Actinobacterial diversity in limestone deposit sites in Hundung, Manipur (India) and their antimicrobial activities.
ABSTRACT: Studies on actinobacterial diversity in limestone habitats are scarce. This paper reports profiling of actinobacteria isolated from Hundung limestone samples in Manipur, India using ARDRA as the molecular tool for preliminary classification. A total of 137 actinobacteria were clustered into 31 phylotypic groups based on the ARDRA pattern generated and representative of each group was subjected to 16S rRNA gene sequencing. Generic diversity of the limestone isolates consisted of Streptomyces (15 phylotypic groups), Micromonospora (4), Amycolatopsis (3), Arthrobacter (3), Kitasatospora (2), Janibacter (1), Nocardia (1), Pseudonocardia (1) and Rhodococcus (1). Considering the antimicrobial potential of these actinobacteria, 19 showed antimicrobial activities against at least one of the bacterial and candidal test pathogens, while 45 exhibit biocontrol activities against at least one of the rice fungal pathogens. Out of the 137 actinobacterial isolates, 118 were found to have at least one of the three biosynthetic gene clusters (PKS-I, PKS-II, NRPS). The results indicate that 86% of the strains isolated from Hundung limestone deposit sites possessed biosynthetic gene clusters of which 40% exhibited antimicrobial activities. It can, therefore, be concluded that limestone habitat is a promising source for search of novel secondary metabolites.
Project description:Moonmilk speleothems of limestone caves host a rich microbiome, among which Actinobacteria represent one of the most abundant phyla. Ancient medical texts reported that moonmilk had therapeutical properties, thereby suggesting that its filamentous endemic actinobacterial population might be a source of natural products useful in human treatment. In this work, a screening approach was undertaken in order to isolate cultivable Actinobacteria from moonmilk of the Grotte des Collemboles in Belgium, to evaluate their taxonomic profile, and to assess their potential in biosynthesis of antimicrobials. Phylogenetic analysis revealed that all 78 isolates were exclusively affiliated to the genus <i>Streptomyces</i> and clustered into 31 distinct phylotypes displaying various pigmentation patterns and morphological features. Phylotype representatives were tested for antibacterial and antifungal activities and their genomes were mined for secondary metabolite biosynthetic genes coding for non-ribosomal peptide synthetases (NRPSs), and polyketide synthases (PKS). The moonmilk <i>Streptomyces</i> collection was found to display strong inhibitory activities against a wide range of reference organisms, as 94, 71, and 94% of the isolates inhibited or impaired the growth of Gram-positive, Gram-negative bacteria, and fungi, respectively. Interestingly, 90% of the cave strains induced strong growth suppression against the multi-drug resistant <i>Rasamsonia argillacea</i>, a causative agent of invasive mycosis in cystic fibrosis and chronic granulomatous diseases. No correlation was observed between the global antimicrobial activity of an individual strain and the number of NRPS and PKS genes predicted in its genome, suggesting that approaches for awakening cryptic metabolites biosynthesis should be applied to isolates with no antimicrobial phenotype. Overall, our work supports the common belief that moonmilk might effectively treat various infectious diseases thanks to the presence of a highly diverse population of prolific antimicrobial producing <i>Streptomyces</i>, and thus may indeed constitute a promising reservoir of potentially novel active natural compounds.
Project description:Plant associated endophytic actinobacteria may contribute to plant growth and defense by direct or indirect methods. Our aim was to evaluate the plant growth promoting and antifungal activities of endophytic actinobacteria associated with Camellia spp. and related genera, Eurya to find potent plant growth promoting strains that could be applied in future microbe based bioformulations. We isolated 46 endophytic actinobacteria based on morphological characteristics of the isolates. 16S rRNA gene sequence analysis showed that the strains represented nine actinobacterial genera, Nocardia, Amycolatopsis, Streptomyces, Pseudonocardia, Kribbella, Actinomadura, Microbispora, Rothia and Saccharomonospora. In vitro functional characterization of the isolates for plant growth promoting (PGP) traits revealed many potent PGP isolates such as, SA1 and S43 which showed all the tested PGP traits, i.e., phosphate solubilization, indole-3-acetic acid (IAA), ammonia, siderophore and chitinase production. Out of the 46 endophytic actinobacteria isolates, 21 showed inhibition against atleast one test fungal phytopathogen and, isolates SA25 and SA29 exhibited broad spectrum antifungal activity against all the tested phytopathogens. Most of the endophytic actinobacteria isolates having antifungal activity were positive for the presence of chitinase, NRPS (Non-ribosomal peptides synthetase) or PKS-1 (Polyketide Synthase) gene, suggesting the presence of distinctive mechanisms to inhibit the growth of pathogenic plant fungi. ARDRA (Amplified Ribosomal DNA Restriction analysis) and BOX-PCR fingerprinting analysis of the potent isolates with antagonistic activity grouped the isolates into 5 and 4 separate clusters, respectively. In addition, an assessment using bonitur scale revealed the top ranked isolates based on their PGP and biocontrol traits. Further detection of IAA production by the top ranked actinobacterial isolates namely, SA1, T1LA3 and S85 by using thin-layer chromatography (TLC), high-performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LC-MS) was done. Endophytic actinobacteria isolates, namely, SA1, T1LA3, and SA14 were further tested for their efficacy in promoting the growth of commercial tea clones, namely, TV1, TV9, TV18, and TV22 in nursery conditions. All the endophytic isolates tested showed significant differences (P ≤ 0.05) in terms of plant growth promoting parameters in the treated plants compared to untreated control and may, thus be, deemed as potential candidates for application in bioformulations for tea growth.
Project description:Marine Actinobacteria, particularly coral-associated Actinobacteria, have attracted attention recently. In this study, the abundance and diversity of Actinobacteria associated with three types of coral thriving in a thermally stressed coral reef system north of the Arabian Gulf were investigated. Coscinaraea columna, Platygyra daedalea and Porites harrisoni have been found to harbor equivalent numbers of culturable Actinobacteria in their tissues but not in their mucus. However, different culturable actinobacterial communities have been found to be associated with different coral hosts. Differences in the abundance and diversity of Actinobacteria were detected between the mucus and tissue of the same coral host. In addition, temporal and spatial variations in the abundance and diversity of the cultivable actinobacterial communities were detected. In total, 19 different actinobacterial genera, namely Micrococcus, Brachybacterium, Brevibacterium, Streptomyces, Micromonospora, Renibacterium, Nocardia, Microbacterium, Dietzia, Cellulomonas, Ornithinimicrobium, Rhodococcus, Agrococcus, Kineococcus, Dermacoccus, Devriesea, Kocuria, Marmoricola, and Arthrobacter, were isolated from the coral tissue and mucus samples. Furthermore, 82 isolates related to Micromonospora, Brachybacterium, Nocardia, Micrococcus, Arthrobacter, Rhodococcus, and Streptomyces showed antimicrobial activities against representative Gram-positive and/or Gram-negative bacteria. Even though Brevibacterium and Kocuria were the most dominant actinobacterial isolates, they failed to show any antimicrobial activity, whereas less dominant genera, such as Streptomyces, did show antimicrobial activity. Focusing on the diversity of coral-associated Actinobacteria may help to understand how corals thrive under harsh environmental conditions and may lead to the discovery of novel antimicrobial metabolites with potential biotechnological applications.
Project description:Many bacteria belonging to the phylum of Actinobacteria are known as antagonists against phytpathogenic microbes. This study aimed to analyze the effect of ozone on the actinobacterial community of the rhizosphere of four years old European beech (Fagus sylvatica L.) trees during different time points of the vegetation period. Effects of ozone on the total community structure of Actinobacteria were studied based on the analysis of 16S rRNA gene amplicons. In addition effects of the ozone treatment on the diversity of potential biocontrol active Actionobacteria being able to produce antibiotics were characterized by using the type II polyketide synthases (PKS) genes as marker. Season as well as ozone treatments had a significant effect on parts of the actinobacterial rhizosphere community of European beech. However on the basis of the performed analysis, the diversity of Actinobacteria possessing type II PKS genes is neither affected by seasonal changes nor by the ozone treatments, indicating no influence of the investigated treatments on the biocontrol active part of the actinobacterial community.
Project description:Actinobacteria is found to have a potent metabolic activity against pathogens. The present study reveals the assessment of potent antifungal secondary metabolites from actinobacteria isolated from Indian marine mangrove sediments. The samples were collected from the coastal regions of Muthupet, Andaman and the Nicobar Islands. Identification was carried out using 16S rRNA analysis and biosynthetic genes (Polyketide synthase type I/II and Non-ribosomal peptide synthase) were screened. Actinobacteria were assayed for their antifungal activity against 16 clinical Candida albicans and the compound analysis was performed using gas chromatography-mass spectrometry GC-MS. The 31 actinobacterial strains were isolated and 16S rRNA gene sequencing revealed that this ecosystem is rich on actinobacteria, with Streptomyces as the predominant genus. The PCR based screening of biosynthetic genes revealed the presence of PKS-I in six strains, PKS-II in four strains and NRPS in 11 strains. The isolated actinobacteria VITGAP240 and VITGAP241 (two isolates) were found to have a potential antifungal activity against all the tested C. albicans. GC-MS results revealed that the actinobacterial compounds were belonging to heterocyclic, polyketides and peptides. Overall, the strains possess a wide spectrum of antifungal properties which affords the production of significant bioactive metabolites as potential antibiotics.
Project description:Actinobacteria occur in many environments and have the capacity to produce secondary metabolites with antibiotic potential. Identification and taxonomy of actinobacteria that produce antimicrobial substances is essential for the screening of new compounds, and sequencing of the 16S region of ribosomal DNA (rDNA), which is conserved and present in all bacteria, is an important method of identification. Melanized fungi are free-living organisms, which can also be pathogens of clinical importance. This work aimed to evaluate growth inhibition of melanized fungi by actinobacteria and to identify the latter to the species level. In this study, antimicrobial activity of 13 actinobacterial isolates from the genus Streptomyces was evaluated against seven melanized fungi of the genera Exophiala, Cladosporium, and Rhinocladiella. In all tests, all actinobacterial isolates showed inhibitory activity against all isolates of melanized fungi, and only one actinobacterial isolate had less efficient inhibitory activity. The 16S rDNA region of five previously unidentified actinobacterial isolates from Ilha do Mel, Paraná, Brazil, was sequenced; four of the isolates were identified as Streptomyces globisporus subsp. globisporus, and one isolate was identified as Streptomyces aureus. This work highlights the potential of actinobacteria with antifungal activity and their role in the pursuit of novel antimicrobial substances.
Project description:BACKGROUND: Bacterial aromatic polyketides are a pharmacologically important group of natural products synthesized by type II polyketide synthases (type II PKSs) in actinobacteria. Isolation of novel aromatic polyketides from microbial sources is currently impeded because of the lack of knowledge about prolific taxa for polyketide synthesis and the difficulties in finding and optimizing target microorganisms. Comprehensive analysis of type II PKSs and the prediction of possible polyketide chemotypes in various actinobacterial genomes will thus enable the discovery or synthesis of novel polyketides in the most plausible microorganisms. DESCRIPTION: We performed a comprehensive computational analysis of type II PKSs and their gene clusters in actinobacterial genomes. By identifying type II PKS subclasses from the sequence analysis of 280 known type II PKSs, we developed highly accurate domain classifiers for these subclasses and derived prediction rules for aromatic polyketide chemotypes generated by different combinations of type II PKS domains. Using 319 available actinobacterial genomes, we predicted 231 type II PKSs from 40 PKS gene clusters in 25 actinobacterial genomes, and polyketide chemotypes corresponding to 22 novel PKS gene clusters in 16 genomes. These results showed that the microorganisms capable of producing aromatic polyketides are specifically distributed within a certain suborder of Actinomycetales such as Catenulisporineae, Frankineae, Micrococcineae, Micromonosporineae, Pseudonocardineae, Streptomycineae, and Streptosporangineae. CONCLUSIONS: We could identify the novel candidates of type II PKS gene clusters and their polyketide chemotypes in actinobacterial genomes by comprehensive analysis of type II PKSs and prediction of aromatic polyketides. The genome analysis results indicated that the specific suborders in actinomycetes could be used as prolific taxa for polyketide synthesis. The chemotype-prediction rules with the suggested type II PKS modules derived using this resource can be used further for microbial engineering to produce various aromatic polyketides. All these resources, together with the results of the analysis, are organized into an easy-to-use database PKMiner, which is accessible at the following URL: http://pks.kaist.ac.kr/pkminer. We believe that this web-based tool would be useful for research in the discovery of novel bacterial aromatic polyketides.
Project description:Mangrove is a rich and underexploited ecosystem with great microbial diversity for discovery of novel and chemically diverse antimicrobial compounds. The goal of the study was to explore the pharmaceutical actinobacterial resources from mangrove soil and gain insight into the diversity and novelty of cultivable actinobacteria. Consequently, 10 mangrove soil samples were collected from Futian and Maoweihai of China, and the culture-dependent method was employed to obtain actinobacteria. A total of 539 cultivable actinobacteria were isolated and distributed in 39 genera affiliated to 18 families of 8 orders by comparison analysis of partial 16S rRNA gene sequences. The dominant genus was <i>Streptomyces</i> (16.0 %), followed by <i>Microbacterium</i> (14.5 %), <i>Agromyces</i> (14.3 %), and <i>Rhodococcus</i> (11.9 %). Other 35 rare actinobacterial genera accounted for minor proportions. Notably, 11 strains showed relatively low 16S rRNA gene sequence similarities (< 98.65 %) with validly described species. Based on genotypic analyses and phenotypic characteristics, 115 out of the 539 actinobacterial strains were chosen as representative strains to test their antibacterial activities against "ESKAPE" bacteria by agar well diffusion method and antibacterial mechanism by the double fluorescent protein reporter system. Fifty-four strains in 23 genera, including 2 potential new species, displayed antagonistic activity in antibacterial assay. Meanwhile, 5 strains in 3 genera exhibited inhibitory activity on protein biosynthesis due to ribosome stalling. These results demonstrate that cultivable actinobacteria from mangrove soil are potentially rich sources for discovery of new antibacterial metabolites and new actinobacterial taxa.
Project description:Endophytic actinobacteria exist widely in plant tissues and are considered as a potential bioresource library of natural products. Tea plants play important roles in human health and in the lifestyles of Asians, especially the Chinese. However, little is known about the endophytic actinobacteria of tea plants. In this study, 16 actinobacteria of 7 different genera and 28 actinobacteria of 8 genera were isolated and analyzed by 16S rRNA gene sequencing from tea cultivars of Zijuan and Yunkang-10 (Camellia sinensis var. assamica), respectively. The diversity of actinobacteria species from Zijuan were higher in July than December (6 vs. 3 genera), but the diversity of species from Yunkang-10 were higher in December than July (7 vs. 3 genera). No actinobacteria isolates were obtained from any tea cultivar in September. Ten isolates from Yunkang-10 exhibited antimicrobial activity against at least one human pathogenic microorganism (Staphylococcus epidermidis, Shigella flexneri, and Escherichia coli), but none of the isolates from Zijuan exhibited antimicrobial activities. Fourteen strains were further exammined the genes of polyketide synthetase (PKS)-I and PKS-II and non-ribosomal peptide synthetase (NRPS). Brevibacterium sp. YXT131 from Yunkang-10 showed strong inhibitory activity against S. epidermidis, Sh. flexneri, and E. coli, and PKS-I and PKS-II and NRPS genes were obtained from the strain. In in vitro assays, extracts from 14 actinobacteria that were tested for antibiotic biosynthetic genes showed no inhibition of concanavalin A (ConA)-induced murine splenocyte proliferation. In in vivo assays, the crude extract of YXT131 modulated the immune response by decreasing the proinflammatory cytokines interleukin (IL)-12/IL-23 p40 and tumor necrosis factor (TNF)-? in the serum of mice. These results confirm that endophytic actinobacteria from tea plants might be an undeveloped bioresource library for active compounds.
Project description:The important biological macromolecules, such as lipopeptide and glycolipid biosurfactant producing marine actinobacteria were analyzed and their potential linkage between type II polyketide synthase (PKS) genes was explored. A unique feature of type II PKS genes is their high amino acid (AA) sequence homology and conserved gene organization. These enzymes mediate the biosynthesis of polyketide natural products with enormous structural complexity and chemical nature by combinatorial use of various domains. Therefore, deciphering the order of AA sequence encoded by PKS domains tailored the chemical structure of polyketide analogs still remains a great challenge. The present work deals with an in vitro and in silico analysis of PKS type II genes from five actinobacterial species to correlate KS domain architecture and structural features. Our present analysis reveals the unique protein domain organization of iterative type II PKS and KS domain of marine actinobacteria. The findings of this study would have implications in metabolic pathway reconstruction and design of semi-synthetic genomes to achieve rational design of novel natural products.