Recruitment of a double bond isomerase to serve as a reductive dehalogenase during biodegradation of pentachlorophenol

Tetrachlorohydroquinone dehalogenase catalyzes the replacement of chlorine atoms on tetrachlorohydroquinone and trichlorohydroquinone with hydrogen atoms during the biodegradation of pentachlorophenol by Sphingomonas chlorophenolica. The sequence of the active site region of tetrachlorohydroquinone dehalogenase is very similar to those of the corresponding regions of maleylacetoacetate isomerases, enzymes that catalyze the glutathione-dependent isomerization of a cis double bond in maleylacetoacetate to the trans configuration during the catabolism of phenylalanine and tyrosine. Furthermore, tetrachlorohydroquinone dehalogenase catalyzes the isomerization of maleylacetone (an analogue of maleylacetoacetate) at a rate nearly comparable to that of a bona fide bacterial maleylacetoacetate isomerase. Since maleylacetoacetate isomerase is involved in a common and presumably ancient pathway for catabolism of tyrosine, while tetrachlorohydroquinone dehalogenase catalyzes a more specialized reaction, it is likely that tetrachlorohydroquinone dehalogenase arose from a maleylacetoacetate isomerase. The substrates and overall transformations involved in the dehalogenation and isomerization reactions are strikingly different. This enzyme provides a remarkable example of Nature's ability to recruit an enzyme with a useful structural scaffold and elaborate upon its basic catalytic capabilities to generate a catalyst for a newly needed reaction.     

Sialidase-like Asp-boxes: sequence-similar structures within different protein folds

Sequence similarity is the most common measure currently used to infer homology between proteins. Typically, homologous protein domains show sequence similarity over their entire lengths. Here we identify Asp box motifs, initially found as repeats in sialidases and neuraminidases, in new structural and sequence contexts. These motifs represent significantly similar sequences, localized to beta hairpins within proteins that are otherwise different in sequence and three-dimensional structure. By performing a combined sequence- and structure-based analysis we detect Asp boxes in more than nine protein families, including bacterial ribonucleases, sulfite oxidases, reelin, netrins, some lipoprotein receptors, and a variety of glycosyl hydrolases. Although the function common to each of these proteins, if any, remains unclear, we discuss possible functions of Asp boxes on the basis of previously determined experimental results and discuss different evolutionary scenarios for the origin of Asp-box containing proteins.     

High-throughput analysis of single hematopoietic stem cell proliferation in microfluidic cell culture arrays

Heterogeneity in cell populations poses a major obstacle to understanding complex biological processes. Here we present a microfluidic platform containing thousands of nanoliter-scale chambers suitable for live-cell imaging studies of clonal cultures of nonadherent cells with precise control of the conditions, capabilities for in situ immunostaining and recovery of viable cells. We show that this platform mimics conventional cultures in reproducing the responses of various types of primitive mouse hematopoietic cells with retention of their functional properties, as demonstrated by subsequent in vitro and in vivo (transplantation) assays of recovered cells. The automated medium exchange of this system made it possible to define when Steel factor stimulation is first required by adult hematopoietic stem cells in vitro as the point of exit from quiescence. This technology will offer many new avenues to interrogate otherwise inaccessible mechanisms governing mammalian cell growth and fate decisions.     

Influenza Aerosols in UK Hospitals during the H1N1 (2009) Pandemic – The Risk of Aerosol Generation during Medical Procedures

Background

Nosocomial infection of health-care workers (HCWs) during outbreaks of respiratory infections (e.g. Influenza A H1N1 (2009)) is a significant concern for public health policy makers. World Health Organization (WHO)-defined ‘aerosol generating procedures’ (AGPs) are thought to increase the risk of aerosol transmission to HCWs, but there are presently insufficient data to quantify risk accurately or establish a hierarchy of risk-prone procedures.

Methodology/Principal Findings

This study measured the amount of H1N1 (2009) RNA in aerosols in the vicinity of H1N1 positive patients undergoing AGPs to help quantify the potential risk of transmission to HCWs. There were 99 sampling occasions (windows) producing a total of 198 May stages for analysis in the size ranges 0.86–7.3 µm. Considering stages 2 (4–7.3 µm) and 3 (0.86–4 µm) as comprising one sample, viral RNA was detected in 14 (14.1%) air samples from 10 (25.6%) patients. Twenty three air samples were collected while potential AGPs were being performed of which 6 (26.1%) contained viral RNA; in contrast, 76 May samples were collected when no WHO 2009 defined AGP was being performed of which 8 (10.5%) contained viral RNA (unadjusted OR = 2.84 (95% CI 1.11–7.24) adjusted OR = 4.31 (0.83–22.5)).

Conclusions/Significance

With our small sample size we found that AGPs do not significantly increase the probability of sampling an H1N1 (2009) positive aerosol (OR (95% CI) = 4.31 (0.83–22.5). Although the probability of detecting positive H1N1 (2009) positive aerosols when performing various AGPs on intensive care patients above the baseline rate (i.e. in the absence of AGPs) did not reach significance, there was a trend towards hierarchy of AGPs, placing bronchoscopy and respiratory and airway suctioning above baseline (background) values. Further, larger studies are required but these preliminary findings may be of benefit to infection control teams.     

DNA barcodes for species identification of euphausiids (Euphausiacea, Crustacea)

Many species of euphausiids (Euphausiacea, Crustacea) are distinguishedby subtle or geographically variable morphological characters,and erroneous identification of euphausiid species may be morefrequent than currently acknowledged. DNA barcodes (short DNAsequences that discriminate species and aid in recognition ofunknown species) are of use for this group. A 650 bp regionof mitochondrial cytochrome oxidase I (mtCOI) was sequencedfor 40 species of 10 euphausiid genera: Bentheuphausia, Euphausia,Meganyctiphanes, Nematobrachion, Nematoscelis, Nyctiphanes,Stylocheiron, Tessarabrachion, Thyssanoessa and Thysanopoda.mtCOI sequence variation discriminated all species; pairwisedifferences averaged 16.4% (range 7–24%); mean generalizedtime reversible (GTR) genetic distance was 26.7%. mtCOI reliablyidentified euphausiid species: variation within species wastypically < 1% and GTR distance was typically < 2%. Atlanticand Pacific Ocean populations of Euphausia brevis differed by13% (GTR genetic distance = 28%) and may deserve status as distinctspecies. mtCOI gene trees were reconstructed for five generausing maximum parsimony, maximum likelihood and Bayesian algorithms;best-fit models of nucleotide evolution were determined foreach genus. The mtCOI gene tree for 20 species of Euphausiareproduced one of three morphologically defined species groups.mtCOI resolved relationships among closely related species ofmost genera, usually in accord with morphological groupings.A comprehensive DNA barcode database for euphausiids will helpensure accurate species identification, recognition of crypticspecies and evaluation of taxonomically meaningful geographicvariation.     

Pre-steady-state kinetic studies of the reductive dehalogenation catalyzed by tetrachlorohydroquinone dehalogenase

Tetrachlorohydroquinone dehalogenase catalyzes two successive reductive dehalogenation reactions in the pathway for degradation of pentachlorophenol in the soil bacterium Sphingobium chlorophenolicum. We have used pre-steady-state kinetic methods to probe both the mechanism and the rates of elementary steps in the initial stages of the reductive dehalogenation reaction. Binding of trichlorohydroquinone (TriCHQ) to the active site is followed by rapid deprotonation to form TriCHQ-2 and subsequent formation of 3,5,6-trichloro-4-hydroxycyclohexa-2,4-dienone (TriCHQ*). Further conversion of TriCHQ* to 2,6-dichlorohydroquinone (DCHQ) proceeds only in the presence of glutathione. Conversion of TriCHQ to DCHQ during the first turnover is quite rapid, occurring at about 25 s-1 when the enzyme is saturated with TriCHQ and glutathione. The rate of subsequent turnovers is limited by the rate of the thiol-disulfide exchange reaction required to regenerate the free enzyme after turnover, a reaction that is intrinsically less difficult, but is hampered by premature binding of the aromatic substrate to the active site before the catalytic cycle is completed.     

Structural biology

A selection of interesting papers that were published in the two months before our press date in major journals most likely to report significant results in structural biology.     

Microdistribution of Faunal Assemblages at Deep-Sea Hydrothermal Vents in the Southern Ocean

Chemosynthetic primary production by microbes supports abundant faunal assemblages at deep-sea hydrothermal vents, with zonation of invertebrate species typically occurring along physico-chemical gradients. Recently discovered vent fields on the East Scotia Ridge (ESR) in the Southern Ocean represent a new province of vent biogeography, but the spatial dynamics of their distinct fauna have yet to be elucidated. This study determines patterns of faunal zonation, species associations, and relationships between faunal microdistribution and hydrothermal activity in a vent field at a depth of 2,400 m on the ESR. Remotely operated vehicle (ROV) dives obtained high-definition imagery of three chimney structures with varying levels of hydrothermal activity, and a mosaic image of >250 m² of seafloor co-registered with temperature measurements. Analysis of faunal microdistribution within the mosaiced seafloor reveals a consistent pattern of faunal zonation with increasing distance from vent sources and peak temperatures. Assemblages closest to vent sources are visibly dominated by a new species of anomuran crab, Kiwa n. sp. (abundance >700 individuals m⁻²), followed by a peltospiroid gastropod (>1,500 individuals m⁻²), eolepadid barnacle (>1,500 individuals m⁻²), and carnivorous actinostolid anemone (>30 individuals m⁻²). Peripheral fauna are not dominated by a single taxon, but include predatory and scavenger taxa such as stichasterid seastars, pycnogonids and octopus. Variation in faunal microdistribution on chimneys with differing levels of activity suggests a possible successional sequence for vent fauna in this new biogeographic province. An increase in δ³⁴S values of primary consumers with distance from vent sources, and variation in their δ¹³C values also indicate possible zonation of nutritional modes of the vent fauna. By using ROV videography to obtain a high-resolution representation of a vent environment over a greater extent than previous studies, these results provide a baseline for determining temporal change and investigations of processes structuring faunal assemblages at Southern Ocean vents.