A Sensitive Method to Monitor <Emphasis Type="Italic">Bacillus subtilis</Emphasis> and <Emphasis Type="Italic">Streptomyces coelicor</Emphasis>-related Bacteria in Maize Rhizobacterial Communities: The Use of Genome-Wide Microarrays

Commercially available DNA microarrays containing genome-wide spotted oligonucleotides encompass the soil bacteria Bacillus subtilis or Streptomyces coelicolor genomes. These have been used to analyse potential differences in rhizobacterial communities of transgenic maize engineered to express the Bacillus thuringensis Cry toxin (Bt maize) in three different agricultural soils. No differences in hybridisation were observed between genetically and non-genetically modified maize rhizobacteria from two Bt lines with a detection sensitivity of five copies of a particular gene above the background. Soil-specific hybridisation results were obtained when rhizobacterial DNA was compared to the corresponding genomic DNA spotted in the microarrays suggesting that the use of genome-wide DNA arrays could serve as a useful tool for the molecular monitoring of rhizobacterial communities. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. An erratum to this article can be found at     

Sporicidal Activity of Synthetic Antifungal Undecapeptides and Control of Penicillium Rot of Apples

The antifungal activity of cecropin A(2-8)-melittin(6-9) hybrid undecapeptides, previously reported as active against plant pathogenic bacteria, was studied. A set of 15 sequences was screened in vitro against Fusarium oxysporum, Penicillium expansum, Aspergillus niger, and Rhizopus stolonifer. Most compounds were highly active against F. oxysporum (MIC < 2.5 μM) but were less active against the other fungi. The best peptides were studied for their sporicidal activity and for Sytox green uptake in F. oxysporum microconidia. A significant inverse linear relationship was observed between survival and fluorescence, indicating membrane disruption. Next, we evaluated the in vitro activity against P. expansum of a 125-member peptide library with the general structure R-X¹KLFKKILKX¹⁰L-NH₂, where X¹ and X¹⁰ corresponded to amino acids with various degrees of hydrophobicity and hydrophilicity and R included different N-terminal derivatizations. Fifteen sequences with MICs below 12.5 μM were identified. The most active compounds were BP21 {Ac,F,V} and BP34 {Ac,L,V} (MIC < 6.25 μM), where the braces denote R, X¹, and X¹⁰ positions and where Ac is an acetyl group. The peptides had sporicidal activity against P. expansum conidia. Seven of these peptides were tested in vivo by evaluating their preventative effect of inhibition of P. expansum infection in apple fruits. The peptide Ts-FKLFKKILKVL-NH₂ (BP22), where Ts is a tosyl group, was the most active with an average efficacy of 56% disease reduction, which was slightly lower than that of a commercial formulation of the fungicide imazalil.The discovery of antimicrobial compounds to treat plant diseases of economical importance in agriculture remains a major scientific challenge (1). Antimicrobial peptides are being considered as a good alternative to current fungicides and a great deal of scientific effort has been invested in studying their application in plant disease control (29, 34, 35).Antimicrobial peptides have been reported to display interesting activities against pathogenic microbes that are resistant to conventional antibiotics and to exhibit a broad spectrum of activity against bacteria, fungi, enveloped viruses, parasites, and tumor cells (7-10, 19, 20, 40, 49). The mechanism of action of these peptides against fungi consists of cell lysis by binding to the membrane surface and disrupting its structure, interference with the synthesis of essential cell wall components, or interaction with specific internal targets (12, 13, 15, 23, 29).Despite their good lytic activity, major concerns about the use of antimicrobial peptides as pesticides in plant protection are the high production cost associated with synthetic procedures and their low stability toward protease degradation. Several design strategies have been devised in order to find shorter and more stable peptides, while maintaining or increasing the activity with a low cytotoxicity. These strategies include the juxtaposition of fragments of natural antimicrobial peptides, the modification of natural peptides, and the de novo design of sequences maintaining the crucial features of native antimicrobial peptides (2, 3, 11, 24, 32, 38, 42). However, the process involved in the development of lead candidates is time consuming and limited by the number of individual compounds that can be synthesized. Combinatorial chemistry has allowed the rapid preparation of synthetic libraries and their screening has led to the identification of peptides with high activity against selected phytopathogenic bacteria and fungi (4, 26, 27, 33).During our current research oriented to the development of new antimicrobial agents for use in plant protection, we designed linear undecapeptides (CECMEL11) derived from the cecropin A-melittin hybrid peptide WKLFKKILKVL-NH₂ (Pep3) (5, 17). Using a combinatorial approach, we identified peptides with high activity against plant pathogenic bacteria, such as Erwinia amylovora, Xanthomonas vesicatoria, and Pseudomonas syringae, and with low susceptibility to protease degradation (4, 5).In order to broaden the study, we decided to test the CECMEL11 peptides against the plant pathogenic fungi Fusarium oxysporum, Aspergillus niger, Rhizopus stolonifer, and Penicillium expansum. The fungus F. oxysporum causes Fusarium wilt in more than a hundred species of plants, and it is an important pathogen in horticultural crops (44). Several Rhizopus and Penicillium species cause soft rot and blue mold rot, respectively, which are important postharvest diseases in stone and pome fruits (6, 18, 22, 39). Apart from the economic losses, Aspergillus and Penicillium species are also of interest from a public health point of view due to the production of mycotoxins (45, 47). The importance of Penicillium species in the postharvest of fruits emphasizes the interest to develop antimicrobial peptides to control this fungus.Taking into account the relevance of these pathogens, the aim of the present study was the analysis of the antifungal activity profile of the CECMEL11 peptides in order to identify sporicidal sequences against the above fungi. As a proof of concept, the feasibility of using such peptides to protect fruits from fungal spoilage was evaluated using a P. expansum/apple model.     

Internalization of the receptor for advanced glycation end products (RAGE) is required to mediate intracellular responses

To dissect the rat receptor for advanced glycation end products (RAGE) subcellular distribution and trafficking in eukaryotic cells, an expression system coding for a fusion protein between the RAGE and an enhanced green fluorescent protein (EGFP) has been used. The RAGE-EGFP protein is expressed at the plasma membrane of CHO-k1 and Neuro-2a (N2a) cells and retains the capacity to bind Texas Red-labelled advanced glycation end products (AGEs). AGEs addition to the cell cultures induced a change in the subcellular distribution of the fluorescent RAGE-EGFP protein compatible with an internalization of the AGEs-RAGE complex. Furthermore, while N2a cells expressing the RAGE-EGFP showed an increase in ERK1/2 phosphorylation and NF-kappaB DNA binding in response to AGEs, pre-incubation with dansyl-cadaverine or phenylarsine oxide, inhibitors of receptors internalization, blocked the activation of ERKs and other intracellular responses mediated by AGEs. These results suggest that internalization plays a key role in the signal transduction mediated by RAGE.     

Structural and Biophysical Characterization of BoxC from Burkholderia xenovorans LB400: A NOVEL RING-CLEAVING ENZYME IN THE CROTONASE SUPERFAMILY*

The mineralization of aromatic compounds by microorganisms relies on a structurally and functionally diverse group of ring-cleaving enzymes. The recently discovered benzoate oxidation pathway in Burkholderia xenovorans LB400 encodes a novel such ring-cleaving enzyme, termed BoxC, that catalyzes the conversion of 2,3-dihydro-2,3-dihydroxybenzoyl-CoA to 3,4-dehydroadipyl-CoA without the requirement for molecular oxygen. Sequence analysis indicates that BoxC is a highly divergent member of the crotonase superfamily and nearly double the size of the average superfamily member. The structure of BoxC determined to 1.5 Å resolution reveals an intriguing structural demarcation. A highly divergent region in the C terminus probably serves as a structural scaffold for the conserved N terminus that encompasses the active site and, in conjunction with a conserved C-terminal helix, mediates dimer formation. Isothermal titration calorimetry and molecular docking simulations contribute to a detailed view of the active site, resulting in a compelling mechanistic model where a pair of conserved glutamate residues (Glu¹⁴⁶ and Glu¹⁶⁸) work in tandem to deprotonate the dihydroxylated ring substrate, leading to cleavage. A final deformylation step incorporating a water molecule and Cys¹¹¹ as a general base completes the formation of 3,4-dehydroadipyl-CoA product. Overall, this study establishes the basis for BoxC as one of the most divergent members of the crotonase superfamily and provides the first structural insight into the mechanism of this novel class of ring-cleaving enzymes.Aromatic compounds comprise approximately one-quarter of the earth''s biomass (1) and are the second most abundant natural product next to carbohydrates. The majority of aromatic compounds in the environment are in the form of the organic polymer lignin that plays a structural role in cross-linking cell wall polysaccharides in plants. Despite the inherent thermostability of the aromatic ring, these naturally occurring compounds are efficiently mineralized by various microorganisms. Human-made aromatic compounds, such as those used in industrial processes, however, are often recalcitrant to microbial degradation due to their chemical complexity, decreased bioavailability, and increased thermostability. Moreover, bacteria have only been exposed to these compounds for a relatively short period of time. As a result, these compounds persist in the environment, where they can increase to toxic levels and cause irreversible damage to the biosphere.The common structural blueprint shared by natural and human-made aromatic compounds is the resonance-stabilized planar ring system. Microorganisms overcome the stability of these aromatic structures by employing specific ring-cleaving enzymes that form part of complex catabolic pathways. Until recently, two general classes of microbial processes were characterized that catalyze the degradation of aromatic compounds. These classifications, termed the aerobic and anaerobic pathways, were based primarily on the mode of initial activation and subsequent cleavage of the aromatic ring. The aerobic pathway, exemplified by the peripheral biphenyl and the central ben-cat pathway, relies on the extensive use of molecular oxygen for both the hydroxylation (activation) and cleavage of the aromatic ring (2–4). The anaerobic pathway, however, mediates a reductive dearomatization followed by a hydrolytic ring cleavage, as observed in the classical benzoate pathway (5–7). In both cases, the underlying mechanism incorporates an activation step that renders the ring susceptible to cleavage.Recently, a third aromatic degradation pathway was identified in Burkholderia xenovorans strain LB400 (LB400) (8–10) and Azoarcus evansii (11–13). This novel pathway, termed the box (benzoate oxidation) pathway, incorporates features of both the aerobic and anaerobic pathways, resulting in a hybrid pathway. Microarray analysis of the 9.7-Mb genome of LB400 revealed two paralogous copies of the box pathway, one encoded on chromosome 1 (box_c) and the second on the megaplasmid (box_m) (9). Knock-out studies confirm that both box pathways are capable of assimilating benzoate (10) yet are differentially regulated based on available carbon source and growth phase of the organism (9). Recent structural and biochemical characterization of benzoate CoA ligase (14) and aldeheyde dehydrogenase (15) from the box pathway in LB400 have provided valuable insight into the basis of substrate specificity and details describing the molecular mechanisms.A unique feature of the hybrid box pathway is the incorporation of both CoA ligation and hydroxylation prior to ring cleavage (16), suggesting that both strategies are important for ring activation. It is noteworthy that although CoA ligation is common in the activation of aromatic acids under anaerobic conditions, it has thus far been unseen in the aerobic degradation of aromatic compounds. Furthermore, investigation of the box pathway intermediates from the related A. evansii demonstrated that the thioesterified dihydrodiol intermediate was not oxidized and rearomatized as normally occurs in aerobic aromatic metabolism (11). Instead, it was shown to be directly cleaved without the requirement of molecular oxygen in a reaction that resulted in the loss of one unit of carbon and oxygen as formate (11). This critical ring cleavage step in the box pathway is catalyzed by BoxC (2,3-dihydro-2,3-dihydroxybenzoyl-CoA lyase/hydrolase) (11), which differs from traditional aerobic and anaerobic ring-cleaving enzymes in that oxygen is not used in catalysis, and the ring substrate is only partially reduced. Based on sequence analysis, BoxC is assigned to the crotonase superfamily. The cleavage reaction catalyzed by BoxC, however, suggests that BoxC defines a new mechanistic niche and intriguingly is one of the four outstanding crotonase superfamily members for which no structural information exists (17).A mechanism for BoxC from A. evansii was recently proposed based on the identification of chemical species using NMR and mass spectrometry (11). In the absence of structural information of BoxC, however, the mechanistic details, including the identity of the catalytic residues, remain undefined. To investigate the detailed molecular mechanism of BoxC, we carried out a structural and biophysical analysis complemented with molecular docking. The resulting data provide a compelling mechanistic model with the identification of key catalytic residues and active site structure that stabilize proposed transition state intermediates. Furthermore, the 1.5 Å resolution structure of BoxC reveals intriguing divergent architectural features with respect to other members of the crotonase superfamily. Overall, this study provides the first structural characterization of the novel BoxC family of enzymes and is interpreted with respect to the proposed molecular mechanism and divergence within the crotonase superfamily.     

Discovery,characterization and comparison of inhibitors of Bacillus anthracis and Staphylococcus aureus replicative DNA helicases

Antibacterial compounds with new mechanisms of action are needed for effective therapy against drug-resistant pathogens in the clinic and in biodefense. Screens for inhibitors of the essential replicative helicases of Bacillus anthracis and Staphylococcus aureus yielded 18 confirmed hits (IC₅₀ ? 25 μM). Several (5 of 18) of the inhibitors were also shown to inhibit DNA replication in permeabilized polA-deficient B. anthracis cells. One of the most potent inhibitors also displayed antibacterial activity (MIC ～5 μg/ml against a range of Gram-positive species including bacilli and staphylococci) together with good selectivity for bacterial versus mammalian cells (CC₅₀/MIC > 16) suitable for further optimization. This compound shares the bicyclic ring of the clinically proven aminocoumarin scaffold, but is not a gyrase inhibitor. It exhibits a mixed mode of helicase inhibition including a component of competitive inhibition with the DNA substrate (K_i = 8 μM) and is rapidly bactericidal at 4 × MIC.     

Immobilization–stabilization of the lipase from Thermomyces lanuginosus: Critical role of chemical amination

This paper describes the immobilization and stabilization of the lipase from Thermomyces lanuginosus (TLL) on glyoxyl agarose. Enzymes attach to this support only by the reaction between several aldehyde groups of the support and several Lys residues on the external surface of the enzyme molecules at pH 10. However, this standard immobilization procedure is unsuitable for TLL lipase due to the low stability of TLL at pH 10 and its low content on Lys groups that makes that the immobilization process was quite slow. The chemical amination of TLL, after reversible immobilization on hydrophobic supports, has been shown to be a simple and efficient way to improve the multipoint covalent attachment of this enzyme. The modification enriches the enzyme surface in primary amino groups with low pKb, thus allowing the immobilization of the enzyme at lower pH values. The aminated enzyme was rapidly immobilized at pH 9 and 10, with activities recovery of approximately 70%. The immobilization of the chemically modified enzyme improved its stability by 5-fold when compared to the non-modified enzyme during thermal inactivation and by hundreds of times when the enzyme was inactivated in the presence of organic solvents, being both glyoxyl preparations more stable than the enzyme immobilized on bromocyanogen.     

Rethinking species selection for restoration of arid shrublands

Restoration is playing an increasingly important role in ecology as natural habitats become scarcer and chances to restore ecosystems damaged by human activities are more common. However, restoration of degraded Mediterranean arid ecosystems is hampered by drought and poor soils, which cause many establishment failures. To compare how species belonging to different successional stages establish in a very stressful site, we carried out a field experiment with 14 tree and shrub species differing in functional traits. After three growing seasons, mid-successional shrubs such as the leafless Ephedra fragilis and the C₄ Salsola oppositifolia, or green-stemmed legumes like Coronilla juncea, Genista umbellat, and Retama sphaerocarpa, showed survival rates up to 93%, while late-successional species like Tetraclinis articulata, Pinus halepensis, Olea europaea, and Pistacia lentiscus, frequently used and recommended in regular restoration projects, hardly reached 55%. We found that survival was highest for legumes, followed by leafless species, and C₄ shrubs, traits that are believed to maximize resource uptake in cleared and infertile areas while reducing water losses. Thus, selection of mid-successional species having such traits should be considered for successful restoration. These species would increase the success of restoration programs, but also would increase soil fertility, reduce soil erosion processes, and eventually facilitate establishment of other species, therefore accelerating secondary succession. We suggest a new approach for the restoration for arid shrublands in which species are carefully selected based on traits that best suit the environmental conditions.     

Kinetics of transport and phosphorylation of glucose in cancer cells

Metabolic control analysis of tumor glycolysis has indicated that hexokinase (HK) and glucose transporter (GLUT) exert the main flux control (71%). To understand why they are the main controlling steps, the GLUT and HK kinetics and the contents of GLUT1, GLUT2, GLUT3, GLUT4, HKI, and HKII were analyzed in rat hepatocarcinoma AS‐30D and HeLa human cervix cancer. An improved protocol to determine the kinetic parameters of GLUT was developed with D ‐[2‐³H‐glucose] as physiological substrate. Kinetic analysis revealed two components at low‐ and high‐glucose concentrations in both tumor cells. At low glucose and 37°C, the V_max was 55 ± 20 and 17.2 ± 6 nmol (min × mg protein)^?1, whereas the K_m was 0.52 ± 0.7 and 9.3 ± 3 mM for hepatoma and HeLa cells, respectively. GLUT activity was partially inhibited by cytochalasin B (IC₅₀ = 0.44 ± 0.1; K_i = 0.3 ± 0.1 µM) and phloretin (IC₅₀ = 8.7 µM) in AS‐30D hepatocarcinoma. At physiological glucose, GLUT1 and GLUT3 were the predominant active isoforms in HeLa cells and AS‐30D cells, respectively. HK activity in HeLa cells was much lower (60 mU/mg protein) than that in AS‐30D cells (700 mU/mg protein), but both HKs were strongly inhibited by G6P. HKII was the predominant isoform in AS‐30D carcinoma and HeLa cells. The much lower GLUT V_max and catalytic efficiency (V_max/K_m) values in comparison to those of G6P‐sensitive HK suggested the transporter exerts higher control on the glycolytic flux than HK in cancer cells. Thus, GLUT seems a more adequate therapeutic target. J. Cell. Physiol. 221: 552–559, 2009. © 2009 Wiley‐Liss, Inc.     

Mobilization of Neural Stem Cells and Generation of New Neurons in 6-OHDA�Clesioned Rats by Intracerebroventricular Infusion of Liver Growth Factor

Neural stem cells with self-renewal and multilineage potential persist in the subventricular zone of the adult mammalian forebrain. These cells remain relatively quiescent but, under certain conditions, can be stimulated, giving rise to new neurons. Liver growth factor (LGF) is a mitogen for liver cells that shows biological activity in extrahepatic sites and is useful for neuroregenerative therapies. The aim of this study was to investigate the potential neurogenic activity of LGF in the 6-hydroxydopamine rat model of Parkinson''s disease. Proliferation was significantly increased in the subventricular zone and denervated striatum of rats receiving ICV LGF infusions, and 25% of the proliferating cells were doublecortin-positive neurons. Doublecortin-positive cells with the morphology of migrating neuroblasts were also observed in the dorsal and ventral regions of the striatum of LGF-infused animals. Moreover, some newly generated cells were neuronal nuclei-positive mature neurons. LGF also stimulated microglia and induced astrogliosis, both phenomena associated with generation and migration of new neurons in the adult brain. In summary, our study shows that LGF stimulates neurogenesis when applied intraventricularly in 6-hydroxydopamine–lesioned rats. Considering that this factor also promotes neuronal migration into damaged tissue, we propose LGF as a novel factor useful for neuronal replacement in neurodegenerative diseases. (J Histochem Cytochem 57:491–502, 2009)