Rational redesign of the 4-chlorobenzoate binding site of 4-chlorobenzoate: coenzyme a ligase for expanded substrate range

Environmental aromatic acids are transformed to chemical energy in bacteria that possess the requisite secondary pathways. Some of these pathways rely on the activation of the aromatic acid by coenzyme A (CoA) thioesterification catalyzed by an aromatic acid: CoA ligase. Adaptation of such pathways to the bioremediation of man-made pollutants such as polychlorinated biphenyl (PCB) and dichlorodiphenyltrichloroethane (DDT) requires that the chlorinated benzoic acid byproduct that is formed be able to be eliminated by further degradation. To take advantage of natural benzoic acid degrading pathways requiring initial ring activation by thioesterification, the pathway aromatic acid:CoA ligase must be an effective catalyst with the chlorinated benzoic acid. This study, which focuses on the 4-chlorobenzoate:CoA ligase (CBL) of the 4-monochlorobiphenyl degrading bacterium Alcaligenes sp. strain ALP83, was carried out to determine if the 4-chlorobenzoate binding site of this enzyme can be transformed by rational design to recognize the chlorobenzoic acids formed in the course of breakdown of other environmental PCB congeners. The fundamental question addressed in this study is whether it is possible to add or subtract space from the substrate-binding pocket of this ligase (to complement the topology of the unnatural aromatic substrate) without causing disruption of the ligase catalytic machinery. Herein, we report the results of a substrate specificity analysis that, when interpreted within the context of the X-ray crystal structures, set the stage for the rational design of the ligase for thioesterification of two PCB-derived chlorobenzoic acids. The ligase was first optimized to catalyze CoA thioesterification of 3,4-dichlorobenzoic acid, a poor substrate, by truncating Ile303, a large hydrophobic residue that packs against the ring meta-C(H) group. The structural basis for the approximately 100-fold enhancement in the rate of 3,4-dichlorobenzoate thioesterification catalyzed by the I303A and I303G CBL mutants was validated by determination of the crystal structure of the 3,4-dichlorobenzoate-bound enzymes. Determinations of the structures of I303 mutant complexes of 3-chlorobenzoate, a very poor substrate, revealed nonproductive binding as a result of the inability of the substrate ring C(4)H group to fill the pocket that binds the C(4)Cl group of the native substrate. The C(4)Cl pocket of the CBL I303A mutant was then reduced in size by strategic amino acid replacement. A 54-fold improvement in catalytic efficiency was observed for the CBL F184W/I303A/V209T triple mutant. The results of this investigation are interpreted as evidence that the plasticity of the ligase catalytic scaffold is sufficient to allow expansion of substrate range by rational design. The combination of structural and kinetic analyses of the constructed mutants proved to be an effective approach to engineering the ligase for novel substrates.     

Reproductive biology of the endemic goby, Lentipes concolor, from Makamaka'ole Stream, Maui and Waikolu Stream, Moloka'i

Constant pressure in Hawai'i to use limited freshwater resources has resulted in increasing concern for the future of the native stream fauna. Hawaiian freshwater gobies have an amphidromous life cycle with a marine larva period and require streams which flow continuously to the ocean for the critical reproductive periods and during recruitment. As such, the stream fauna is particularly sensitive to any anthropogenic perturbations which disrupt the continuity of stream flows. The objective of this 2-year study was to compare the life cycles of the goby, Lentipes concolor, from a heavily diverted stream on Moloka'i and a relatively undisturbed stream on Maui. In Makamaka'ole Stream, Maui, the population of L. concolor was reproductively active all year with females potentially spawning 2–3 times annually. The timing of spawning did not occur consistently during the wet or dry season but coincided with high stream flow conditions regardless of time-of-year. In Waikolu Stream, Moloka'i, the reproductive pattern was more variable with the number of reproductively active females ranging from 0% to 100%. In general the number of eggs was greater and egg size smaller for female L. concolor in Waikolu Stream than in Makamaka'ole Stream. However, female reproductive condition of L. concolor from Maui was consistently higher than from fish on Moloka'i. Reproduction of L. concolor in Makamaka'ole Stream was correlated with the seasonal pattern of flow rates with peaks in female reproductive condition associated with periods of elevated discharge. No correlation between reproduction and discharge occurred in Waikolu Stream. There were considerable differences between the magnitude of discharge in the two streams. Waikolu Stream experienced prolonged periods of extremely low flows which have become common since the Moloka'i Irrigation System began diverting water from the stream in 1960. In Makamaka'ole Stream, L. concolor was capable of reproducing throughout the year and adjusting fecundity in response to stream flow conditions. In contrast, the population in Waikolu Stream appeared to have a ‘boom or bust’ reproductive pattern; the population had reduced or no reproduction when stream flow conditions reached extreme low levels, but the population succesfully reproduced during higher flow months. The diversion structure in Waikolu Stream has dampened the natural seasonal discharge cycle, exacerbated natural low flow conditions, and increased the likelihood of prolonged periods of extremely low flow. Stream management practices in the Hawaiian Islands must take into account the complex life cycles and sensitivity to variable stream flow conditions of the native fauna. This revised version was published online in July 2006 with corrections to the Cover Date.     

Microbial interactions in alcoholic beverages

International Microbiology - This review examines the different types of interactions between the microorganisms involved in the fermentation processes of alcoholic beverages produced all over the...     

Using Manifold Learning for Atlas Selection in Multi-Atlas Segmentation

Multi-atlas segmentation has been widely used to segment various anatomical structures. The success of this technique partly relies on the selection of atlases that are best mapped to a new target image after registration. Recently, manifold learning has been proposed as a method for atlas selection. Each manifold learning technique seeks to optimize a unique objective function. Therefore, different techniques produce different embeddings even when applied to the same data set. Previous studies used a single technique in their method and gave no reason for the choice of the manifold learning technique employed nor the theoretical grounds for the choice of the manifold parameters. In this study, we compare side-by-side the results given by 3 manifold learning techniques (Isomap, Laplacian Eigenmaps and Locally Linear Embedding) on the same data set. We assess the ability of those 3 different techniques to select the best atlases to combine in the framework of multi-atlas segmentation. First, a leave-one-out experiment is used to optimize our method on a set of 110 manually segmented atlases of hippocampi and find the manifold learning technique and associated manifold parameters that give the best segmentation accuracy. Then, the optimal parameters are used to automatically segment 30 subjects from the Alzheimer’s Disease Neuroimaging Initiative (ADNI). For our dataset, the selection of atlases with Locally Linear Embedding gives the best results. Our findings show that selection of atlases with manifold learning leads to segmentation accuracy close to or significantly higher than the state-of-the-art method and that accuracy can be increased by fine tuning the manifold learning process.     

Molecular Imaging Reveals a Progressive Pulmonary Inflammation in Lower Airways in Ferrets Infected with 2009 H1N1 Pandemic Influenza Virus

Molecular imaging has gained attention as a possible approach for the study of the progression of inflammation and disease dynamics. Herein we used [¹⁸F]-2-deoxy-2-fluoro-D-glucose ([¹⁸F]-FDG) as a radiotracer for PET imaging coupled with CT (FDG-PET/CT) to gain insight into the spatiotemporal progression of the inflammatory response of ferrets infected with a clinical isolate of a pandemic influenza virus, H1N1 (H1N1pdm). The thoracic regions of mock- and H1N1pdm-infected ferrets were imaged prior to infection and at 1, 2, 3 and 6 days post-infection (DPI). On 1 DPI, FDG-PET/CT imaging revealed areas of consolidation in the right caudal lobe which corresponded with elevated [¹⁸F]-FDG uptake (maximum standardized uptake values (SUVMax), 4.7–7.0). By days 2 and 3, consolidation (CT) and inflammation ([¹⁸F]-FDG) appeared in the left caudal lobe. By 6 DPI, CT images showed extensive areas of patchy ground-glass opacities (GGO) and consolidations with the largest lesions having high SUVMax (6.0–7.6). Viral shedding and replication were detected in most nasal, throat and rectal swabs and nasal turbinates and lungs on 1, 2 and 3 DPI, but not on day 7, respectively. In conclusion, molecular imaging of infected ferrets revealed a progressive consolidation on CT with corresponding [¹⁸F]-FDG uptake. Strong positive correlations were measured between SUVMax and bronchiolitis-related pathologic scoring (Spearman’s ρ = 0.75). Importantly, the extensive areas of patchy GGO and consolidation seen on CT in the ferret model at 6 DPI are similar to that reported for human H1N1pdm infections. In summary, these first molecular imaging studies of lower respiratory infection with H1N1pdm show that FDG-PET can give insight into the spatiotemporal progression of the inflammation in real-time.     

Structure of the ribosome associating GTPase HflX

The HflX‐family is a widely distributed but poorly characterized family of translation factor‐related guanosine triphosphatases (GTPases) that interact with the large ribosomal subunit. This study describes the crystal structure of HflX from Sulfolobus solfataricus solved to 2.0‐Å resolution in apo‐ and GDP‐bound forms. The enzyme displays a two‐domain architecture with a novel “HflX domain” at the N‐terminus, and a classical G‐domain at the C‐terminus. The HflX domain is composed of a four‐stranded parallel β‐sheet flanked by two α‐helices on either side, and an anti‐parallel coiled coil of two long α‐helices that lead to the G‐domain. The cleft between the two domains accommodates the nucleotide binding site as well as the switch II region, which mediates interactions between the two domains. Conformational changes of the switch regions are therefore anticipated to reposition the HflX‐domain upon GTP‐binding. Slow GTPase activity has been confirmed, with an HflX domain deletion mutant exhibiting a 24‐fold enhanced turnover rate, suggesting a regulatory role for the HflX domain. The conserved positively charged surface patches of the HflX‐domain may mediate interaction with the large ribosomal subunit. The present study provides a structural basis to uncover the functional role of this GTPases family whose function is largely unknown. Proteins 2010. © 2009 Wiley‐Liss, Inc.     

Sulfur assimilation by Oxyrrhis marina feeding on a 35S‐DMSP‐labelled prey

A laboratory grazing experiment was conducted with the aim of quantifying the sulfur assimilation by a herbivore protist feeding on a dimethylsulfoniopropionate (DMSP)‐containing phytoplankter. When supplied with dissolved ³⁵S‐DMSP, cultures of an axenic strain of the diatom Thalassiosira pseudonana took up 60–95% of the added radioisotope and accumulated it untransformed in the cytoplasm. Radiolabelled diatom cells were offered as prey to the heterotrophic dinoflagellate Oxyrrhis marina. After 32 h in the dark, all the prey had been grazed and digested, leaving only radiolabelled O. marina in the grazing bottles and thus providing an estimate of the percentage of DMSP‐sulfur retained by the predator. Subsequent precipitation with cold trichloroacetic acid (TCA) provided the fraction of retained DMSP‐S that had been assimilated into the micrograzer macromolecules. In parallel incubations with predator and dissolved ³⁵S‐DMSP only (no prey), O. marina (and their closely associated bacteria) took up the radiolabelled substrate osmotrophically to an activity of 0.04 dpm cell^?1 and assimilated it all into macromolecules. By correcting grazing ³⁵S‐DMSP assimilation for osmotrophic ³⁵S‐DMSP assimilation, and comparing it with the ingested radioisotope, the percentage of ingested DMSP‐sulfur retained and assimilated by the predator was determined to be 32 ± 4%. This is the first study that provides direct evidence that ingestion of a DMSP‐containing prey supplies structural sulfur to a herbivore protist and that quantifies this assimilative supply at one‐third of ingested DMSP.     

Cutting edge: Chk1 directs senescence and mitotic catastrophe in recovery from G2 checkpoint arrest

Besides the well‐understood DNA damage response via establishment of G₂ checkpoint arrest, novel studies focus on the recovery from arrest by checkpoint override to monitor cell cycle re‐entry. The aim of this study was to investigate the role of Chk1 in the recovery from G₂ checkpoint arrest in HCT116 (human colorectal cancer) wt, p53^–/– and p21^–/– cell lines following H₂O₂ treatment. Firstly, DNA damage caused G₂ checkpoint activation via Chk1. Secondly, overriding G₂ checkpoint led to (i) mitotic slippage, cell cycle re‐entry in G₁ and subsequent G₁ arrest associated with senescence or (ii) premature mitotic entry in the absence of p53/p21^WAF1 causing mitotic catastrophe. We revealed subtle differences in the initial Chk1‐involved G₂ arrest with respect to p53/p21^WAF1: absence of either protein led to late G₂ arrest instead of the classic G₂ arrest during checkpoint initiation, and this impacted the release back into the cell cycle. Thus, G₂ arrest correlated with downstream senescence, but late G₂ arrest led to mitotic catastrophe, although both cell cycle re‐entries were linked to upstream Chk1 signalling. Chk1 knockdown deciphered that Chk1 defines long‐term DNA damage responses causing cell cycle re‐entry. We propose that recovery from oxidative DNA damage‐induced G₂ arrest requires Chk1. It works as cutting edge and navigates cells to senescence or mitotic catastrophe. The decision, however, seems to depend on p53/p21^WAF1. The general relevance of Chk1 as an important determinant of recovery from G₂ checkpoint arrest was verified in HT29 colorectal cancer cells.