Unlocking the genome of the non-sourdough Kazachstania humilis MAW1: insights into inhibitory factors and phenotypic properties.

BACKGROUND: Ascomycetous budding yeasts are ubiquitous environmental microorganisms important in food production and medicine. Due to recent intensive genomic research, the taxonomy of yeast is becoming more organized based on the identification of monophyletic taxa. This includes genera important to humans, such as Kazachstania. Until now, Kazachstania humilis (previously Candida humilis) was regarded as a sourdough-specific yeast. In addition, any antibacterial activity has not been associated with this species. RESULTS: Previously, we isolated a yeast strain that impaired bio-hydrogen production in a dark fermentation bioreactor and inhibited the growth of Gram-positive and Gram-negative bacteria. Here, using next generation sequencing technologies, we sequenced the genome of this strain named K. humilis MAW1. This is the first genome of a K. humilis isolate not originating from a fermented food. We used novel phylogenetic approach employing the 18 S-ITS-D1-D2 region to show the placement of the K. humilis MAW1 among other members of the Kazachstania genus. This strain was examined by global phenotypic profiling, including carbon sources utilized and the influence of stress conditions on growth. Using the well-recognized bacterial model Escherichia coli AB1157, we show that K. humilis MAW1 cultivated in an acidic medium inhibits bacterial growth by the disturbance of cell division, manifested by filament formation. To gain a greater understanding of the inhibitory effect of K. humilis MAW1, we selected 23 yeast proteins with recognized toxic activity against bacteria and used them for Blast searches of the K. humilis MAW1 genome assembly. The resulting panel of genes present in the K. humilis MAW1 genome included those encoding the 1,3-ß-glucan glycosidase and the 1,3-ß-glucan synthesis inhibitor that might disturb the bacterial cell envelope structures. CONCLUSIONS: We characterized a non-sourdough-derived strain of K. humilis, including its genome sequence and physiological aspects. The MAW1, together with other K. humilis strains, shows the new organization of the mating-type locus. The revealed here pH-dependent ability to inhibit bacterial growth has not been previously recognized in this species. Our study contributes to the building of genome sequence-based classification systems; better understanding of K.humilis as a cell factory in fermentation processes and exploring bacteria-yeast interactions in microbial communities.

Contamination of aquatic environment with anticancer reagents influences Daphnia magna - Ecotoxicogenomics approach.

Pharmaceuticals used in human medicine contaminate freshwater ecosystems. Chemotherapeutics applied in cancer treatment are found in freshwaters at low concentrations (in the range of ng L-1) which, however, can be toxic or mutagenic to aquatic organisms. The aim of this study was to determine the impact of the alkylating/crosslinking anticancer agents, cyclophosphamide (CP) and cisplatin (CDDP), at the concentration detected in water, on Daphnia magna life history, transcriptome, and proteome. This filter feeding cladoceran is an important member of the aquatic food webs controlling algal biomass and forming basic food for planktivorous fish. Here, observations of the D. magna growth rate, age at first reproduction, and the number of eggs produced were performed in the presence of CP or CDDP. The D. magna proteins and RNA were isolated and analysed by mass spectrometry and the mRNA-seq method, respectively. Five generations of contact with the pharmaceuticals in question significantly influenced the D. magna life history parameters with the growth rate and number of laid eggs decreased, whereas age at first reproduction was increased. A decrease in survivorship was observed when daphnids were exposed to CP. These changes are the result of modifications in the gene/transcript expression followed by differences in the proteome profile in comparison to the untreated control. The proteome changes were generally in accordance with the modified transcriptome. The ecotoxicogenomics approach makes it possible to get closer to a complete picture of the influence of CP and CDDP on Daphnia. We have gathered evidence that animals in the presence of anticancer pharmaceuticals attempt to cope with permanent stress by changing their proteome and transcriptome profile. Additionally, our analyses indicate that CDDP showed a stronger effect on tested organisms than CP.

On the use of adenovirus dodecahedron as a carrier for glycoconjugate vaccines.

Virus-Like Particles (VLPs) have been used as immunogenic molecules in numerous recombinant vaccines. VLPs can also serve as vaccine platform to exogenous antigens, usually peptides incorporated within the protein sequences which compose the VLPs or conjugated to them. We herein described the conjugation of a synthetic tetrasaccharide mimicking the Streptococcus pneumoniae serotype 14 capsular polysaccharide to recombinant adenoviral type 3 dodecahedron, formed by the self-assembling of twelve penton bases and investigated the induced immune response when administered subcutaneously (s.c.). Whether formulated in the form of a dodecahedron or disassembled, the glycoconjugate induced an anti-protein response after two and three immunizations equivalent to that observed when the native dodecahedron was administered. On the other hand, the glycoconjugate induced a weak anti-IgM response which diminishes after two doses but no IgM-to-IgG switch was observed in mice against the serotype 14 capsular polysaccharide. In definitive, the whole conjugation process preserved both particulate nature and immunogenicity of the adenoviral dodecahedron. Further studies are needed to fully exploit adenoviral dodecahedron potential in terms of plasticity towards sequence engineering and of its capacity to stimulate the immune system via the intranasal route of administration as well as to shift the response to the carbohydrate antigen by playing both with the carbohydrate to protein ratio and the length of the synthetic carbohydrate antigen.

Effect of Posttranslational Modifications on the Structure and Activity of FTO Demethylase.

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.

Human and Arabidopsis alpha-ketoglutarate-dependent dioxygenase homolog proteins-New players in important regulatory processes.

The family of AlkB homolog (ALKBH) proteins, the homologs of Escherichia coli AlkB 2-oxoglutarate (2OG), and Fe(II)-dependent dioxygenase are involved in a number of important regulatory processes in eukaryotic cells including repair of alkylation lesions in DNA, RNA, and nucleoprotein complexes. There are nine human and thirteen Arabidopsis thaliana ALKBH proteins described, which exhibit diversified functions. Among them, human ALKBH5 and FaT mass and Obesity-associated (FTO) protein and Arabidopsis ALKBH9B and ALKBH10B have been recognized as N6 methyladenine (N6 meA) demethylases, the most abundant posttranscriptional modification in mRNA. The FTO protein is reported to be associated with obesity and type 2 diabetes, and involved in multiple other processes, while ALKBH5 is induced by hypoxia. Arabidopsis ALKBH9B is an N6 meA demethylase influencing plant susceptibility to viral infections via m6 A/A ratio control in viral RNA. ALKBH10B has been discovered to be a functional Arabidopsis homolog of FTO; thus, it is also an RNA N6 meA demethylase involved in plant flowering and several other regulatory processes including control of metabolism. High-throughput mass spectrometry showed multiple sites of human ALKBH phosphorylation. In the case of FTO, the type of modified residue decides about the further processing of the protein. This modification may result in subsequent protein ubiquitination and proteolysis, or in the blocking of these processes. However, the impact of phosphorylation on the other ALKBH function and their downstream pathways remains nearly unexplored in both human and Arabidopsis. Therefore, the investigation of evolutionarily conserved functions of ALKBH proteins and their regulatory impact on important cellular processes is clearly called for.

Unsymmetrically Substituted Dibenzo[b,f][1,5]-diazocine-6,12(5H,11H)dione-A Convenient Scaffold for Bioactive Molecule Design.

A novel approach for the synthesis of unsymmetrically substituted dibenzo[b,f][1,5]diazocine-6,12(5H,11H)diones has been developed. This facile three-step method uses variously substituted 1H-benzo[d][1,3]oxazine-2,4-diones (isatoic anhydrides) and 2-aminobenzoic acids as a starting materials. The obtained products were further transformed into N-alkyl-, N-acetyl- and dithio analogues. Developed procedures allowed the synthesis of unsymmetrical dibenzo[b,f][1,5]diazocine-6,12(5H,11H)diones and three novel heterocyclic scaffolds: benzo[b]naphtho[2,3-f][1,5]diazocine-6,14(5H,13H)dione, pyrido[3,2-c][1,5]benzodiazocine-5,11(6H,12H)-dione and pyrazino[3,2-c][1,5]benzodiazocine-6,12(5H,11H)dione. For 11 of the compounds crystal structures were obtained. The preliminary cytotoxic effect against two cancer (HeLa, U87) and two normal lines (HEK293, EUFA30) as well as antibacterial activity were determined. The obtained dibenzo[b,f][1,5]diazocine(5H,11H)6,12-dione framework could serve as a privileged structure for the drug design and development.

Cell factories converting lactate and acetate to butyrate: Clostridium butyricum and microbial communities from dark fermentation bioreactors.

BACKGROUND: Interactions between microorganisms during specific steps of anaerobic digestion determine metabolic pathways in bioreactors and consequently the efficiency of fermentation processes. This study focuses on conversion of lactate and acetate to butyrate by bacteria of dark fermentation. The recently recognized flavin-based electron bifurcation as a mode of energy coupling by anaerobes increases our knowledge of anaerobic lactate oxidation and butyrate formation. RESULTS: Microbial communities from dark fermentation bioreactors or pure culture of Clostridium butyricum are able to convert lactate and acetate to butyrate in batch experiments. The ability of C. butyricum to transform lactate and acetate to butyrate was shown for the first time, with ethanol identified as an additional end product of this process. A search for genes encoding EtfAB complexes and their gene neighbourhood in C. butyricum and other bacteria capable of lactate and acetate conversion to butyrate as well as butyrate-producers only and the lactate oxidiser Acetobacterium woodii, revealed that the Etf complexes involved in (i) lactate oxidation and (ii) butyrate synthesis, form separate clusters. There is a more extent similarity between Etf subunits that are involved in lactate oxidation in various species (e.g. A. woodii and C. butyricum) than between the different etf gene products within the same species of butyrate producers. A scheme for the metabolic pathway of lactate and acetate transformation to butyrate in C. butyricum was constructed. CONCLUSIONS: Studies on the conversion of lactate and acetate to butyrate by microbial communities from dark fermentation bioreactors or Clostridium butyricum suggest that a phenomenon analogous to cross-feeding of lactate in gastrointestinal tract also occurs in hydrogen-yielding reactors. A scheme of lactate and acetate transformation pathway is proposed, based on the example of C. butyricum, which employs flavin-based electron bifurcation. This process utilizes electron-transferring flavoprotein (Etf) complexes specific for (i) lactate oxidation and (ii) butyrate formation. Phylogenetic analysis revealed that such complexes are encoded in the genomes of other bacteria capable of lactate and acetate conversion to butyrate. These findings contribute significantly to our understanding of the metabolic pathways and symbiotic interactions between bacteria during the acidogenic step of anaerobic digestion.

Hyperbaric oxygen therapy for the treatment of hypoxic/ischemic injury upon perinatal asphyxia-are we there yet?

Birth asphyxia and its main sequel, hypoxic-ischemic encephalopathy, are one of the leading causes of children's deaths worldwide and can potentially worsen the quality of life in subsequent years. Despite extensive research efforts, efficient therapy against the consequences of hypoxia-ischemia occurring in the perinatal period of life is still lacking. The use of hyperbaric oxygen, improving such vital consequences of birth asphyxia as lowered partial oxygen pressure in tissue, apoptosis of neuronal cells, and impaired angiogenesis, is a promising approach. This review focused on the selected aspects of mainly experimental hyperbaric oxygen therapy. The therapeutic window for the treatment of perinatal asphyxia is very narrow, but administering hyperbaric oxygen within those days improves outcomes. Several miRNAs (e.g., mir-107) mediate the therapeutic effect of hyperbaric oxygen by modulating the Wnt pathway, inhibiting apoptosis, increasing angiogenesis, or inducing neural stem cells. Combining hyperbaric oxygen therapy with drugs, such as memantine or ephedrine, produced promising results. A separate aspect is the use of preconditioning with hyperbaric oxygen. Overall, preliminary clinical trials with hyperbaric oxygen therapy used in perinatal asphyxia give auspicious results.

Is Feto-Maternal Transfusion after Cesarean Delivery Different in Singleton and Twin Pregnancy?

Background: The aim of the study was to investigate if feto-maternal transfusion was related to the size of the fetal-maternal interface, and, therefore, was larger in twin pregnancy in comparison with singleton pregnancy. Methods: Blood samples from women with singleton (n = 11), and monochorionic (n = 11) and dichorionic (n = 13) twin gestations were tested. Flow cytometry tests with hemoglobin F, glycophorin A, and hemoglobin F and carbonic anhydrase simultaneous staining were used to detect fetal red blood cells and maternal F cells. Results: In all cases, the volume of feto-maternal transfusion was estimated to be low. The highest rate of fetal red blood cells in the maternal circulation was observed in the blood of women with dichorionic twin gestations both before and after delivery. An increase in fetal red blood cells was observed after cesarean section in singletons and twins. The median rate of maternal F cells was 2.23% in singleton, 2.1% in monochorionic and 3.95% in dichorionic pregnancy. Conclusions: Feto-maternal transfusion during pregnancy may be related to the multiplicity and chorionicity of pregnancy.

Resolving the role of podoplanin in the motility of papillary thyroid carcinoma-derived cells using RNA sequencing.

The intracellular level of podoplanin (PDPN), a transmembrane protein of still unclear function, is frequently altered in metastatic tumors. High expression of PDPN is frequently observed in papillary thyroid cancer (PTC) specimens. Similarly, PTC-derived cell lines (BCPAP and TPC1, harboring the BRAF V600E mutation and RET/PTC1 fusion, respectively), also present enhanced PDPN yield. We previously reported that depletion of PDPN impairs migration of TPC1 cells, but augments metastasis of BCPAP cells. Interestingly, this phenomenon stays in contrast to the migratory pattern observed for wild-type cells, where TPC1 exhibited higher motility than BCPAP cells. Here, we aimed to elucidate the potential role of PDPN in regulation of molecular mechanisms leading to the diverse metastatic features of the studied PTC-derived cells. We consider that this phenomenon may be caused by alternative regulation of signaling pathways due to the presence of the mutated BRAF allele or RET/PTC1 fusion. The high-throughput RNA sequencing (RNA-seq) technique was used to uncover the genes and signaling pathways affected in wild-type and PDPN-depleted TPC1 and BCPAP cells. We found that changes in the expression of various factors of signaling pathways, like RHOA and RAC1 GTPases and their regulators, are linked with both high PDPN levels and presence of the BRAF V600E mutation. We imply that the suppressed motility of wild-type BCPAP cells results from overactivation of RHOA through natively high PDPN expression. This process is accompanied by inhibition of the PI3K kinase and consequently RAC1, due to overactivation of RAS-mediated signaling and the PTEN regulator.

Lethal and mutagenic properties of MMS-generated DNA lesions in Escherichia coli cells deficient in BER and AlkB-directed DNA repair.

Methylmethane sulphonate (MMS), an S(N)2-type alkylating agent, generates DNA methylated bases exhibiting cytotoxic and mutagenic properties. Such damaged bases can be removed by a system of base excision repair (BER) and by oxidative DNA demethylation catalysed by AlkB protein. Here, we have shown that the lack of the BER system and functional AlkB dioxygenase results in (i) increased sensitivity to MMS, (ii) elevated level of spontaneous and MMS-induced mutations (measured by argE3 --> Arg(+) reversion) and (iii) induction of the SOS response shown by visualization of filamentous growth of bacteria. In the xth nth nfo strain additionally mutated in alkB gene, all these effects were extreme and led to 'error catastrophe', resulting from the presence of unrepaired apurinic/apyrimidinic (AP) sites and 1-methyladenine (1meA)/3-methylcytosine (3meC) lesions caused by deficiency in, respectively, BER and AlkB dioxygenase. The decreased level of MMS-induced Arg(+) revertants in the strains deficient in polymerase V (PolV) (bearing the deletion of the umuDC operon), and the increased frequency of these revertants in bacteria overproducing PolV (harbouring the pRW134 plasmid) indicate the involvement of PolV in the error-prone repair of 1meA/3meC and AP sites. Comparison of the sensitivity to MMS and the induction of Arg(+) revertants in the double nfo alkB and xth alkB, and the quadruple xth nth nfo alkB mutants showed that the more AP sites there are in DNA, the stronger the effect of the lack of AlkB protein. Since the sum of MMS-induced Arg(+) revertants in xth, nfo and nth xth nfo and alkB mutants is smaller than the frequency of these revertants in the BER(-) alkB(-) strain, we consider two possibilities: (i) the presence of AP sites in DNA results in relaxation of its structure that facilitates methylation and (ii) additional AP sites are formed in the BER(-) alkB(-) mutants.

Contribution of transcription-coupled DNA repair to MMS-induced mutagenesis in E. coli strains deficient in functional AlkB protein.

In Escherichia coli the alkylating agent methyl methanesulfonate (MMS) induces defense systems (adaptive and SOS responses), DNA repair pathways, and mutagenesis. We have previously found that AlkB protein induced as part of the adaptive (Ada) response protects cells from the genotoxic and mutagenic activity of MMS. AlkB is a non-heme iron (II), alpha-ketoglutarate-dependent dioxygenase that oxidatively demethylates 1meA and 3meC lesions in DNA, with recovery of A and C. Here, we studied the impact of transcription-coupled DNA repair (TCR) on MMS-induced mutagenesis in E. coli strain deficient in functional AlkB protein. Measuring the decline in the frequency of MMS-induced argE3-->Arg(+) revertants under transient amino acid starvation (conditions for TCR induction), we have found a less effective TCR in the BS87 (alkB(-)) strain in comparison with the AB1157 (alkB(+)) counterpart. Mutation in the mfd gene encoding the transcription-repair coupling factor Mfd, resulted in weaker TCR in MMS-treated and starved AB1157 mfd-1 cells in comparison to AB1157 mfd(+), and no repair in BS87 mfd(-) cells. Determination of specificity of Arg(+) revertants allowed to conclude that MMS-induced 1meA and 3meC lesions, unrepaired in bacteria deficient in AlkB, are the source of mutations. These include AT-->TA transversions by supL suppressor formation (1meA) and GC-->AT transitions by supB or supE(oc) formation (3meC). The repair of these lesions is partly Mfd-dependent in the AB1157 mfd-1 and totally Mfd-dependent in the BS87 mfd-1 strain. The nucleotide sequence of the mfd-1 allele shows that the mutated Mfd-1 protein, deprived of the C-terminal translocase domain, is unable to initiate TCR. It strongly enhances the SOS response in the alkB(-)mfd(-) bacteria but not in the alkB(+)mfd(-) counterpart.

Anticancer agents found in environment affect Daphnia at population, individual and molecular levels.

Pharmaceuticals are used in medical treatment on a large scale and as a waste contaminate freshwater ecosystems. Growing amount of so-called civilization diseases, such as different type of cancer, significantly contribute to this form of pollution. The aim of the present study was to determine how the exposure to chemotherapeutics: cyclophosphamide (CP) and cisplatin (CDDP), at detected in environment concentrations, influence proteome profile, life history and population parameters of naturally setting surface waters Daphnia pulex and Daphnia pulicaria. The parameters important for crustaceans, survivorship and population growth rate, were importantly decreased by CDDP treatment but not influenced by CP. On the contrary, the individual growth rate was affected only by CP and exclusively in the case of D. pulicaria. In both clones treated with CP or CDDP, decreased number of eggs was observed. Interestingly, Daphnia males were less sensitive to tested chemotherapeutic than females. Proteome profile revealed that tested anticancer pharmaceuticals modified expression of some proteins involved in Daphnia metabolism. Moreover, males exposed to CDDP showed increased level of enzymes participating in DNA repair. Summing up, the contaminating environment chemotherapeutics reduced fitness of naturally occurring Daphnia species. In consequence this may affect functioning of the aquatic food webs.

ALKBH overexpression in head and neck cancer: potential target for novel anticancer therapy.

The nine identified human homologues of E. coli AlkB 2-oxoglutarate (2OG) and Fe(II)-dependent dioxygenase, ALKBH1-8 and FTO, display different substrate specificities and diverse biological functions. Here we discovered the combined overexpression of members of the ALKBH family in head and neck squamous cell carcinomas (HNSCC). We found direct correlation of ALKBH3 and FTO expression with primary HNSCC tumor size. We observed unidentified thus far cytoplasmic localization of ALKBH2 and 5 in HNSCC, suggesting abnormal role(s) of ALKBH proteins in cancer. Further, high expression of ALKBHs was observed not only in HNSCC, but also in several cancerous cell lines and silencing ALKBH expression in HeLa cancer cells resulted in dramatically decreased survival. Considering the discovered impact of high expression of ALKBH proteins on HNSCC development, we screened for ALKBH blockers among newly synthetized anthraquinone derivatives and demonstrated their potential to support standard anticancer therapy.

How cyclophosphamide at environmentally relevant concentration influences Daphnia magna life history and its proteome.

The waste of commonly used medicines is known to contaminate freshwater ecosystems. Pharmaceuticals can be toxic, mutagenic, or modifying to freshwater organisms even at low concentrations if consider their permanent presence in the environment. Chemotherapeutics used to treat cancer, and in particular alkylating agents, contribute significantly to this form of pollution, the latter introducing cytotoxic and/or mutagenic lesions to the DNA and RNA of organisms which can be disruptive to their cells. The aim of the present study was to investigate the influence of the alkylating anticancer agent cyclophosphamide (CP) on Daphnia magna clones. We evaluated the life history parameters and protein profiles of this crustacean following exposure to environmentally relevant CP concentration of 10 ng L-1. Even at this low concentration, the alkylating agent caused modification of the life history parameters and proteome profile of the Daphnia. These changes were clone-specific and involved growth rate, age at first reproduction, neonate number, and proteins related to cell cycle and redox state regulation. The disturbance caused by pharmaceuticals contaminating freshwater ecosystem is probably weaker and unlikely to be cytotoxic in character due to the high dilution of these substances in the water. However, our results indicate that prolonged exposure of organisms to these toxins may lead to modifications on the organismal and molecular levels with unpredictable significance for the entire ecosystem.

Correction: How cyclophosphamide at environmentally relevant concentration influences Daphnia magna life history and its proteome.

[This corrects the article DOI: 10.1371/journal.pone.0195366.].

Evaluation of Anti-cancer Activity of Stilbene and Methoxydibenzo[b,f] oxepin Derivatives.

BACKGROUND: Stilbenes, 1,2-diphenylethen derivatives, including resveratrol and combretastatins, show anticancer features especially against tumor angiogenesis. Fosbretabulin, CA-4, in combination with carboplatin, is in the last stages of clinical tests as an inhibitor of thyroid cancer. The mode of action of these compounds involves suppression of angiogenesis through interfering with tubulin (de)polymerization. OBJECTIVE: We have previously synthesized five E-2-hydroxystilbenes and seven dibenzo [b,f]oxepins in Z configuration, with methyl or nitro groups at varied positions. The aim of the present work was to evaluate the anticancer activity and molecular mechanism(s) of action of these compounds. RESULTS: Two healthy, EUFA30 and HEK293, and two cancerous, HeLa and U87, cell lines were treated with four newly synthetized stilbenes and seven oxepins. Two of these compounds, JJR5 and JJR6, showed the strongest cytotoxic effect against cancerous cells tested and these two were selected for further investigations. They induced apoptosis with sub-G1 or S cell cycle arrest and PARP cleavage, with no visible activation of caspases 3 and 7. Proteomic differential analysis of stilbene-treated cells led to the identification of proteins involved almost exclusively in cell cycle management, apoptosis, DNA repair and stress response, e.g. oxidative stress. CONCLUSION: Among the newly synthesized stilbene derivatives, we selected two as potent anticancer compounds triggering late apoptosis/necrosis in cancerous cells through sub-G1 phase cell cycle arrest. They changed cyclin expression, induced DNA repair mechanisms, enzymes involved in apoptosis and oxidative stress response. Compounds JJR5 and JJR6 can be a base for structure modification(s) to obtain even more active derivatives.

Methane-yielding microbial communities processing lactate-rich substrates: a piece of the anaerobic digestion puzzle.

BACKGROUND: Anaerobic digestion, whose final products are methane and carbon dioxide, ensures energy flow and circulation of matter in ecosystems. This naturally occurring process is used for the production of renewable energy from biomass. Lactate, a common product of acidic fermentation, is a key intermediate in anaerobic digestion of biomass in the environment and biogas plants. Effective utilization of lactate has been observed in many experimental approaches used to study anaerobic digestion. Interestingly, anaerobic lactate oxidation and lactate oxidizers as a physiological group in methane-yielding microbial communities have not received enough attention in the context of the acetogenic step of anaerobic digestion. This study focuses on metabolic transformation of lactate during the acetogenic and methanogenic steps of anaerobic digestion in methane-yielding bioreactors. RESULTS: Methane-yielding microbial communities instead of pure cultures of acetate producers were used to process artificial lactate-rich media to methane and carbon dioxide in up-flow anaerobic sludge blanket reactors. The media imitated the mixture of acidic products found in anaerobic environments/digesters where lactate fermentation dominates in acidogenesis. Effective utilization of lactate and biogas production was observed. 16S rRNA profiling was used to examine the selected methane-yielding communities. Among Archaea present in the bioreactors, the order Methanosarcinales predominated. The acetoclastic pathway of methane formation was further confirmed by analysis of the stable carbon isotope composition of methane and carbon dioxide. The domain Bacteria was represented by Bacteroidetes, Firmicutes, Proteobacteria, Synergistetes, Actinobacteria, Spirochaetes, Tenericutes, Caldithrix, Verrucomicrobia, Thermotogae, Chloroflexi, Nitrospirae, and Cyanobacteria. Available genome sequences of species and/or genera identified in the microbial communities were searched for genes encoding the lactate-oxidizing metabolic machinery homologous to those of Acetobacterium woodii and Desulfovibrio vulgaris. Furthermore, genes for enzymes of the reductive acetyl-CoA pathway were present in the microbial communities. CONCLUSIONS: The results indicate that lactate is oxidized mainly to acetate during the acetogenic step of AD and this comprises the acetotrophic pathway of methanogenesis. The genes for lactate utilization under anaerobic conditions are widespread in the domain Bacteria. Lactate oxidation to the substrates for methanogens is the most energetically attractive process in comparison to butyrate, propionate, or ethanol oxidation.

Inducible repair of alkylated DNA in microorganisms.

Alkylating agents, which are widespread in the environment, also occur endogenously as primary and secondary metabolites. Such compounds have intrinsically extremely cytotoxic and frequently mutagenic effects, to which organisms have developed resistance by evolving multiple repair mechanisms to protect cellular DNA. One such defense against alkylation lesions is an inducible Adaptive (Ada) response. In Escherichia coli, the Ada response enhances cell resistance by the biosynthesis of four proteins: Ada, AlkA, AlkB, and AidB. The glycosidic bonds of the most cytotoxic lesion, N3-methyladenine (3meA), together with N3-methylguanine (3meG), O(2)-methylthymine (O(2)-meT), and O(2)-methylcytosine (O(2)-meC), are cleaved by AlkA DNA glycosylase. Lesions such as N1-methyladenine (1meA) and N3-methylcytosine (3meC) are removed from DNA and RNA by AlkB dioxygenase. Cytotoxic and mutagenic O(6)-methylguanine (O(6)meG) is repaired by Ada DNA methyltransferase, which transfers the methyl group onto its own cysteine residue from the methylated oxygen. We review (i) the individual Ada proteins Ada, AlkA, AlkB, AidB, and COG3826, with emphasis on the ubiquitous and versatile AlkB and its prokaryotic and eukaryotic homologs; (ii) the organization of the Ada regulon in several bacterial species; (iii) the mechanisms underlying activation of Ada transcription. In vivo and in silico analysis of various microorganisms shows the widespread existence and versatile organization of Ada regulon genes, including not only ada, alkA, alkB, and aidB but also COG3826, alkD, and other genes whose roles in repair of alkylated DNA remain to be elucidated. This review explores the comparative organization of Ada response and protein functions among bacterial species beyond the classical E. coli model.

Ada response - a strategy for repair of alkylated DNA in bacteria.

Alkylating agents are widespread in the environment and also occur endogenously. They can be cytotoxic or mutagenic to the cells introducing alkylated bases to DNA or RNA. All organisms have evolved multiple DNA repair mechanisms to counteract the effects of DNA alkylation: the most cytotoxic lesion, N(3)-methyladenine (3meA), is excised by AlkA glycosylase initiating base excision repair (BER); toxic N(1)-methyladenine (1meA) and N(3)-methylcytosine (3meC), induced in DNA and RNA, are removed by AlkB dioxygenase; and mutagenic and cytotoxic O(6)-methylguanine (O(6) meG) is repaired by Ada methyltransferase. In Escherichia coli, Ada response involves the expression of four genes, ada, alkA, alkB, and aidB, encoding respective proteins Ada, AlkA, AlkB, and AidB. The Ada response is conserved among many bacterial species; however, it can be organized differently, with diverse substrate specificity of the particular proteins. Here, an overview of the organization of the Ada regulon and function of individual proteins is presented. We put special effort into the characterization of AlkB dioxygenases, their substrate specificity, and function in the repair of alkylation lesions in DNA/RNA.