Single TRAM domain RNA-binding proteins in Archaea: functional insight from Ctr3 from the Antarctic methanogen Methanococcoides burtonii.

TRAM domain proteins present in Archaea and Bacteria have a ß-barrel shape with anti-parallel ß-sheets that form a nucleic acid binding surface; a structure also present in cold shock proteins (Csps). Aside from protein structures, experimental data defining the function of TRAM domains is lacking. Here, we explore the possible functional properties of a single TRAM domain protein, Ctr3 (cold-responsive TRAM domain protein 3) from the Antarctic archaeon Methanococcoides burtonii that has increased abundance during low temperature growth. Ribonucleic acid (RNA) bound by Ctr3 in vitro was determined using RNA-seq. Ctr3-bound M. burtonii RNA with a preference for transfer (t)RNA and 5S ribosomal RNA, and a potential binding motif was identified. In tRNA, the motif represented the C loop; a region that is conserved in tRNA from all domains of life and appears to be solvent exposed, potentially providing access for Ctr3 to bind. Ctr3 and Csps are structurally similar and are both inferred to function in low temperature translation. The broad representation of single TRAM domain proteins within Archaea compared with their apparent absence in Bacteria, and scarcity of Csps in Archaea but prevalence in Bacteria, suggests they represent distinct evolutionary lineages of functionally equivalent RNA-binding proteins.

Temperature-dependent global gene expression in the Antarctic archaeon Methanococcoides burtonii.

Methanococcoides burtonii is a member of the Archaea that was isolated from Ace Lake in Antarctica and is a valuable model for studying cold adaptation. Low temperature transcriptional regulation of global gene expression, and the arrangement of transcriptional units in cold-adapted archaea has not been studied. We developed a microarray for determining which genes are expressed in operons, and which are differentially expressed at low (4°C) or high (23°C) temperature. Approximately 55% of genes were found to be arranged in operons that range in length from 2 to 23 genes, and mRNA abundance tended to increase with operon length. Analysing microarray data previously obtained by others for Halobacterium salinarum revealed a similar correlation between operon length and mRNA abundance, suggesting that operons may play a similar role more broadly in the Archaea. More than 500 genes were differentially expressed at levels up to ≈ 24-fold. A notable feature was the upregulation of genes involved in maintaining RNA in a state suitable for translation in the cold. Comparison between microarray experiments and results previously obtained using proteomics indicates that transcriptional regulation (rather than translation) is primarily responsible for controlling gene expression in M. burtonii. In addition, certain genes (e.g. involved in ribosome structure and methanogenesis) appear to be regulated post-transcriptionally. This is one of few experimental studies describing the genome-wide distribution and regulation of operons in archaea.

GPU acceleration of a model-based iterative method for Digital Breast Tomosynthesis.

Digital Breast Tomosynthesis (DBT) is a modern 3D Computed Tomography X-ray technique for the early detection of breast tumors, which is receiving growing interest in the medical and scientific community. Since DBT performs incomplete sampling of data, the image reconstruction approaches based on iterative methods are preferable to the classical analytic techniques, such as the Filtered Back Projection algorithm, providing fewer artifacts. In this work, we consider a Model-Based Iterative Reconstruction (MBIR) method well suited to describe the DBT data acquisition process and to include prior information on the reconstructed image. We propose a gradient-based solver named Scaled Gradient Projection (SGP) for the solution of the constrained optimization problem arising in the considered MBIR method. Even if the SGP algorithm exhibits fast convergence, the time required on a serial computer for the reconstruction of a real DBT data set is too long for the clinical needs. In this paper we propose a parallel SGP version designed to perform the most expensive computations of each iteration on Graphics Processing Unit (GPU). We apply the proposed parallel approach on three different GPU boards, with computational performance comparable with that of the boards usually installed in commercial DBT systems. The numerical results show that the proposed GPU-based MBIR method provides accurate reconstructions in a time suitable for clinical trials.

The response of the marine bacterium Sphingopyxis alaskensis to solar radiation assessed by quantitative proteomics.

The adaptive response of the marine bacterium Sphingopyxis alaskensis RB2256 to solar radiation (both visible and ultraviolet) was assessed by a quantitative proteomic approach using iTRAQ (isobaric tags for relative and absolute quantification). Both growth phase (mid-log and stationary phase) and duration (80 min or 8 h) of different light treatments (combinations of visible light, UV-A and UV-B) were assessed relative to cultures maintained in the dark. Rates of total protein synthesis and viability were also assessed. Integrating knowledge from the physiological experiments with quantitative proteomics of the 12 conditions tested provided unique insight into the adaptation biology of UV and visible light responses of S. alaskensis. High confidence identifications were obtained for 811 proteins (27% of the genome), 119 of which displayed significant quantitative differences. Mid-log-phase cultures produced twice as many proteomic changes as stationary-phase cultures, while extending the duration of irradiation exposure of stationary-phase cultures did not increase the total number of quantitative changes. Proteins with significant quantitative differences were identified that were characteristic of growth phase and light treatment, and cellular processes, pathways and interaction networks were determined. Key factors of the solar radiation adaptive response included DNA-binding proteins implicated in reducing DNA damage, detoxification of toxic compounds such as glyoxal and reactive oxygen species, iron sequestration to minimize oxidative stress, chaperones to control protein re/folding, alterations to nitrogen metabolism, and specific changes to transcriptional and translational processes.

Characterization of a temperature-responsive two component regulatory system from the Antarctic archaeon, Methanococcoides burtonii.

Cold environments dominate the Earth's biosphere and the resident microorganisms play critical roles in fulfilling global biogeochemical cycles. However, only few studies have examined the molecular basis of thermosensing; an ability that microorganisms must possess in order to respond to environmental temperature and regulate cellular processes. Two component regulatory systems have been inferred to function in thermal regulation of gene expression, but biochemical studies assessing these systems in Bacteria are rare, and none have been performed in Archaea or psychrophiles. Here we examined the LtrK/LtrR two component regulatory system from the Antarctic archaeon, Methanococcoides burtonii, assessing kinase and phosphatase activities of wild-type and mutant proteins. LtrK was thermally unstable and had optimal phosphorylation activity at 10 °C (the lowest optimum activity for any psychrophilic enzyme), high activity at 0 °C and was rapidly thermally inactivated at 30 °C. These biochemical properties match well with normal environmental temperatures of M. burtonii (0-4 °C) and the temperature this psychrophile is capable of growing at in the laboratory (-2 to 28 °C). Our findings are consistent with a role for LtrK in performing phosphotransfer reactions with LtrR that could lead to temperature-dependent gene regulation.

Aptamer based peptide enrichment for quantitative analysis of gonadotropin-releasing hormone by LC-MS/MS.

Over recent years threats to racing have expanded to include naturally occurring biological molecules, such as peptides and proteins, and their synthetic analogues. Traditionally, antibodies have been used to enable detection of these compounds as they allow purification and concentration of the analyte of interest. The rapid expansion of peptide-based therapeutics necessitates a similarly rapid development of suitable antibodies or other means of enrichment. Potential alternative enrichment strategies include the use of aptamers, which offer the significant advantage of chemical synthesis once the nucleic acid sequence is known. A method was developed for the enrichment, detection and quantitation of gonadotropin-releasing hormone (GnRH) in equine urine using aptamer-based enrichment and LC-MS/MS. The method achieved comparable limits of detection (1 pg/mL) and quantification (2.5 pg/mL) to previously published antibody-based enrichment methods. The intra- and inter-assay precision achieved was less than 10% at both 5 and 20 pg/mL, and displayed a working dynamic range of 2.5-100 pg/mL. Significant matrix enhancement (170 ± 8%) and low analytical recovery (29 ± 15%) was observed, although the use of an isotopically heavy labelled GnRH peptide, GnRH (Pro(13)C5,(15)N), as the internal standard provides compensation for these parameters. Within the current limits of detection GnRH was detectable up to 1h post administration in urine and identification of a urinary catabolite extended this detection window to 4h. Based on the results of this preliminary investigation we propose the use of aptamers as a viable alternative to antibodies in the enrichment of peptide targets from equine urine.

Morphological and proteomic analysis of biofilms from the Antarctic archaeon, Halorubrum lacusprofundi.

Biofilms enhance rates of gene exchange, access to specific nutrients, and cell survivability. Haloarchaea in Deep Lake, Antarctica, are characterized by high rates of intergenera gene exchange, metabolic specialization that promotes niche adaptation, and are exposed to high levels of UV-irradiation in summer. Halorubrum lacusprofundi from Deep Lake has previously been reported to form biofilms. Here we defined growth conditions that promoted the formation of biofilms and used microscopy and enzymatic digestion of extracellular material to characterize biofilm structures. Extracellular DNA was found to be critical to biofilms, with cell surface proteins and quorum sensing also implicated in biofilm formation. Quantitative proteomics was used to define pathways and cellular processes involved in forming biofilms; these included enhanced purine synthesis and specific cell surface proteins involved in DNA metabolism; post-translational modification of cell surface proteins; specific pathways of carbon metabolism involving acetyl-CoA; and specific responses to oxidative stress. The study provides a new level of understanding about the molecular mechanisms involved in biofilm formation of this important member of the Deep Lake community.

Developing a genetic manipulation system for the Antarctic archaeon, Halorubrum lacusprofundi: investigating acetamidase gene function.

No systems have been reported for genetic manipulation of cold-adapted Archaea. Halorubrum lacusprofundi is an important member of Deep Lake, Antarctica (~10% of the population), and is amendable to laboratory cultivation. Here we report the development of a shuttle-vector and targeted gene-knockout system for this species. To investigate the function of acetamidase/formamidase genes, a class of genes not experimentally studied in Archaea, the acetamidase gene, amd3, was disrupted. The wild-type grew on acetamide as a sole source of carbon and nitrogen, but the mutant did not. Acetamidase/formamidase genes were found to form three distinct clades within a broad distribution of Archaea and Bacteria. Genes were present within lineages characterized by aerobic growth in low nutrient environments (e.g. haloarchaea, Starkeya) but absent from lineages containing anaerobes or facultative anaerobes (e.g. methanogens, Epsilonproteobacteria) or parasites of animals and plants (e.g. Chlamydiae). While acetamide is not a well characterized natural substrate, the build-up of plastic pollutants in the environment provides a potential source of introduced acetamide. In view of the extent and pattern of distribution of acetamidase/formamidase sequences within Archaea and Bacteria, we speculate that acetamide from plastics may promote the selection of amd/fmd genes in an increasing number of environmental microorganisms.

Low temperature regulated DEAD-box RNA helicase from the Antarctic archaeon, Methanococcoides burtonii.

DEAD-box RNA helicases, by unwinding duplex RNA in bacteria and eukaryotes, are involved in essential cellular processes, including translation initiation and ribosome biogenesis, and have recently been implicated in enabling bacteria to survive cold-shock and grow at low temperature. Despite these critical physiological roles, they have not been characterized in archaea. Due to their presumed importance in removing cold-stabilised secondary structures in mRNA, we have characterised a putative DEAD-box RNA helicase gene (deaD) from the Antarctic methanogen, Methanococcoides burtonii. The encoded protein, DeaD is predicted to contain a core element involved in ATP hydrolysis and RNA-binding, and an unusual C-terminal domain that contains seven perfect, trideca-peptide, direct repeats that may be involved in RNA binding. Alignment and phylogenetic analyses were performed on the core regions of the M. burtonii and other DEAD-box RNA helicases. These revealed a loose but consistent clustering of archaeal and bacterial sequences and enabled the generation of a prokaryotic-specific consensus sequence. The consensus highlights the importance of residues other than the eight motifs that are often associated with DEAD-box RNA helicases, as well as de-emphasising the importance of the "A" residue within the "DEAD" motif. Cells growing at 4 degrees C contained abundant levels of deaD mRNA, however no mRNA was detected in cells growing at 23 degrees C (the optimal temperature for growth). The transcription initiation site was mapped downstream from an archaeal box-A element (TATA box), which preceded a long (113 nucleotides) 5'-untranslated region (5'-UTR). Within the 5'-UTR was an 11 bp sequence that closely matches (nine out of 11) cold-box elements that are present in the 5'-UTRs of cold-shock induced genes from bacteria. To determine if the archaeal 5'-UTR performs an analagous function to the bacterial 5'-UTRs, the archaeal deaD 5'-UTR was transcribed in E. coli under the control of the cspA promoter and transcriptional terminator. It has previously been reported that overexpression of the cspA 5'-UTR leads to an extended cold-shock response due to the 5'-UTR titrating cellular levels of a cold-shock repressor protein(s). In our hands, the cold-shock protein profiles resulting from overexpression of Escherichia coli cspA and M. burtonii deaD 5'-UTRs were similar, however they did not differ from those for the overexpression of a control plasmid lacking a 5'-UTR. In association with other recent data from E. coli, our results indicate that the role of the 5'-UTR in gene regulation is presently unclear. Irrespective of the mechanisms, it is striking that highly similar 5'-UTRs with cold-box elements are present in cold induced genes from E. coli, Anabaena and M. burtonii. This is the first study examining low temperature regulation in archaea and provides initial evidence that gene expression from a cold adapted archaeon involves a bacterial-like transcriptional regulatory mechanism. In addition, it provides the foundation for further studies into the function and regulation of DEAD-box RNA helicases in archaea, and in particular, their roles in low temperature adaptation.

Loss of heat-shock acquisition of thermotolerance in yeast is not correlated with loss of heat-shock proteins.

Yeast cells when subjected to a primary heat shock, defined as a temperature shift from 23 to 37 degrees C for 30 min, acquired tolerance to heat stress (52 degrees C/5 min). Primary heat shocked cells incubated at 23 degrees C for up to 3 h, progressively lost thermotolerance but retained high levels of the major heat-shock proteins as observed on polyacrylamide gels. On the other hand, a temperature shift back up to 37 degrees C for 30 min fully restored thermotolerance. The major high-molecular-mass heat-shock proteins (hsp) identified were of approximate molecular mass 100 kDa (hsp 100), 80 kDa (hsp 80) and 70 kDa (hsp 70). The results indicate that loss of heat-shock acquisition of thermotolerance is not correlated with loss of heat-shock proteins.

Archaeal cold-adapted proteins: structural and evolutionary analysis of the elongation factor 2 proteins from psychrophilic, mesophilic and thermophilic methanogens.

To identify structural features important for low temperature activity in archaeal proteins, elongation factor 2 (EF-2) genes (aef2) were sequenced from psychrophilic, mesophilic and thermophilic methanogens. Scatter plots were used to compare evolutionary distances for EF-2 amino acid sequences vs. 16S-rRNA sequences from methanogens growing at diverse temperatures. The absence of a temperature bias for the rate of protein vs. nucleic acid evolution demonstrated the importance of comparing closely related proteins in order to identify changes indicative of thermal adaptation. Three-dimensional modelling of the new EF-2 sequences enabled the identification of amino acid residues that may be important for conferring low temperature activity and included greater structural flexibility produced by fewer salt bridges, less packed hydrophobic cores and the reduction of proline residues in loop structures.

Mitochondrial and cytoplasmic protein syntheses are not required for heat shock acquisition of ethanol and thermotolerance in yeast.

Heat shock acquisition of ethanol- and thermotolerance in Saccharomyces cerevisiae was not inhibited in cells incubated in the presence of cycloheximide or chloramphenicol. Respiratory-deficient (rho-) mutants also characteristically exhibited the heat shock response. It was concluded that mitochondrial and cytoplasmic protein syntheses are not required for heat shock acquisition of ethanol and thermotolerance in yeast.

Sphingomonads from marine environments.

Sphingomonas species play an important role in the ecology of a range of marine habitats. Isolates and 16S-rRNA clones have been obtained from corals, natural and artificial sources of marine hydrocarbons and eutrophic and oligotrophic waters, and have been isolated as hosts for marine phages. In addition they are found in oceans spanning temperature ranges from polar to temperate waters. While less is known about marine sphingomonads in comparison to their terrestrial counterparts, their importance in microbial ecology is evident. This is illustrated by, for example, the numerical dominance of strain RB2256 in oligotrophic waters. Furthermore, the known marine sphingomonads represent a phylogenetic cross-section of the Sphingomonas genus. This review focuses on our present knowledge of cultured isolates and 16S-rDNA clones from marine environments.

Towards real-time image deconvolution: application to confocal and STED microscopy.

Although deconvolution can improve the quality of any type of microscope, the high computational time required has so far limited its massive spreading. Here we demonstrate the ability of the scaled-gradient-projection (SGP) method to provide accelerated versions of the most used algorithms in microscopy. To achieve further increases in efficiency, we also consider implementations on graphic processing units (GPUs). We test the proposed algorithms both on synthetic and real data of confocal and STED microscopy. Combining the SGP method with the GPU implementation we achieve a speed-up factor from about a factor 25 to 690 (with respect the conventional algorithm). The excellent results obtained on STED microscopy images demonstrate the synergy between super-resolution techniques and image-deconvolution. Further, the real-time processing allows conserving one of the most important property of STED microscopy, i.e the ability to provide fast sub-diffraction resolution recordings.

Biotechnological uses of enzymes from psychrophiles.

The bulk of the Earth's biosphere is cold (e.g. 90% of the ocean's waters are ≤ 5°C), sustaining a broad diversity of microbial life. The permanently cold environments vary from the deep ocean to alpine reaches and to polar regions. Commensurate with the extent and diversity of the ecosystems that harbour psychrophilic life, the functional capacity of the microorganisms that inhabitat the cold biosphere are equally diverse. As a result, indigenous psychrophilic microorganisms provide an enormous natural resource of enzymes that function effectively in the cold, and these cold-adapted enzymes have been targeted for their biotechnological potential. In this review we describe the main properties of enzymes from psychrophiles and describe some of their known biotechnological applications and ways to potentially improve their value for biotechnology. The review also covers the use of metagenomics for enzyme screening, the development of psychrophilic gene expression systems and the use of enzymes for cleaning.

The involvement of transcriptional read-through from internal promoters in the expression of a novel endoglucanase gene FSendA, from Fibrobacter succinogenes AR1.

Two distinct mRNA transcripts were synthesized in Escherichia coli during expression of FSendA, an endoglucanase gene from Fibrobacter succinogenes AR1. Expression of FSendA required a ribosomal frameshift between open reading frame 1 (ORF1) and ORF2 to allow contiguous translation of a 453 amino acid protein (1). The primary transcript initiated upstream of ORF1 and the secondary transcript from within ORF1. Both transcripts terminated downstream of ORF2 and termination was essential for endoglucanase expression. Deletion of the primary transcript promoter region allowed read-through of the secondary transcript beyond the terminator region, indicating that a component of the intact FSendA gene allowed efficient transcription termination. The possibility of autogenous regulation by translation products is suggested.

Molecular cloning, expression, and characterization of endoglucanase genes from Fibrobacter succinogenes AR1.

A cosmid gene library was constructed in Escherichia coli from genomic DNA isolated from the ruminal anaerobe Fibrobacter succinogenes AR1. Clones were screened on carboxymethyl cellulose, and 8 colonies that produced large clearing zones and 25 colonies that produced small clearing zones were identified. Southern blot hybridization revealed the existence of at least three separate genes encoding cellulase activity. pRC093, which is representative of cosmid clones that produce large clearing zones, was subcloned in pGem-1, and the resulting hybrid pRCEH directed synthesis of endoglucanase activity localized on a 2.1-kb EcoRI-HindIII insert. Activity was expressed from this fragment when it was cloned in both orientations in pGem-1 and pGem-2, indicating that F. succinogenes promoters functioned successfully in E. coli. A high level of endoglucanase activity was detected on acid-swollen cellulose, ball-milled cellulose, and carboxymethyl cellulose; and a moderate level was detected on filter paper, Avicel, lichenan, and xylan. Most activity (80%) was localized in the periplasm of E. coli, with low but significant levels (16%) being detected in the extracellular medium. The periplasmic endoglucanase had an estimated molecular weight of 46,500, had an optimum temperature of 39 degrees C, and exhibited activity over a broad pH range, with a maximum at pH 5.0.

Physiological responses to starvation in the marine oligotrophic ultramicrobacterium Sphingomonas sp. strain RB2256.

Sphingomonas sp. strain RB2256 is representative of the ultramicrobacteria that proliferate in oligotrophic marine waters. While this class of bacteria is well adapted for growth with low concentrations of nutrients, their ability to respond to complete nutrient deprivation has not previously been investigated. In this study, we examined two-dimensional protein profiles for logarithmic and stationary-phase cells and found that protein spot intensity was regulated by up to 70-fold. A total of 72 and 177 spots showed increased or decreased intensity, respectively, by at least twofold during starvation. The large number of protein spots (1,500) relative to the small genome size (ca. 1.5 Mb) indicates that gene expression may involve co- and posttranslational modifications of proteins. Rates of protein and RNA synthesis were examined throughout the growth phase and up to 7 days of starvation and revealed that synthesis was highly regulated. Rates of protein synthesis and cellular protein content were compared to ribosome content, demonstrating that ribosome synthesis was not directly linked to protein synthesis and that the function of ribosomes may not be limited to translation. By comparing the genetic capacity and physiological responses to starvation of RB2256 to those of the copiotrophic marine bacterium Vibrio angustum S14 (J. Ostling, L. Holmquist, and S. Kjelleberg, J. Bacteriol. 178:4901-4908, 1996), the characteristics of a distinct starvation response were defined for Sphingomonas strain RB2256. The capacity of this ultramicrobacterium to respond to starvation is discussed in terms of the ecological relevance of complete nutrient deprivation in an oligotrophic marine environment. These studies provide the first evidence that marine oligotrophic ultramicrobacteria may be expected to include a starvation response and the capacity for a high degree of gene regulation.

Effect of temperature on stability and activity of elongation factor 2 proteins from Antarctic and thermophilic methanogens.

Despite the presence and abundance of archaea in low-temperature environments, little information is available regarding their physiological and biochemical properties. In order to investigate the adaptation of archaeal proteins to low temperatures, we purified and characterized the elongation factor 2 (EF-2) protein from the Antarctic methanogen Methanococcoides burtonii, which was expressed in Escherichia coli, and compared it to the recombinant EF-2 protein from a phylogenetically related thermophile, Methanosarcina thermophila. Using differential scanning calorimetry to assess protein stability and enzyme assays for the intrinsic GTPase activity, we identified biochemical and biophysical properties that are characteristic of the cold-adapted protein. This includes a higher activity at low temperatures caused by a decrease of the activation energy necessary for GTP hydrolysis and a decreased activation energy for the irreversible denaturation of the protein, which indicates a less thermostable structure. Comparison of the in vitro properties of the proteins with the temperature-dependent characteristics of growth of the organisms indicates that additional cytoplasmic factors are likely to be important for the complete thermal adaptation of the proteins in vivo. This is the first study to address thermal adaptation of proteins from a free-living, cold-adapted archaeon, and our results indicate that the ability of the Antarctic methanogen to adapt to the cold is likely to involve protein structural changes.