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.

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.

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.

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.

Anaerobic Benzene Mineralization by Nitrate-Reducing and Sulfate-Reducing Microbial Consortia Enriched From the Same Site: Comparison of Community Composition and Degradation Characteristics.

Benzene mineralization under nitrate-reducing conditions was successfully established in an on-site reactor continuously fed with nitrate and sulfidic, benzene-containing groundwater extracted from a contaminated aquifer. Filling material from the reactor columns was used to set up anoxic enrichment cultures in mineral medium with benzene as electron donor and sole organic carbon source and nitrate as electron acceptor. Benzene degradation characteristics and community composition under nitrate-reducing conditions were monitored and compared to those of a well-investigated benzene-mineralizing consortium enriched from the same column system under sulfate-reducing conditions. The nitrate-reducing cultures degraded benzene at a rate of 10.1 ± 1.7 µM d-1, accompanied by simultaneous reduction of nitrate to nitrite. The previously studied sulfate-reducing culture degraded benzene at similar rates. Carbon and hydrogen stable isotope enrichment factors determined for nitrate-dependent benzene degradation differed significantly from those of the sulfate-reducing culture (ΛH/C nitrate = 12 ± 3 compared to ΛH/C sulfate = 28 ± 3), indicating different benzene activation mechanisms under the two conditions. The nitrate-reducing community was mainly composed of Betaproteobacteria, Ignavibacteria, and Anaerolineae. Azoarcus and a phylotype related to clone Dok59 (Rhodocyclaceae) were the dominant genera, indicating an involvement in nitrate-dependent benzene degradation. The primary benzene degrader of the sulfate-reducing consortium, a phylotype belonging to the Peptococcaceae, was absent in the nitrate-reducing consortium.

Functional redundancy ensures performance robustness in 3-stage PHA-producing mixed cultures under variable feed operation.

Polyhydroxyalkanoates (PHA) are biopolymers that can be produced by mixed microbial cultures using wastes or industrial by-products, which represent an economical and environmental advantage over pure culture processes. The use of alternate feedstocks enables using seasonal by-products, providing that the process is resilient to transient conditions. The mixed microbial communities of a 3-stage PHA producing system fed initially with molasses and then cheese whey were investigated through amplicon sequencing of the 16S rRNA gene. The transition in feedstock resulted in an adaptation of the acidogenic community, where Actinobacteria dominated with sugarcane molasses (up to 93% of the operational taxonomic units) and Firmicutes, with cheese whey (up to 97%). The resulting fermentation products profile also changed, with a higher fraction of HV precursors obtained with molasses than cheese whey (7.1±0.5 and 1.7±0.7 gCOD/L, respectively). As for the PHA storing culture, the genera Azoarcus, Thauera and Paracoccus were enriched with fermented molasses (average 89% of Bacteria). Later, fermented cheese whey fostered a higher diversity, including some less characterised PHA-storers such as the genera Paenibacillus and Lysinibacillus. Although the microbial community structure was significantly affected by the feedstock shift, the acidogenic and PHA storing performance of the 3-stage system was very similar once a pseudo steady state was attained, showing that a reliable level of functional redundancy was attained in both mixed cultures.

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 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.

Anaerobic co-metabolic biodegradation of tetrabromobisphenol A using a bioelectrochemical system.

Tetrabromobisphenol A(TBBPA), a pollutant in industrial wastewaters, needs to be removed due to its high toxicity and persistence. The main biodegradation pathway for TBBPA has been studied, and bisphenol A(BPA), which is toxic to the environment, is recognized as the general terminal product. In this study, we explored a new approach for the anaerobic biodegradation of TBBPA in a bioelectrochemical system (BES) through co-metabolic degradation of TBBPA with glucose. The half-life of TBBPA was significantly reduced to 13.5h-1 at 25µg/l of TBBPA. With an increase in the concentration of TBBPA, the removal rates of TBBPA rose to more than eighty percent. Based on the analysis of the products, we found that the degradation products of TBBPA were 2,6-dibromo-4-(1-methyl-1-phenylethyl) phenol, (double-benzenes product) and 2,6-dibromo-4-(prop-1-en-2-yl) phenol (single-benzene product), rather than BPA. Simultaneously, we proposed two degradation pathways for TBBPA in a BES system. According to the microbial diversity analysis of the anode biofilm, we speculated that the microorganism responsible for the biodegradation of TBBPA was Azoarcus. Additionally, we briefly analyzed the effect of TBBPA on the performance of BES system to pave the way for the further analysis of the interaction between the TBBPA and the BES system.

Degradation of cyclic diguanosine monophosphate by a hybrid two-component protein protects Azoarcus sp. strain CIB from toluene toxicity.

Cyclic diguanosine monophosphate (c-di-GMP) is a second messenger that controls diverse functions in bacteria, including transitions from planktonic to biofilm lifestyles, virulence, motility, and cell cycle. Here we describe TolR, a hybrid two-component system (HTCS), from the ß-proteobacterium Azoarcus sp. strain CIB that degrades c-di-GMP in response to aromatic hydrocarbons, including toluene. This response protects cells from toluene toxicity during anaerobic growth. Whereas wild-type cells tolerated a sudden exposure to a toxic concentration of toluene, a tolR mutant strain or a strain overexpressing a diguanylate cyclase gene lost viability upon toluene shock. TolR comprises an N-terminal aromatic hydrocarbon-sensing Per-Arnt-Sim (PAS) domain, followed by an autokinase domain, a response regulator domain, and a C-terminal c-di-GMP phosphodiesterase (PDE) domain. Autophosphorylation of TolR in response to toluene exposure initiated an intramolecular phosphotransfer to the response regulator domain that resulted in c-di-GMP degradation. The TolR protein was engineered as a functional sensor histidine kinase (TolRSK) and an independent response regulator (TolRRR). This classic two-component system (CTCS) operated less efficiently than TolR, suggesting that TolR was evolved as a HTCS to optimize signal transduction. Our results suggest that TolR enables Azoarcus sp. CIB to adapt to toxic aromatic hydrocarbons under anaerobic conditions by modulating cellular levels of c-di-GMP. This is an additional role for c-di-GMP in bacterial physiology.

The ICEXTD of Azoarcus sp. CIB, an integrative and conjugative element with aerobic and anaerobic catabolic properties.

Integrative and conjugative elements (ICE) play a major role in aerobic degradation of aromatic compounds, but they have not yet been shown to be involved in anaerobic degradation. We have characterized here the ICEXTD element which endows to the beta-proteobacterium Azoarcus sp. CIB with the ability to utilize aromatic hydrocarbons. The core region of ICEXTD , which shows a remarkable synteny with that of ICEclc-like elements, allows its own intracellular and intercellular mobility. ICEXTD integrates at the tRNAGly of the host chromosome, but it can also excise to produce a ready to transfer circular form. The adaptation modules of ICEXTD represent a unique combination of gene clusters for aerobic (tod genes) and anaerobic (bss-bbs and mbd genes) degradation of certain aromatic hydrocarbons, e.g., toluene, m-xylene and cumene. Transfer of ICEXTD to other Azoarcus strains, e.g., A. evansii, confers them the ability to degrade aromatic hydrocarbons both aerobically and anaerobically. Interestingly, ICEXTD allows Cupriavidus pinatubonensis, a bacterium unable to degrade anaerobically aromatic compounds, to grow with m-xylene under anoxic conditions. Thus, ICEXTD constitutes the first mobile genetic element able to expand the catabolic abilities of certain bacteria for the removal of aromatic hydrocarbons either in the presence or absence of oxygen.

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.

Efficient regulation of elemental sulfur recovery through optimizing working height of upflow anaerobic sludge blanket reactor during denitrifying sulfide removal process.

In this study, two lab-scale UASB reactors were established to testify S(0) recovery efficiency, and one of which (M-UASB) was improved from the previous T-UASB by shortening reactor height once S(2-) over oxidation was observed. After the height was shortened from 60 to 30cm, S(0) recovery rate was improved from 7.4% to 78.8%, and while, complete removal of acetate, nitrate and S(2-) was simultaneously maintained. Meanwhile, bacterial community distribution was homogenous throughout the reactor, with denitrifying sulfide oxidization bacteria predominant, such as Thauera and Azoarcus spp., indicating the optimized condition for S(0) recovery. The effective control of working height/volume in reactors plays important roles for the efficient regulation of S(0) recovery during DSR process.