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Green waste composting materials and finished composts were collected from different commercial ex situ composting sites all treating source segregated green waste feedstocks. Stability of each material was determined using the standard ORG0020 dynamic respiration test. To assess whether stability could be used as an indicator for the potential suitability of green waste composting materials and finished composts as amendments for soil bioremediation, comparison was made with alkane and aromatic hydrocarbon degrader abundance determined using a quantitative PCR (qPCR) approach. Specifically, primers targeting alkB and, polyaromatic hydrocarbon ring-hydroxylating dioxygenases genes (PAH-RHD) of Gram positive (GP) and Gram negative (GN) populations were used for qPCR analysis. The results showed no direct correction between compost stability and gene abundance. Further, increase in alkB gene abundance was not linked to PAH-RHD gene abundance. The results support the use of qPCR as a tool for screening organic amendments on a site by site basis for soil bioremediation treatment.

RNA contains diverse modifications that exert important influences in a variety of cellular processes. So far more than 150 modifications have been identified in various RNA species, mainly in rRNA...

Aim of present work was to assess in-planta association potential of isolated endophytic bacterial strain Pseudomonas sp. (J10) (KY608252) with two cultivars of Lolium perenne L. (small & jumbo) and Arabidopsis thaliana L. for total petroleum hydrocarbon (TPH) degradation, alkane monooxygenase (alkb) gene expression and phytotoxicity analysis. A plant-microbe phytoremediation system was established to investigate the bacteria's ability to colonize the plant body and quantification of alkb gene to help withstand TPH stress in soil as well as in hydroponics. A real-time PCR method was developed to analyze bacterial colonization and survival within the plant body. Analysis revealed that J10 efficiently colonized all the tested plant species and expressed alkb gene under hydrocarbon stress ranging between 3.7 × 102–3.9 × 106 in A. thaliana and L. perenne (small), respectively. The colonization was more pronounced in soil as compared to hydroponic system. J10 inoculation reduced phytotoxicity and suggested that inoculation had a positive effect on plant growth under stress conditions as compared to control. L. perenne (small) showed significant TPH removal efficiency (45.6%) followed by L. perenne jumbo (24.5%) and A. thaliana (6.2%). In hydroponics, L. perenne (small) degraded about 28.2% TPH followed by L. perenne (jumbo) as 24.4%. Potential of the indigenously isolated plant endophytes may be exploited further for phytoremediation efficiency and industrial applications.

5-Methylcytosine (5mC) in DNA CpG islands is an important epigenetic biomarker for mammalian gene regulation. It is oxidized to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC) by the ten-eleven translocation (TET) family enzymes, which are α-ketoglutarate (α-KG)/Fe(II)-dependent dioxygenases. In this work, we demonstrate that the epigenetic marker 5mC is modified to 5hmC, 5fC, and 5caC in vitro by another class of α-KG/Fe(II)-dependent proteins—the DNA repair enzymes in the AlkB family, which include ALKBH2, ALKBH3 in huamn and AlkB in Escherichia coli. Theoretical calculations indicate that these enzymes may bind 5mC in the syn-conformation, placing the methyl group comparable to 3-methylcytosine, the prototypic substrate of AlkB. This is the first demonstration of the AlkB proteins to oxidize a methyl group attached to carbon, instead of nitrogen, on a DNA base. These observations suggest a broader role in epigenetics for these DNA repair proteins.

The annual production of plastics has doubled over the past 15 years and, consequently, a large amount of plastic has accumulated in the environment generating ecological problems. In this study, a Paenibacillus sp. isolate was obtained from a landfill from Brazil and it presented the alkane hydroxylase gene (alkB). Weight loss of low-density polyethylene (LDPE) was measured and a significant difference in final weight compared to initial weight was assessed. Some chemical characteristics, such as bond scissions and formation of new functional groups [carboxylic acids (3300–2500 cm−1), esters (1210–1163 cm−1), and ethers (1075–1020 cm−1)], were detected by Fourier-transform infrared spectroscopy. Bacterial colonization on the plastic surface and physical changes, as formation of cracks and pits, was visualized by scanning electron microscopy. This isolate was susceptible to all the antimicrobials tested. Therefore, this isolate possesses great potential to degrade polyethylene and become an option for LDPE bioremediation.

With the intensive development of the sheep industry and increasing global temperatures, heat stress in sheep has become an increasingly severe and important issue in recent years. The level of N6-methyladenosine (m6A) RNA methylation changes in response to stress plays important roles in stress responses. However, the role of m6A in the heat stress response of sheep remains unclear. To explore this issue, we measured heat stress protein (HSP) expression, liver function indexes, m6A on RNA, m6A-related enzyme expression, and tissue damage in sheep that had been subjected to heat stress. At the transcriptome level, our results showed significant increases in m6A on RNA and increased mRNA levels of HSPs (HSP70, HSP90, and HSP110) and m6A-related enzymes [METTL3 (methyltransferase-like 3), METTL14 (methyltransferase-like 14), WTAP (wilms tumor 1-associated protein), FTO (fat mass and obesity-associated protein), ALKBH5 (alkB homologue 5), YTHDF1-3 (YTH domain family proteins), and YTHDC1-2 (YTH domain-containing proteins)] following heat stress. At the protein level, the expression of METTL3, YTHDF1-2, and YTHDC2 showed no significant differences following heat stress. However, in contrast to its mRNA level after heat stress, the protein expression of YTHDF3 was reduced, while the expression of HSPs (HSP70, HSP90, and HSP110), METTL14, WTAP, FTO, ALKBH5, YTHDF3, and YTHDC1 increased in line with their measured mRNA levels. Histological experiments revealed that heat stress caused varying degrees of damage to sheep liver tissue. Moreover, immunohistochemical staining indicated that the m6A-related enzymes were expressed in sheep hepatocytes, and differences in expression patterns were observed between the control and heat stress groups. In summary, differences in the level of m6A and the expression of m6A-related enzymes in the liver of sheep were observed after heat stress. This indicates that m6A is involved in the regulation of heat stress in sheep. Our findings provide a new avenue for studying the responses to heat stress in sheep.

Studding the diversity of soil indigenous microorganisms, and monitoring effect of contaminants on microbial population, is very critical for understanding microbial activity during bioremediation and selecting successful remediation strategy. To simulate the natural environment, four microcosms were prepared by artificially contaminating clean soil with defined amounts of petroleum hydrocarbons including alkanes mixture (C13–C20), polyaromatic hydrocarbons (PAHs) mixture (anthracene, phenanthrene, fluoranthene, pyrene and benzo (α) pyrene) and both alkanes and PAHs. Contaminants degradation and heterotrophic bacterial count were measured during a 6-month study. Copy number of alkB and C23DO genes was studied using real-time PCR, and bacterial diversity was monitored by 16S rRNA gene PCR and denaturing gradient gel electrophoresis (DGGE). Results indicated that all types of contaminants (except the five ring benzo (α) pyrene) were totally degraded after 6 months and the increase in hydrocarbon degradation rate coincided with the enhancement of total heterotrophic bacterial count in each microcosm. Real-time PCR results showed a significant increase in the copy number of both alkB and C23DO genes in alkane- and PAHs-contaminated microcosm comparing with the control microcosm, indicating selection for special hydrocarbon degraders in hydrocarbon-amended microcosms. The results of DGGE revealed that the type of contaminant in the same soil has a remarkable influence on soil bacterial community structure. Sequencing of DGGE bands suggested that most of the dominant members of the microbial community of contaminated soil are unculturable bacteria from Proteobacteria and the genus Bacillus.

Aims The Algerian coastline is exposed to several types of pollution, including hydrocarbons. The aim of this work was to isolate oil-degrading bacteria and to explore the intrinsic bioremediation potential of part of its contaminated harbour. Methods and results A collection of 119 strains, capable to grow on mineral medium supplemented with hydrocarbons, were obtained from polluted sediment and seawater collected from Sidi Fredj harbour (Algiers). Twenty-three strains were selected for further studies. Sequencing of the 16S rRNA gene showed that most isolates belong to genera of hydrocarbonoclastic bacteria (Alcanivorax), generalist hydrocarbons degraders (Marinobacter, Pseudomonas, Gordonia, Halomonas, Erythrobacter and Brevibacterium) and other bacteria not known as hydrocarbon degraders (Xanthomarina) but were able to degrade hydrocarbons. Strains related to Marinobacter and Alcanivorax were frequently isolated from our samples and resulted the most effective in degrading crude oil. Screening of catabolic genes alkB and xylA revealed the presence of alkB gene in several bacterial strains; one isolate harboured both catabolic genes while other isolates carried none of the studied genes. However, they grew in the presence of crude oil implying the existence of other biodegradation pathways. Conclusions The samples of seawater and sediment from the Algerian coast contain high level of hydrocarbon-degrading bacteria that could be interesting and useful for future bioremediation purposes. Significance and impact of the study This investigation demonstrates the diversity of hydrocarbon-degrading bacteria from a marine-contaminated area in Algeria, and their variable biodegradation abilities.

N6-methyladenosine (m6A) is the most prevalent internal modification of eukaryotic messenger RNA (mRNA). Until now, two RNA demethylases have been identified, including FTO (fat mass and obesity-associated protein) and ALKBH5 (α-ketoglutarate-dependent dioxygenase alkB homologue 5). As a mammalian m6A demethylase, ALKBH5 significantly affects mRNA export and RNA metabolism as well as the assembly of mRNA processing factors in nuclear speckles, and ALKBH5 may play a significant role in these biological processes. Nevertheless, no modulator of ALKBH5 has been reported. The reason for that may be the lack of in vitro assays for ALKBH5 inhibitor screening. Herein, we describe the development of two homogeneous assays for ALKBH5 using N6-methyladenosine as substrate with different principles. Using ALKBH5 recombinant, we developed a formaldehyde dehydrogenase coupled fluorescence based assay and an antibody based assay for the activity evaluation of ALKBH5. These robust coupled assays are suitable for screening ALKBH5 inhibitors in 384-well format (Z' factors of 0.74), facilitating the discovery of modulators in the quest for the regulation of biological processes.

Recent studies suggested that the widespread presence of N1-methyladenosine (m1A) plays a very important role in environmental stress, ribosome biogenesis and antibiotic resistance. The RNA-guided, RNA-targeting CRISPR Cas13a exhibits a "collateral effect" of promiscuous RNase activity upon target recognition with high sensitivity. Inspired by the advantage of CRISPR Cas13a, we designed a system to detect m1A induced mismatch, providing a rapid, simple and fluorescence-based m1A detection. For A-ssRNA, the Cas13a-based molecular detection platform showed a high fluorescence signal. For m1A-ssRNA, there is an about 90% decline of fluorescence. Moreover, this approach can also be used to quantify m1A in RNAs and applied for the analysis of dynamic m1A demethylation of 28S rRNA with AlkB.

To investigate the hydrocarbon-degrading ability, biosurfactant-producing capacity, and alkane monooxygenase system of Leclercia spp. A bacterial strain classified as Leclercia sp. B45 was isolated, and its biosurfactant-producing capacity, hydrocarbon-degrading ability, and alkane hydroxylase (alkB) gene transcriptional patterns were evaluated by TLC, FTIR, GC-MS, and RT-qPCR, respectively. Strain B45 showed active biosurfactant-producing ability, which was preferentially induced by C16. The extracted biosurfactant tolerated a wide range of salinity, pH, and temperature. The degradation rate of n-decane (C10), n-hexadecane (C16), and octacosane (C28) by strain B45 could reach 92.6%, 94.1%, and 67.8%, respectively. Furthermore, the alkB transcription levels in the strain B45 with C10, C16, or C28 as a carbon source were distinctly higher than those of the control group during the late exponential and stationary phases. The relative alkB transcript copy number decreased with the increase in alkane chain length, which is consistent with B45 strain biodegradation kinetics. Leclercia sp. B45 showed excellent n-alkane degradation performance and biosurfactant-producing capacity. Meanwhile, the alkB gene in Leclercia sp. B45 is likely to represent a novel gene, whose transcription level was significantly upregulated when induced by n-alkane. These results provide new insights into alkane metabolism mechanism in Leclercia sp. B45.

AlkBH1 is a member of the AlkB superfamily which are kinds of Fe (II) and α-ketoglutarate (α-KG)-dependent dioxygenases. At present, only demethyltransferases FTO and AlkBH5 have relatively clear substrate studies among these members, the types and mechanisms of substrates catalysis of other members are not clear, especially the demethyltransferase AlkBH1. AlkBH1, as a demethylase, has important functions of reversing DNA methylation and repairing DNA damage. And it has become a promising target for the treatment of many cancers, the regulation of neurological and genetic related diseases. Many scholars have made important discoveries in the diversity of AlkBH1 substrates, but there is no comprehensive summary, which affects the design inhibitor target of AlkBH1. Herein, We are absorbed in the latest progress in the study of AlkBH1 substrate diversity and its relationship with human diseases. Besides, we also discuss future research directions and suggest other studies to reveal the specific catalytic effect of AlkBH1 on cancer substrates.

The combination of phage display technology with high-throughput sequencing enables in-depth analysis of library diversity and selection-driven dynamics. We applied short-read sequencing of the mutagenized region on focused display libraries of two homologous nucleic acid modification eraser proteins-AlkB and FTO-biopanned against methylated DNA. This revealed enriched genotypes with small indels and concomitant doubtful amino acid motifs within the FTO library. Nanopore sequencing of the entire display vector showed additional enrichment of large deletions overlooked by region-specific sequencing, and further impacted the interpretation of the obtained amino acid motifs. We could attribute enrichment of these corrupted clones to amplification bias due to arduous FTO display slowing down host cell growth as well as phage production. This amplification bias appeared to be stronger than affinity-based target selection. Recommendations are provided for proper sequence analysis of phage display data, which can improve motive discovery in libraries of proteins that are difficult to display. ispartof: INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES vol:22 issue:11 ispartof: location:Switzerland status: published

tRNAs can exist in distinct isoforms because of different chemical modifications, which confounds attempts to accurately sequence individual tRNA species using next generation sequencing approaches or to quantify different RNA modifications at specific sites on a tRNA strand. Herein, we develop a mass spectrometric (MS) ladder complementation sequencing (MLC-Seq), allowing for direct and simultaneous sequencing of full-length tRNA molecules, including those with low abundance. MLC-seq is achieved by improved instrumentation, and advanced algorithms that identify each tRNA species and related isoforms in an RNA mixture, and assemble full MS ladders from partial ladders with missing ladder components. Using MLC-Seq, we successfully obtained the sequence of tRNA-Phe from yeast and tRNA-Glu from mouse hepatocytes, and simultaneously revealed new tRNA isoforms derived from nucleotide modifications. Importantly, MLC-Seq pinpointed the location and stoichiometry changes of RNA modifications in tRNA-Glu upon the treatment of dealkylated enzyme AlkB, which confirmed its known enzymatic activity and suggested previously unidentified effects in RNA editing.

AlkB homolog 5 (ALKBH5) has been reported as a key m6A demethylase that is involved in development and diseases; however, the function of ALKBH5 in osteogenesis remains unknown. In this study, we report that ALKBH5 mRNA and protein expression were upregulated during osteoblast differentiation and that ALKBH5 knockdown suppressed osteoblast differentiation, mineralization, and the expression of osteogenic biomarkers. Conversely, ALKBH5 overexpression promoted osteogenesis. Moreover, the expression of wild-type ALKBH5, but not the m6A-modified active site mutant ALKBH5, could rescue ALKBH5 knockdown-induced osteogenesis inhibition. Furthermore, knockdown of ALKBH5 significantly impaired the mRNA stability of the transcription factor Runx2, which plays a key role in osteoblast differentiation. Taken together, our results suggest that ALKBH5 promotes osteogenesis through modulating Runx2 mRNA stability.

Institutional Review Board Statement: The experimental procedures were approved and conducted according to guidelines of the appropriate Polish Local Ethics Commission for Experiments on Animals No. 9 in Lodz (Agreement 9/ŁB87/2018). Acknowledgments: We thank Jeremy Rock and Sarah Fortune for providing us with the pLJR965 vector and detailed instructions for the generation of Cas9-regulated strains in M. tuberculosis. The authors thank the mass spectrometry service at the Institute of Biochemistry and Biophysics PAS in Warsaw for MS analysis. The MS analysis equipment used for the analysis was sponsored in part by the Centre for Preclinical Research and Technology (CePT), a project cosponsored by the European Regional Development Fund and Innovative Economy, the National Cohesion Strategy of Poland. Mycobacteria exploit at least two independent global systems in response to DNA damage: the LexA/RecA-dependent SOS response and the PafBC-regulated pathway. Intracellular pathogens, such as Mycobacterium tuberculosis, are exposed to oxidative and nitrosative stress during the course of infection while residing inside host macrophages. The current understanding of RecA-independent responses to DNA damage is based on the saprophytic model of Mycobacterium smegmatis, a free-living and nonpathogenic mycobacterium. The aim of the present study was to identify elements of RecA-independent responses to DNA damage in pathogenic intracellular mycobacteria. With the help of global transcriptional profiling, we were able to dissect RecA-dependent and RecA-independent pathways. We profiled the DNA damage responses of an M. tuberculosis strain lacking the recA gene, a strain with an undetectable level of the PafBC regulatory system, and a strain with both systems tuned down simultaneously. RNA-Seq profiling was correlated with the evaluation of cell survival in response to DNA damage to estimate the relevance of each system to the overall sensitivity to genotoxic agents. We also carried out whole-cell proteomics analysis of the M. tuberculosis strains in response to mitomycin C. This approach highlighted that LexA, a well-defined key element of the SOS system, is proteolytically inactivated during RecA-dependent DNA repair, which we found to be transcriptionally repressed in response to DNA-damaging agents in the absence of RecA. Proteomics profiling revealed that AlkB was significantly overproduced in the ΔrecA pafBCCRISPRi/dCas9 strain and that Holliday junction resolvase RuvX was a DNA damage response factor that was significantly upregulated regardless of the presence of functional RecA and PafBC systems, thus falling into a third category of DNA damage factors: RecA- and PafBC-independent. While invisible to the mass spectrometer, the genes encoding alkA, dnaB, and dnaE2 were significantly overexpressed in the ΔrecA pafBCCRISPRi/dCas9 strain at the transcript level. A.B. was supported by grant “OPUS” from the National Science Centre, Poland, UMO2015/19/B/NZ6/02978. P.P. was supported by grant “OPUS” from the National Science Centre, Poland, UMO-2019/33/B/NZ1/02770.

usc This study assessed the ability of phosphorus (P) fertilizer to remediate the rhizosphere of three wild plant species (Banksia seminuda, a tree; Chloris truncata, a grass; and Hakea prostrata, a shrub) growing in a soil contaminated with total (aliphatic) petroleum hydrocarbon (TPH). Plant growth, photosynthesis (via chlorophyll fluorescence), soil microbial activity, alkane hydroxylase AlkB (aliphatic hydrocarbon-degrading) gene abundance, and TPH removal were evaluated 120 days after planting. Overall, although TPH served as an additional carbon source for soil microorganisms, the presence of TPH in soil resulted in decreased plant growth and photosynthesis. However, growth, photosynthesis, microbial activities, and AlkB gene abundance were enhanced by the application of P fertilizer, thereby increasing TPH removal rates, although the extent and optimum P dosage varied among the plant species. The highest TPH removal (64.66%) was observed in soil planted with the Poaceae species, C. truncata, and amended with 100 mg P kg−1 soil, while H. prostrata showed higher TPH removal compared to the plant belonging to the same Proteaceae family, B. seminuda. The presence of plants resulted in higher AlkB gene abundance and TPH removal relative to the unplanted control. The removal of TPH was associated directly with AlkB gene abundance (R2 > 0.9, p < 0.001), which was affected by plant identity and P levels. The results indicated that an integrated approach involving wild plant species and optimum P amendment, which was determined through experimentation using different plant species, was an efficient way to remediate soil contaminated with TPH. Refereed/Peer-reviewed

Polyethylene-degrading bacteria have been emerging as a rational and safe alternative in bioremediation strategies. In this context, some Paenibacillus species produce enzymes involved in the biodegradation of pollutants. Among the enzymes involved in the biodegradation of polyethylene, the alkane hydroxylases, encoded by alkB homologous genes, play a key role in this process. Therefore, this study aimed to identify and perform a genomic investigation of the first polyethylene-degrading Paenibacillus sp. strain, named DK1. The whole-genome sequence-based analysis revealed that the DK1 strain belonged to the species Paenibacillus aquistagni and shared a total of 4327 CDSs with P. aquistagni strain 11. On the other hand, a comparison of the gene clusters showed that DK1 strain harbored a genetic context surrounding the alkB-like gene similar to that found in Pseudomonas sp. strains. The percentage of similarity ranged from 47.88 to 99.76% among all complete amino acid sequences of AlkB-like proteins analyzed. Nevertheless, the predicted amino acid sequences of AlkB-like contained typical structural motifs of alkane hydroxylases, such as His boxes and the HYG motif. These findings associated with the previously reported phenotypic results highlighted the potential of P. aquistagni strain DK1 to biodegrade polyethylene. Therefore, further studies focusing on the biochemical and structural properties of the AlkB-like protein from Paenibacillus may also contribute to the development of sustainable bioremediation strategies.

Alpha-ketoglutarate-dependent dioxygenase (ALKBH) is a DNA repair gene involved in the repair of alkylating DNA damage. There are nine types of ALKBH (ALKBH1-8 and FTO) identified in humans. In particular, certain types of ALKBH enzymes are dioxygenases that directly reverse DNA methylation damage via transfer of a methyl group from the DNA adduct onto α-ketoglutarate and release of metabolic products including succinate and formaldehyde. Here, we tested whether ALKBH6 plays a significant role in preventing alkylating DNA damage and decreasing genomic instability in pancreatic cancer cells. Using anE. colistrain deficient with ALKB, we found that ALKBH6 complements ALKB deficiency and increases resistance after alkylating agent treatment. In particular, the loss of ALKBH6 in human pancreatic cancer cells increases alkylating agent-induced DNA damage and significantly decreases cell survival. Furthermore,in silicoanalysis from The Cancer Genome Atlas (TCGA) database suggests that overexpression of ALKBH6 provides better survival outcomes in patients with pancreatic cancer. Overall, our data suggest that ALKBH6 is required to maintain the integrity of the genome and promote cell survival of pancreatic cancer cells.

The FTO protein is involved in a wide range of physiological processes, including adipogenesis and osteogenesis. This two-domain protein belongs to the AlkB family of 2-oxoglutarate (2-OG)- and Fe(II)-dependent dioxygenases, displaying N6-methyladenosine (N6-meA) demethylase activity. The aim of the study was to characterize the relationships between the structure and activity of FTO. The effect of cofactors (Fe2+/Mn2+ and 2-OG), Ca2+ that do not bind at the catalytic site, and protein concentration on FTO properties expressed in either E. coli (ECFTO) or baculovirus (BESFTO) system were determined using biophysical methods (DSF, MST, SAXS) and biochemical techniques (size-exclusion chromatography, enzymatic assay). We found that BESFTO carries three phosphoserines (S184, S256, S260), while there were no such modifications in ECFTO. The S256D mutation mimicking the S256 phosphorylation moderately decreased FTO catalytic activity. In the presence of Ca2+, a slight stabilization of the FTO structure was observed, accompanied by a decrease in catalytic activity. Size exclusion chromatography and MST data confirmed the ability of FTO from both expression systems to form homodimers. The MST-determined dissociation constant of the FTO homodimer was consistent with their in vivo formation in human cells. Finally, a low-resolution structure of the FTO homodimer was built based on SAXS data.

Polystyrene (PS), a major plastic waste, is difficult to biodegrade due to its unique chemical structure that comprises phenyl moieties attached to long linear alkanes. In this study, we investigated the biodegradation of PS by mesophilic bacterial cultures obtained from various soils in common environments. Two new strains, Pseudomonas lini JNU01 and Acinetobacter johnsonii JNU01, were specifically enriched in non-carbonaceous nutrient medium, with PS as the only source of carbon. Their growth after culturing in basal media increased more than 3-fold in the presence of PS. Fourier transform infrared spectroscopy analysis, used to confirm the formation of hydroxyl groups and potentially additional chemical bond groups, showed an increase in the amount of oxidized PS samples. Moreover, field emission scanning electron microcopy analysis confirmed PS biodegradation by biofilms of the screened microbes. Water contact angle measurement additionally offered insights into the increased hydrophilic characteristics of PS films. Bioinformatics and transcriptional analysis of A. johnsonii JNU01 revealed alkane-1-monooxygenase (AlkB) to be involved in PS biodegradation, which was confirmed by the hydroxylation of PS using recombinant AlkB. These results provide significant insights into the discovery of novel functions of Pseudomonas sp. and Acinetobacter sp., as well as their potential as PS decomposers.

High-throughput sequencing reveals the complex landscape of small noncoding RNAs (sRNAs). However, it is limited by requiring 5′-monophosphate and 3′-hydroxyl in RNAs for adapter ligation and hindered by methylated nucleosides that interfere with reverse transcription. Here we develop Cap-Clip acid pyrophosphatase (Cap-Clip), T4 polynucleotide kinase (PNK) and AlkB/AlkB(D135S)-facilitated small ncRNA sequencing (CPA-seq) to detect and quantify sRNAs with terminus multiplicities and nucleoside methylations. CPA-seq identified a large number of previously undetected sRNAs. Comparison of sRNAs with or without AlkB/AlkB(D135S) treatment reveals nucleoside methylations on sRNAs. Using CPA-seq, we profiled the sRNA transcriptomes (sRNomes) of nine mouse tissues and reported the extensive tissue-specific differences of sRNAs. We also observed the transition of sRNomes during hepatic reprogramming. Knockdown of mesenchymal stem cell-enriched U1-5′ snsRNA promoted hepatic reprogramming. CPA-seq is a powerful tool with high sensitivity and specificity for profiling sRNAs with methylated nucleosides and diverse termini.

Douglas Channel and the adjacent Hecate Strait (British Columbia, Canada) are part of a proposed route to ship diluted bitumen (dilbit). This study presents how two types of dilbit naturally degrade in this environment by using an in situ microcosm design based on dilbit-coated beads. We show that dilbit-associated n-alkanes were microbially biodegraded with estimated half-lives of 57–69 days. n-Alkanes appeared to be primarily degraded using the aerobic alkB, ladA and CYP153 pathways. The loss of dilbit polycyclic aromatic hydrocarbons (PAHs) was slower than of n-alkanes, with half-lives of 89–439 days. A biodegradation of PAHs could not be conclusively determined, although a significant enrichment of the phnAc gene (a marker for aerobic PAH biodegradation) was observed. PAH degradation appeared to be slower in Hecate Strait than in Douglas Channel. Microcosm-associated microbial communities were shaped by the presence of dilbit, deployment location and incubation time but not by dilbit type. Metagenome-assembled genomes of putative dilbit-degraders were obtained and could be divided into populations of early, late and continuous degraders. The majority of the identified MAGs could be assigned to the orders Flavobacteriales, Methylococcales, Pseudomonadales and Rhodobacterales. A high proportion of the MAGs represent currently unknown lineages or lineages with currently no cultured representative., This study investigated the biodegradation of diluted bitumen and the associated microbial communities by using a novel in situ microcosm design deployed at the coast of British Columbia (Canada).

Alkylated DNA repair protein AlkB homolog 8 (ALKBH8) is a member of the AlkB family of dioxygenases. ALKBH8 is a methyltransferase of the highly variable wobble nucleoside position in the anticodon loop of tRNA and thus plays a critical role in tRNA modification by preserving codon recognition and preventing errors in amino acid incorporation during translation. Moreover, its activity catalyzes uridine modifications that are proposed to be critical for accurate protein translation. Previously, two distinct homozygous truncating variants in the final exon of ALKBH8 were described in two unrelated large Saudi Arabian kindreds with intellectual developmental disorder and autosomal recessive 71 (MRT71) syndrome (MIM# 618504). Here, we report a third family-of Egyptian descent-harboring a novel homozygous frame-shift variant in the last exon of ALKBH8. Two affected siblings in this family exhibit global developmental delay and intellectual disability as shared characteristic features of MRT71 syndrome, and we further characterize their observed dysmorphic features and brain MRI findings. This description of a third family with a truncating ALKBH8 variant from a distinct population broadens the phenotypic and genotypic spectrum of MRT71 syndrome, affirms that perturbations in tRNA biogenesis can contribute to neurogenetic disease traits, and firmly establishes ALKBH8 as a novel neurodevelopmental disease gene.

DNA alkylation damage, emanating from the exposure to environmental alkylating agents or produced by certain endogenous metabolic processes, affects cell viability and genomic stability. Fe(II)/2-oxoglutarate-dependent dioxygenase enzymes, such as Escherichia coli AlkB, are involved in protecting DNA from alkylation damage. Inspired by the natural product indenone derivatives reported to inhibit this class of enzymes, and a set of 2-chloro-3-amino indenone derivatives was synthesized and screened for their inhibitory properties against AlkB. The synthesis of 2-chloro-3-amino indenone derivatives was achieved from 2,3-dichloro indenones through addition-elimination method using alkyl/aryl amines under catalyst-free conditions. Using an in vitro reconstituted DNA repair assay, we have identified a 2-chloro-3-amino indenone compound 3o to be an inhibitor of AlkB. We have determined the binding affinity, mode of interaction, and kinetic parameters of inhibition of 3o and tested its ability to sensitize cells to methyl methanesulfonate that mainly produce DNA alkylation damage. This study established the potential of indenone-derived compounds as inhibitors of Fe(II)/2-oxoglutarate-dependent dioxygenase AlkB.

Alkylated DNA lesions, induced by both exogenous chemical agents and endogenous metabolites, represent a major form of DNA damage in cells. The repair of alkylation damage is critical in all cells because such damage is cytotoxic and potentially mutagenic. Alkylation chemotherapy is a major therapeutic modality for many tumors, underscoring the importance of the repair pathways in cancer cells. Several different pathways exist for alkylation repair, including base excision and nucleotide excision repair, direct reversal by methyl-guanine methyltransferase (MGMT), and dealkylation by the AlkB homolog (ALKBH) protein family. However, maintaining a proper balance between these pathways is crucial for the favorable response of an organism to alkylating agents. Here, we summarize the progress in the field of DNA alkylation lesion repair and describe the implications for cancer chemotherapy.

DNA and RNA have various methylation modifications or damage that are directly related to some human diseases and physiological regulation. Most of these methylation modifications are reversible and can be dynamically repaired by RNA or DNA demethylases. Over the past few decades, enzymes from the ALKB and TET families have been shown to have the ability to demethylate nucleic acids, which involves intermediates in the oxidative repair process. These intermediates can be accurately captured by advanced methods such as HPLC, LC-MS, TLC, and crystallization, which can significantly promote our understanding of the dynamic mechanism of demethylation. In this review, we discuss recent research advances in this area and raise open questions and constructive opinions about the capture of nucleic acid demethylation intermediates.

Floating treatment wetlands (FTWs) are cost-effective systems for the remediation of polluted water. In FTWs, the metabolic activity of microorganisms associated with plants is fundamental to treatment efficiency. Bioaugmentation, the addition of microorganisms with pollutant-degrading capabilities, appears to be a promising means to enhance the treatment efficiency of FTWs. Here, we quantified the effect of bioaugmentation with a four-membered bacterial consortium on the remediation of water contaminated with crude oil in pilot-scale FTWs planted with Phragmites australis or Typha domingensis. The bacteria had been isolated from the endosphere and rhizosphere of various plants and carry the alkane hydroxylase gene, alkB, involved in aerobic hydrocarbon degradation. During a treatment period of 36 days, FTWs planted with P. australis achieved a reduction in hydrocarbon concentration from 300 mg/L to 16 mg/L with and 56 mg/L without bioaugmentation. In the FTWs planted with T. domingensis, respective hydrocarbon concentrations were 46 mg/L and 84 mg/L. The inoculated bacteria proliferated in the rhizoplane and in the plant interior. Copy numbers of the alkB gene and its mRNA increased over time in plant-associated samples, suggesting increased bacterial hydrocarbon degradation. The results show that bioaugmentation improved the treatment of oil-contaminated water in FTWs by at least a factor of two, indicating that the performance of full-scale systems can be improved at only small costs.

Ozdemir, Guven/0000-0002-7577-4233; SAHAR, Umut/0000-0002-2200-6986; Vural, Caner/0000-0003-1400-6377; DIALLO, Mamadou Malick/0000-0001-8264-2960, WOS: 000497848100001, PubMed: 31752596, Biodegradation is a cost-effective process commonly used to eliminate many xenobiotic hydrocarbons such as diesel oils. However, their hydrophobic character reduces the biodegradation efficiency. in order to overcome this hurdle, kurstakins isolated from Bacillus thuringiensis strain 7SA were used as emulsifying agents. the influence of kurstakin molecules on diesel oil degradation by Acinetobacter haemolyticus strain 2SA was evaluated in the presence and absence of the aforementioned lipopeptide. the degradation rates and gene expressions of alkane hydroxylases were evaluated at days 4, 10, 14 and 21. Results showed that kurstakin molecules increased the hydrophobicity of 2SA. Moreover, diesel oil degradation activities were higher in the presence of kurstakin with 29%, 35%, 29% and 23% improvement at 4th, 10th, 14th and 21st day respectively. Statistical analysis indicated that the difference between the degradation rates in the presence and absence of kurstakin was significant with p = 0.03. the detection of three different hydroxylase genes namely alkB, almA and cyp153 in 2SA genome, might have allowed more efficient degradability of alkanes. According to the real-time PCR results, cyp153 was the most induced gene during diesel oil degradation in the presence and absence of kurstakin. Yet, the three genes demonstrated higher levels of expression in the presence of kurstakin when compared to its absence. This study showed that kurstakins enhance the diesel oil biodegradation rate by increasing the hydrophobicity of 2SA. in addition to their anti-fungal activities, kurstakins can be used as biosurfactant to increase biodegradation of diesel oil.

AlkB is a DNA/RNA repair enzyme that removes base alkylations such as N1-methyladenosine (m1A) or N3-methylcytosine (m3C) from DNA and RNA. The AlkB enzyme has been used as a critical tool to facilitate tRNA sequencing and identification of mRNA modifications. As a tool, AlkB mutants with better reactivity and new functionalities are highly desired; however, previous identification of such AlkB mutants was based on the classical approach of targeted mutagenesis. Here, we introduce a high-throughput screening method to evaluate libraries of AlkB variants for demethylation activity on RNA and DNA substrates. This method is based on a fluorogenic RNA aptamer with an internal modified RNA/DNA residue which can block reverse transcription or introduce mutations leading to loss of fluorescence inherent in the cDNA product. Demethylation by an AlkB variant eliminates the blockage or mutation thereby restores the fluorescence signals. We applied our screening method to sites D135 and R210 in the Escherichia coli AlkB protein and identified a variant with improved activity beyond a previously known hyperactive mutant toward N1-methylguanosine (m1G) in RNA. We also applied our method to O6-methylguanosine (O6mG) modified DNA substrates and identified candidate AlkB variants with demethylating activity. Our study provides a high-throughput screening method for in vitro evolution of any demethylase enzyme.

DNA integrity is challenged by both exogenous and endogenous alkylating agents. DNA repair proteins such as Escherichia coli AlkB family of enzymes can repair 1-methyladenine and 3-methylcytosine adducts by oxidative demethylation. Human AlkB homologue 5 (ALKBH5) is RNA N6-methyladenine demethylase and not known to be involved in DNA repair. Herein we show that ALKBH5 also has weak DNA repair activity and it can demethylate DNA 3-methylcytosine. The mutation of the amino acid residues involved in demethylation also abolishes the DNA repair activity of ALKBH5. Overexpression of ALKBH5 decreases the 3-methylcytosine level in genomic DNA and reduces the cytotoxic effects of the DNA damaging alkylating agent methyl methanesulfonate. Thus, demethylation by ALKBH5 might play a supporting role in maintaining genome integrity.

Many aquatic environments are at risk for oil contamination and alkanes are one of the primary constituents of oil. The alkane hydroxylase (AlkB) is a common enzyme used by microorganisms to initiate the process of alkane-degradation. While many aspects of alkane bioremediation have been studied, the diversity and evolution of genes involved in hydrocarbon degradation from environmental settings is relatively understudied. The majority of work done to-date has focused on the marine environment. Here we sought to better understand the phylogenetic diversity of alkB genes across marine and freshwater settings using culture-independent methods. We hypothesized that there would be distinct phylogenetic diversity of alkB genes in freshwater relative to the marine environment. Our results confirm that alkB has distinct variants based on environment while our diversity analyses demonstrate that freshwater and marine alkB communities have unique responses to oil amendments. Our results also demonstrate that in the marine environment, depth is a key factor impacting diversity of alkB genes.

Iron-dependent dioxygenases of the AlkB protein family found in most organisms throughout the tree of life play a major role in oxidative dealkylation processes. Many of these enzymes have attracted the attention of researchers across different fields and have been subjected to thorough biochemical characterization because of their link to human health and disease. For example, several mammalian AlkB homologues are involved in the direct reversal of alkylation damage in DNA, while others have been shown to play a regulatory role in epigenetic or epitranscriptomic nucleic acid methylation or in post-translational modifications such as acetylation of actin filaments. These studies show that that divergence in amino acid sequence and structure leads to different characteristics and substrate specificities. In this review, we aim to summarize current insights in the structural features involved in the substrate selection of AlkB homologues, with focus on nucleic acid interactions. ispartof: Dna Repair vol:96 ispartof: location:Netherlands status: Published online

The AlkB family of non-heme-Fe(II)/2-oxoglutarate(2OG)-dependent oxygenases are essential regulators of RNA epigenetics by serving as erasers of one-carbon marks on RNA with release of formaldehyde (FA). Two major human AlkB family members, FTO and ALKBH5, both act as oxidative demethylases of N6 methyladenosine (m6A) but furnish different major products, N6 hydroxymethyladenosine (hm6A) and adenosine (A), respectively. Here we identify foundational mechanistic differences between FTO and ALKBH5 that promote these distinct biochemical outcomes. In contrast to FTO, which follows a traditional oxidative N-demethylation pathway to catalyze conversion of m6A to hm6A with subsequent slow release of A and FA, we find that ALKBH5 catalyzes a directm6A-to-A transformation with rapid FA release. We identify a catalytic R130/K132/Y139 triad within ALKBH5 that facilitates release of FA via an unprecedented covalent-based demethylation mechanism with direct detection of a covalent intermediate. Importantly, a K132Q mutant furnishes an ALKBH5 enzyme with an m6A demethylation profile that resembles that of FTO, establishing the importance of this residue in the proposed covalent mechanism. Finally, we show that ALKBH5 is an endogenous source of FA in the cell by activity-based sensing of FA fluxes perturbed via ALKBH5 knockdown. This work provides a fundamental biochemical rationale for non-redundant roles of these RNA demethylases beyond different substrate preferences and cellular localization, where m6A demethylation by ALKBH5 versus FTO results in release of FA, an endogenous one-carbon unit but potential genotoxin, at different rates in living systems.Significance StatementNon-heme iron enzymes FTO and ALKBH5 play central roles in epigenetic RNA regulation by catalyzing the oxidation of N6-methyladenosine (m6A) to produce N6-hydroxymethyladenosine (hm6A) and adenosine (A), respectively. Here, we provide a mechanistic rationale for these distinct biochemical outcomes by identifying that ALKBH5 performs m6A demethylation via an unprecedented covalent-based mechanism with concomitant and rapid release of A and formaldehyde (FA), whereas FTO liberates hm6A to release A and FA over longer timescales. This work reveals foundational biochemical differences between these closely related but non-redundant epigenetic enzymes and identifies ALKBH5 as an endogenous source of rapid formaldehyde generation in cells.

Alkb homolog 7 (ALKBH7) is a mitochondrial α-ketoglutarate dioxygenase required for necrotic cell death in response to DNA alkylating agents, but its physiologic role within tissues remains unclear. Herein, we show that ALKBH7 plays a key role in the regulation of dialdehyde metabolism, which impacts cardiac survival in response to ischemia-reperfusion (IR) injury. Using a multi-omics approach, we do not find evidence that ALKBH7 functions as a prolyl-hydroxylase. However, we do find that mice lacking ALKBH7 exhibit a significant increase in glyoxalase I (GLO-1), a dialdehyde detoxifying enzyme. Consistent with increased dialdehyde production, metabolomics analysis reveals rewiring of metabolic pathways related to the toxic glycolytic by-product methylglyoxal (MGO), as well as accelerated glycolysis and elevated levels of MGO protein adducts, in mice lacking ALKBH7. Consistent with roles for both necrosis and glycative stress in cardiac IR injury, hearts from male but not femaleAlkbh7-/-mice are protected against IR, although somewhat unexpectedly this protection does not appear to involve modulation of the mitochondrial permeability transition pore. Highlighting the importance of MGO metabolism for the observed protection, removal of glucose as a metabolic substrate or pharmacologic inhibition of GLO-1 both abrogate cardioprotection in ALKBH7 deficient mice. Integrating these observations, we propose that ALKBH7 plays a role in the regulation of glyoxal metabolism, and that protection against necrosis and IR injury bought on by ALKBH7 deficiency originates from hormetic signaling in response to elevated MGO stress.

Class-II fumarases (Fumarate Hydratase, FH) are dual targeted enzymes, occurring in the mitochondria and cytosol of all eukaryotes. They are essential components in the DNA damage response (DDR) and more specifically, protecting cells from DNA double strand breaks. Similarly, the Gram-positive Bacterium Bacillus subtilis Class-II fumarase, in addition to its role in the TCA cycle, also participates in the DDR. Escherichia coli, harbors three fumarase genes; Class-I fumA and fumB and Class-II fumC. Notably, Class-I fumarases, show no sequence similarity to Class-II fumarases and are of different evolutionary origin. Strikingly, here we show that E. coli fumarase functions are distributed between Class-I fumarases which participate in the DDR, and the Class-II fumarase which participates in respiration. In E. coli, we discover that the signaling molecule, alpha-ketoglutarate (α-KG), has a novel function, complementing DNA damage sensitivity of fum null mutants. Excitingly, we identify the E. coli α-KG dependent DNA repair enzyme AlkB, as the target of this interplay of metabolite signaling. In addition to α-KG, fumarate (fumaric acid) is shown to affect DNA damage repair on two different levels, first by directly inhibiting the DNA damage repair enzyme AlkB demethylase activity, both in vitro and in vivo (countering α-KG). The second is a more global effect on transcription, as fum null mutants exhibit a decrease in transcription of key DNA damage repair genes. Together these results show evolutionary adaptable metabolic signaling of the DDR, in which fumarases and different metabolites are recruited regardless of the evolutionary enzyme Class preforming the function.Significance StatementClass-II fumarases have been shown to participate in cellular respiration and the DNA damage response. Here we show, for the first time, that in the model prokaryote, Escherichia coli, which harbors both Class-I and Class-II fumarases, it is the Class-I fumarases that participate in DNA damage repair by a mechanism which is different than those described for other fumarases. Strikingly, this mechanism employs a novel signaling molecule, alpha-ketoglutarate (α-KG), and its target is the DNA damage repair enzyme AlkB. In addition, we show that fumarase precursor metabolites, fumarate and succinate, can inhibit the α-KG-dependent DNA damage repair enzyme, AlkB, both in vitro and in vivo. This study provides a new perspective on the function and evolution of metabolic signaling.