Growth and Detachment of Cell Clusters from Mature Mixed-Species Biofilms

Detachment from biofilms is an important consideration in the dissemination of infection and the contamination of industrial systems but is the least-studied biofilm process. By using digital time-lapse microscopy and biofilm flow cells, we visualized localized growth and detachment of discrete cell clusters in mature mixed-species biofilms growing under steady conditions in turbulent flow in situ. The detaching biomass ranged from single cells to an aggregate with a diameter of approximately 500 μm. Direct evidence of local cell cluster detachment from the biofilms was supported by microscopic examination of filtered effluent. Single cells and small clusters detached more frequently, but larger aggregates contained a disproportionately high fraction of total detached biomass. These results have significance in the establishment of an infectious dose and public health risk assessment.     

Naked Amoebae in Biofilms Collected from a Temperate Freshwater Pond

Biofilms collected on Plexiglass substrates, from a freshwater pond in northern New York State, were examined microscopically for naked amoebae densities, sizes, diversity, and estimated C‐biomass. Five samples were obtained during summer 2006 and 2007. The densities ranged from 109 to 136/cm² biofilm surface and 285 to 550/mg biofilm dry weight. Sizes ranged from 13 to 200 μm. Diversities ranged from 4.23 to 4.55. C‐biomass ranged from 64 to 543 ng C/cm² and 125 to 1,700 μg C/g dry weight. Thirty morphospecies were identified among the five samples, including very large amoebae in the range of 100–200 μm. Large amoebae (≥ 50 μm) accounted for the largest proportion of the C‐biomass.     

Formation of In Vitro Mixed-Species Biofilms by Lactobacillus pentosus and Yeasts Isolated from Spanish-Style Green Table Olive Fermentations

The present work details the in vitro interactions between Lactobacillus pentosus and yeast strains isolated from table olive processing to form mixed biofilms. Among the different pairs assayed, the strongest biofilms were obtained from L. pentosus and Candida boidinii strain cocultures. However, biofilm formation was inhibited in the presence of d-(+)-mannose. In addition, biofilm formation by C. boidinii monoculture was stimulated in the absence of cell-cell contact with L. pentosus. Scanning electron microscopy revealed that a sort of “sticky” material formed by the yeasts contributed to substrate adherence. Hence, the data obtained in this work suggest that yeast-lactobacilli biofilms may be favored by the presence of a specific mate of yeast and L. pentosus, and that more than one mechanism might be implicated in the biofilm formation. This knowledge will help in the design of appropriate mixed starter cultures of L. pentosus-yeast species pairs that are able to improve the quality and safety of Spanish-style green table olive processing.     

Naturally Saline Boreal Communities as Models for Reclamation of Saline Oil Sand Tailings

Reclaimed landscapes after oil sands mining have saline soils; yet, they are required to have similar biodiversity and productivity as the predisturbance nonsaline landscape. Given that many species in the boreal forest are not tolerant of salinity, we studied the effects of soil salinity on plant communities in natural saline landscapes to understand potential plant responses during the reclamation process. Vegetation–soil relationships were measured along transects from flooded wetlands to upland forest vegetation in strongly saline, slightly saline, nonsaline, and reclaimed boreal landscapes. In strongly saline landscapes, surface soil salinity was high (>10 dS/m) in flooded, wet‐meadow, and dry‐meadow vegetation zones as compared to slightly saline (<5 dS/m) and nonsaline (<2 dS/m) landscapes. Plant communities in these vegetation zones were quite different from nonsaline boreal landscapes and were dominated by halophytes common to saline habitats of the Great Plains. In the shrub and forest vegetation zones, surface soil salinity was similar between saline and nonsaline landscapes, resulting in similar plant communities. In strongly saline landscapes, soils remained saline at depth through the shrub and forest vegetation zones (>10 dS/m), suggesting that forest vegetation can establish over saline soils as long as the salts are below the rooting zone. The reclaimed landscape was intermediate between slightly saline and nonsaline landscapes in terms of soil salinity but more similar to nonsaline habitats with respect to species composition. Results from this study suggest it may be unrealistic to expect that plant communities similar to those found on the predisturbance landscape can be established on all reclaimed landscapes after oil sands mining.     

Bioleaching of Heavy Metals from Mine Tailings by Aspergillus fumigatus

The bioleaching experiment was conducted for the removal of heavy metals from mine tailings. A fungal strain was isolated from the gold mine tailings and it has been identified as Aspergillus fumigatus based on its 18S rDNA analysis. Bioleaching using A. fumigatus was carried out in bioleaching step processes (one-step and two-step) at various tailings concentrations (1%, 2%, 4%, and 8% [w/v]). In the one-step bioleaching process where fungi were cultivated in the presence of the tailings, concentration of oxalic acid was the highest among the organic acids produced. On the other hand, in the two-step bioleaching process where the metabolic products of fungal growth, which have been separated from its biomass, were used, citric acid was dominant. In the one-step process, the highest As (62%), Fe (58%), Mn (100%), and Zn (54%) removals were observed at the lowest tailings concentration (1%). The removal of Pb at 1% tailings concentration in the one-step process was 56%, whereas 88% removal was achieved in the two-step process where citric acid was dominant. In general, heavy metals removal efficiency decreased with increased tailings of the concentration in both bioleaching processes. This study shows the possibility of using A. fumigatus to bioleach hazardous heavy meals from gold mine tailings.     

Extraction of Arsenic and Heavy Metals from Contaminated Mine Tailings by Soil Washing

In this study, the soil washing technique has been used to treat mine tailings contaminated heavily with arsenic and heavy metals at Jingok mine, which is one of the abandoned mines in Korea. The results showed that phosphoric acid, citric acid, oxalic acid, and sodium metabisulfite were highly effective in extracting arsenic and heavy metals. Among them, oxalic acid was the most effective (especially for Pb, Cu, and Zn), as even a residual fraction of arsenic was partly extracted. The optimum concentration of washing reagent and the ratio between the mine tailings and washing reagent were found to be 0.5 M and 1:20, respectively. In addition, the extraction kinetics of arsenic and heavy metals was fast, in which the reaction time of 30 minutes was deemed to be a sufficient contact time. From the results, it may be concluded that the low pH of washing solution and the amount of dissolved Fe may be considered as the most important factors in the extraction of arsenic and heavy metals.     

Bioaccumulation of Metals in Cultured Carp (Cyprinus carpio) from Lake Chapala,Mexico

Biological Trace Element Research - Fluoride accumulates and is toxic to bones. Clinical bone lesions occur in a phased manner, being less severe early in the natural course of skeletal fluorosis....     

Development of an mlrA Gene-Directed TaqMan PCR Assay for Quantitative Assessment of Microcystin-Degrading Bacteria within Water Treatment Plant Sand Filter Biofilms

We report for the first time a quantitative mlrA gene-directed TaqMan PCR assay for the rapid detection of microcystin-degrading bacteria. This was applied, in combination with 16S ribosomal DNA-directed quantitative PCR and denaturing gradient gel electrophoresis, to study virgin sand filter column biofilm development and to correlate mlrA gene abundance with microcystin removal efficiency.It has been predicted that the changing climatic conditions around the world are likely to increase both the occurrence and the intensity of blue-green algal (cyanobacterial) blooms (16). Of particular concern to the water industry are the blooms of Microcystis, Anabaena, Nostoc, and Planktothrix species, which are capable of producing microcystin toxins within surface water storages used for potable water supply (4, 5, 12). In dissolved (extracellular) form, microcystins are not efficiently removed by conventional water treatment processes (6), and more advanced treatment options, such as activated carbon application or ozonation, are usually employed. However, these are expensive alternatives, and removal efficiencies are often compromised by the presence of natural organic matter (15). Biological filtration of microcystins is now recognized as an alternative treatment barrier (1, 7, 8, 14) and is favored by water utilities, as the process is generally low technology, chemical free, and requires little maintenance, where retrofitting of the process into existing water treatment plant (WTP) infrastructure is often feasible.To date, 10 different microcystin-degrading bacteria have been isolated from rivers, lakes, and biofilters (7), and the mlr gene cluster has been demonstrated to encode proteins involved in the initial steps of microcystin biodegradation by such organisms (2, 3). The MlrA protein is responsible for the initial hydrolytic cleavage of the cyclic microcystin structure, and conventional mlrA gene-directed PCR has been employed for qualitative detection of microcystin-degrading bacteria from lakes (18) and within the biofilm of biofilters (1, 7, 8). However, these conventional PCR assays do not allow for accurate quantitation of mlrA gene abundance and have not been designed with degenerate primer sequences to allow for variations that exist between different mlrA homologues. In this study, we report for the first time a quantitative mlrA gene-directed TaqMan PCR assay, including degenerate oligonucleotides targeting conserved DNA regions, for the rapid detection of microcystin-degrading bacteria.Using all available mlrA nucleotide sequences to date (GenBank accession numbers {"type":"entrez-nucleotide","attrs":{"text":"DQ112243","term_id":"70987104","term_text":"DQ112243"}}DQ112243, {"type":"entrez-nucleotide","attrs":{"text":"AF411068","term_id":"345808052","term_text":"AF411068"}}AF411068, {"type":"entrez-nucleotide","attrs":{"text":"AB114203","term_id":"34392799","term_text":"AB114203"}}AB114203, {"type":"entrez-nucleotide","attrs":{"text":"AB161685","term_id":"42415302","term_text":"AB161685"}}AB161685, and {"type":"entrez-nucleotide","attrs":{"text":"AB114202","term_id":"34392797","term_text":"AB114202"}}AB114202), primers qmlrAf (5′-AGCCCKGGCCCRCTGC-3′) and qmlrAr (5′-ATGCCARGCCCACCACAT-3′) and the TaqMan probe qmlrA-tm, which was labeled with 6-carboxyfluorescein (FAM) at the 5′ end and labeled with black hole quencher 1 (BHQ1) at the 3′ end (5′-FAM-TGCCSCAGCTSCTCAAGAAGTTTG-BHQ1-3′), were designed to target highly conserved regions of the mlrA gene for quantitative TaqMan PCR. Reactions resulted in the amplification of a 120-bp product and were carried out in quadruplicate on a Rotor Gene 6000 (Corbett Research, New South Wales, Australia) thermal cycling system. Each 25-μl reaction mixture contained 200 μM of each deoxynucleoside triphosphate, 2.0 mM of MgCl₂, 1× PCR buffer, 0.5 μM of primers qmlrAf and qmlrAr, 0.25 μM of TaqMan probe qmlrA-tm, 400 μg/ml bovine serum albumin, 0.5 U of Platinum Taq DNA polymerase (Invitrogen, Carlsbad, CA), and 2.5 μl of either a DNA standard or sample template. Thermal cycling conditions consisted of an initial denaturation at 95°C for 2 min followed by 45 cycles of denaturation at 95°C for 5 s and annealing/extension at 62°C for 25 s. Data were collected in the FAM channel (gain set to 6) at the end of each annealing/extension step.Quantitation was performed using DNA standard curves constructed from a serial dilution, in Milli-Q water, of an 807-bp mlrA gene fragment (18) from the two microcystin-degrading bacteria Sphingomonas sp. strain ACM-3962 (2) and Sphingopyxis sp. strain LH21 (7). Results were linear over the range of 1 × 10¹ to 1 × 10⁹ mlrA gene copies/μl with linear coefficient values (R²) for ACM-3962 and LH21 of 0.999 and 0.998 and reaction efficiencies of 0.97 and 0.99, respectively. Specificity of the mlrA TaqMan assay was verified by the absence of signals for genomic DNA from the non-microcystin-degrading bacteria Aeromonas hydrophila (ATCC 7966), Bacillus subtilis (ATCC 10145), Escherichia coli (ATCC 11775), Pseudomonas aeruginosa (ATCC 10145), and Staphylococcus epidermidis (ATCC 12228).Determination of the DNA extraction efficiency, linear range, and the limit of detection of the TaqMan PCR assay under environmentally relevant conditions was achieved by the spiking of known amounts of Sphingopyxis sp. strain LH21 into Myponga reservoir water and Morgan WTP filter sand (containing a biofilm) in log₁₀-fold increments ranging from 0 to 1 × 10⁸ cells/ml or g, respectively. DNA from each water (0.5 ml) and sand preparation (0.5g) was then extracted in triplicate using the UltraClean soil DNA isolation kit (MoBio Laboratories Inc., Carlsbad, CA). For reservoir water, the TaqMan assay was linear over 6 log₁₀ orders of magnitude, with an R² of 0.998 and a limit of detection equivalent to 10² mlrA copies/ml. For sand filter medium, the TaqMan assay was linear over 5 log₁₀ orders of magnitude, with an R² equivalent to 0.998 and a limit of detection equivalent to 10³ mlrA copies/g. These results are comparable to other quantitative PCR (qPCR) data cited in the literature, where the detection limit was 2 × 10² cells/ml for Methylocystis sp. from water (13) and approximately 10³ cells/g for Escherichia coli O157:H7 (11) and Rhodococcus sp. (17) cells from soil.The mlrA TaqMan assay was then applied, in conjunction with 16S rDNA qPCR (10) and 16S rDNA PCR-denaturing gradient gel electrophoresis (DGGE) cluster analysis (9), to investigate the attachment and subsequent biofilm formation upon virgin sand particles within a laboratory scale sand filter and also to investigate the previously unknown relationship between mlrA gene copy abundance and microcystin removal through biofiltration processes. Virgin sand (effective particle size, 1.25 mm; uniformity coefficient, 1.4; particle density, 1.62 g/cm³) (Riversands Pty Ltd, Carbrook, Queensland, Australia) was washed and sterilized by autoclaving. Sand was then packed into a glass column (length, 30 cm; internal diameter, 2.5 cm) at a bed height of 15 cm, and the column was continually fed with Myponga reservoir water (empty bed contact time, 15 min; UV₂₅₄/cm, 0.412; dissolved organic carbon, 11.8 mg/liter; specific UV absorbance, 3.5 liters/mg-m; and pH, 6.7; South Australia) and spiked daily with microcystin-LR at a target concentration of 5 μg/liter. Full details of the sand column apparatus are described by Ho et al. (8). Influent and effluent water samples were taken at regular intervals for microcystin-LR determination by high-performance liquid chromatography (8). Sand samples (0.5 g) were sampled in triplicate from the top surface of the sand bed, and the biofilm DNA samples were extracted using the UltraClean soil DNA isolation kit (Mo Bio).As shown in Fig. ​Fig.1a,1a, bacterial biofilm development (determined by 16S rDNA qPCR) appeared to begin within the first 24 h of start-up, and following day 3, the 16S bacterial abundance remained steady at approximately 1 × 10⁸ copies/g. Despite the limited change in total bacterial abundance following day 3, 16S rDNA PCR-DGGE cluster analysis revealed a dynamic shift in the overall bacterial community composition throughout the 18-day study (Fig. ​(Fig.2).2). There was also a 3-day period before removals of microcystin were observed through the column (Fig. ​(Fig.1b).1b). At day 4, there was a 12.6% removal of microcystin, although the abundance of mlrA-containing bacteria on day 4 remained below the limit of detection of the TaqMan assay (Fig. ​(Fig.1b).1b). By day 7, the removal of microcystin had increased to 30.8% and was accompanied by the detection of 1.17 × 10³ mlrA gene copies/g of sand. For the remainder of the study, there was a close association between the trend of microcystin removal and the abundance of mlrA gene copies/g of sand (Fig. ​(Fig.1b).1b). These data suggested that the efficiency of microcystin removals by biofiltration processes was directly related to the abundance of microcystin-degrading bacteria within the sand filter biofilm. Within biofilters that have had no preexposure to microcystins, lag periods have been reported to be as short as 2 to 4 days (1, 8), but they have been reported to be up to 211 days for a filter containing virgin sand (19). In most cases, 100% removal of microcystin through biofilters usually occurs rapidly within several days following the lag period (8, 19), although complete removal of microcystin in this study was achieved 15 days following the 3-day lag period (Fig. ​(Fig.1b1b).Open in a separate windowFIG. 1.(a) Abundance of 16S rDNA (closed circles) and mlrA gene copies (closed squares) within the sand filter column biofilm. (b) Abundance of mlrA gene copies (closed squares) within the sand filter column biofilm and the percentage of microcystin-LR removed (open triangles) through the column. Error bars represent standard deviations of the results for triplicate analyses.Open in a separate windowFIG. 2.Cluster analysis of DGGE band profiles for the sand filter column study. Scale bar represents two band differences.A clone library was then constructed to investigate the diversity of mlrA homologues detected by the mlrA TaqMan PCR assay within the sand filter column on day 18. Following sequence analysis of 50 cloned DNA fragments, three unique mlrA gene sequences, MC-A, MC-B, and MC-C, were obtained. Each sequence had greater than 98% similarity to previously described mlrA gene sequences, where MC-A, MC-B, and MC-C represented 22, 72, and 6% of the clone library, respectively.In summary, this study has demonstrated the development of an mlrA gene-directed TaqMan PCR assay for the assessment of microcystin-degrading bacteria within biologically active sand filter biofilm. The data presented here revealed that during the early phase of operation of a sand filter column, the removal of microcystin was directly related to the abundance of microcystin-degrading bacteria within the biofilm, although removals began only once an adequate number of microcystin-degrading bacteria were established. It is envisaged that the TaqMan PCR assay will be valuable for WTP operators who wish to investigate the abundance of microcystin-degrading bacteria within their biofilters and, in turn, assess the capacity of the biofilters for removing microcystin toxins—especially as such episodes are particularly transient in nature.     

Mobility and Translocation of Heavy Metals from Mine Tailings in Three Plant Species after Amendment with Compost and Biosurfactant

An experiment was performed to determine the effects of mine tailings alone mixed with compost or with compost plus crude biosurfactant on the accumulation of heavy metals (Pb, Zn, Cu, Cr, Cd, and Ni) in Acacia retinodes, Nicotiana glauca, and Echinochloa polystachya. The plants were grown in soil, mine tailings, and mine tailings containing compost over a period of seven and five months for shrubs or grass, respectively. The plants Acacia retinodes and Nicotiana glauca grown in mine tailings containing compost showed increases in dry biomass (from 62 to 79%) compared with plants in only tailings. Heavy metals accumulated in the roots and leaves showed high translocation rates of Cr in N. glauca, Cd in A. retinodes, and Ni in E. polystachya. Concentrations of heavy metals in the three plants irrigated with crude biosurfactant were not significantly different from those irrigated with water. Zn and Cd fractions within mine tailings containing compost were bound to carbonate, Pb was bound to residues, and Cu was bound to Fe-oxides. Cd had the highest mobility factor followed in order by Zn, Pb, and Cu. The elevated concentrations of Pb in roots and the low translocation rate for N. glauca and A. retinodes indicate that they are suitable for phytostabilizing Pb and Zn.     

Bioprocessing-Based Approach for Bitumen/Water/Fines Separation and Hydrocarbon Recovery from Oil Sands Tailings

Oil sands are a major source of oil, but their industrial processing generates tailings ponds that are an environmental hazard. The main concerns are mature fine tailings (MFT) composed of residual hydrocarbons, water, and fine clay. Tailings ponds include toxic contaminants such as heavy metals, and toxic organics including naphthenics. Naphthenic acids and polyaromatic hydrocarbons (PAHs) degrade very slowly and pose a long-term threat to surface and groundwater, as they can be transported in the MFT. Research into improved technologies that would enable densification and settling of the suspended particles is ongoing. In batch tests, BioTiger?, a microbial consortium that can metabolize PAHs, demonstrated improved oil sands tailings settling from a Canadian tailings pond. Results also showed, depending on the timing of the measurements, lower suspended solids and turbidity. Elevated total organic carbon was observed in the first 48 hours in the BioTiger?-treated columns and then decreased in overlying water. Oil sands tailings mixed with BioTiger? showed a two-fold reduction in suspended solids within 24 hours as compared to abiotic controls. The tailings treated with BioTiger? increased in microbial densities three orders of magnitude from 8.5 × 105 CFU/mL to 1.2 × 108 CFU/mL without any other carbon or energy source added, indicating metabolism of hydrocarbons and other available nutrients. Results demonstrated that bioaugmentation of BioTiger? increased separation of organic carbon from particles in oil sands and enhanced settling with tailings with improved water quality. Journal style is for Abstract to be less than 200 words, and contain no citations to other sources; please edit as needed     

Bioremediation of Vegetable Oil and Grease from Polluted Wastewater Using a Sand Biofilm System

Pseudomonas sp. (L1), P. diminuta(L2) were among eight bacterial strains isolated from vegetable grease and oil-contaminated industrial wastewater, four of which only were found to have the ability to degrade oil and grease. They were identified and investigated for oil and grease degradation either individually or in combinations in previous unpublished work by the authors. Since the combination M1 (Pseudomonas sp. andP. diminuta) produced the highest degradative activity, it was used in the present study in a biofilm sand filter system for vegetable oil and grease removal. This system was tested either as one unit or two units in sequence where different flow rates (30, 50, 100 ml/h) were applied compared to a control unit(s). Results showed that both biofilm systems reduced oily wastewater, even in cases of high degree of pollution (fat, oil & grease (FOG), 7535 ppm; biochemical oxygen demand (BOD₅), 525 ppm; chemical oxygen demand (COD), 1660 ppm). Results also showed a removal of FOG with efficiency at 100%; BOD₅ at 95.9% and COD at 96%, at 50 ml/h flow rate using one unit of biofilm system. On using two units in sequence, a complete removal of FOG, BOD₅ and COD with efficiency 100%, at flow rate 100 ml/h was achieved. In conclusion, the previous biofilm results indicated the efficiency of such a system in treating oily polluted wastewater (vegetable oil origin) on the basis of bacterial isolates being used, the optimum flow rate, and the number of biofilm units used in sequence to obtain the highest removal capacity of such a system. This revised version was published online in July 2006 with corrections to the Cover Date.     

Cyclothiazide Unmasks an AMPA-Evoked Release of Arachidonic Acid from Cultured Striatal Neurones

Abstract: The joint, but not independent, activation of α-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) and metabotropic glutamate receptors induces liberation of arachidonic acid from cultured mouse striatal neurones. We examined whether blocking AMPA receptor desensitisation with cyclothiazide would modify this response. Cyclothiazide strongly potentiated the combined AMPA/(1 S ,3 R )-1-aminocyclopentane-1,3-dicarboxylic acid (ACPD)-evoked release of arachidonic acid (EC₅₀ of ∼7 µ M ) but did not modulate the basal, ACPD, or NMDA response. The enhanced liberation of arachidonic acid, observed in the presence of cyclothiazide, was due to the appearance of a genuine AMPA response that was independent of an associative activation of metabotropic receptors. The potentiated and nonpotentiated AMPA responses were inhibited by both competitive [2,3-dihydroxy-6-nitro-7-sulphamoylbenzo( f )quinoxaline] and 2,3-benzodiazepine noncompetitive (GYKI 53655 and GYKI 52466) receptor antagonists. Cyclothiazide was equally effective at potentiating the AMPA response in either the presence or absence of glucose, suggesting that the increased glutamate-evoked arachidonic acid release observed in these cells under conditions of glucose deprivation is not due to reduced AMPA receptor desensitisation. The enhanced liberation of arachidonic acid measured in the presence of cyclothiazide appeared to result from a large (fourfold) elevation of the AMPA-induced increase in intracellular calcium level. Therefore, an AMPA-evoked mobilisation of arachidonic acid could potentially contribute to non-NMDA receptor-mediated neurotoxicity, which has been observed in neuronal cells in the presence of cyclothiazide.     

Metals in Tortula muralis from sandstone buildings in an urban agglomeration

Acrocarpous mosses present on stonewalls in urban areas may be used for monitoring purposes. Therefore for this investigation we selected Tortula muralis expecting that this species with an ability to colonise buildings in polluted agglomerations where other species have vanished may provide important information to monitor environmental quality. Concentrations of Cd, Co, Cr, Cu, Fe, Mn, Ni, Pb, V and Zn in T. muralis, in dust deposited on sandstone surfaces, and in the sandstones on which the moss grew were measured. T. muralis appeared to be a good bioindicator of airborne pollution which accumulated especially extremely high Cu, Fe and Zn concentrations reaching values much higher than harmful for plants. There was no relation between metal concentrations in underlying sandstones and T. ruralis. There was a relation between metal concentrations in dust and T. muralis. Cd, Co, Cr, Mn and V concentrations in T. muralis were higher than in dust. The Self-Organizing Feature Map identifying groups of sampling sites with similar concentrations of metals in mosses was able to classify the pollution level by distinguishing groups of highly, medium and less polluted sites. Once trained, SOFM can be applied in ecological investigations and could form a future basis for recognizing e.g. the type of pollution in urban environments by analysing the concentrations of elements in T. muralis.     

The aminopeptidase from Aeromonas proteolytica can function as an esterase

The aminopeptidase from Aeromonas proteolytica (AAP) can catalyze the hydrolysis of L-leucine ethyl ester ( L-Leu-OEt) with a rate of 96 +/- 5 s-1 and a Km of 700 microM. The observed turnover number for L-Leu-OEt hydrolysis by AAP is similar to that observed for peptide hydrolysis, which is 67 +/- 5 s-1. The k(cat) values for the hydrolysis of L-Leu-OEt and L-leucine- p-nitroanilide ( L- pNA) catalyzed by AAP were determined at different pH values under saturating substrate concentrations. Construction of an Arrhenius plot from the temperature dependence of AAP-catalyzed ester hydrolysis indicates that the rate-limiting step does not change as a function of temperature and is product formation. The activation energy ( Ea) for the activated ES ester complex is 13.7 kJ mol-1, while the enthalpy and entropy of activation at 25 degrees C calculated over the temperature range 298-338 K are 11.2 kJ mol-1 and -175 J K-1 mol-1, respectively. The free energy of activation at 25 degrees C was found to be 63.4 kJ mol-1. The enthalpy of ionization was also measured and was found to be very similar for both peptide and ester substrates, yielding values of 20 kJ mol-1 for L-Leu-OEt and 25 kJ mol-1 for L- pNA. For peptide and L-amino acid ester cleavage reactions catalyzed by AAP, and 6.07, respectively. Proton inventory data suggest that two protons are transferred in the rate-limiting step of ester hydrolysis while only one is transferred in peptide hydrolysis. The combination of these data with the available X-ray crystallographic, kinetic, spectroscopic, and thermodynamic data for AAP provides new insight into the catalytic mechanism of AAP.     

How bacteria can block xenophagy: an insight from Salmonella

ABSTRACT

Xenophagy, a unique type of selective macroautophagy/autophagy, targets invading pathogens as part of the host immune response. In order to survive within the host, bacteria have established various self-defense mechanisms. In a recent paper from Feng Shao’s lab, the Salmonella effector protein SopF has been demonstrated to block xenophagy by interrupting the vacuolar type H⁺-translocating (v-) ATPase-ATG16L1 axis, which is important for antibacterial autophagy initiation. SopF can specifically ADP-ribosylate Gln124 on ATP6V0C, a v-ATPase component, thus influencing recruitment of ATG16L1 onto the bacteria-containing vacuole within the host cytosol.

Abbreviations: ATG: autophagy-related; S. Typhimurium: Salmonella enterica serovar Typhimurium; T3SS: type III secretion system     

Assessment of Polycyclic Aromatic Hydrocarbons (PAH) and Heavy Metals in the Vicinity of an Oil Refinery in India

Petroleum products are one of the major sources of energy for industry and daily life. Growth of the petroleum industry and shipping of petroleum products has resulted in the pollution. Populations living in the vicinity of oil refinery waste sites may be at greater risk of potential exposure to polycyclic aromatic hydrocarbons (PAH) through inhalation, ingestion, and direct contact with contaminated media. PAH have often been found to coexist with environmental pollutants including heavy metals due to similar pollution sources. The levels and distribution patterns of Σ16 PAH (sum of the 16 PAH) and heavy metals (lead, copper, nickel, cobalt, and chromium) were determined in soil and sediment in the vicinity (5 km radius) of an oil refinery in India. Concentrations of Σ16 PAH in the soils and sediments were found to be 60.36 and 241.23 ppm, respectively. Higher amount of PAH in sediments as compared to soil is due to low water solubility of PAH, settled in the bottom of aquatic bodies. The levels of lead, copper, nickel, cobalt, and chromium (total) in soil were 12.52, 13.52, 18.78, 4.84, and 8.29 ppm, while the concentrations of these metals in sediments were 16.38, 47.88, 50.15, 7.07, and 13.25 ppm, respectively. Molecular diagnostics indices of PAH (Ratio of Phenanthrene/Anthracene, Fluranthene/Pyrene) calculated for soil and sediment samples indicate that the oil refinery environment is contaminated with PAH from petrogenic as well as pyrolytic origin and heavy vehicular traffic on the Agra- Delhi National highway. Sixteen PAH priority pollutants were detected in the United States in entire samples collected near oil refinery areas and concentrations of Σ16 PAH in soil was found to be 1.20 times higher than the threshold value for PAH in soil by ICRCL (Inter-Departmental Committee on the Redevelopment of Contaminated Land). This concentration could lead to disastrous consequences for the biotic and abiotic components of the ecosystem and may affect the soil quality, thus impairing plant growth and its bioaccumulation in food chain.     

Quartz Sand as an Adsorbent for Purification of Extracellular Glucose Oxidase from Penicillium funiculosum 46.1

A procedure for purification of extracellular glucose oxidase (GO, EC 1.1.3.4) from a filtrate of culture liquid (CLF) of the fungus Penicillium funiculosum 46.1 has been developed using alluvial quartz sand as an adsorbent. Modifying the sand by changing the charge and polarity did not lead to a significant increase in its adsorption capacity towards GO. The effectiveness of sand and aluminum oxide used as adsorbents for GO isolation from CLF has been compared. Glucose oxidase isolated from CLF by adsorption on sand exhibited a greater catalytic activity than enzyme preparations obtained by column chromatography on CLF. Glucose oxidase from P. funiculosum 46.1 was adsorbed on sand more effectively than on aluminum oxide. It is concluded that sand may be used for fractionation of partially purified GO.     

Mucor hiemalis endo-beta-N-acetylglucosaminidase can transglycosylate a bisecting hybrid-type oligosaccharide from an ovalbumin glycopeptide

We found that the recombinant endo-beta-N-acetylglucosaminidase of Mucor hiemalis (Endo-M) expressed in Candida boidinii had the transglycosylation activity of transferring a bisecting hybrid-type oligosaccharide from an ovalbumin glycopeptide to the acceptor (p-nitrophenyl 2-acetamido-2-deoxy-beta-D-glucopyranoside) in a good yield of 43%.