Metagenomic Insights Unveil the Dominance of Undescribed Actinobacteria in Pond Ecosystem of an Indian Shrine

Metagenomic analysis holds immense potential for identifying rare and uncharacterized microorganisms from many ecological habitats. Actinobacteria have been proved to be an excellent source of novel antibiotics for several decades. The present study was designed to delineate and understand the bacterial diversity with special focus on Actinobacteria from pond sediment collected from Sanjeeviraya Hanuman Temple, Ayyangarkulam, Kanchipuram, Tamil Nadu, India. The sediment had an average temperature (25.32%), pH (7.13), salinity (0.960 mmhos/cm) and high organic content (10.7%) posing minimal stress on growth condition of the microbial community. Subsequent molecular manipulations, sequencing and bioinformatics analysis of V3 and V4 region of 16S rRNA metagenomics analysis confirmed the presence of 40 phyla, 100 classes, 223 orders, 319 families and 308 genera in the sediment sample dominated by Acidobacteria (18.14%), Proteobacteria (15.13%), Chloroflexi (12.34), Actinobacteria (10.84%), Cyanobacteria (5.58%), Verrucomicrobia (3.37%), Firmicutes (2.28%), and, Gemmatimonadetes (1.63%). Among the Actinobacteria phylum, Acidothermus (29.68%) was the predominant genus followed by Actinospica (17.65%), Streptomyces (14.64%), Nocardia (4.55%) and Sinomonas (2.9%). Culture-dependent isolation of Actinobacteria yielded all strains of similar morphology to that of Streptomyces genus which clearly indicating that the traditional based technique is incapable of isolating majority of the non-Streptomyces or the so called rare Actinobacteria. Although Actinobacteria were among the dominant phylum, a close look at the species level indicated that only 15.2% within the Actinobacterial phylum could be assigned to cultured species. This leaves a vast majority of the Actinobacterial species yet to be explored with possible novel metabolites have special pharmaceutical and industrial application. It also indicates that the microbial ecology of pond sediment is neglected fields which need attention.


INTRODUCTION
forward and reverse primer sequences (Supplementary data 1) were implemented to create a 117 single amplicon of approximately 250bp. Concentrations were adjusted to 4uM and prepared for 118 loading on the Illumina Miseq according to Illumina's 16S metagenomics protocol. Samples 119 were pooled, denatured, and loaded on the Illumina Miseq at 8pM and sequenced paired end (2 x 120 300) using a MiSeq® Reagent Kitv3. 121 Bioinformatics and Statistical Analysis 122 QIIME 1.9.1 [31] pipeline was used for the entire downstream analysis. Quality check was 123 performed using FastQC0.11.7 [36] and PHRED score reads with >Q30 were considered for 124 further analysis. High quality reads were adapter trimmed and the paired-end reads were stitched 125 using FLASH 1.2.11 [32] to make consensus FASTA sequences. Consensus reads were formed 126 with 0 mismatch having an average contig length of 350 to 450bp and queried to UCHIME [33] 127 to remove all the chimeric sequence which was subsequently pooled and clustered into 128 Operational Taxonomic Units (OTUs) based on their sequence similarity using Uclust (similarity 129 cutoff = 0.97) [35]. Representative sequence was identified for each OTU against SILVA OTUs mycelial fragmentation were observed under the bright field microscope at 40x magnification. Klebsiella pneumoniae. The bacterial strains were grown in nutrient agar medium at 37°C.

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Actinobacterial cultures were grown on YEME (Yeast Extract-Malt Extract) agar plates for 10 150 days at 28°C. Bacterial pathogens were spread on LB (Luria bertani) modified agar plate using 151 sterile cotton swab. Agar plug with 5mm diameter which contains the secreted Actinobacteria 152 metabolites were cut from the YEME agar medium in sterile condition and placed over LB 153 modified agar seeded with test pathogens. All the plates were incubated at 37ºC for 24 hours.

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Zone of inhibition was measured after incubation and expressed in millimetre in diameter.

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Pond sediment physico-chemical properties 158 The physico-chemical property of the studied sediment sample is summarized in Table 1. The 159 overall physico-chemical properties of the sample was moderate with an electrical conductivity 160 (EC) and total salinity of the sediment of 0.952 mmhos/cm and 0.960 mmhos/cm respectively 161 and the water content of the sediment at the sampling moments was medium. Although calcium 162 and zinc were the dominant cation in sample, the sampling area showed to be dynamic with 163 respect to salinity contributions from different anions and cations from one year to the other. We 164 observed a neutral pH (7.13) and higher organic matter (10.7%) content in the sample retrieved 165 in January 2018.
The AKTS-1 microbiota resulted in 514249 high-quality reads with an average read length of 168 250 bp and 55.09 % total GC content. Base quality score of each cycle in more than 80% of the 169 total reads had phred score greater than 30 (>Q30; error-probability >=0.001). We observed 170 513710 reads passed mismatch filter which were further processed for removal of chimera and  Table 2). On ISP2 agar medium, Actinobacterial strains produced powdery growth (n 10) 190 whereas the remaining eight strains produced rough (n 5) and leather (n 3)  Freshwater bodies account merely 1% of the total water on earth with uneven distribution around 199 the globe. However, freshwater is necessary for majority of plants, animals and for our survival.    Table 2). It is apparent that the discovery of novel rare Actinobacteria can be expected to 271 provide new bioactive compounds [26,37,42].

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In the past years, focus on natural product discovery from Actinobacteria shifted from The study shows the presence of unexplored rare and uncultured Actinobacteria through 291 metagenomics. The work also clearly demonstrates that most of the actinobacteria strains have 292 high antimicrobial activity. However, the current traditional selective media is not suitable for 293 harvesting rare and uncultured Actinobacteria community. Hence, potential novel secondary 294 metabolite from rare Actinobacteria, especially from exotic environmental habitats, is still 295 underexplored. Focus has to be highlighted to develop novel insolation strategies and media to 296 isolate such rare microbes. Neglecting the rare isolates would understate the immense potential 297 of novel bioactive compounds and their role as a keystone species in the environment.