Existence of rare actinobacterial forms in the Indian sector of Southern Ocean: 16 S rRNA based metabarcoding study.

The significance of the Southern Ocean (SO) as a sink of atmospheric CO2 and other greenhouse gases is well established. Earlier studies have highlighted the role of microbes in various SO ecosystem processes. However, the diversity and role of actinobacteria in the Indian sector of SO (ISO) water and sediments are unknown. This study aimed to analyze the diversity of actinobacteria in water and sediment samples of SO based on amplicon microbiome analyses. The taxonomic analysis identified a total number of 27 phyla of which Proteobacteria (40.2%), Actinobacteria (13.6%), and Firmicutes (8.7%) were found to be dominant. The comparative study of water and sediment samples revealed the dominance of different actinobacteria in water and sediments. While the order Streptomycetales was dominant in the water samples, Micrococcales was found to be dominant in the sediment samples. The genus level analysis found the presence of eight and seventeen genera in the sediment and water samples, respectively. The genus Streptomyces, Saccharopolyspora, Nocardioides, Sva0996 marine group, and Mycobacterium were seen both in sediment and water samples. Marmoricola, Ilumatobacter, and Glaciihabitans were observed only in sediment samples whereas Rhodococcus, Corynebacterium, Micrococcus, Turicella, Pseudonocardia, Bifidobacterium, Nesterenkonia, Collinsella, Knoellia, Cadidatus, Actinomarina, Libanicoccus and Cutibacterium were noticed exclusively in water samples. Our study also emphasizes the need for further detailed study to understand the links between actinobacterial diversity and their ecological functions in the ISO. The available metabarcoding data paves the way for future research in cultivable forms of novel and rare Actinobacteria for their bioprospecting applications.

Bioactive compound from marine seagrass Streptomyces argenteolus TMA13: combatting fish pathogens with time-kill kinetics and live-dead cell imaging.

Actinobacteria, pervasive in aquatic and terrestrial environments, exhibit a filamentous morphology, possess DNA with a specific G + C content and production of numerous secondary metabolites. This study, focused on actinobacteria isolated from marine seagrass, investigating their antibacterial activity against fish pathogens. Among 28 isolates, Streptomyces argenteolus TMA13 displayed the maximum zone of inhibition against fish pathogens-Aeromonas hydrophila (10 mm), Aeromonas caviae (22 mm), Edwardsiella tarda (17 mm), Vibrio harveyi (22 mm) and Vibrio anguillarum (12 mm) using the agar plug method. Optimization of this potent strain involved with various factors, including pH, temperature, carbon source and salt condition to enhance both yield production and antibacterial efficacy. In anti-biofilm assay shows the maximum percentage of inhibition while increasing concentration of TMA13 extract. Minimal Inhibitory Concentration (MIC) and Minimal Bactericidal Concentration (MBC) assays with TMA13 crude extract demonstrated potent activity against fish pathogens at remarkably low concentrations. Time-kill kinetics assay showcased growth curve variations over different time intervals for all fish pathogens treated with a 100 µg/ml concentration of crude extract, indicating a decline in cells viability and progression into the death phase. Additionally, fluorescence microscopic visualization of bacterial cells exposed to the extracts emitting green and red fluorescence, enabling live-dead cell differentiation was also studied. Further characterization of the crude extract through GC-MS and FT-IR analyses performed and identified secondary metabolites with functional groups exhibiting significant antibacterial activity. This study elucidates the capacity of Streptomyces argenteolus TMA13 to enhance the production of antibiotic compounds effective against fish pathogens.

Exploration of fish gut-associated actinobacteria for its antifouling activity.

An attempt has been made to screen the fish gut-associated actinobacterial cultures for antifouling compounds. Fifteen morphologically distinct bacterial cultures were recovered from the biofouling samples scraped from the boat surfaces and other maritime structures in the Kovalam beach (Lat. 12.7870°N; Long. 80.2504°E) coastal areas in Tamil Nadu, India. All the bacterial isolates were identified at generic level from which two isolates namely KB6 and KB7 as Staphylococcus sp. were found the exhibit strong biofilm formation. Four actinobacterial strains viz., IM20, PYA9, F8, and SQA4 were evaluated for inhibitory properties against biofouling bacteria using the agar plug method. Strain IM20 which strongly inhibited the biofouling bacteria was chosen as prospective strain for further studies. When compared to submerged fermentation, IM20 produced a high amount of antifouling chemicals on the agar surface fermentation. Among the solvents tested, better extraction of antifouling compounds was seen in ethyl acetate extract. Antifouling compound production by the strain IM20 was found to be influenced by a number of variables such as glucose, fructose, glutamine, malt extract, pH 7 and 9, temperature 30 and 40° C and NaCl at 2.5% and 5% concentrations. Gas chromatography-mass spectrometry (GC-MS) analysis of the strain IM20 extract revealed the presence of pyrrolo (1,2-a]pyrazine-1,4-dione, hexahydro) in significant amount. In the present study, the fish gut-associated Streptomyces sp. IM20 was identified as an unusual and newly added source for the isolation of antifouling compounds.

Antibacterial and Anti-HIV Metabolites from Marine Streptomyces albus MAB56 Isolated from Andaman and Nicobar Islands, India.

Marine-derived actinobacteria have tremendous potential to produce novel metabolites with diverse biological activities. The Andaman coast of India has a lot of microbial diversity, but it is still a relatively unknown ecology for isolating novel actinobacteria with beneficial bioactive compounds. We have isolated 568 actinobacterial strains from mangrove rhizosphere sediments and sponge samples. Crude extracts from 75 distinct strains were produced by agar surface fermentation and extracted using ethyl acetate. In the disc diffusion method, 25 actinobacterial strains showed antimicrobial activity; notably, the strain MAB56 demonstrated promising broad-spectrum activity. Strain MAB56 was identified as Streptomyces albus by cultural, microscopic, and molecular methods. Conditions for bioactive metabolites from MAB56 were optimized and produced in a lab-scale fermenter. Three active metabolites (C1, C2, and C3) that showed promising broad-spectrum antimicrobial activity were isolated through HPLC-based purification. Based on the UV, FT-IR, NMR, and LC-MS analysis, the chemical nature of the active compounds was confirmed as 12-methyltetradecanoic acid (C1), palmitic acid (C2), and tridecanoic acid (C3) with molecular formulae C14H28O2, C16H32O2, and C13H26O2, respectively. Interestingly, palmitic acid (C2) also exhibited anti-HIV activity with an IC50 value of < 1 µg/ml. Our findings reveal that the actinobacteria from the Andaman marine ecosystems are promising for isolating anti-infective metabolites.

A Novel Finding: 2,4-Di-tert-butylphenol from Streptomyces bacillaris ANS2 Effective Against Mycobacterium tuberculosis and Cancer Cell Lines.

The aim of the present study is to identify actinobacteria Streptomyces bacillaris ANS2 as the source of the potentially beneficial compound 2,4-di-tert-butylphenol, describe its chemical components, and assess its anti-tubercular (TB) and anti-cancer properties. Ethyl acetate was used in the agar surface fermentation of S. bacillaris ANS2 to produce the bioactive metabolites. Using various chromatographic and spectroscopy analyses, the potential bioactive metabolite separated and identified as 2,4-di-tert-butylphenol (2,4-DTBP). The lead compound 2,4-DTBP inhibited 78% and 74% of relative light unit (RLU) decrease against MDR Mycobacterium tuberculosis at 100ug/ml and 50ug/ml concentrations, respectively. The Wayne model was used to assess the latent/dormant potential in M. tuberculosis H37RV at various doses, and the MIC for the isolated molecule was found to be 100ug/ml. Furthermore, the molecular docking of 2,4-DTBP was docked using Autodock Vinasuite onto the substrate binding site of the target Mycobacterium lysine aminotransferase (LAT) and the grid box was configured for the docking run to cover the whole LAT dimer interface. At a dosage of 1 mg/ml, the anti-cancer activity of the compound 2,4-DTBP was 88% and 89% inhibited against the HT 29 (colon cancer) and HeLa (cervical cancer) cell lines. According to our literature survey, this present finding may be the first report on anti-TB activity of 2,4-DTBP and has the potential to become an effective natural source and the promising pharmaceutical drug in the future.

In-vivo studies on Transitmycin, a potent Mycobacterium tuberculosis inhibitor.

This study involves the in-vitro and in-vivo anti-TB potency and in-vivo safety of Transitmycin (TR) (PubChem CID:90659753)- identified to be a novel secondary metabolite derived from Streptomyces sp (R2). TR was tested in-vitro against drug resistant TB clinical isolates (n = 49). 94% of DR-TB strains (n = 49) were inhibited by TR at 10µg ml-1. In-vivo safety and efficacy studies showed that 0.005mg kg-1 of TR is toxic to mice, rats and guinea pigs, while 0.001mg kg-1 is safe, infection load did not reduce. TR is a potent DNA intercalator and also targets RecA and methionine aminopeptidases of Mycobacterium. Analogue 47 of TR was designed using in-silico based molecule detoxification approaches and SAR analysis. The multiple targeting nature of the TR brightens the chances of the analogues of TR to be a potent TB therapeutic molecule even though the parental compound is toxic. Analog 47 of TR is proposed to have non-DNA intercalating property and lesser in-vivo toxicity with high functional potency. This study attempts to develop a novel anti-TB molecule from microbial sources. Though the parental compound is toxic, its analogs are designed to be safe through in-silico approaches. However, further laboratory validations on this claim need to be carried out before labelling it as a promising anti-TB molecule.

Actinobacteria as a source of biofertilizer/biocontrol agents for bio-organic agriculture.

The global human population keeps growing and natural energy supplies are depleting, creating a threat to environmental demands, food security, and energy supply. As a result, increased agricultural output is required to accomplish the rising population's food demands. A strong reliance on chemical fertilizers to boost food production has harmed the environment and human health, and it is becoming too expensive as well. One of the potential solution to this problem is to use beneficial microorganisms as a substitute for artificial fertilizers in food production. Actinobacteria have been used as the most successful and long-lasting microorganisms throughout evolution. They are thought to be one of the most primordial living forms on our planet. Actinobacteria, particularly Streptomyces, have proved their ability to formulate biofertilizers in the agricultural sector by supplying nutrients to plants for better growth, increasing yield, managing abiotic and biotic stress, and resisting phytopathogen assault. This review describes the mechanism of actinobacterial biofertilizers used in the current agricultural market and their challenges and future importance to sustainable agriculture.

Exploring Newer Biosynthetic Gene Clusters in Marine Microbial Prospecting.

Marine microbes genetically evolved to survive varying salinity, temperature, pH, and other stress factors by producing different bioactive metabolites. These microbial secondary metabolites (SMs) are novel, have high potential, and could be used as lead molecule. Genome sequencing of microbes revealed that they have the capability to produce numerous novel bioactive metabolites than observed under standard in vitro culture conditions. Microbial genome has specific regions responsible for SM assembly, termed biosynthetic gene clusters (BGCs), possessing all the necessary genes to encode different enzymes required to generate SM. In order to augment the microbial chemo diversity and to activate these gene clusters, various tools and techniques are developed. Metagenomics with functional gene expression studies aids in classifying novel peptides and enzymes and also in understanding the biosynthetic pathways. Genome shuffling is a high-throughput screening approach to improve the development of SMs by incorporating genomic recombination. Transcriptionally silent or lower level BGCs can be triggered by artificially knocking promoter of target BGC. Additionally, bioinformatic tools like antiSMASH, ClustScan, NAPDOS, and ClusterFinder are effective in identifying BGCs of existing class for annotation in genomes. This review summarizes the significance of BGCs and the different approaches for detecting and elucidating BGCs from marine microbes.

Biocontrol Streptomyces Induces Resistance to Bacterial Wilt by Increasing Defense-Related Enzyme Activity in Solanum melongena L.

Streptomyces strains were isolated from rhizosphere soil and evaluated for in vitro plant growth and antagonistic potential against Ralstonia solanacearum. Based on their in vitro screening, seven Streptomyces were evaluated for plant growth promotion (PGP) and biocontrol efficacy by in-planta and pot culture study. In the in-planta study, Streptomyces-treated eggplant seeds showed better germination percentage, plant growth, and disease occurrence against R. solanacearum than the control treatment. Hence, all seven Streptomyces cultures were developed as a bioformulation by farmyard manure and used for pot culture study. The highest plant growth, weight, and total chlorophyll content were observed in UP1A-1-treated eggplant followed by UP1A-4, UT4A-49, and UT6A-57. Similarly, the maximum biocontrol efficacy was observed in UP1A-1-treated eggplants against bacterial wilt. The biocontrol potential of Streptomyces is also confirmed through metabolic responses by assessing the activities of the defense-related enzymes peroxidase (POX), polyphenol oxidase (PPO), and phenylalanine ammonia-lyase (PAL) and as well as the levels of total phenol. Treatment with UP1A-1/ UT4A-49 and challenge with R. solanacearum led to maximum changes in the activities of POX, PPO, and PAL and the levels of total phenol in the eggplants at different time intervals. Alterations in enzymes of UP1A-1 treatment were related to early defense responses in eggplant. Therefore, the treatment with UP1A-1 significantly delayed the establishment of bacterial wilt in eggplant. Altogether, the present study suggested that the treatment of Streptomyces maritimus UP1A-1 fortified farmyard manure has improved the plant growth and stronger disease control against R. solanacearum on eggplant.

Anti-Biofilm Activity and Biocontrol Potential of Streptomyces Cultures Against Ralstonia solanacearum on Tomato Plants.

Biological control of phytopathogen is a promising approach when compared to the use of chemical agents. In the present study, seven Streptomyces cultures showing promising anti biofilm activity against Ralstonia solanacearum was mixed individually with farmyard manure. All the Streptomyces fortified farmyard manure (SFYM) were screened for plant growth promotion and control of bacterial wilt caused by R. solanacearum on tomato. Further, the ability of SFYM on stimulating the production of defense-related enzymes in R. solanacearum-inoculated tomato plants was investigated. When compared to the control tomato plants, the SFYM-treated plants had longer shoot and root length along with higher fresh and dry weight. The maximum level of chlorophyll was observed in the plants treated with strain UP1A-1 (2.21 ± 0.18 mg g-1). Strain UP1A-1 also showed maximum of 96.8 ± 1.4% biocontrol efficacy in tomato plants challenged with R. solanacearum. In addition, the UP1A-1 treated tomato plants showed maximum accumulation of total phenolics (3.02 ± 0.09 mg g-1) after 6 days of pathogen inoculation (DPI). Similarly, tomato plants treated with UP1A-1 showed highest level of peroxides, polyphenol oxidase and phenylalanine ammonia lyase during 1-9 DPI. Findings of present study revealed that the Streptomyces culture UP1A-1 fortified farm yard manure could be applied as an eco-friendly alternative to synthetic agents for controlling bacterial wilt in tomato plants.

Anti freeze proteins (Afp): Properties, sources and applications - A review.

Extreme cold marine and freshwater temperatures (below 4 °C) induce massive deterioration to the cell membranes of organisms resulting in the formation of ice crystals, consequently causing organelle damage or cell death. One of the adaptive mechanisms organisms have evolved to thrive in cold environments is the production of antifreeze proteins with the functional capabilities to withstand frigid temperatures. Antifreeze proteins are extensively identified in different cold-tolerant species and they facilitate the persistence of cold-adapted organisms by decreasing the freezing point of their body fluids. Various structurally diverse types of antifreeze proteins detected possess the ability to modify ice crystal growth by thermal hysteresis and ice recrystallization inhibition. The unique properties of antifreeze proteins have made them a promising resource in industry, biomedicine, food storage and cryobiology. This review collates the findings of the various studies carried out in the past and the recent developments observed in the properties, functional mechanisms, classification, distinct sources and the ever-increasing applications of antifreeze proteins. This review also summarizes the possibilities of the way forward to identify new avenues of research on anti-freeze proteins.

Metagenomic 16S rDNA amplicon data of microbial diversity and its predicted metabolic functions in the Southern Ocean (Antarctic).

Antarctica holds about 70% of all the freshwater on the planet in the form of ice. The seawater, it chills, affect the currents and temperature everywhere. Global warming risks the melting of the icecaps as it has already increased the ocean temperature by 1 °C to the West Antarctic peninsula since 1955. A better understanding of the microbial community in this extreme environment of utmost importance is of interest to the scientific community. Herein, we document our metagenomics analysis of the microbial diversity and abundance in the Southern Ocean [Lat 55″ 33' 396 S; Lon 55″ 31' 448 E] using Next Generation Sequencing (NGS), QIIME 1.9.1, Silvangs and a naïve Bayesian classifier. Such metagenomics data hold the potential to aid predictive analysis, which is critical to our understanding of the dynamics of the microbial communities and their role in the Southern Ocean at present and in the future.

Bioactive Potential of Actinomycetes from Less Explored Ecosystems against Mycobacterium tuberculosis and Other Nonmycobacterial Pathogens.

Bioactive potential of actinomycetes isolated from certain less explored Indian ecosystems against Mycobacterium tuberculosis and other nonmycobacterial pathogens was investigated. Actinomycetes were isolated from the soil samples collected from desert, coffee plantation, rubber forest, and hill area and their cultural and micromorphological characteristics were studied. Crude extracts were prepared by agar surface fermentation and tested against M. tuberculosis isolates by luciferase reporter phage (LRP) assay at 100 µg/mL. Activity against nonmycobacterial pathogens was studied by agar plug method. Totally 54 purified cultures of actinomycetes including 43 Streptomyces and 11 non-Streptomyces were isolated. While screening for antitubercular activity, extracts of 39 actinomycetes showed activity against one or more M. tuberculosis isolates whereas 27 isolates exhibited antagonistic activity against nonmycobacterial pathogens. In particular crude extracts from sixteen actinomycete isolates inhibited all the three M. tuberculosis isolates tested. Findings of the present study concluded that less explored ecosystems investigated in this study are the potential resource for bioactive actinomycetes. Further purification and characterization of active molecule from the potential extracts will pave the way for determination of MIC, toxicity, and specificity studies.