Studies on the Preparation,Isolation, and Reactions of Adenosine Schiff Bases

Abstract

A systematic study of the preparation, isolation, and reactions of adenosine Schiff bases is presented. Schiff bases of nucleosides can be prepared and isolated, but the reaction appears to be specific for 2′, 3′-0-isopropylidene adenosines. The use of nucleoside Schiff bases as synthetic intermediates is not yet a viable process.     

CRITICA: coding region identification tool invoking comparative analysis.

Gene recognition is essential to understanding existing and future DNA sequence data. CRITICA (Coding Region Identification Tool Invoking Comparative Analysis) is a suite of programs for identifying likely protein-coding sequences in DNA by combining comparative analysis of DNA sequences with more common noncomparative methods. In the comparative component of the analysis, regions of DNA are aligned with related sequences from the DNA databases; if the translation of the aligned sequences has greater amino acid identity than expected for the observed percentage nucleotide identity, this is interpreted as evidence for coding. CRITICA also incorporates noncomparative information derived from the relative frequencies of hexanucleotides in coding frames versus other contexts (i.e., dicodon bias). The dicodon usage information is derived by iterative analysis of the data, such that CRITICA is not dependent on the existence or accuracy of coding sequence annotations in the databases. This independence makes the method particularly well suited for the analysis of novel genomes. CRITICA was tested by analyzing the available Salmonella typhimurium DNA sequences. Its predictions were compared with the DNA sequence annotations and with the predictions of GenMark. CRITICA proved to be more accurate than GenMark, and moreover, many of its predictions that would seem to be errors instead reflect problems in the sequence databases. The source code of CRITICA is freely available by anonymous FTP (rdp.life.uiuc.edu in/pub/critica) and on the World Wide Web (http:/(/)rdpwww.life.uiuc.edu).     

Complete Genome Sequence of the Aerobic CO-Oxidizing Thermophile Thermomicrobium roseum

In order to enrich the phylogenetic diversity represented in the available sequenced bacterial genomes and as part of an “Assembling the Tree of Life” project, we determined the genome sequence of Thermomicrobium roseum DSM 5159. T. roseum DSM 5159 is a red-pigmented, rod-shaped, Gram-negative extreme thermophile isolated from a hot spring that possesses both an atypical cell wall composition and an unusual cell membrane that is composed entirely of long-chain 1,2-diols. Its genome is composed of two circular DNA elements, one of 2,006,217 bp (referred to as the chromosome) and one of 919,596 bp (referred to as the megaplasmid). Strikingly, though few standard housekeeping genes are found on the megaplasmid, it does encode a complete system for chemotaxis including both chemosensory components and an entire flagellar apparatus. This is the first known example of a complete flagellar system being encoded on a plasmid and suggests a straightforward means for lateral transfer of flagellum-based motility. Phylogenomic analyses support the recent rRNA-based analyses that led to T. roseum being removed from the phylum Thermomicrobia and assigned to the phylum Chloroflexi. Because T. roseum is a deep-branching member of this phylum, analysis of its genome provides insights into the evolution of the Chloroflexi. In addition, even though this species is not photosynthetic, analysis of the genome provides some insight into the origins of photosynthesis in the Chloroflexi. Metabolic pathway reconstructions and experimental studies revealed new aspects of the biology of this species. For example, we present evidence that T. roseum oxidizes CO aerobically, making it the first thermophile known to do so. In addition, we propose that glycosylation of its carotenoids plays a crucial role in the adaptation of the cell membrane to this bacterium''s thermophilic lifestyle. Analyses of published metagenomic sequences from two hot springs similar to the one from which this strain was isolated, show that close relatives of T. roseum DSM 5159 are present but have some key differences from the strain sequenced.     

The MetabolomeExpress Project: enabling web-based processing,analysis and transparent dissemination of GC/MS metabolomics datasets

Background  

Standardization of analytical approaches and reporting methods via community-wide collaboration can work synergistically with web-tool development to result in rapid community-driven expansion of online data repositories suitable for data mining and meta-analysis. In metabolomics, the inter-laboratory reproducibility of gas-chromatography/mass-spectrometry (GC/MS) makes it an obvious target for such development. While a number of web-tools offer access to datasets and/or tools for raw data processing and statistical analysis, none of these systems are currently set up to act as a public repository by easily accepting, processing and presenting publicly submitted GC/MS metabolomics datasets for public re-analysis.     

Comparing the in Vivo Function of α-Carboxysomes and β-Carboxysomes in Two Model Cyanobacteria

The carbon dioxide (CO₂)-concentrating mechanism of cyanobacteria is characterized by the occurrence of Rubisco-containing microcompartments called carboxysomes within cells. The encapsulation of Rubisco allows for high-CO₂ concentrations at the site of fixation, providing an advantage in low-CO₂ environments. Cyanobacteria with Form-IA Rubisco contain α-carboxysomes, and cyanobacteria with Form-IB Rubisco contain β-carboxysomes. The two carboxysome types have arisen through convergent evolution, and α-cyanobacteria and β-cyanobacteria occupy different ecological niches. Here, we present, to our knowledge, the first direct comparison of the carboxysome function from α-cyanobacteria (Cyanobium spp. PCC7001) and β-cyanobacteria (Synechococcus spp. PCC7942) with similar inorganic carbon (C_i; as CO₂ and HCO₃⁻) transporter systems. Despite evolutionary and structural differences between α-carboxysomes and β-carboxysomes, we found that the two strains are remarkably similar in many physiological parameters, particularly the response of photosynthesis to light and external C_i and their modulation of internal ribulose-1,5-bisphosphate, phosphoglycerate, and C_i pools when grown under comparable conditions. In addition, the different Rubisco forms present in each carboxysome had almost identical kinetic parameters. The conclusions indicate that the possession of different carboxysome types does not significantly influence the physiological function of these species and that similar carboxysome function may be possessed by each carboxysome type. Interestingly, both carboxysome types showed a response to cytosolic C_i, which is of higher affinity than predicted by current models, being saturated by 5 to 15 mm C_i. This finding has bearing on the viability of transplanting functional carboxysomes into the C₃ chloroplast.Cyanobacteria inhabit a diverse range of ecological habitats, including both freshwater and marine ecosystems. The flexibility to occupy these different habitats is thought to come in part from the carbon-concentrating mechanism (CCM) present in all species (Badger et al., 2006). The CCM comprises inorganic carbon (C_i; as carbon dioxide [CO₂] and HCO₃⁻) transporters for C_i uptake and protein microbodies called carboxysomes for CO₂ concentration and fixation by Rubisco (Badger and Price, 2003). The CCM is believed to have evolved in response to changes in the absolute and relative levels of CO₂ and oxygen (O₂) in the atmosphere during the evolution of oxygenic photosynthesis in cyanobacteria (Price et al., 2008).There are two main phylogenetic groups within the cyanobacteria based on Rubisco and carboxysome phylogenies; α-cyanobacteria have α-carboxysomes with Form-IA Rubisco, whereas β-cyanobacteria have β-carboxysomes with Form-IB Rubisco (Tabita, 1999; Badger et al., 2002). Rubisco large subunit protein sequences from these two groups are closely related but nevertheless, distinguishable (Supplemental Fig. S1). In general, α-cyanobacteria and β-cyanobacteria occupy a quite different range of ecological habitats. The α-cyanobacteria are mostly marine organisms, with the majority of species living in the open ocean (Badger et al., 2006). Marine α-cyanobacteria live in very stable environments with high pH (pH 8.2) and dissolved carbon levels but low nutrients. They are characterized by small cells, very small genomes (1.6–2.8 Mb), and a few constitutively expressed carbon uptake transporters (Rae et al., 2011; Beck et al., 2012). They have been described as low flux, low energy cyanobacteria with a minimal CCM (Badger et al., 2006). Although these species are slow growing, oceanic cyanobacteria contribute as much as one-half of oceanic primary productivity (Liu et al., 1997, 1999; Field et al., 1998), suggesting that they may contribute up to 25% to net global productivity every year.In comparison, β-cyanobacteria occupy a much more diverse range of habitats, including freshwater, estuarine, and hot springs and never reach the same levels of global abundance (Badger et al., 2006). They are characterized by larger cells, larger genomes (2.2–3.6 Mb), and an array of carbon uptake transporters, including those transporters induced under low C_i (Rae et al., 2011, 2013). In addition to these broadly defined α-groups and β-groups, there are small numbers of α-cyanobacteria that have been termed transitional strains (Price, 2011; Rae et al., 2011). These species (e.g. Cyanobium spp. PCC7001, Synechococcus spp. WH5701, and Cyanobium spp. PCC6307; Supplemental Fig. S1) live in marginal marine and freshwater environments and have a number of characteristics similar to β-cyanobacteria. For example, they have a more diverse range of C_i uptake systems and a significantly larger genome than closely related α-cyanobacteria, and it has been suggested that the additional genes encoding transport systems were acquired by horizontal gene transfer (HGT) from β-cyanobacteria (Rae et al., 2011).Although the carboxysomes from α-cyanobacteria and β-cyanobacteria are very similar in overall structure, in that they share an outer protein shell of common phylogenetic origin (Kerfeld et al., 2005), they are distinguished from each other largely by differences in the proteins, which seem to make up or interact with the interior of the carboxysome compartment (Supplemental Table S1). This finding suggests that their different structures today have arisen through periods of common and convergent evolution. Certain carboxysome shell proteins from α-carboxysomes and β-carboxysomes show regions of significant sequence homology. These proteins are denoted as CsoS1 to CsoS4 (in α-cyanobacteria) and CcmKLO (in β-cyanobacteria), and the homologous regions have been termed bacterial microcompartment domains (Kerfeld et al., 2010; Rae et al., 2013). Proteins with these domains are also found in bacterial microcompartments in proteobacteria. However, other identified carboxysome proteins do not show any sequence homology between α-carboxysomes and β-carboxysomes but may perform similar functional roles. For example, carbonic anhydrase activity is essential for carboxysome function, but its activity seems to be provided by a range of different proteins (β-CcaA, β-CcmM, and α-CsoSCA; Kupriyanova et al., 2013). Similarly, β-CcmM and α-CsoS2 could play similar roles in organizing the interface between the shell and Rubisco within the carboxysomes (Gonzales et al., 2005; Long et al., 2007).The functioning of a carboxysome relies on a number of biochemical properties associated with the protein microbody structure. These properties include the biochemical/kinetic properties of Rubisco contained within carboxysomes, the conductance of the carboxysome shell to the influx of substrate ribulose-1,5-bisphosphate (RuBP) and the efflux of the carboxylation product phosphoglycerate (PGA), the conductance of the shell to the influx of bicarbonate and the efflux of CO₂, and lastly, the manner in which bicarbonate is converted to CO₂ within the carboxysomes. α-Carboxysomes and β-carboxysomes have the potential to differ in each of these properties. The flux of phosphorylated sugars across the shell has been postulated to be mediated by the pores in the hexameric shell proteins (Yeates et al., 2010; Kinney et al., 2011), which although similar, do differ between the two carboxysomes types. Bicarbonate and CO₂ uptake processes are less well-defined but probably involve aspects of the way in which unique shell interface proteins interact with Rubisco, which also differs in that CsoS2 and CsoSCA are probably the interacting proteins involved in α-carboxysomes (Espie and Kimber, 2011), whereas CcmM and β-carboxysomal CA are variably involved in β-carboxysomes (Long et al., 2010). Finally, the Form-IA and Form-IB Rubisco proteins at the heart of carboxylation, although similar, have the potential to show different kinetic properties. Although Form-IB Rubiscos from β-cyanobacteria are well-characterized, the Form-IA counterparts have received very little attention. In addition, the CCM of very few strains of cyanobacteria have been studied at the level of biochemistry and physiology, and they have been almost exclusively β-cyanobacteria. As a result, there are significant gaps in our knowledge about the similarities and differences in functional traits between α-cyanobacterial and β-cyanobacterial strains. One important question that remains to be answered is whether α-carboxysomes and β-carboxysomes have intrinsic differences in their biochemical properties that influence the nature of the CCM, which is established within each broad cell type.Because of the difficulties in isolating and assaying intact carboxysomes in vitro, the characterization of biochemical properties of carboxysomes is not easily addressed. One way forward is to study the properties of the CCM in detail in a model representative strain from each group and compare their characteristics to contrast the intracellular function of α-cell types and β-cell types. In the past, it has been restricted because of the difficulties in growing many of the open ocean α-cyanobacteria and their very different natures in relation to inorganic transporter composition. However, the availability of α-cyanobacteria transition strains, which grow well in the laboratory, has provided an opportunity to address this question. The α-cyanobacteria Cyanobium spp. PCC7001 (hereafter Cyanobium spp.), in particular, grows in standard freshwater media (BG11) and has growth and photosynthetic performance properties that closely match the model β-cyanobacteria, Synechococcus spp. PCC7942 (hereafter Synechococcus spp.); for this reason, Cyanobium spp. is ideal for a balanced comparison of the in vivo physiological properties of α-carboxysomes and β-carboxysomes in two species with relatively similar C_i-uptake properties.Genome analysis of both strains indicates that Cyanobium spp. have many of the same carbon uptake systems present in Synechococcus spp. (Rae et al., 2011). In using two strains with such similar transport capacities, we aimed to shed light on aspects of the functional properties of carboxysomes in each strain and how these properties affect the operation of the CCM in α-cyanobacteria and β-cyanobacteria. Using both membrane inlet mass spectrometry (MIMS) and silicon oil centrifugation, we investigated C_i pool sizes and CO₂ uptake rates in both species for cells grown at high and low CO₂. Comparative Rubisco properties and photosynthetic rates of each species were determined, and intracellular pools of RuBP and PGA were measured. In addition, we characterized a number of cellular properties to determine differences in the biochemical environments in which each carboxysome type exists. Together, the results provide a unique functional comparison of two distinct carboxysome types from phylogenetically disparate cyanobacteria.