Thauera sp. for efficient nitrate removal in continuous denitrifying moving bed biofilm reactor.

Thauera is the most widely found dominant denitrifying genus in wastewater. In earlier study, MBBR augmented with a specially developed denitrifying five-membered bacterial consortium (DC5) where Thauera was found to be the most abundant and persistent genus. Therefore, to check the functional potential of Thauera in the removal of nitrate-containing wastewater in the present study Thauera sp.V14 one of the member of the consortium DC5 was used as the model organism. Thauera sp.V14 exhibited strong hydrophobicity, auto-aggregation ability, biofilm formation and denitrification ability, which indicated its robust adaptability short colonization and nitrate removal efficiency. Continuous reactor studies with Thauera sp.V14 in 10 L dMBBR showed 91% of denitrification efficiency with an initial nitrate concentration of 620 mg L-1 within 3 h of HRT. Thus, it revealed that Thauera can be employed as an effective microorganism for nitrate removal from wastewater based on its performance in the present studies.

Genes and enzymes involved in the biodegradation of the quaternary carbon compound pivalate in the denitrifying Thauera humireducens strain PIV-1.

Quaternary carbon-containing compounds exist in natural and fossil oil-derived products and are used in chemical and pharmaceutical applications up to industrial scale. Due to the inaccessibility of the quaternary carbon atom for a direct oxidative or reductive attack, they are considered as persistent in the environment. Here, we investigated the unknown degradation of the quaternary carbon-containing model compound pivalate (2,2-dimethyl-propionate) in the denitrifying bacterium Thauera humireducens strain PIV-1 (formerly Thauera pivalivorans). We provide multiple evidence for a pathway comprising the activation to pivalyl-CoA and the carbon skeleton rearrangement to isovaleryl-CoA. Subsequent reactions proceed similar to the catabolic leucine degradation pathway such as the carboxylation to 3-methylglutaconyl-CoA and the cleavage of 3-methyl-3-hydroxyglutaryl-CoA to acetyl-CoA and acetoacetate. The completed genome of Thauera humireducens strain PIV-1 together with proteomic data was used to identify pivalate-upregulated gene clusters including genes putatively encoding pivalate CoA ligase and adenosylcobalamin-dependent pivalyl-CoA mutase. A pivalate-induced gene encoding a putative carboxylic acid CoA ligase was heterologously expressed, and its highly enriched product exhibited pivalate CoA ligase activity. The results provide the first experimental insights into the biodegradation pathway of a quaternary carbon-containing model compound that serves as a blueprint for the degradation of related quaternary carbon-containing compounds.

IncP-type plasmids carrying genes for antibiotic resistance or for aromatic compound degradation are prevalent in sequenced Aromatoleum and Thauera strains.

Self-transferable plasmids of the incompatibility group P-1 (IncP-1) are considered important carriers of genes for antibiotic resistance and other adaptive functions. In the laboratory, these plasmids have a broad host range; however, little is known about their in situ host profile. In this study, we discovered that Thauera aromatica K172T , a facultative denitrifying microorganism capable of degrading various aromatic compounds, contains a plasmid highly similar to the IncP-1 ε archetype pKJK5. The plasmid harbours multiple antibiotic resistance genes and is maintained in strain K172T for at least 1000 generations without selection pressure from antibiotics. In a subsequent search, we found additional nine IncP-type plasmids in a total of 40 sequenced genomes of the closely related genera Aromatoleum and Thauera. Six of these plasmids form a novel IncP-1 subgroup designated θ, four of which carry genes for anaerobic or aerobic degradation of aromatic compounds. Pentanucleotide sequence analyses (k-mer profiling) indicated that Aromatoleum spp. and Thauera spp. are among the most suitable hosts for the θ plasmids. Our results highlight the importance of IncP-1 plasmids for the genetic adaptation of these common facultative denitrifying bacteria and provide novel insights into the in situ host profile of these plasmids.

Advanced Insights into Catalytic and Structural Features of the Zinc-Dependent Alcohol Dehydrogenase from Thauera aromatica.

The asymmetric reduction of ketones to chiral hydroxyl compounds by alcohol dehydrogenases (ADHs) is an established strategy for the provision of valuable precursors for fine chemicals and pharmaceutics. However, most ADHs favor linear aliphatic and aromatic carbonyl compounds, and suitable biocatalysts with preference for cyclic ketones and diketones are still scarce. Among the few candidates, the alcohol dehydrogenase from Thauera aromatica (ThaADH) stands out with a high activity for the reduction of the cyclic α-diketone 1,2-cyclohexanedione to the corresponding α-hydroxy ketone. This study elucidates catalytic and structural features of the enzyme. ThaADH showed a remarkable thermal and pH stability as well as stability in the presence of polar solvents. A thorough description of the substrate scope combined with the resolution and description of the crystal structure, demonstrated a strong preference of ThaADH for cyclic α-substituted cyclohexanones, and indicated structural determinants responsible for the unique substrate acceptance.

Thauera sedimentorum sp. nov., Isolated from Coastal Sediment.

A Gram-negative, strictly aerobic, rod-shaped, non-spore-forming bacterium, designated CAU 1555T, was isolated from a sediment sample collected on Jeju Island, Republic of Korea. Growth of the isolate was observed at 20-37 °C (optimum at 30 °C) and pH 5.5-10.0 (optimum at 8.0). Phylogenetic analysis based on the result of 16S rRNA gene sequences revealed that strain CAU 1555T belonged to the genus Thauera and was closely related to Thauera hydrothermalis GD-2T (98.4% sequence similarity), Thauera lacus D20T (96.6%), and Thauera linaloolentis 47LolT (95.5%). Strain CAU 1555T possessed phosphatidylglycerol, diphosphatidylglycerol, phosphatidylethanolamine, one unidentified phospholipid, and one aminophospholipid as the major polar lipids; Q-8 as the predominant respiratory quinone; and C16:0, summed feature 3 (comprising C16:1ω6c and/or C16:1ω7c), and summed feature 8 (comprising C18:1 ω7c/ C18:1 ω6c) as the major fatty acids. The average nucleotide identity (ANI), average amino acid identity (AAI), and digital DNA-DNA hybridization (dDDH) values between the new isolate and T. hydrothermalis GD-2T were 84.5%, 86.4%, and 28.0%, respectively. Whole-genome sequencing of strain CAU 1555T revealed 3,955,289 bp with a DNA G + C content of 68.0 mol%. Based on the results of its polyphasic properties and genomic analysis, the isolate represents a novel species within the genus Thauera, for which the name Thauera sedimentorum sp. nov. is proposed, with CAU 1555T (= KCTC 72546T = MCCC 1K04065T) as the type strain.

Genome analysis of Thauera chlorobenzoica strain 3CB-1T, a halobenzoate-degrading bacterium isolated from aquatic sediment.

The genus Thauera is characterized by several species and strains with the ability to degrade a variety of aromatic compounds under denitrifying conditions. Thauera chlorobenzoica strain 3CB-1T, isolated from river sediment, has the unique ability to degrade a variety of halobenzoates, such as 3-chlorobenzoate, 3-bromobenzoate, 3-iodobenzoate, and 2-fluorobenzoate, coupled to nitrate reduction. The genome of T. chlorobenzoica strain 3CB-1T has been sequenced, allowing us to gain insights into the molecular basis for the anaerobic degradation of (halo)aromatic compounds. The 3.77-Mb genome contains 3584 genes; 3514 are protein-coding genes of which 198 are likely associated with degradation of aromatic compounds. It has a G + C content of 67.25%. The genome contains two sets of CoA reductase gene clusters, both belonging to class I benzoate-CoA reductases (BCRs). The genes in one of the two clusters differ from the typical BCRs, with low sequence identities, suggesting they might have different substrate specificities. The genome also contains four benzoate-CoA ligase genes. One likely encodes a 3-hydroxybenzoate-CoA ligase, and two others group together with benzoate-CoA ligases from Thauera aromatica. The fourth has a 77% identity to the mbdA gene from Azoarcus sp. CIB, is absent in the T. aromatica genome, and potentially encodes a halobenzoate-CoA ligase. 3-Chlorobenzoate is reductively dechlorinated in T. chlorobenzoica by a benzoyl-CoA reductase.

Cloning, expression and biochemical characterization of a novel amidase from Thauera sinica K11.

A novel amidase (TAM) was identified and cloned from the genome of Thauera sinica K11. The recombinant protein was purified to homogeneity by one-step affinity chromatography for up to 26.4-fold with a yield of 38.1%. Gel filtration chromatography and SDS-PAGE revealed that the enzyme was a tetramer with a subunit of approximately 37.5 kDa. The amidase exhibited the maximum acyl transfer activity at 45 °C and pH 7.0, and it was highly stable over a wide pH range of 6.0-11.0. Inhibition of enzyme activity was observed in the presence of metal ions, thiol reagents and organic solvents. TAM showed a broad substrate spectrum toward aliphatic, aromatic and heterocyclic amides. For linear aliphatic monoamides, the acyl transfer activity of TAM was decreased with the extension of the carbon chain length, and thus the highest activity of 228.2 U/mg was obtained when formamide was used as substrate. This distinct selectivity of amidase to linear aliphatic monoamides expanded the findings of signature amidases to substrate specificity.

An Aerobic Hybrid Phthalate Degradation Pathway via Phthaloyl-Coenzyme A in Denitrifying Bacteria.

The degradation of the xenobiotic phthalic acid esters by microorganisms is initiated by the hydrolysis to the respective alcohols and ortho-phthalate (hereafter, phthalate). In aerobic bacteria and fungi, oxygenases are involved in the conversion of phthalate to protocatechuate, the substrate for ring-cleaving dioxygenases. In contrast, anaerobic bacteria activate phthalate to the extremely unstable phthaloyl-coenzyme A (CoA), which is decarboxylated by oxygen-sensitive UbiD-like phthaloyl-CoA decarboxylase (PCD) to the central benzoyl-CoA intermediate. Here, we demonstrate that the facultatively anaerobic, denitrifying Thauera chlorobenzoica 3CB-1 and Aromatoleum evansii KB740 strains use phthalate as a growth substrate under aerobic and denitrifying conditions. In vitro assays with extracts from cells grown aerobically with phthalate demonstrated the succinyl-CoA-dependent activation of phthalate followed by decarboxylation to benzoyl-CoA. In T. chlorobenzoica 3CB-1, we identified PCD as a highly abundant enzyme in both aerobically and anaerobically grown cells, whereas genes for phthalate dioxygenases are missing in the genome. PCD was highly enriched from aerobically grown T. chlorobenzoica cells and was identified as an identical enzyme produced under denitrifying conditions. These results indicate that the initial steps of aerobic phthalate degradation in denitrifying bacteria are accomplished by the anaerobic enzyme inventory, whereas the benzoyl-CoA oxygenase-dependent pathway is used for further conversion to central intermediates. Such a hybrid pathway requires intracellular oxygen homeostasis at concentrations low enough to prevent PCD inactivation but sufficiently high to supply benzoyl-CoA oxygenase with its cosubstrate.IMPORTANCE Phthalic acid esters (PAEs) are industrially produced on a million-ton scale per year and are predominantly used as plasticizers. They are classified as environmentally relevant xenobiotics with a number of adverse health effects, including endocrine-disrupting activity. Biodegradation by microorganisms is considered the most effective process to eliminate PAEs from the environment. It is usually initiated by the hydrolysis of PAEs to alcohols and o-phthalic acid. Degradation of o-phthalic acid fundamentally differs in aerobic and anaerobic microorganisms; aerobic phthalate degradation heavily depends on dioxygenase-dependent reactions, whereas anaerobic degradation employs the oxygen-sensitive key enzyme phthaloyl-CoA decarboxylase. We demonstrate that aerobic phthalate degradation in facultatively anaerobic bacteria proceeds via a previously unknown hybrid degradation pathway involving oxygen-sensitive and oxygen-dependent key enzymes. Such a strategy is essential for facultatively anaerobic bacteria that frequently switch between oxic and anoxic environments.

Comparative Metagenomic and Metatranscriptomic Analyses Reveal the Functional Species and Metabolic Characteristics of an Enriched Denitratation Community.

Nitrite supply for mainstream anammox via denitratation has attracted increasing attention. The functional species responsible for denitratation and their metabolic characteristics were unravelled in this study. A highly stable denitratation community was enriched from activated sludge by combined control of C/N and pH. Nitrite accumulation and nitrate removal efficiencies were both higher than 80% during long-term operation (>100 d). The genotypic complete denitrifier Thauera aminoaromatica MZ1T was identified to be mainly responsible for acetate consumption, polyhydroxybutyrate (PHB) accumulation, and nitrate reduction. The presence of nitrate restricted the transcription and electron allocation of downstream denitrifying enzymes due to low expression of their electron transport modules (cytochrome bc1 and cytochrome c). Metabolic reconstruction of this strain indicated that the reducing power generated via the tricarboxylic acid (TCA) cycle was mainly provided for PHB synthesis and nitrate reduction in the exogenous feast phase. After the depletion of acetate, PHB was degraded and then entered the TCA cycle, providing reducing power for nitrate reduction. This allocation strategy of reducing power with priority given to carbon storage instead of nitrite reduction might favor their survival in oligotrophic and weak alkaline habitats. These results updated our understanding of the causes underlying nitrite accumulation and its physiological benefits.

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.

A catalytically versatile benzoyl-CoA reductase, key enzyme in the degradation of methyl- and halobenzoates in denitrifying bacteria.

Class I benzoyl-CoA (BzCoA) reductases (BCRs) are key enzymes in the anaerobic degradation of aromatic compounds. They catalyze the ATP-dependent reduction of the central BzCoA intermediate and analogues of it to conjugated cyclic 1,5-dienoyl-CoAs probably by a radical-based, Birch-like reduction mechanism. Discovered in 1995, the enzyme from the denitrifying bacterium Thauera aromatica (BCRTar) has so far remained the only isolated and biochemically accessible BCR, mainly because BCRs are extremely labile, and their heterologous production has largely failed so far. Here, we describe a platform for the heterologous expression of the four structural genes encoding a designated 3-methylbenzoyl-CoA reductase from the related denitrifying species Thauera chlorobenzoica (MBRTcl) in Escherichia coli This reductase represents the prototype of a distinct subclass of ATP-dependent BCRs that were proposed to be involved in the degradation of methyl-substituted BzCoA analogues. The recombinant MBRTcl had an αßγδ-subunit architecture, contained three low-potential [4Fe-4S] clusters, and was highly oxygen-labile. It catalyzed the ATP-dependent reductive dearomatization of BzCoA with 2.3-2.8 ATPs hydrolyzed per two electrons transferred and preferentially dearomatized methyl- and chloro-substituted analogues in meta- and para-positions. NMR analyses revealed that 3-methylbenzoyl-CoA is regioselectively reduced to 3-methyl-1,5-dienoyl-CoA. The unprecedented reductive dechlorination of 4-chloro-BzCoA to BzCoA probably via HCl elimination from a reduced intermediate allowed for the previously unreported growth of T. chlorobenzoica on 4-chlorobenzoate. The heterologous expression platform established in this work enables the production, isolation, and characterization of bacterial and archaeal BCR and BCR-like radical enzymes, for many of which the function has remained unknown.

DbdR, a New Member of the LysR Family of Transcriptional Regulators, Coordinately Controls Four Promoters in the Thauera aromatica AR-1 3,5-Dihydroxybenzoate Anaerobic Degradation Pathway.

The facultative anaerobe Thauera aromatica strain AR-1 uses 3,5-dihydroxybenzoate (3,5-DHB) as a sole carbon and energy source under anoxic conditions using an unusual oxidative strategy to overcome aromatic ring stability. A 25-kb gene cluster organized in four main operons encodes the anaerobic degradation pathway for this aromatic. The dbdR gene coding for a LysR-type transcriptional regulator (LTTR), which is present at the foremost end of the cluster, is required for anaerobic growth on 3,5-DHB and for the expression of the main pathway operons. A model structure of DbdR showed conserved key residues for effector binding with its closest relative TsaR for p-toluenesulfonate degradation. We found that DbdR controlled expression of three promoters upstream from the operons coding for the three main steps of the pathway. While one of them (P orf20 ) was only active in the presence of 3,5-DHB, the other two (P dbhL and P orf18 ) showed moderate basal levels that were further induced in the presence of the pathway substrate, which needed be converted to hydroxyhydroquinone to activate transcription. Both basal and induced activities were strictly dependent on DbdR, which was also required for transcription from its own promoter. DbdR basal expression was moderately high and, unlike most LTTR, increased 2-fold in response to the presence of the effector. DbdR was found to be a tetramer in solution, producing a single retardation complex in binding assays with the three enzymatic promoters, consistent with its tetrameric structure. The three promoters had a conserved organization with a clear putative primary (regulatory) binding site and a putative secondary (activating) binding site positioned at the expected distances from the transcription start site. In contrast, two protein-DNA complexes were observed for the P dbdR promoter, which also showed significant sequence divergence from those of the three other promoters. Taken together, our results show that a single LTTR coordinately controls expression of the entire 3,5-DHB anaerobic degradation pathway in Thauera aromatica AR-1, allowing a fast and optimized response to the presence of the aromatic.IMPORTANCEThauera aromatica AR-1 is a facultative anaerobe that is able to use 3,5-dihydroxybenzoat (3,5-DHB) as the sole carbon and energy source in a process that is dependent on nitrate respiration. We have shown that a single LysR-type regulator with unusual properties, DbdR, controls the expression of the pathway in response to the presence of the substrate; unlike other regulators of the family, DbdR does not repress but activates its own synthesis and is able to bind and activate three promoters directing the synthesis of the pathway enzymes. The promoter architecture is conserved among the three promoters but deviates from that of typical LTTR-dependent promoters. The substrate must be metabolized to an intermediate compound to activate transcription, which requires basal enzyme levels to always be present. The regulatory network present in this strain is designed to allow basal expression of the enzymatic machinery, which would rapidly metabolize the substrate when exposed to it, thus rendering the effector molecule. Once activated, the regulator induces the synthesis of the entire pathway through a positive feedback, increasing expression from all the target promoters to allow maximum growth.

Stereochemical Insights into the Anaerobic Degradation of 4-Isopropylbenzoyl-CoA in the Denitrifying Bacterium Strain pCyN1.

The constitutions and absolute configurations of two previously unknown intermediates, (1S,2S,4S)-2-hydroxy-4-isopropylcyclohexane-1-carboxylate and (S)-3-isopropylpimelate, of anaerobic degradation of p-cymene in the bacterium Aromatoleum aromaticum pCyN1 are reported. These intermediates (as CoA esters) are involved in the further degradation of 4-isopropylbenzoyl-CoA formed by methyl group hydroxylation and subsequent oxidation of p-cymene. Proteogenomics indicated 4-isopropylbenzoyl-CoA degradation involves (i) a novel member of class I benzoyl-CoA reductase (BCR) as known from Thauera aromatica K172 and (ii) a modified ß-oxidation pathway yielding 3-isopropylpimeloyl-CoA analogously to benzoyl-CoA degradation in Rhodopseudomonas palustris. Reference standards of all four diastereoisomers of 2-hydroxy-4-isopropylcyclohexane-1-carboxylate as well as both enantiomers of 3-isopropylpimelate were obtained by stereoselective syntheses via methyl 4-isopropyl-2-oxocyclohexane-1-carboxylate. The stereogenic center carrying the isopropyl group was established using a rhodium-catalyzed asymmetric conjugate addition. X-ray crystallography revealed that the thermodynamically most stable stereoisomer of 2-hydroxy-4-isopropylcyclohexane-1-carboxylate is formed during p-cymene degradation. Our findings imply that the reductive dearomatization of 4-isopropylbenzoyl-CoA by the BCR of A. aromaticum pCyN1 stereospecifically forms (S)-4-isopropyl-1,5-cyclohexadiene-1-carbonyl-CoA.

Thauera lacus sp. nov., isolated from a saline lake in Inner Mongolia.

A Gram-stain-negative, facultatively anaerobic, motile and rod-shaped bacterium, designated D20T, was isolated from the saline Lake Dai in Inner Mongolia, PR China. Growth of strain D20T occurred at 25-45 °C (optimum, 40 °C), pH 4.0-12.0 (optimum, 8.0) and with 0-3 % NaCl (w/v); (optimum, 0-1 %). The results of 16S rRNA gene sequence analysis revealed that strain D20T was most closely related to three Thauera species, Thaueraselenatis AXT, Thaueraaminoaromatica S2T and Thaueraaromatica K172T, with a similarity value of 96.2 %. The major respiratory quinone of strain D20T was ubiquinone-8 (Q-8), and the dominant fatty acids (>10 %) were summed feature 3 (C16 : 1ω6c and/or C16 : 1ω7c; 39.8 %), C16 : 0 (30.9 %) and summed feature 8 (C18 : 1ω6c and/or C18 : 1ω7c; 13.5 %). The polar lipid profile contained phosphatidylethanolamine, diphosphatidylglycerol, phosphatidylglycerol, one aminophospholipid and five unidentified lipids. The DNA G+C content was 67.2 mol% (data from the genome sequence). The estimated genome size was 3.7 Mb. The phenotypic, genotypic and chemotaxonomic differences between strain D20T and its phylogenetic relatives indicated that strain D20T should be regarded as a novel species in the genus Thauera, for which the name Thaueralacus sp. nov. is proposed. The type strain is D20T (=MCCC 1H00305T=KCTC 62586T).

Establishment of BmoR-based biosensor to screen isobutanol overproducer.

BACKGROUND: Isobutanol, a C4 branched-chain higher alcohol, is regarded as an attractive next-generation transport fuel. Metabolic engineering for efficient isobutanol production has been achieved in many studies. BmoR, an alcohol-regulated transcription factor, mediates a σ54-dependent promoter Pbmo of alkane monooxygenase in n-alkane metabolism of Thauera butanivorans and displays high sensitivity to C4-C6 linear alcohols and C3-C5 branched-chain alcohols. In this study, to achieve the high-level production of isobutanol, we established a screening system which relied on the combination of BmoR-based biosensor and isobutanol biosynthetic pathway and then employed it to screen isobutanol overproduction strains from an ARTP mutagenesis library. RESULTS: Firstly, we constructed and verified a GFP-based BmoR-Pbmo device responding to the isobutanol produced by the host. Then, this screening system was employed to select three mutants which exhibited higher GFP/OD600 values than that of wild type. Significantly, GFP/OD600 of mutant 10 was 190.7 ± 4.8, a 1.4-fold higher value than that of wild type. Correspondingly, the isobutanol titer of that strain was 1597.6 ± 129.6 mg/L, 2.0-fold higher than the wild type. With the overexpression of upstream pathway genes, the isobutanol production from mutant 10 reached 14.0 ± 1.0 g/L after medium optimization in shake flask. The isobutanol titer reached 56.5 ± 1.8 g/L in a fed-batch production experiment. CONCLUSIONS: This work screened out isobutanol overproduction strains from a mutagenesis library by using a screening system which depended on the combination of BmoR-based biosensor and isobutanol biosynthetic pathway. Optimizing fermentation condition and reinforcing upstream pathway could realize the increase of isobutanol production from the overproducer. Lastly, fed-batch fermentation of the mutant enhanced the isobutanol production to 56.5 ± 1.8 g/L.

Impact of phenol on the performance, kinetics, microbial communities and functional genes of an autotrophic denitrification system.

Nitrate and phenol often co-occur in wastewater because of the complex industrial and agricultural processes, while the impacts of phenol on autotrophic denitrification remain unclear. Here, a sulfur and hydrogen-oxidizing autotrophic denitrification reactor was established, and the effects of different concentrations of phenol on the nitrate removal performance, kinetics, microbial communities, and functional genes were investigated. Increasing concentrations of phenol significantly decreased the denitrification efficiency in the reactor. The kinetic data indicate the limitation of nitrate diffusion may be one of reasons. Increasing phenol concentrations declined the activities of nitrate and nitrite reductases and induced the production of reactive oxygen species (ROS) and the release of lactate dehydrogenase (LDH), suggesting potential toxicity to the denitrifying consortium. Denitrifying gene nirK was most sensitive to phenol stresses in the reactor. In addition, Thauera was the predominant genus in system with and without phenol, Bacillus was enriched under high phenol concentrations.

Enzymes involved in the anaerobic degradation of phenol by the sulfate-reducing bacterium Desulfatiglans anilini.

BACKGROUND: The sulfate-reducing bacterium Desulfatiglans anilini can grow with phenol as sole source of carbon and energy under strictly anaerobic, sulfate-reducing conditions. In the nitrate-reducing bacterium Thauera aromatica, the enzymes involved in phenol degradation have been well elucidated, whereas the anaerobic phenol degradation pathway by D. anilini was not studied in detail yet. RESULTS: The pathway of anaerobic phenol degradation by the sulfate-reducing bacterium Desulfatiglans anilini was studied by identification of genes coding for phenylphosphate synthase (encoded by pps genes) and phenylphosphate carboxylase (encoded by ppc genes) in the genome of D. anilini, by analysis of the transcription and translation of pps-ppc genes, and by measurement of phenylphosphate synthase activity in cell-free extracts of phenol-grown cells. The majority of genes involved in phenol degradation were found to be organized in one gene cluster. The gene cluster contained genes ppsα (phenylphosphate synthase alpha subunit), ppsß (phenylphosphate synthase beta subunit), ppcß (phenylphosphate carboxylase beta subunit), as well as 4-hydroxybenzoyl-CoA ligase and 4-hydroxylbenzoyl-CoA reductase-encoding genes. The genes ppsγ (phenylphosphate synthase gamma subunit), ppcα (phenylphosphate carboxylase alpha subunit) and ppcδ (phenylphosphate carboxylase delta subunit) were located elsewhere in the genome of D. anilini, and no obvious homologue of ppcγ (phenylphosphate carboxylase gamma subunit) was found in the genome. Induction of genes pps and ppc during growth on phenol was confirmed by reverse transcription polymerase chain reaction. Total proteome analysis revealed that the abundance of enzymes encoded by the gene cluster under study was much higher in phenol-grown cells than that in benzoate-grown cells. In in-vitro enzyme assays with cell-free extracts of phenol-grown cells, phenylphosphate was formed from phenol in the presence of ATP, Mg2+, Mn2+, K+ as co-factors. CONCLUSIONS: The genes coding for enzymes involved in the anaerobic phenol degradation pathway were identified in the sulfate-reducing bacterium D. anilini. The results indicate that the first steps of anaerobic phenol degradation in D. anilini are phosphorylation of phenol to phenylphosphate by phenylphosphate synthase and carboxylation of phenylphosphate by phenylphosphate carboxylase.

Thauera propionica sp. nov., isolated from downstream sediment sample of the river Ganges, Kanpur, India.

A Gram-stain-negative, non-endospore-producing, short-rod strain, KNDSS-Mac4T, was isolated from a downstream sediment sample of the river Ganges, Kanpur, India and studied by using the polyphasic taxonomic approach. 16S rRNA gene sequence analysis uncovered that the strain had similarity to species of the genus Thauera and formed a distinct phylogenetic cluster with Thauera humireducens KACC16524T. However, KNDSS-Mac4T showed closest phylogenetic affiliation to Thauera aminoaromatica DSM 14742T with 16S rRNA gene sequence similarity of 98.7 % followed by Thauera phenylacetica DSM 14743T (98.6 %), Thauera chlorobenzoica (98.2 %), T. humireducens KACC16524T (98.2 %), Thauera selenatis ATCC 55363T (98.2 %) and Thauera mechernichensis DSM 12266T (98.0 %). The digital DNA-DNA hybridization and average nucleotide identity values between strain KNDSS-Mac4T and the two most closely related taxa, T. aminoaromatica DSM 14742T and T. phenylacetica DSM 14743T, were 26.0, 26.7 and 84.0, 84.3 % respectively. Major lipids present were phosphatidylglycerol, three unknown aminophospholipids, phosphatidylmethylethanolamine, two unidentified lipids and Q-8 as the only ubiquonone. The major cellular fatty acids present were C16 : 1 ω6c/C16 : 1ω7c and C16 : 0. The DNA G+C content of strain KNDSS-Mac4T was 65.9 %. Based on data from phenotypic tests and the genotypic differences of strain KNDSS-Mac4T from its closest phylogenetic relatives, it is evident that this isolate should be regarded as a new species. It is proposed that strain KNDSS-Mac4T should be classified in the genus Thauera as a novel species, Thauerapropionica sp. nov. The type strain is KNDSS-Mac4T (=KCTC 52820T=VTCC-B-910017T).

Thauera hydrothermalis sp. nov., a thermophilic bacterium isolated from hot spring.

A Gram-stain-negative, non-spore-forming, rod-shaped, motile bacterial strain, designated GD-2T, was isolated from a sediment sample collected from a hot spring in the Tibet Autonomous Region, China. Strain GD-2T grew at a temperature range of 37-55 °C (optimum, 45-50 °C), a pH range of 5.5-11.0 (pH 7.0-7.5) and a NaCl concentration range of 0-4.0 % (0 %). The phylogenetic analysis based on 16S rRNA gene sequencing showed that strain GD-2T represented a member of the genus Thauera within the family Zoogloeaceae. Strain GD-2T was closely related to Thauera linaloolentis 47LolT with the highest 16S rRNA gene sequence similarity of 95.5 %. The whole genomic average nucleotide identity value for GD-2T and 47LolT was 75.3 %. The predominant cellular fatty acids of the strain were C16 : 0, summed feature 3 (C16 : 1ω6c and/or C16 : 1ω7c), C10 : 0 3-OH and C12 : 0. The main polar lipids were phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, three unidentified phospholipids and two unidentified aminolipids. The major isoprenoid quinone was ubiquinone 8. Genome sequencing revealed that the genome size of GD-2T was 3 059 321 bp with a G+C content of 63.57 mol%. On the basis of phylogenetic, phenotypic and chemotaxonomic characteristics, strain GD-2T is considered to represent a novel species of the genus Thauera, for which the name Thauera hydrothermalis sp. nov. is proposed. The type strain is GD-2T (=NBRC 112472T=CGMCC 1.15527T).

Liquid chromatography/isotope ratio mass spectrometry analysis of halogenated benzoates for characterization of the underlying degradation reaction in Thauera chlorobenzoica CB-1T.

RATIONALE: Halogenated benzoic acids occur in the environment due to their widespread agricultural and pharmaceutical use. Compound-specific stable isotope analysis (CSIA) has developed over the last decades for investigation of in situ transformation and reaction mechanisms of environmental pollutants amenable by gas chromatography (GC). As polar compounds are unsuitable for GC analysis we developed a method to perform liquid chromatography (LC)/CSIA for halogenated benzoates. METHODS: LC/isotope ratio mass spectrometry (IRMS) utilizing a LC-Surveyor pump coupled to a MAT 253 isotope ratio mass spectrometer via a LC-Isolink interface was applied. For chromatographic separation a YMC-Triart C18 column and a potassium hydrogen phosphate buffer (150 mM, pH 7.0, 40°C, 200 µL mL-1 ) were used, followed by wet oxidation deploying 1.5 mol L-1 ortho-phosphoric acid and 200 g L-1 sodium peroxodisulfate at 75 µL mL-1 . RESULTS: Separation of benzoate and halogenated benzoates could be achieved in less than 40 min over a concentration range of 2 orders of magnitude. Under these conditions the dehalogenation reaction of Thauera chlorobenzoica 3CB-1T using 3-chloro-, 3-bromo- and 4-chlorobenzoic acid was investigated resulting in inverse carbon isotope fractionation for meta-substituted benzoic acids and minor normal fractionation for para-substituted benzoic acids. Together with the respective growth rates this led to the assumption that dehalogenation of para-halobenzoic acids follows a different mechanism from that of meta-halobenzoic acids. CONCLUSIONS: A new LC/IRMS method for the quantitative determination of halogenated benzoates was developed and used to investigate the in vivo transformation pathways of these compounds, providing some insights into degradation and removal of these widespread compounds by T. chlorobenzoica 3CB-1T .