Bacterial Diversity and Composition in Oylat Cave (Turkey) with Combined Sanger/Pyrosequencing Approach

Abstract

A b s t r a c tThe microbiology of caves is an important topic for better understanding subsurface biosphere diversity. The diversity and taxonomic composition of bacterial communities associated with cave walls of the Oylat Cave was studied first time by molecular cloning based on Sanger/pyrosequencing approach. Results showed an average of 1,822 operational taxonomic units per sample. Clones analyzed from Oylat Cave were found to belong to 10 common phyla within the domain Bacteria. Proteobacteria dominated the phyla, followed by Actino bacteria, Acidobacteria and Nitrospirae. Shannon diversity index was between to 3.76 and 5.35. The robust analysis conducted for this study demon strated high bacterial diversity on cave rock wall surfaces. This copy is for personal use only -distribution prohibited.Gulecal-Pektas Y. 70After collection, the samples were frozen on dry ice on site, and stored at -20°C upon return to the laboratory (Groth et al., 1999). Environmental DNA was extracted from samples using Fast DNA Spin kit for soil (MP biomedicals, Solon, OH USA).Clone library construction and Sanger sequence analysis. Total genomic DNA was used as a template for 16S rRNA PCR amplification using bacteria 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-GGTTACCTTGTTACGACTT-3') universal pri mers (Weisburg et al., 1991). Each 20 µl reaction mixture contained: l µl environmental DNA template, 2.25 mM MgCl 2 , 2 µl GeneAmp 10X PCR Buffer II (Applied Biosystems, Foster City, CA, USA), 100 µM dNTPs (SigmaAldrich, Saint Louis, MO, USA), 0.2 µM each primer, 2.5 U AmpliTaq Gold DNA Polymerase (Applied Biosystems, Carlsbad, CA, USA). Thermal cycling was as follows: initial denaturation 5 min at 94°C, 25 cycles of 94°C for 1 min, hybridization at 50°C for 25 s and elongation at 72°C for 2 min followed by a final elongation at 72°C for 20 min. PCR products were purified using a QIAquick kit (QIAGEN, Valencia, CA, USA), and were cloned into Escherichia coli hosts using the TOPO TA Cloning kit with the pCR 2.1 Vector (Invitrogen Corporation, Carlsbad, CA, USA). Plasmid DNA was extracted and purified using the Ultra Clean Standard Mini Plasmid Prep Kit (MoBio Laboratories). Cloning products were sequenced by TUBITAK MAM DNA Services Facility at Gebze, Turkey, using standard M13 primers.Partial sequences were assembled with CodonCode Aligner v.1.2.4 (CodonCode, USA) and manually checked. Assembled sequences were checked for chimera by Bellerophon server (Huber et al., 2004) and Chimera_Check v 2.7 (Cole et al., 2005). Sample sequences were aligned by BioEdit (Ibis Biosciences, Carlsbad, CA, USA). Phylogenetic analysis was performed in PAUP (Sinauer Associates, Sunderland, MA) using parsimony, neighbor-joining, and maximum likelihood analyses. The 16S rRNA gene sequences were submitted to the NCBI Gen Bank database under accession numbers JQ065958-JQ065959 and JQ219081-JQ219137.454 pyrosequencing and sequence analysis. For the pyrosequencing, the V6 region of the 16S rRNA gene was amplified using PCR with a bacterial primer set 967f...

Co-occurrence pattern and function prediction of bacterial community in Karst cave

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“…The Proteobacteria was the predominant group across all the three sample types. Proteobacteria, as a group of microbes responding to unstable carbon sources, was found to be the most abundant in some other cave ecosystems and various environments as well [25,[34][35][36][37]. The Firmicutes was the second abundant phylum found in soil samples in Zhijin cave, possibly due to their resistance towards nutrient stress and capability of survival under most extreme habitats [25,38].…”

Diversity, Distribution and Co-occurrence Patterns of Bacterial Communities in a Karst Cave System

“…Our results might be justified for two reasons: (1) Some of the aforementioned caves are so-called show-caves, with pre-historic paintings on their wall, whose increased availability of nutrients may lead to the dominance of Proteobacteria , as proposed by Tomczyk-Żak and Zielenkiewicz (2016). (2) Previous studies mainly focus on the surface of cave rock (Pasić et al, 2010; Engel et al, 2013; Ivanova et al, 2013; Gulecal-Pektas, 2016), while in our study, the surface of rock samples was first washed and attention was instead focused on bacteria living inside the rock or tightly attached to it. Remarkably, Pseudonocardia was identified as an indicator genus of rock bacterial community in our study.…”

“…Several studies have revealed that Proteobacteria can dominate cave rock ecosystems (Pasić et al, 2010; Engel et al, 2013; Ivanova et al, 2013; Gulecal-Pektas, 2016). We found that Actinobacteria was mostly predominant.…”

A Study on actinobacterial diversity of Hampoeil cave and screening of their biological activities

“…Normally, caves are ecosystems that has been considered as oligotrophic conditions (Airoldi and Cinelli, 1997) as well as less-explored microbial ecosystems (Barton and Jurado, 2007). Various microorganisms have been isolated from various caves worldwide, including Gram positive and Gram negative bacteria (Gulecal-Pektas, 2016, Yasir, 2018), protozoa (Kajtezović and Rubinić, 2013) and fungi (Belyagoubi et al, 2018, Cunningham et al, 1995).…”