Construction of Aerobic/Anaerobic-Substrate-Induced Gene Expression Procedure for Exploration of Metagenomes From Subseafloor Sediments.

Taisuke Wakamatsu,Takeshi Terada,Fumiaki Mori,Saki Mizobuchi,Taiki Futagami,Yuki Morono

doi:10.3389/fmicb.2021.726024

Abstract

Substrate-induced gene expression (SIGEX) is a high-throughput promoter-trap method. It is a function-based metagenomic screening tool that relies on transcriptional activation of a reporter gene green fluorescence protein (gfp) by a metagenomic DNA library upon induction with a substrate. However, its use is limited because of the relatively small size of metagenomic DNA libraries and incompatibility with screening metagenomes from anaerobic environments. In this study, these limitations of SIGEX were addressed by fine-tuning metagenome DNA library construction protocol and by using Evoglow, a green fluorescent protein that forms a chromophore even under anaerobic conditions. Two metagenomic libraries were constructed for subseafloor sediments offshore Shimokita Peninsula (Pacific Ocean) and offshore Joetsu (Japan Sea). The library construction protocol was improved by (a) eliminating short DNA fragments, (b) applying topoisomerase-based high-efficiency ligation, (c) optimizing insert DNA concentration, and (d) column-based DNA enrichment. This led to a successful construction of metagenome DNA libraries of approximately 6 Gbp for both samples. SIGEX screening using five aromatic compounds (benzoate, 3-chlorobenzoate, 3-hydroxybenzoate, phenol, and 2,4-dichlorophenol) under aerobic and anaerobic conditions revealed significant differences in the inducible clone ratios under these conditions. 3-Chlorobenzoate and 2,4-dichlorophenol led to a higher induction ratio than that for the other non-chlorinated aromatic compounds under both aerobic and anaerobic conditions. After the further screening of induced clones, a clone induced by 3-chlorobenzoate only under anaerobic conditions was isolated and characterized. The clone harbors a DNA insert that encodes putative open reading frames of unknown function. Previous aerobic SIGEX attempts succeeded in the isolation of gene fragments from anaerobes. This study demonstrated that some gene fragments require a strict in vivo reducing environment to function and may be potentially missed when screened by aerobic induction. The newly developed anaerobic SIGEX scheme will facilitate functional exploration of metagenomes from the anaerobic biosphere.

Highlights

The remarkable advances of DNA sequencing technology (Mardis, 2017) have led to the accumulation of a vast amount of DNA sequence information, including that for uncultured microbes, i.e., metagenome-assembled genomes (Papudeshi et al, 2017) or single-cell genomes (Xu and Zhao, 2018)
To expand the environmental metagenome coverage, we here optimized the steps of the protocol for Substrate-induced gene expression (SIGEX) metagenome library construction
This study presents the development of an improved SIGEX approach for anaerobic screening of genes from microbes dwelling in extreme habitats

Summary

Introduction

The remarkable advances of DNA sequencing technology (Mardis, 2017) have led to the accumulation of a vast amount of DNA sequence information, including that for uncultured microbes, i.e., metagenome-assembled genomes (Papudeshi et al, 2017) or single-cell genomes (Xu and Zhao, 2018). As an advantage of these methods, the screening yields positive clones that contain functional genes Such phenotypic screens result in a false-negative loss of positive clones that fail to function because of the formation of inclusion bodies or the absence of appropriate cofactors. Such screens are generally labor-intensive and time-consuming, because of a manual selection of positive clones with the desired enzymatic or other activity from the numerous clones from a metagenomic library (Berini et al, 2017)

Objectives

Methods

Results

Discussion

Conclusion