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.

Nitrogeniibacter mangrovi gen. nov., sp. nov., a novel anaerobic and aerobic denitrifying betaproteobacterium and reclassification of Azoarcus pumilus as Aromatoleum pumilum comb. nov.

Denitrification is a vital link in the global bio-nitrogen cycle. Here, we isolated a strain (M9-3-2T) that is a novel benzo[a]pyrene (BaP)-tolerant, anaerobic and aerobic denitrifying bacterium from a continuous BaP-enrichment cultured mangrove sediment. In silico comparative genomics and taxonomic analysis clearly revealed that strain M9-3-2T (=MCCC 1K03313T=JCM 32045T) represents a novel species of a novel genus named as Nitrogeniibacter mangrovi gen. nov., sp. nov., belonging to family Zoogloeaceae, order Rhodocyclales. In addition, the species Azoarcus pumilus is transferred into genus Aromatoleum and named Aromatoleum pumilum comb. nov. The predominant respiratory quinone of strain M9-3-2T was ubiquinone-8 and the major polar lipids were diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, three unidentified phospholipids and three unidentified aminophospholipids. In this study, the capacity of strain M9-3-2T to eliminate nitrate was detected under anaerobic and aerobic conditions, and the removal rates of nitrate were 6.1×10-6 µg N/l/h/cell and 3×10-7 µg N/l/h/cell, respectively. Our results suggested that strain M9-3-2T could play an important role in the nitrogen removal regardless of the presence of oxygen in natural or/and man-made ecosystems.

RNA diversification by a self-reproducing ribozyme revealed by deep sequencing and kinetic modelling.

We demonstrate that a recombinase ribozyme achieves multiple functions in the same reaction network: self-reproduction, iterative elongation and circularization of other RNAs, leading to synthesis of diverse products predicted by a kinetic model. This shows that key mechanisms can be integrated and controlled toward Darwinian evolution in RNA reaction networks.

Denitrifying bio-cathodes developed from constructed wetland sediments exhibit electroactive nitrate reducing biofilms dominated by the genera Azoarcus and Pontibacter.

To limit the nitrate contamination of ground and surface water, stimulation of denitrification by electrochemical approach is an innovative way to be explored. Two nitrate reducing bio-cathodes were developed under constant polarization (-0.5 V vs SCE) using sediments and water from a constructed wetland (Rampillon, Seine-et-Marne, France). The bio-cathodes responded to nitrate addition on chronoamperometry through an increase of the reductive current. The denitrification efficiency of the pilots increased by 47% compared to the negative controls without electrodes after polarization. 16S rRNA gene sequencing of the biofilms and sediments evidenced the significant and discriminating presence of the Azoarcus and Pontibacter genera in the biofilms from biocathodes active for nitrate reduction. Our study shows the possibility to promote the development of efficient Azoarcus-dominated biocathodes from freshwater sediment to enhance nitrate removal from surface waters.

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.

Two-stage enrichment of hydrogen-oxidizing bacteria as biofertilizers.

Biofertilizers can replace chemical fertilizers to promote the plant growth without causing any pollution. The study of hydrogen-oxidizing bacteria (HOB) enrichment as biofertilizers from mixed culture is scarce. Our recent study shows that biofertilizing HOB are successfully enriched in a short hydraulic retention time of 10 h. While, the mechanism is unknown. This study intentionally used a two-stage method to enrich biofertilizing HOB specifically with nitrate as nitrogen source in Stage 1 and then 1-aminocyclopropane-1-carboxylate (ACC) as nitrogen source in Stage 2. It was found Pseudomonas (34.46%, reported HOB) predominated in Stage 1, while Azospirillum (59.35%), Azoarcus (36%) were dominant genera and Azospirillum lipoferum strain DSM 1691 (50%), Azoarcus olearius strain DQS-4 (50%) were dominant species in Stage 2. The enriched HOB of Stage 2 showed ACC deaminase activity. Furthermore, they could also fix N2 and consume Ca3(PO4)2. Thus, the two-stage method can be used as a specific enrichment for HOB as biofertilizers, which extends the application of HOB in agriculture.

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.

Azoarcus halotolerans sp. nov., a novel member of Rhodocyclaceae isolated from activated sludge collected in Hong Kong.

A floc-forming bacterial strain, designated HKLI-1T, was isolated from the activated sludge of a municipal sewage treatment plant in Hong Kong SAR, PR China. Cells of this strain were Gram-stain-negative, strictly aerobic, catalase- and oxidase-positive, rod-shaped and motile by means of a single polar flagellum. Growth occurred at 18-37 °C (optimum, 28 °C), pH 5.5-9.0 (optimum, pH 7.5) and with 0-8.0 % (w/v) NaCl (optimum, 1-1.5 %) concentration. The major fatty acids of strain HKLI-1T were C16 : 0 and summed feature 3 (C16 : 1 ω7c and/or C16 : 1 ω6c). The polar lipid profile contained diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol and three unidentified lipids. The DNA G+C content was 63.5 mol% from whole genomic sequence analysis. Based on the results of 16S rRNA gene sequences analysis, this strain should be assigned to the genus Azoarcus and is closely related to Azoarcus olearius DQS-4T (94.93 % 16S rRNA gene sequence pairwise similarity), Azoarcus toluclasticus MF63T (94.91 %) and Azoarcus communis SWub3T (94.01 %), but separate from them by large distances in different phylogenetic trees. Based on whole genome analysis, the orthologous average nucleotide identity and in silico DNA-DNA hybridization values against four of the closest relatives were 73.03-74.83 and 17.2-23.0 %, respectively. The phylogenetic, genotypic, phenotypic and chemotaxonomic data demonstrated that strain HKLI-1T could be distinguished from its phylogenetically related species, and that this strain represented a novel species within the genus Azoarcus, for which the name Azoarcus halotolerans sp. nov. is proposed. The type strain is HKLI-1T (= 72659T=CCTCC AB 2019312T).

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.

Discovery of an Inducible Toluene Monooxygenase That Cooxidizes 1,4-Dioxane and 1,1-Dichloroethylene in Propanotrophic Azoarcus sp. Strain DD4.

Cometabolic degradation plays a prominent role in bioremediation of commingled groundwater contamination (e.g., chlorinated solvents and the solvent stabilizer 1,4-dioxane [dioxane]). In this study, we untangled the diversity and catalytic functions of multicomponent monooxygenases in Azoarcus sp. strain DD4, a Gram-negative propanotroph that is effective in degrading dioxane and 1,1-dichloroethylene (1,1-DCE). Using a combination of knockout mutagenesis and heterologous expression, a toluene monooxygenase (MO) encoded by the tmoABCDEF gene cluster was unequivocally proved to be the key enzyme responsible for the cometabolism of both dioxane and 1,1-DCE. Interestingly, in addition to utilizing toluene as a primary substrate, this toluene MO can also oxidize propane into 1-propanol. Expression of this toluene MO in DD4 appears inducible by both substrates (toluene and propane) and their primary hydroxylation products (m-cresol, p-cresol, and 1-propanol). These findings coherently explain why DD4 can grow on propane and express toluene MO for active cooxidation of dioxane and 1,1-DCE. Furthermore, upregulation of tmo transcription by 1-propanol underlines the implication potential of using 1-propanol as an alternative auxiliary substrate for DD4 bioaugmentation. The discovery of this toluene MO in DD4 and its degradation and induction versatility can lead to broad applications, spanning from environmental remediation and water treatment to biocatalysis in green chemistry.IMPORTANCE Toluene MOs have been well recognized given their robust abilities to degrade a variety of environmental pollutants. Built upon previous research efforts, this study ascertained the untapped capability of a toluene MO in DD4 for effective cooxidation of dioxane and 1,1-DCE, two of the most prevailing yet challenging groundwater contaminants. This report also aligns the induction of a toluene MO with nontoxic and commercially accessible chemicals (e.g., propane and 1-propanol), extending its implications in the field of environmental microbiology and beyond.

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.

Azoarcus pumilus sp. nov., isolated from seawater in Sanya, China.

A novel Gram-stain-negative, motile, rod-shaped (0.4-0.5×1.0-2.0 µm) strain with one polar flagellum, designated SY39T, was isolated from seawater in Sanya, China. Strain SY39T was able to grow at 15-40 °C (optimum, 35-37 °C), pH 6.5-8.5 (pH 8.0) and 0.5-6.0 % (w/v) NaCl (3.5 %). Chemotaxonomic analysis showed that the isoprenoid quinones were Q-8 (88.6 %) and Q-7 (11.4 %). The dominant fatty acids were C16 : 0 and summed feature 3 (C16 : 1ω7c/C16 : 1ω6c). The polar lipids of strain SY39T consisted of diphosphatidyglycerol, phosphatidylglycerol, phosphatidylethanolamine, one unknown phosphoglycolipid, one unknown glycolipid and two unknown aminophosphoglycolipids. The DNA G+C content of the genomic DNA was 66.5 mol%. The phylogenetic analysis of 16S rRNA gene sequences showed that strain SY39T belongs to the genus Azoarcus with similarity ranging from 92.3 to 95.2 %. Based on the phenotypic, chemotaxonomic and phylogenetic features, strain SY39T is concluded to represent a novel species of the genus Azoarcus, for which the name Azoarcus pumilus sp. nov. is proposed. The type strain is SY39T (=KCTC 62157T=MCCC 1K03430T).

Aromatoleum gen. nov., a novel genus accommodating the phylogenetic lineage including Azoarcus evansii and related species, and proposal of Aromatoleum aromaticum sp. nov., Aromatoleum petrolei sp. nov., Aromatoleum bremense sp. nov., Aromatoleum toluolicum sp. nov. and Aromatoleum diolicum sp. nov.

Comparative 16S rRNA gene sequence analysis and major physiological differences indicate two distinct sublineages within the genus Azoarcus: the Azoarcus evansii lineage, comprising Azoarcusevansii (type strain KB740T=DSM 6898T=CIP 109473T=NBRC 107771T), Azoarcusbuckelii (type strain U120T=DSM 14744T=LMG 26916T), Azoarcusanaerobius (type strain LuFRes1T=DSM 12081T=LMG 30943T), Azoarcustolulyticus (type strain Tol-4T=ATCC 51758T=CIP 109470T), Azoarcustoluvorans (type strain Td21T=ATCC 700604T=DSM 15124T) and Azoarcustoluclasticus (type strain MF63T=ATCC 700605T), and the Azoarcus indigens lineage, comprising Azoarcusindigens (type strain VB32T=ATCC 51398T=LMG 9092T), Azoarcus communis (type strain SWub3T=ATCC 51397T=LMG 9095T) and Azoarcusolearius (type strain DQS-4T=BCRC 80407T=KCTC 23918T=LMG 26893T). Az. evansii lineage members have remarkable anaerobic degradation capacities encompassing a multitude of alkylbenzenes, aromatic compounds and monoterpenes, often involving novel biochemical reactions. In contrast, Az. indigens lineage members are diazotrophic endophytes lacking these catabolic capacities. It is proposed that species of the Az. evansii lineage should be classified in a novel genus, Aromatoleum gen. nov. Finally, based on the literature and new growth, DNA-DNA hybridization and proteomic data, the following five new species are proposed: Aromatoleum aromaticum sp. nov. (type strain EbN1T=DSM 19018T=LMG 30748T and strain pCyN1=DSM 19016=LMG 31004), Aromatoleum petrolei sp. nov. (type strain ToN1T=DSM 19019T=LMG 30746T), Aromatoleumbremense sp. nov. (type strain PbN1T=DSM 19017T=LMG 31005T), Aromatoleum toluolicum sp. nov. (type strain TT=DSM 19020T=LMG 30751T) and Aromatoleum diolicum sp. nov. (type strain 22LinT=DSM 15408T=LMG 30750T).

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.

Community dynamics in a nitrate-reducing microbial consortium cultivated with p-alkylated vs. non-p-alkylated aromatic compounds.

In this study, we established the nitrate-reducing, aromatic compound-degrading enrichment culture pMB18. Its community structure was controlled by the aromatic substrate applied. In the presence of a p-alkylated substrate, microorganisms related to Sulfuritalea, Ignavibacterium and Comamonadaceae were abundant. Non-p-alkylated structural analogues promoted the enrichment of Azoarcus, which was probably favored by the excretion of nitrite. The analysis of the bamA gene, which is a functional marker for anaerobic aromatic compound degradation, as well as a differential abundance analysis suggested the involvement of Sulfuritalea and Comamonadaceae in the degradation of p-alkylated substrates. Members of the genus Azoarcus were assumed to be the key players for the degradation of the non-p-alkylated substrates. A gene cluster encoding a putative 4-methylbenzoyl-CoA reductase, which is supposed to be specific for the dearomatization of p-alkylated benzoyl-CoA intermediates, was detected in culture pMB18 dominated by Sulfuritalea, Ignavibacterium and Comamonadaceae, but not in an Azoarcus-dominated culture. This study allowed insight into a microbial community, whose composition was guided by the aromatic substrate applied.

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.