Putative P1B-type ATPase from the bacterium Achromobacter xylosoxidans A8 alters Pb2 +/Zn2 +/Cd2 +-resistance and accumulation in Saccharomyces cerevisiae

PbtA, a putative P_1B-type ATPase from the Gram-negative soil bacterium Achromobacter xylosoxidans A8 responsible for Pb^2 +/Zn^2 +/Cd^2 +-resistance in Escherichia coli, was heterologously expressed in Saccharomyces cerevisiae. When present in Zn^2 +- and Pb^2 +/Cd^2 +-hypersensitive S. cerevisiae strains CM137 and DTY168, respectively, PbtA was able to restore Zn^2 +- and Pb^2 +-resistant phenotype. At the same time, the increase of Pb, Zn, and Cd accumulation in yeast was observed. However, Cd^2 +-tolerance of the pbtA-bearing yeasts dramatically decreased. The PbtA-eGFP fusion protein was localized primarily in the tonoplast and also in the plasma membrane and the perinuclear region corresponding to the endoplasmic reticulum at later growth stages. This indicates that PbtA protein is successfully incorporated into membranes in yeasts. Since PbtA caused a substantial increase of Pb^2 +/Zn^2 +-resistance and accumulation in baker's yeast, we propose its further use for the genetic modification of suitable plant species in order to obtain an effective tool for the phytoremediation of sites polluted by toxic transition metals.     

Allelopathic effects of Microcystis aeruginosa on green algae and a diatom: Evidence from exudates addition and co-culturing

Allelopathic interactions among phytoplankton species are regarded as one of the important factors contributing to phytoplankton species competition and succession. The role and extent of allelopathic effects of blooming freshwater cyanobacteria on other phytoplankton species in eutrophied waters, however, are still unknown. We examined the allelopathic effect of Microcystis aeruginosa on two common green algae (Scenedesmus quadricauda, Chlorella pyrenoidosa) and a diatom (Cyclotella meneghiniana) by adding exudates from different growth phases and in co-culture tests. Exudates of M. aeruginosa from the exponential growth phase and the stationary phase significantly inhibited the growth of S. quadricauda, C. pyrenoidosa and C. meneghiniana, whereas those from the decline phase increased their growth. The presence of M. aeruginosa extremely inhibited the growth of all tested species in co-cultures within 24 h. Our results indicate that under the tested environmental conditions (25 °C, light 80 μmol quanta m⁻² s⁻¹, manual shaking twice a day), allelopathic effects of M. aeruginosa on other phytoplankton species can significantly contribute to their competitive success.     

An in vitro urinary tract catheter system to investigate biofilm development in catheter-associated urinary tract infections

Biofilm development in urinary tract catheters is an often underestimated problem. However, this form of infection leads to high mortality rates and causes significant costs in health care. Therefore, it is important to analyze these biofilms and establish avoiding strategies. In this study a continuous flow-through system for the cultivation of biofilms under catheter-associated urinary tract infection conditions was established and validated. The in vitro urinary tract catheter system implies the composition of urine (artificial urine medium), the mean volume of urine of adults (1 mL min^–1), the frequently used silicone catheter (foley silicon catheter) as well as the infection with uropathogenic microorganisms like Pseudomonas aeruginosa. Three clinical isolates from urine of catheterized patients were chosen due to their ability to form biofilms, their mobility and their cell surface hydrophobicity. As reference strain P. aeruginosa PA14 has been used. Characteristic parameters as biofilm thickness, specific biofilm growth rate and substrate consumption were observed. Biofilm thicknesses varied from 105 ± 16 μm up to 246 ± 67 μm for the different isolates. The specific biofilm growth rate could be determined with a non invasive optical biomass sensor. This sensor allows online monitoring of the biofilm growth in the progress of the cultivation.     

Availability of N and S affect nutrient acquisition efficiencies differently by Trifolium repens and Lolium perenne when grown in monoculture or in mixture

Sulphur (S) deficiency is recognized as a limiting factor for crop production in many regions in the world. In grasslands, S availability has been shown to alter the biomass production of Trifolium repens and Lolium perenne and their specific interactions. To establish the role of N and S availabilities on the competitive interaction for these minerals by T. repens and L. perenne when grown together, two S rates (0 and 30 kg S ha^?1) combined with three N rates (0, 50 and 180 kg N ha^?1) were investigated in a cut/regrowth experiment over a period of 4 months under glasshouse conditions. N was applied as ¹⁵NH₄ ¹⁵NO₃ to determine their actual N fertilizer recovery in the harvested fraction of the shoot. S yields were used to estimate their apparent S fertilizer recovery. At final harvest, N reserves of T. repens stolons were analyzed to estimate their implication in the regrowth process. In monoculture and in both cuts (1 and 2), N benefited both species by increasing their N and S yields. S benefited only T. repens. In mixture, at cut 1, L. perenne behaved as a better competitor than T. repens thanks to N, while at cut 2, T. repens dominated the community thanks to strong positive S effect. N recovery of L. perenne grown in mixture was greatly improved by S supply. For T. repens, S enhanced its ability to fix N₂ and improved the accumulation of soluble proteins in its stolons. It is clear that the N:S ratio of soil may affect the functionality of grassland plant communities and their structure. Results suggest that (i) the limitations in the availability of soil S could restrict leguminous species growth in high N soil conditions, and (ii) the modulation of S level could be used as a tool to modify the composition of grassland communities.     

Biodegradation of organochlorine pesticides by bacteria grown in microniches of the porous structure of green bean coffee

In this paper, the authors propose a model for DDT biodegradation by bacteria grown in microniches created in the porous structure of green bean coffee. Five bacteria isolated from coffee beans, identified as Pseudomonas aeruginosa, P. putida, Stenotrophomonas maltophilia, Flavimonas oryzihabitans, and Morganella morganii. P. aeruginosa and F. oryzihabitans, were selected for pesticide degradation. Bacteria were selected according to their ability to grow on mineral media amended with: (a) glucose (10 g l⁻¹), (b) peptone (2 g l⁻¹), and (c) ground coffee beans (2 g l⁻¹). These three media were supplemented with 50 mg l⁻¹ of 1,1,1-trichloro-2,2-bis (4-chlorophenyl) ethane (DDT) and endosulfan. GC/MS analysis demonstrated that the greatest DDT removal was obtained in the medium supplemented with coffee beans, where 1,1-dichloro-2,2′-bis (4-chlorophenyl)ethylene (DDE), 1-chloro-2,2-bis (4-chlorophenyl) ethane (DDMU) and 2,2′-bis (p-chlorophenyl)ethanol (DDOH) were detected. DDMU is a product of the reductive dechlorination of DDE, which in this system could be carried out under the anaerobic conditions in microniches present in the porous structure of the coffee bean. This was supported by scanning electron microscopy. Green bean coffee could be used as a nutrient source and as a support for bacterial growth in pesticide degradation.     

Esculentin-2CHa: A host-defense peptide with differential cytotoxicity against bacteria,erythrocytes and tumor cells

The host-defense peptide, esculentin-2CHa (GFSSIFRGVA¹⁰KFASKGLGK D²⁰LAKLGVDLVA³⁰ CKISKQC) shows potent (MIC ≤ 6 μM) growth inhibitory activity against clinical isolates of multidrug-resistant strains of Staphylococcus aureus, Acinetobacter baumannii, and Stenotrophomonas maltophilia and differential cytotoxic activity against human erythrocytes (LC₅₀ = 150 μM) and human non-small cell lung adenocarcinoma A549 cells (LC₅₀ = 10 μM). Esculentin-2CHa significantly (P < 0.01) stimulates the release of the anti-inflammatory cytokine IL-10 by mouse lymphoid cells and elevates its production after stimulation with concanavalin A and significantly (P < 0.05) stimulates TNF-α production by peritoneal macrophages. Effects on IL-6 and IL-1β production were not significant. Removal of the hydrophobic N-terminal hexapeptide (GFSSIF) from esculentin-2CHa results in abolition of growth inhibitory activity against S. aureus and cytotoxic activity against erythrocytes and A549 cells as well as a marked (≥16-fold) reduction in potency against A. baumannii and S. maltophilia. The primary structure of esculentin-2 has been poorly conserved between frog species but evolutionary pressure has acted to maintain the hydrophobic character of this N-terminal hexapeptide sequence. Removal of the cyclic C-terminal domain (CKISKQC) and replacement of the Cys³¹ and Cys³⁷ residues by serine resulted in appreciable decreases in cytotoxicity against all microorganisms and against mammalian cells. The more cationic [D20K, D27K] analog showed a modest increase in potency against all microorganisms (up to 4-fold) but a marked increase in cytotoxicity against erythrocytes (LC₅₀ = 11 μM) and A549 cells (LC₅₀ = 3 μM).     

Molecular characterization of Rifr mutations in Pseudomonas aeruginosa and Pseudomonas putida

The rpoB gene encoding for β subunit of RNA polymerase is a target of mutations leading to rifampicin resistant (Rif^r) phenotype of bacteria. Here we have characterized rpoB/Rif^r system in Pseudomonas aeruginosa and Pseudomonas putida as a test system for studying mutational processes. We found that in addition to the appearance of large colonies which were clearly visible on Rif selective plates already after 24 h of plating, small colonies grew up on these plates for 48 h. The time-dependent appearance of the mutant colonies onto selective plates was caused by different levels of Rif resistance of the mutants. The Rif^r clusters of the rpoB gene were sequenced and analyzed for 360 mutants of P. aeruginosa and for 167 mutants of P. putida. The spectrum of Rif^r mutations characterized for P. aeruginosa grown at 37 °C and that characterized for P. putida grown at 30 °C were dissimilar but the differences almost disappeared when the mutants of both strain were isolated at the same temperature, at 30 °C. The strong Rif^r phenotype of P. aeruginosa and P. putida was accompanied only with substitutions of these residues which belong to the putative Rif-binding pocket. Approximately 70% of P. aeruginosa mutants, which were isolated at 37 °C and expressed weak Rif^r phenotype, contained base substitutions in the N-terminal cluster of the rpoB gene. The differences in the spectra of mutations at 30 °C and 37 °C can be explained by temperature-sensitive growth of several mutants in the presence of rifampicin. Thus, our results imply that both the temperature for the growth of bacteria and the time for isolation of Rif^r mutants from selective plates are critical when the rpoB/Rif^r test system is employed for comparative studies of mutagenic processes in Pseudomonas species which are conventionally cultivated at different temperatures.     

Interspecific allelopathy in cyanobacteria: Cylindrospermopsin and Cylindrospermopsis raciborskii effect on the growth and metabolism of Microcystis aeruginosa

The biological role of cyanobacteria secondary metabolites is relatively unknown although several possible hypotheses have been discussed. In the following study the effect of cylindrospermopsin (CYN) and metabolites of non-CYN producing Cylindrospermopsis raciborskii strain on growth, alkaline phosphatase (ALP) activity and microcystin-LR (MC-LR) production in Microcystis aeruginosa was evaluated. Higher concentrations of CYN (10 and 50 μg L⁻¹) induced toxicity effects demonstrated by significant growth inhibition and M. aeruginosa cell necrosis. Lower concentrations of CYN (1 and 5 μg L⁻¹) slightly decreased growth rates but significantly up-regulated ALP activity. Moreover, under all studied CYN concentrations MC-LR production strongly decreased. Spent C. raciborskii medium mimicked the CYN action by inducing strong inhibition of M. aeruginosa growth and MC-LR production and through up-regulation of ALP activity. On the other hand, spent M. aeruginosa medium did not affect C. raciborskii growth and no alterations in ALP activity were observed. Co-culturing of these two species resulted in an increase of C. raciborskii contribution at the expense of M. aeruginosa. From the results we conclude that CYN can be involved in interspecific competition in cyanobacteria and that non-CYN producing C. raciborskii strains may produce a hitherto unknown bioactive compound(s) which can mimic CYN action.     

Biochemical and biophysical combined study of bicarinalin,an ant venom antimicrobial peptide

We have recently characterized bicarinalin as the most abundant peptide from the venom of the ant Tetramorium bicarinatum. This antimicrobial peptide is active against Staphylococcus and Enterobacteriaceae. To further investigate the antimicrobial properties of this cationic and cysteine-free peptide, we have studied its antibacterial, antifungal and antiparasitic activities on a large array of microorganisms. Bicarinalin was active against fifteen microorganisms with minimal inhibitory concentrations ranging from 2 and 25 μmol L⁻¹. Cronobacter sakazakii, Salmonella enterica, Candida albicans, Aspergilus niger and Saccharomyces cerevisiae were particularly susceptible to this novel antimicrobial peptide. Resistant strains of Staphylococcus aureus, Pseudomonas aeruginosa and C. albicans were as susceptible as the canonical strains. Interestingly, bicarinalin was also active against the parasite Leishmania infantum with a minimal inhibitory concentrations of 2 μmol L⁻¹. The bicarinalin pre-propeptide cDNA sequence has been determined using a combination of degenerated primers with RACE PCR strategy. Interestingly, the N-terminal domain of bicarinalin pre-propeptide exhibited sequence similarity with the pilosulin antimicrobial peptide family previously described in the Myrmecia venoms. Moreover, using SYTOX green uptake assay, we showed that, for all the tested microorganisms, bicarinalin acted through a membrane permeabilization mechanism. Two dimensional-NMR experiments showed that bicarinalin displayed a 10 residue-long α-helical structure flanked by two N- and C-terminal disordered regions. This partially amphipathic helix may explain the membrane permeabilization mechanism of bicarinalin observed in this study. Finally, therapeutic value of bicarinalin was highlighted by its low cytotoxicity against human lymphocytes at bactericidal concentrations and its long half-life in human serum which was around 15 h.     

Optimisation and scale-up of α-glucuronidase production by recombinant Saccharomyces cerevisiae in aerobic fed-batch culture with constant growth rate

α-Glucuronidase (EC 3.2.1.139) of family GH 115 from Scheffersomyces stipitis is a valuable enzyme for the modification of water-soluble xylan into insoluble biopolymers, due to its unique ability to act on polymeric xylans. The influence of growth rate on the production of α-glucuronidase by recombinant Saccharomyces cerevisiae MH1000pbk10D-glu in glucose-limited fed-batch culture was studied at 14 and 100 L scale. At and below the critical specific growth rate (μ_crit) of 0.12 h⁻¹ at 14 L scale, the biomass yield coefficient (Y_x/s) remained constant at 0.4 g g⁻¹ with no ethanol production, whereas ethanol yields relative to biomass (k_eth/x) of up to 0.54 g g⁻¹ and a steady decrease in Y_x/s were observed at μ > 0.12 h⁻¹. Production of α-glucuronidase was growth associated at a product yield (k_α-glu/x) of 0.45 mg g⁻¹, with the highest biomass (37.35 g/L) and α-glucuronidase (14.03 mg/L) concentrations, were recorded during fed-batch culture at or near to μ_crit. Scale-up with constant k_La from 14 to 100 L resulted in ethanol concentrations of up to 2.5 g/L at μ = 0.12 h⁻¹. At this scale, α-glucuronidase yield could be maximised at growth rates below μ_crit, to prevent localised high glucose concentration pockets at the feed entry zone that would induce oxido-reductive metabolism. This is the first report where recombinant production of α-glucuronidase (EC 3.2.1.139) by S. cerevisiae was optimised for application at pilot scale.     

Antibiotic resistance modulation by natural products obtained from Nasutitermes corniger (Motschulsky, 1855) and its nest

Insects and their products are included in the traditional pharmacopoeia of various ethnic groups worldwide. In the Brazilian semiarid region can be highlighted the use of the termite Nasutitermes corniger for the treatment of various diseases. This study evaluated the ethanol extract of N. corniger and its nest as an antimicrobial agent and as a modulator of bacterial resistance against multidrug strains. The Minimum Inhibitory Concentration (MIC) of the extract on Staphylococcus aureus and Escherichia coli by microdilution was determined, as well as MIC of antibiotics in the presence and absence of extract. Despite having no significant antimicrobial activity (MIC ⩾ 1000 μg mL⁻¹), the extract showed additive activity to the antibiotic efficacy, significantly reducing its MIC. These results suggest that N. corniger and its nest are promising natural products for use in antimicrobial therapy.     

Bioconversion of post harvest crop residues and cattle shed manure into value-added products using earthworm Eudrilus eugeniae Kinberg

The post-harvest residues of some local crops, e.g. wheat (Triticum aestivum), millets (Penniseum typhoides and Sorghum vulgare), and a pulse (Vigna radiata) were subjected to recycle through vermicomposting by using the epigeic earthworm Eudrilus eugeniae Kinberg, under laboratory conditions. The crop residues were amended with animal dung; and three types of vermibeds were prepared: (i) millet straw (S. vulgare + Pennisenum typhoides in equal quantity) + sheep manure (1:2 ratio) (MS), (ii) pulse bran (Vigna radiata) + wheat straw (Triticum aestivum) + cow dung (1:1:2 ratio) (PWC), and (iii) mixed crop residues (mixing of all types crop residues, used in this study) + cow dung in 1:1 ratio (MCR + CD). The fourth treatment was cattle shed manure (CSM). At the end, ready vermicompost showed lower organic C content and higher concentrations of other important plant nutrients. Organic C content decreased in the order: MCR + CD (27.6%) > PWC (22.8%) > CMS (22.6%) > MS (19.4%). The ready vermicompost obtained from MCR + CD vermibed showed the maximum increase (% of initial level) in content of total N (143.4%), available P (111.1%) and exchangeable K (100.0%). The end product showed reduction in C:N ration between the ranges of 60.7% (CSM) and 70.3% (MCR + CD), at the end. The composting earthworm E. eugeniae exhibited the highest values of biological parameters: maximum mean individual biomass (1261.25 ± 7.0 mg), biomass gain (955.84 ± 11.03 mg), growth rate (10.62 ± 0.10 mg wt. worm⁻¹ day⁻¹), cocoon numbers (87.67 ± 6.51), and reproduction rate (0.66 ± 0.01 cocoons worm⁻¹ day⁻¹) in CSM container, while MS vermibeds showed the lowest values of these parameters. During experimentation, the maximum mortality for E. eugeniae was recorded in MS (16.67 ± 7.63%) followed by CSM > PWC > MCR + CD. Results indicated that the C:N ratio of the substrate drastically influenced the growth parameters of E. eugeniae, and it showed the close relations with maximum individual biomass gain (R² = 0.96), individual growth rate (R² = 0.82), and reproduction rate (cocoon worm⁻¹ day⁻¹) (R² = 0.72), in different treatments. This study clearly indicates that vermicomposting of crop residues and cattle shed wastes can not only produce a value-added product (vermicomposting) but at the same time acts as best culture medium for large-scale production of earthworms.     

In vitro estimations of the rate and extent of ruminal digestion of starch-rich feed fractions compared to in vivo data

An experiment was conducted to study the rumen digestion characteristics of whole feeds (WF) and the neutral detergent fibre (aNDF) and neutral detergent soluble (NDS) fractions of a range of starch-rich feeds using an automated in vitro gas production (GP) technique. In addition, the ruminal digestibility values predicted from the GP data were compared to previously acquired in vivo data. Nine feeds with starch concentrations ranging from 389 to 712 g/kg dry matter and with known in vivo digestibilities were subjected to neutral detergent extraction. The GP for each WF and the corresponding aNDF fractions were measured in duplicate in buffered rumen fluid during 72 h on two occasions. The fermentation residues were collected and analyzed for aNDF concentration to estimate their true organic matter (OM) and NDF digestibility. The GP from the NDS fraction was calculated by subtracting the GP from the aNDF fraction from the GP of the WF. A three-pool Gompertz model was fitted to the GP profiles (R² = 0.99) and a two compartment, mechanistic and dynamic rumen model was used to predict the digestibility of the potentially digestible feed fraction and the effective digestion rate (k_d). The true OM and NDF digestibility determined for the WF ranged from 0.804 to 1.011 and from 0.362 to 1.107, respectively. The NDF digestibility determined for the aNDF fraction ranged from 0.410 to 0.985. The effective k_d values estimated using GP data varied from 0.118 to 0.282/h for the WF and from 0.123 to 0.301/h for the NDS fraction, and were less (P<0.05) for maize compared to small grains (SG) but did not differ between barley and wheat (P>0.05). The effective k_d values for the aNDF fraction ranged from 0.039 to 0.082/h and did not differ (P>0.05) either between maize and SG or between barley and wheat. The predicted ruminal NDS digestibility determined using GP data closely matched the in vivo data describing starch digestion (R² = 0.81). The effective k_d values for the WF were strongly related (R² = 0.94) to those for the NDS fractions. The results indicate that when measured with the GP technique, the differences in the digestion characteristics of maize and small grains are less than those previously reported in studies using the in situ method. It is concluded that the predicted NDS digestibility determined using GP data corresponded well to the in vivo starch digestibility. Our results also suggest that the first order digestion rates of NDS (starch) in starch-rich feeds can be accurately determined by incubating WF samples in the GP system and using the GP kinetic data in a dynamic, mechanistic rumen model.     

Crystal structure of 3-isopropylmalate dehydrogenase in complex with NAD+ and a designed inhibitor

Isopropylmalate dehydrogenase (IPMDH) is the third enzyme specific to leucine biosynthesis in microorganisms and plants, and catalyzes the oxidative decarboxylation of (2R,3S)-3-isopropylmalate to α-ketoisocaproate using NAD⁺ as an oxidizing agent. In this study, a thia-analogue of the substrate was designed and synthesized as an inhibitor for IPMDH. The analogue showed strong competitive inhibitory activity with K_i = 62 nM toward IPMDH derived from Thermus thermophilus. Moreover, the crystal structure of T. thermophilus IPMDH in a ternary complex with NAD⁺ and the inhibitor has been determined at 2.8 Å resolution. The inhibitor exists as a decarboxylated product with an enol/enolate form in the active site. The product interacts with Arg 94, Asn 102, Ser 259, Glu 270, and a water molecule hydrogen-bonding with Arg 132. All interactions between the product and the enzyme were observed in the position associated with keto-enol tautomerization. This result implies that the tautomerization step of the thia-analogue during the IPMDH reaction is involved in the inhibition.     

Ecosystem functions as indicators for heathland responses to nitrogen fertilisation

Anthropogenic deposition of reactive nitrogen (N) has increased during the 20th century, and is considered an important driver of shifts in ecosystem functions and biodiversity loss. The objective of the present study was to identify those ecosystem functions that best evidence a target ecosystem’s sensitivity to N deposition, taking coastal heathlands as an example. We conducted a three-year field experiment in heathlands of the island Fehmarn (Baltic Sea, North Germany), which currently are subject to a background deposition of 9 kg N ha⁻¹ yr⁻¹. We experimentally applied six levels of N fertilisation (application of 0, 2.5, 5, 10, 20, and 50 kg N ha⁻¹ yr⁻¹), and quantified the growth responses of different plant species of different life forms (dwarf shrubs, graminoids, bryophytes, lichens) as well as shifts in the C:N ratios of plant tissue and humus horizons. For an applicability of the experimental findings (in terms of heathland management and critical load assessment) fertilisation effects on response variables were visualised by calculating the treatment ‘effect sizes’. The current year’s shoot increment of the dominant dwarf shrub Calluna vulgaris proved to be the most sensitive indicator to N fertilisation. Shoot increment significantly responded to additions of ≥ 5 kg N ha⁻¹ yr⁻¹ already in the first year, whereas flower formation of Calluna vulgaris increased only in the high-N treatments. Similarly, tissue C:N ratios of vascular plants (Calluna vulgaris and the graminoids Carex arenaria and Festuca ovina agg.) only decreased in the highest N treatments (50 and 20 kg N ha⁻¹ yr⁻¹, respectively). In contrast, tissue C:N ratios of cryptogams responded more quickly and sensitively than vascular plants. For example, Cladonia spp. tissue C:N ratios responded to N additions ≥ 5 kg N ha⁻¹ yr⁻¹ in the second study year. After three years we observed an increase in cover of graminoids and a corresponding decrease of cryptogams at N fertilisation rates of ≥ 10 kg N ha⁻¹ yr⁻¹. Soil C:N ratios proved to be an inappropriate indicator for N fertilisation at least within our three-year study period. Although current critical N loads for heathlands (10−20 kg N ha⁻¹ yr⁻¹) were confirmed in our experiment, the immediate and highly sensitive response of the current year’s shoots of Calluna vulgaris suggests that at least some ecosystem functions (e.g. dwarf shrub growth) also might respond to low (i.e. < 10 kg N ha⁻¹ yr⁻¹) but chronic inputs of N.     

Improved PCR performance and fidelity of double mutant Neq A523R/N540R DNA polymerase

We previously reported that Neq A523R DNA polymerase is more efficient in PCR than wild-type Neq DNA polymerase, and amplifies products more rapidly. Neq A523R DNA polymerase also amplifies templates more rapidly than Pfu DNA polymerase, but has a lower fidelity than Pfu DNA polymerase. To improve product yield and the fidelity of amplification simultaneously, we constructed and characterized the double mutant Neq A523R/N540R. The yield of PCR products was greater for Neq A523R/N540R DNA polymerase than wild-type and other mutant DNA polymerases, and the Neq double mutant catalyzed amplification of a 12-kb PCR product from a lambda template with an extension time of 3 min. The PCR error rate of Neq A523R/N540R DNA polymerase (6.3 × 10⁻⁵) was roughly similar to that of Pfu DNA polymerase (4.8 × 10⁻⁵), but much lower than those of wild-type Neq DNA polymerase (57.2 × 10⁻⁵), Neq A523R DNA polymerase (13.1 × 10⁻⁵), and Neq N540R DNA polymerase (37.7 × 10⁻⁵). These results indicated that A523R and N540R mutations of Neq DNA polymerase had synergistic effects on its fidelity.     

Degradation of long-chain n-alkanes (C36 and C40) by Pseudomonas aeruginosa strain WatG

Pseudomonas aeruginosa strain WatG was unable to utilize either n-hexatriacontane (C₃₆) or n-tetracontane (C₄₀), which are both insoluble in a mineral salts medium (MSM), as a sole carbon source. However, when C₃₆ and C₄₀ were added to MSM containing crude oil, more than 25% of each of the compounds was degraded by this strain after 2 weeks at 30 °C. These results demonstrate that P. aeruginosa strain WatG has the ability to degrade long-chain alkanes up to C₄₀, when they are solubilized by crude oil components.     

The effects of NH4+ and NO3− on growth,resource allocation and nitrogen uptake kinetics of Phragmites australis and Glyceria maxima

The effects of NH₄⁺ or NO₃⁻ on growth, resource allocation and nitrogen (N) uptake kinetics of two common helophytes Phragmites australis (Cav.) Trin. ex Steudel and Glyceria maxima (Hartm.) Holmb. were studied in semi steady-state hydroponic cultures. At a steady-state nitrogen availability of 34 μM the growth rate of Phragmites was not affected by the N form (mean RGR = 35.4 mg g⁻¹ d⁻¹), whereas the growth rate of Glyceria was 16% higher in NH₄⁺-N cultures than in NO₃⁻-N cultures (mean = 66.7 and 57.4 mg g⁻¹ d⁻¹ of NH₄⁺ and NO₃⁻ treated plants, respectively). Phragmites and Glyceria had higher S/R ratio in NH₄⁺ cultures than in NO₃⁻ cultures, 123.5 and 129.7%, respectively.Species differed in the nitrogen utilisation. In Glyceria, the relative tissue N content was higher than in Phragmites and was increased in NH₄⁺ treated plants by 16%. The tissue NH₄⁺ concentration (mean = 1.6 μmol g fresh wt⁻¹) was not affected by N treatment, whereas NO₃⁻ contents were higher in NO₃⁻ (mean = 1.5 μmol g fresh wt⁻¹) than in NH₄⁺ (mean = 0.4 μmol g fresh wt⁻¹) treated plants. In Phragmites, NH₄⁺ (mean = 1.6 μmol g fresh wt⁻¹) and NO₃⁻ (mean = 0.2 μmol g fresh wt⁻¹) contents were not affected by the N regime. Species did not differ in NH₄⁺ (mean = 56.5 μmol g⁻¹ root dry wt h⁻¹) and NO₃⁻ (mean = 34.5 μmol g⁻¹ root dry wt h⁻¹) maximum uptake rates (V_max), and V_max for NH₄⁺ uptake was not affected by N treatment. The uptake rate of NO₃⁻ was low in NH₄⁺ treated plants, and an induction phase for NO₃⁻ was observed in NH₄⁺ treated Phragmites but not in Glyceria. Phragmites had low K_m (mean = 4.5 μM) and high affinity (10.3 l g⁻¹ root dry wt h⁻¹) for both ions compared to Glyceria (K_m = 6.3 μM, affinity = 8.0 l g⁻¹ root dry wt h⁻¹). The results showed different plasticity of Phragmites and Glyceria toward N source. The positive response to NH₄⁺-N source may participates in the observed success of Glyceria at NH₄⁺ rich sites, although other factors have to be considered. Higher plasticity of Phragmites toward low nutrient availability may favour this species at oligotrophic sites.     

Uptake and inhibition kinetics of nitrogen in Microcystis aeruginosa: Results from cultures and field assemblages collected in the San Francisco Bay Delta,CA

The nitrogen (N) uptake kinetic parameters for Microcystis field assemblages collected from the San Francisco Bay Delta (Delta) in 2012 and non-toxic and toxic laboratory culture strains of M. aeruginosa were assessed. The ¹⁵N tracer technique was used to investigate uptake of ammonium (NH₄⁺), nitrate (NO₃⁻), urea and glutamic acid over short-term incubations (0.5–1 h), and to study inhibition of NO₃⁻, NH₄⁺ and urea uptake by NH₄⁺, NO₃⁻ and NH₄⁺, respectively. This study demonstrates that Delta Microcystis can utilize different forms of inorganic and organic N, with the greatest capacity for NH₄⁺ uptake and the least for glutamic acid uptake, although N uptake did not always follow the classic Michaelis–Menten hyperbolic relationship at substrate concentrations up to 67 μmol N L⁻¹. Current ambient N concentrations in the Delta may be at sub-saturating levels for N uptake, indicating that if N loading (especially NH₄⁺) were to increase, Delta Microcystis assemblages have the potential for increased N uptake rates. Delta Microcystis had the highest specific affinity, α, for NH₄⁺ and the lowest for NO₃⁻. In culture, N uptake by non-toxic and toxic M. aeruginosa strains was much higher than from the field, but followed similar N utilization trends to those in the field. Neither strain showed severe inhibition of NO₃⁻ uptake by NH₄⁺ or inhibition of NH₄⁺ uptake on NO₃⁻, but both strains showed some inhibition of urea uptake by NH₄⁺.     

Thermophilic α-glucan phosphorylase from Clostridium thermocellum: Cloning,characterization and enhanced thermostability

ORF Cthe0357 from the thermophilic bacterium Clostridium thermocellum ATCC 27405 that encodes a putative α-glucan phosphorylase (αGP) was cloned and expressed in Escherichia coli. The protein with a C-terminal His-tag was purified by Ni²⁺ affinity chromatography; the tag-free protein obtained from a cellulose-binding module–intein–αGP fusion protein was purified through affinity adsorption on amorphous cellulose followed by intein self-cleavage. Both purified enzymes had molecular weights of ca. 81,000 and similar specific activities. The optimal conditions were pH 6.0–6.5 and 60 °C for the synthesis direction and pH 7.0–7.5 and 80 °C for the degradation direction. This enzyme had broad substrate specificities for different chain length dextrins and soluble starch. The thermal inactivation of this enzyme strongly depended on temperature, protein concentration, and certain addictives that were shown previously to benefit the protein thermostability. The half lifetime of 0.05 mg αGP/mL at 50 °C was extended by 45-fold to 90 h through a combined addition of 0.1 mM Mg²⁺, 5 mM DTT, 1% NaCl, 0.1% Triton X-100, and 1 mg/mL BSA. The enzyme with prolonged stability would work as a building block for cell-free synthetic enzymatic pathway biotransformations, which can implement complicated biocatalysis through assembly of a number of enzymes and coenzymes.