Characterization of Uncultured Genome Fragment from Soil Metagenomic Library Exposed Rare Mismatch of Internal Tetranucleotide Frequency.

Yunpeng Liu,Zhongli Cui,Ruifu Zhang,Nan Zhang,Lin Chen,Dongqing Yang,Qirong Shen

doi:10.3389/fmicb.2016.02081

Abstract

Exploring the genomic information of a specific uncultured soil bacterium is vital to understand its function in the ecosystem but is still a challenge due to the lack of culture techniques. To examine the genomes of uncultured bacteria, a metagenomic bacterial artificial chromosome library derived from a soil sample was screened for 16S rDNA-containing clones. Five clones (4C6, 5E7, 5G4, 5G12, and 5H7) containing uncultured soil bacteria genome fragment (with low 16S rDNA similarity to isolated bacteria) were selected for sequencing. Clone 5E7 and 5G4 showed only 82 and 83% of 16S rDNA identity to known sequences. Phylogenetic analysis of 16S rDNA indicated that 5E7 and 5G4 were potentially from new class of Chloroflexi. Only one-third of the 5G4 open reading frames have significant hits against HMMER. Internal tetranucleotide frequency analysis indicated that the unknown region of 5G4 was poorly correlated with other parts of the clone, indicating that this section might be obtained through lateral transfer. It was suggested that this region rich for unknown genes is under fast evolution.

Highlights

Soils are dominated by immensely diverse populations of microorganisms that remain largely unexplored (Torsvik and Øvreås, 2002)
Subsequent sequencing of the 16S rDNA confirmed that five clones, 4C6, 5E7, 5G4, 5G12, and 5H7, were originated from uncultured bacteria
By using an restriction fragment length polymorphism (RFLP) method, the metagenomic library containing 3,024 bacterial artificial chromosome (BAC) clones from the DNA sample from red soil in South China constructed in a previous study (Liu et al, 2011) was screened for uncultured bacterial insertions

Summary

Introduction

Soils are dominated by immensely diverse populations of microorganisms that remain largely unexplored (Torsvik and Øvreås, 2002). It is estimated that more than 99% of the microorganisms present in natural environments are not readily cultivable with known cultivation techniques, and this situation will not change until new culture technologies are developed (Streit and Schmitz, 2004; Urich et al, 2008; Yamada and Sekiguchi, 2009). Fingerprinting techniques, including denaturing gradient gel electrophoresis (DGGE) and temperature gradient gel electrophoresis (TGGE), have been successfully used in many diversity studies and shown to be powerful methods for detecting uncultured microbes in soil (Lalande et al, 2013). DGGE/TGGE fails to detect minority populations due to inadequate sensitivity.

Methods

Results

Conclusion