozyme AHAS III. Eukaryotic AHASs have more complex regulatory mechanisms and larger regulatory subunits. Such AHASs have two separate ACT sequence domains on the same regulatory polypeptide and can simultaneously bind two amino acids with synergistic effects. Yeast and fungal AHASs have ATP-binding sequence inserts in their regulatory subunits and are activated by MgATP in addition to being inhibited by valine.

Significant catalytic roles for Glu47 and Gln 110 in all four of the C-C bond-making and -breaking steps of the reactions of acetohydroxyacid synthase II.

Acetohydroxy acid synthase (AHAS) is a thiamin diphosphate (ThDP)-dependent enzyme that catalyzes the first common step in the biosynthesis of branched-chain amino acids, condensation of pyruvate with a second 2-ketoacid to form either acetolactate or acetohydroxybutyrate. AHAS isozyme II from Escherichia coli is specific for pyruvate as the first donor substrate but exhibits a 60-fold higher specificity for 2-ketobutyrate (2-KB) over pyruvate as an acceptor substrate. In previous studies relying on steady state and transient kinetics, substrate competition and detailed analysis of the distribution of intermediates in the steady-state, we have identified several residues which confer specificity for the donor and acceptor substrates, respectively. Here, we examine the roles of active site polar residues Glu47, Gln110, Lys159, and His251 for elementary steps of catalysis using similar approaches. While Glu47, the conserved essential glutamate conserved in all ThDP-dependent enzymes whose carboxylate is in H-bonding distance of the ThDP iminopyrimidine N1', is involved as expected in cofactor activation, substrate binding, and product elimination, our studies further suggest a crucial catalytic role for it in the carboligation of the acceptor and the hydroxyethyl-ThDP enamine intermediate. The Glu47-cofactor proton shuttle acts in concert with Gln110 in the carboligation. We suggest that either the transient oxyanion on the acceptor carbonyl is stabilized by H-bonding to the glutamine side chain, or carboligation involves glutamine tautomerization and the elementary reactions of addition and protonation occur in a concerted manner. This is in contrast to the situation in other ThDP enzymes that catalyze a carboligation, such as, e.g., transketolase or benzaldehyde lyase, where histidines act as general acid/base catalysts. Our studies further suggest global catalytic roles for Gln110 and Glu47, which are engaged in all major bond-breaking and bond-making steps. In contrast to earlier suggestions, Lys159 has a minor effect on the kinetics and specificity of AHAS II, far less than does Arg276, previously shown to influence the specificity for a 2-ketoacid as a second substrate. His251 has a large effect on donor substrate binding, but this effect masks any other effects of replacement of His251.

Role of the C-terminal domain of the regulatory subunit of AHAS isozyme III: use of random mutagenesis with in vivo reconstitution (REM-ivrs).

In order to clarify the role of the C-terminal domain of the ilvH protein (the regulatory subunit of enterobacterial AHAS isozyme III, whose structure has been solved and reported by Kaplun et al., J Mol Biol 357, 951, 2006) in the process of valine inhibition of AHAS III, we developed a procedure that randomly mutagenizes a specific segment of a gene through error-prone PCR and screens for mutants on the basis of the properties of the holoenzymes reconstituted in vivo (REM-ivrs). Previous work showed that the N-terminal domain includes the valine-binding ACT domain of the regulatory subunit and is sufficient to completely activate the catalytic subunit, but that this domain cannot confer valine sensitivity on the reconstituted enzyme. It appeared that the C-terminal domain of the ilvH is involved in some way in "signal transmission" of the inhibition by valine. As knowledge of the structure of AHAS holoenzymes and the interactions between the catalytic and regulatory subunits is very limited, a procedure that focuses on the C-terminal domain in the ilvH gene could add to the understanding of the mechanism by which the binding of valine to the regulatory subunit is coupled to inhibition of the catalytic activity. In the REM-ivrs procedure, a medium copy (~40 copies) plasmid expressing ilvH with a Val(r) mutation confers the Val(r) phenotype upon bacteria. All the single missense mutations produced by REM-ivrs were found to be localized to the interface between the C-terminal domains of two monomers in the ilvH dimer. The loss of specific contacts involved in inter-monomer interactions in this region might conceivably disrupt the structure of the C-terminal domain itself. Biochemical study of an isolated Val(r) mutant elicited by the REM-ivrs method detected no binding of radioactively labeled valine, as previously found in a truncation mutant. The idea that the C-terminal domain has a specific "signal-transmission" role was also contradicted by examination of the thermal stability of the Val(r) REM-ivrs variants by the Thermofluor method, which does not detect any signs of biphasic melting behavior for any of the mutants. We propose that the mutants of ilvH isolated by the REM-ivrs method differ from the wild-type in the equilibrium between two states of the enzyme. Without the specific interdomain contacts of the wild-type ilvH protein, the holoenzyme reconstituted from mutant regulatory subunits is apparently in a state with uninhibited activity and low affinity for valine.

Interactions between large and small subunits of different acetohydroxyacid synthase isozymes of Escherichia coli.

The large, catalytic subunits (LSUs; ilvB, ilvG and ilvI, respectively) of enterobacterial acetohydroxyacid synthases isozymes (AHAS I, II and III) have molecular weights approximately 60 kDa and are paralogous with a family of other thiamin diphosphate dependent enzymes. The small, regulatory subunits (SSUs) of AHAS I and AHAS III (ilvN and ilvH) are required for valine inhibition, but ilvN and ilvH can only confer valine sensitivity on their own LSUs. AHAS II is valine resistant. The LSUs have only approximately 15, <<1 and approximately 3%, respectively, of the activity of their respective holoenzymes, but the holoenzymes can be reconstituted with complete recovery of activity. We have examined the activation of each of the LSUs by SSUs from different isozymes and ask to what extent such activation is specific; that is, is effective nonspecific interaction possible between LSUs and SSUs of different isozymes? To our surprise, the AHAS II SSU ilvM is able to activate the LSUs of all three of the isozymes, and the truncated AHAS III SSUs ilvH-Delta80, ilvH-Delta86 and ilvH-Delta89 are able to activate the LSUs of both AHAS I and AHAS III. However, none of the heterologously activated enzymes have any feedback sensitivity. Our results imply the existence of a common region in all three LSUs to which regulatory subunits may bind, as well as a similarity between the surfaces of ilvM and the other SSUs. This surface must be included within the N-terminal betaalphabetabetaalphabeta-domain of the SSUs, probably on the helical face of this domain. We suggest hypotheses for the mechanism of valine inhibition, and reject one involving induced dissociation of subunits.

Allosteric regulation of Bacillus subtilis threonine deaminase, a biosynthetic threonine deaminase with a single regulatory domain.

The enzyme threonine deaminase (TD) is a key regulatory enzyme in the pathway for the biosynthesis of isoleucine. TD is inhibited by its end product, isoleucine, and this effect is countered by valine, the product of a competing biosynthetic pathway. Sequence and structure analyses have revealed that the protomers of many TDs have C-terminal regulatory domains, composed of two ACT-like subdomains, which bind isoleucine and valine, while others have regulatory domains of approximately half the length, composed of only a single ACT-like domain. The regulatory responses of TDs from both long and short sequence varieties appear to have many similarities, but there are significant differences. We describe here the allosteric properties of Bacillus subtilis TD ( bsTD), which belongs to the short variety of TD sequences. We also examine the effects of several mutations in the regulatory domain on the kinetics of the enzyme and its response to effectors. The behavior of bsTD can be analyzed and rationalized using a modified Monod-Wyman-Changeux model. This analysis suggests that isoleucine is a negative effector, and valine is a very weak positive effector, but that at high concentrations valine inhibits activity by competing with threonine for binding to the active site. The behavior of bsTD is contrasted with the allosteric behavior reported for TDs from Escherichia coli and Arabidopsis thaliana, TDs with two subdomains. We suggest a possible evolutionary pathway to the more complex regulatory effects of valine on the activity of TDs of the long sequence variety, e.g., E. coli TD.

A new Thraustochytrid, strain Fng1, isolated from the surface mucus of the hermatypic coral Fungia granulosa.

Recent evidence suggests that there is a dynamic microbial biota living on the surface and in the mucus layer of many hermatypic coral species that plays an essential role in coral well-being. Most of the studies published to date emphasize the importance of prokaryotic communities associated with the coral mucus in coral health and disease. In this study, we report the presence of a protist (Fng1) in the mucus of the hermatypic coral Fungia granulosa from the Gulf of Eilat. This protist was identified morphologically and molecularly as belonging to the family Thraustochytridae (phylum Stramenopile, order Labyrinthulida), a group of heterotrophs widely distributed in the marine environment. Morphological examination of this strain revealed a nonmotile organism c. 35 mum in diameter, which is able to thrive on carbon-deprived media, and whose growth and morphology are inoculum dependent. Its fatty acid production profile revealed an array of polyunsaturated fatty acids. A similar protist was also isolated from the mucus of the coral Favia sp. In light of these findings, its possible contribution to the coral holobiont is discussed.

Glyoxylate carboligase lacks the canonical active site glutamate of thiamine-dependent enzymes.

Thiamine diphosphate (ThDP), a derivative of vitamin B1, is an enzymatic cofactor whose special chemical properties allow it to play critical mechanistic roles in a number of essential metabolic enzymes. It has been assumed that all ThDP-dependent enzymes exploit a polar interaction between a strictly conserved glutamate and the N1' of the ThDP moiety. The crystal structure of glyoxylate carboligase challenges this paradigm by revealing that valine replaces the conserved glutamate. Through kinetic, spectroscopic and site-directed mutagenesis studies, we show that although this extreme change lowers the rate of the initial step of the enzymatic reaction, it ensures efficient progress through subsequent steps. Glyoxylate carboligase thus provides a unique illustration of the fine tuning between catalytic stages imposed during evolution on enzymes catalyzing multistep processes.

Coral mucus-associated bacterial communities from natural and aquarium environments.

The microbial biota dwelling in the mucus, on the surface, and in the tissues of many coral species may have an important role in holobiont physiology and health. This microbiota differs with coral species, water depth, and geographic location. Here we compare the surface mucus microbiota of the coral Fungia granulosa from the natural environment with that from individuals maintained in aquaria. Molecular analysis revealed that the microbial community of the mucus microlayer of the coral F. granulosa includes a wide range of bacteria and that these change with environment. Coral mucus from the natural environment contained a significantly higher diversity of microorganisms than did mucus from corals maintained in the closed-system aquaria. A microbial community shift, with the loss of several groups, including actinobacterial and cyanobacterial groups, was observed in corals maintained in aquaria. The most abundant bacterial class in F. granulosa mucus was the Alphaproteobacteria, regardless of whether the corals were from aquaria or freshly collected from their natural environment. A significantly higher percentage of bacteria from the Betaproteobacteria class was evident in aquarium corals (24%) when compared with corals from the natural environment (3%). The differences in mucus-inhabiting microbial communities between corals from captive and natural environments suggest an adaptation of the mucus bacterial communities to the different conditions.

Structure of the regulatory subunit of acetohydroxyacid synthase isozyme III from Escherichia coli.

The enzyme acetohydroxyacid synthase (AHAS) catalyses the first common step in the biosynthesis of the three branched-chain amino acids. Enzymes in the AHAS family generally consist of regulatory and catalytic subunits. Here, we describe the first crystal structure of an AHAS regulatory subunit, the ilvH polypeptide, determined at a resolution of 1.75 A. IlvH is the regulatory subunit of one of three AHAS isozymes expressed in Escherichia coli, AHAS III. The protein is a dimer, with two beta alpha beta beta alpha beta ferredoxin domains in each monomer. The two N-terminal domains assemble to form an ACT domain structure remarkably close to the one predicted by us on the basis of the regulatory domain of 3-phosphoglycerate dehydrogenase (3PGDH). The two C-terminal domains combine so that their beta-sheets are roughly positioned back-to-back and perpendicular to the extended beta-sheet of the N-terminal ACT domain. On the basis of the properties of mutants and a comparison with 3PGDH, the effector (valine) binding sites can be located tentatively in two symmetrically related positions in the interface between a pair of N-terminal domains. The properties of mutants of the ilvH polypeptide outside the putative effector-binding site provide further insight into the functioning of the holoenzyme. The results of this study open avenues for further studies aimed at understanding the mechanism of regulation of AHAS by small-molecule effectors.

Acetohydroxyacid synthase isozyme I from Escherichia coli has unique catalytic and regulatory properties.

AHAS I is an isozyme of acetohydroxyacid synthase which is apparently unique to enterobacteria. It has been known for over 20 years that it has many properties which are quite different from those of the other two enterobacterial AHASs isozymes, as well as from those of "typical" AHASs which are single enzymes in a given organism. These include a unique mechanism for regulation of expression and the absence of a preference for forming acetohydroxybutyrate. We have cloned the two subunits, ilvB and ilvN, of this Escherichia coli isoenzyme and examined the enzymatic properties of the purified holoenzyme and the enzyme reconstituted from purified subunits. Unlike other AHASs, AHAS I demonstrates cooperative feedback inhibition by valine, and the kinetics fit closely to an exclusive binding model. The formation of acetolactate by AHAS I is readily reversible and acetolactate can act as substrate for alternative AHAS I-catalyzed reactions.