Isolation and characterization of environmental bacteria capable of extracellular biosorption of mercury

Accumulation of toxic metals in the environment represents a public health and wildlife concern. Bacteria resistant to toxic metals constitute an attractive biomass for the development of systems to decontaminate soils, sediments, or waters. In particular, biosorption of metals within the bacterial cell wall or secreted extracellular polymeric substances (EPS) is an emerging process for the bioremediation of contaminated water. Here the isolation of bacteria from soil, effluents, and river sediments contaminated with toxic metals permitted the selection of seven bacterial isolates tolerant to mercury and associated with a mucoid phenotype indicative of the production of EPS. Inductively coupled plasma-optical emission spectroscopy and transmission electron microscopy in conjunction with X-ray energy dispersive spectrometry revealed that bacteria incubated in the presence of HgCl2 sequestered mercury extracellularly as spherical or amorphous deposits. Killed bacterial biomass incubated in the presence of HgCl2 also generated spherical extracellular mercury deposits, with a sequestration capacity (40 to 120 mg mercury per g [dry weight] of biomass) superior to that of live bacteria (1 to 2 mg mercury per g [dry weight] of biomass). The seven strains were shown to produce EPS, which were characterized by Fourier transform-infrared (FT-IR) spectroscopy and chemical analysis of neutral-carbohydrate, uronic acid, and protein contents. The results highlight the high potential of Hg-tolerant bacteria for applications in the bioremediation of mercury through biosorption onto the biomass surface or secreted EPS.     

The roles of Tyr(CD1) and Trp(G8) in Mycobacterium tuberculosis truncated hemoglobin O in ligand binding and on the heme distal site architecture

The crystal structure of the cyano-met form of Mt-trHbO revealed two unusual distal residues Y(CD1) and W(G8) forming a hydrogen-bond network with the heme-bound ligand [Milani, M., et al. (2003) Proc. Natl. Acad. Sci. U.S.A. 100, 5766-5771]. W(G8) is an invariant residue in group II and group III trHbs and has no counterpart in other globins. A previous study reported that changing Y(CD1) for a Phe causes a significant increase in the O2 combination rate, but almost no change in the O2 dissociation rate [Ouellet, H., et al. (2003) Biochemistry 42, 5764-5774]. Here we investigated the role of the W(G8) in ligand binding by using resonance Raman spectroscopy, stopped-flow spectrophotometry, and X-ray crystallography. For this purpose, W(G8) was changed, by site-directed mutagenesis, to a Phe in both the wild-type protein and the mutant Y(CD1)F to create the single mutant W(G8)F and the double mutant Y(CD1)F/W(G8)F, respectively. Resonance Raman results suggest that W(G8) interacts with the heme-bound O2 and CO, as evidenced by the increase of the Fe-O2 stretching mode from 559 to 564 cm-1 and by the lower frequency of the Fe-CO stretching modes (514 and 497 cm-1) compared to that of the wild-type protein. Mutation of W(G8) to Phe indicates that this residue controls ligand binding, as evidenced by a dramatic increase of the combination rates of both O2 and CO. Also, the rate of O2 dissociation showed a 90-1000-fold increase in the W(G8)F and Y(CD1)F/W(G8)F mutants, that is in sharp contrast with the values obtained for the other distal mutants Y(B10)F and Y(CD1)F [Ouellet, H., et al. (2003) Biochemistry 42, 5764-5774]. Taken together, these data indicate a pivotal role for the W(G8) residue in O2 binding and stabilization.     

Relationship between amperometric pre-spike feet and secretion granule composition in chromaffin cells: an overview

Amperometry is a simple and powerful technique to study exocytosis at the single cell level. By positioning and polarizing (at an appropriate potential at which the molecules released by the cell can be oxidized) a carbon fiber microelectrode at the top of the cell, each exocytotic event is detected as an amperometric spike. More particularly, a portion of these spikes has previously been shown to present a foot, i.e. a small pedestal of current that precedes the spike itself. Among the important number of works dealing with the monitoring of exocytosis by amperometry under different conditions, only a few studies focus on amperometric spikes with a foot. In this work, by coupling our previous and recent experiments on chromaffin cells (that release catecholamines after stimulation) with literature data, we bring more light on what an amperometric foot and particularly its features, represents.     

A Multicompartment Approach - Diatoms,Macrophytes, Benthic Macroinvertebrates and Fish - To Assess the Impact of Toxic Industrial Releases on a Small French River

The River Luzou flows through a sandy substrate in the South West of France. According to the results of two assessment surveys, the Water Agency appraised that this river may not achieve the good ecological status by 2015 as required by the Water Framework Directive (2000/60/EC). This ecosystem is impacted by industrial effluents (organic matter, metals and aromatic compounds). In order to assess and characterize the impact, this study aimed to combine a set of taxonomic and non-taxonomic metrics for diatoms, macrophytes, macroinvertebrates and fish along the up- to downstream gradient of the river. Diversity metrics, biological indices, biological and ecological traits were determined for the four biological quality elements (BQE). Various quantitative metrics (biomass estimates) were also calculated for diatom communities. The results were compared to physicochemical analysis. Biological measurements were more informative than physicochemical analysis, in the context of the study. Biological responses indicated both the contamination of water and its intensity. Diversity metrics and biological indices strongly decreased with pollution for all BQE but diatoms. Convergent trait selection with pollution was observed among BQE: reproduction, colonization strategies, or trophic regime were clearly modified at impaired sites. Taxon size and relation to the substrate diverged among biological compartments. Multiple anthropogenic pollution calls for alternate assessment methods of rivers'' health. Our study exemplifies the fact that, in the case of complex contaminations, biological indicators can be more informative for environmental risk, than a wide screening of contaminants by chemical analysis alone. The combination of diverse biological compartments provided a refined diagnostic about the nature (general mode of action) and intensity of the contamination.     

Enhanced Biodegradation of Petroleum Hydrocarbons in Contaminated Soil

Soil samples taken from a contaminated site in Northern Quebec, Canada, exhibited a low capacity for biodegradation of total petroleum hydrocarbons (TPH), despite a high capacity for the mineralization of aromatic hydrocarbons and a low toxicity of soil leachates as measured by Microtox assay. Toxicity assays directly performed on surface soil, including earthworm mortality and barley seedling emergence, indicated moderate to high levels of toxicity. Soil biostimulation did not improve the removal of petroleum hydrocarbons, while bioaugmentation of soil with a developed enrichment culture increased the efficiency of hydrocarbon removal from 20.4% to 49.2%. A considerable increase in the removal of TPH was obtained in a bioslurry process, enhancing the mass transfer of hydrocarbons from soil to the aqueous phase and increasing the efficiency of hydrocarbon removal to over 70% after 45 days of incubation. The addition of ionic or nonionic surfactants did not have a significant impact on biodegradation of hydrocarbons. The extent of hydrocarbon mineralization during the bioslurry process after 45 days of incubation ranged from 41.3% to 58.9%, indicating that 62.7% to 83.1% of the eliminated TPH were transformed into CO₂ and water.     

Membrane Association and Multimerization of Secreton Component PulC

The PulC component of the Klebsiella oxytoca pullulanase secretion machinery (the secreton) was found by subcellular fractionation to be associated with both the cytoplasmic (inner) and outer membranes. Association with the outer membrane was independent of other secreton components, including the outer membrane protein PulD (secretin). The association of PulC with the inner membrane is mediated by the signal anchor sequence located close to its N terminus. These results suggest that PulC forms a bridge between the two membranes that is disrupted when bacteria are broken open for fractionation. Neither the signal anchor sequence nor the cytoplasmic N-terminal region that precedes it was found to be required for PulC function, indicating that PulC does not undergo sequence-specific interactions with other cytoplasmic membrane proteins. Cross-linking of whole cells resulted in the formation of a ca. 110-kDa band that reacted with PulC-specific serum and whose detection depended on the presence of PulD. However, antibodies against PulD failed to react with this band, suggesting that it could be a homo-PulC trimer whose formation requires PulD. The data are discussed in terms of the possible role of PulC in energy transduction for exoprotein secretion.     

Different Patterns of Punctate White Matter Lesions in Serially Scanned Preterm Infants

Background and Purpose

With the increased use of MRI in preterm infants, punctate white matter lesions (PWML) are more often recognized. The aim of this study was to describe the incidence and characteristics of these lesions as well as short-term outcome in a cohort of serially scanned preterm infants, using both conventional imaging, diffusion (DWI) and susceptibility (SWI) weighted imaging.

Materials and Methods

112 preterm infants with 2 MRIs in the neonatal period, with evidence of punctate white matter lesions, were included. Appearance, lesion load, location, and abnormalities on DWI and SWI were scored and outcome data were collected.

Results

Different patterns of punctate white matter lesions did appear: a linear appearance associated with signal loss on SWI, and a cluster appearance associated with restricted diffusion on DWI on the first MRI. Cluster and mixed lesions on the first scan changed in appearance in over 50% on the second scan, whereas linear lesions generally kept their appearance. Lesions were only visible on the early scan in 33%, and were only seen at term equivalent age in 20%. Nine infants developed cerebral palsy, due to additional overt white matter lesions in six.

Conclusion

Two patterns of punctate white matter lesions were identified: one with loss of signal on SWI in a linear appearance, and the other with DWI lesions with restricted diffusion in a cluster appearance. These different patterns are suggestive of a difference in underlying pathophysiology. To reliably classify PWML in the preterm infant in either pattern, an early MRI with DWI and SWI sequences is required.     

The second respiratory chain of Candida parapsilosis: a comprehensive study

The yeast C. parapsilosis CBS7157 is strictly dependent on oxidative metabolism for growth since it lacks a fermentative pathway. It is nevertheless able to grow on high glucose concentrations and also on a glycerol medium supplemented with antimycin A or drugs acting at the level of mitochondrial protein synthesis. Besides its normal respiratory chain C. parapsilosis develops a second electron transfer chain antimycin A-insensitive which allows the oxidation of cytoplasmic NAD(P)H resulting from glycolytic and hexose monophosphate pathways functioning through a route different from the NADH-coenzyme Q oxidoreductase described in S. cerevisiae or from the alternative pathways described in numerous plants and microorganisms. The second respiratory chain of C. parapsilosis involves 2 dehydrogenases specific for NADH and NADPH respectively, which are amytal and mersalyl sensitive and located on the outer face of the inner membrane. Since this antimycin A-insensitive pathway is fully inhibited by myxothiazol, it was hypothesized that electrons are transferred to a quinone pool that is different from the classical coenzyme Q-cytochrome b cycle. Two inhibitory sites were evidenced with myxothiazol, one related to the classical pathway, the other to the second pathway and thus, the second quinone pool could bind to a Q-binding protein at a specific site. Elimination of this second pool leads to a fully antimycin A-sensitive NADH oxidation, whereas its reincorporation in mitochondria allows recovery of an antimycin A-insensitive, myxothiazol sensitive NADH oxidation. The third step in this second respiratory chain involves a specific pool of cytochrome c which can deliver electrons either to a third phosphorylation site or to an alternative oxidase, cytochrome 590. This cytochrome is inhibited by high cyanide concentrations and salicylhydroxamates.