Rimosus Research Articles

Initial pH determines the morphological characteristics and secondary metabolite production in Aspergillus terreus and Streptomyces rimosus cocultures

The influence of the initial pH on the morphology and secondary metabolite production in cocultures and axenic cultures of Aspergillus terreus and Streptomyces rimosus was investigated. The detected secondary metabolites (6 of bacterial and 4 of fungal origin) were not found in the cultures initiated at pH values less than or equal to 4.0. The highest mean levels of oxytetracycline were recorded in S. rimosus axenic culture at pH 5.0. Initiating the axenic culture at pH 5.9 led to visibly lower product levels, yet the presence of A. terreus reduced the negative effect of non-optimal pH and led to higher oxytetracycline titer than in the corresponding S. rimosus axenic culture. The cocultivation initiated at pH 5.0 or 5.9 triggered the formation of oxidized rimocidin. The products of A. terreus were absent in the cocultures. At pH 4.0, the striking morphological differences between the coculture and the axenic cultures were recorded.

The repertoire and levels of secondary metabolites in microbial cocultures depend on the inoculation ratio: a case study involving Aspergillus terreus and Streptomyces rimosus

ObjectiveThe aim of this study was to determine the influence of the inoculation volume ratio on the production of secondary metabolites in submerged cocultures of Aspergillus terreus and Streptomyces rimosus.ResultsThe shake flask cocultures were initiated by using 23 inoculum variants that included different volumes of A. terreus and S. rimosus precultures. In addition, the axenic controls were propagated in parallel with the cocultures. UPLC‒MS analysis revealed the presence of 15 secondary metabolites, 12 of which were found both in the “A. terreus vs. S. rimosus” cocultures and axenic cultures of either A. terreus or S. rimosus. The production of the remaining 3 molecules was recorded solely in the cocultures. The repertoire and quantity of secondary metabolites were evidently dependent on the inoculation ratio. It was also noted that detecting filamentous structures resembling typical morphological forms of a given species was insufficient to predict the presence of a given metabolite.ConclusionsThe modification of the inoculation ratio is an effective strategy for awakening and enhancing the production of secondary metabolites that are not biosynthesized under axenic conditions.

Oxytetracycline hyper-production through targeted genome reduction of Streptomyces rimosus.

Most biosynthetic gene clusters (BGC) encoding the synthesis of important microbial secondary metabolites, such as antibiotics, are either silent or poorly expressed; therefore, to ensure a strong pipeline of novel antibiotics, there is a need to develop rapid and efficient strain development approaches. This study uses comparative genome analysis to instruct rational strain improvement, using Streptomyces rimosus, the producer of the important antibiotic oxytetracycline (OTC) as a model system. Sequencing of the genomes of two industrial strains M4018 and R6-500, developed independently from a common ancestor, identified large DNA rearrangements located at the chromosome end. We evaluated the effect of these genome deletions on the parental S. rimosus Type Strain (ATCC 10970) genome where introduction of a 145 kb deletion close to the OTC BGC in the Type Strain resulted in massive OTC overproduction, achieving titers that were equivalent to M4018 and R6-500. Transcriptome data supported the hypothesis that the reason for such an increase in OTC biosynthesis was due to enhanced transcription of the OTC BGC and not due to enhanced substrate supply. We also observed changes in the expression of other cryptic BGCs; some metabolites, undetectable in ATCC 10970, were now produced at high titers. This study demonstrated for the first time that the main force behind BGC overexpression is genome rearrangement. This new approach demonstrates great potential to activate cryptic gene clusters of yet unexplored natural products of medical and industrial value.IMPORTANCEThere is a critical need to develop novel antibiotics to combat antimicrobial resistance. Streptomyces species are very rich source of antibiotics, typically encoding 20-60 biosynthetic gene clusters (BGCs). However, under laboratory conditions, most are either silent or poorly expressed so that their products are only detectable at nanogram quantities, which hampers drug development efforts. To address this subject, we used comparative genome analysis of industrial Streptomyces rimosus strains producing high titers of a broad spectrum antibiotic oxytetracycline (OTC), developed during decades of industrial strain improvement. Interestingly, large-scale chromosomal deletions were observed. Based on this information, we carried out targeted genome deletions in the native strain S. rimosus ATCC 10970, and we show that a targeted deletion in the vicinity of the OTC BGC significantly induced expression of the OTC BGC, as well as some other silent BGCs, thus suggesting that this approach may be a useful way to identify new natural products.

Evaluation of the Structure-Function Relationship of SGNH Lipase from Streptomyces rimosus by Site-Directed Mutagenesis and Computational Approach.

Streptomyces rimosus extracellular lipase (SrL) is a multifunctional hydrolase belonging to the SGNH family. Here site-directed mutagenesis (SDM) was used for the first time to investigate the functional significance of the conserved amino acid residues Ser10, Gly54, Asn82, Asn213, and His216 in the active site of SrL. The hydrolytic activity of SrL variants was determined using para-nitrophenyl (pNP) esters with C4, C8, and C16 fatty acid chains. Mutation of Ser10, Asn82, or His216, but not Gly54, to Ala abolished lipase activity for all substrates. In contrast, the Asn213Ala variant showed increased enzymatic activity for C8 and C16 pNP esters. Molecular dynamics (MD) simulations showed that the interactions between the long alkyl chain substrate (C16) and Ser10 and Asn82 were strongest in Asn213Ala SrL. In addition to Asn82, Gly54, and Ser10, several new constituents of the substrate binding site were recognized (Lys28, Ser53, Thr89, and Glu212), as well as strong electrostatic interactions between Lys28 and Glu212. In addition to the H bonds Ser10-His216 and His216-Ser214, Tyr11 interacted strongly with Ser10 and His216 in all complexes with an active enzyme form. A previously unknown strong H bond between the catalytically important Asn82 and Gly54 was uncovered, which stabilizes the substrate in an orientation suitable for the enzyme reaction.

Effects of the Coculture Initiation Method on the Production of Secondary Metabolites in Bioreactor Cocultures of Penicillium rubens and Streptomyces rimosus.

Bioreactor cocultures involving Penicillium rubens and Streptomyces rimosus were investigated with regard to secondary metabolite production, morphological development, dissolved oxygen levels, and carbon substrate utilization. The production profiles of 22 secondary metabolites were analyzed, including penicillin G and oxytetracycline. Three inoculation approaches were tested, i.e., the simultaneous inoculation of P. rubens with S. rimosus and the inoculation of S. rimosus delayed by 24 or 48 h relative to P. rubens. The delayed inoculation of S. rimosus into the P. rubens culture did not prevent the actinomycete from proliferating and displaying its biosynthetic repertoire. Although a period of prolonged adaptation was needed, S. rimosus exhibited growth and the production of secondary metabolites regardless of the chosen delay period (24 or 48 h). This promising method of coculture initiation resulted in increased levels of metabolites tentatively identified as rimocidin B, 2-methylthio-cis-zeatin, chrysogine, benzylpenicilloic acid, and preaustinoid D relative to the values recorded for the monocultures. This study demonstrates the usefulness of the delayed inoculation approach in uncovering the metabolic landscape of filamentous microorganisms and altering the levels of secondary metabolites.

Resistom <i>Streptomyces rimosus</i> - a reservoir of resistance genes to aminoglycoside antibiotics

The study of aminoglycoside acetyltransferases in actinobacteria of the genus Streptomyces is an integral part of the study of soil bacteria as the main reservoir and possible source of drug resistance genes. Previously, in the strain Streptomyces rimosus ATCC 10970 (producing oxyteteracycline), which is resistant to most natural aminoglycoside antibiotics, we have identified and biochemically characterized 3 aminoglycoside phosphotransferases, which cause resistance to kanamycin, neomycin, paromomycin, streptomycin, and hygromycin B. In the presented work, it was shown that resistance to other AGs in this strain is associated with the presence of the enzyme aminoglycoside acetyltransferase, belonging to the AAC(2′) subfamily. Induction of the expression of the gene, designated by us as aac(2′)-If, in Escherichia coli cells determines resistance to a wide range of natural aminoglycoside antibiotics (neomycin, gentamicin, tobramycin, sisomycin, and paromomycin) and to an increase in the minimum inhibitory concentrations of these antibiotics.

Resistome in Streptomyces rimosus - A Reservoir of Aminoglycoside Antibiotics Resistance Genes.

Investigation of aminoglycoside acetyltransferases in actinobacteria of the genus Streptomyces is an integral part of the study of soil bacteria as the main reservoir and possible source of drug resistance genes. Previously, we have identified and biochemically characterized three aminoglycoside phosphotransferases, which cause resistance to kanamycin, neomycin, paromomycin, streptomycin, and hygromycin B in the strain Streptomyces rimosus ATCC 10970 (producing oxytetracycline), which is resistant to most natural aminoglycoside antibiotics. In the presented work, it was shown that the resistance of this strain to other AGs is associated with the presence of the enzyme aminoglycoside acetyltransferase, belonging to the AAC(2') subfamily. Induction of the expression of the gene, designated by us as aac(2')-If, in Escherichia coli cells determines resistance to a wide range of natural aminoglycoside antibiotics (neomycin, gentamicin, tobramycin, sisomycin, and paromomycin) and increases minimum inhibitory concentrations of these antibiotics.

Biological Management of Bacterial Leaf Blight of Rice (Xanthomonas oryzae pv. oryzae) through Rice Rhizosphere Antagonist Actinobacteria

Rice (Oryza sativa L.) is a staple food crop for more than two billion people around the world. Bacterial leaf blight (BLB) caused by Xanthomonas oryzae pv. oryzae (Xoo) (Ishimiya) Swings et al. is one of the most destructive diseases of rice in all the major rice-growing. Use of antibiotics is the common practice for the management of BLB, but it has detrimental effect on human health and environment. Hence other eco-friendly strategies like biological control and host plant resistance (HPR) should be effectively exploited for BLB management. An attempt was made to manage the disease using actinobacteria isolated from rice rhizosphere. Among sixteen actinobacterial isolates, six were selected for secondary screening based on disc diffusion assay. Out of six isolates AUDT-502 produced maximum zone of inhibition (1.81 cm) followed by AUDT-508 (1.62 cm) and were evaluated under glasshouse condition. AUDT-502 (Streptomyces rimosus) appeared as the most promising bio agent which was able to inhibit the pathogen (Xoo) both in in vitro and in vivo condition. AUDT-502 increased the plant growth parameters like number of tillers (5.73), plant height (56.85 cm), root length (34.33cm) and dry root weight (3.90g) and also reduced the leaf blight lesion length and per cent disease index (4.43 per cent) significantly when compared to other treatments. The current study thus showed that there was a good correlation between in vitro inhibition of the pathogen and the in vivo control of the disease and AUDT-502 (Streptomyces rimosus) appears to be a hopeful bioagent for the management of bacterial leaf blight of rice.

Double-reporter-guided targeted activation of the oxytetracycline silent gene cluster in Streptomyces rimosus M527.

In Streptomyces rimosus M527, the oxytetracycline (OTC) biosynthetic gene cluster is not expressed under laboratory conditions. In this study a reported-guided mutant selection (RGMS) procedure was used to activate the cluster. The double-reporter plasmid pAGT was constructed in which gusA encoding a β-glucuronidase and tsr encoding a thiostrepton resistance methyltransferase were placed under the control of the native promoter of oxyA gene (PoxyA ). Plasmid pAGT was introduced and integrated into the chromosome of S. rimosus M527 by conjugation, yielding initial strain M527-pAGT. Subsequently, mutants of M527-pAGT were generated by using ribosome engineering technology. The mutants harboring activated OTC gene cluster were selected based on visual observation of GUS activity and thiostrepton resistance. Finally, mutant M527-pAGT-R7 was selected producing OTC in a concentration of 235.2 mg/L. In this mutant transcriptional levels of oxysr genes especial oxyAsr gene were increased compared to wild-type strain S. rimosus M527. The mutant M527-pAGT-R7 showed antagonistic activities against Gram-negative and Gram-positive strains. All data indicate that the OTC gene cluster was successfully activated using the RGMS method.

Psychophysiological Responses of Humans during Seed-Sowing Activity Using Soil Inoculated with Streptomyces rimosus.

Electroencephalogram (EEG) responses and serum metabolite levels were used to investigate the effects of horticultural activities (seed-sowing) on the psychophysiological aspects of adults based on the presence or absence of the soil microorganism Streptomyces rimosus. In this case, 31 adults were subjected to seed-sowing activities using S. rimosus inoculated (experimental group) and medium (control group) soils. EEG was measured to analyze the resulting psychophysiological response, and blood samples (5 mL) were collected. The relative gamma power (RG), relative high beta (RHB), and SEF 50 and SEF 90 were significantly higher in the right than in the left occipital lobe (p < 0.05). In both occipital lobes, ratios of SMR to theta (RST), mid beta to theta (RMT), and SMR-mid beta to theta (RSMT) were high (p < 0.05). GC-TOF-MS-based serum metabolite analysis detected 33 metabolites. Compared to the control group, the experimental group showed a lower content of amino acids (except aspartic acid), lipids, and C6 sugar monomers after the activity (p < 0.05). Aminomalonic acid was decreased, and aspartic acid was increased (p < 0.05). This study confirmed a positive effect on improving the concentration and attention of adults when seed-sowing activity was performed using S. rimosus-inoculated soil.

(Un)coupling the factors contributing to the interfacial activation of Streptomyces rimosus lipase: computational and spectrophotometric study

Streptomyces rimosus lipases (SrLip) present an enzyme class that catalyzes the hydrolysis of triacylglycerols producing fatty acids and glycerols. The abrupt increase in catalytic activity of SrLip when found at water-oil interface is assigned as interfacial activation (IA). Since the latter is a complex function of interface charge, chemical composition, as well as substrate orientation and concentration, the role of individual factors and their combinations that trigger IA remains rather elusive. The aim of this work is to unravel the contribution of particular factor in IA by examining SrLip activity toward p-nitrophenylpalmitate (p-NPP) as a substrate incorporated in three chemically different self-assembled aggregates: gum Arabic, sodium deoxycholate and liposomes made of 1,2-dioleyl-sn-glycero-3-phosphocholine (DOPC) lipids, and at two different pH values (pH = 6.8 and pH = 10.0). Spectrophotometric (UV/Vis) study revealed that SrLip catalyzes p-NPP hydrolysis when the substrate is embedded in sodium deoxycholate at both pH values, suggesting that the coupling of favorable interface charge and substrate orientation drives the interfacial activation. Moreover, especially in alkaline conditions (pH = 10.0), hydrolysis of the ester bond of the substrate incorporated in DOPC bilayers occurs when appropriate orientation, concentration and arrangement are accomplished. All experiments were conducted in parallel on an inactive mutant SrLipS10/A as a reference. The molecular-level details of particular interface and accompanied substrates examined by all-atom molecular dynamics simulations revealed that the availability of p-NPP at these three interfaces decreases in the following order: sodium-deoxycholate > DOPC > model gum Arabic.

Streptomyces rimosus rhizobacteria and Glomus mosseae mycorrhizal fungus inoculation alleviate salinity stress in grapevine through morphophysiological changes and nutritional balance

Arbuscular mycorrhizal fungi (AMF) and plant growth-promoting rhizobacteria (PGPR) have positive impacts on growth and production of plants under saline condition. However, their interactive effects on plant behavior under salt stress remained unclear. To address this, a glasshouse experiment was conducted to elucidate the effect of the single and combined (co-inoculation) application of Glomus mosseae (Gm) AMF and Streptomyces rimosus (Sr) PGPR on salt tolerance of Sultana grapevine (Vitis vinifera L.). Based on results, the combined inoculation with Gm AMF and Sr PGPR markedly improved shoot and root morphometric traits and also soluble carbohydrate, soluble proteins, proline content compared to those vines inoculated with these symbionts alone or uninoculated vines under saline condition. The highest main root length was related to inoculated vines with Sr PGPR, which was 18.5% higher than uninoculated control plants under saline condition. Under saline condition, co-inoculation with Gm AMF and Sr PGPR decreased leaf necrotic, leaf ion leakage and malondialdehyde content by 31%, 43% and 38% but increased SPAD index, relative water content, catalase and guaiacol peroxidase activity by 45%, 26%, 30% and 53% compared with control uninoculated plant. Among salt stressed plants, the single inoculated vines with Gm AMF showed the highest total phenol and flavonoid content compared to control plants. In vines grown under saline condition, the highest content of leaf P, Mg, Fe, Zn and Mn were related to Gm plus Sr -inoculated vines. Leaf NO−3 and K of all inoculated vines did not showed statistically significant difference with each other's. Under saline condition, root NO3, K, Mg, Fe, Zn and Mn did not showed significant difference among inoculated plants but differed significantly with control vines. Therefore, combined application of Gm AMF and Sr PGPR assisted host plants to uptake higher nutrients during salt stress; confer salt tolerance to vines by increase in osmoregulants, phenolic compounds and antioxidant enzymes activities in their tissues.

Psychophysiological and Metabolomics Responses of Adults during Horticultural Activities Using Soil Inoculated with Streptomyces rimosus: A Pilot Study.

This study compared the physiological effects at a metabolomics level with autonomic nervous system responses in adults during soil mixing activities, based on the presence or absence of Streptomyces rimosus in the soil. Thirty adult participants performed soil mixing activities for 5 min using sterilized soil with culture media and Streptomyces rimosus, respectively. Blood samples were drawn twice from each participant after each activity. Electroencephalograms were measured during the activity. Serum metabolites underwent metabolite profiling by gas chromatography, followed by multivariate analyses. Serum brain-derived neurotrophic factor and C-reactive protein levels were measured by Enzyme-Linked Immunosorbent Assay. Soil-emitted volatile organic compounds were identified via solid-phase microextraction and gas chromatography–mass spectroscopy, followed by multivariate analyses. The volatile compound analysis revealed that the terpenoid and benzoid compounds, geosmin, and 2-methylisoborneol were greater in soil with Streptomyces rimosus. Serum metabolomics revealed that the treatment group (soil inoculated with Streptomyces rimosus) possessed relatively higher levels of serotonin compared to the control group (soil mixed with culture media), and serum C-reactive protein levels were significantly lower in the treatment group. In the treatment group, the electroencephalogram revealed that alpha band activity of the occipital lobe increased. This study concludes that Streptomyces rimosus soil contact can positively affect human metabolic and autonomic reactions. Therefore, this pilot study confirmed the possible role of soil microorganisms in horticultural activities for psychophysiological effects in humans.

Phosphoproteome Dynamics of Streptomyces rimosus during Submerged Growth and Antibiotic Production.

ABSTRACTStreptomyces rimosus is an industrial streptomycete, best known as a producer of oxytetracycline, one of the most widely used antibiotics. Despite the significant contribution of Streptomyces species to the pharmaceutical industry, most omics analyses have only been conducted on the model organism Streptomyces coelicolor. In recent years, protein phosphorylation on serine, threonine, and tyrosine (Ser, Thr, and Tyr, respectively) has been shown to play a crucial role in the regulation of numerous cellular processes, including metabolic changes leading to antibiotic production and morphological changes. In this study, we performed a comprehensive quantitative (phospho)proteomic analysis during the growth of S. rimosus under conditions of oxytetracycline production and pellet fragmentation. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis combined with phosphopeptide enrichment detected a total of 3,725 proteins, corresponding to 45.6% of the proteome and 417 phosphorylation sites from 230 phosphoproteins. Significant changes in abundance during three distinct growth phases were determined for 494 proteins and 98 phosphorylation sites. Functional analysis revealed changes in phosphorylation events of proteins involved in important cellular processes, including regulatory mechanisms, primary and secondary metabolism, cell division, and stress response. About 80% of the phosphoproteins detected during submerged growth of S. rimosus have not yet been reported in streptomycetes, and 55 phosphoproteins were not reported in any prokaryote studied so far. This enabled the creation of a unique resource that provides novel insights into the dynamics of (phospho)proteins and reveals many potential regulatory events during antibiotic production in liquid culture of an industrially important bacterium.IMPORTANCE Streptomyces rimosus is best known as a primary source of oxytetracycline (OTC). The significant global market value of OTC highlights the need for a better understanding of the regulatory mechanisms that lead to production of this antibiotic. Our study provides, for the first time, a detailed insight into the dynamics of (phospho)proteomic profiles during growth and antibiotic production in liquid culture of S. rimosus. Significant changes in protein synthesis and phosphorylation have been revealed for a number of important cellular proteins during the growth stages that coincide with OTC production and morphological changes of this industrially important bacterium. Most of these proteins have not been detected in previous studies. Therefore, our results significantly expand the insight into phosphorylation events associated with important cellular processes and antibiotic production; they also greatly increase the phosphoproteome of streptomycetes and contribute with newly discovered phosphoproteins to the database of prokaryotic phosphoproteomes. This can consequently lead to the design of novel research directions in elucidation of the complex regulatory network in Streptomyces.

Momomycin, an Antiproliferative Cryptic Metabolite from the Oxytetracycline Producer Streptomyces rimosus.

Natural products provide an important source of pharmaceuticals and chemical tools. Traditionally, assessment of unexplored microbial phyla has led to new natural products. However, with every new microbe, the number of orphan biosynthetic gene clusters (BGC) grows. As such, the more difficult proposition is finding new molecules from well-studied strains. Herein, we targeted Streptomyces rimosus, the widely-used oxytetracycline producer, for the discovery of new natural products. Using MALDI-MS-guided high-throughput elicitor screening (HiTES), we mapped the global secondary metabolome of S. rimosus and structurally characterized products of three cryptic BGCs, including momomycin, an unusual cyclic peptide natural product with backbone modifications and several non-canonical amino acids. We elucidated important aspects of its biosynthesis and evaluated its bioactivity. Our studies showcase HiTES as an effective approach for unearthing new chemical matter from "drained" strains.

Momomycin, an Antiproliferative Cryptic Metabolite from the Oxytetracycline Producer Streptomyces rimosus

Natural products provide an important source of pharmaceuticals and chemical tools. Traditionally, assessment of unexplored microbial phyla has led to new natural products. However, with every new microbe, the number of orphan biosynthetic gene clusters (BGC) grows. As such, the more difficult proposition is finding new molecules from well-studied strains. Herein, we targeted Streptomyces rimosus, the widely-used oxytetracycline producer, for the discovery of new natural products. Using MALDI-MS-guided high-throughput elicitor screening (HiTES), we mapped the global secondary metabolome of S. rimosus and structurally characterized products of three cryptic BGCs, including momomycin, an unusual cyclic peptide natural product with back-bone modifications and several non-canonical amino acids. We elucidated important aspects of its biosynthesis and evaluated its bioactivity. Our studies showcase HiTES as an effective approach for unearthing new chemical matter from “drained” strains

Co-cultivation of filamentous microorganisms in the presence of aluminum oxide microparticles

In the present work, the approaches of submerged co-cultivation and microparticle-enhanced cultivation (MPEC) were combined and evaluated over the course of three case studies. The filamentous fungus Aspergillus terreus was co-cultivated with Penicillium rubens, Streptomyces rimosus, or Cerrena unicolor in shake flasks with or without the addition of aluminum oxide microparticles. The influence of microparticles on the production of lovastatin, penicillin G, oxytetracycline, and laccase in co-cultures was compared with the effects recorded for the corresponding monocultures. In addition, the quantitative analyses of morphological parameters, sugars consumption, and by-products formation were performed. The study demonstrated that the influence of microparticles on the production of a given molecule in mono- and co-culture may differ considerably, e.g., the biosynthesis of oxytetracycline was shown to be inhibited due to the presence of aluminum oxide in “A. terreus vs. S. rimosus” co-cultivation variants but not in S. rimosus monocultures. The differences were also observed regarding the morphological characteristics, e.g., the microparticles-induced changes of projected area in the co-cultures and the corresponding monocultures were not always comparable. In addition, the study showed the importance of medium composition on the outcomes of MPEC, as exemplified by lovastatin production in A. terreus monocultures. Finally, the co-cultures of A. terreus with a white-rot fungus C. unicolor were described here for the first time.Key points• Aluminum oxide affects secondary metabolites production in submerged co-cultures.• Mono- and co-cultures are differently impacted by the addition of aluminum oxide.• Effect of aluminum oxide on metabolites production depends on medium composition.

Antifungal activity of chitinase produced by Streptomyces species isolated from grassland soils in Futa Area, Akure

BackgroundThis study was carried out to investigate the antifungal activity of chitinase produced by Streptomyces species isolated from grassland soils against selected pathogenic fungi, namely: Magnaporthe oryzae, Fusarium graminearum, Rhizoctonia solani, Puccinia species and Botyrtis cinerea. Chitinase produced by Streptomyces albus was purified consecutively with dialysis, gel filtration and ion- exchange chromatography, the test isolate can be exploited for large scale production of chitinase.ResultsSeven Streptomyces species (Streptomyces rimosus, S. albus, S. riseoflavus, S. fumosus, S. spiralis, S. aureofaciens and S. flavogriseus) isolated from three different grassland soils were screened based on the morphology and their ability to produce chitinase. S. rimosus, S. fumosus, S. albus and S. flavogriseus were able to produce chitinase, although the enzyme chitinase was found to be higher in Streptomyces albus. The effect of environmental factors were determined on Streptomyces albus being the best chitinolytic producing isolate. Temperature of 30οC was favourable for Streptomyces albus and pH 6.0 was a growth factor for maximum chitinase production. The activity of the enzyme was stimulated by the addition of metal ions like Mn2+, K+, Na+, Mg+, Fe2+, Ca2+ but Zn2+ inhibited chitinase activity. Chitinase activity was favoured by maltose as carbon source and NH4 (SO4)2 as nitrogen source. Culture filtrate of Streptomyces albus was able to inhibit the growth of all the selected pathogenic fungi namely Magnaporthe oryzae, Fusarium graminearum, Rhizoctonia solani, Puccinia species and Botyrtis cinerea whereas Streptomyces rimosus has the least inhibitory activity.ConclusionsStreptomyces albus has the highest ability to produce chitinase which can act as antifungal for degradation of the cell wall of many phytopathogenic fungi.

PREPARATION, OPTIMIZATION, CHARACTERIZATION AND ANTIMICROBIAL ACTIVITY OF CHITOSAN AND CALCIUM NANOPARTICLES LOADED WITH STREPTOMYCES RIMOSUS EXTRACTED COMPOUNDS AS DRUG DELIVERY SYSTEMS

The present study aims to search for a nano-delivery system that enables access of antimicrobial compounds to the targeting site of the microbial pathogens. To achieve this objective, the antimicrobial compounds extracted from the local isolate Streptomyces rimosus were loaded on chitosan (CS) and calcium phosphate (CaP) nanoparticles (NPs) to facilitate drug delivery to some species of bacteria (Escherichia coli ATCC25922, Staphylococcus aureus ATCC25923 and Bacillus cereus ATCC6633) and the yeast Candida albicans ATCC10231. The best incubation period for the production of antifungal and antibacterial compounds, pH, temperature, carbon sources and nitrogen sources were around the third day, 7.0, 30°C, starch and potassium nitrate, respectively. The extracted compounds were identified using gas chromatography-mass spectrometry (GC-MS) analysis which revealed to the identification of nine antimicrobial organic acids. The prepared NPs were characterized using transmission electron microscopy (TEM) and Zeta potential analyser. Results showed those chitosan nanoparticles (CSNPs) were spherical shaped NPs with an average size ≈22 ± 2 nm. The CSNPs loaded with the antimicrobial molecule’s diameter was ranged from 55 upto 100 nm. Calcium phosphates nanoparticles (CaPNPs) were also spherical shaped NPs with diameter range from 70 to 95 nm. Loading of antimicrobial compounds onto CaPNPs made them appeared in rod shape with width varying from 14 to 27 nm and length from 274 to 397 nm. The synthesized NPs showed negative values confirming their high dispersity and long-term stability. The tested strains were resistant to solo NPs; but the loaded NPs with the extracted antimicrobial compounds, particularly the CaPNPs, augmented the potency of the isolated antimicrobial compounds, with emergence of differential antimicrobial activity. The activity of the NPs-bound antimicrobial compounds was more evident against bacteria than fungi, Gram-positive bacteria than Gram-negative bacteria and against B. cereus than S. aureus.

Enhanced Oxytetracycline Production by Streptomyces rimosus in Submerged Co-Cultures with Streptomyces noursei.

In the present study, Streptomyces rimosus was confronted with Streptomyces noursei, Penicillium rubens, Aspergillus niger, Chaetomium globosum, or Mucor racemosus in two-species submerged co-cultures in shake flasks with the goal of evaluating the oxytetracycline production and morphological development. The co-culture of S. rimosus with S. noursei exhibited stimulation in oxytetracycline biosynthesis compared with the S. rimosus monoculture, whereas the presence of M. racemosus resulted in a delay in antibiotic production. Different strategies of initiating the “S. rimosus + S. noursei” co-cultures were tested. The improvement in terms of oxytetracycline titers was recorded in the cases where S. noursei was co-inoculated with S. rimosus in the form of spores. As the observed morphological changes were not unique to the co-culture involving S. noursei, there was no evidence that the improvement of oxytetracycline levels could be attributed mainly to morphology-related characteristics.