Aerobic/Anaerobic-Substrate-Induced Gene Expression for Exploration of Metagenomes from Subseafloor Sediments

Phenotypic metagenome screening tool, the substrate-induced gene expression (SIGEX) is the high-throughput promoter-trap method that relies on transcriptional activation of a reporter gene (green fluorescence protein, gfp) of metagenomic DNA library by the induction of a substrate compound. Despite of the advantages (independence from genomic context or cultivation) of the phenotypic SIGEX approach, its use was limited because of the relatively small size of metagenomic DNA library, and the mismatch of aerobic induction condition for induction of metagenomes from anaerobic environments. In this study, we aimed to overcome the challenges by fine-tuning genomic library construction protocols and applying green fluorescent protein gene that can form chromophore in anaerobic condition (evoglow). In total, 2 metagenomic libraries were constructed from the subseafloor sediment of offshore Joetsu, Japan Sea and offshore Shimokita Peninshula, Pacific ocean of three different depths each. Through the improvements in library construction protocol (applying topo isomerase-based high efficiency ligation, optimization of insert DNA concentration, and intense elimination of short DNA fragments) we successfully constructed metagenome DNA library of ~6 Gbp for each of the samples. 16S rRNA gene analyses of those metagenomic DNA libraries showed that Desulfobacterota including several genera was one of the dominant phyla (20.0-25.4%) in both metagenomes. SIGEX screening by using five aromatic compounds (benzoate, 3-chlorobenzoate, 3-hydroxyhbenzoate, phenol, or 2,4-dichlorophenol) in aerobic and anaerobic conditions showed differences in the ratio of inducible clones in aerobic and anaerobic inductions. In general, 3-chlorobenzoate and 2,4-dichlorophenol than the other aromatic compounds in both aerobic and anaerobic condition. Further screening of induced clones resulted in isolation of a clone that was induced by 3-chlorobenzoate only in anaerobic induction. Previous SIGEX attempts succeeded in isolation of gene fragments originating from anaerobes, suggesting the intracellular stability of inducing factor inside of E. coli host cells, however, our results confirmed that part of the gene fragments requires stricter reducing environment to be functionable. Our newly developed anaerobic SIGEX scheme will have high potential in exploring metagenomes in anaerobic biosphere.