Use of Substrate-Induced Gene Expression in Metagenomic Analysis of an Aromatic Hydrocarbon-Contaminated Soil

Abstract

Metagenomics allows the study of genes related to xenobiotic degradation in a culture-independent manner, but many of these studies are limited by the lack of genomic context for metagenomic sequences. This study combined a phenotypic screen known as substrate-induced gene expression (SIGEX) with whole-metagenome shotgun sequencing. SIGEX is a high-throughput promoter-trap method that relies on transcriptional activation of a green fluorescent protein (GFP) reporter gene in response to an inducing compound and subsequent fluorescence-activated cell sorting to isolate individual inducible clones from a metagenomic DNA library. We describe a SIGEX procedure with improved library construction from fragmented metagenomic DNA and improved flow cytometry sorting procedures. We used SIGEX to interrogate an aromatic hydrocarbon (AH)-contaminated soil metagenome. The recovered clones contained sequences with various degrees of similarity to genes (or partial genes) involved in aromatic metabolism, for example, nahG (salicylate oxygenase) family genes and their respective upstream nahR regulators. To obtain a broader context for the recovered fragments, clones were mapped to contigs derived from de novo assembly of shotgun-sequenced metagenomic DNA which, in most cases, contained complete operons involved in aromatic metabolism, providing greater insight into the origin of the metagenomic fragments. A comparable set of contigs was generated using a significantly less computationally intensive procedure in which assembly of shotgun-sequenced metagenomic DNA was directed by the SIGEX-recovered sequences. This methodology may have broad applicability in identifying biologically relevant subsets of metagenomes (including both novel and known sequences) that can be targeted computationally by in silico assembly and prediction tools.T he massive influx of novel sequence data derived from nextgeneration sequencing (NGS) technologies, in the context of both individual genomes (1) and metagenomes (2, 3), has far outstripped efforts to link those sequences to specific organisms and biological functions (4). Techniques such as quantitative PCR (qPCR) (5), microarrays (6), clone libraries (7), and stable-isotope probing (8) have been used successfully to identify organisms potentially involved in biodegradation. Substrate-induced gene expression (SIGEX) was proposed as a method for uncovering novel catabolic operons from metagenomes (9-12). SIGEX is a promoter trap method based on single-cell sorting of clones from a plasmid library using flow cytometry (FCM), where metagenomic clones of interest are identified by the increased expression of a downstream fluorescent reporter gene in the presence, but not in the absence, of an inducing compound. SIGEX was initially perceived as having great potential for mining genes from metagenomic samples in a high-throughput manner, without requiring prior knowledge of the sequences being screened for (13-15). However, SIGEX, and metagenomic promoter traps in general, has not lived up to this pote...

Metal-ion-induced expression of gene fragments from subseafloor micro-organisms in the Kumano forearc basin, Nankai Trough

“…The approach to finding downstream sequence information by sequencing the source metagenome and choosing SIGEX‐induced fragments (Meier et al . ) will complement currently limited information on the function(s) of these DNA fragments.…”

“…Based on the rapid development of sequencing technologies, Meier et al . () demonstrated the use of SIGEX for complementing limitations associated with database‐dependent gene functional prediction for the soil microbiome. Additionally, Uchiyama and Miyazaki () utilized the SIGEX method to discover a novel transcriptional regulator induced by salicylate, which is non‐metabolizable in standard laboratory strains of Escherichia coli .…”

Hydrocarbon-associated substrates reveal promising fungi for poly (ethylene terephthalate) (PET) depolymerization

“…From these, only M. arundinis CBMAI 2109 was able to convert 2.0% ± 0.4 of the PET nanoparticle to the terephthalic acid (monomer), as confirmed by the calculations from Eq. 2, which compares the fluorescence of the positive control (total fluorescence of HOTP) with that of the assay [52]. This strain also converted 43.3% ± 2.7 of long-chain ester probe in umbelliferone.…”

Metagenomics of Thermophiles with a Focus on Discovery of Novel Thermozymes

“…HTS techniques increase the success of function-based metagenomic screens since they compensate for the often low hit rates in such screens (Ekkers et al, 2012 ). Apart from conventional high throughput screens, which use microtiter plate wells to store a large number of clones (Ko et al, 2013 ), microarray-based technologies coupled with microfluidic devices, cell compartmentalization, flow cytometry, and cell sorting are arising as promising new technologies for this purpose (Najah et al, 2014 ; Meier et al, 2015 ; Vidal-Melgosa et al, 2015 ). Microfluidic technologies are of undeniable interest when it comes to reaching screening rates of a million clones per day (Ufarté et al, 2015 ).…”