Streptomyces Olivaceoviridis Research Articles

Genome sequence of bioactive streptomycete isolated from mangrove forest in East Malaysia, Streptomyces monashensis MUSC 1JT

Members of Streptomyces are known to be prolific producers of bioactive compounds, including antibacterials, antioxidants, neuroprotective agents as well as immunomodulatory compounds. Isolated from East Malaysia, Streptomyces monashensis MUSC 1JT (=DSM 103626T =MCCC 1K03221T) was initially described as a novel streptomycete using a polyphasic approach. Based on phylogenetic analyses, the closely strains related to MUSC 1JT were identified as Streptomyces corchorusii DSM 40340T (98.7 %), Streptomyces olivaceoviridis NBRC 3066T (98.7 %), Streptomyces canarius NBRC 13431T (98.6 %), and Streptomyces coacervatus AS-0823T (98.4 %). Seven day fermentative extracts of MUSC 1JT exhibited potent antioxidant activity and significant cytotoxic activity against colon cancer cell lines, thus the strain was selected for whole genome sequencing. The genome size of MUSC 1JT is described to be 10.3 Mbps with G + C content of 71.50%. Based on antiSMASH analysis, the strain possessess great genomic potential, having fifty nine biosynthetic gene clusters related to production of secondary metabolites and antibiotics. Therefore, these results serve a foundation for further in-depth investigation to harness its bioactive potential for the development of highly valuable pharmaceutical products.

Streptomyces monashensis sp. nov., a novel mangrove soil actinobacterium from East Malaysia with antioxidative potential

A new Streptomyces species discovered from Sarawak mangrove soil is described, with the proposed name – Streptomyces monashensis sp. nov. (strain MUSC 1JT). Taxonomy status of MUSC 1JT was determined via polyphasic approach. Phylogenetic and chemotaxonomic properties of strain MUSC 1JT were in accordance with those known for genus Streptomyces. Based on phylogenetic analyses, the strains closely related to MUSC 1JT were Streptomyces corchorusii DSM 40340T (98.7%), Streptomyces olivaceoviridis NBRC 13066T (98.7%), Streptomyces canarius NBRC 13431T (98.6%) and Streptomyces coacervatus AS-0823T (98.4%). Outcomes of DNA–DNA relatedness between strain MUSC 1JT and its closely related type strains covered from 19.7 ± 2.8% to 49.1 ± 4.3%. Strain MUSC 1JT has genome size of 10,254,857 bp with DNA G + C content of 71 mol%. MUSC 1JT extract exhibited strong antioxidative activity up to 83.80 ± 4.80% in the SOD assay, with significant cytotoxic effect against colon cancer cell lines HCT-116 and SW480. Streptomyces monashensis MUSC 1JT (=DSM 103626T = MCCC 1K03219T) could potentially be a producer of novel bioactive metabolites; hence discovery of this new species may be highly significant to the biopharmaceutical industry as it could lead to development of new and useful chemo-preventive drugs.

Functional Characterization of the GH10 and GH11 Xylanases from Streptomyces olivaceoviridis E-86 Provide Insights into the Advantage of GH11 Xylanase in Catalyzing Biomass Degradation.

We functionally characterized the GH10 xylanase (SoXyn10A) and the GH11 xylanase (SoXyn11B) derived from the actinomycete Streptomyces olivaceoviridis E-86. Each enzyme exhibited differences in the produced reducing power upon degradation of xylan substrates. SoXyn10A produced higher reducing power than SoXyn11B. Gel filtration of the hydrolysates generated by both enzymes revealed that the original substrate was completely decomposed. Enzyme mixtures of SoXyn10A and SoXyn11B produced the same level of reducing power as SoXyn10A alone. These observations were in good agreement with the composition of the hydrolysis products. The hydrolysis products derived from the incubation of soluble birchwood xylan with a mixture of SoXyn10A and SoXyn11B produced the same products as SoXyn10A alone with similar compositions. Furthermore, the addition of SoXyn10A following SoXyn11B-mediated digestion of xylan produced the same products as SoXyn10A alone with similar compositions. Thus, it was hypothesized that SoXyn10A could degrade xylans to a smaller size than SoXyn11B. In contrast to the soluble xylans as the substrate, the produced reducing power generated by both enzymes was not significantly different when pretreated milled bagasses were used as substrates. Quantification of the pentose content in the milled bagasse residues after the enzyme digestions revealed that SoXyn11B hydrolyzed xylans in pretreated milled bagasses much more efficiently than SoXyn10A. These data suggested that the GH10 xylanases can degrade soluble xylans smaller than the GH11 xylanases. However, the GH11 xylanases may be more efficient at catalyzing xylan degradation in natural environments (e.g. biomass) where xylans interact with celluloses and lignins.

Potensi Bacillus licheniformis dan Streptomyces olivaceoviridis sebagai Penghambat Pertumbuhan Jamur Saprolegnia sp. Penyebab Saprolegniasis pada Ikan secara In Vitro <Br><I>[Potential of Bacillus licheniformis and Streptomyces olivaceoviridis as Inhibiting The Growth of Fungus Saprolegnia sp, Cause Saprolegniasis on Fish by Using In Vitro]<I>

Abstract Saprolegniasis is a fungal disease on fish caused by Saprolegnia sp, which is saprophyte, damaging healthy tissue and makes the immune system in fish deacreased. The uniquennes of Saprolegnia sp has the main components of the cell wall in the form of chitin that was instrumental in shaping the structure of the tip growth of fungal hyphae. Control of fungal pathogenic Saprolegnia sp can use chitinolytic microorganisms based on ability to produce chitinase for example using bacteria. This study aims to potential of Bacillus licheniformis and Streptomyces olivaceoviridis as inhibiting the growth of fungus Saprolegnia sp, cause saprolegniasis on fish by using in vitro. The research method is experimental with completely randomized design (CRD), which consists of four treatments with five replications. The experimental used is A (Saprolegnia sp and Bacillus licheniformis), B (Saprolegnia sp and Streptomyces olivaceoviridis), C negative control (Saprolegnia sp) and D positive control (Saprolegnia sp and Ketokonazol 2 %). The main parameters measured were observed inhibition zone on each treatment. Supporting parameters were observed is an observation of abnormal hpyphae structure after being induced by bacteria Bacillus licheniformis dan Streptomyces olivaceoviridis. Data were analyzed using analysis of variants (ANOVA) and to know the difference between treatments were determined by Tukey honestly significant difference (Tukey HSD) Test. The results showed that the potential of chitinolytic Bacillus licheniformis can provide a good barrier of 4,62 cm by 5,48 cm compared Streptomyces olivaceoviridis in inhibiting the growth Saprolegnia sp. Suggestions in this research is the need to further research on the value of chitinase Bacillus licheniformis and Streptomyces olivaceoviridis. Further research by using in vivo.

Streptomyces lactacystinicus sp. nov. and Streptomyces cyslabdanicus sp. nov., producing lactacystin and cyslabdan, respectively.

Actinomycete strains OM-6519(T) and K04-0144(T) produce the bioactive compounds lactacystin and cyslabdan, respectively. Here, the taxonomic positions of these two strains were determined. The morphological and chemical features of strains OM-6519(T) and K04-0144(T) indicated that they belonged to the genus Streptomyces. Strain OM-6519(T) showed the highest 16S rRNA gene sequence similarities with Streptomyces xanthocidicus NBRC 13469(T) (99.7%), Streptomyces chrysomallus subsp. fumigatus NBRC 15394(T) (99.6%) and Streptomyces aburaviensis NRRL B-2218(T) (99.5%). However, the DNA-DNA relatedness values between strain OM-6519(T) and the three related strains were below 70%. Strain K04-0144(T) showed the highest 16S rRNA gene sequence similarities with Streptomyces corchorusii NBRC 13032(T) (99.4%), Streptomyces olivaceoviridis NBRC 15394(T) (99.4%) and Streptomyces canarius NRRL B-2218(T) (99.3%). However, the DNA-DNA relatedness values between strain K04-0144(T) and the three related strains were also below 70%. Based on morphological, cultural and physiological characteristics and DNA-DNA relatedness data, strains OM-6519(T) and K04-0144(T) should be classified as new species of the genus Streptomyces, for which the names Streptomyces lactacystinicus sp. nov. and Streptomyces cyslabdanicus sp. nov. are proposed. The type strain of S. lactacystinicus is OM-6519(T) (=NBRC 110082(T), DSM 43136(T)). The type strain of S. cyslabdanicus is K04-0144(T) (=NBRC 110081(T), DSM 42135(T)).

Concerted responses between the chitin-binding protein secreting Streptomyces olivaceoviridis and Aspergillus proliferans

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.

Introduction of a disulfide bridge enhances the thermostability of a Streptomyces olivaceoviridis xylanase mutant

Substitution of the N-terminus of Streptomyces olivaceoviridis xylanase XYNB to generate mutant TB has been previously shown to increase the thermostability of the enzyme. To further improve the stability of this mutant, we introduced a disulfide bridge (C109-C153) into the TB mutant, generating TS. To assess the effect of the disulfide bridge in the wild-type enzyme, the S109C-N153C mutation was also introduced into XYNB, resulting in XS. The mutants were expressed in Pichia pastoris, the recombinant enzymes were purified, and the effect of temperature and pH on enzymatic activity was characterized. Introduction of the disulfide bridge (C109-C153) into XYNB (XS variant) and TB (TS variant) increased the thermostability up to 2.8-fold and 12.4-fold, respectively, relative to XYNB, after incubation at 70 degrees C, pH 6.0, for 20 min. In addition, a synergistic effect of the disulfide bridge and the N-terminus replacement was observed, which extended the half-life of XYNB from 3 to 150 min. Moreover, XS and TS displayed better resistance to acidic conditions compared with the respective enzymes that did not contain a disulfide bridge.

An Enzyme-Linked Immunosorbent Assay (ELISA) to Determine the Specificity of the Sugar-Binding Protein NgcE, a Component of the ABC Transporter forN-Acetylglucosamine inStreptomyces olivaceoviridis

The ATP-binding cassette (ABC) transporter Ngc for N-acetylglucosamine (GlcNAc) of the chitin-degrader Streptomyces olivaceoviridis comprises the solute-binding protein NgcE, which has highest affinity for GlcNAc and N,N'-diacetylchitobiose {(GlcNAc)2} and reduced affinity for longer chitooligomers. NgcE was used to develop a generally applicable enzyme-linked immunosorbent assay (ELISA) system. As a prerequisite, the reducing end of (GlcNAc)2 was coupled with the ethylamino group of 2-(4-aminophenyl)ethylamine. The resulting conjugate was linked with amino groups of bovine serum albumin (BSA) to gain the neoglycoprotein BSA-APEA-(GlcNAc)2, which was fixed to wells in microtitre-plates. The NgcE protein was shown to bind efficiently to the immobilized BSA-APEA-(GlcNAc)2. In competition assays, the affinity of NgcE was 1,000-fold higher for GlcNAc and (GlcNAc)2 than for (GlcNAc)3 and (GlcNAc)4. These results are consistent with those previously obtained by surface plasmon resonance. Since the ELISA method can be performed very rapidly at low cost, it should be an efficient general tool to determine the affinity of a ligand to its cognate solute-binding protein.

Improvement of the Thermostability of Xylanase by N-terminus Replacement

The hybrid xylanase TB was constructed by the substitution of the N-terminus segment of the Streptomyces olivaceoviridis xylanase XYNB with the corresponding region of Thermomonospora fusca xylanase TfxA. The hybrid gene tb, encoding the TB, was correctly expressed in E. coli BL21 and Pichia pastoris GS115. The TB was purified and its enzymatic properties were determined. The results revealed that the optimal temperature and optimal pH of TB were at 70 °C and 6.0, which have been obviously improved compared with those of XYNB. The thermostability of the TB was all about six-fold of XYNB' s after incubating the properly diluted enzyme solutions at 80 °C and 90 °C for 3 min, respectively. The pH stability of TB was 5-9 (pH range in which the relative activity was above 50 %), which was narrower than that of XYNB. Still, TB remains a high specific activity, as does XYNB. Analysis of a homology modeling and sequence similarity were used to reveal the factors influencing the enzymatic properties of TB and the relationship between structure and function of xylanase was discussed.

Mutational analysis of the binding affinity and transport activity for N-acetylglucosamine of the novel ABC transporter Ngc in the chitin-degrader Streptomyces olivaceoviridis.

The highly differentiated bacterium Streptomyces olivaceoviridis efficiently hydrolyses chitin, a highly abundant natural polysaccharide, to low molecular weight products including N-acetylglucosamine (NAG) and N,N' -diacetylchitobiose (chitobiose). NAG is taken up by a PTS (phosphoenolpyruvate-dependent phosphotransferase system) which includes the PtsC2 protein, and via the ABC (ATP-binding cassette) transporter Ngc, which itself includes the substrate-binding protein NgcE. This is at present the only ABC transporter which is known to mediate specific uptake of NAG (K(m) 0.48 microM, V(max) 1.3 nmol/min/mg dry weight) and is competitively inhibited by chitobiose (K(i) 0.68 microM). The latter finding suggests that the Ngc system transports both NAG and chitobiose efficiently. To identify amino acid residues required for the function of NgcE, either the wild-type or one of several mutant forms of the ngcE gene was introduced into the strain S. olivaceoviridis DeltaNgcE/DeltaPtsC1/DeltaPtsC2, which lacks both functional transport systems for NAG, and chromosomal recombinants were selected. Based on the in vivo transport parameters of the recombinants, and the in vitro binding characteristics of the corresponding purified proteins, the following conclusions can be drawn. (1) Replacement of the C-terminally located residue Y396 by A (Y396A) has little effect on ligand-binding or transport parameters. The W395A mutation also induced little change in the substrate affinity in vitro, but it led in vivo to a marked increase (11 fold) in K(m), and enhanced V(max) (by 1.5 fold). (2) The amino acids Y201 and W280 both contribute (51% and 38%) to the ligand-binding capacity of NgcE. They are both very important for the in vivo function of the complete transport apparatus; strains expressing either Y201A or W280A show drastically (100 or 150 times) enhanced K(m) values. (3) The concomitant presence of either Y200 and W280 or Y201 and W280 is essential for the function of NgcE. (4) Y201 is located within a tyrosyl-rich motif. This has been found to share some features with the ligand-binding site of amelogenins (enamel matrix proteins), which interact with NAG residues in glycoconjugates. In addition, it is distantly related to the ligand-binding site(s) in the plant-lectins UDA ( Urtica dioicaagglutinin, specific for NAG and its oligomers) and WGA (wheat germ agglutinin, which recognises a motif comprising three consecutive NAG residues).

Streptomyces olivaceoviridis possesses a phosphotransferase system that mediates specific, phosphoenolpyruvate-dependent uptake of N-acetylglucosamine.

We recently described the ABC transporter Ngc (encoded by the ncgEFG operon) from Streptomyces olivaceoviridis, the first of its kind to be shown to transport N-acetylglucosamine and N,N'-diacetylchitobiose (chitobiose). A chromosomal mutant carrying a disruption of the ngcE gene, which encodes the sugar binding protein, was still able to transport N-acetylglucosamine. This phenotype can now be attributed to a functional phosphoenolpyruvate (PEP)-dependent phosphotransferase system (PTS). Two adjacent homologous genes, ptsC1 and ptsC2, were identified, and deduced to encode proteins which are 56% identical and can be predicted to contain eight transmembrane regions. PtsC1 (432 amino acids) and PtsC2 (403 residues) each correspond to a single EIIC domain; such domains are otherwise known only in several bacterial multidomain permeases for glucose/mannose or N-acetylglucosamine. The C-terminal sequences of PtsC1 and PtsC2 correspond to the motifs LKTPGREP and LPTRGRES, respectively. The ptsB gene located upstream of ptsC1 is predicted to encode a homologue of the EIIB domains usually found in bacterial multidomain permeases. Physiological and biochemical analyses of ngcE mutants carrying disruptive insertions in ptsC1 or ptsC2 or both revealed that, when grown on N-acetylglucosamine, the membrane component PtsC2, unlike PtsC1, mediates PEP-dependent, specific (K(m)=5 micro M) transport of N-acetylglucosamine, but not of other hexoses. Cross-complementation of membrane and cytoplasmic fractions from the various mutants led to the conclusion that S. olivaceoviridis also expresses the functional soluble components HPr, EI and EIIA of the PTS system. During growth on xylose, uptake of this pentose occurred if ptsC1 or ptsC2 was intact, but not in a mutant containing disrupted forms of both genes.

The novel Streptomyces olivaceoviridis ABC transporter Ngc mediates uptake of N-acetylglucosamine and N,N'-diacetylchitobiose.

During cultivation in the presence of N-acetylglucosamine or chitin, Streptomyces olivaceoviridis mycelium efficiently takes up [(14)C]-labelled N-acetylglucosamine. Uptake of the labelled compound can be completely inhibited by unlabelled N-acetylglucosamine and partially by chitobiose. After extraction of the membrane with Triton X-100, two forms of a protein that binds to N-acetylglucosamine and N, N'-diacetylchitobiose (chitobiose) were purified to homogeneity by two consecutive rounds of anionic exchange chromatography. The protein was named NgcE. Using surface plasmon resonance, its binding parameters were determined. It showed highest affinity for N-acetylglucosamine (K(D)=8.28 x 10(-9) M) and for chitobiose (K(D)=2.87 x 10(-8) M). Varying equilibrium dissociation constants in the micromolecular range were ascertained for chitotetraose (K(D)=4.5 x 10(-6) M), chitopentaose (K(D)=1.03 x 10(-6) M) and chitohexaose (K(D)=3.02 x 10(-6) M); the lowest value was measured for chitotriose (K(D)=19.4 x 10(-6) M). After having determined the sequences of several internal peptides from the binding protein by Edman degradation, the corresponding ngcE gene, which encodes a predicted lipid-anchored protein, was identified by reverse genetics. Using a genomic phage library of S. olivaceoviridis genes encoding two other membrane proteins (named NgcF and NgcG) were identified adjacent to ngcE. Each of these is predicted to have six membrane-spanning helices and a consensus motif for integral membrane proteins characteristic of ABC transporters. In addition, the gene for a predicted regulator protein (NgcR) was detected. The ngcEFG operon lacks a gene for an ATP-hydrolysing protein. NgcE is a new member of the CUT-1 family of ABC transporters for carbohydrates. Comparative studies of the wild-type and a mutant strain carrying an insertion within the ngc operon clearly demonstrate that the Ngc system mediates the uptake of N-acetylglucosamine and chitobiose in vivo.

Effect of the culture filtrates ofStreptomyces on growth and productivity of wheat plants

Streptomyces olivaceoviridis, S. rimosus andS. rochei proved to possess a high capacity for the production of auxins, gibberellins and cytokinin-like substances, together with substantial levels of α-amylase and proteinase. Grain priming with culture filtrates ofS. olivaceoviridis, S. rimosus orS. rochei appeared to enhance growth vigor and crop yield of wheat plants. In the majority of cases, the culture filtrate ofS. olivaceoviridis appeared to be the most effective in this respect. The present results are discussed in relation to the indirect role played by these bacteria in producing plant growth-regulating substances and their effects on growth and yield of wheat.

Specific interaction of the Streptomyces chitin-binding protein CHB1 with alpha-chitin--the role of individual tryptophan residues.

Streptomyces olivaceoviridis secretes a so far unique protein of 18.7 kDa (CHB1) which lacks catalytic activity. It interacts highly specifically with alpha-chitin, but not with beta-chitin, chitosan, or cellulose. Each of the five codons for tryptophan (Trp) in the chb1 gene was replaced by those for leucine (Leu) or tyrosine (Tyr). Eight corresponding mutant proteins and the wild-type protein were purified to homogeneity and their binding capacity to alpha-chitin was determined. The relative affinities to anti-CHB1 antibodies, the kinetics of binding, the dissociation constants, circular dichroism, and fluorescence emission spectra for three mutant types were compared to the characteristics of CHB1. The presented data lead to the following conclusions. (a) CHBI presents a highly flexible protein lacking alpha-helices. (b) Replacement of each of the buried Trp residues (Trp134 and Trp184) leads to conformational alterations and, in due course, to a considerably reduced binding affinity of the protein. (c) The exchange of the exposed Trp 57 by either Leu or Tyr results in relatively slight topological changes, but entails a loss of binding capacity of about 90%. (d) The dissociation constant was highest for the mutant protein [L57]CHB1 (2.17 microM), followed by [L134]CHB1 (0.91 microM) and [L184]CHB1 (0.26 microM), and lowest for the progenitor CHB1 (0.11 microM), indicating its strong affinity to the unsoluble substrate. (e) The data suggest that the exposed Trp57 contributes directly and significantly to the interaction of CHB1 with alpha-chitin.

Production and processing of a 59-kilodalton exochitinase during growth of Streptomyces lividans carrying pCHIO12 in soil microcosms amended with crab or fungal chitin.

Streptomyces lividans (pCHIO12), which carries the previously cloned Streptomyces olivaceoviridis exo-chiO1 gene on a multicopy vector, secretes a 59-kDa exochitinase, consisting of a catalytic domain (40 kDa), a central fibronectin type III-like module, and a chitin-binding domain (12 kDa). The propagation rate of S. lividans (pCHIO12) was higher in soil microcosms amended with fungal mycelia than in those containing crab chitin. Comparative biochemical and immunological studies allowed the following conclusions to be drawn. Within soil microcosm systems amended with crab shell chitin or chitin-containing Aspergillus proliferans mycelia, the strain expressed the clones exo-chiO1 gene and produced high quantities of a 59-kDa exochitinase. The enzyme was preferentially attached via its binding domain to the pellet from soil or liquid cultures. In contrast, truncated forms of 47, 40, and 25 kDa could be easily extracted from soil. The relative proportions of the 59-kDa enzyme and its truncated forms varied depending on the source of chitin and differed in soil and in liquid cultures.

Binding and Substrate Specificities of a Streptomyces olivaceoviridis Chitinase in Comparison with Its Proteolytically Processed Form

Binding and Substrate Specificities of a Streptomyces olivaceoviridis Chitinase in Comparison with Its Proteolytically Processed Form

The novel lectin-like protein CHB1 is encoded by a chitin-inducible Streptomyces olivaceoviridis gene and binds specifically to crystalline alpha-chitin of fungi and other organisms.

The chb1 gene, which encodes the unique lectin-like alpha-chitin-binding protein CHB1 of Streptomyces olivaceoviridis, was cloned. Transformants of Streptomyces lividans harbouring the plasmid pCHB10 overproduced the extracellular CHB1 protein; the protein showed neither enzymatic nor antifungal activity. Biochemical analyses and immunofluorescence microscopy indicated that CHB1 binds strongly to alpha-chitin, but neither to chitosan and beta-chitin, nor to various types of cellulose. Within hyphae of fungi, the relative location of crystalline chitin was visualized with fluorescein-labelled CHB1. These studies suggest that the new protein could serve as a tool to identify alpha-chitin within different organisms. The chb1 gene consists of a reading frame of 603 bp and its transcription occurred only if the Streptomyces strain was cultivated with chitin as the sole carbon source. The deduced mature CHB1 protein (18.7 kDa) shows no apparent similarity to any known protein. Within a region containing 100 residues of the deduced CHB1 protein, four tryptophan and two asparagine residues as well as one glycine and one cysteine residue were identified, the relative positions of which are analogous to those of several cellulose-binding domains of bacterial glycohydrolases. The results of spectroscopical studies suggest a possible involvement of tryptophan residues in the interaction of CHB1 with alpha-chitin.