Formation of Biogenic Manganese Oxide Nodules on Hyphae of a New Fungal Isolate of Periconia That Immobilizes Aqueous Copper.

Mn(II)-oxidizing microorganisms are considered to play significant roles in the natural geochemical cycles of Mn and other heavy metals because the insoluble biogenic Mn oxides (BMOs) that are produced by these microorganisms adsorb other dissolved heavy metals and immobilize them as precipitates. In the present study, a new Mn(II)-oxidizing fungal strain belonging to the ascomycete genus Periconia, a well-studied plant-associating fungal genus with Mn(II)-oxidizing activity that has not yet been exami-ned in detail, was isolated from natural groundwater outflow sediment. This isolate, named strain TS-2, was confirmed to oxidize dissolved Mn(II) and produce insoluble BMOs that formed characteristic, separately-located nodules on their hyphae while leaving major areas of the hyphae free from encrustation. These BMO nodules also adsorbed and immobilized dissolved Cu(II), a model analyte of heavy metals, as evidenced by elemental mapping ana-lyses of fungal hyphae-BMO assemblages using a scanning electron microscope with energy-dispersive X-ray spectroscopy (SEM-EDX). Analyses of functional genes within the whole genome of strain TS-2 further revealed the presence of multiple genes predicted to encode laccases/multicopper oxidases that were potentially responsible for Mn(II) oxidation by this strain. The formation of BMO nodules may have functioned to prevent the complete encrustation of fungal hyphae, thereby enabling the control of heavy metal concentrations in their local microenvironments while maintaining hyphal functionality. The present results will expand our knowledge of the physiological and morphological traits of Mn(II)-oxidizing Periconia, which may affect the natural cycle of heavy metals through their immobilization.

Whole-genome sequence of Periconia sp. strain TS-2, an ascomycete fungus isolated from a freshwater outflow and capable of Mn(II) oxidation.

Members of the genus Periconia are commonly found as plant-associated filamentous fungi. Here, the first draft genome sequence of a new Periconia strain, TS-2, that was isolated from freshwater outflow sediment and possesses the ability to oxidize dissolved Mn(II), was obtained and has an estimated size of 40.7 Mb.

Genomic analysis of a marine alphaproteobacterium Sagittula sp. strain MA-2 that carried eight plasmids.

Bacteria that belong to the family Roseobacteraceae in the Alphaproteobacteria class are widely distributed in marine environments with remarkable physiological diversity, which is considered to be attributed to their genomic plasticity. In this study, a novel isolate of the genus Sagittula within Roseobacteraceae, strain MA-2, was obtained from a coastal marine bacterial consortium enriched with aromatic hydrocarbons, and its complete genome was sequenced. The genome with a total size of 5.69 Mbp was revealed to consist of a 4.67-Mbp circular chromosome and eight circular plasmids ranging in size from 19.5 to 361.5 kbp. Further analyses of functional genes in the strain MA-2 genome identified homologous genes responsible for the biotransformation of gentisic acid, which were located on one of its plasmids and were not found in genomes of other Sagittula strains available from databases. This suggested that strain MA-2 had acquired these genes via horizontal gene transfers that enabled them to degrade and utilize gentisic acid as a growth substrate. This study provided the second complete genome sequence of the genus Sagittula and supports the hypothesis that acquisition of ecologically relevant genes in extrachromosomal replicons allows Roseobacteraceae to be highly adaptable to diverse lifestyles.

The GGDEF protein Dgc2 suppresses both motility and biofilm formation in the filamentous cyanobacterium Leptolyngbya boryana.

Colony pattern formations of bacteria with motility manifest complicated morphological self-organization phenomena. Leptolyngbya boryana is a filamentous cyanobacterium, which has been used as a genetic model organism for studying metabolism including photosynthesis and nitrogen fixation. A widely used type strain [wild type (WT) in this article] of this species has not been reported to show any motile activity. However, we isolated a spontaneous mutant strain that shows active motility (gliding activity) to give rise to complicated colony patterns, including comet-like wandering clusters and disk-like rotating vortices on solid media. Whole-genome resequencing identified multiple mutations in the genome of the mutant strain. We confirmed that inactivation of the candidate gene dgc2 (LBDG_02920) in the WT background was sufficient to give rise to motility and morphologically complex colony patterns. This gene encodes a protein containing the GGDEF motif which is conserved at the catalytic domain of diguanylate cyclase (DGC). Although DGC has been reported to be involved in biofilm formation, the dgc2 mutant significantly facilitated biofilm formation, suggesting a role for the dgc2 gene in suppressing both gliding motility and biofilm formation. Thus, Leptolyngbya is expected to be an excellent genetic model for studying dynamic colony pattern formation and to provide novel insights into the role of DGC family genes in biofilm formation. IMPORTANCE Self-propelled bacteria often exhibit complex collective behaviors, such as formation of dense-moving clusters, which are exemplified by wandering comet-like and rotating disk-like colonies; however, the molecular details of how these structures are formed are scant. We found that a strain of the filamentous cyanobacterium Leptolyngbya deficient in the GGDEF protein gene dgc2 elicits motility and complex and dynamic colony pattern formation, including comet-like and disk-like clusters. Although c-di-GMP has been reported to activate biofilm formation in some bacterial species, disruption of dgc2 unexpectedly enhanced it, suggesting a novel role for this GGDEF protein for inhibiting both colony pattern formation and biofilm formation.

Nondesulfurizing benzothiophene biotransformation to hetero and homodimeric ortho-substituted diaryl disulfides by the model PAH-degrading Sphingobium barthaii.

Understanding the biotransformation mechanisms of toxic sulfur-containing polycyclic aromatic hydrocarbon (PASH) pollutants such as benzothiophene (BT) is useful for predicting their environmental fates. In the natural environment, nondesulfurizing hydrocarbon-degrading bacteria are major active contributors to PASH biodegradation at petroleum-contaminated sites; however, BT biotransformation pathways by this group of bacteria are less explored when compared to desulfurizing organisms. When a model nondesulfurizing polycyclic aromatic hydrocarbon-degrading soil bacterium, Sphingobium barthaii KK22, was investigated for its ability to cometabolically biotransform BT by quantitative and qualitative methods, BT was depleted from culture media but was biotransformed into mostly high molar mass (HMM) hetero and homodimeric ortho-substituted diaryl disulfides (diaryl disulfanes). HMM diaryl disulfides have not been reported as biotransformation products of BT. Chemical structures were proposed for the diaryl disulfides by comprehensive mass spectrometry analyses of the chromatographically separated products and were supported by the identification of transient upstream BT biotransformation products, which included benzenethiols. Thiophenic acid products were also identified, and pathways that described BT biotransformation and novel HMM diaryl disulfide formation were constructed. This work shows that nondesulfurizing hydrocarbon-degrading organisms produce HMM diaryl disulfides from low molar mass polyaromatic sulfur heterocycles, and this may be taken into consideration when predicting the environmental fates of BT pollutants.

Comprehensive Genomic Characterization of Marine Bacteria Thalassospira spp. Provides Insights into Their Ecological Roles in Aromatic Hydrocarbon-Exposed Environments.

The marine bacterial genus Thalassospira has often been identified as an abundant member of polycyclic aromatic hydrocarbon (PAH)-exposed microbial communities. However, despite their potential usability for biotechnological applications, functional genes that are conserved in their genomes have barely been investigated. Thus, the goal of this study was to comprehensively examine the functional genes that were potentially responsible for aromatic hydrocarbon biodegradation in the Thalassospira genomes available from databases, including a new isolate of Thalassospira, strain GO-4, isolated from a phenanthrene-enriched marine bacterial consortium. Strain GO-4 was used in this study as a model organism to link the genomic data and their metabolic functions. Strain GO-4 growth assays confirmed that it utilized a common phenanthrene biodegradation intermediate 2-carboxybenzaldehyde (CBA) as the sole source of carbon and energy, but did not utilize phenanthrene. Consistently, strain GO-4 was found to possess homologous genes of phdK, pht, and pca that encode enzymes for biodegradation of CBA, phthalic acid, and protocatechuic acid, respectively. Further comprehensive genomic analyses for 33 Thalassospira genomes from databases showed that a gene cluster that consisted of phdK and pht homologs was conserved in 13 of the 33 strains. pca gene homologs were found in all examined genomes; however, homologs of the known PAH-degrading genes, such as the pah, phn, or nah genes, were not found. Possibly Thalassospira spp. co-occupy niches with other PAH-degrading bacteria that provide them with PAH degradation intermediates and facilitated their inhabitation in PAH-exposed microbial ecosystems. IMPORTANCE Comprehensive investigation of multiple genomic data sets from targeted microbial taxa deposited in databases may provide substantial information to predict metabolic capabilities and ecological roles in different environments. This study is the first report that details the functional profiling of Thalassospira spp. that have repeatedly been found in polycyclic aromatic hydrocarbon (PAH)-exposed marine bacterial communities by using genomic data from a new isolate, Thalassospira strain GO-4, and other strains in databases. Through screening of functional genes potentially involved in aromatic hydrocarbon biodegradation across 33 Thalassospira genomes and growth assays for strain GO-4, it was suggested that Thalassospira spp. unexceptionally conserved the ability to metabolize single-ring, PAH biodegradation intermediates, while being incapable of utilizing PAHs. This expanded our understanding of this potentially important contributing member to PAH-degrading microbial ecosystems; such species are considered to be specialized in driving downstream reactions of PAH biodegradation.

Complete Genome Sequence of Thalassospira sp. Strain GO-4, a Marine Bacterium Isolated from a Phenanthrene-Enriched Bacterial Consortium.

The genus Thalassospira has often been studied as a potential major contributing member of aromatic hydrocarbon-exposed microbial communities. Here, the complete genome sequence of a new isolate of Thalassospira, strain GO-4, was obtained and was confirmed to possess functional genes that are responsible for its metabolism of phthalic acid.

Quantification of cyanobacterial cyclic di-guanosine monophosphate (c-di-GMP) by liquid chromatography electrospray ionization tandem mass spectrometry.

Cyclic di-guanosine monophosphate (c-di-GMP) is a second messenger found ubiquitously in bacteria. This signaling molecule regulates a variety of physiological activities such as phototaxis and flocculation in cyanobacteria and is critical for their environmental adaptation. Although genes encoding the enzymes for synthesis and/or degradation of c-di-GMP are found in the genomes of both multicellular and unicellular cyanobacteria, little is known about the biological functions of these enzymes in cyanobacterial cells. Here we have established a robust and highly sensitive liquid chromatography tandem mass spectrometry (LC-MS/MS)-based method for c-di-GMP quantification using a cost-effective solvent, methanol. Quantification methods were validated by measuring c-di-GMP in the cyanobacterium Synechococcus elongatus PCC 7942 through spiking and recovery assays after which the method was applied to examine short-term changes in cellular levels of c-di-GMP in response to a transition from light to dark or from dark to light in S. elongatus. Results showed that a transient increase in c-di-GMP upon transitioning from light to dark was occurring which resembled responses involving cyclic adenosine monophosphate and other second messengers in cyanobacteria. These findings demonstrated that our method enabled relatively specific and sensitive quantification of c-di-GMP in cyanobacteria at lower cost.

Natural Chromosome-Chromid Fusion across rRNA Operons in a Burkholderiaceae Bacterium.

Chromids (secondary chromosomes) in bacterial genomes that are present in addition to the main chromosome appear to be evolutionarily conserved in some specific bacterial groups. In rare cases among these groups, a small number of strains from Rhizobiales and Vibrionales were shown to possess a naturally fused single chromosome that was reported to have been generated through intragenomic homologous recombination between repeated sequences on the chromosome and chromid. Similar examples have never been reported in the family Burkholderiaceae, a well-documented group that conserves chromids. Here, an in-depth genomic characterization was performed on a Burkholderiaceae bacterium that was isolated from a soil bacterial consortium maintained on diesel fuel and mutagenic benzo[a]pyrene. This organism, Cupriavidus necator strain KK10, was revealed to carry a single chromosome with unexpectedly large size (>6.6 Mb), and results of comparative genomics with the genome of C. necator N-1T indicated that the single chromosome of KK10 was generated through fusion of the prototypical chromosome and chromid at the rRNA operons. This fusion hypothetically occurred through homologous recombination with a crossover between repeated rRNA operons on the chromosome and chromid. Some metabolic functions that were likely expressed from genes on the prototypical chromid region were indicated to be retained. If this phenomenon-the bacterial chromosome-chromid fusion across the rRNA operons through homologous recombination-occurs universally in prokaryotes, the multiple rRNA operons in bacterial genomes may not only contribute to the robustness of ribosome function, but also provide more opportunities for genomic rearrangements through frequent recombination. IMPORTANCE A bacterial chromosome that was naturally fused with the secondary chromosome, or "chromid," and presented as an unexpectedly large single replicon was discovered in the genome of Cupriavidus necator strain KK10, a biotechnologically useful member of the family Burkholderiaceae. Although Burkholderiaceae is a well-documented group that conserves chromids in their genomes, this chromosomal fusion event has not been previously reported for this family. This fusion has hypothetically occurred through intragenomic homologous recombination between repeated rRNA operons and, if so, provides novel insight into the potential of multiple rRNA operons in bacterial genomes to lead to chromosome-chromid fusion. The harsh conditions under which strain KK10 was maintained-a genotoxic hydrocarbon-enriched milieu-may have provided this genotype with a niche in which to survive.

Complete Genome Sequence of Cupriavidus necator KK10, an Azaarene-Degrading and Polyhydroxyalkanoate-Producing Soil Bacterium.

Cupriavidus necator KK10 has been investigated in azaarene and diesel fuel biodegradation studies and is capable of polyhydroxyalkanoate (PHA) production. Its complete genome sequence revealed two closed circular sequences, the chromosome (6.68 Mb) and plasmid (1.67 Mb). The KK10 genome carries functional genes potentially responsible for azaarene biodegradation and polyhydroxyalkanoate biosynthesis.

Identification of a Diguanylate Cyclase That Facilitates Biofilm Formation on Electrodes by Shewanella oneidensis MR-1.

In many bacteria, cyclic diguanosine monophosphate (c-di-GMP), synthesized by diguanylate cyclase (DGC), serves as a second messenger involved in the regulation of biofilm formation. Although studies have suggested that c-di-GMP also regulates the formation of electrochemically active biofilms (EABFs) by Shewanella oneidensis MR-1, DGCs involved in this process remained to be identified. Here, we report that the SO_1646 gene, hereafter named dgcS, is upregulated under medium flow conditions in electrochemical flow cells (EFCs), and its product (DgcS) functions as a major DGC in MR-1. In vitro assays demonstrated that purified DgcS catalyzed the synthesis of c-di-GMP from GTP. Comparisons of intracellular c-di-GMP levels in the wild-type strain and a dgcS deletion mutant (ΔdgcS mutant) showed that production of c-di-GMP was markedly reduced in the ΔdgcS mutant when cells were grown in batch cultures and on electrodes in EFCs. Cultivation of the ΔdgcS mutant in EFCs also revealed that the loss of DgcS resulted in impaired biofilm formation and decreased current generation. These findings demonstrate that MR-1 uses DgcS to synthesize c-di-GMP under medium flow conditions, thereby activating biofilm formation on electrodes.IMPORTANCE Bioelectrochemical systems (BESs) have attracted wide attention owing to their utility in sustainable biotechnology processes, such as microbial fuel cells and electrofermentation systems. In BESs, electrochemically active bacteria (EAB) form biofilms on electrode surfaces, thereby serving as effective catalysts for the interconversion between chemical and electric energy. It is therefore important to understand mechanisms for the formation of biofilm by EAB grown on electrodes. Here, we show that a model EAB, S. oneidensis MR-1, expresses DgcS as a major DGC, thereby activating the formation of biofilms on electrodes via c-di-GMP-dependent signal transduction cascades. The findings presented herein provide the molecular basis for improving electrochemical interactions between EAB and electrodes in BESs. The results also offer molecular insights into how Shewanella regulates biofilm formation on solid surfaces in the natural environment.

Complete Genome Sequence of Sphingobium barthaii KK22, a High-Molecular-Weight Polycyclic Aromatic Hydrocarbon-Degrading Soil Bacterium.

Sphingobium barthaii KK22T is a high-molecular-weight polycyclic aromatic hydrocarbon-degrading soil bacterium that has been investigated in biotransformation, microbial ecology, and DNA damage studies. The complete genome sequence of S. barthaii revealed four closed circular sequences, including two chromosomes, a megaplasmid, and a smaller plasmid, by hybrid assembly using short- and long-read sequencing technologies.

Multispecies Diesel Fuel Biodegradation and Niche Formation Are Ignited by Pioneer Hydrocarbon-Utilizing Proteobacteria in a Soil Bacterial Consortium.

A soil bacterial consortium that was grown on diesel fuel and consisted of more than 10 members from different genera was maintained through repetitive subculturing and was utilized as a practical model to investigate a bacterial community that was continuously exposed to petroleum hydrocarbons. Through metagenomics analyses, consortium member isolation, growth assays, and metabolite identification which supported the linkage of genomic data and functionality, two pioneering genera, Sphingobium and Pseudomonas, whose catabolic capabilities were differentiated, were found to be responsible for the creation of specialized ecological niches that were apparently occupied by other bacterial members for survival within the consortium. Coexisting genera Achromobacter and Cupriavidus maintained their existence in the consortium through metabolic dependencies by utilizing hydrocarbon biotransformation products of pioneer metabolism, which was confirmed through growth tests and identification of biotransformation products of the isolated strains. Pioneering Sphingobium and Pseudomonas spp. utilized relatively water-insoluble hydrocarbon parent compounds and facilitated the development of a consortium community structure that resulted in the creation of niches in response to diesel fuel exposure which were created through the production of more-water-soluble biotransformation products available to cocolonizers. That these and other organisms were still present in the consortium after multiple transfers spanning 15 years provided evidence for these ecological niches. Member survival through occupation of these niches led to robustness of each group within the multispecies bacterial community. Overall, these results contribute to our understanding of the complex ecological relationships that may evolve during prokaryotic hydrocarbon pollutant biodegradation.IMPORTANCE There are few metagenome studies that have explored soil consortia maintained on a complex hydrocarbon substrate after the community interrelationships were formed. A soil bacterial consortium maintained on diesel fuel was utilized as a practical model to investigate bacterial community relationships through metagenomics analyses, consortium member isolation, growth assays, and metabolite identification, which supported the linkage of genomic data and functionality. Two pioneering genera were responsible for the biodegradation of aromatics and alkanes by initiating biotransformation and thereby created specialized niches that were populated by other members. A model that represents these relationships was constructed, which contributes to our understanding of the complex ecological relationships that evolve during prokaryotic hydrocarbon pollutant biodegradation.

Characterization of N-(2,6-dimethylphenyl)hydroxylamine adducts of 2'-deoxyguanosine under weakly basic conditions.

Aromatic amines are a class of chemical carcinogens that are activated by cytochrome P450 enzymes to form arylhydroxylamines that are conjugated to form N-acetoxyarylamines or N-sulfonyloxyarylamines. These conjugates undergo N-O bond cleavage to become reactive nitrenium ions that may form DNA adducts. Numerous studies in the past using N-acetoxyarylamines to investigate DNA adduct formation were conducted, however, less is known in regard to DNA adduct formation directly from arylhydroxylamines - especially under conditions that mimic the physiological conditions of cells such as weakly basic conditions. In this study, 2'-deoxyguanosine (dG) was exposed to N-(2,6-dimethylphenyl)hydroxylamine (2,6-DMPHA) and N-phenylhydroxylamine (PHA) at pH 7.4 without enzymes and analyzed by liquid chromatography high resolution mass spectrometry (LC-HRMS). 2,6-DMPHA exposure resulted in the production of relatively low amounts of adducts however the identities of at least six different adducts that were formed through reactions with carbon, nitrogen and oxygen of 2'-deoxyguanosine were proposed based upon different analytical approaches including HRMS CID fragmentation and NMR analyses. Contrastively, PHA exposure under identical conditions resulted in one adduct at the C8 position. It was concluded from these results and results of theoretical calculations that nitrenium ions produced from 2,6-DMPHA were relatively more stable resulting in longer nitrenium ion lifetimes which ultimately led to greater potential for 2,6-DMPHA nitrenium ions to react with multiple sites on dG.

Formation of Bulky DNA Adducts by Non-Enzymatic Production of 1,2-Naphthoquinone-Epoxide from 1,2-Naphthoquinone under Physiological Conditions.

Quinones may be formed metabolically or abiotically from environmental pollutants and polycyclic aromatic hydrocarbons (PAHs); many are recognized as toxicological intermediates that cause a variety of deleterious cellular effects including mutagenicity. The PAH-o-quinone, 1,2-naphthoquinone (1,2-NQ), may exert its genotoxic effects through interactions with cellular nucleophiles such as DNA, however, the mechanisms of 1,2-NQ adduct formation are still under investigation. With the aim to further understand these mechanisms, the chemical structures of adducts formed from the reaction of 2'-deoxyguanosine (dG) with 1,2-NQ under physiological conditions were investigated by liquid chromatography electrospray ionization tandem mass spectrometry and 1H NMR analyses. Results showed that 1,2-NQ underwent non-enzymatic oxidation to form a 1,2-NQ-epoxide which in turn formed at least four bulky adducts with dG, and these adducts were more likely to be formed under physiological conditions. A mechanism was proposed whereby hydration of 1,2-NQ to form unstable naphthohydroquinones and 2-hydroxy-1,4-naphthoquinone resulted in formation of hydrogen peroxide that oxidized 1,2-NQ. These results suggest that the genotoxicity of 1,2-NQ may not only be caused through oxidative DNA damage and adduct formation through Michael addition but also through non-enzymatic oxidative transformation of 1,2-NQ itself to form an intermediate PAH-epoxide which covalently binds to DNA.

In vitro DNA/RNA Adductomics to Confirm DNA Damage Caused by Benzo[a]pyrene in the Hep G2 Cell Line.

In the development of new chemical substances, genetic toxicity evaluations are a high priority for safety risk management. Evaluation of the possibility of compound carcinogenicity with accuracy and at reasonable cost in the early stages of development by in vitro techniques is preferred. Currently, DNA damage-related in vitro genotoxicity tests are widely-used screening tools after which next generation toxicity testing may be applied to confirm DNA damage. DNA adductomics may be used to evaluate DNA damage in vitro, however confirmation of DNA adduct identities through comparison to authentic standards may be time-consuming and expensive processes. Considering this, a streamlined method for confirming putative DNA adducts that are detected by DNA adductomics may be useful. With this aim, in vitro DNA adductome methods in conjunction with in vitro RNA adductome methods may be proposed as a DNA adductome verification approach by which to eliminate false positive annotations. Such an approach was evaluated by conducting in vitro assays whereby Hep G2 cell lines that were exposed to or not exposed to benzo[a]pyrene were digested to their respective 2'-deoxynucleosides or ribonucleosides and analyzed by liquid chromatography electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) by comparative DNA and RNA adductomics through neutral loss targeting of the [M + H]+ > [M + H - 116]+ or [M + H]+ > [M + H -132]+ transitions over predetermined ranges. Comparisons of DNA adductome maps revealed putative DNA adducts that were detected in exposed cells but not in unexposed cells. Similarly, comparisons of RNA adductome maps revealed putative RNA adducts in exposed cells but not in unexposed cells. Taken together these experiments revealed that analogous forms of putative damage had occurred in both DNA and RNA which supported that putative DNA adducts detected by DNA adductomics were DNA adducts. High resolution mass spectrometry (HRMS) was utilized to confirm that putative nucleic acid adducts detected in both DNA and RNA were derived from benzo[a]pyrene exposure and these putative adducts were identified as 7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene- (B[a]PDE)-type adducts. Overall, this study demonstrates the usefulness of utilizing DNA/RNA adductomics to screen for nucleic acid damage.

Triple quadrupole mass spectrometry comparative DNA adductomics of Hep G2 cells following exposure to safrole.

A DNA adduct screening pipeline was constructed to apply triple quadrupole mass spectrometry comparative DNA adductomics to investigate the effects of the naturally-occurring plant constituent, safrole (4-allyl-1,2-methylenedioxybenzene), on human hepatoma cells, Hep G2. DNA from Hep G2 cells that were exposed to or not exposed to safrole were digested to 2'-deoxynucleosides and analyzed by liquid chromatography electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) whereby the neutral loss of 2'-deoxyribose was targeted by monitoring the [M+H]+ > [M+H - 116]+ transition over a defined range. Comparative analyses through construction of DNA adductome maps revealed numerous putative DNA adduct candidates. Targeted product ion scan investigations allowed for detailed fragmentation ion analyses and the identities of at least five bulky alkylated adducts of 2'-deoxyguanosine and 2'-deoxyadenosine with molar masses greater than 400 Da each were proposed. All adducts were derived from safrole exposure and pathways to explain the occurrence of these adducts in Hep G2 cells through metabolism of safrole are discussed. This study demonstrates the potential utility of constructing triple quadrupole MS comparative DNA adductomics pipelines to screen chemicals for DNA adducts by using human cell lines.

RAMA casein zymography: Time-saving and highly sensitive casein zymography for MMP7 and trypsin.

To detect metalloproteinase-7 (MMP7), zymography is conducted using a casein substrate and conventional CBB stain. It has disadvantages because it is time consuming and has low sensitivity. Previously, a sensitive method to detect MMP7 up to 30 pg was reported, however it required special substrates and complicated handlings. RAMA casein zymography described herein is rapid, sensitive, and reproducible. By applying high-sensitivity staining with low substrate conditions, the staining process is completed within 1 h and sensitivity was increased 100-fold. The method can detect 10 pg MMP7 by using commercially available casein without complicated handlings. Moreover, it increases detection sensitivity for trypsin.

Analysis of the Fine-Tuning of Cyanobacterial Circadian Phase by Monochromatic Light and Long-Day Conditions.

The cyanobacterial circadian-related protein, Pex, accumulates in the dark period of the diurnal light-dark cycle. After the diurnal cycle, an approximately 3 h advance in the phase of the circadian bioluminescence rhythm is observed in pex-deficient mutants, as compared with the wild type. However, it is unclear what type of photosensing mechanism regulates the accumulation and the phase change. In monochromatic light irradiation experiments, Pex accumulation was strongly repressed under blue light conditions; however, only small reductions in Pex accumulation were observed under red or green light conditions. After the diurnal cycle of 12 h of white fluorescent light and 12 h of blue light, the phase advance was repressed more than that of the cycle of 12 h red (or green) light. The phase advance also occurred after 16 h light/8 h dark cycles (long-day cycles) but did not occur after 8 h light/16 h dark cycles (short-day cycles). While Pex is a unique winged helix transcription factor harboring secondary structures (α0 and α4 helices), the importance of the structures is not understood. In in vivo experiments with site-directed mutations in the α0 helix, the obtained mutants, in which Pex was missing the hydrophobic side chain at the 28th or 32nd amino acid residue, exhibited no phase delay after the light/dark cycle. In in vitro DNA binding assays, the mutant proteins showed no binding to the promoter region of the clock gene kaiA. From these results, we propose a molecular model which describes the phase delay in cyanobacteria.