Biochemical and biophysical characterisation yields insights into the mechanism of a Cd/Zn transporting ATPase purified from the hyperaccumulator plant Thlaspi caerulescens

TcHMA4 (GenBank no. AJ567384), a Cd/Zn transporting ATPase of the P(1B)-type (=CPx-type) was isolated and purified from roots of the Cd/Zn hyperaccumulator Thlaspi caerulescens. Optimisation of the purification protocol, based on binding of the natural C-terminal His-tag of the protein to a Ni-IDA metal affinity column, yielded pure, active TcHMA4 in quantities sufficient for its biochemical and biophysical characterisation with various techniques. TcHMA4 showed activity with Cu(2+), Zn(2+) and Cd(2+) under various concentrations (tested from 30nM to 10μM), and all three metal ions activated the ATPase at a concentration of 0.3μM. Notably, the enzyme worked best at rather high temperatures, with an activity optimum at 42°C. Arrhenius plots yielded interesting differences in activation energy. In the presence of zinc it remained constant (E(A)=38kJ?mol(-1)) over the whole concentration range while it increased from 17 to 42kJ?mol(-1) with rising copper concentration and decreased from 39 to 23kJ?mol(-1) with rising cadmium concentration. According to EXAFS the TcHMA4 appeared to bind Cd(2+) mainly by thiolate sulphur from cysteine, and not by imidazole nitrogen from histidine.     

Bacterial chitinase with phytopathogen control capacity from suppressive soil revealed by functional metagenomics

Plant disease caused by fungal pathogens results in vast crop damage globally. Microbial communities of soil that is suppressive to fungal crop disease provide a source for the identification of novel enzymes functioning as bioshields against plant pathogens. In this study, we targeted chitin-degrading enzymes of the uncultured bacterial community through a functional metagenomics approach, using a fosmid library of a suppressive soil metagenome. We identified a novel bacterial chitinase, Chi18H8, with antifungal activity against several important crop pathogens. Sequence analyses show that the chi18H8 gene encodes a 425-amino acid protein of 46 kDa with an N-terminal signal peptide, a catalytic domain with the conserved active site F¹⁷⁵DGIDIDWE¹⁸³, and a chitinase insertion domain. Chi18H8 was expressed (pGEX-6P-3 vector) in Escherichia coli and purified. Enzyme characterization shows that Chi18H8 has a prevalent chitobiosidase activity with a maximum activity at 35 °C at pH lower than 6, suggesting a role as exochitinase on native chitin. To our knowledge, Chi18H8 is the first chitinase isolated from a metagenome library obtained in pure form and which has the potential to be used as a candidate agent for controlling fungal crop diseases. Furthermore, Chi18H8 may also answer to the demand for novel chitin-degrading enzymes for a broad range of other industrial processes and medical purposes.     

An in vitro model of human neocortical development using pluripotent stem cells: cocaine-induced cytoarchitectural alterations

Neocortical development involves ordered specification of forebrain cortical progenitors to various neuronal subtypes, ultimately forming the layered cortical structure. Modeling of this process using human pluripotent stem cells (hPSCs) would enable mechanistic studies of human neocortical development, while providing new avenues for exploration of developmental neocortical abnormalities. Here, we show that preserving hPSCs aggregates – allowing embryoid body formation – while adding basic fibroblast growth factor (bFGF) during neuroepithelial development generates neural rosettes showing dorsal forebrain identity, including Mash1⁺ dorsal telencephalic GABAergic progenitors. Structures that mirrored the organization of the cerebral cortex formed after rosettes were seeded and cultured for 3 weeks in the presence of FGF18, BDNF and NT3. Neurons migrated along radial glia scaffolding, with deep-layer CTIP2⁺ cortical neurons appearing after 1 week and upper-layer SATB2⁺ cortical neurons forming during the second and third weeks. At the end of differentiation, these structures contained both glutamatergic and GABAergic neurons, with glutamatergic neurons being most abundant. Thus, this differentiation protocol generated an hPSC-based model that exhibits temporal patterning and a neuronal subtype ratio similar to that of the developing human neocortex. This model was used to examine the effects of cocaine during neocorticogenesis. Cocaine caused premature neuronal differentiation and enhanced neurogenesis of various cortical neuronal subtypes. These cocaine-induced changes were inhibited by the cytochrome P450 inhibitor cimetidine. This in vitro model enables mechanistic studies of neocorticogenesis, and can be used to examine the mechanisms through which cocaine alters the development of the human neocortex.KEY WORDS: Neocortical development, Dorsal forebrain model, hPSCs, Cocaine, Premature neuronal differentiation     

Development of Stable Vibrio cholerae O1 Hikojima Type Vaccine Strains Co–Expressing the Inaba and Ogawa Lipopolysaccharide Antigens

We describe here the development of stable classical and El Tor V. cholerae O1 strains of the Hikojima serotype that co–express the Inaba and Ogawa antigens of O1 lipopolysaccharide (LPS). Mutation of the wbeT gene reduced LPS perosamine methylation and thereby gave only partial transformation into Ogawa LPS on the cell surface. The strains express approximately equal amounts of Inaba– and Ogawa–LPS antigens which are preserved after formalin–inactivation of the bacteria. Oral immunizations of both inbred and outbred mice with formalin–inactivated whole–cell vaccine preparations of these strains elicited strong intestinal IgA anti–LPS as well as serum vibriocidal antibody responses against both Inaba and Ogawa that were fully comparable to the responses induced by the licensed Dukoral vaccine. Passive protection studies in infant mice showed that immune sera raised against either of the novel Hikojima vaccine strains protected baby mice against infection with virulent strains of both serotypes. This study illustrates the power of using genetic manipulation to improve the properties of bacteria strains for use in killed whole–cell vaccines.     

Clay minerals and metal oxides strongly influence the structure of alkane-degrading microbial communities during soil maturation

Clay minerals, charcoal and metal oxides are essential parts of the soil matrix and strongly influence the formation of biogeochemical interfaces in soil. We investigated the role of these parental materials for the development of functional microbial guilds using the example of alkane-degrading bacteria harbouring the alkane monooxygenase gene (alkB) in artificial mixtures composed of different minerals and charcoal, sterile manure and a microbial inoculum extracted from an agricultural soil. We followed changes in abundance and community structure of alkane-degrading microbial communities after 3 and 12 months of soil maturation and in response to a subsequent 2-week plant litter addition. During maturation we observed an overall increasing divergence in community composition. The impact of metal oxides on alkane-degrading community structure increased during soil maturation, whereas the charcoal impact decreased from 3 to 12 months. Among the clay minerals illite influenced the community structure of alkB-harbouring bacteria significantly, but not montmorillonite. The litter application induced strong community shifts in soils, maturated for 12 months, towards functional guilds typical for younger maturation stages pointing to a resilience of the alkane-degradation function potentially fostered by an extant ‘seed bank''.The tremendous microbial diversity in soils is, among other causes, a result of the huge structural heterogeneity and the enormous variety of biogeochemical interfaces (BGIs) present (Totsche et al., 2010). It has been postulated that during soil development different parental materials shape individual BGIs. They determine to a large extent the specific surface area and charge of BGIs and control for example, the water availability for microorganisms (Young and Crawford, 2004; Chorover et al., 2007). Such abiotic properties of BGIs may strongly influence microbial community structure and function. For instance, Vogel et al. (2014) demonstrated that organic carbon preferentially attaches to rough surfaces of mineral clusters, turning them into hotspots for microbial activity. In this study, we investigated the abundance and community structure of a functional microbial guild (alkane degraders harbouring the alkane monooxygenase gene alkB) at two different time points during the maturation of artificial soils formed from different parental materials. Thereby we compared artificial soils after a shorter period of maturation (namely, 3 months) and more developed soil structures (namely, 12 months of maturation), in each case before and after the addition of alkane-containing substrate (plant litter). We hypothesized that the functional guild studied will be largely influenced by the availability of alkane substrates, essential nutrients (for example, nitrogen) and water, which are largely controlled by differing sorption coefficients of the used parental materials that undergo an alteration process over time. Furthermore we wanted to understand if the ‘dormant or rare biosphere'' could act as a seed bank and ensure process stabilization after substrate addition (Giebler et al., 2013).We used eight artificial soils (Pronk et al., 2012) of identical texture, yet differing in composition regarding the presence of clay minerals (montmorillonite (MT) and illite (IL), metal oxides (ferrihydrite (FH) and boehmite (B)) and charcoal (CH). These materials were chosen as representatives of common soil components with large and reactive surface areas, and can be expected to take part in the formation of BGIs in natural soils (Totsche et al., 2010). Initially, the development of microbial communities had been stimulated by adding a water-extracted fraction of microorganisms from an agricultural soil, classified as a Eutric Cambisol (Ultuna, Sweden) together with sterile manure to the sterile soil components (for details see Supplementary Material S1.1). We took soil samples after maturation phases of either 3 (T3) or 12 months (T12), in which we analysed the abundance (quantitative PCR, qPCR, Supplementary Material S1.2) and community structure (terminal restriction fragment length polymorphism analysis, T-RFLP, Supplementary Material S1.3) of alkane-degrading microbes, as well as response patterns towards the addition of plant litter from winter wheat after 2 weeks of incubation subsequent to maturation. Schulz et al. (2012) described the alkane monooxygenase gene (alkB) as a sensitive marker for the molecular analysis of alkane-degrading bacteria responding to subtle changes in substrate availability that is, fresh plant material, which in general contains large amounts of easily available alkanes in form of waxes. The litter was added on top of the microcosms to create a vertical substrate gradient. Thus, soil samples were taken from the litter–soil interface (top 1 mm of the soil column; T3-I, T12-I) and ‘bulk'' soil (10–11 mm of the soil column; T3-B, T12-B). All details on the Materials and Methods can be found in the Supplementary Material S1.We detected higher alkB gene copy numbers in soils matured for 3 than in those matured for 12 months (Figure 1), regardless whether we analysed them before or after litter addition. Solely the soil where MT and CH had been added together exhibited stable alkB abundance patterns at both time points of maturation. However, the relative response towards litter addition was often stronger in soils with a longer maturation history (T12), particularly when soils contained illite mixtures or ferrihydrite (Figure 1 and Supplementary Material S2). The effects were more pronounced in samples from the litter–soil interface (T3-I and T12-I) than in bulk soil samples (T3-B and T12-B). A previous study revealed that the overall CO₂ respiration rates for these artificial soils after 3 and 12 months of maturation were comparable (Pronk et al., 2012). Several studies therefore concluded that the amount of organic substrates and nutrients present was enough to support activity of the total microflora (Pronk et al., 2012; Babin et al., 2013; Pronk et al., 2013). However, the lower abundance of alkane degraders in soils, maturated for 12 months, in conjunction with the respective stronger response towards litter addition might reflect a depletion of easily bioavailable specific substrates (namely, alkanes) in these soils by degradation and/or maturation-driven reallocation of nutrients to smaller, poorly accessible soil structures (Kögel-Knabner et al., 2008; Pronk et al., 2012; Vogel et al., 2014).Open in a separate windowFigure 1Abundance patterns of alkB genes in artificial soils after 3 (T3) and 12 months (T12). Diamonds (left, y axis) represent alkB gene abundances in matured soils (T3 in black and T12 in grey; s.e. of n=3). Bars (right, y axis) show a relative comparison between the response patterns to litter addition after 12 months compared with 3 months in the different artificial soils (litter–soil interface: black pattern; bulk soil: grey pattern). A value of 1 indicates an identically strong response at T12 and T3 to litter addition, a value >1 indicates a stronger and a value <1 a weaker litter response at T12- compared with T3-litter treated samples. The response values were calculated as quotients of alkB gene abundances after litter treatment vs after maturation, but without litter (for detailed information on calculation see Supplementary Material S1.4; for absolute alkB gene copy numbers of all treatments and correlation with environmental factors see Supplementary Material S2).The community structure of alkB-harbouring bacteria diverged significantly between 3 and 12 months of soil maturation (before litter addition; Figure 2a) with strong differences in some of the dominant T-RFs (for example typically present at T3: 88, 95, 123, 266, 347, 360, 468, 469, 527; more specific for T12: 80, 82, 83, 106; for details see Supplementary Material S1.3 and S3). Moreover, the replicates of the different artificial soil mixtures showed higher variability at T12 when compared with T3 (that is, greater distance between triplicates in the non-metric multidimensional scaling (NMDS)-plot, Figure 2a; P=0.002, statistical calculation Supplementary Material S1.4). This may indicate a higher variability of the alkB community structure with longer maturation time, even within the same artificial soil mixture. When correlating the alkB community structure with the ‘soil complexity'' (that is, the artificial soils contained 1 versus 2 or 3 components in addition to texture-providing quartz), we found that indeed the ‘soil complexity'' significantly influenced the community structure after 3 and 12 months of maturation (P<0.05; Supplementary Material S4-D). Altogether these findings indicate an increasing heterogeneity of the BGIs with maturation time and/or soil complexity, which was accompanied by an increasing formation of microaggregates (Pronk et al., 2012). One may speculate, that the resulting higher hierarchical structure in more matured soils probably creates diverse niches favouring the development of diverging microbial communities in response to their particular microenvironment as also described by Vos et al. (2013).Open in a separate windowFigure 2Community structure of alkB genes in artificial soils illustrated by NMDS. Solid ellipses indicate 95%, dashed ellipses 99% of community spreads of T3 (red) or T12 (blue; for details see Supplementary Material S4). (a) community differences between matured soil without litter at T3 (open symbols) and T12 (closed symbols). The eight artificial soils are indicated by colour (for legend see a; n=1: MT and IL, n=2: FH, n=3: all others). Exemplary for selected soils, distances of replicates to their group centroid are represented by dashed lines, whereby greater distance indicates greater dissimilarity of community structure. (b) alkB community structure before (red or blue ellipse) and after litter addition (green ellipses). The blue arrow indicates strong community shift of T12. (c, d) show selected soil components significantly affecting the alkB communities in all T3 or T12 treatments, respectively (P<at least 0.05; for details see Supplementary Material S4).The addition of fresh plant litter induced a pronounced shift of alkB community patterns in soils, maturated for 12 months (T12-I and T12-B compared with T12; Figure 2b, Supplementary Material S4), whereas no significant changes were observed after 3 months of maturation. Interestingly, the T12-I and T12-B communities after litter addition converged with those of the younger maturation stage, supporting the conclusion that the alkanes might have been depleted during the longer maturation without refeeding. The convergence points to the resilience of the alkane-degradation function through a potential ‘seed bank'' formed by dormant or rare alkane degraders in times of low substrate availability (Giebler et al., 2013). These microbes might be reactivated when the system experiences more favourable environmental conditions (Epstein, 2009; Shade et al., 2012). Their existence is furthermore indicated by high-abundant T-RFs formerly not detected in matured T12 soils (for example, T-RF 86, 88, 94, 95, 239, 463, 523 when comparing T12 with T12-I and T12-B; Supplementary Material S1.3 and S3).When analysing the data sets of 3 and 12 months of maturation individually, the presence of illite and charcoal as soil components in the different soil mixtures were found to be the main factors controlling alkB community structure in all treatments (stronger significance for T3; Figure 2c and Supplementary Material S4) as also observed for the bacterial community in general (Ding et al., 2013). The role of clay minerals like illite might be to sequester large amounts of the soil organic matter (Kaiser and Guggenberger, 2003; Vogel et al., 2014) thereby affecting the nutrient availability (Pronk et al., 2013). The assumption of the reduced availability of nutrients after 3 months is furthermore supported by the significant impact of charcoal, which may adsorb nutrients and reduce their bioavailability (Sohi et al., 2010; Lehmann et al., 2011). A leaching of alkanes by the charcoal used in this study can be excluded (Pronk et al., 2012). However, the charcoal effect receded at later maturation stages, when the reactive sites of minerals and charcoal surfaces gradually became covered with organic matter (Pronk et al., 2012).After 12 months of soil maturation, an emerging relevance of metal oxides as drivers for the community structure of alkB-harbouring microbes (Figure 2e) became evident. Metal oxides strongly interact with the organic matter of natural soils (Eusterhues et al., 2003; Kaiser and Guggenberger, 2003; Wagai and Mayer, 2007). However, a weak affinity and low sorption of organic matter had been reported for our artificial soils owing to constantly neutral pH (Heister et al., 2012; Pronk et al., 2013). Thus, factors other than nutrient availability might be responsible for the influence of metal oxides on the community structure as the increasing heterogeneity of the habitat creates more microniches. Potentially the noncharged surfaces of metal oxides in our experiment might function as valuable ‘safe havens'' still available for colonisation by microbes when other surfaces are already occupied. Thus, their role might become manifest when a soil reaches a higher hierarchical structure.Overall, our results support the hypothesis that complex structured BGIs as generated by clay minerals or metal oxides and charcoal are important drivers for the development of microbial community structures and their functional traits. It seems that structure–diversity and structure–function relationships are emerging properties, which go beyond the characteristics of individual components, that is, they are the result of a dynamic interplay of the BGIs and microbial communities responding to these microhabitats.     

Degradation of the amyloid beta-protein by the novel mitochondrial peptidasome, PreP

Recently we have identified the novel mitochondrial peptidase responsible for degrading presequences and other short unstructured peptides in mitochondria, the presequence peptidase, which we named PreP peptidasome. In the present study we have identified and characterized the human PreP homologue, hPreP, in brain mitochondria, and we show its capacity to degrade the amyloid beta-protein (Abeta). PreP belongs to the pitrilysin oligopeptidase family M16C containing an inverted zinc-binding motif. We show that hPreP is localized to the mitochondrial matrix. In situ immuno-inactivation studies in human brain mitochondria using anti-hPreP antibodies showed complete inhibition of proteolytic activity against Abeta. We have cloned, overexpressed, and purified recombinant hPreP and its mutant with catalytic base Glu(78) in the inverted zinc-binding motif replaced by Gln. In vitro studies using recombinant hPreP and liquid chromatography nanospray tandem mass spectrometry revealed novel cleavage specificities against Abeta-(1-42), Abeta-(1-40), and Abeta Arctic, a protein that causes increased protofibril formation an early onset familial variant of Alzheimer disease. In contrast to insulin degrading enzyme, which is a functional analogue of hPreP, hPreP does not degrade insulin but does degrade insulin B-chain. Molecular modeling of hPreP based on the crystal structure at 2.1 A resolution of AtPreP allowed us to identify Cys(90) and Cys(527) that form disulfide bridges under oxidized conditions and might be involved in redox regulation of the enzyme. Degradation of the mitochondrial Abeta by hPreP may potentially be of importance in the pathology of Alzheimer disease.     

The antimicrobial peptide cathelicidin protects the urinary tract against invasive bacterial infection

The urinary tract functions in close proximity to the outside environment, yet must remain free of microbial colonization to avoid disease. The mechanisms for establishing an antimicrobial barrier in this area are not completely understood. Here, we describe the production and function of the cathelicidin antimicrobial peptides LL-37, its precursor hCAP-18 and its ortholog CRAMP in epithelial cells of human and mouse urinary tract, respectively. Bacterial contact with epithelial cells resulted in rapid production and secretion of the respective peptides, and in humans LL-37/hCAP-18 was released into urine. Epithelium-derived cathelicidin substantially contributed to the protection of the urinary tract against infection, as shown using CRAMP-deficient and neutrophil-depleted mice. In addition, clinical E. coli strains that were more resistant to LL-37 caused more severe urinary tract infections than did susceptible strains. Thus, cathelicidin seems to be a key factor in mucosal immunity of the urinary tract.     

Genetic transformation of the oilseed crop <Emphasis Type="Italic">Crambe abyssinica</Emphasis>

Engineering oilseed crops for industrial purposes requires a suitable crop that does not outcross to any food oilseed crop, thus eliminating problems of gene flow. Crambe abyssinica is such a dedicated crop as it does not hybridize with any existing food oilseed crops. However, lack of regeneration and transformation protocols has limited the use of C. abyssinica in genetic manipulation studies. In this study, efficient regeneration and transformation protocols for Crambe have been developed. Hypocotyls of C. abyssinica cv. Galactica were incubated on a Murashige and Skoog medium supplemented with various plant growth regulators (PGRs). Among the different PGR combinations tested, 10 μM thidiazuron and 2.7 μM α-naphthaleneacetic acid promoted highest frequency of regeneration, up to 60%. Among six Agrobacterium stains evaluated, each harbouring the cloning vector containing the neomycin phosphotransferase (nptII) and β-glucuronidase (gus) genes. EHA101 and AGL-1 yielded the highest transformation frequencies of 1.3 and 2.1%, respectively. Putative transgenic lines were recovered, and confirmed as transgenic by Southern blot analysis. Subsequently, Agrobacterium-mediated transformation of hypocotyls of cv. Galactica with constructs harbouring the wax synthase and fatty acid reductase genes have also successfully recovered confirmed transgenic plants carrying these transgenes.