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Degradation of oil hydrocarbons artificially introduced into bottom sediments in a bioelectrochemical system of a membrane-free (silt) type was studied. Passive bioelectrochemical stimulation by means of electrodes connected by an external circuit with a resistance of 1 kΩ, with an average electric current of ~85 µA was found to cause an increase in degradation during two months from 23.0 to 57.9%. Contamination of bottom sediments with oil (1.32 g/kg) slightly decreased the current in the external circuit of the bioelectrochemical system. The relationship was revealed between the degree of oil degradation and predominant utilization of the lighter n-alkanes in the C14H30–C30H62 series, compared with both the original oil and the residual hydrocarbons of the control. An increase in the representation of the alkB alkane monooxygenase genes relative to the 16S rRNA gene in the total DNA isolated from the sediments was induced by the introduction of hexadecane, both in the case of electrochemical stimulation and in the control. The results may be of interest for the development of new methods of bioelectrochemical removal of organic pollutants from anaerobic environments. [ABSTRACT FROM AUTHOR]

Biotreatment of oily sludge and the involved microbial communities, particularly in saline environments, have been rarely investigated. We enriched a halophilic bacterial consortium (OS-100) from petroleum refining oily sludge, which degraded almost 86% of the aliphatic hydrocarbon (C10-C30) fraction of the oily sludge within 7 days in the presence of 100 g/L NaCl. Two halophilic hydrocarbon-degrading bacteria related to the genera Chromohalobacter and Halomonas were isolated from the OS-100 consortium. Hydrocarbon degradation by the OS-100 consortium was relatively higher compared to the isolated bacteria, indicating potential synergistic interactions among the OS-100 community members. Exclusion of FeCl2, MgCl2, CaCl2, trace elements, and vitamins from the culture medium did not significantly affect the hydrocarbon degradation efficiency of the OS-100 consortium. To the contrary, hydrocarbon biodegradation dropped from 94.1 to 54.4% and 5% when the OS-100 consortium was deprived from phosphate and nitrogen sources in the culture medium, respectively. Quantitative PCR revealed that alkB gene expression increased up to the 3rd day of incubation with 11.277-fold, consistent with the observed increments in hydrocarbon degradation. Illumina-MiSeq sequencing of 16 S rRNA gene fragments revealed that the OS-100 consortium was mainly composed of the genera Halomonas, Idiomarina, Alcanivorax and Chromohalobacter. This community structure changed depending on the culturing conditions. However, remarkable changes in the community structure were not always associated with remarkable shifts in the hydrocarbonoclastic activity and vice versa. The results show that probably synergistic interactions between community members and different subpopulations of the OS-100 consortium contributed to salinity tolerance and hydrocarbon degradation. Key points: • A halophilic bacterial consortium efficiently degrades oily sludge hydrocarbons. • Nitrogen starvation and high salinity have the greatest impact on hydrocarbon loss. • Change in the consortium structure is not a prerequisite to shift biodegradation. [ABSTRACT FROM AUTHOR]

ALKBH proteins, the homologs of Escherichia coli AlkB dioxygenase, constitute a single-protein repair system that safeguards cellular DNA and RNA against the harmful effects of alkylating agents. ALKBH10B, the first discovered N6-methyladenosine (m6A) demethylase in Arabidopsis (Arabidopsis thaliana), has been shown to regulate plant growth, development, and stress responses. However, until now, the functional role of the plant ALKBH10B has solely been reported in arabidopsis, cotton, and poplar, leaving its functional implications in other plant species shrouded in mystery. In this study, we identified the AlkB homolog SlALKBH10B in tomato (Solanum lycopersicum) through phylogenetic and gene expression analyses. SlALKBH10B exhibited a wide range of expression patterns and was induced by exogenous abscisic acid (ABA) and abiotic stresses. By employing CRISPR/Cas9 gene editing techniques to knock out SlALKBH10B, we observed an increased sensitivity of mutants to ABA treatment and upregulation of gene expression related to ABA synthesis and response. Furthermore, the Slalkbh10b mutants displayed an enhanced tolerance to drought and salt stress, characterized by higher water retention, accumulation of photosynthetic products, proline accumulation, and lower levels of reactive oxygen species and cellular damage. Collectively, these findings provide insights into the negative impact of SlALKBH10B on drought and salt tolerance in tomato plant, expanding our understanding of the biological functionality of SlALKBH10B. [ABSTRACT FROM AUTHOR]

Nucleic acid methylations are important genetic and epigenetic biomarkers. The formation and removal of these markers is related to either methylation or demethylation. In this review, we focus on the demethylation or oxidative modification that is mediated by the 2-oxoglutarate (2-OG)/Fe(II)-dependent AlkB/TET family enzymes. In the catalytic process, most enzymes oxidize 2-OG to succinate, in the meantime oxidizing methyl to hydroxymethyl, leaving formaldehyde and generating demethylated base. The AlkB enzyme from Escherichia coli has nine human homologs (ALKBH1-8 and FTO) and the TET family includes three members, TET1 to 3. Among them, some enzymes have been carefully studied, but for certain enzymes, few studies have been carried out. This review focuses on the kinetic properties of those 2-OG/Fe(II)-dependent enzymes and their alkyl substrates. We also provide some discussions on the future directions of this field.

Nucleic acid methylations are important genetic and epigenetic biomarkers. The formation and removal of these markers is related to either methylation or demethylation. In this review, we focus on the demethylation or oxidative modification that is mediated by the 2-oxoglutarate (2-OG)/Fe(II)-dependent AlkB/TET family enzymes. In the catalytic process, most enzymes oxidize 2-OG to succinate, in the meantime oxidizing methyl to hydroxymethyl, leaving formaldehyde and generating demethylated base. The AlkB enzyme from Escherichia coli has nine human homologs (ALKBH1-8 and FTO) and the TET family includes three members, TET1 to 3. Among them, some enzymes have been carefully studied, but for certain enzymes, few studies have been carried out. This review focuses on the kinetic properties of those 2-OG/Fe(II)-dependent enzymes and their alkyl substrates. We also provide some discussions on the future directions of this field. [ABSTRACT FROM AUTHOR]

The human DNA repair enzyme AlkB homologue-2 (ALKBH2) repairs methyl adducts from genomic DNA. Overexpression of ALKBH2 has been implicated in both tumorigenesis and chemotherapy resistance in some cancers, including glioblastoma and renal cancer rendering it a potential therapeutic target and a diagnostic marker. However, no inhibitor is available against these important DNA repair proteins. Intending to repurpose a drug as an inhibitor of ALKBH2, we performed in silico evaluation of HIV protease inhibitors and identified Ritonavir as an ALKBH2-interacting molecule. Using molecular dynamics simulation, we elucidated the molecular details of Ritonavir-ALKBH2 interaction. The present work highlights that Ritonavir might be used to target the ALKBH2-mediated DNA alkylation repair. [ABSTRACT FROM AUTHOR]

ALKBH proteins, the homologs of Escherichia coli AlkB dioxygenase, constitute a single-protein repair system that safeguards cellular DNA and RNA against the harmful effects of alkylating agents. ALKBH10B, the first discovered N6-methyladenosine (m6A) demethylase in Arabidopsis (Arabidopsis thaliana), has been shown to regulate plant growth, development, and stress responses. However, until now, the functional role of the plant ALKBH10B has solely been reported in arabidopsis, cotton, and poplar, leaving its functional implications in other plant species shrouded in mystery. In this study, we identified the AlkB homolog SlALKBH10B in tomato (Solanum lycopersicum) through phylogenetic and gene expression analyses. SlALKBH10B exhibited a wide range of expression patterns and was induced by exogenous abscisic acid (ABA) and abiotic stresses. By employing CRISPR/Cas9 gene editing techniques to knock out SlALKBH10B, we observed an increased sensitivity of mutants to ABA treatment and upregulation of gene expression related to ABA synthesis and response. Furthermore, the Slalkbh10b mutants displayed an enhanced tolerance to drought and salt stress, characterized by higher water retention, accumulation of photosynthetic products, proline accumulation, and lower levels of reactive oxygen species and cellular damage. Collectively, these findings provide insights into the negative impact of SlALKBH10B on drought and salt tolerance in tomato plant, expanding our understanding of the biological functionality of SlALKBH10B.

The alpha-ketoglutarate-dependent dioxygenase (AlkB) gene family plays an essential role in regulating plant development and stress response. However, the AlkB gene family is still not well understood in cotton. In this study, 40 AlkB genes in cotton and Arabidopsis are identified and classified into three classes based on phylogenetic analysis. Their protein motifs and exon/intron structures are highly conserved. Chromosomal localization and synteny analysis suggested that segmental or whole-genome duplication and polyploidization events contributed to the expansion of the cotton AlkB gene family. Furthermore, the AlkB genes showed dynamic spatiotemporal expression patterns and diverse responses to abiotic stresses. Among them, GhALKBH10 was down-regulated under various abiotic stresses and its subcellular expression was localized in cytoplasm and nucleus. Silencing GhALKBH10 in cotton increased antioxidant capacity and reduced cytoplasmic Na+ concentration, thereby improved the plant tolerance to salinity. Conversely, overexpression (OE) of GhALKBH10 in Arabidopsis markedly weakened the plant tolerance to salinity. The global m6A levels measured in VIGS and OE transgenic lines showed that they were significantly higher in TRV: GhALKBH10 plants (VIGS) than in TRV: 00 plants but significantly lower in OE plants than wild-type plants under salt stress, which could be considered as a potential m6A demethylase in cotton. Our results suggest that the GhALKBH10 gene negatively regulates salt tolerance in plants, which provides information of the cotton AlkB family and an understanding of GhALKBH10 function under salt condition as well as a new gene for salt-tolerant cotton breeding. • AlkB genes have conserved structure and functions and unevenly distribute in cotton. • ALKBH10-silenced cotton enhanced salt tolerance and increased global m6A levels. • ALKBH10-overexpressed Arabidopsis weakened salt tolerance and decreased m6A levels. • ALKBH10 is reported as an m6A eraser in response to salt stress for the first time. [ABSTRACT FROM AUTHOR]

The human DNA repair enzyme AlkB homologue-2 (ALKBH2) repairs methyl adducts from genomic DNA and is overexpressed in several cancers. However, there are no known inhibitors available for this crucial DNA repair enzyme. The aim of this study was to examine whether the first-generation HIV protease inhibitors having strong anti-cancer activity can be repurposed as inhibitors of ALKBH2. We selected four such inhibitors and performed in vitro binding analysis against ALKBH2 based on alterations of its intrinsic tryptophan fluorescence and differential scanning fluorimetry. The effect of these HIV protease inhibitors on the DNA repair activity of ALKBH2 was also evaluated. Interestingly, we observed that one of the inhibitors, ritonavir, could inhibit ALKBH2-mediated DNA repair significantly via competitive inhibition and sensitized cancer cells to alkylating agent methylmethane sulfonate (MMS). This work may provide new insights into the possibilities of utilizing HIV protease inhibitor ritonavir as a DNA repair antagonist. • Four HIV drugs with anti-cancer properties were evaluated as ALKBH2 antagonist. • Intrinsic Trp fluorescence quenching showed that ritonavir binds to ALKBH2. • Thermal unfolding showed ritonavir binding requires ALKBH2 R110 residue. • Enzyme kinetics results suggested that ritonavir competitively inhibits ALKBH2. • Ritonavir exposure reduced cancer cell survival following MMS treatment. [ABSTRACT FROM AUTHOR]

Background. Alkanes are important components of fossil energy, such as crude oil. The alkane monooxygenase encoded by alkB gene performs the initial step of alkane degradation under aerobic conditions. The alkB gene is well studied due to its ubiquity as well as the availability of experimentally functional evidence. The alkBFGHJKL and alkST clusters are special kind of alkB-type alkane hydroxylase system, which encode all proteins necessary for converting alkanes into corresponding fatty acids. Methods. To explore whether the alkBFGHJKL and alkST clusters were widely distributed, we performed a large-scale analysis of isolate and metagenome assembled genome data (>390,000 genomes) to identify these clusters, together with distributions of corresponding taxonomy and niches. The set of alk-genes (including but not limited to alkBGHJ) located near each other on a DNA sequence was defined as an alk-gene cluster in this study. The alkB genes with alkGHJ located nearby on a DNA sequence were picked up for the investigation of putative alk-clusters. Results. Atotal of 120 alk-gene clusters were found in 117 genomes. All the 117 genomes are from strains located only in - and -proteobacteria. The alkB genes located in alk-gene sets were clustered into a deeply branched mono-clade. Further analysis showed similarity organization types of alk-genes were observed within closely related species. Although a large number of IS elements were observed nearby, they did not lead to the wide spread of the alk-gene cluster. The uneven distribution of these elements indicated that there might be other factors affecting the transmission of alk-gene clusters. Conclusions. We conducted systematic bioinformatics research on alk-genes located near each other on a DNA sequence. This benchmark dataset of alk-genes can provide base line for exploring its evolutional and ecological importance in future studies. [ABSTRACT FROM AUTHOR]

The human alkylation B (AlkB) homologs, ALKBH2 and ALKBH3, respond to methylation damage to maintain genomic integrity and cellular viability. Both ALKBH2 and ALKBH3 are direct reversal repair enzymes that remove 1-methyladenine (1meA) and 3-methylcytosine (3meC) lesions commonly generated by alkylating chemotherapeutic agents. Thus, the existence of deficiencies in ALKBH proteins can be exploited in synergy with chemotherapy. In this study, we investigated possible interactions between ALKBH2 and ALKBH3 with other proteins that could alter damage response and discovered an interaction with the mismatch repair (MMR) system. To test whether the lack of active MMR impacts ALKBH2 and/or ALKBH3 response to methylating agents, we generated cells deficient in ALKBH2, ALKBH3, or both in addition to Mlh homolog 1 (MLH1), another MMR protein. We found that MLH1 ko ALKBH3 ko cells showed enhanced resistance toward S N 1- and S N 2-type methylating agents, whereas MLH1 ko ALKBH2 ko cells were only resistant to S N 1-type methylating agents. Concomitant loss of ALKBH2 and ALKBH3 (ALKBH2 ko 3 ko ) rendered cells sensitive to S N 1- and S N 2-agents, but the additional loss of MLH1 enhanced resistance to both types of damage. We also showed that ALKBH2 ko 3 ko cells have an ATR-dependent arrest at the G 2 /M checkpoint, increased apoptotic signaling, and replication fork stress in response to methylation. However, these responses were not observed with the loss of functional MLH1 in MLH1 ko ALKBH2 ko 3 ko cells. Finally, in MLH1 ko ALKBH2 ko 3 ko cells, we observed elevated mutant frequency in untreated and temozolomide treated cells. These results suggest that obtaining a more accurate prognosis of chemotherapeutic outcome requires information on the functionality of ALKBH2, ALKBH3, and MLH1., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

The Tsukamurella tyrosinosolvens PS2 strain was isolated from hydrocarbons-contaminated petrochemical sludge as a long chain alkane-utilizing bacteria. Complete genome analysis showed the presence of two alkane oxidation systems: alkane 1-monooxygenase (alkB) and cytochrome P450 monooxygenase (P450) genes with established high homology to the well-known alkane-degrading actinobacteria. According to the comparative genome analysis, both systems have a wide distribution among environmental and clinical isolates of the genus Tsukamurella and other members of Actinobacteria. We compared the expression of different proteins during the growth of Tsukamurella on sucrose and on hexadecane. Both alkane monooxygenases were upregulated on hexadecane: AlkB—up to 2.5 times, P450—up to 276 times. All proteins of the hexadecane oxidation pathway to acetyl-CoA were also upregulated. Accompanying proteins for alkane degradation involved in biosurfactant synthesis and transport of organic and inorganic molecules were increased. The change in the carbon source affected the pathways for the regulation of translation and transcription. The proteomic profile showed that hexadecane is an adverse factor causing activation of general and universal stress proteins as well as shock and resistance proteins. Differently expressed proteins of Tsukamurella tyrosinosolvens PS2 shed light on the alkane degradation in other members of Actinobacteria class. Key Points: • alkB and P450 systems have a wide distribution among the genus Tsukamurella. • alkB and P450 systems have coexpression with the predominant role of P450 protein. • Hexadecane causes significant changes in bacterial proteome. [ABSTRACT FROM AUTHOR]

N 1-methyladenosine (m1A), N 3-methylcytidine (m3C), and N 1-methylguanosine (m1G) are common in transfer RNA (tRNA) and tRNA-derived fragments. These modifications alter Watson-Crick base-pairing, and cause pauses or stops during reverse transcription required for most high-throughput RNA sequencing protocols, resulting in inefficient detection of methyl-modified RNAs. Here, we describe a procedure to demethylate RNAs containing m1A, m3C, or m1G using the Escherichia coli dealkylating enzyme AlkB, along with instructions for subsequent processing with widely used protocols for small RNA sequencing.

Alkane-oxidizing enzymes play an important role in the global carbon cycle. Alkane monooxygenase (AlkB) oxidizes most of the medium-chain length alkanes in the environment. The first AlkB identified was from P. putida GPo1 (initially known as P. oleovorans) in the early 1970s, and it continues to be the family member about which the most is known. This AlkB is found as part of the OCT operon, in which all of the key proteins required for growth on alkanes are present. The AlkB catalytic cycle requires that the diiron active site be reduced. In P. putida GPo1, electrons originate from NADH and arrive at AlkB via the intermediacy of a flavin reductase and an iron–sulfur protein (a rubredoxin). In this Mini Review, we will review what is known about the canonical arrangement of electron-transfer proteins that activate AlkB and, more importantly, point to several other arrangements that are possible. These other arrangements include the presence of a simpler rubredoxin than what is found in the canonical arrangement, as well as two other classes of AlkBs with fused electron-transfer partners. In one class, a rubredoxin is fused to the hydroxylase and in another less well-explored class, a ferredoxin reductase and a ferredoxin are fused to the hydroxylase. We review what is known about the biochemistry of these electron-transfer proteins, speculate on the biological significance of this diversity, and point to key questions for future research. [ABSTRACT FROM AUTHOR]

In eukaryotes, N6-methyladenosine (m6A) is the most abundant and highly conserved RNA modification. In vivo, m6A demethylase dynamically regulates the m6A level by removing the m6A marker where it plays an important role in plant growth, develop- ment and response to abiotic stress. The confirmed m6A demethylases in Arabidopsis thaliana include ALKBH9B and ALKBH10B, both belonging to the ALKB family. In this study, BvALKB family members were identified in sugar beet genome-wide database, and their conserved domains, gene structures, chromosomal locations, phylogeny, conserved motifs and expression of BvALKB genes were analyzed. Almost all BvALKB proteins contained the conserved domain of 2OG-Fe II-Oxy. Phylogenetic analysis suggested that the ten proteins were clustered into five groups, each of which had similar motifs and gene structures. Three Arabidopsis m6A demethylase-homologous proteins (BvALKBH6B, BvALKBH8B and BvALKBH10B) were of particular interest in our study. Expression profile analysis showed that almost all genes were up-regulated or down-regulated to varying degrees under salt stress. More specifically, BvALKBH10B homologous to AtALKBH10B was significantly up-regulated, suggesting that the transcriptional activity of this gene is responsive to salt stress. This study provides a theoretical basis for further screening of m6A demethylase in sugar beet, and also lays a foundation for studying the role of ALKB family proteins in growth, development and response to salinity stress. [ABSTRACT FROM AUTHOR]

Alkane-oxidizing enzymes play an important role in the global carbon cycle. Alkane monooxygenase (AlkB) oxidizes most of the medium-chain length alkanes in the environment. The first AlkB identified was from P. putida GPo1 (initially known as P. oleovorans) in the early 1970s, and it continues to be the family member about which the most is known. This AlkB is found as part of the OCT operon, in which all of the key proteins required for growth on alkanes are present. The AlkB catalytic cycle requires that the diiron active site be reduced. In P. putida GPo1, electrons originate from NADH and arrive at AlkB via the intermediacy of a flavin reductase and an iron–sulfur protein (a rubredoxin). In this Mini Review, we will review what is known about the canonical arrangement of electron-transfer proteins that activate AlkB and, more importantly, point to several other arrangements that are possible. These other arrangements include the presence of a simpler rubredoxin than what is found in the canonical arrangement, as well as two other classes of AlkBs with fused electron-transfer partners. In one class, a rubredoxin is fused to the hydroxylase and in another less well-explored class, a ferredoxin reductase and a ferredoxin are fused to the hydroxylase. We review what is known about the biochemistry of these electron-transfer proteins, speculate on the biological significance of this diversity, and point to key questions for future research.

In eukaryotes, N6-methyladenosine (m6A) is the most abundant and highly conserved RNA modification. In vivo, m6A demethylase dynamically regulates the m6A level by removing the m6A marker where it plays an important role in plant growth, development and response to abiotic stress. The confirmed m6A demethylases in Arabidopsis thaliana include ALKBH9B and ALKBH10B, both belonging to the ALKB family. In this study, BvALKB family members were identified in sugar beet genome-wide database, and their conserved domains, gene structures, chromosomal locations, phylogeny, conserved motifs and expression of BvALKB genes were analyzed. Almost all BvALKB proteins contained the conserved domain of 2OG-Fe II-Oxy. Phylogenetic analysis suggested that the ten proteins were clustered into five groups, each of which had similar motifs and gene structures. Three Arabidopsis m6A demethylase-homologous proteins (BvALKBH6B, BvALKBH8B and BvALKBH10B) were of particular interest in our study. Expression profile analysis showed that almost all genes were up-regulated or down-regulated to varying degrees under salt stress. More specifically, BvALKBH10B homologous to AtALKBH10B was significantly up-regulated, suggesting that the transcriptional activity of this gene is responsive to salt stress. This study provides a theoretical basis for further screening of m6A demethylase in sugar beet, and also lays a foundation for studying the role of ALKB family proteins in growth, development and response to salinity stress.

The epigenetic modifications control the pathogenicity of human pathogenic fungi, which have been poorly studied in Mucorales, causative agents of mucormycosis. This order belongs to a group referred to as early-diverging fungi that are characterized by high levels of N6-methyldeoxy adenine (6mA) in their genome with dense 6mA clusters associated with actively expressed genes. AlkB enzymes can act as demethylases of 6mA in DNA, with the most remarkable eukaryotic examples being mammalian ALKBH1 and Caenorhabditis elegans NMAD-1. The Mucor lusitanicus (formerly M. circinelloides f. lusitanicus) genome contains one gene, dmt1, and two genes, dmt2 and dmt3, encoding proteins similar to C. elegans NMAD-1 and ALKBH1, respectively. The function of these three genes was analyzed by the generation of single and double deletion mutants for each gene. Multiple processes were studied in the mutants, but defects were only found in single and double deletion mutants for dmt1. In contrast to the wild-type strain, dmt1 mutants showed an increase in 6mA levels during the dimorphic transition, suggesting that 6mA is associated with dimorphism in M. lusitanicus. Furthermore, the spores of dmt1 mutants challenged with macrophages underwent a reduction in polar growth, suggesting that 6mA also has a role during the spore–macrophage interaction that could be important in the infection process. [ABSTRACT FROM AUTHOR]

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, protect cells from DNA double-strand breaks. Similarly, the gram-positive bacterium Bacillus subtilis class-II fumarase, in addition to its role in the tricarboxylic acid cycle, 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, alphaketoglutarate (α-KG), has a 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, because 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 performing the function. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

DNA damaging agents have been a cornerstone of cancer therapy for nearly a century. The discovery of many of these chemicals, particularly the alkylating agents, are deeply entwined with the development of poisonous materials originally intended for use in warfare. Over the last decades, their anti-proliferative effects have focused on the specific mechanisms by which they damage DNA, and the factors involved in the repair of such damage. Due to the variety of aberrant adducts created even for the simplest alkylating agents, numerous pathways of repair are engaged as a defense against this damage. More recent work has underscored the role of RNA damage in the cellular response to these agents, although the understanding of their role in relation to established DNA repair pathways is still in its infancy. In this review, we discuss the chemistry of alkylating agents, the numerous ways in which they damage nucleic acids, as well as the specific DNA and RNA repair pathways which are engaged to counter their effects. [ABSTRACT FROM AUTHOR]

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. • Fe(II)/2OG-dependent dioxygenase ALKBH5 can demethylate 3meC in vitro. • ALKBH5 rescues MMS sensitivity in heterologous system. • ALKBH5 overexpression can demethylate 3meC from genomic DNA in vivo. [ABSTRACT FROM AUTHOR]

Differences in tRNA expression have been implicated in a remarkable number of biological processes. There is growing evidence that tRNA genes can play dramatically different roles depending on both expression and post-transcriptional modification, yet sequencing tRNAs to measure abundance and detect modifications remains challenging. Their secondary structure and extensive post-transcriptional modifications interfere with RNA-seq library preparation methods and have limited the utility of high-throughput sequencing technologies. Here, we combine two modifications to standard RNA-seq methods by treating with the demethylating enzyme AlkB and ligating with tRNA-specific adapters in order to sequence tRNAs from four species of flowering plants, a group that has been shown to have some of the most extensive rates of post-transcriptional tRNA modifications. This protocol has the advantage of detecting full-length tRNAs and sequence variants that can be used to infer many post-transcriptional modifications. We used the resulting data to produce a modification index of almost all unique reference tRNAs in Arabidopsis thaliana, which exhibited many anciently conserved similarities with humans but also positions that appear to be 'hot spots' for modifications in angiosperm tRNAs. We also found evidence based on northern blot analysis and droplet digital PCR that, even after demethylation treatment, tRNA-seq can produce highly biased estimates of absolute expression levels most likely due to biased reverse transcription. Nevertheless, the generation of full-length tRNA sequences with modification data is still promising for assessing differences in relative tRNA expression across treatments, tissues or subcellular fractions and help elucidate the functional roles of tRNA modifications. [ABSTRACT FROM AUTHOR]

Cultured hydrocarbon-oxidizing bacteria have been isolated for the first time from bacterioplankton in urban Lake Beloye (Moscow). The taxonomic positions of two bacterial strains (2012B and 2012C) isolated from this lake have been determined. Lipids of the strain 2012B comprise C14:0–C19:0 fatty acids, the most abundant of them being C15:0 (54%), C16:0 (17%), C17:0 (10%), and 10-methyl С18:0 (3.5%). Lipids of the strain 2012C comprise C14:0–C19:0 fatty acids, the most abundant of them being C15:0 (45%), C16:0 (32%), and C17:0 (9%). A phylogenetic analysis of strain 2012B is performed using the nucleotide sequences of the 16S rRNA (KP779654.1) and alkB (KR422620.1) genes and the strain is identified as a typical member of the genus Rhodococcus spp. (Actinobacteria, Nocardiaceae). The combination of molecular identification and analysis of biochemical and physiological properties makes it possible to identify strain 2012B as Rhodococcusqingshengii 2012B. A phylogenetic analysis of strain 2012C is performed using the nucleotide sequences of 16S rRNA (MG966152) and shows the highest identity (99.57%) of strain 2012С with Pseudomonas psychrotolerans and Pseudomonas oryzihabitans [ABSTRACT FROM AUTHOR]

The aim of the present study was to reveal how different microbial communities evolve in diesel fuel/crude oil-contaminated environments under aerobic and microaerobic conditions. To investigate this question, aerobic and microaerobic bacterial enrichments amended with a diesel fuel/crude oil mixture were established and analysed. The representative aerobic enrichment community was dominated by Gammaproteobacteria (64.5%) with high an abundance of Betaproteobacteriales (36.5%), followed by Alphaproteobacteria (8.7%), Actinobacteria (5.6%), and Candidatus Saccharibacteria (4.5%). The most abundant alkane monooxygenase (alkB) genotypes in this enrichment could be linked to members of the genus Rhodococcus and to a novel Gammaproteobacterium, for which we generated a high-quality draft genome using genome-resolved metagenomics of the enrichment culture. Contrarily, in the microaerobic enrichment, Gammaproteobacteria (99%) overwhelmingly dominated the microbial community with a high abundance of the genera Acinetobacter (66.3%), Pseudomonas (11%) and Acidovorax (11%). Under microaerobic conditions, the vast majority of alkB gene sequences could be linked to Pseudomonas veronii. Consequently, results shed light on the fact that the excellent aliphatic hydrocarbon degrading Rhodococcus species favour clear aerobic conditions, while oxygen-limited conditions can facilitate the high abundance of Acinetobacter species in aliphatic hydrocarbon-contaminated subsurface environments. [ABSTRACT FROM AUTHOR]

[Objectives] The paper aimed to screen different kinds and properties of diesel strains with high degradation capacity and study their degrading mechanism in different environmental media.[Methods] A high-efficiency diesel degrading bacteria named as strain J-3 was isolated from the sludge sediments of Nanjing Yangzi Petrochemical Plant. The physiological and biochemical test together with 16S rRNA gene sequencing analysis indicated that the strain J-3 belonged to Serratia sp.. The degradation efficiency of diesel and the degradation mechanism were studied by hydroponic and soil culture methods combined with gas chromatography-mass spectrometric analysis(GC-MS)and genome comparison techniques.[Results] The strain could efficiently grow using diesel as its sole carbon and energy source. It could produce surfactant,which showed it had a stable emulsifying property. The strain grew well under the temperature of 20-40℃,and the pH of 5-9 conditions,it could tolerate 15 g·L-1 salinity and 5 g·L-1 diesel concentration. The degradation efficiency of diesel under the optimal growth conditions(pH7,25℃,1 g·L-1 salinity and 0.5 g·L-1 diesel concentration)was 62.0%. GC-MS spectrum analysis confirmed that J-3 had the ability to degrade all components in diesel oil,and the degradation of some media and long straight-chain alkanes exceeded 99%. Based on the whole-genome DNA sequence analysis of the strain J-3,the two genes associated with alkane oxidation were found,namely alkB,an alkane hydroxylase gene,and almA,a long-chain alkane monooxygenase gene. These two oxidase genes might play a role in the degradation of diesel oil by strain J-3. In addition,when the strain J-3 was applied to the soil remediation test of diesel pollution,it could degrade all diesel components in the soil. The degradation of strain J-3 under different treatments was up to 33.8%-49.0%,which could effectively degrade diesel in soil.[Conclusions] The strain J-3 was a high-efficiency diesel degradation bacterial strain. It can be well used for growth in both water and soil,and can be applied to the actual bioremediation project of diesel oil pollution. [ABSTRACT FROM AUTHOR]

The epigenetic modifications control the pathogenicity of human pathogenic fungi, which have been poorly studied in Mucorales, causative agents of mucormycosis. This order belongs to a group referred to as early-diverging fungi that are characterized by high levels of N6-methyldeoxy adenine (6mA) in their genome with dense 6mA clusters associated with actively expressed genes. AlkB enzymes can act as demethylases of 6mA in DNA, with the most remarkable eukaryotic examples being mammalian ALKBH1 and Caenorhabditis elegans NMAD-1. The Mucor lusitanicus (formerly M. circinelloides f. lusitanicus) genome contains one gene, dmt1, and two genes, dmt2 and dmt3, encoding proteins similar to C. elegans NMAD-1 and ALKBH1, respectively. The function of these three genes was analyzed by the generation of single and double deletion mutants for each gene. Multiple processes were studied in the mutants, but defects were only found in single and double deletion mutants for dmt1. In contrast to the wild-type strain, dmt1 mutants showed an increase in 6mA levels during the dimorphic transition, suggesting that 6mA is associated with dimorphism in M. lusitanicus. Furthermore, the spores of dmt1 mutants challenged with macrophages underwent a reduction in polar growth, suggesting that 6mA also has a role during the spore–macrophage interaction that could be important in the infection process.

Mutation patterns of DNA adducts, such as mutational spectra and signatures, are useful tools for diagnostic and prognostic purposes. Mutational spectra of carcinogens derive from three sources: adduct formation, replication bypass, and repair. Here, we consider the repair aspect of 1,N6-ethenoadenine (εA) by the 2-oxoglutarate/Fe(II)-dependent AlkB family enzymes. Specifically, we investigated εA repair across 16 possible sequence contexts (5′/3′ flanking base to εA varied as G/A/T/C). The results revealed that repair efficiency is altered according to sequence, enzyme, and strand context (ss- versus ds-DNA). The methods can be used to study other aspects of mutational spectra or other pathways of repair.

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.

ALKBH proteins, the homologs of Escherichia coli AlkB dioxygenase, constitute a single-protein repair system that safeguards cellular DNA and RNA against the harmful effects of alkylating agents. ALKBH10B, the first discovered N 6 -methyladenosine (m 6 ), has been shown to regulate plant growth, development, and stress responses. However, until now, the functional role of the plant ALKBH10B has solely been reported in arabidopsis, cotton, and poplar, leaving its functional implications in other plant species shrouded in mystery. In this study, we identified the AlkB homolog SlALKBH10B in tomato ( Arabidopsis thaliana ), has been shown to regulate plant growth, development, and stress responses. However, until now, the functional role of the plant ALKBH10B has solely been reported in arabidopsis, cotton, and poplar, leaving its functional implications in other plant species shrouded in mystery. In this study, we identified the AlkB homolog SlALKBH10B in tomato ( Solanum lycopersicum ) through phylogenetic and gene expression analyses. SlALKBH10B exhibited a wide range of expression patterns and was induced by exogenous abscisic acid (ABA) and abiotic stresses. By employing CRISPR/Cas9 gene editing techniques to knock out SlALKBH10B , we observed an increased sensitivity of mutants to ABA treatment and upregulation of gene expression related to ABA synthesis and response. Furthermore, the Slalkbh10b mutants displayed an enhanced tolerance to drought and salt stress, characterized by higher water retention, accumulation of photosynthetic products, proline accumulation, and lower levels of reactive oxygen species and cellular damage. Collectively, these findings provide insights into the negative impact of SlALKBH10B on drought and salt tolerance in tomato plant, expanding our understanding of the biological functionality of SlALKBH10B .

The zero- and double-quantum methyl TROSY Hahn-echo and the methyl 1H–1H dipole–dipole cross-correlation nuclear magnetic resonance experiments enable estimation of multiple quantum chemical exchange broadening in methyl groups in proteins. The two relaxation rate constants are established to be linearly dependent using molecular dynamics simulations and empirical analysis of experimental data. This relationship allows chemical exchange broadening to be recognized as an increase in the Hahn-echo relaxation rate constant. The approach is illustrated by analyzing relaxation data collected at three temperatures for E. coli ribonuclease HI and by analyzing relaxation data collected for different cofactor and substrate complexes of E. coli AlkB. [ABSTRACT FROM AUTHOR]

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 [ABSTRACT FROM AUTHOR]

Abstract Repair of DNA alkylation damage is essential for maintaining genome integrity and Fe(II)/2-oxoglutarate(2OG)-dependent dioxygenase family of enzymes play crucial role in repairing some of the alkylation damages. Alkylation repair protein-B (AlkB) of Escherichia coli belongs to Fe(II)/2OG-dependent dioxygenase family and carries out DNA dealkylation repair. We report here identification of a hypothetical Mycobacterium leprae protein (accession no. ML0190) from the genomic database and show that this 615-bp open reading frame encodes a protein with sequence and structural similarity to Fe(II)/2OG-dependent dioxygenase AlkB. We identified mRNA transcript of this gene in the M. leprae infected clinical skin biopsy samples isolated from the leprosy patients. Heterologous expression of ML0190 in methyl methane sulfonate (MMS) sensitive and DNA repair deficient strain of Saccharomyces cerevisiae and Escherichia coli resulted in resistance to alkylating agent MM. The results of the present study imply that Mycobacterium leprae ML0190 is involved in protecting the bacterial genome from DNA alkylation damage. Highlights • M leprae hypothetical gene ML0190 mRNA is present in leprosy tissue. • Predicted structure reveals that ML0190 is a DNA-alkylation repair protein. • Heterologous expression of ML0190 results in resistance to alkylating agent MMS. [ABSTRACT FROM AUTHOR]

Abstract Small fringing marshes are ecologically important habitats often impacted by petroleum. We characterized the phylogenetic structure (16S rRNA) and petroleum hydrocarbon degrading alkane hydroxylase genes (alkB and CYP 153A1) in a sediment microbial community from a New Hampshire fringing marsh, using alkane-exposed dilution cultures to enrich for petroleum degrading bacteria. 16S rRNA and alkB analysis demonstrated that the initial sediment community was dominated by Betaproteobacteria (mainly Comamonadaceae) and Gammaproteobacteria (mainly Pseudomonas), while CYP 153A1 sequences predominantly matched Rhizobiales. 24 h of exposure to n -hexane, gasoline, dodecane, or dilution culture alone reduced functional and phylogenetic diversity, enriching for Gammaproteobacteria, especially Pseudomonas. Gammaproteobacteria continued to dominate for 10 days in the n -hexane and no alkane exposed samples, while dodecane and gasoline exposure selected for gram-positive bacteria. The data demonstrate that small fringing marshes in New England harbor petroleum-degrading bacteria, suggesting that petroleum degradation may be an important fringing marsh ecosystem function. Highlights • A fringing tidal marsh microbial community is dominated by Beta- and Gammaproteobacteria. • Pseudomonad and Rhizobiales type alkB and CYP 153A1 alkane hydroxylase genes were detected. • Pseudomonads dominated communities enriched with shorter chain n-alkanes. • Rhodococci dominated a community enriched with dodecane. • AlkB alkane hydroxylases were more abundant than CYP 153A1 hydroxylases in marsh sediments. [ABSTRACT FROM AUTHOR]

Intrinsically disordered regions (IDRs) of proteins often regulate function through interactions with folded domains. Escherichia coli single-stranded DNA binding protein SSB binds and stabilizes single-stranded DNA (ssDNA). The N-terminal of SSB contains characteristic OB (oligonucleotide/oligosaccharide-binding) fold which binds ssDNA tightly but non-specifically. SSB also forms complexes with a large number proteins via the C-terminal interaction domain consisting mostly of acidic amino acid residues. The amino acid residues located between the OB-fold and C-terminal acidic domain are known to constitute an IDR and no functional significance has been attributed to this region. Although SSB is known to bind many DNA repair protein, it is not known whether it binds to DNA dealkylation repair protein AlkB. Here, we characterize AlkB SSB interaction and demonstrate that SSB binds to AlkB via the IDR. We have established that AlkB-SSB interaction by in vitro pull-down and yeast two-hybrid analysis. We mapped the site of contact to be the residues 152-169 of SSB. Unlike most of the SSB-binding proteins which utilize C-terminal acidic domain for interaction, IDR of SSB is necessary and sufficient for AlkB interaction. [ABSTRACT FROM AUTHOR]

Human AlkB homolog 3 (ALKBH3), a homolog of the Escherichia coli protein AlkB, demethylates 1-methyladenine and 3-methylcytosine (3-meC) in single-stranded DNA and RNA by oxidative demethylation. Immunohistochemical analyses on clinical cancer specimens and knockdown experiments using RNA interference in vitro and in vivo indicate that ALKBH3 is a promising molecular target for the treatment of prostate, pancreatic, and non-small cell lung cancer. Therefore, an inhibitor for ALKBH3 demethylase is expected to be a first-in-class molecular-targeted drug for cancer treatment. Here, we report the development of a novel, quantitative real-time PCR-based assay for ALKBH3 demethylase activity against 3-meC by highly active recombinant ALKBH3 protein using a silkworm expression system. This assay enables us to screen for inhibitors of ALKBH3 demethylase, which may result in the development of a novel molecular-targeted drug for cancer therapy.

Background/Aims: ALKBH1, an AlkB homologue in the 2-oxoglutarate and Fe2+ dependent hydroxylase family, is a histone dioxygenase that removes methyl groups from histone H2A. Studies of transgenic mice lacking Alkbh1 reveal that most Alkbh1-/- embryos die during embryonic development. Embryonic stem cells (ESCs) derived from these mice have prolonged expression of pluripotency markers and delayed induction of genes involved in neural differentiation, indicating that ALKBH1 is involved in regulation of pluripotency and differentiation. The aim of this study was to further investigate the role ALKBH1 in early development. Methods: Double-filter methods for nitrocellulose-filter binding, dot blot, enzyme-linked immunosorbent assay (ELISA), immonocytochemistry, cell culture and differentiation of mouse ESCs, Co-IP and miRNA analysis. Results: We found that SOX2 and NANOG bind the ALKBH1 promoter, and we identified protein-protein interactions between ALKBH1 and these core transcription factors of the pluripotency network. Furthermore, lack of ALKBH1 affected the expression of developmentally important miRNAs, which are involved in the regulation of NANOG, SOX2 and neural differentiation. Conclusion: Our results suggest that ALKBH1 interacts with the core transcriptional pluripotency network of ESCs and is involved in regulation of pluripotency and differentiation.

Repair of alkylation damage in DNA is essential for maintaining genome integrity. Escherichia Coli ( E.coli ) DNA repair enzyme AlkB removes methyl adducts including 1-methyladenine and 3-methylcytosine present in DNA by oxidative demethylation from single-stranded DNA (ssDNA). E. coli single-stranded DNA binding protein (SSB) selectively binds ssDNA in a sequence-independent manner. We have recently shown that AlkB can repair methyl adduct present in SSB-coated ssDNA. In this study, we aimed to elucidate details of AlkB-mediated DNA repair of SSB-bound DNA substrate. Therefore, we generated a structural model of AlkB-SSB-ssDNA and using Molecular Dynamics simulation analysis we show that flexibility of SSB-bound DNA allows AlkB to bind in multiple ways. Our docking analysis of AlkB-SSB-ssDNA structure revealed that the Cyt109 base is present in the hydrophobic cavity of AlkB active site pocket. The characterization of AlkB-SSB interaction pattern would likely to help in understanding the mode of alkylated DNA adduct recognition by AlkB. [ABSTRACT FROM AUTHOR]

Iron deprivation induces transcription of genes required for iron uptake, and transcription factor Aft1 and Aft2 mediate this by regulating transcriptional program in Saccharomyces cerevisiae. Iron-dependent Fe(II) and 2-oxoglutarate-dependent dioxygenase family proteins are involved in various cellular pathways including DNA alkylation damage repair. Whether Aft1/Aft2 are required for DNA alkylation repair is currently unknown. In this report, we have analyzed DNA alkylation repair under iron-deprived condition. Saccharomyces cerevisiae Tpa1 is a member of Fe(II) and 2-oxoglutarate-dependent dioxygenase family, and we show that deletion of AFT1 and AFT2 genes affects Tpa1 function resulting in sensitivity to alkylating agent methyl methane sulfonate (MMS). Deletion of AFT1 and AFT2 along with base excision repair pathway DNA glycosylase MAG1 renders the aft1Δaft2Δmag1Δ mutant highly sensitive to MMS. We have further studied effect of iron depletion by replacing S. cerevisiae Tpa1 with Escherichia coli AlkB and human AlkBH3. We observed that the activity of AlkB and AlkBH3 is also diminished similarly when present in aft1Δaft2Δ background as evident by sensitivity to MMS. [ABSTRACT FROM AUTHOR]

Abstract Repair of alkylation damage in DNA is essential for maintaining genome integrity. Escherichia coli (E.coli) protein AlkB removes various alkyl DNA adducts including N 1-methyladenine (N 1meA) and N 3-methylcytosine (N 3meC) by oxidative demethylation. Previous studies showed that AlkB preferentially removes N 1meA and N 3meC from single-stranded DNA (ssDNA). It can also remove N 1meA and N 3meC from double-stranded DNA by base-flipping. Notably, ssDNA produced during DNA replication and recombination, remains bound to E. coli single-stranded DNA binding protein SSB and it is not known whether AlkB can repair methyl adduct present in SSB-coated DNA. Here we have studied AlkB-mediated DNA repair using SSB-bound DNA as substrate. In vitro repair reaction revealed that AlkB could efficiently remove N 3meC adducts inasmuch as DNA length is shorter than 20 nucleotides. However, when longer N 3meC-containing oligonuleotides were used as the substrate, efficiency of AlkB catalyzed reaction was abated compared to SSB-bound DNA substrate of identical length. Truncated SSB containing only the DNA binding domain could also support the stimulation of AlkB activity, suggesting the importance of SSB-DNA interaction for AlkB function. Using 70-mer oligonucleotide containing single N 3meC we demonstrate that SSB-AlkB interaction promotes faster repair of the methyl DNA adducts. Highlights • E. coli single stranded DNA binding protein (SSB) wraps single-stranded DNA (ssDNA). • SSB is known to be involved in base excision repair and recombination repair but not in DNA dealkylation repair. • The alkyl-adduct positioned at the 5′ of ssDNA is more accessible to AlkB compared to the internally located alkyl-adducts. • The presence of SSB lowers the K M of AlkB-catalyzed reaction and promote repair of the longer substrate. • AlkB-mediated repair of methyl adducts from very short ssDNA is rather efficient and SSB-independent. [ABSTRACT FROM AUTHOR]

DNA aptamers are attractive capture probes for affinity chromatography since, in contrast to antibodies, they can be chemically synthesized and, in contrast to tag-specific capture probes (such as Nickel-NTA or Glutathione), they can be used for purification of proteins free of genetic modifications (such as His or GST tags). Despite these attractive features of aptamers as capture probes, there are only a few reports on aptamer-based protein purification and none of them includes a test of the purified protein's activity, thus, leaving discouraging doubts about method’s ability to purify proteins in their active state. The goal of this work was to prove that aptamers could facilitate isolation of active proteins. We refined a complete aptamer-based affinity purification procedure, which takes 4 h to complete. We further applied this procedure to purify two recombinant proteins, MutS and AlkB, from bacterial cell culture: 0.21 mg of 85%-pure AlkB from 4 mL of culture and 0.24 mg of 82%-pure MutS from 0.5 mL of culture. Finally, we proved protein activity by two capillary electrophoresis based assays: an enzymatic assay for AlkB and a DNA-binding assay for MutS. We suggest that in combination with aptamer selection for non-purified protein targets in crude cell lysate, aptamer-based purification provides a means of fast isolation of tag-free recombinant proteins in their native state without the use of antibodies. [ABSTRACT FROM AUTHOR]

Diesel fuel is one of the most important sources of hydrocarbon contamination worldwide. Its composition consists of a complex mixture of n-alkanes, branched alkanes and aromatic compounds. Hydrocarbon degradation in Pseudomonas species has been mostly studied under aerobic conditions; however, a dynamic spectrum of oxygen availability can be found in the environment. Pseudomonas extremaustralis, an Antarctic bacterium isolated from a pristine environment, is able to degrade diesel fuel and presents a wide microaerophilic metabolism. In this work RNA-deep sequence experiments were analyzed comparing the expression profile in aerobic and microaerophilic cultures. Interestingly, genes involved in alkane degradation, including alkB, were over-expressed in micro-aerobiosis in absence of hydrocarbon compounds. In minimal media supplemented with diesel fuel, n-alkanes degradation (C13-C19) after 7 days was observed under low oxygen conditions but not in aerobiosis. In-silico analysis of the alkB promoter zone showed a putative binding sequence for the anaerobic global regulator, Anr. Our results indicate that some diesel fuel components can be utilized as sole carbon source under microaerophilic conditions for cell maintenance or slow growth in a Pseudomonas species and this metabolism could represent an adaptive advantage in polluted environments. [ABSTRACT FROM AUTHOR]