Substrate-induced Gene Expression Research Articles

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

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.

A plasmid-based genomic screening system for transcriptional regulators of non-adjacent xenobiotic catabolism genes.

Bacteria play an important role in the catabolism of environmental xenobiotics. The study of transcriptional regulation has greatly enhanced our understanding of the molecular mechanisms associated with these processes. However, genes encoding transcription factors (TFs) for xenobiotic catabolism are usually not located in the immediate vicinity of the catabolic genes that they regulate; therefore, functional identification of these TFs is difficult. Significantly modified from a metagenome screening method substrate-induced gene expression (SIGEX), here we propose a synthetic pSRGFP-18 plasmid-based tool as a TF reporter system. In short, two multiple cloning sites (MCS) were designed; one in front of an egfp reporter gene was constructed for promoter insertion, and the other MCS was used for shotgun cloning of genomic DNA. Based on the regulatory relationship between TFs and the promoter of their associated catabolic genes, this approach allowed us to screen for TF genes using a genome shotgun approach. This system performed well when testing the known operons. Following statistical analysis of known catabolic operons in Escherichia coli and Bacillus subtilis, the suggested region of the target promoter for this system was from - 250 to + 150. Furthermore, to broaden the applicability of this plasmid, a series of pSRGFP-18 and pBBR1-based pSRGFP-X plasmids were constructed, which had broad host ranges and contained different antibiotic markers. This study outlines a powerful tool to enable functional identification of TFs for bacterial xenobiotic catabolism.

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

Using substrate-induced gene-expression (SIGEX) screening on subseafloor sediment samples from the Nankai Trough, Japan, we identified gene fragments showing an induction response to metal ions. Environmental DNA libraries in Escherichia coli host cells were tested by the addition of metal ions (Ni2+ , Co2+ , Ga3+ or Mo6+ ), followed by cell sorting of clones exhibiting green fluorescence upon co-expression of green fluorescence protein downstream of the inserted gene fragments. One clone displayed Ni2+ -specific induction, three clones displayed Ga3+ -specific induction and three clones displayed an induction response to multiple metal ions. DNA sequence analysis showed that a variety of genes showed induction responses in the screened clones. Using the SIGEX approach, we retrieved gene fragments with no previously identified response to metal ions that exhibited metal-ion-induced expression. This method has the potential to promote exploration of gene function through gene-induction response. We successfully linked gene-induction response with sequence information for gene fragments of previously unknown function. The SIGEX-based approach exhibited the potential to identify genetic function in unknown gene pools from the deep subseafloor biosphere, as well as novel genetic components for future biotechnological applications.

Molecular perspectives and recent advances in microbial remediation of persistent organic pollutants.

Nutrition and pollution stress stimulate genetic adaptation in microorganisms and assist in evolution of diverse metabolic pathways for their survival on several complex organic compounds. Persistent organic pollutants (POPs) are highly lipophilic in nature and cause adverse effects to the environment and human health by biomagnification through the food chain. Diverse microorganisms, harboring numerous plasmids and catabolic genes, acclimatize to these environmentally unfavorable conditions by gene duplication, mutational drift, hypermutation, and recombination. Genetic aspects of some major POP catabolic genes such as biphenyl dioxygenase (bph), DDT 2,3-dioxygenase, and angular dioxygenase assist in degradation of biphenyl, organochlorine pesticides, and dioxins/furans, respectively. Microbial metagenome constitutes the largest genetic reservoir with miscellaneous enzymatic activities implicated in degradation. To tap the metabolic potential of microorganisms, recent techniques like sequence and function-based screening and substrate-induced gene expression are proficient in tracing out novel catabolic genes from the entire metagenome for utilization in enhanced biodegradation. The major endeavor of today's scientific world is to characterize the exact genetic mechanisms of microbes for bioremediation of these toxic compounds by excavating into the uncultured plethora. This review entails the effect of POPs on the environment and involvement of microbial catabolic genes for their removal with the advanced techniques of bioremediation.

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

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.

Distribution of dehalogenation activity in subseafloor sediments of the Nankai Trough subduction zone

Halogenated organic matter buried in marine subsurface sediment may serve as a source of electron acceptors for anaerobic respiration of subseafloor microbes. Detection of a diverse array of reductive dehalogenase-homologous (rdhA) genes suggests that subseafloor organohalide-respiring microbial communities may play significant ecological roles in the biogeochemical carbon and halogen cycle in the subseafloor biosphere. We report here the spatial distribution of dehalogenation activity in the Nankai Trough plate-subduction zone of the northwest Pacific off the Kii Peninsula of Japan. Incubation experiments with slurries of sediment collected at various depths and locations showed that degradation of several organohalides tested only occurred in the shallow sedimentary basin, down to 4.7 metres below the seafloor, despite detection of rdhA in the deeper sediments. We studied the phylogenetic diversity of the metabolically active microbes in positive enrichment cultures by extracting RNA, and found that Desulfuromonadales bacteria predominate. In addition, for the isolation of genes involved in the dehalogenation reaction, we performed a substrate-induced gene expression screening on DNA extracted from the enrichment cultures. Diverse DNA fragments were obtained and some of them showed best BLAST hit to known organohalide respirers such as Dehalococcoides, whereas no functionally known dehalogenation-related genes such as rdhA were found, indicating the need to improve the molecular approach to assess functional genes for organohalide respiration.

Substrate-induced gene expression (SIGEX) screening of metagenome libraries

Substrate-induced gene-expression screening (SIGEX) has been developed for isolating novel catabolic genes from environmental metagenomes, particularly genes that are difficult to obtain using conventional gene-cloning methods. In SIGEX, restriction enzyme-digested metagenome fragments are ligated into an operon-trap vector (e.g., p18GFP), and a library is constructed in a liquid culture by transforming a cloning host (e.g., Escherichia coli). The library is subjected to a substrate-dependent gene-induction assay, and positive cells are selected by detecting activity of a co-expressed marker (e.g., GFP) encoded in the vector. High-throughput screening is possible if fluorescence-activated cell sorting (FACS) is used to select GFP-expressing cells. The abovementioned SIGEX procedure requires approximately 17 d. In this protocol, a widely applicable SIGEX scheme is presented along with typical experimental results.

Screening for novel enzymes from metagenome and SIGEX, as a way to improve it

Metagenomics has been successfully applied to isolate novel biocatalysts from the uncultured microbiota in the environment. Two types of screening have been used to identify clones carrying desired traits from metagenomic libraries: function-based screening, and sequence-based screening. Both function- and sequence- based screening have individual advantages and disadvantages, and they have been applied successfully to discover biocatalysts from metagenome. However, both strategies are laborious and tedious because of the low frequency of screening hits. A recent paper introduced a high throughput screening strategy, termed substrate-induced gene-expression screening (SIGEX). SIGEX is designed to select the clones harboring catabolic genes induced by various substrates in concert with fluorescence activated cell sorting (FACS). This method was applied successfully to isolate aromatic hydrocarbon-induced genes from a metagenomic library. Although SIGEX has many limitations, it is expected to provide economic advantages, especially to industry.

Substrate-induced gene-expression screening of environmental metagenome libraries for isolation of catabolic genes

Recent awareness that most microorganisms in the environment are resistant to cultivation has prompted scientists to directly clone useful genes from environmental metagenomes. Two screening methods are currently available for the metagenome approach, namely, nucleotide sequence-based screening and enzyme activity-based screening. Here we have introduced and optimized a third option for the isolation of novel catabolic operons, that is, substrate-induced gene expression screening (SIGEX). This method is based on the knowledge that catabolic-gene expression is generally induced by relevant substrates and, in many cases, controlled by regulatory elements situated proximate to catabolic genes. For SIGEX to be high throughput, we constructed an operon-trap gfp-expression vector available for shotgun cloning that allows for the selection of positive clones in liquid cultures by fluorescence-activated cell sorting. The utility of SIGEX was demonstrated by the cloning of aromatic hydrocarbon-induced genes from a groundwater metagenome library and subsequent genome-informatics analysis.