Form IA Research Articles

Error estimates for quadrature rules based on the Arnoldi process

The Arnoldi process can be applied to inexpensively approximate matrix functionals of the form IA(f)=uTf(A)v, where A∈RN×N is a large nonsymmetric matrix, u,v∈RN, and the superscript T denotes transposition. Here f is a function such that f(A) is a well defined matrix function. The computed approximations may be regarded as quadrature rules. This paper presents a new inexpensive approach, based on an extension of quadrature rules introduced by Spalević (2007), to compute estimates of the error in quadrature rules for the approximation of IA(f). Knowledge of the quadrature error is helpful for determining the number of nodes of the quadrature rule to be used. Numerical experiments illustrate the accuracy of the computed error estimates.

Impact of Processing Methods on the Physico-chemical Properties of Posaconazole Amorphous Solid Dispersions.

Different methods have been exploited to generate amorphous solid dispersions (ASDs) of poorly water-soluble drugs. However, the impact of processing methods on drug stability and dissolution hasn't been studied extensively. The purpose of the current study is to investigate the impact of the two common ASD processing methods, hot-melt extrusion (HME) and spray drying, on the chemical/physical stability and supersaturation of Posaconazole (Posa) based ASDs. ASDs with 25% drug loading in hydroxypropylmethylcellulose acetate succinate were prepared using HME, and two types of spray dryers, a Procept Sprayer (ASD-Procept) and a Nano Sprayer (ASD-Nano). The relative physical stability of these ASDs upon exposure to heat and crystalline API seeding followed the order: ASD-Nano > ASD-Procept ≈HME. ASD-Procept and ASD-Nano showed similar chemical stability, slightly less stable than HME under 40°C/75%RH. All three ASDs demonstrated similar supersaturation induction times, and de-supersaturation kinetics with or without crystalline seeds. Posa ASDs prepared via spray drying were chemically less stable compared with HME, which can be attributed to their smaller particle size and hollow structure allowing oxygen penetration. For ASD-Procept and HME, the detailed phase changes involving recrystallization of amorphous Posa and a solid-solid phase transition from Posa Form I to Form Ia during the seed-induced studies were proposed. Similar dissolution and supersaturation-precipitation kinetics of three Posa ASDs indicated that any residual nanocrystals in the bulk ASDs were not enough to induce crystallization to differentiate ASDs made by three processing methods.

Polymorphs, Solvatomorphs, Hydrate, and Perhydrate of Dabrafenib

Crystal form screening of the anticancer drug dabrafenib (DBF) was performed using a wide range of nonpolar aprotic, polar aprotic, and polar protic solvents. Extensive crystallization in these solvent systems produced three crystal forms (I, II, and III) of the drug, a monohydrate, an isomorphous peroxo solvate, and eight different solvates with ethyl acetate, dichloromethane, chloroform, carbon tetrachloride, 1,1,2,2-tetrachloroethane, tetrachloroethylene, benzene, and anisole of DBF. Surprisingly no solvates were crystallized with the alcohol solvents attempted. The novel crystalline forms were characterized by single-crystal X-ray diffraction (SC-XRD) or structure determination from powder data (SDPD). The neutral drug derived from the mesylate salt of DBF by neutralization (form Ia) and the crystallized form I exhibited almost a similar powder XRD line pattern but their differential scanning calorimetry (DSC) thermograms comparison showed significant differences. Thus, the structure of microcrystalline powder form Ia was solved from SDPD reflections and showed the same molecular packing but different conformational strain compared to form I (structure by SC-XRD). The relative stability of the drug polymorphs was calculated using dispersion-corrected density functional theory with an intramolecular conformational energy correction. The calculations suggest that form II is the most stable polymorph and form III, form I, and form Ia are progressively less stable. Residual water content analysis of the isomorphous peroxosolvate showed that it is a mixed hydrate/peroxosolvate with ∼9.5% water occupancy quantified by 31P nuclear magnetic resonance analysis of the peroxosolvate content using triphenylphosphine oxide. The supramolecular analysis and molecular packing of DBF crystal forms suggest that the molecule has a propensity to form solvates with aprotic solvents and more specifically with chlorohydrocarbons. With its high molecular flexibility, prolific solvate potential, and drug polymorphism, the present work opens the opportunity for further solid-form landscape study of the popular anticancer drug DBF.

Atypical Carboxysome Loci: JEEPs or Junk?

Carboxysomes, responsible for a substantial fraction of CO2 fixation on Earth, are proteinaceous microcompartments found in many autotrophic members of domain Bacteria, primarily from the phyla Proteobacteria and Cyanobacteria. Carboxysomes facilitate CO2 fixation by the Calvin-Benson-Bassham (CBB) cycle, particularly under conditions where the CO2 concentration is variable or low, or O2 is abundant. These microcompartments are composed of an icosahedral shell containing the enzymes ribulose 1,5-carboxylase/oxygenase (RubisCO) and carbonic anhydrase. They function as part of a CO2 concentrating mechanism, in which cells accumulate HCO3− in the cytoplasm via active transport, HCO3− enters the carboxysomes through pores in the carboxysomal shell proteins, and carboxysomal carbonic anhydrase facilitates the conversion of HCO3− to CO2, which RubisCO fixes. Two forms of carboxysomes have been described: α-carboxysomes and β-carboxysomes, which arose independently from ancestral microcompartments. The α-carboxysomes present in Proteobacteria and some Cyanobacteria have shells comprised of four types of proteins [CsoS1 hexamers, CsoS4 pentamers, CsoS2 assembly proteins, and α-carboxysomal carbonic anhydrase (CsoSCA)], and contain form IA RubisCO (CbbL and CbbS). In the majority of cases, these components are encoded in the genome near each other in a gene locus, and transcribed together as an operon. Interestingly, genome sequencing has revealed some α-carboxysome loci that are missing genes encoding one or more of these components. Some loci lack the genes encoding RubisCO, others lack a gene encoding carbonic anhydrase, some loci are missing shell protein genes, and in some organisms, genes homologous to those encoding the carboxysome-associated carbonic anhydrase are the only carboxysome-related genes present in the genome. Given that RubisCO, assembly factors, carbonic anhydrase, and shell proteins are all essential for carboxysome function, these absences are quite intriguing. In this review, we provide an overview of the most recent studies of the structural components of carboxysomes, describe the genomic context and taxonomic distribution of atypical carboxysome loci, and propose functions for these variants. We suggest that these atypical loci are JEEPs, which have modified functions based on the presence of Just Enough Essential Parts.

The CbbQO-type rubisco activases encoded in carboxysome gene clusters can activate carboxysomal form IA rubiscos

The CO2-fixing enzyme rubisco is responsible for almost all carbon fixation. This process frequently requires rubisco activase (Rca) machinery, which couples ATP hydrolysis to the removal of inhibitory sugar phosphates, including the rubisco substrate ribulose 1,5-bisphosphate (RuBP). Rubisco is sometimes compartmentalized in carboxysomes, bacterial microcompartments that enable a carbon dioxide concentrating mechanism (CCM). Characterized carboxysomal rubiscos, however, are not prone to inhibition, and often no activase machinery is associated with these enzymes. Here, we characterize two carboxysomal rubiscos of the form IAC clade that are associated with CbbQO-type Rcas. These enzymes release RuBP at a much lower rate than the canonical carboxysomal rubisco from Synechococcus PCC6301. We found that CbbQO-type Rcas encoded in carboxysome gene clusters can remove RuBP and the tight-binding transition state analog carboxy-arabinitol 1,5-bisphosphate from cognate rubiscos. The Acidithiobacillus ferrooxidans genome encodes two form IA rubiscos associated with two sets of cbbQ and cbbO genes. We show that the two CbbQO activase systems display specificity for the rubisco enzyme encoded in the same gene cluster, and this property can be switched by substituting the C-terminal three residues of the large subunit. Our findings indicate that the kinetic and inhibitory properties of proteobacterial form IA rubiscos are diverse and predict that Rcas may be necessary for some α-carboxysomal CCMs. These findings will have implications for efforts aiming to introduce biophysical CCMs into plants and other hosts for improvement of carbon fixation of crops.

Dissolved inorganic carbon determines the abundance of microbial primary producers and primary production in Tibetan Plateau lakes.

Climate change globally accelerates the shrinkage of inland lakes, resulting in increases in both water salinity and dissolved inorganic carbon (DIC). The increases of salinity and DIC generate contrasting effects on microbial primary producers and primary production, however, their combined effects remain unclear in aquatic ecosystems. We hypothesized that increased DIC mitigates the constraints of enhanced salinity on microbial primary producers and primary production. To test this, we employed isotope labeling and molecular methods to explore primary production and four dominant types of microbial primary producers (form IA, IB, IC and ID) in lakes on the Tibetan Plateau. Results showed that DIC was positively correlated with the abundance of the form IAB and ID microbial primary producers and primary production (all P <0.001) and offset salinity constraints. Structural equation models elucidated that DIC substantially enhanced primary production by stimulating the abundance of form ID microbial primary producers. The abundance of form ID primary producers explained more variations (14.6%) of primary production than form IAB (6%) and physicochemical factors (6.8%). Diatoms (form ID) played a determinant role in primary production in the lakes by adapting to high DIC and high salinity. Our findings suggest that inland lakes may support higher primary productivity in future climate change scenarios.

Spatiotemporal dynamics of different CO2 fixation strategies used by prokaryotes in a dimictic lake

The Calvin-Benson-Bassham (CBB) cycle and the 3-hydroxypropionate/4-hydroxybutyrate (HP/HB) cycle are two inorganic carbon assimilation pathways widely used by prokaryotic autotrophs in lakes. We investigated the effect of mixing periods and stable water stratification patterns on the trajectories of both CO2 fixation strategies in a dimictic lake (Piburger See), because information on the spatiotemporal dynamics of prokaryotes using these pathways in freshwater ecosystems is far from complete. Based on a quantitative approach (droplet digital PCR) of genes coding for key enzymes in different CO2 assimilation pathways, nine depths covering the entire water column were investigated on a monthly basis for one year. Our data show that the abundance of photoautotrophs and obligate chemolithoautotrophs preferentially using form IA RubisCO was determined by seasonal variations. Highest numbers were observed in summer, while a strong decline of prokrayotes using RubisCO form IA was measured between December and May, the period where the lake was mostly covered by ice. The spatiotemporal distribution patterns of genes coding for RubisCO form IC genes, an enzyme usually used by facultative autotrophs for CO2 assimilation, were less pronounced. Bacteria harboring RubisCO form II were dominating the oxygen limited hypolimnion, while nitrifying Thaumarchaeota using the HP/HB cycle were of minor importance in the lake. Our data reveal that the seasonal heterogeneity, which is determined by the dimictic thermal regime of the lake, results in pronounced spatiotemporal changes of different CO2 assimilation pathways with depth-dependent environmental parameters as key factors for their distribution.

Quantitative investigation of intermolecular interactions in dimorphs of 3-Chloro-N-(2-fluorophenyl)benzamide and 2-Iodo-N-(4- bromophenyl)benzamide

In this study, we have analyzed the role of different intermolecular interactions in the polymorphic modifications of 3-chloro-N-(2-fluorophenyl)benzamide (I) and 2-iodo-N-(4- bromophenyl)benzamide (II). The crystals were obtained via slow evaporation method with the alteration of solvents for crystallization. The already reported form [Cryst. Growth Des. (2011) 11:1578] crystallizes in P2 $$_{1}$$ /c with Z $$^\prime $$ =2 [Form IA], while the new form crystallizes in Pna2 $$_{1}$$ with Z $$^\prime $$ =1 [Form IB]. The latter compound crystallizes in P2 $$_{1}$$ /n with Z $$^\prime $$ =1 [Form IIA] and in Pbca with Z $$^\prime $$ =1 [Form IIB]. Weak Cl $$\cdot \cdot \cdot $$ Cl and C-H $$\cdot \cdot \cdot $$ F interactions in the former (IA, IB) and weak I $$\cdot \cdot \cdot $$ I and C-H $$\cdot \cdot \cdot \uppi $$ interactions in the latter (IIA, IIB) play a vital role in the formation of different polymorphic modifications leading to the observation of conformational and packing polymorphs, respectively. PIXEL calculations show that most of the interactions present in the crystal structures are dispersive in nature. 2D Fingerprint plots revealed that the relative contribution of different intermolecular interactions in different polymorphic forms was different. SYNOPSIS We have analyzed the role of different intermolecular interactions and evaluated their contributions in the polymorphic modifications of 3-chloro-N-(2-fluorophenyl)benzamide (I) and 2-iodo-N-(4- bromophenyl)benzamide (II).

Soil Carbon-Fixation Rates and Associated Bacterial Diversity and Abundance in Three Natural Ecosystems.

CO2 assimilation by autotrophic microbes is an important process in soil carbon cycling, and our understanding of the community composition of autotrophs in natural soils and their role in carbon sequestration of these soils is still limited. Here, we investigated the autotrophic C incorporation in soils from three natural ecosystems, i.e., wetland (WL), grassland (GR), and forest (FO) based on the incorporation of labeled C into the microbial biomass. Microbial assimilation of 14C (14C-MBC) differed among the soils from three ecosystems, accounting for 14.2-20.2% of 14C-labeled soil organic carbon (14C-SOC). We observed a positive correlation between the cbbL (ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) large-subunit gene) abundance, 14C-SOC level, and 14C-MBC concentration confirming the role of autotrophic bacteria in soil carbon sequestration. Distinct cbbL-bearing bacterial communities were present in each soil type; form IA and form IC RubisCO-bearing bacteria were most abundant in WL, followed by GR soils, with sequences from FO soils exclusively derived from the form IC clade. Phylogenetically, the diversity of CO2-fixing autotrophs and CO oxidizers differed significantly with soil type, whereas cbbL-bearing bacterial communities were similar when assessed using coxL. We demonstrate that local edaphic factors such as pH and salinity affect the C-fixation rate as well as cbbL and coxL gene abundance and diversity. Such insights into the effect of soil type on the autotrophic bacterial capacity and subsequent carbon cycling of natural ecosystems will provide information to enhance the sustainable management of these important natural ecosystems.

Structural Characterization of a Newly Identified Component of α-Carboxysomes: The AAA+ Domain Protein CsoCbbQ.

Carboxysomes are bacterial microcompartments that enhance carbon fixation by concentrating ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) and its substrate CO2 within a proteinaceous shell. They are found in all cyanobacteria, some purple photoautotrophs and many chemoautotrophic bacteria. Carboxysomes consist of a protein shell that encapsulates several hundred molecules of RuBisCO, and contain carbonic anhydrase and other accessory proteins. Genes coding for carboxysome shell components and the encapsulated proteins are typically found together in an operon. The α-carboxysome operon is embedded in a cluster of additional, conserved genes that are presumably related to its function. In many chemoautotrophs, products of the expanded carboxysome locus include CbbO and CbbQ, a member of the AAA+ domain superfamily. We bioinformatically identified subtypes of CbbQ proteins and show that their genes frequently co-occur with both Form IA and Form II RuBisCO. The α-carboxysome-associated ortholog, CsoCbbQ, from Halothiobacillus neapolitanus forms a hexamer in solution and hydrolyzes ATP. The crystal structure shows that CsoCbbQ is a hexamer of the typical AAA+ domain; the additional C-terminal domain, diagnostic of the CbbQ subfamily, structurally fills the inter-monomer gaps, resulting in a distinctly hexagonal shape. We show that CsoCbbQ interacts with CsoCbbO and is a component of the carboxysome shell, the first example of ATPase activity associated with a bacterial microcompartment.

New conformational polymorph of hydrochlorothiazide with improved solubility

Context: To characterize a new conformation of hydrochlorothiazide (HCT) with better solubility and establishing its relationship with previously reported form I, obtained during attempted crystallization experiments.Objective: The aim of present investigation is to unveil a new conformational polymorph (form IA) having a higher solubility compared to commercially available form I.Materials and methods: New form (IA) was obtained from slow evaporation as well as by solvent–antisolvent method and was then characterized by DSC, FTIR, PXRD and SCXRD. Equilibrium solubility profile shows that it is more soluble than form I.Results: Appearance of phase transition endotherm at 215.87 °C in DSC spectra indicated the existence of new polymorph which was further confirmed by FTIR and PXRD. Single crystal study showed significant difference in various bond angles and torsion angles of the two forms. The solubility exhibited by form IA was (938 µg/mL) compared to form I (791 µg/mL) in water.Discussion: Complete structural analysis and molecular arrangements in the unit cell along with the DSC and FTIR data confirm the existence of new conformer of HCT.Conclusion: This study reveals the existence of a new conformational polymorph of HCT molecule having higher solubility could prove to be promising in pre-formulation.

Advances in Understanding Carboxysome Assembly in Prochlorococcus and Synechococcus Implicate CsoS2 as a Critical Component.

The marine Synechococcus and Prochlorococcus are the numerically dominant cyanobacteria in the ocean and important in global carbon fixation. They have evolved a CO2-concentrating-mechanism, of which the central component is the carboxysome, a self-assembling proteinaceous organelle. Two types of carboxysome, α and β, encapsulating form IA and form IB d-ribulose-1,5-bisphosphate carboxylase/oxygenase, respectively, differ in gene organization and associated proteins. In contrast to the β-carboxysome, the assembly process of the α-carboxysome is enigmatic. Moreover, an absolutely conserved α-carboxysome protein, CsoS2, is of unknown function and has proven recalcitrant to crystallization. Here, we present studies on the CsoS2 protein in three model organisms and show that CsoS2 is vital for α-carboxysome biogenesis. The primary structure of CsoS2 appears tripartite, composed of an N-terminal, middle (M)-, and C-terminal region. Repetitive motifs can be identified in the N- and M-regions. Multiple lines of evidence suggest CsoS2 is highly flexible, possibly an intrinsically disordered protein. Based on our results from bioinformatic, biophysical, genetic and biochemical approaches, including peptide array scanning for protein-protein interactions, we propose a model for CsoS2 function and its spatial location in the α-carboxysome. Analogies between the pathway for β-carboxysome biogenesis and our model for α-carboxysome assembly are discussed.

Energy costs of carbon dioxide concentrating mechanisms in aquatic organisms

Minimum energy (as photon) costs are predicted for core reactions of photosynthesis, for photorespiratory metabolism in algae lacking CO2 concentrating mechanisms (CCMs) and for various types of CCMs; in algae, with CCMs; allowance was made for leakage of CO2 from the internal pool. These predicted values are just compatible with the minimum measured photon costs of photosynthesis in microalgae and macroalgae lacking or expressing CCMs. More energy-expensive photorespiration, for example for organisms using Rubiscos with lower CO2-O2 selectivity coefficients, would be less readily accommodated within the lowest measured photon costs of photosynthesis by algae lacking CCMs. The same applies to the cases of CCMs with higher energy costs of active transport of protons or inorganic carbon species, or greaterallowance for significant leakage from the accumulated intracellular pool of CO2. High energetic efficiency can involve a higher concentration of catalyst to achieve a given rate of reaction, adding to the resource costs of growth. There are no obvious mechanistic interpretations of the occurrence of CCMs algae adapted to low light and low temperatures using the rationales adopted for the occurrence of C4 photosynthesis in terrestrial flowering plants. There is an exception for cyanobacteria with low-selectivity Form IA or IB Rubiscos, and those dinoflagellates with low-selectivity Form II Rubiscos, for which very few natural environments have high enough CO2:O2 ratios to allow photosynthesis in the absence of CCMs.

RubisCO gene clusters found in a metagenome microarray from acid mine drainage.

The enzyme responsible for carbon dioxide fixation in the Calvin cycle, ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO), is always detected as a phylogenetic marker to analyze the distribution and activity of autotrophic bacteria. However, such an approach provides no indication as to the significance of genomic content and organization. Horizontal transfers of RubisCO genes occurring in eubacteria and plastids may seriously affect the credibility of this approach. Here, we presented a new method to analyze the diversity and genomic content of RubisCO genes in acid mine drainage (AMD). A metagenome microarray containing 7,776 large-insertion fosmids was constructed to quickly screen genome fragments containing RubisCO form I large-subunit genes (cbbL). Forty-six cbbL-containing fosmids were detected, and six fosmids were fully sequenced. To evaluate the reliability of the metagenome microarray and understand the microbial community in AMD, the diversities of cbbL and the 16S rRNA gene were analyzed. Fosmid sequences revealed that the form I RubisCO gene cluster could be subdivided into form IA and IB RubisCO gene clusters in AMD, because of significant divergences in molecular phylogenetics and conservative genomic organization. Interestingly, the form I RubisCO gene cluster coexisted with the form II RubisCO gene cluster in one fosmid genomic fragment. Phylogenetic analyses revealed that horizontal transfers of RubisCO genes may occur widely in AMD, which makes the evolutionary history of RubisCO difficult to reconcile with organismal phylogeny.

Comparative molecular analysis of chemolithoautotrophic bacterial diversity and community structure from coastal saline soils, Gujarat, India

BackgroundSoils harbour high diversity of obligate as well as facultative chemolithoautotrophic bacteria that contribute significantly to CO2 dynamics in soil. In this study, we used culture dependent and independent methods to assess the community structure and diversity of chemolithoautotrophs in agricultural and coastal barren saline soils (low and high salinity). We studied the composition and distribution of chemolithoautotrophs by means of functional marker gene cbbL encoding large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase and a phylogenetic marker 16S rRNA gene. The cbbL form IA and IC genes associated with carbon fixation were analyzed to gain insight into metabolic potential of chemolithoautotrophs in three soil types of coastal ecosystems which had a very different salt load and sulphur content.ResultsIn cbbL libraries, the cbbL form IA was retrieved only from high saline soil whereas form IC was found in all three soil types. The form IC cbbL was also amplified from bacterial isolates obtained from all soil types. A number of novel monophyletic lineages affiliated with form IA and IC phylogenetic trees were found. These were distantly related to the known cbbL sequences from agroecosystem, volcanic ashes and marine environments. In 16S rRNA clone libraries, the agricultural soil was dominated by chemolithoautotrophs (Betaproteobacteria) whereas photoautotrophic Chloroflexi and sulphide oxidizers dominated saline ecosystems. Environmental specificity was apparently visible at both higher taxonomic levels (phylum) and lower taxonomic levels (genus and species). The differentiation in community structure and diversity in three soil ecosystems was supported by LIBSHUFF (P = 0.001) and UniFrac.ConclusionThis study may provide fundamentally new insights into the role of chemolithoautotrophic and photoautotrophic bacterial diversity in biochemical carbon cycling in barren saline soils. The bacterial communities varied greatly among the three sites, probably because of differences in salinity, carbon and sulphur contents. The cbbL form IA-containing sulphide-oxidizing chemolithotrophs were found only in high saline soil clone library, thus giving the indication of sulphide availability in this soil ecosystem. This is the first comparative study of the community structure and diversity of chemolithoautotrophic bacteria in coastal agricultural and saline barren soils using functional (cbbL) and phylogenetic (16S rDNA) marker genes.

Diversity of RuBisCO gene responsible for CO2 fixation in an Antarctic moss pillar

Antarctic “moss pillars” are lake-bottom biocenoses that are primarily comprised of aquatic mosses. The pillars consist of distinct redox-affected sections: oxidative exteriors and reductive interiors. Batteries of SSU rRNA genotypes of eukaryotes, eubacteria, and cyanobacteria, but no archaea, have been identified in these pillars. However, rRNA-based phylogenetic analysis provides limited information on metabolic capabilities. To investigate the microorganisms that have the potential for CO2 fixation in the pillars, we studied the genetic diversity of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO, EC 4.1.1.39)—an enzyme involved in CO2 fixation. PCR clone libraries targeting all forms of the RuBisCO large subunit-encoding gene were constructed and 1,092 clones were randomly sequenced. Phylogenetic analysis indicated that proteobacterial form IA operational RuBisCO units (ORUs) were detected at the same frequency as the cyanobacterial form IB ORUs. Surprisingly, the form IA ORU, which was closely related to the sequences from deep-sea environments, was detected from all moss pillar sections. The form IB ORU related to Bryophyta, considered to be derived from moss, was identical to the sequence of Leptobryum sp. isolated from Lake Hotoke-Ike where the pillars were found. Moreover, certain cyanobacterial ORUs were found exclusively in the exterior of the pillar, whereas form II ORUs related to chemolithoautotrophic sulfur oxidizers and purple sulfur bacteria were found exclusively in the interior. No forms IC, ID, or III RuBisCO genes were detected. This is the first report demonstrating that bacteria with the potential for CO2 fixation and chemoautotrophy are present in the Antarctic moss pillar ecosystem.

Diversity and expression of different forms of RubisCO genes in polluted groundwater under different redox conditions

Groundwater polluted with methyl-tert-butyl ether (MTBE) and ammonium was investigated for chemolithoautotrophic CO(2) fixation capabilities based on detailed analyses of ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) large subunit genes. Samples retrieved from a groundwater conditioning unit, characterized by different redox conditions, were examined for the presence of form IA, form IC (cbbL) and form II (cbbM) RubisCO genes and transcripts obtained from DNA- and RNA-extracts. Form IA RubisCO sequences, which revealed a complex and distinct variety in different sampling stations, were expressed in the original groundwater and in samples amended with oxygen, but not in the aquifer groundwater enriched with nitrate. Form IC RubisCO genes were exclusively detected in groundwater supplied with oxygen and sequences were affiliated with cbbL genes in nitrifying bacteria. cbbM genes were not expressed in the oxygen-amended groundwater, probably due to the low CO(2) /O(2) substrate specificity of this enzyme. Most form II RubisCO transcripts were affiliated with RubisCO genes of denitrifiers, which are important residents in the groundwater supplied with nitrate. The distinct distribution pattern and diversity of RubisCO genes and transcripts obtained in this study suggest that the induction of different RubisCO enzymes is highly regulated and closely linked to the actual environmental conditions.

Two Polymorphs of 1-(2-Methyl-3-oxoisoindolin-5-yl)urea

4-Ureidophthalimide or ureido isoindolin-1-one based ligands were shown by NMR spectroscopy and theoretical studies to bind the CG base pairs in the major groove. To examine this hydrogen-bonded complex, we cocrystallized 5-fluorocytosine, 9-methylguanine and 1-(2-methyl-3-oxoisoindolin-5-yl)urea. Two polymorphs of the latter compound were obtained during the cocrystallization attempts. Both crystallized in monoclinic space groups: form Ia in P21/c with cell parameters of a = 11.382(2), b = 6.042(1), c = 14.102(2) A and β = 96.51(1)°, and form Ib in P21/n with cell parameters of a = 7.092(1), b = 11.643(2), c = 11.580(2) A and β = 93.48(1)°. An identical molecular conformation of the two polymorphs is observed. Both polymorphs have an R 2 2 (8) N–H···O interaction linking the urea fragments of two molecules to a centrosymmetric dimer. Nevertheless, the crystal packing for both forms is completely different. In Ia, two dimers are connected by two R 2 1 (6) N–H···O bonds simultaneously to a shifted ribbon motif, whereas in Ib the two R 2 1 (6) interactions link two molecules from two different dimers. Two polymorphs of 1-(2-methyl-3-oxoisoindolin-5-yl)urea show similar R 2 2 (8) and R 2 1 (6) hydrogen-bond patterns but one completely different crystal packing.

Functional Prokaryotic RubisCO from an Oceanic Metagenomic Library

Culture-independent studies have indicated that there is significant diversity in the ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO) enzymes used by marine, freshwater, and terrestrial autotrophic bacteria. Surprisingly, little is known about the catalytic properties of many environmentally significant RubisCO enzymes. Because one of the goals of RubisCO research is to somehow modify or select for RubisCO molecules with improved kinetic properties, a facile means to isolate functional and novel RubisCO molecules directly from the environment was developed. In this report, we describe the first example of functional RubisCO proteins obtained from genes cloned and characterized from metagenomic libraries derived from DNA isolated from environmental samples. Two form IA marine RubisCO genes were cloned, and each gene supported both photoheterotrophic and photoautotrophic growth of a RubisCO deletion strain of Rhodobacter capsulatus, strain SBI/II(-), indicating that catalytically active recombinant RubisCO was synthesized. The catalytic properties of the metagenomic RubisCO molecules were further characterized. These experiments demonstrated the feasibility of studying the functional diversity and enzymatic properties of RubisCO enzymes without first cultivating the host organisms. Further, this "proof of concept" experiment opens the way for development of a simple functional screen to examine the properties of diverse RubisCO genes isolated from any environment, and subsequent further bioselection may be possible if the growth conditions of complemented R. capsulatus strain SBI/II(-) are varied.

What Do We Know about Determination of Amino Acids with Orthophthalaldehyde?

AbstractMechanisms of the formation of the fluorescent species widely used for determination of amino acids, based on reaction with orthophthalaldehyde (OPA) were unknown. Attempts to elucidate this mechanism based solely on product identification were unsuccessful. Analytical methods based on reaction of OPA with amino acids in the presence of RS− or CN− ions were developed empirically. The reaction conditions reported in more than 700 papers differed considerably. In aqueous solutions OPA exists in three different forms in equilibrium – the unhydrated form Ia (20%), monohydrated acyclic form Ib (8%) and cyclic hemiacetal form Ic (72%). These forms are in equilibria, the rate of establishment of which is acid and base catalyzed. Reactions of OPA and NH3 and 2‐aminoethanol (colamine) take place in a series of equilibria depicted in Scheme 1. The isoindole derivatives are formed in cyclization of the carbinolamine. Formation of an imine is a side reaction.