Reductase Homolog Research Articles

In-depth analysis of erythrose reductase homologs in Yarrowia lipolytica

The unconventional yeast Yarrowia lipolytica produces erythritol as an osmoprotectant to adapt to osmotic stress. In this study, the array of putative erythrose reductases, responsible for the conversion of d-erythrose to erythritol, was analyzed. Single knockout and multiple knockout strains were tested for their ability to produce polyols in osmotic stress conditions. Lack of six of the reductase genes does not affect erythritol significantly, as the production of this polyol is comparable to the control strain. Deletion of eight of the homologous erythrose reductase genes resulted in a 91% decrease in erythritol synthesis, a 53% increase in mannitol synthesis, and an almost 8-fold increase in arabitol synthesis as compared to the control strain. Additionally, the utilization of glycerol was impaired in the media with induced higher osmotic pressure. The results of this research may shed new light on the production of arabitol and mannitol from glycerol by Y. lipolytica and help to develop strategies for further modification in polyol pathways in these microorganisms.

The identification of viral ribonucleotide reductase encoded by ORF23 and ORF141 genes and effect on CyHV-2 replication.

Ribonucleotide reductase (RR) is essential for the replication of the double-stranded DNA virus CyHV-2 due to its ability to catalyze the conversion of ribonucleotides to deoxyribonucleotides, and is a potential target for the development of antiviral drugs to control CyHV-2 infection. Bioinformatic analysis was conducted to identify potential homologues of RR in CyHV-2. The transcription and translation levels of ORF23 and ORF141, which showed high homology to RR, were measured during CyHV-2 replication in GICF. Co-localization experiments and immunoprecipitation were performed to investigate the interaction between ORF23 and ORF141. siRNA interference experiments were conducted to evaluate the effect of silencing ORF23 and ORF141 on CyHV-2 replication. The inhibitory effect of hydroxyurea, a nucleotide reductase inhibitor, on CyHV-2 replication in GICF cells and RR enzymatic activity in vitro was also evaluated. ORF23 and ORF141 were identified as potential viral ribonucleotide reductase homologues in CyHV-2, and their transcription and translation levels increased with CyHV-2 replication. Co-localization experiments and immunoprecipitation suggested an interaction between the two proteins. Simultaneous silencing of ORF23 and ORF141 effectively inhibited the replication of CyHV-2. Additionally, hydroxyurea inhibited the replication of CyHV-2 in GICF cells and the in vitro enzymatic activity of RR. These results suggest that the CyHV-2 proteins ORF23 and ORF141 function as viral ribonucleotide reductase and their function makes an effect to CyHV-2 replication. Targeting ribonucleotide reductase could be a crucial strategy for developing new antiviral drugs against CyHV-2 and other herpesviruses.

Linearolactone Induces Necrotic-like Death in Giardia intestinalis Trophozoites: Prediction of a Likely Target.

Linearolactone (LL) is a neo-clerodane type diterpene that has been shown to exert giardicidal effects; however, its mechanism of action is unknown. This work analyzes the cytotoxic effect of LL on Giardia intestinalis trophozoites and identifies proteins that could be targeted by this active natural product. Increasing concentrations of LL and albendazole (ABZ) were used as test and reference drugs, respectively. Cell cycle progression, determination of reactive oxygen species (ROS) and apoptosis/necrosis events were evaluated by flow cytometry (FCM). Ultrastructural alterations were analyzed by transmission electron microscopy (TEM). Ligand–protein docking analyses were carried out using the LL structure raised from a drug library and the crystal structure of an aldose reductase homologue (GdAldRed) from G. intestinalis. LL induced partial arrest at the S phase of trophozoite cell cycle without evidence of ROS production. LL induced pronecrotic death in addition to inducing ultrastructural alterations as changes in vacuole abundances, appearance of perinuclear and periplasmic spaces, and deposition of glycogen granules. On the other hand, the in silico study predicted that GdAldRed is a likely target of LL because it showed a favored change in Gibbs free energy for this complex.

A Structural View of Alkyl-Coenzyme M Reductases, the First Step of Alkane Anaerobic Oxidation Catalyzed by Archaea.

Microbial anaerobic oxidation of alkanes intrigues the scientific community by way of its impact on the global carbon cycle, and its biotechnological applications. Archaea are proposed to degrade short- and long-chain alkanes to CO2 by reversing methanogenesis, a theoretically reversible process. The pathway would start with alkane activation, an endergonic step catalyzed by methyl-coenzyme M reductase (MCR) homologues that would generate alkyl-thiols carried by coenzyme M. While the methane-generating MCR found in methanogens has been well characterized, the enzymatic activity of the putative alkane-fixing counterparts has not been validated so far. Such an absence of biochemical investigations contrasts with the current explosion of metagenomics data, which draws new potential alkane-oxidizing pathways in various archaeal phyla. Therefore, validating the physiological function of these putative alkane-fixing machines and investigating how their structures, catalytic mechanisms, and cofactors vary depending on the targeted alkane have become urgent needs. The first structural insights into the methane- and ethane-capturing MCRs highlighted unsuspected differences and proposed some explanations for their substrate specificity. This Perspective reviews the current physiological, biochemical, and structural knowledge of alkyl-CoM reductases and offers fresh ideas about the expected mechanistic and chemical differences among members of this broad family. We conclude with the challenges of the investigation of these particular enzymes, which might one day generate biofuels for our modern society.

Reduced protein sequence patterns in identifying key structural elements of dissimilatory sulfite reductase homologs

Methanogenic archaea carry homologs of dissimilatory sulfite reductase (Dsr), called Dsr Like proteins (DsrLP). Dsr reduces sulfite to sulfide, a key step in an Earth’s ancient metabolic process called dissimilatory sulfate reduction. The DsrLPs do not function as Dsr, and a computational approach is needed to develop hypotheses for guiding wet bench investigations on DsrLP’s function. To make the computational analysis process efficient, the DsrLP amino acid sequences were transformed using only eight alphabets functionally representing twenty amino acids. The resultant reduced amino acid sequences were analyzed to identify conserved signature patterns in DsrLPs. Many of these patterns mapped on critical structural elements of Dsr and some were associated tightly with particular DsrLP groups. A search into the UniProtKB database identified several proteins carrying DsrLP’s signature patterns; cysteine desulfurase, nucleosidase, and uroporphyrinogen III methylase were such matches. These outcomes provided clues to the functions of DsrLPs and highlighted the utility of the computational approach used.

Genomic Features of the Bundle-Forming Heliobacterium Heliophilum fasciatum.

Eight species of heliobacteria have had their genomes sequenced. However, only two of these genomes have been analyzed in detail, those from the thermophilic Heliomicrobium (Hmi.) modesticaldum and the alkaliphilic Heliorestis (Hrs.) convoluta. Here we present analyses of the draft genome sequence of a species of heliobacterium that grows optimally at a moderate temperature and neutral pH. The organism, Heliophilum (Hph.) fasciatum, is phylogenetically unique among cultured heliobacteria and was isolated from rice soil, a common habitat for heliobacteria. The Hph. fasciatum genome contains 3.14 Mbp—similar to that of other reported heliobacteria—but has a G+C base ratio that lies between that of Hmi. modesticaldum and Hrs. convoluta. Many of the genomic features of Hmi. modesticaldum and Hrs. convoluta, such as the absence of genes encoding autotrophic pathways, the presence of a superoperonal cluster of photosynthesis-related genes, and genes encoding endospore-specific proteins, are also characteristic of the Hph. fasciatum genome. However, despite the fact that Hph. fasciatum is diazotrophic, classical nif genes encoding the alpha and beta subunits of dinitrogenase (nifDK) present in other heliobacteria could not be identified. Instead, genes encoding several highly divergent NifDK homologs were present, at least one of which likely encodes a functional dinitrogenase and another a methylthio-alkane reductase (MarDK) for sulfur assimilation. A classical NifH (dinitrogenase reductase) homolog was also absent in Hph. fasciatum, but a related protein was identified that likely carries out this function as well as electron delivery to MarDK. The N2-fixing system of Hph. fasciatum is therefore distinct from that of other heliobacteria and may have unusual properties.

The Overexpression of YALI0B07117g Results in Enhanced Erythritol Synthesis from Glycerol by the Yeast Yarrowia lipolytica.

The unconventional yeast Yarrowia lipolytica is used to produce erythritol from glycerol. In this study, the role of the erythrose reductase (ER) homolog YALI0B07117g in erythritol synthesis was analyzed. The deletion of the gene resulted in an increased production of mannitol (308%) and arabitol (204%) before the utilization of these polyols began. The strain overexpressing the YALI0B07117g gene was used to increase the erythritol yield from glycerol as a sole carbon source in batch cultures, resulting in a yield of 0.4 g/g. The specific consumption rate (qs) increased from 5.83 g/g/L for the WT strain to 8.49 g/g/L for the modified strain and the productivity of erythritol increased from 0.28 g/(L h) for the A101 strain to 0.41 g/(L h) for the modified strain. The application of the research may prove positive for shortening the cultivation time due to the increased rate of consumption of the substrate combined with the increased parameters of erythritol synthesis.

A Potential Role of Coumestrol in Soybean Leaf Senescence and Its Interaction With Phytohormones.

Coumestrol is a natural organic compound synthesized in soy leaves and functions as a phytoalexin. The coumestrol levels in plants are reported to increase upon insect attack. This study investigates the correlation between coumestrol, senescence, and the effect of phytohormones on the coumestrol levels in soybean leaves. Our analysis involving high-performance liquid chromatography and 2-D gel electrophoresis indicated a significant difference in the biochemical composition of soybean leaves at various young and mature growth stages. Eight chemical compounds were specifically detected in young leaves (V1) only, whereas three different coumestans isotrifoliol, coumestrol, and phaseol were detected only in mature, yellow leaves of the R6 and R7 growth stage. MALDI-TOF-MS analysis was used to identify two proteins 3,9 -dihydroxypterocarpan 6A-monooxygenase (CYP93A1) and isoflavone reductase homolog 2 (IFR2) only in mature leaves, which are key components of the coumestrol biosynthetic pathway. This indicates that senescence in soybean is linked to the accumulation of coumestrol. Following the external application of coumestrol, the detached V1-stage young leaves turned yellow and showed an interesting development of roots at the base of the midrib. Additionally, the application of phytohormones, including SA, methyl jasmonate (MeJA), and ethephon alone and in various combinations induced yellowing within 5 days of the application with a concomitant significant increase in endogenous coumestrol accumulation. This was also accompanied by a significant increase in the expression of genes CYP81E28 (Gm08G089500), CYP81E22 (Gm16G149300), GmIFS1, and GmIFS2. These results indicate that various coumestans, especially coumestrol, accumulate during leaf maturity, or senescence in soybean.

Possible Involvement of a Tetrathionate Reductase Homolog in Dissimilatory Arsenate Reduction by Anaeromyxobacter sp. Strain PSR-1.

Anaeromyxobacter sp. strain PSR-1, a dissimilatory arsenate [As(V)]-reducing bacterium, can utilize As(V) as a terminal electron acceptor for anaerobic respiration. A previous draft genome analysis revealed that strain PSR-1 lacks typical respiratory As(V) reductase genes (arrAB), which suggested the involvement of another protein in As(V) respiration. Dissimilatory As(V) reductase activity of strain PSR-1 was induced under As(V)-respiring conditions and was localized predominantly in the periplasmic fraction. The activity was visualized by partially denaturing gel electrophoresis, and liquid chromatography-tandem mass spectrometry analysis identified proteins involved in the active band. Among these proteins, a protein annotated as molybdopterin-dependent oxidoreductase (PSR1_00330) exhibited the highest sequence coverage, 76%. Phylogenetic analysis revealed that this protein was a homolog of tetrathionate reductase catalytic subunit TtrA. However, the crude extract of strain PSR-1 did not show significant tetrathionate reductase enzyme activity. Comparative proteomic analysis revealed that the protein PSR1_00330 and a homolog of tetrathionate reductase electron transfer subunit TtrB (PSR1_00329) were expressed abundantly and specifically under As(V)-respiring conditions, respectively. The genes encoding PSR1_00330 and PSR1_00329 formed an operon-like structure along with a gene encoding a c-type cytochrome (cyt c), and their transcription was upregulated under As(V)-respiring conditions. These results suggest that the protein PSR1_00330, which lacks tetrathionate reductase activity, functions as a dissimilatory As(V) reductase in strain PSR-1. Considering the wide distribution of TtrA homologs among bacteria and archaea, they may play a hitherto unknown role along with conventional respiratory As(V) reductase (Arr) in the biogeochemical cycling of arsenic in nature.IMPORTANCE Dissimilatory As(V)-reducing prokaryotes play significant roles in arsenic release and contamination in groundwater and threaten the health of people worldwide. Generally, such prokaryotes reduce As(V) by means of a respiratory As(V) reductase designated Arr. However, some dissimilatory As(V)-reducing prokaryotes such as Anaeromyxobacter sp. strain PSR-1 lack genes encoding Arr, suggesting the involvement of other protein in As(V) reduction. In this study, using multiple proteomic and transcriptional analyses, it was found that the dissimilatory As(V) reductase of strain PSR-1 was a protein closely related to the tetrathionate reductase catalytic subunit (TtrA). Tetrathionate reductase is known to play a role in anaerobic respiration of Salmonella on tetrathionate, but strain PSR-1 showed neither growth on tetrathionate nor significant tetrathionate reductase enzyme activity. These results suggest the possibility that TtrA homologs encoded in a wide variety of archaeal and bacterial genomes might function as dissimilatory As(V) reductases.

Molecular and functional characterization of a mitochondrial glutathione reductase homolog from redlip mullet (Liza haematocheila): Disclosing its antioxidant properties in the fish immune response mechanism

Glutathione reductase (GSHR) is a biologically important enzyme involved in the conversion of oxidized glutathione (GSSG) into its reduced form, reduced glutathione (GSH), with the catalytic activity of NADPH. Most animals and aquatic organisms, including fish, possess high levels of this enzyme system to neutralize oxidative stress in cells. The current study was conducted to broaden our knowledge of GSHR in fish by identifying a mitochondrial isoform of this enzyme (LhGSHRm) in redlip mullet, Liza haematocheila, and clarifying its structure and function. The complete open reading frame of LhGSHRm consists of 1527 base pairs, encoding 508 amino acids, with a predicted molecular weight of 55.43 kDa. Multiple sequence alignment revealed the conservation of important amino acids in this fish. Phylogenetic analysis demonstrated the closest evolutionary relationship between LhGSHRm and other fish GSHRm counterparts. In tissue distribution analysis, the highest mRNA expression of LhGSHRm was observed in the gill tissue under normal physiological conditions. Following pathogenic challenges, the LhGSHRm transcription level was upregulated in a time-dependent manner in the gill and liver tissues, which may modulate the immune reaction against pathogens. rLhGSHRm showed considerable glutathione reductase activity in an enzyme assay. Further, the biological activity of rLhGSHRm in balancing cellular oxidative stress was observed in both disk diffusion and DPPH assays. Collectively, these results support that LhGSHRm has profound effects on modulating the immune reaction in fish to sustain precise redox homeostasis.

Altering potato isoprenoid metabolism increases biomass and induces early flowering

Isoprenoids constitute the largest class of plant natural products and have diverse biological functions including in plant growth and development. In potato (Solanum tuberosum), the regulatory mechanism underlying the biosynthesis of isoprenoids through the mevalonate pathway is unclear. We assessed the role of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) homologs in potato development and in the metabolic regulation of isoprenoid biosynthesis by generating transgenic lines with down-regulated expression (RNAi-hmgr) or overexpression (OE) of one (StHMGR1 or StHMGR3) or two genes, HMGR and farnesyl diphosphate synthase (FPS; StHMGR1/StFPS1 or StHMGR3/StFPS1). Levels of sterols, steroidal glycoalkaloids (SGAs), and plastidial isoprenoids were elevated in the OE-HMGR1, OE-HMGR1/FPS1, and OE-HMGR3/FPS1 lines, and these plants exhibited early flowering, increased stem height, increased biomass, and increased total tuber weight. However, OE-HMGR3 lines showed dwarfism and had the highest sterol amounts, but without an increase in SGA levels, supporting a rate-limiting role for HMGR3 in the accumulation of sterols. Potato RNAi-hmgr lines showed inhibited growth and reduced cytosolic isoprenoid levels. We also determined the relative importance of transcriptional control at regulatory points of isoprenoid precursor biosynthesis by assessing gene-metabolite correlations. These findings provide novel insights into specific end-products of the sterol pathway and could be important for crop yield and bioenergy crops.

Pinoresinol‐lariciresinol reductase: Substrate versatility, enantiospecificity, and kinetic properties

Two western red cedar pinoresinol-lariciresinol reductase (PLR) homologues were studied to determine their enantioselective, substrate versatility, and kinetic properties. PLRs are downstream of dirigent protein engendered, coniferyl alcohol derived, stereoselective coupling to afford entry into the 8- and 8'-linked furofuran lignan, pinoresinol. Our investigations showed that each PLR homolog can enantiospecifically metabolize different furofuran lignans with modified aromatic ring substituents, but where phenolic groups at both C4/C4' are essential for catalysis. These results are consistent with quinone methide intermediate formation in the PLR active site. Site-directed mutagenesis and kinetic measurements provided additional insight into factors affecting enantioselectivity and kinetic properties. From these data, PLRs can be envisaged to allow for the biotechnological potential of generation of various lignan skeleta, that could be differentially "decorated" on their aromatic ring substituents, via the action of upstream dirigent proteins.

A Variation in FGF14 Is Associated with Downbeat Nystagmus in a Genome-Wide Association Study

Downbeat nystagmus (DBN) is a frequent form of acquired persisting central fixation nystagmus, often associated with other cerebellar ocular signs, such as saccadic smooth pursuit or gaze-holding deficits. Despite its distinct clinical features, the underlying etiology of DBN often remains unclear. Therefore, a genome-wide association study (GWAS) was conducted in 106 patients and 2609 healthy controls of European ancestry to identify genetic variants associated with DBN. A genome-wide significant association (p < 5 × 10−8) with DBN was found for a variation on chromosome 13 located within the fibroblast growth factor 14 gene (FGF14). FGF14 is expressed in Purkinje cells (PCs) and a reduction leads to a decreased spontaneous firing rate and excitability of PCs, compatible with the pathophysiology of DBN. In addition, mutations in the FGF14 gene cause spinocerebellar ataxia type 27. Suggestive associations (p < 1 × 10−05) could be detected for 15 additional LD-independent loci, one of which is also located in the FGF14 gene. An association of a region containing the dihydrofolate reductase (DHFR) and MutS Homolog 3 (MSH3) genes on chromosome 5 was slightly below the genome-wide significance threshold. DHFR is relevant for neuronal regulation, and a dysfunction is known to induce cerebellar damage. Among the remaining twelve suggestive associations, four genes (MAST4, TPPP, FTMT, and IDS) seem to be involved in cerebral pathological processes. Thus, this GWAS analysis has identified a potential genetic contribution to idiopathic DBN, including suggestive associations to several genes involved in postulated pathological mechanisms of DBN (i.e., impaired function of cerebellar PCs).

Efficient and Low-Cost Error Removal in DNA Synthesis by a High-Durability MutS.

Enzyme-based error correction is a key step in de novo DNA synthesis, yet the inherent instability of error-correction enzymes such as MutS has hindered the throughput and efficiency of DNA synthesis workflows. Here we introduce a process called Improved MICC (iMICC), in which all error-correction steps of oligos and fragments within a complete gene-synthesis cycle are completed in a simple, efficient, and low-cost manner via a MutS protein engineered for high durability. By establishing a disulfide bond of L157C-G233C, full-activity shelf life of E. coli MutS (eMutS) was prolonged from 7 to 49 days and was further extended to 63 days via cellulose-bound 4 °C storage. In synthesis of 10 Cas9 homologues in-solution and 10 xylose reductase (XR) homologues on-chip, iMICC reduced error frequency to 0.64/Kb and 0.41/Kb, respectively, with 72.1% and 86.4% of assembled fragments being error-free. By elevating base accuracy by 37.6-fold while avoiding repetitive preparation of fresh enzymes, iMICC is more efficient and robust than the wild-type eMutS, and it is 6.6-fold more accurate and 26.7-fold cheaper than CorrectASE. These advantages promise its broad applications in industrial DNA synthesis.

Phylogenomic analysis of novel Diaforarchaea is consistent with sulfite but not sulfate reduction in volcanic environments on early Earth

The origin(s) of dissimilatory sulfate and/or (bi)sulfite reducing organisms (SRO) remains enigmatic despite their importance in global carbon and sulfur cycling since at least 3.4 Ga. Here, we describe novel, deep-branching archaeal SRO populations distantly related to other Diaforarchaea from two moderately acidic thermal springs. Dissimilatory (bi)sulfite reductase homologs, DsrABC, encoded in metagenome assembled genomes (MAGs) from spring sediments comprise one of the earliest evolving Dsr lineages. DsrA homologs were expressed in situ under moderately acidic conditions. MAGs lacked genes encoding proteins that activate sulfate prior to (bi)sulfite reduction. This is consistent with sulfide production in enrichment cultures provided sulfite but not sulfate. We suggest input of volcanic sulfur dioxide to anoxic spring-water yields (bi)sulfite and moderately acidic conditions that favor its stability and bioavailability. The presence of similar volcanic springs at the time SRO are thought to have originated (>3.4 Ga) may have supplied (bi)sulfite that supported ancestral SRO. These observations coincide with the lack of inferred SO42− reduction capacity in nearly all organisms with early-branching DsrAB and which are near universally found in hydrothermal environments.

Ultrastructural and proapoptotic-like effects of kaempferol in Giardia duodenalis trophozoites and bioinformatics prediction of its potential protein target.

BACKGROUND Kaempferol (KPF) is a flavonoid with antiparasitic activity including experimental giardiasis which mechanism of action is unknown.OBJECTIVE To analyse the cytotoxic effects of KPF on Giardia duodenalis trophozoites and to identify a likely parasite target of this compound.METHODS We used inhibitory concentrations of KPF (IC25, IC50 and IC100) and albendazole (ABZ) as reference drug. The ultrastructure of the trophozoites was analysed by transmission electron microscopy (TEM) whilst apoptosis/necrosis, production of reactive oxygen species (ROS) and cell cycle progression were assessed by flow cytometry (FCM) and confocal laser microscopy (CLM). Ligand-protein docking analyses were carried out using KPF structure from a drug library and crystal structure of a G. duodenalis aldose reductase (GdAldRed) homolog.RESULTS KPF provoked appearance of perinuclear and periplasmic spaces devoid of cytosolic content and multilamellar structures. KPF induced proapoptotic death associated with partial arrest in the S phase without ROS production. Bioinformatics approaches predicted that GdAldRed is a viable KPF target (ΔG = -7.09 kCal/mol), exhibiting 92% structural identity and a similar coupling pattern as its human homolog.CONCLUSIONS KPF exerted a proapoptotic effect on G. duodenalis trophozoites involving partial interruption of DNA synthesis without oxidative stress or structure damage to chromatin and cytoskeletal structures. GdAldRed is a likely target underlying its antigiardial activity.

Can Indirect Herbicide Exposure Modify the Response of the Colorado Potato Beetle to an Organophosphate Insecticide?

Organisms live in complex multivariate environments. In agroecosystems, this complexity is often human-induced as pest individuals can be exposed to many xenobiotics simultaneously. Predicting the effects of multiple stressors can be problematic, as two or more stressors can have interactive effects. Our objective was to investigate whether indirect glyphosate-based herbicide (GBH) exposure of the host plant has interactive effects in combination with an insecticide (azinphos-methyl) on an invasive pest Colorado potato beetle (Leptinotarsa decemlineata Say). We tested the effects of GBH and insecticide on the survival, insecticide target genes expression (acetylcholinesterase genes) and oxidative status biomarkers (glutathione S-transferase [GST], glucose-6-phosphate dehydrogenase [G6PDH], glutathione reductase homolog [GR], glutathione peroxidase homolog [GPx], total glutathione [totGSH], glutathione reduced-oxidized [GSH: GSSG], catalase [CAT], superoxide dismutase [SOD], lipid hydroperoxides). We found that exposure to indirect GBH has no single or interactive effects in combination with the insecticide on larval survival. However, prior exposure to GBH inhibits Ldace1 gene expression by 0.55-fold, which is the target site for the organophosphate and carbamate insecticides. This difference disappears when individuals are exposed to both GBH and insecticide, suggesting an antagonistic effect. On the other hand, oxidative status biomarker scores (PCAs of GPx, GR, and CAT) were decreased when exposed to both stressors, indicating a synergistic effect. Overall, we found that indirect GBH exposure can have both antagonistic and synergistic effects in combination with an insecticide, which should be considered when aiming for an ecologically relevant risk assessment of multiple human-induced stressors.

New insight into the nitrogen metabolism of simultaneous heterotrophic nitrification-aerobic denitrification bacterium in mRNA expression.

Here, the draft genome of simultaneous nitrification-denitrification strain (SND) Klebsiella sp. KSND revealed possible existence of genes involved in N-assimilation and -dissimilation pathways. The change levels of genes under defined N-sources were analyzed by Quantitative Real-Time PCR. It suggested that NH4+-assimilation via NADP-glutamate dehydrogenase pathway would occur preferentially. NirBD genes were tightly regulated in a lower level, so that nitrite was rapidly consumed for detoxication by denitrification. Three types of nitrate reductase homologues are surprisingly present in KSND, whereas the dominant nitrate reduction for assimilation and denitrification processes mediates by NapA-type nitrate reductase. Nitric oxide reductase homologues FlRd and FlRd-red provide an adequate capacity for NO detoxification. The recombinant hydroxylamine reductase showed high activity in hydroxylamine to generate ammonium, which might contribute to detoxification mechanism in nitrogen cycling. Overall, this study firstly provides valuable insights into the genes expression and enzyme action, which helps understanding the mechanism of SND processes.

Efficient nitrogen removal by simultaneous heterotrophic nitrifying-aerobic denitrifying bacterium in a purification tank bioreactor amended with two-stage dissolved oxygen control

Nitrogen removal performance of a simultaneous heterotrophic nitrifying-aerobic denitrifying (SND) bacterium (KSND) in a purification tank bioreactor (PTBR) amended with two-stage dissolved oxygen (DO) control was investigated. NH4+–N and total nitrogen (TN) removal efficiencies under aerobic conditions for domestic wastewater treatment were 97.12% and 52.64%, respectively. Under serial aerobic (DO > 4.0 mg/L) and anaerobic (DO < 0.5 mg/L) phases, average TN removal efficiency from effluent was 95.45%, without nitrate and nitrite accumulation. DO control assay demonstrated that anaerobic condition adversely affected nitrification (46.13%), but was conducive to denitrification (93.52%). Transcriptional analysis revealed 2.72-fold increase in hydroxylamine reductase expression under aerobic condition as compared to anaerobic condition. Nitrate reductase and nitric oxide reductase homologs had the additional activity of supporting anaerobic or aerobic denitrification in SND bacteria. Under two-stage DO control, KSND maintained high abundance in oligotrophic PTBR, removing 87.88% TN from low-carbon to nitrogen domestic sewage in 180-days.

Substrate Specificity Analysis of Dihydrofolate/Dihydromethanopterin Reductase Homologs in Methylotrophic α-Proteobacteria.

Methane-producing archaea and methylotrophic bacteria use tetrahydromethanopterin (H4MPT) and/or tetrahydrofolate (H4F) as coenzymes in one-carbon (C1) transfer pathways. The α-proteobacterium Methylobacterium extorquens AM1 contains a dihydromethanopterin reductase (DmrA) and two annotated dihydrofolate reductases (DfrA and DfrB). DmrA has been shown to catalyze the final step of H4MPT biosynthesis; however, the functions of DfrA and DfrB have not been examined biochemically. Moreover, sequence alignment (BLAST) searches have recognized scores of proteins that share up to 99% identity with DmrA but are annotated as diacylglycerol kinases (DAGK). In this work, we used bioinformatics and enzyme assays to provide insight into the phylogeny and substrate specificity of selected Dfr and DmrA homologs. In a phylogenetic tree, DmrA and homologs annotated as DAGKs grouped together in one clade. Purified histidine-tagged versions of the annotated DAGKs from Hyphomicrobium nitrativorans and M. nodulans (respectively, sharing 69 and 84% identity with DmrA) showed only low activity in phosphorylating 1,2-dihexanoyl-sn-glycerol when compared with a commercial DAGK from Escherichia coli. However, the annotated DAGKs successfully reduced a dihydromethanopterin analog (dihydrosarcinapterin, H2SPT) with kinetic values similar to those determined for M. extorquens AM1 DmrA. DfrA and DfrB showed little or no ability to reduce H2SPT under the conditions studied; however, both catalyzed the NADPH-dependent reduction of dihydrofolate. These results provide the first evidence that DfrA and DfrB function as authentic dihydrofolate reductases, while DAGKs with greater than 69% identity to DmrA may be misannotated and are likely to function in H4MPT biosynthesis.