Spontaneous Covalent Self-Assembly of the Azoarcus Ribozyme from Five Fragments.

Spontaneous covalent assembly of short RNA fragments has been proposed as a plausible prebiotically relevant pathway to a self-reproducing system. We previously showed that the Azoarcus group I intron could self-assemble from four RNA fragments. Here, we extended this fragmentation to five RNAs that averaged <40 nucleotides in length. We optimized this reaction and showed that a dehydration-rehydration sequence was the most effective means to date to shift the self-assembly equilibrium from reactants to products.

Elemental sulfur recovery and spatial distribution of functional bacteria and expressed genes under different carbon/nitrate/sulfide loadings in up-flow anaerobic sludge blanket reactors.

To characterize the impact of influent loading on elemental sulfur (S0) recovery during the denitrifying and sulfide oxidation process, three identical, lab-scale UASB reactors (30cm in length) were established in parallel under different influent acetate/nitrate/sulfide loadings, and the reactor performance and functional community structure were investigated. The highest S0 recovery was achieved at 77.9% when the acetate/nitrate/sulfide loading was set to 1.9/1.6/0.7kgd-1m-3. Under this condition, the genera Thauera, Sulfurimonas, and Azoarcus were predominant at 0-30, 0-10 and 20-30cm, respectively; meanwhile, the sqr gene was highly expressed at 0-30cm. However, as the influent loading was halved and doubled, S0 recovery was decreased to 27.9% and 45.1%, respectively. As the loading was halved, the bacterial distribution became heterogeneous, and certain autotrophic sulfide oxidation genera, such as Thiobacillus, dominated, especially at 20-30cm. As the loading doubled, the bacterial distribution was relatively homogeneous with Thauera and Azoarcus being predominant, and the nirK and sox genes were highly expressed. The study verified the importance of influent loading to regulate S0 recovery, which could be achieved as Thauera and Sulfurimonas dominated. An influent loading that was too low or too high gave rise to insufficient oxidation or over-oxidation of the sulfide and low S0 recovery performance.

Enzymes involved in the anaerobic degradation of ortho-phthalate by the nitrate-reducing bacterium Azoarcus sp. strain PA01.

The pathway of anaerobic degradation of o-phthalate was studied in the nitrate-reducing bacterium Azoarcus sp. strain PA01. Differential two-dimensional protein gel profiling allowed the identification of specifically induced proteins in o-phthalate-grown compared to benzoate-grown cells. The genes encoding o-phthalate-induced proteins were found in a 9.9 kb gene cluster in the genome of Azoarcus sp. strain PA01. The o-phthalate-induced gene cluster codes for proteins homologous to a dicarboxylic acid transporter, putative CoA-transferases and a UbiD-like decarboxylase that were assigned to be specifically involved in the initial steps of anaerobic o-phthalate degradation. We propose that o-phthalate is first activated to o-phthalyl-CoA by a putative succinyl-CoA-dependent succinyl-CoA:o-phthalate CoA-transferase, and o-phthalyl-CoA is subsequently decarboxylated to benzoyl-CoA by a putative o-phthalyl-CoA decarboxylase. Results from in vitro enzyme assays with cell-free extracts of o-phthalate-grown cells demonstrated the formation of o-phthalyl-CoA from o-phthalate and succinyl-CoA as CoA donor, and its subsequent decarboxylation to benzoyl-CoA. The putative succinyl-CoA:o-phthalate CoA-transferase showed high substrate specificity for o-phthalate and did not accept isophthalate, terephthalate or 3-fluoro-o-phthalate whereas the putative o-phthalyl-CoA decarboxylase converted fluoro-o-phthalyl-CoA to fluoro-benzoyl-CoA. No decarboxylase activity was observed with isophthalyl-CoA or terephthalyl-CoA. Both enzyme activities were oxygen-insensitive and inducible only after growth with o-phthalate. Further degradation of benzoyl-CoA proceeds analogous to the well-established anaerobic benzoyl-CoA degradation pathway of nitrate-reducing bacteria.

Biosynthesis of selenium nanoparticles by Azoarcus sp. CIB.

BACKGROUND: Different bacteria have been reported so far that link selenite resistance to the production of metallic selenium nanoparticles (SeNPs). Although SeNPs have many biotechnological applications in diverse areas, the molecular mechanisms involved in their microbial genesis are not fully understood. The Azoarcus genus is a physiologically versatile group of beta-proteobacteria of great environmental relevance. Azoarcus sp. CIB is a facultative anaerobe that combines the ability to degrade under aerobic and/or anaerobic conditions a wide range of aromatic compounds, including some toxic hydrocarbons such as toluene and m-xylene, with an endophytic life style in the root of rice. We unravel here an additional physiological feature of the strain CIB that is related to its resistance to selenium oxyanions and the formation of SeNPs. RESULTS: This work is the first report of a member of the Azoarcus genus that is able to anaerobically grow in the presence of selenite. Electron microscopy preparations and X-ray spectroscopy analyses demonstrate the reduction of selenite to spherical electron-dense SeNPs whose average size was 123 ± 35 nm of diameter. Our data suggest that the main molecular mechanism of selenite resistance resides on an energy-dependent selenite exporter. Azoarcus cells trigger the synthesis of SeNPs when they reach the stationary-phase of growth, and either the exhaustion of electron donor or acceptor, both of which lead to starvation conditions, produce the reduction of selenite to red elemental selenium. Azoarcus becomes a promising biocatalyst, either as whole cells or cellular extracts, for the anaerobic and/or aerobic green synthesis of SeNPs. CONCLUSIONS: Azoarcus turns out to be a new eco-friendly system to reduce selenite and produce spherical SeNPs. Moreover, this is the first report of a rice endophyte able to produce SeNPs. Since Azoarcus is also able to degrade both aerobically and anaerobically toxic aromatic compounds of great environmental concern, it becomes a suitable candidate for a more sustainable agricultural practice and for bioremediation strategies.

Identification and biosynthesis of a novel xanthomonadin-dialkylresorcinol-hybrid from Azoarcus sp. BH72.

A novel xanthomonadin-dialkylresorcinol hybrid named arcuflavin was identified in Azoarcus sp. BH72 by a combination of feeding experiments, HPLC-MS and MALDI-MS and gene clusters encoding the biosynthesis of this non-isoprenoid aryl-polyene containing pigment are reported. A chorismate-utilizing enzyme from the XanB2-type producing 3- and 4-hydroxybenzoic acid and an AMP-ligase encoded by these gene clusters were characterized, that might perform the first two steps of the polyene biosynthesis. Furthermore, a detailed analysis of the already known or novel biosynthesis gene clusters involved in the biosynthesis of polyene containing pigments like arcuflavin, flexirubin and xanthomonadin revealed the presence of similar gene clusters in a wide range of bacterial taxa, suggesting that polyene and polyene-dialkylresorcinol pigments are more widespread than previously realized.

Cyclohexane-1,2-dione hydrolase from denitrifying Azoarcus sp. strain 22Lin, a novel member of the thiamine diphosphate enzyme family.

Alicyclic compounds with hydroxyl groups represent common structures in numerous natural compounds, such as terpenes and steroids. Their degradation by microorganisms in the absence of dioxygen may involve a C-C bond ring cleavage to form an aliphatic intermediate that can be further oxidized. The cyclohexane-1,2-dione hydrolase (CDH) (EC 3.7.1.11) from denitrifying Azoarcus sp. strain 22Lin, grown on cyclohexane-1,2-diol as a sole electron donor and carbon source, is the first thiamine diphosphate (ThDP)-dependent enzyme characterized to date that cleaves a cyclic aliphatic compound. The degradation of cyclohexane-1,2-dione (CDO) to 6-oxohexanoate comprises the cleavage of a C-C bond adjacent to a carbonyl group, a typical feature of reactions catalyzed by ThDP-dependent enzymes. In the subsequent NAD(+)-dependent reaction, 6-oxohexanoate is oxidized to adipate. CDH has been purified to homogeneity by the criteria of gel electrophoresis (a single band at ∼59 kDa; calculated molecular mass, 64.5 kDa); in solution, the enzyme is a homodimer (∼105 kDa; gel filtration). As isolated, CDH contains 0.8 ± 0.05 ThDP, 1.0 ± 0.02 Mg(2+), and 1.0 ± 0.015 flavin adenine dinucleotide (FAD) per monomer as a second organic cofactor, the role of which remains unclear. Strong reductants, Ti(III)-citrate, Na(+)-dithionite, and the photochemical 5-deazaflavin/oxalate system, led to a partial reduction of the FAD chromophore. The cleavage product of CDO, 6-oxohexanoate, was also a substrate; the corresponding cyclic 1,3- and 1,4-diones did not react with CDH, nor did the cis- and trans-cyclohexane diols. The enzymes acetohydroxyacid synthase (AHAS) from Saccharomyces cerevisiae, pyruvate oxidase (POX) from Lactobacillus plantarum, benzoylformate decarboxylase from Pseudomonas putida, and pyruvate decarboxylase from Zymomonas mobilis were identified as the closest relatives of CDH by comparative amino acid sequence analysis, and a ThDP binding motif and a 2-fold Rossmann fold for FAD binding could be localized at the C-terminal end and central region of CDH, respectively. A first mechanism for the ring cleavage of CDO is presented, and it is suggested that the FAD cofactor in CDH is an evolutionary relict.

The reducing component BoxA of benzoyl-coenzyme A epoxidase from Azoarcus evansii is a [4Fe-4S] protein.

BoxA is the reductase component of the benzoyl-coenzyme A (CoA) oxidizing epoxidase enzyme system BoxAB. The enzyme catalyzes the key step of an hitherto unknown aerobic, CoA-dependent pathway of benzoate metabolism, which is the epoxidation of benzoyl-CoA to the non-aromatic 2,3-epoxybenzoyl-CoA. The function of BoxA is the transfer of two electrons from NADPH to the epoxidase component BoxB. We could show recently that BoxB is a diiron enzyme, whereas here we demonstrate that BoxA harbors an FAD and two [4Fe-4S] clusters per protein monomer. The characterization of BoxA was hampered by severe oxygen sensitivity; the cubane [4Fe-4S] clusters degrade already with traces of oxygen. Interestingly, the adventitiously formed [3Fe-4S] centers could be reconstituted in vitro by adding Fe(II) and sulfide to retrieve the native cubane centers. BoxA is the first example of a reductase of this type that has an FAD and two bacterial ferredoxin-type [4Fe-4S] clusters. In other cases within the catalytically versatile family of diiron enzymes, the related reductases have plant-type ferredoxin or Rieske-type [2Fe-2S] centers only.

Predicting RNA structure by multiple template homology modeling.

Despite the importance of 3D structure to understand the myriad functions of RNAs in cells, most RNA molecules remain out of reach of crystallographic and NMR methods. However, certain structural information such as base pairing and some tertiary contacts can be determined readily for many RNAs by bioinformatics or relatively low cost experiments. Further, because RNA structure is highly modular, it is possible to deduce local 3D structure from the solved structures of evolutionarily related RNAs or even unrelated RNAs that share the same module. RNABuilder is a software package that generates model RNA structures by treating the kinematics and forces at separate, multiple levels of resolution. Kinematically, bonds in bases, certain stretches of residues, and some entire molecules are rigid while other bonds remain flexible. Forces act on the rigid bases and selected individual atoms. Here we use RNABuilder to predict the structure of the 200-nucleotide Azoarcus group I intron by homology modeling against fragments of the distantly-related Twort and Tetrahymena group I introns and by incorporating base pairing forces where necessary. In the absence of any information from the solved Azoarcus intron crystal structure, the model accurately depicts the global topology, secondary and tertiary connections, and gives an overall RMSD value of 4.6 A relative to the crystal structure. The accuracy of the model is even higher in the intron core (RMSD = 3.5 A), whereas deviations are modestly larger for peripheral regions that differ more substantially between the different introns. These results lay the groundwork for using this approach for larger and more diverse group I introns, as well for still larger RNAs and RNA-protein complexes such as group II introns and the ribosomal subunits.

Metal ion dependence of cooperative collapse transitions in RNA.

Positively charged counterions drive RNA molecules into compact configurations that lead to their biologically active structures. To understand how the valence and size of the cations influences the collapse transition in RNA, small-angle X-ray scattering was used to follow the decrease in the radius of gyration (R(g)) of the Azoarcus and Tetrahymena ribozymes in different cations. Small, multivalent cations induced the collapse of both ribozymes more efficiently than did monovalent ions. Thus, the cooperativity of the collapse transition depends on the counterion charge density. Singular value decomposition of the scattering curves showed that folding of the smaller and more thermostable Azoarcus ribozyme is well described by two components, whereas collapse of the larger Tetrahymena ribozyme involves at least one intermediate. The ion-dependent persistence length, extracted from the distance distribution of the scattering vectors, shows that the Azoarcus ribozyme is less flexible at the midpoint of transition in low-charge-density ions than in high-charge-density ions. We conclude that the formation of sequence-specific tertiary interactions in the Azoarcus ribozyme overlaps with neutralization of the phosphate charge, while tertiary folding of the Tetrahymena ribozyme requires additional counterions. Thus, the stability of the RNA structure determines its sensitivity to the valence and size of the counterions.

Oxygen-dependent regulation of the central pathway for the anaerobic catabolism of aromatic compounds in Azoarcus sp. strain CIB.

The role of oxygen in the transcriptional regulation of the PN promoter that controls the bzd operon involved in the anaerobic catabolism of benzoate in the denitrifying Azoarcus sp. strain CIB has been investigated. In vivo experiments using PN::lacZ translational fusions, in both Azoarcus sp. strain CIB and Escherichia coli cells, have shown an oxygen-dependent repression effect on the transcription of the bzd catabolic genes. E. coli Fnr was required for the anaerobic induction of the PN promoter, and the oxygen-dependent repression of the bzd genes could be bypassed by the expression of a constitutively active Fnr* protein. In vitro experiments revealed that Fnr binds to the PN promoter at a consensus sequence centered at position -41.5 from the transcription start site overlapping the -35 box, suggesting that PN belongs to the class II Fnr-dependent promoters. Fnr interacts with RNA polymerase (RNAP) and is strictly required for transcription initiation after formation of the RNAP-PN complex. An fnr ortholog, the acpR gene, was identified in the genome of Azoarcus sp. strain CIB. The Azoarcus sp. strain CIB acpR mutant was unable to grow anaerobically on aromatic compounds and it did not drive the expression of the PN::lacZ fusion, suggesting that AcpR is the cognate transcriptional activator of the PN promoter. Since the lack of AcpR in Azoarcus sp. strain CIB did not affect growth on nonaromatic carbon sources, AcpR can be considered a transcriptional regulator of the Fnr/Crp superfamily that has evolved to specifically control the central pathway for the anaerobic catabolism of aromatic compounds in Azoarcus.

RNA tertiary interactions mediate native collapse of a bacterial group I ribozyme.

Large RNAs collapse into compact intermediates in the presence of counterions before folding to the native state. We previously found that collapse of a bacterial group I ribozyme correlates with the formation of helices within the ribozyme core, but occurs at Mg2+ concentrations too low to support stable tertiary structure and catalytic activity. Here, using small-angle X-ray scattering, we show that Mg2+-induced collapse is a cooperative folding transition that can be fit by a two-state model. The Mg2+ dependence of collapse is similar to the Mg2+ dependence of helix assembly measured by partial ribonuclease T1 digestion and of an unfolding transition measured by UV hypochromicity. The correspondence between multiple probes of RNA structure further supports a two-state model. A mutation that disrupts tertiary contacts between the L9 tetraloop and its helical receptor destabilized the compact state by 0.8 kcal/mol, while mutations in the central triplex were less destabilizing. These results show that native tertiary interactions stabilize the compact folding intermediates under conditions in which the RNA backbone remains accessible to solvent.

RNA kink turns to the left and to the right.

A helix-loop-helix within the group I intron has most of the canonical sequence elements of a kink turn (K-turn), yet it bends in the opposite direction. The reverse K-turn kinks toward the major rather than the minor grooves of the flanking helices. This suggests that there are two distinct subclasses of tertiary structures that a K-turn secondary structure can adopt. The final structure may be specified by external factors, such as protein binding or the tertiary structural context, rather than the intrinsic conformation of the RNA.

Crystal structure of a group I intron splicing intermediate.

A recently reported crystal structure of an intact bacterial group I self-splicing intron in complex with both its exons provided the first molecular view into the mechanism of RNA splicing. This intron structure, which was trapped in the state prior to the exon ligation reaction, also reveals the architecture of a complex RNA fold. The majority of the intron is contained within three internally stacked, but sequence discontinuous, helical domains. Here the tertiary hydrogen bonding and stacking interactions between the domains, and the single-stranded joiner segments that bridge between them, are fully described. Features of the structure include: (1) A pseudoknot belt that circumscribes the molecule at its longitudinal midpoint; (2) two tetraloop-tetraloop receptor motifs at the peripheral edges of the structure; (3) an extensive minor groove triplex between the paired and joiner segments, P6-J6/6a and P3-J3/4, which provides the major interaction interface between the intron's two primary domains (P4-P6 and P3-P9.0); (4) a six-nucleotide J8/7 single stranded element that adopts a mu-shaped structure and twists through the active site, making critical contacts to all three helical domains; and (5) an extensive base stacking architecture that realizes 90% of all possible stacking interactions. The intron structure was validated by hydroxyl radical footprinting, where strong correlation was observed between experimental and predicted solvent accessibility. Models of the pre-first and pre-second steps of intron splicing are proposed with full-sized tRNA exons. They suggest that the tRNA undergoes substantial angular motion relative to the intron between the two steps of splicing.

Small structural costs for evolution from RNA to RNP-based catalysis.

Typical RNA-based cellular catalysts achieve their active structures only as complexes with protein cofactors, implying that protein binding compensates for some structural deficiencies in the RNA. An unresolved question was the extent to which protein-facilitation imposes additional structural costs, by requiring that an RNA maintain structures required for protein binding, beyond those required for catalysis. We used nucleotide analog interference to identify initially 71 functional group substitutions at phosphate, 2'-ribose, and adenosine base positions that compromise RNA self-splicing in the bI5 group I intron. Protein-facilitated splicing by CBP2 suppresses 11 of 30 interfering substitutions at the RNA backbone and a greater fraction, 27 of 41, at the adenosine base, including at structures conserved among group I introns. Only one substitution directly interferes with protein binding but not with self-splicing. This substitution, plus three adenosine base modifications that interfere more strongly in CBP2-dependent splicing than in self-splicing, yield a cost for protein facilitation of only four functional groups, as approximated by this set of analogs. The small observed structural cost provides a strong physical rationale for the evolutionary drive from RNA to RNP-based function in biology. Remarkably, the four extra requirements do not appear to report disruption of direct protein-RNA contacts and instead likely reflect design against misfolding rather than for maintenance of a protein-binding site.