Mitigation of Paddy Field Soil Methane Emissions by Betaproteobacterium Azoarcus Inoculation of Rice Seeds.

Paddy fields are a major source of atmospheric methane, a greenhouse gas produced by methanogens and consumed by methanotrophs in flooded soil. The inoculation of rice seeds with the bacterium Azoarcus sp. KH32C alters the rice root-associated soil bacterial community composition. The present study investigated the effects of KH32C-inoculated rice cultivation on soil methanogens and methanotrophs involved in methane emissions from a rice paddy field. KH32C-inoculated and non-inoculated rice (cv. Nipponbare) were cultivated in a Japanese rice paddy with and without nitrogen fertilizer. Measurements of methane emissions and soil solution chemical properties revealed increases in methane flux over the waterlogged period with elevations in the concentrations of dissolved methane, dissolved organic carbon, and ferrous iron, which is an indicator of soil reduction levels. Reverse transcription quantitative PCR and amplicon sequencing were used to assess the transcription of the methyl-coenzyme M reductase gene (mcrA) from methanogens and the particulate methane monooxygenase gene (pmoA) from methanotrophs in paddy soil. The results obtained showed not only the transcript copy numbers, but also the compositions of mcrA and pmoA transcripts were related to methane flux. KH32C-inoculated rice cultivation recruited soil methanogens and methanotrophs that suppressed high methane synthesis, increased methane consumption, and decreased methane emissions by 23.5 and 17.2% under non-fertilized and nitrogen-fertilized conditions, respectively, while maintaining rice grain yield. The present study demonstrated the mitigation of paddy field methane emissions arising from the use of KH32C in rice cultivation due to its influence on the compositions of soil methanogen and methanotroph populations.

Elucidation of Essential Genes and Mutant Fitness during Adaptation toward Nitrogen Fixation Conditions in the Endophyte Azoarcus olearius BH72 Revealed by Tn-Seq.

Azoarcus olearius BH72 is a diazotrophic model endophyte that contributes fixed nitrogen to its host plant, Kallar grass, and expresses nitrogenase genes endophytically. Despite extensive studies on biological nitrogen fixation (BNF) of diazotrophic endophytes, little is known about global genetic players involved in survival under respective physiological conditions. Here, we report a global genomic screen for putatively essential genes of A. olearius employing Tn5 transposon mutagenesis with a modified transposon combined with high-throughput sequencing (Tn-Seq). A large Tn5 master library of ~6 × 105 insertion mutants of strain BH72 was obtained. Next-generation sequencing identified 183,437 unique insertion sites into the 4,376,040-bp genome, displaying one insertion every 24 bp on average. Applying stringent criteria, we describe 616 genes as putatively essential for growth on rich medium. COG (Clusters of Orthologous Groups) assignment of the 564 identified protein-coding genes revealed enrichment of genes related to core cellular functions and cell viability. To mimic gradual adaptations toward BNF conditions, the Tn5 mutant library was grown aerobically in synthetic medium or microaerobically on either combined or atmospheric nitrogen. Enrichment and depletion analysis of Tn5 mutants not only demonstrated the role of BNF- and metabolism-related proteins but also revealed that, strikingly, many genes relevant for plant-microbe interactions decrease bacterial competitiveness in pure culture, such type IV pilus- and bacterial envelope-associated genes. IMPORTANCE A constantly growing world population and the daunting challenge of climate change demand new strategies in agricultural crop production. Intensive usage of chemical fertilizers, overloading the world's fields with organic input, threaten terrestrial and marine ecosystems as well as human health. Long overlooked, the beneficial interaction of endophytic bacteria and grasses has attracted ever-growing interest in research in the last decade. Capable of biological nitrogen fixation, diazotrophic endophytes not only provide a valuable source of combined nitrogen but also are known for diverse plant growth-promoting effects, thereby contributing to plant productivity. Elucidation of an essential gene set for a prominent model endophyte such as A. olearius BH72 provides us with powerful insights into its basic lifestyle. Knowledge about genes detrimental or advantageous under defined physiological conditions may point out a way of manipulating key steps in the bacterium's lifestyle and plant interaction toward a more sustainable agriculture.

Engineering Escherichia coli for anaerobic alkane activation: Biosynthesis of (1-methylalkyl)succinates.

In anoxic environments, microbial activation of alkanes for subsequent metabolism occurs most commonly through the addition of fumarate to a subterminal carbon, producing an alkylsuccinate. Alkylsuccinate synthases are complex, multi-subunit enzymes that utilize a catalytic glycyl radical and require a partner, activating enzyme for hydrogen abstraction. While many genes encoding putative alkylsuccinate synthases have been identified, primarily from nitrate- and sulfate-reducing bacteria, few have been characterized and none have been reported to be functionally expressed in a heterologous host. Here, we describe the functional expression of the (1-methylalkyl)succinate synthase (Mas) system from Azoarcus sp. strain HxN1 in recombinant Escherichia coli. Mass spectrometry confirms anaerobic biosynthesis of the expected products of fumarate addition to hexane, butane, and propane. Maximum production of (1-methylpentyl)succinate is observed when masC, masD, masE, masB, and masG are all present on the expression plasmid; omitting masC reduces production by 66% while omitting any other gene eliminates production. Meanwhile, deleting iscR (encoding the repressor of the E. coli iron-sulfur cluster operon) improves product titer, as does performing the biotransformation at reduced temperature (18°C), both suggesting alkylsuccinate biosynthesis is largely limited by functional expression of this enzyme system.

Comparative Genomics Provides Insights into the Taxonomy of Azoarcus and Reveals Separate Origins of Nif Genes in the Proposed Azoarcus and Aromatoleum Genera.

Among other attributes, the Betaproteobacterial genus Azoarcus has biotechnological importance for plant growth-promotion and remediation of petroleum waste-polluted water and soils. It comprises at least two phylogenetically distinct groups. The "plant-associated" group includes strains that are isolated from the rhizosphere or root interior of the C4 plant Kallar Grass, but also strains from soil and/or water; all are considered to be obligate aerobes and all are diazotrophic. The other group (now partly incorporated into the new genus Aromatoleum) comprises a diverse range of species and strains that live in water or soil that is contaminated with petroleum and/or aromatic compounds; all are facultative or obligate anaerobes. Some are diazotrophs. A comparative genome analysis of 32 genomes from 30 Azoarcus-Aromatoleum strains was performed in order to delineate generic boundaries more precisely than the single gene, 16S rRNA, that has been commonly used in bacterial taxonomy. The origin of diazotrophy in Azoarcus-Aromatoleum was also investigated by comparing full-length sequences of nif genes, and by physiological measurements of nitrogenase activity using the acetylene reduction assay. Based on average nucleotide identity (ANI) and whole genome analyses, three major groups could be discerned: (i) Azoarcus comprising Az. communis, Az. indigens and Az. olearius, and two unnamed species complexes, (ii) Aromatoleum Group 1 comprising Ar. anaerobium, Ar. aromaticum, Ar. bremense, and Ar. buckelii, and (iii) Aromatoleum Group 2 comprising Ar. diolicum, Ar. evansii, Ar. petrolei, Ar. toluclasticum, Ar. tolulyticum, Ar. toluolicum, and Ar. toluvorans. Single strain lineages such as Azoarcus sp. KH32C, Az. pumilus, and Az. taiwanensis were also revealed. Full length sequences of nif-cluster genes revealed two groups of diazotrophs in Azoarcus-Aromatoleum with nif being derived from Dechloromonas in Azoarcus sensu stricto (and two Thauera strains) and from Azospira in Aromatoleum Group 2. Diazotrophy was confirmed in several strains, and for the first time in Az. communis LMG5514, Azoarcus sp. TTM-91 and Ar. toluolicum TT. In terms of ecology, with the exception of a few plant-associated strains in Azoarcus (s.s.), across the group, most strains/species are found in soil and water (often contaminated with petroleum or related aromatic compounds), sewage sludge, and seawater. The possession of nar, nap, nir, nor, and nos genes by most Azoarcus-Aromatoleum strains suggests that they have the potential to derive energy through anaerobic nitrate respiration, so this ability cannot be usefully used as a phenotypic marker to distinguish genera. However, the possession of bzd genes indicating the ability to degrade benzoate anaerobically plus the type of diazotrophy (aerobic vs. anaerobic) could, after confirmation of their functionality, be considered as distinguishing phenotypes in any new generic delineations. The taxonomy of the Azoarcus-Aromatoleum group should be revisited; retaining the generic name Azoarcus for its entirety, or creating additional genera are both possible outcomes.

Diverse Bacterial Genes Modulate Plant Root Association by Beneficial Bacteria.

The plant rhizosphere harbors a diverse population of microorganisms, including beneficial plant growth-promoting bacteria (PGPB), that colonize plant roots and enhance growth and productivity. In order to specifically define bacterial traits that contribute to this beneficial interaction, we used high-throughput transposon mutagenesis sequencing (TnSeq) in two model root-bacterium systems associated with Setaria viridis: Azoarcus olearius DQS4T and Herbaspirillum seropedicae SmR1. This approach identified ∼100 significant genes for each bacterium that appeared to confer a competitive advantage for root colonization. Most of the genes identified specifically in A. olearius encoded metabolism functions, whereas genes identified in H. seropedicae were motility related, suggesting that each strain requires unique functions for competitive root colonization. Genes were experimentally validated by site-directed mutagenesis, followed by inoculation of the mutated bacteria onto S. viridis roots individually, as well as in competition with the wild-type strain. The results identify key bacterial functions involved in iron uptake, polyhydroxybutyrate metabolism, and regulation of aromatic metabolism as important for root colonization. The hope is that by improving our understanding of the molecular mechanisms used by PGPB to colonize plants, we can increase the adoption of these bacteria in agriculture to improve the sustainability of modern cropping systems.IMPORTANCE There is growing interest in the use of associative, plant growth-promoting bacteria (PGPB) as biofertilizers to serve as a sustainable alternative for agriculture application. While a variety of mechanisms have been proposed to explain bacterial plant growth promotion, the molecular details of this process remain unclear. The current research supports the idea that PGPB use in agriculture will be promoted by gaining more knowledge as to how these bacteria colonize plants, promote growth, and do so consistently. Specifically, the research seeks to identify those bacterial genes involved in the ability of two, PGPB strains, Azoarcus olearius and Herbaspirillum seropedicae, to colonize the roots of the C4 model grass Setaria viridis. Applying a transposon mutagenesis (TnSeq) approach, we assigned phenotypes and function to genes that affect bacterial competitiveness during root colonization. The results suggest that each bacterial strain requires unique functions for root colonization but also suggests that a few, critical functions are needed by both bacteria, pointing to some common mechanisms. The hope is that such information can be exploited to improve the use and performance of PGPB in agriculture.

Resorcinol Hydroxylase of Azoarcus anaerobius: Molybdenum Dependence, Activity, and Heterologous Expression.

The obligately anaerobic, denitrifying bacterium Azoarcus anaerobius strain LuFRes1 grows with resorcinol (1,3-dihydroxybenzene) as sole carbon and energy source. Resorcinol is oxidized to hydroxyhydroquinone (1,2,4-trihydroxybenzene) by resorcinol hydroxylase (RH), an inducible membrane-bound enzyme. Sequence comparison places resorcinol hydroxylase into the group of anaerobic molybdopterin oxidoreductases and dimethyl sulfoxide reductase-like enzymes. In the large subunit, a molybdopterin-binding domain was predicted, and the small subunit most likely contains two [4Fe-4S] centers. Growth of molybdate-starved cells was inhibited by tungstate, and in vitro resorcinol hydroxylase activity was inhibited by arsenite and selenite that are known to inhibit molybdenum-containing enzymes. The two genes encoding resorcinol hydroxylase could be expressed in Escherichia coli but the products remained in inclusion bodies. All attempts to purify RH from A. anaerobius or to produce soluble, active RH in E. coli failed. Nevertheless, RH was produced as a C-terminally Strep-tagged protein from plasmid pSKM1 in Thauera aromatica AR1 transconjugants carrying a transposon insertion in the coding gene for the large (ΔrhL) or the small subunit (ΔrhS) of RH from cosmid R+. RH in the membrane fraction of wild-type transconjugant T. aromatica AR1/R+ showed a specific activity of 80 mU mg-1, and the specific activity of RH in the membranes of the complemented mutants was in the same range (80-95 mU mg-1). We conclude that RH of A. anaerobius is a membrane-bound molybdoenzyme consisting of two subunits which might require a further loosely bound subunit as membrane anchor.

Description of Azoarcus nasutitermitis sp. nov. and Azoarcus rhizosphaerae sp. nov., two nitrogen-fixing species isolated from termite nest and rhizosphere of Ficus religiosa.

A polyphasic taxonomic approach was used to characterise two presumably novel bacteria, designated strains CC-YHH838T and CC-YHH848T isolated from termite nest and rhizosphere of Ficus religiosa, respectively. These two nitrogen-fixing strains were observed to be Gram-staining-negative, aerobic rod, and colonies were yellowish in color. Growth of strains was observed at 20-37 °C, pH 7-8, and in the presence of 1-2% NaCl. Phylogenetic analyses based on 16S rRNA genes revealed a distinct taxonomic position attained by strain CC-YHH838T and CC-YHH848T associated with Thauera hydrothermalis (97.1% sequence identity), and formed a separate branch with Azoarcus indigens (95.4%), Aromatoleum aromaticum (96.2%), and lower sequence similarity to other species. The calculation of OrthoANI values pointed out strains CC-YHH838T and CC-YHH848T gave 78.9% and 79.8% compared to Thauera hydrothermalis, respectively. The major fatty acids (> 5%) were C16:0, C17:0 cyclo, C10:0 3-OH, C16:1ω7c/C16:1ω6c and C18:1ω7c/C18:1ω6c. The polar lipid profile comprised phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol and unidentified aminophospholipid and phospholipids; the predominant polyamines were putrescine and spermidine. The predominant respiratory system was ubiquinone (Q-8) and the DNA G + C contents were 61.4 ± 0.1 mol% and 60.2 ± 1.3 mol%, respectively. Based on the phylogenetic and polyphasic comparisons, strains CC-YHH838T and CC-YHH848T are proposed to represent two novel species within the genus Azoarcus in the family Rhodocyclaceae, for which the name Azoarcus nasutitermitis sp. nov. (type strain CC-YHH838T = BCRC 81059T = JCM 32001T) and Azoarcus rhizosphaerae sp. nov. (type strain CC-YHH848T = BCRC 81060T = JCM 32002T) were proposed.

Further Insights into the Architecture of the PN Promoter That Controls the Expression of the bzd Genes in Azoarcus.

The anaerobic degradation of benzoate in bacteria involves the benzoyl-CoA central pathway. Azoarcus/Aromatoleum strains are a major group of anaerobic benzoate degraders, and the transcriptional regulation of the bzd genes was extensively studied in Azoarcus sp. CIB. In this work, we show that the bzdR regulatory gene and the PN promoter can also be identified upstream of the catabolic bzd operon in all benzoate-degrader Azoarcus/Aromatoleum strains whose genome sequences are currently available. All the PN promoters from Azoarcus/Aromatoleum strains described here show a conserved architecture including three operator regions (ORs), i.e., OR1 to OR3, for binding to the BzdR transcriptional repressor. Here, we demonstrate that, whereas OR1 is sufficient for the BzdR-mediated repression of the PN promoter, the presence of OR2 and OR3 is required for de-repression promoted by the benzoyl-CoA inducer molecule. Our results reveal that BzdR binds to the PN promoter in the form of four dimers, two of them binding to OR1. The BzdR/PN complex formed induces a DNA loop that wraps around the BzdR dimers and generates a superstructure that was observed by atomic force microscopy. This work provides further insights into the existence of a conserved BzdR-dependent mechanism to control the expression of the bzd genes in Azoarcus strains.

A Novel Redox-Sensing Histidine Kinase That Controls Carbon Catabolite Repression in Azoarcus sp. CIB.

We have identified and characterized the AccS multidomain sensor kinase that mediates the activation of the AccR master regulator involved in carbon catabolite repression (CCR) of the anaerobic catabolism of aromatic compounds in Azoarcus sp. CIB. A truncated AccS protein that contains only the soluble C-terminal autokinase module (AccS') accounts for the succinate-dependent CCR control. In vitro assays with purified AccS' revealed its autophosphorylation, phosphotransfer from AccS'∼P to the Asp60 residue of AccR, and the phosphatase activity toward its phosphorylated response regulator, indicating that the equilibrium between the kinase and phosphatase activities of AccS' may control the phosphorylation state of the AccR transcriptional regulator. Oxidized quinones, e.g., ubiquinone 0 and menadione, switched the AccS' autokinase activity off, and three conserved Cys residues, which are not essential for catalysis, are involved in such inhibition. Thiol oxidation by quinones caused a change in the oligomeric state of the AccS' dimer resulting in the formation of an inactive monomer. This thiol-based redox switch is tuned by the cellular energy state, which can change depending on the carbon source that the cells are using. This work expands the functional diversity of redox-sensitive sensor kinases, showing that they can control new bacterial processes such as CCR of the anaerobic catabolism of aromatic compounds. The AccSR two-component system is conserved in the genomes of some betaproteobacteria, where it might play a more general role in controlling the global metabolic state according to carbon availability.IMPORTANCE Two-component signal transduction systems comprise a sensor histidine kinase and its cognate response regulator, and some have evolved to sense and convert redox signals into regulatory outputs that allow bacteria to adapt to the altered redox environment. The work presented here expands knowledge of the functional diversity of redox-sensing kinases to control carbon catabolite repression (CCR), a phenomenon that allows the selective assimilation of a preferred compound among a mixture of several carbon sources. The newly characterized AccS sensor kinase is responsible for the phosphorylation and activation of the AccR master regulator involved in CCR of the anaerobic degradation of aromatic compounds in the betaproteobacterium Azoarcus sp. CIB. AccS seems to have a thiol-based redox switch that is modulated by the redox state of the quinone pool. The AccSR system is conserved in several betaproteobacteria, where it might play a more general role controlling their global metabolic state.

The 7α-hydroxysteroid dehydratase Hsh2 is essential for anaerobic degradation of the steroid skeleton of 7α-hydroxyl bile salts in the novel denitrifying bacterium Azoarcus sp. strain Aa7.

Bile salts are steroid compounds from the digestive tract of vertebrates and enter the environment via defecation. Many aerobic bile-salt degrading bacteria are known but no bacteria that completely degrade bile salts under anoxic conditions have been isolated so far. In this study, the facultatively anaerobic Betaproteobacterium Azoarcus sp. strain Aa7 was isolated that grew with bile salts as sole carbon source under anoxic conditions with nitrate as electron acceptor. Phenotypic and genomic characterization revealed that strain Aa7 used the 2,3-seco pathway for the degradation of bile salts as found in other denitrifying steroid-degrading bacteria such as Sterolibacterium denitrificans. Under oxic conditions strain Aa7 used the 9,10-seco pathway as found in, for example, Pseudomonas stutzeri Chol1. Metabolite analysis during anaerobic growth indicated a reductive dehydroxylation of 7α-hydroxyl bile salts. Deletion of the gene hsh2 Aa7 encoding a 7-hydroxysteroid dehydratase led to strongly impaired growth with cholate and chenodeoxycholate but not with deoxycholate lacking a hydroxyl group at C7. The hsh2 Aa7 deletion mutant degraded cholate and chenodeoxycholate to the corresponding C19 -androstadienediones only while no phenotype change was observed during aerobic degradation of cholate. These results showed that removal of the 7α-hydroxyl group was essential for cleavage of the steroid skeleton under anoxic conditions.

Effects of Preferential Counterion Interactions on the Specificity of RNA Folding.

The real-time search for native RNA structure is essential for the operation of regulatory RNAs. We previously reported that a fraction of the Azoarcus ribozyme achieves a compact structure in less than a millisecond. To scrutinize the forces that drive initial folding steps, we used time-resolved SAXS to compare the folding dynamics of this ribozyme in thermodynamically isostable concentrations of different counterions. The results show that the size of the fast-folding population increases with the number of available counterions and correlates with the flexibility of initial RNA structures. Within 1 ms of folding, Mg2+ exhibits a smaller preferential interaction coefficient per charge, ΔΓ+/ Z, than Na+ or [Co(NH3)6]3+. The lower ΔΓ+/ Z corresponds to a smaller yield of folded RNA, although Mg2+ stabilizes native RNA more efficiently than other ions at equilibrium. These results suggest that strong Mg2+-RNA interactions impede the search for globally native structure during early folding stages.

Coupled catabolism and anabolism in autocatalytic RNA sets.

The ability to process molecules available in the environment into useable building blocks characterizes catabolism in contemporary cells and was probably critical for the initiation of life. Here we show that a catabolic process in collectively autocatalytic sets of RNAs allows diversified substrates to be assimilated. We modify fragments of the Azoarcus group I intron and find that the system is able to restore the original native fragments by a multi-step reaction pathway. This allows in turn the formation of catalysts by an anabolic process, eventually leading to the accumulation of ribozymes. These results demonstrate that rudimentary self-reproducing RNA systems based on recombination possess an inherent capacity to assimilate an expanded repertoire of chemical resources and suggest that coupled catabolism and anabolism could have arisen at a very early stage in primordial living systems.

Microbial community of a gasworks aquifer and identification of nitrate-reducing Azoarcus and Georgfuchsia as key players in BTEX degradation.

We analyzed a coal tar polluted aquifer of a former gasworks site in Thuringia (Germany) for the presence and function of aromatic compound-degrading bacteria (ACDB) by 16S rRNA Illumina sequencing, bamA clone library sequencing and cultivation attempts. The relative abundance of ACDB was highest close to the source of contamination. Up to 44% of total 16S rRNA sequences were affiliated to ACDB including genera such as Azoarcus, Georgfuchsia, Rhodoferax, Sulfuritalea (all Betaproteobacteria) and Pelotomaculum (Firmicutes). Sequencing of bamA, a functional gene marker for the anaerobic benzoyl-CoA pathway, allowed further insights into electron-accepting processes in the aquifer: bamA sequences of mainly nitrate-reducing Betaproteobacteria were abundant in all groundwater samples, whereas an additional sulfate-reducing and/or fermenting microbial community (Deltaproteobacteria, Firmicutes) was restricted to a highly contaminated, sulfate-depleted groundwater sampling well. By conducting growth experiments with groundwater as inoculum and nitrate as electron acceptor, organisms related to Azoarcus spp. were identified as key players in the degradation of toluene and ethylbenzene. An organism highly related to Georgfuchsia toluolica G5G6 was enriched with p-xylene, a particularly recalcitrant compound. The anaerobic degradation of p-xylene requires a metabolic trait that was not described for members of the genus Georgfuchsia before. In line with this, we were able to identify a putative 4-methylbenzoyl-CoA reductase gene cluster in the respective enrichment culture, which is possibly involved in the anaerobic degradation of p-xylene.

Engineering a bzd cassette for the anaerobic bioconversion of aromatic compounds.

Microorganisms able to degrade aromatic contaminants constitute potential valuable biocatalysts to deal with a significant reusable carbon fraction suitable for eco-efficient valorization processes. Metabolic engineering of anaerobic pathways for degradation and recycling of aromatic compounds is an almost unexplored field. In this work, we present the construction of a functional bzd cassette encoding the benzoyl-CoA central pathway for the anaerobic degradation of benzoate. The bzd cassette has been used to expand the ability of some denitrifying bacteria to use benzoate as sole carbon source under anaerobic conditions, and it paves the way for future pathway engineering of efficient anaerobic biodegraders of aromatic compounds whose degradation generates benzoyl-CoA as central intermediate. Moreover, a recombinant Azoarcus sp. CIB strain harbouring the bzd cassette was shown to behave as a valuable biocatalyst for anaerobic toluene valorization towards the synthesis of poly-3-hydroxybutyrate (PHB), a biodegradable and biocompatible polyester of increasing biotechnological interest as a sustainable alternative to classical oil-derived polymers.

The Regulatory Network Controlling Ethanol-Induced Expression of Alcohol Dehydrogenase in the Endophyte Azoarcus sp. Strain BH72.

The habitat of the nitrogen-fixing endophyte Azoarcus sp. strain BH72 is grass roots grown under waterlogged conditions that produce, under these conditions, ethanol. Strain BH72 is well equipped to metabolize ethanol, with eight alcohol dehydrogenases (ADHs), of which ExaA2 and ExaA3 are the most relevant ones. exaA2 and exaA3 cluster and are surrounded by genes encoding two-component regulatory systems (TCSs) termed ExaS-ExaR and ElmS-GacA. Functional genomic analyses revealed that i) expression of the corresponding genes was induced by ethanol, ii) the genes were also expressed in the rhizoplane or even inside of rice roots, iii) both TCSs were indispensable for growth on ethanol, and iv) they were important for competitiveness during rice root colonization. Both TCSs form a hierarchically organized ethanol-responsive signal transduction cascade with ExaS-ExaR as the highest level, essential for effective expression of the ethanol oxidation system based on ExaA2. Transcript and expression levels of exaA3 increased in tcs deletion mutants, suggesting no direct influence of both TCSs on its ethanol-induced expression. In conclusion, this underscores the importance of ethanol for the endophytic lifestyle of Azoarcus sp. strain BH72 and indicates a tight regulation of the ethanol oxidation system during root colonization.

Topological and thermodynamic factors that influence the evolution of small networks of catalytic RNA species.

An RNA-directed recombination reaction can result in a network of interacting RNA species. It is now becoming increasingly apparent that such networks could have been an important feature of the RNA world during the nascent evolution of life on the Earth. However, the means by which such small RNA networks assimilate other available genotypes in the environment to grow and evolve into the more complex networks that are thought to have existed in the prebiotic milieu are not known. Here, we used the ability of fragments of the Azoarcus group I intron ribozyme to covalently self-assemble via genotype-selfish and genotype-cooperative interactions into full-length ribozymes to investigate the dynamics of small (three- and four-membered) networks. We focused on the influence of a three-membered core network on the incorporation of additional nodes, and on the degree and direction of connectivity as single new nodes are added to this core. We confirmed experimentally the predictions that additional links to a core should enhance overall network growth rates, but that the directionality of the link (a "giver" or a "receiver") impacts the growth of the core itself. Additionally, we used a simple mathematical model based on the first-order effects of lower-level interactions to predict the growth of more complex networks, and find that such a model can, to a first approximation, predict the ordinal rankings of nodes once a steady-state distribution has been reached.

The Azoarcus anaerobius 1,3-Dihydroxybenzene (Resorcinol) Anaerobic Degradation Pathway Is Controlled by the Coordinated Activity of Two Enhancer-Binding Proteins.

The anaerobic resorcinol degradation pathway in Azoarcus anaerobius is unique in that it uses an oxidative rather than a reductive strategy to overcome the aromatic ring stability in degradation of this compound, in a process that is dependent on nitrate respiration. We show that the pathway is organized in five transcriptional units, three of which are inducible by the presence of the substrate. Three σ54-dependent promoters located upstream from the three operons coding for the main pathway enzymes were identified, which shared a similar structure with conserved upstream activating sequences (UASs) located at 103 to 111 bp from the transcription start site. Expression of the pathway is controlled by the bacterial enhancer-binding proteins (bEBPs) RedR1 and RedR2, two homologous regulators that, despite their high sequence identity (97%), have nonredundant functions: RedR2, the master regulator which also controls RedR1 expression, is itself able to promote transcription from two of the promoters, while RedR1 activity is strictly dependent on the presence of RedR2. The two regulators were shown to interact with each other, suggesting that the natural mode of activation is by forming heterodimers, which become active in the presence of the substrate after its metabolization to hydroxybenzoquinone through the pathway enzymes. The model structure of the N-terminal domain of the proteins is composed of tandem GAF and PAS motifs; the possible mechanisms controlling the activity of the regulators are discussed.IMPORTANCEAzoarcus anaerobius is a strict anaerobe that is able to use 1,3-dihydroxybenzene as the sole carbon source in a process that is dependent on nitrate respiration. We have shown that expression of the pathway is controlled by two regulators of almost identical sequences: the bEBPs RedR1 and RedR2, which share 97% identity. These regulators control three promoters with similar structure. Despite their sequence identity, the two bEBPs are not redundant and are both required for maximum pathway expression. In fact, the two proteins function as heterodimers and require activation by the pathway intermediate hydroxyhydroquinone. The structure of the domain sensing the activation signal resembles that of regulators that are known to interact with other proteins.

The oil-contaminated soil diazotroph Azoarcus olearius DQS-4T is genetically and phenotypically similar to the model grass endophyte Azoarcus sp. BH72.

The genome of Azoarcus olearius DQS-4T , a N2 -fixing Betaproteobacterium isolated from oil-contaminated soil in Taiwan, was sequenced and compared with other Azoarcus strains. The genome sequence showed high synteny with Azoarcus sp. BH72, a model endophytic diazotroph, but low synteny with five non-plant-associated strains (Azoarcus CIB, Azoarcus EBN1, Azoarcus KH32C, A. toluclasticus MF63T and Azoarcus PA01). Average Nucleotide Identity (ANI) revealed that DQS-4T shares 98.98% identity with Azoarcus BH72, which should now be included in the species A. olearius. The genome of DQS-4T contained several genes related to plant colonization and plant growth promotion, such as nitrogen fixation, plant adhesion and root surface colonization. In accordance with the presence of these genes, DQS-4T colonized rice (Oryza sativa) and Setaria viridis, where it was observed within the intercellular spaces and aerenchyma mainly of the roots. Although they promote the growth of grasses, the mechanism(s) of plant growth promotion by A. olearius strains is unknown, as the genomes of DQS-4T and BH72 do not contain genes for indole acetic acid (IAA) synthesis nor phosphate solubilization. In spite of its original source, both the genome and behaviour of DQS-4T suggest that it has the capacity to be an endophytic, nitrogen-fixing plant growth-promoting bacterium.

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.

Global expression analysis of the response to microaerobiosis reveals an important cue for endophytic establishment of Azoarcus sp. BH72.

The endophyte Azoarcus sp. BH72, fixing nitrogen microaerobically, encounters low O2 tensions in flooded roots. Therefore, its transcriptome upon shift to microaerobiosis was analyzed using oligonucleotide microarrays. A total of 8.7% of the protein-coding genes were significantly modulated. Aerobic conditions induced expression of genes involved in oxidative stress protection, while under microaerobiosis, 233 genes were upregulated, encoding hypothetical proteins, transcriptional regulators, and proteins involved in energy metabolism, among them a cbb3 -type terminal oxidase contributing to but not essential for N2 fixation. A newly established sensitive transcriptional reporter system using tdTomato allowed to visualize even relatively low bacterial gene expression in association with roots. Beyond metabolic changes, low oxygen concentrations seemed to prime transcription for plant colonization: Several genes known to be required for endophytic rice interaction were induced, and novel bacterial colonization factors were identified, such as azo1653. The cargo of the type V autotransporter Azo1653 had similarities to the attachment factor pertactin. Although for short term swarming-dependent colonization, it conferred a competitive disadvantage, it contributed to endophytic long-term establishment inside roots. Proteins sharing such opposing roles in the colonization process appear to occur more generally, as we demonstrated a very similar phenotype for another attachment protein, Azo1684. This suggests distinct cellular strategies for endophyte establishment.