Evolution by leaps: gene duplication in bacteria

Background  

Sequence related families of genes and proteins are common in bacterial genomes. In Escherichia coli they constitute over half of the genome. The presence of families and superfamilies of proteins suggest a history of gene duplication and divergence during evolution. Genome encoded protein families, their size and functional composition, reflect metabolic potentials of the organisms they are found in. Comparing protein families of different organisms give insight into functional differences and similarities.     

The synaptic acetylcholinesterase tetramer assembles around a polyproline II helix

Functional localization of acetylcholinesterase (AChE) in vertebrate muscle and brain depends on interaction of the tryptophan amphiphilic tetramerization (WAT) sequence, at the C-terminus of its major splice variant (T), with a proline-rich attachment domain (PRAD), of the anchoring proteins, collagenous (ColQ) and proline-rich membrane anchor. The crystal structure of the WAT/PRAD complex reveals a novel supercoil structure in which four parallel WAT chains form a left-handed superhelix around an antiparallel left-handed PRAD helix resembling polyproline II. The WAT coiled coils possess a WWW motif making repetitive hydrophobic stacking and hydrogen-bond interactions with the PRAD. The WAT chains are related by an approximately 4-fold screw axis around the PRAD. Each WAT makes similar but unique interactions, consistent with an asymmetric pattern of disulfide linkages between the AChE tetramer subunits and ColQ. The P59Q mutation in ColQ, which causes congenital endplate AChE deficiency, and is located within the PRAD, disrupts crucial WAT-WAT and WAT-PRAD interactions. A model is proposed for the synaptic AChE(T) tetramer.     

The impact of crystallization conditions on structure‐based drug design: A case study on the methylene blue/acetylcholinesterase complex

Structure‐based drug design utilizes apoprotein or complex structures retrieved from the PDB. >57% of crystallographic PDB entries were obtained with polyethylene glycols (PEGs) as precipitant and/or as cryoprotectant, but <6% of these report presence of individual ethyleneglycol oligomers. We report a case in which ethyleneglycol oligomers' presence in a crystal structure markedly affected the bound ligand's position. Specifically, we compared the positions of methylene blue and decamethonium in acetylcholinesterase complexes obtained using isomorphous crystals precipitated with PEG200 or ammonium sulfate. The ligands' positions within the active‐site gorge in complexes obtained using PEG200 are influenced by presence of ethyleneglycol oligomers in both cases bound to W84 at the gorge's bottom, preventing interaction of the ligand's proximal quaternary group with its indole. Consequently, both ligands are ～3.0Å further up the gorge than in complexes obtained using crystals precipitated with ammonium sulfate, in which the quaternary groups make direct π‐cation interactions with the indole. These findings have implications for structure‐based drug design, since data for ligand‐protein complexes with polyethylene glycol as precipitant may not reflect the ligand's position in its absence, and could result in selecting incorrect drug discovery leads. Docking methylene blue into the structure obtained with PEG200, but omitting the ethyleneglycols, yields results agreeing poorly with the crystal structure; excellent agreement is obtained if they are included. Many proteins display features in which precipitants might lodge. It will be important to investigate presence of precipitants in published crystal structures, and whether it has resulted in misinterpreting electron density maps, adversely affecting drug design.     

Anaerobic calorimetry of Zymomonas mobilis using a heat-flux sensor

A relatively simple and inexpensive anaerobic calorimeter was designed and evaluated by using a Hy-Cal Engineering BI-7 bidirectional heat-flux sensor to measure heat output from magnetically stirred I-L flask fermentations. The production of ethanol and cumulative heat output by Zymomonas mobilis were both linearly proportional to glucose concentrations up to 160 g/L.     

Continuous estimation of viscosity,fluid density,surface tension,and heat output during fermentations

Currently, sensors for measuring fluid density, surface tension, viscosity, and heat output are rarely if ever used in estimating conditions continuously in microbial cultures. Measurement of fluid density and surface tension can be accomplished with differential air pressure-based sensors. Fluid viscosity can be estimated by measuring differential fluid pressures. These three sensors, as tested with cultures, were comprised of glass and rubber tubing, a gas supply for density and surface tension and a peristaltic pump for viscosity. The heat output was estimated with an infrared detector spaced two inches from the bottom surface of a solid-state culture flask.The mention of firm names or trade products does not imply that they are endorsed or recommended by the U.S. Department of Agriculture over other firms or similar products not mentioned.     

Schiff and pseudo-Schiff reagents: the reactions and reagents of Hugo Schiff,including a classification of various kinds of histochemical reagents used to detect aldehydes

During the 1860’s, Hugo Schiff studied many reactions between amines and aldehydes, some of which have been used in histochemistry, at times without credit to Schiff. Much controversy has surrounded the chemical structures and reaction mechanisms of the compounds involved, but modern analytical techniques have clarified the picture. I review these reactions here. I used molecular modeling software to investigate dyes that contain primary amines representing eight chemical families. All dyes were known to perform satisfactorily for detecting aldehydes in tissue sections. The models verified the correct chemical structures at various points in their reactions and also determined how decolorization occurred in those with “leuco” forms. Decolorization in the presence of sulfurous acid can occur by either adduction or reduction depending on the dye. The final condensation product with aldehyde was determined to be either a C-sulfonic acid adduct on the carbonyl carbon atom or an aminal at the same atom. Based on the various outcomes, I have placed the dyes and their reactions into five categories. Because Hugo Schiff studied the reactions between aldehydes and amines with and without various acids or alcohol, it is only proper to call each of them Schiff reactions that used various types of Schiff reagents.     

Temporal Expression of Peripheral Blood Leukocyte Biomarkers in a Macaca fascicularis Infection Model of Tuberculosis; Comparison with Human Datasets and Analysis with Parametric/Non-parametric Tools for Improved Diagnostic Biomarker Identification

A temporal study of gene expression in peripheral blood leukocytes (PBLs) from a Mycobacterium tuberculosis primary, pulmonary challenge model Macaca fascicularis has been conducted. PBL samples were taken prior to challenge and at one, two, four and six weeks post-challenge and labelled, purified RNAs hybridised to Operon Human Genome AROS V4.0 slides. Data analyses revealed a large number of differentially regulated gene entities, which exhibited temporal profiles of expression across the time course study. Further data refinements identified groups of key markers showing group-specific expression patterns, with a substantial reprogramming event evident at the four to six week interval. Selected statistically-significant gene entities from this study and other immune and apoptotic markers were validated using qPCR, which confirmed many of the results obtained using microarray hybridisation. These showed evidence of a step-change in gene expression from an ‘early’ FOS-associated response, to a ‘late’ predominantly type I interferon-driven response, with coincident reduction of expression of other markers. Loss of T-cell-associate marker expression was observed in responsive animals, with concordant elevation of markers which may be associated with a myeloid suppressor cell phenotype e.g. CD163. The animals in the study were of different lineages and these Chinese and Mauritian cynomolgous macaque lines showed clear evidence of differing susceptibilities to Tuberculosis challenge. We determined a number of key differences in response profiles between the groups, particularly in expression of T-cell and apoptotic makers, amongst others. These have provided interesting insights into innate susceptibility related to different host `phenotypes. Using a combination of parametric and non-parametric artificial neural network analyses we have identified key genes and regulatory pathways which may be important in early and adaptive responses to TB. Using comparisons between data outputs of each analytical pipeline and comparisons with previously published Human TB datasets, we have delineated a subset of gene entities which may be of use for biomarker diagnostic test development.     

Direct Correlation between Molecular Dynamics and Enzymatic Stability: A Comparative Neutron Scattering Study of Native Human Butyrylcholinesterase and its “Aged” Soman Conjugate

An incoherent elastic neutron scattering study of the molecular dynamics of native human butyrylcholinesterase and its “aged” soman-inhibited conjugate revealed a significant change in molecular flexibility on an angstrom-nanosecond scale as a function of temperature. The results were related to the stability of each state as established previously by differential scanning calorimetry. A striking relationship was found between the denaturation behavior and the molecular flexibility of the native and inhibited enzymes as a function of temperature. This was reflected in a quantitative correlation between the atomic mean-square displacements on an angstrom-nanosecond scale determined by neutron spectroscopy and the calorimetric specific heat. By the application of a simple two-state model that describes the transition from a folded to a denatured state, the denaturation temperatures of the native and the inhibited enzyme were correctly extracted from the atomic mean-square displacements. Furthermore, the transition entropy and enthalpy extracted from the model fit of the neutron data were, within the experimental accuracy, compatible with the values determined by differential scanning calorimetry.