1874-2858/19 Send Orders for Reprints to [email protected] 154 The Open Microbiology Journal Content list available at: https://openmicrobiologyjournal.com RESEARCH ARTICLE Taxonomic and Functional Annotation of Termite Degraded Butea monosperma (Lam.) Kuntze (Flame of the Forest) Ashwani Kumar1,*, Preeti Vyas1, Muneer A. Malla2 and Anamika Dubey1 1Department of Botany, Metagenomics and Secretomics Research Laboratory, Dr. Harisingh Gour University (A Central University), Sagar-470003, (M.P.), India 2Department of Zoology, Dr. Harisingh Gour University (A Central University), Sagar-470003, (M.P.), India Abstract: Background: Butea monosperma is an economically and medicinally important plant that grows all over India, however, the plant is highly susceptible to termite attack. The present study unravelled the bacterial community composition and their functional attributions from the termite degraded Butea. Methods: Total genomic DNA from termite degraded Butea monosperma samples was extracted and subjected to sequencing on Illumina's Miseq. The raw and unassembled reads obtained from high-throughput sequencing were used for taxonomic and functional profiling using different online and stand-alone softwares. Moreover, to ascertain the effect of different geographical locations and environmental factors, comparative analysis was performed using four other publically available metagenomes. Results: The higher abundance of Actinobacteria (21.27%), Proteobacteria (14.18%), Firmicutes (10.46%), and Bacteroidetes (4.11%) was found at the phylum level. The genus level was dominated by Bacillus (4.33%), Gemmatimonas (3.13%), Mycobacterium (1.82%), Acidimicrobium (1.69%), Thermoleophilum (1.23%), Nocardioides (1.44%), Terrimonas and Acidithermus (1.09%) and Clostridium (1.05%). Functional annotation of the termite degraded B. monosperma metagenome revealed a high abundance of ammonia oxidizers, sulfate reducers, dehalogenators, nitrate reducers, sulfide oxidizers, xylan degraders, nitrogen fixers and chitin degraders. Conclusion: The present study highlights the significance of the inherent microbiome of the degraded Butea shaping the microbial communities for effective degradation of biomass and different environmental toxicants. The unknown bacterial communities present in the sample can serve as enzyme sources for lignocelluloses degradation for biofuel production. Keywords: Butea monosperma, Termites, DNA sequencing, Lignocellulose, Biofuel, Environmental toxicants. Article History Received: March 13, 2019 Revised: May 12, 2019 Accepted: May 16, 2019 1. INTRODUCTION Locally, this plant is known as Palash, Palas, Mutthuga, Butea monosperma (Lam.) Kuntze is native to the tropical Bijasneha, Khakara, Dhak Chichra, Bastard Teak, Bengal and sub-tropical parts of the Indian Subcontinent and Southeast Kino, and Nourouc [2]. The crude extracts of various parts of Asia, and is distributed across India, Bangladesh, Nepal, Sri this plant has been reported to have antifungal, anti- Lanka, Myanmar, Thailand, Laos, Cambodia, Vietnam, inflammatory, antibacterial and hypoglycemic activities. The Malaysia, and western Indonesia. Butea is commonly known as plant has also been found to possess astringent, tonic, diuretics, ‘Flame of the forest’ and belongs to the family Fabaceae [1]. and aphrodisiac properties. The widespread uses of Butea in traditional systems of medicine have resulted in their extensive * Address correspondence to this author at the Department of Botany, chemical analysis for their bio-active components [3]. The Metagenomics and Secretomics Research Laboratory, Dr. Harisingh Gour University (A Central University), Sagar-470003,(M.P.), India; plant holds a significant place because of its medicinal and Tel: +917697432012; Fax: 7582264236; E-mail: [email protected] other miscellaneous uses of economic importance [4]. DOI: 10.2174/1874285801913010154, 2019, 13, 154-163 Taxonomic and Functional Annotation of Termite Degraded The Open Microbiology Journal, 2019, Volume 13 155 Morphologically, the plant exhibits distinct features with large 2. MATERIALS AND METHODS sized flowers that are arranged in a rigid racemes of 15 cm length. 3 flowers together form the tumid nodes of the dark 2.1. Sample Collection and Preparation olive-green velvety rachis. Flowers are used as vegetables by A total of nine samples from termites degraded Butea the tribal's and are also used for the preparation of dye for monosperma tree were randomly sampled from Jhira Ghati, colouring garments and for making skin antiseptic ointments Forest Area, Sagar, M.P. India (Latitude 23°12.070’N and [4]. The above mentioned reasons make Butea an important longitude 079°02.357E’) as shown in Fig. (2). All the collected and much sacred plant species. samples were properly labelled and packed in plastic bags, and Termites are ubiquitous and major constituents of the were immediately transported to the laboratory and stored at forest ecosystem in the tropical and subtropical areas [5]. Apart 4°C till further use. from Antarctica, termites are found in almost all the continents 2.2. DNA Extraction and 16S rRNA Amplicon Sequencing [6] and are well known for their capacity to damage, and destroy wood and wooden products of human homes, building In order to profile the full-scale taxonomic diversity, the materials, forests, agriculture crops and other commercial total bacterial genomic DNA was extracted from 250 products [7]. Termites, besides being small insects, have a mg/sample using the Mo Bio Power soil DNA isolation kit successful evolutionary history mainly because of their (MoBio Laboratories, Inc. CA, USA) as per the manufacturer’s sociality and capability to efficiently digest lignocellulose [8]. instructions. The purity and integrity of the extracted genomic Presently, these tiny creatures contribute more than 95% of the DNA were assessed through agarose gel electrophoresis (1%) insect biomass in the tropical soils [9]. About 3,000 species of and the concentration was quantified via NanoDrop 2000 spec- termites have been described so far, however, comparatively trophotometer (Thermo Scientific) by measuring the absor- only few species pose threat to wooden structures. The bance at 260/280 nm. Total genomic DNA extracted from three principal component of their diet is wood, which is also the replicates was pooled to one sample in order to cover the dominant structural element of the building construction maximum diversity. The DNA samples were stored at -20°C industry. Termites have the ability to degrade 65-99% and till sequencing. Illumina’s MiSeq system was used for the 16S 5-83% of the wood-cellulose and hemicelluloses and lignin rRNA amplicon sequencing of extracted total genomic DNA as within 24 hours under natural conditions [10, 11]. In fact, this instrument is a dominant platform for amplicon sequencing termites possess the inimitable characteristics in order to in microbial ecology studies due to its great flexibility, high- handle lignin-carbohydrate complexes efficiently. A variety of throughput, fast-turnaround time, longer sequence reads and lignocellulolytic enzymes do occur in the termite gut [12, 13]. higher accuracy. Termites, in spite of their small body size, harbor an abundant and astonishingly diverse gut microbiome, which represents 2.3. Taxonomic Profiling of Termite 16S rRNA Gene Using one of the most fascinating examples of symbiosis both within MG-RAST the microbes, as well as between animal and microbes [14]. It In this study, we have analysed the 16S rRNA gene is widely accepted that lignocellulose digestion in termites is sequences using Metagenomic Rapid Annotation Using closely related to the host as well as the specific flora of Subsystem Technology (MG-RAST) server which is a symbiotic microbes [15, 16]. Various evidences do suggest that comprehensive metagenome analysis server and offers a the lignin structure was modified in termites and that the number of advantages like open access, publicly available modification process begins in the foregut and continues in the metagenomes as well as comparative analysis of different mid gut [17, 18]. From the perspectives of both basic research metagenomes. The raw and unassembled sequences generated as well as biotechnology, there arises a substantial interest in from high-throughput sequencing via Illumina were uploaded accomplishing a clear perceptive of the diversity of biological to MG-RAST server v 4.0.3 (http://metagenomics.anl.gov/) for mechanisms that are employed during lignocellulose taxonomic annotation and profiling under MG-RAST ID degradation [19, 20]. Around the globe, termites comprise one number mgm4738027.3 [21]. The low quality regions were of the extremely successful groups of organisms that are trimmed using SolexaQA [22, 23] following downstream capable of degrading wood and are therefore vital for analysis by using RDP as the reference database with the expanding their roles both in carbon turnover and as possible parameters set as default. source of biocatalysts for the efforts that are aimed at converting the wood biomass into biofuels. The main aim of 2.4. Comparative Taxonomic and Functional Diversity for the present study is to unravel the structural composition and Selected samples by QIIME functional attributions of different microbial communities from QIIME is an open-source software pipeline built by the termite degraded B. monosperma and to study the variation making use of PyCogent toolkit [24] and is accessible at in microbial community