[Show abstract][Hide abstract] ABSTRACT: Recent advances in structural biology underlying mechanisms of channel gating have strengthened our knowledge about how K(+) channels can be inter-convertible between conductive and non-conductive states. We have reviewed and combined mutagenesis with biochemical, biophysical and structural information in order to understand the critical roles of the pore residues in stabilizing the pore structure and channel open state. We also discuss how the latest knowledge on the K(+) channel KcsA may provide a step towards better understanding of distinct pore stabilizing differences among diversified K(+) channels.
Journal of Bioenergetics 02/2012; 44(1):199-205. DOI:10.1007/s10863-012-9407-6 · 2.71 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The ascomycete Verticillium dahliae causes worldwide vascular wilt of many field and horticultural plants. The melanized resting structures of this fungus, so-called microsclerotia, survive for many years in soils and continuously re-infect plants. Due to the absence of known fungicides, Verticillium wilt causes immense crop losses. We discovered that the Gram-positive, spore-forming soil bacterium Streptomyces lividans expresses members of the prodiginine family during co-cultivation with V. dahliae. Using HPLC and LC-MS analysis of cultures containing S. lividans alone or grown together with V. dahliae, we found that undecylprodigiosin [394.4 M+H](+) is highly abundant, and streptorubin B [392.4 M+H](+) is present in smaller amounts. Within co-cultures, the quantity of undecylprodigiosin increased considerably and pigment concentrated at and within fungal hyphae. The addition of purified undecylprodigiosin to growing V. dahliae hyphae strongly reduced microsclerotia formation. Undecylprodigiosin was also produced when S. lividans grew on the roots of developing Arabidopsis thaliana plants. Furthermore, the presence of the undecylprodigiosin producer led to an efficient reduction of V. dahliae hyphae and microsclerotia on plant-roots. Based on these novel findings and previous knowledge, we deduce that the prodiginine investigated leads to multiple cellular effects, which ultimately impair specific pathways for signal transduction and apoptosis of the fungal plant pathogen.
[Show abstract][Hide abstract] ABSTRACT: Blue-pigmented exudates arise as droplets on sporulated lawns of Streptomyces coelicolor M110 grown on agar plates. Our electron microscopical and biochemical studies suggest that droplets contain densely packed vesicles with large assemblies of different protein types and/or the polyketide antibiotic actinorhodin. Frozen-hydrated vesicles were unilamellar with a typical bilayer membrane, and ranged from 80 to 400 nm in diameter with a preferred width of 150-300 nm. By means of cryo-electron tomography, three types were reconstructed three-dimensionally: vesicles that were filled with particulate material, likely protein assemblies, those that contained membrane-bound particles, and a vesicle that showed a higher contrast inside, but lacked particles. Our LC/MS analyses of generated tryptic peptides led to the identification of distinct proteins that carry often a predicted N-terminal signal peptide with a twin-arginine motif or lack a canonical signal sequence. The proteins are required for a range of processes: the acquisition of inorganic as well as organic phosphate, iron ions, and of distinct carbon sources, energy metabolism and redox balance, defence against oxidants and tellurites, the tailoring of actinorhodin, folding and assembly of proteins, establishment of turgor, and different signalling cascades. Our novel findings have immense implications for understanding new avenues of environmental biology of streptomycetes and for biotechnological applications.
[Show abstract][Hide abstract] ABSTRACT: A new assay system for chitin has been developed. It comprises the chitin-binding protein ChbB in fusion with a His-tag as well as with a Strep-tag, the latter of which was chemically coupled to horseradish peroxidase. With the resulting complex, minimal quantities of chitin are photometrically detectable. In addition, the assay allows rapid scoring of the activity of chitin-synthases. As a result, a refined procedure for the rapid purification of yeast chitosomes (nano-machineries for chitin biosynthesis) has been established. Immuno-electronmicroscopical studies of purified chitosomes, gained from a yeast strain carrying a chitin-synthase gene fused to that for GFP (green-fluorescence protein), has led to the in situ localization of chitin-synthase-GFP molecules within chitosomes.
International Journal of Molecular Sciences 09/2010; 11(9):3122-37. DOI:10.3390/ijms11093122 · 2.86 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Verticillium wilt, a vascular disease in more than 200 dicotyledonous plants, is due to the ascomycete fungus Verticillium dahliae. As documented by video-microscopy, the soil bacterium Streptomyces lividans strongly reduces the germination of V. dahliae conidia, and the subsequent growth of hyphae. Quantification by the use of DNA-intercalating dyes and Calcofluor-staining revealed that during prolonged co-cultivation, bacterial hyphae proliferate to a dense network, provoke a poor development of V. dahliae vegetative hyphae and lead to an enormous reduction of conidia and microsclerotia. Upon individual application to seeds of the model plant Arabidopsis thaliana, either the bacterial spores or the fungal conidia germinate at or within the mucilage, including its volcano-shaped structures. The extension of hyphae from each individual strain correlates with the reduction of the pectin-containing mucilage-layer. Proliferating hyphae then spread to roots of the emerging seedlings. Plants, which arise in the presence of V. dahliae within agar or soil, have damaged root cells, an atrophied stem and root, as well as poorly developed leaves with chlorosis symptoms. In contrast, S. lividans hyphae settle in bunches preferentially at the outer layer near tips and alongside roots. Resulting plants have a healthy appearance including an intact root system. Arabidopsis thaliana seeds, which are co-inoculated with V. dahliae and S. lividans, have preferentially proliferating bacterial hyphae within the mucilage, and at roots of the outgrowing seedlings. As a result, plants have considerably reduced disease symptoms. As spores of the beneficial S. lividans strain are obtainable in large quantity, its application is highly attractive.
[Show abstract][Hide abstract] ABSTRACT: Streptomycetes, soil-dwelling mycelial bacteria that form sporulating aerial branches, have an exceptionally large number of predicted secreted proteins, including many exported via the twin-arginine transport system. Their use of noncatalytic substrate-binding proteins and hydrolytic enzymes to obtain soluble nutrients from carbohydrates such as chitin and cellulose enables them to interact with other organisms. Some of their numerous secreted proteases participate in developmentally significant extracellular cascades, regulated by inhibitors, which lead to cannibalization of the substrate mycelium biomass to support aerial growth and sporulation. They excrete many secondary metabolites, including important antibiotics. Some of these play roles in interactions with eukaryotes. Surprisingly, some antibiotic biosynthetic enzymes are extracellular. Antibiotic production is often regulated by extracellular signalling molecules, some of which also control morphological differentiation. Amphipathic proteins, assembled with the help of cellulose-like material, are required for both hyphal attachment to surfaces and aerial reproductive growth. Comparative genomic analysis suggests that the acquisition of genes for extracellular processes has played a huge part in speciation. The rare codon TTA, which is present in the key pleiotropic regulatory gene adpA and many pathway-specific regulatory genes for antibiotic production, has a particular influence on extracellular biology.
[Show abstract][Hide abstract] ABSTRACT: Streptomycetes produce many metabolites with medical and biotechnological applications. During fermentations, their hyphae build aggregates, a process in which the newly identified protein HyaS plays an important role. The corresponding hyaS gene is present within all investigated Streptomyces species. Reporter fusions indicate that transcription of hyaS occurs within substrate hyphae of the Streptomyces lividans wild type (WT). The HyaS protein is dominantly associated with the substrate hyphae. The WT strain forms cylindrically shaped clumps of densely packed substrate hyphae, often fusing to higher aggregates (pellets), which remain stably associated during shaking. Investigations by electron microscopy suggest that HyaS induces tight fusion-like contacts among substrate hyphae. In contrast, the pellets of the designed hyaS disruption mutant ΔH are irregular in shape, contain frequently outgrowing bunches of hyphae, and fuse less frequently. ΔH complemented with a plasmid carrying hyaS resembles the WT phenotype. Biochemical studies indicate that the C-terminal region of HyaS has amine oxidase activity. Investigations of ΔH transformants, each carrying a specifically mutated gene, lead to the conclusion that the in situ oxidase activity correlates with the pellet-inducing role of HyaS, and depends on the presence of certain histidine residues. Furthermore, the level of undecylprodigiosin, a red pigment with antibiotic activity, is influenced by the engineered hyaS subtype within a strain. These data present the first molecular basis for future manipulation of pellets, and concomitant production of secondary metabolites during biotechnological processes.
[Show abstract][Hide abstract] ABSTRACT: The SenS/SenR system of Streptomyces reticuli regulates the expression of the redox regulator FurS, the catalase-peroxidase CpeB and the heme-binding protein HbpS. SenS/SenR is also proposed to participate in sensing redox changes, mediated by HbpS. Here, we show in vitro that heme-free HbpS represses the autokinase activity of SenS; whereas hemin-treated HbpS considerably enhances SenS autophosphorylation under redox conditions using either H(2)O(2) or DTT. The presence of iron ions alone or in combination with H(2)O(2) or DTT also leads to significantly increased phosphorylation levels of SenS. Further comparative physiological studies using the S. reticuli WT, a S. reticuli hbpS mutant and a S. reticuli senS-senR mutant corroborates the importance of HbpS and the SenS/SenR system for resistance against high concentrations of iron ions and hemin in vivo. Hence SenS/SenR and HbpS act in concert as a novel three-component system which detects redox stress, mediated by iron ions and heme.
[Show abstract][Hide abstract] ABSTRACT: Streptomyces coelicolor A32 produces a 35.6-kDa carbohydrate-binding protein (named CbpC) in the presence of cellobiose, cellulose or chitin as sole carbon source. The protein was found secreted (a typical signal sequence was present at the N-terminus) and linked to the peptidoglycan layer of the mycelia. At its C-terminal end a putative cell-wall sorting signal was identified, consisting of (1) Streptomyces specific recognition site for a transpeptidase (LAETG instead of generic LPXTP consensus), (2) a hydrophobic region and (3) a tail of positively charged residues. The deletion of this sorting signal abolished the cell-wall attachment because the resulting CbpC-form was found extracellular. After purification this protein was shown to interact strongly with crystalline cellulose; different crystalline chitin-forms were recognised moderately and chitosan not. As demonstrated by analysing further truncated CbpC-forms a glycine-aspartate/serine rich region, which separates the carbohydrate-binding module from the sorting signal, plays an important role in protein stability.
Archives of Microbiology 09/2008; 190(2):119-27. DOI:10.1007/s00203-008-0373-7 · 1.86 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The distribution of polyphosphate (polyP) within the cytoplasmic membrane of Streptomyces lividans hyphae or protoplasts has been determined at high spatial resolution by elemental mapping using energy-filtered electron microscopy (EFTEM). The results revealed that polyP was best traceable after its interaction with lead ions followed by their precipitation as lead sulphide. Concomitant studies of the S.lividans wildtype (WT) strain and its co-embedded mutant DeltaK (lacking a functional kcsA gene) were conducted by labelling as the surface matrix of either one was labelled by cationic colloidal thorium dioxide. Within the WT strain, additional polyP was found to accumulate distinctly at the inner face of the cytoplasmic membrane. After removal of the cell wall (within protoplasts), the polyP-derived lead-sulphide (PbS) precipitate formed clusters of fibrillar material extending up to 50 nm into the cytoplasm. This feature was absent in the DeltaK mutant strain. Together the results revealed that the presence of the KcsA channel and the structured polyP coincide.
[Show abstract][Hide abstract] ABSTRACT: The two-component system SenS-SenR from Streptomyces reticuli has been shown to influence the production of the redox regulator FurS, the mycelium-associated enzyme CpeB, which displays heme-dependent catalase and peroxidase activity as well as heme-independent manganese peroxidase activity, and the extracellular heme-binding protein HbpS. In addition, it was suggested to participate in the sensing of redox changes. In this work, the tagged cytoplasmic domain of SenS (SenS(c)), as well as the full-length differently tagged SenR, and corresponding mutant proteins carrying specific amino acid exchanges were purified after heterologous expression in Escherichia coli. In vitro, SenS(c) is autophosphorylated to SenS(c) approximately P at the histidine residue at position 199, transfers the phosphate group to the aspartic acid residue at position 65 in SenR, and acts as a phosphatase for SenR approximately P. Bandshift and footprinting assays in combination with competition and mutational analyses revealed that only unphosphorylated SenR binds to specific sites upstream of the furS-cpeB operon. Further specific sites within the regulatory region, common to the oppositely orientated senS and hbpS genes, were recognized by SenR. Upon its phosphorylation, the DNA-binding affinity of this area was enhanced. These data, together with previous in vivo studies using mutants lacking functional senS and senR, indicate that the two-component SenS-SenR system governs the transcription of the furS-cpeB operon, senS-senR and the hbpS gene. Comparative analyses reveal that only the genomes of a few actinobacteria encode two-component systems that are closely related to SenS-SenR.
[Show abstract][Hide abstract] ABSTRACT: Streptomycetes belong to the ecologically important bacterial population within soil, which is also inhabited by many fungi. The highly chitinolytic Streptomyces olivaceoviridis and the ascomycete Aspergillus proliferans were chosen as models to test for interactions among bacteria and fungi. In medium lacking a soluble carbon source, individually cultivated spores of the bacterium S. olivaceoviridis and the fungus A. proliferans do not germinate. However, as shown by viability tests, cultivation of a mixture of both spore types provokes successive events: (i) stimulation of the germination of S. olivaceoviridis spores, (ii) initiation of the outgrowth of some fungal spores to which the S. olivaceoviridis chitinase ChiO1 adheres, (iii) massive extension of viable networks of S. olivaceoviridis hyphae at the expense of fungal hyphae and (iv) balanced proliferation of closely interacting fungal and S. olivaceoviridis hyphae. The replacement of the S. olivaceoviridis wild-type strain by a chromosomal disruption mutant (DeltaC), lacking production of the extracellular chitin-binding protein CHB1 but still secreting the chitinase ChiO1, provokes (v) germination of each spore type, (vi) retarded development of both partners, followed by (vii) preferential proliferation of the fungus. Together with biochemical and immunomicroscopy studies, the data support the conclusion that CHB1 molecules aggregate to an extracellular matrix, maintaining a close contact, followed by several concerted responses of the bacterium and the fungus.
[Show abstract][Hide abstract] ABSTRACT: To deepen the knowledge of chitin synthesis, a yeast mutant has been used as a model. Purified chitin synthase I-containing vesicles (chitosomes) with a diameter of 85 to 120 nm are identified by electron microscopy to eject tiny fibers upon addition of UDP-N-acetylglucosamine. The filigree of extruded filaments fused gradually into a large three-dimensional network, which is degradable by a chitinase. The network is targeted and restructured by the Streptomyces chitin-binding protein CHB1, which has a very high affinity only for alpha-chitin. Within the chitosomes, filaments are found to be highly condensed within consecutive oval fibroids, which are specifically targeted by the alpha-chitin-binding protein. The presented data give new insights to the generation of chitin filaments with an antiparallel (alpha) configuration. [image: see text]
[Show abstract][Hide abstract] ABSTRACT: The previous discovery of the Streptomyces lividans kcsA gene and its overexpression followed by the functional reconstitution of the purified gene product has resulted in new strategies to explore this channel protein in vitro. KcsA has evolved as a general model to investigate the structure/function relationship of ion channel proteins. Using specific antibodies raised against a domain of KcsA lacking membrane-spanning regions, KcsA has now been localized within numerous separated clusters between the outer face of the cytoplasm and the cell envelope in substrate hyphae of the S. lividans wild-type strain but not in a designed chromosomal disruption mutant DeltaK, lacking a functional kcsA gene. Previous findings had revealed that caesium ions led to a block of KcsA channel activity within S. lividans protoplasts fused to giant vesicles. As caesium can be scored by electron energy loss spectroscopy better than potassium, this technique was applied to hyphae that had been briefly exposed to caesium instead of potassium ions. Caesium was found preferentially at the cell envelope. Compared to the DeltaK mutant, the relative level of caesium was approximately 30 % enhanced in the wild-type. This is attributed to the presence of KcsA channels. Additional visualization by electron spectroscopic imaging supported this conclusion. The data presented are believed to represent the first demonstration of in vivo monitoring of KcsA in its original host.