First Report on Photodynamic Inactivation of Archaea Including a Novel Method for High-Throughput Reduction Measurement

Archaea are considered third, independent domain of living organisms besides eukaryotic and bacterial cells. To date, no report is available of photodynamic inactivation (PDI) of any archaeal cells. Two commercially available photosensitizers (SAPYR and TMPyP) were used to investigate photodynamic inactivation of Halobacterium salinarum. In addition, a novel high-throughput method was tested to evaluate microbial reduction in vitro. Due to the high salt content of the culture medium, the physical and chemical properties of photosensitizers were analyzed via spectroscopy and fluorescence-based DPBF assays. Attachment or uptake of photosensitizers to or in archaeal cells was investigated. The photodynamic inactivation of Halobacterium salinarum was evaluated via growth curve method allowing a high throughput of samples. The presented results indicate that the photodynamic mechanisms are working even in high salt environments. Either photosensitizer inactivated the archaeal cells with a reduction of 99.9% at least. The growth curves provided a fast and precise measurement of cell viability. The results show for the first time that PDI can kill not only bacterial cells but also robust archaea. The novel method for generating high-throughput growth curves provides benefits for future research regarding antimicrobial substances in general.     

Emerging roles for plant topoisomerase VI

Topoisomerase VI is a unique type II topoisomerase originally identified in archaea. Although lacking in most eukaryotic phyla, topoisomerase VI homologs have been recently identified and characterized in the plant Arabidopsis thaliana. Three new studies of Arabidopsis topoisomerase VI show that this enzyme is important to several processes involving DNA replication and gene expression.     

Chemical Synthesis of Asparagine‐Linked Archaeal N‐Glycan from Methanothermus fervidus

Several N‐linked glycoproteins have been identified in archaea and there is growing evidence that the N‐glycan is involved in survival and functioning of archaea in extreme conditions. Chemical synthesis of the archaeal N‐glycans represents a crucial step towards understanding the putative function of protein glycosylation in archaea. Herein the first total synthesis of the archaeal L ‐asparagine linked hexasaccharide from Methanothermus fervidus is reported using a highly convergent [3+3] glycosylation approach in high overall yields. The synthesis relies on efficient preparation of regioselectively protected thioglycoside building blocks for orthogonal glycosylations and late stage N‐aspartylation.     

Biosynthesis of Hybrid Neutral Lipids with Archaeal and Eukaryotic Characteristics in Engineered Saccharomyces cerevisiae

Discovery of the Asgard superphylum of archaea provides new evidence supporting the two-domain model of life: eukaryotes originated from an Asgard-related archaeon that engulfed a bacterial endosymbiont. However, how eukaryotes acquired bacterial-like membrane lipids with a sn-glycerol-3-phosphate (G3P) backbone instead of the archaeal-like sn-glycerol-1-phosphate (G1P) backbone remains unknown. In this study, we reconstituted archaeal lipid production in Saccharomyces cerevisiae by expressing unsaturated archaeol-synthesizing enzymes. Using Golden Gate cloning for pathway assembly, modular gene replacement was performed, revealing the potential biosynthesis of both G1P- and G3P-based unsaturated archaeol by uncultured Asgard archaea. Unexpectedly, hybrid neutral lipids containing both archaeal isoprenoids and eukaryotic fatty acids were observed in recombinant S. cerevisiae. The ability of yeast and archaeal diacylglycerol acyltransferases to synthesize such hybrid lipids was demonstrated.     

Subcellular localization based comparative study on radioresistant bacteria: A novel approach to mine proteins involve in radioresistance

Radioresistant bacteria (RRB) are among the most radioresistant organisms and has a unique role in evolution. Along with the evolutionary role, radioresistant organisms play important role in paper industries, bioremediation, vaccine development and possibility in anti-aging and anti-cancer treatment. The study of radiation resistance in RRB was mainly focused on cytosolic mechanisms such as DNA repair mechanism, cell cleansing activity and high antioxidant activity. Although it was known that protein localized on outer areas of cell play role in resistance towards extreme condition but the mechanisms/proteins localized on the outer area of cells are not studied for radioresistance. Considering the fact that outer part of cell is more exposed to radiations and proteins present in outer area of the cell may have role in radioresistance. Localization based comparative study of proteome from RRB and non-radio resistant bacteria was carried out. In RRB 20 unique proteins have been identified. Further domain, structural, and pathway analysis of selected proteins were carried out. Out of 20 proteins, 8 proteins were direct involvement in radioresistance and literature study strengthens this, however, 1 proteins had assumed relation in radioresistance. Selected radioresistant proteins may be helpful for optimal use of RRB in industry and health care.     

Convergence of hormones and autoinducers at the host/pathogen interface

Most living organisms possess sophisticated cell-signaling networks in which lipid-based signals modulate biological effects such as cell differentiation, reproduction and immune responses. Acyl homoserine lactone (AHL) autoinducers are fatty acid-based signaling molecules synthesized by several Gram-negative bacteria that are used to coordinate gene expression in a process termed “quorum sensing” (QS). Recent evidence shows that autoinducers not only control gene expression in bacterial cells, but also alter gene expression in mammalian cells. These alterations include modulation of proinflammatory cytokines and induction of apoptosis. Some of these responses may have deleterious effects on the host’s immune response, thereby leading to increased bacterial pathogenesis. Prokaryotes and eukaryotes have cohabited for approximately two billion years, during which time they have been exposed to each others’ soluble signaling molecules. We postulate that organisms from the different kingdoms of nature have acquired mechanisms to sense and respond to each others signaling molecules, and we have named this process interkingdom signaling. We further propose that autoinducers, which exhibit structural and functional similarities to mammalian lipid-based hormones, are excellent candidates for mediating this interkingdom communication. Here we will compare and contrast bacterial QS systems with eukaryotic endocrine systems, and discuss the mechanisms by which autoinducers may exploit mammalian signal transduction pathways.     

DNA Methylation: Site-Specific Methylations Cause Gene Inactivation

In the molecular biology of eukaryotic organisms, the elucidation of mechanisms involved in the regulation of gene expression has assumed an important role. All cells of an organism carry the same genes, but differ in the patterns of genes they express. There is an increasing amount of evidence that cancer cells exhibit a pattern of gene expression which can be very different from that of normal cells. One of the molecular signals that has been recognized in the regulation of gene expression in eukaryotes is the modified nucleotide 5-methylcytosine (5-mC). Through experiments in well-characterized eukaryotic systems, evidence has been adduced that the introduction of 5-mC into highly specific sequences, particularly into the 5′ and promoter regions of a gene, can cause gene inactivation. Viral and other eukaryotic systems have helped in the recognition of this cause-and-effect relationship. Inactive genes are frequently hypermethylated in the promoter region; active genes are hypomethylated. However, these correlations are not always as simple and straightforward. The biochemical mechanisms by which site-specific DNA methylations cause gene inactivation have not yet been determined. It is plausible to postulate that promoter methylations could somehow affect the binding of cellular enzymes involved in recognizing the promoter of a gene. Structural alterations of DNA promoter sequences arising from DNA methylations could also be important. DNA methylation is likely to represent a long-term inactivation signal, since it is presently thought that patterns of DNA methylation can be changed only by DNA replication and specific inhibition of post-replicative maintenance methylation.     

Intramolecular stable carbon isotopic analysis of archaeal glycosyl tetraether lipids

Glycolipids are prominent constituents in the membranes of cells from all domains of life. For example, diglycosyl‐glycerol dibiphytanyl glycerol tetraethers (2Gly‐GDGTs) are associated with methanotrophic ANME‐1 archaea and heterotrophic benthic archaea, two archaeal groups of global biogeochemical importance. The hydrophobic biphytane moieties of 2Gly‐GDGTs from these two uncultivated archaeal groups exhibit distinct carbon isotopic compositions. To explore whether the isotopic compositions of the sugar headgroups provide additional information on the metabolism of their producers, we developed a procedure to analyze the δ¹³C values of glycosidic headgroups. Successful determination was achieved by (1) monitoring the contamination from free sugars during lipid extraction and preparation, (2) optimizing the hydrolytic conditions for glycolipids, and (3) derivatizing the resulting sugars into aldononitrile acetate derivatives, which are stable enough to withstand a subsequent column purification step. First results of δ¹³C values of sugars cleaved from 2Gly‐GDGTs in two marine sediment samples, one containing predominantly ANME‐1 archaea and the other benthic archaea, were obtained and compared with the δ¹³C values of the corresponding biphytanes. In both samples the dominant sugar headgroups were enriched in ¹³C relative to the corresponding major biphytane. This ¹³C enrichment was significantly larger in the putative major glycolipids from ANME‐1 archaea (～15‰) than in those from benthic archaea (<7‰). This method opens a new analytical window for the examination of carbon isotopic relationships between sugars and lipids in uncultivated organisms. Copyright © 2010 John Wiley & Sons, Ltd.     

The Effect of Dia2 Protein Deficiency on the Cell Cycle,Cell Size,and Recruitment of Ctf4 Protein in Saccharomyces cerevisiae

Cells have evolved elaborate mechanisms to regulate DNA replication machinery and cell cycles in response to DNA damage and replication stress in order to prevent genomic instability and cancer. The E3 ubiquitin ligase SCF^Dia2 in S. cerevisiae is involved in the DNA replication and DNA damage stress response, but its effect on cell growth is still unclear. Here, we demonstrate that the absence of Dia2 prolongs the cell cycle by extending both S- and G2/M-phases while, at the same time, activating the S-phase checkpoint. In these conditions, Ctf4—an essential DNA replication protein and substrate of Dia2—prolongs its binding to the chromatin during the extended S- and G2/M-phases. Notably, the prolonged cell cycle when Dia2 is absent is accompanied by a marked increase in cell size. We found that while both DNA replication inhibition and an absence of Dia2 exerts effects on cell cycle duration and cell size, Dia2 deficiency leads to a much more profound increase in cell size and a substantially lesser effect on cell cycle duration compared to DNA replication inhibition. Our results suggest that the increased cell size in dia2∆ involves a complex mechanism in which the prolonged cell cycle is one of the driving forces.     

The Catalytic Mechanism of the Retaining Glycosyltransferase Mannosylglycerate Synthase

To protect their intracellular proteins, extremophile microorganisms synthesize molecules called compatible solutes. These molecules are the result of the attachment of a small negatively charged molecule to a sugar molecule. It has been found that these molecules, not only protect the microorganism against osmotic stress but also against other extreme conditions. They can also confer protection against extreme conditions to isolated enzymes from different organisms making them an exciting prospect for potential biotechnological applications. One of the most widespread compatible solute in hyperthermophile organisms is the molecule 2-O-α-D-mannosyl-D-glycerate (MG). In addition to confer protection to proteins against extreme conditions, MG was found to prevent Alzheimer's β-amyloid aggregation and reduce α-synuclein fibril formation in Parkinson's disease. In this work we studied, using computational methods, the catalytic mechanism of the synthesis of MG by the enzyme mannosylglycerate synthase (MGS) from the thermophilic bacteria Rhodothermus marinus.     

Rolling circle enzymatic replication of a complex multi-crossover DNA nanostructure

Nature has evolved replicable biological molecules, such as DNA, as genetic information carriers. The replication process is tightly controlled by complicated cellular machinery. It is interesting to ask if artificial DNA nano-objects with a complex secondary structure can be replicated in the same way as simple DNA double helices. Here we demonstrate that paranemic crossover DNA, a structurally complicated multi-crossover DNA molecule, can be replicated successfully using Rolling Circle Amplification (RCA). The amplification efficiency is moderate with high fidelity, confirmed by native PAGE, thermal transition study, and Ferguson analysis. The structural details of the DNA structure after the full replication circle are verified by hydroxyl radical autofootprinting. We conclude that RCA can serve as a reliable method to replicate complex DNA structures. We also discuss the possibility of using viruses and bacteria to clone artificial DNA nano-objects. The findings that single stranded paranemic crossover DNA molecules can be replicated by DNA polymerase will not only be useful in nanotechnology but also may have implications for the possible existence of such complicated DNA structures in nature.     

Intact polar membrane lipids in prokaryotes and sediments deciphered by high-performance liquid chromatography/electrospray ionization multistage mass spectrometry--new biomarkers for biogeochemistry and microbial ecology

Lipids from prokaryotic cell membranes can serve as sources of information on the biogeochemistry and microbial ecology of natural ecosystems. Traditionally, apolar derivatives of the intact polar membrane molecules, e.g., fatty acids, have been the major target of lipid-based biogeochemical studies. However, when still intact, i.e., as glycerol esters and ethers with attached polar headgroups, membrane lipids are diagnostic for living prokaryotes, which makes them excellent biomarkers for the study of in situ microbial processes in geological systems such as sediments or soils. Intact polar lipids (IPLs) are attractive analytical targets because they are taxonomically more specific than their apolar derivatives and avoid exclusion of signals from prokaryotes that primarily build their membranes with ether-bound lipids such as archaea and some bacteria. Here we report results from analyses of IPLs in pure cultures of biogeochemically relevant prokaryotes and marine sediments by high-performance liquid chromatography/electrospray ionization ion-trap mass spectrometry (HPLC/ESI-IT-MSn). This technique is suitable for screening of biomass and environmental samples for distinctive taxonomic structural features such as distribution of polar lipid headgroups, types of bonds between alkyl moiety and glycerol backbone, and the chain length and degree of unsaturation in the alkyl moieties. We present analytical protocols to decipher structural information from mass spectral data. The IPL contents in selected archaeal and bacterial species are diverse and qualify as molecular fingerprints. Applied to marine sediments, the approach provided detailed information on the dominant microbial groups. The IPLs from bacterial members of anaerobic methanotrophic communities in surface sediments at Hydrate Ridge resemble those found in Desulfosarcina variabilis. The presence of dietherglycerophospholipids, however, suggests the presence of other bacteria possibly affiliated with the deepest phylogenetic branches in the tree of life. Sediments from approximately 90 m below the seafloor on the Peruvian continental margin are dominated by intact archaeal tetraethers with glycosidically bound hexoses as headgroups, consistent with a significant fraction of the community being archaea. Additional calditol-based tetraethers imply that the sedimentary archaea are taxonomically linked to the crenarchaeal Sulfolobales.     

Assimilation, dissimilation, and detoxification of formaldehyde, a central metabolic intermediate of methylotrophic metabolism

Methanol is a valuable raw material used in the manufacture of useful chemicals as well as a potential source of energy to replace coal and petroleum. Biotechnological interest in the microbial utilization of methanol has increased because it is an ideal carbon source and can be produced from renewable biomass. Formaldehyde, a cytotoxic compound, is a central metabolic intermediate in methanol metabolism. Therefore, microorganisms utilizing methanol have adopted several metabolic strategies to cope with the toxicity of formaldehyde. Formaldehyde is initially detoxified through trapping by some cofactors, such as glutathione, mycothiol, tetrahydrofolate, and tetrahydromethanopterin, before being oxidized to CO2. Alternatively, free formaldehyde can be trapped by sugar phosphates as the first reaction in the C1 assimilation pathways: the xylulose monophosphate pathway for yeasts and the ribulose monophosphate (RuMP) pathway for bacteria. In yeasts, although formaldehyde generation and consumption takes place in the peroxisome, the cytosolic formaldehyde oxidation pathway also plays a role in formaldehyde detoxification as well as energy formation. The key enzymes of the RuMP pathway are found in a variety of microorganisms including bacteria and archaea. Regulation of the genes encoding these enzymes and their catalytic mechanisms depend on the physiological traits of these organisms during evolution.     

Lateral gene transfer in phylogeny of azoreductase enzyme

This paper attempts to reconstruct the phylogeny of azoreductase enzyme from different organisms and compare it with the small subunit rRNA-based phylogeny of the organisms. The two phylogenies were found to be incongruent, indicating several events of lateral transfer of azoreductase gene between phylogenetically diverse organisms. However, the phylogenetic analysis methods have several limitations and a single method may not give the true pattern. Hence, it is necessary to corroborate the results with other complementary analysis tools. We used several tools to test our hypothesis of lateral transfer and found that it was supported not only by the analysis of the whole sequences, but also by the conserved motifs detected in these sequences. There were ample evidences for lateral transfer of azoreductase gene among enteric bacteria. There were also indications that azoreductase probably evolved in prokaryotes and then it was laterally transferred to eukaryotes in multiple events, resulting in some sequence variation among eukaryotic azoreductases. Finally, profile HMMs and conserved motifs extracted from these azoreductase sequences were found to provide sensitive tools for identifying potential azoreductases from the database. The analysis techniques used in this study can be extended to other gene trees to verify their evolutionary histories.     

Archaeal tetraether lipids

The extremely stable biomolecules manufactured by organisms from extreme environments are of great scientific and engineering interest in the development of robust and stable industrial biocatalysts. Identification of molecules that impart stability under extremes will also have a profound impact on our understanding of cellular survival. This review discusses isolation and characterization of archaeal tetraethers as well as target technologies for tetraether lipid application. The isolation and characterization of archaeal tetraether lipids has led to some interesting applications improving on ester lipid technologies. Potential applications include novel lubricants, gene-delivery systems, monolayer lipid matrices for sensor devices, and protein stabilization. Following this review, patent abstracts and additional literature pertaining to the isolation, characterization, and application of archaeal membrane lipids are listed.     

Photoreactive DNA as a Tool to Study Replication Protein A Functioning in DNA Replication and Repair

Replication protein A (RPA), eukaryotic single-stranded DNA-binding protein, is a key player in multiple processes of DNA metabolism including DNA replication, recombination and DNA repair. Human RPA composed of subunits of 70-, 32- and 14-kDa binds ssDNA with high affinity and interacts specifically with multiple proteins. The RPA heterotrimer binds ssDNA in several modes, with occlusion lengths of 8–10, 13–22 and 30 nucleotides corresponding to global, transitional and elongated conformations of protein. Varying the structure of photoreactive DNA, the intermediates of different stages of DNA replication or DNA repair were designed and applied to identify positioning of the RPA subunits on the specific DNA structures. Using this approach, RPA interactions with various types of DNA structures attributed to replication and DNA repair intermediates were examined. This review is dedicated to blessed memory of Prof. Alain Favre who contributed to the development of photoreactive nucleotide derivatives and their application for the study of protein–nucleic acids interactions.