Exploring Active Enzymes Through Functional Metagenomics: Fast Screening of Environmental Samples

The intestinal microbiota has many beneficial roles for its host. However, the precise mechanisms developed by the microbiota to influence the host intestinal cell responses are only partially known. The complexity of the ecosystem and our inability to culture most of these micro-organisms have led to the development of molecular approaches such as functional metagenomics, i.e. the heterologous expression of a metagenome in order to identify functions. This elegant strategy coupled to high throughput screening allowed to identify novel enzymes from different ecosystems where culture methods have not yet been adapted to isolate the candidate microorganisms. We have proposed to use this functional metagenomic approach in order to model the microbiota’s interaction with the host by combining this heterologous expression with intestinal reporter cell lines. The addition of the cellular component to this functional metagenomic approach introduced a second important source of variability resulting in a novel challenge for high throughput screening. First attempts of high throughput screening with various reporter cell-lines showed a high distribution of the response and consequent difficulties to reproduce the response, impairing an easy and clear identification of confirmed hits. In this study, we developed a robust and reproducible methodology to combine these two biological systems for high throughput application. We optimized experimental setups and completed them by appropriate statistical analysis tools allowing the use this innovative approach in a high throughput manner and on a broad range of reporter assays. We herewith present a methodology allowing a high throughput screening combining two biological systems. Therefore ideal conditions for homogeneity, sensitivity and reproducibility of both metagenomic clones as well as reporter cell lines have been identified and validated. We believe that this innovative method will allow the identification of new bioactive microbial molecules and, subsequently, will promote understanding of host-microbiota interactions.

Pollution in the environment due to accumulation of potentially toxic metals results in deterioration of soil and water quality, thus impacting health of all living organisms including microbes. In the present investigation, a functional metagenomics approach was adopted to mine functional genes involved in metal tolerance from potentially toxic metal contaminated site. Eukaryotic cDNA library (1.0-4.0 kb) was screened for the genes providing tolerance to cadmium (Cd) toxicity through a functional complementation assay using Cd-sensitive Saccharomyces cerevisiae mutant ycf1Δ. Out of the 98 clones able to recover growth on Cd-supplemented selective medium, one clone designated as PLCc43 showed more tolerance to Cd along with some other clones. Sequence analysis revealed that cDNA PLCc43 encodes a 284 amino acid protein harbouring four characteristic zinc finger motif repeats (CXXCXGXG) and showing partial homology with heat shock protein (Hsp40) of Acanthamoeba castellanii. qPCR analysis revealed the induction of PLCc43 in the presence of Cd, which was further supported by accumulation of Cd in ycf1Δ/PLCc43 mutant. Cu-sensitive (cup1Δ), Zn-sensitive (zrc1Δ) and Co-sensitive (cot1Δ) yeast mutant strains were rescued from sensitivity when transformed with cDNA PLCc43 indicating its ability to confer tolerance to various potentially toxic metals. Oxidative stress tolerance potential of PLCc43 was also confirmed in the presence of H2O2. Present study results suggest that PLCc43 originating from a functional eukaryotic gene of soil community play an important role in detoxification of potentially toxic metals and may be used as biomarker in various contaminated sites.

In September 2014, a small gastroenteritis outbreak occurred in a Greek restaurant. Primary investigations by official food surveillance revealed significant hygienic problems in the premises. Food samples and environmental samples were analyzed for the presence of bacterial and viral food pathogens. Norovirus genogroup I (GI) was detected in 2 environmental samples and in mixed salad. At the same time, stool samples from patients were analyzed and norovirus GI was detected. Further investigations revealed the presence of norovirus GI on some of the restaurant employees. Comparison of nucleic acid sequences revealed full sequence homology between norovirus RNA genotype 1.2 in food, environmental and stool samples, suggesting a common source of contamination and infection. Sequence analysis of food and environmental samples was facilitated by application of a system for total RNA amplification. Despite the fact that original source of contamination could be determined doubtlessly, observed weaknesses in the food production that caused this outbreak were discussed. The mixed salad could have been contaminated either by the lettuce contaminated at primary production or by one of the food-handlers. The investigation of the path of infection is necessary for the kind of legal consequences to be directed by authorities and may contribute to measures to eliminate possible sources of food contamination.

The majority of microbes in many environments are considered “as yet uncultured” and were traditionally considered inaccessible for study through the microbiological gold standard of pure culture. The emergence of metagenomic approaches has allowed researchers to access and study these microbes in a culture-independent manner through DNA sequencing and functional expression of metagenomic DNA in a heterologous host. Metagenomics has revealed an extraordinary degree of diversity and novelty, not only among microbial communities themselves, but also within the genomes of these microbes. Metagenomic analysis can involve sequence-based or functional approaches (or a combination of both). The continuous improvements to DNA sequencing technologies coupled with dramatic reductions in cost have allowed the field of metagenomics to grow at a rapid rate. Many novel insights on microbial community composition, structure, and functional capacity have been gained from sequence-based metagenomics. Functional metagenomics has been utilized, with much success, to identify many novel genes, proteins, and secondary metabolites such as antibiotics with industrial, biotechnological, pharmaceutical, and medical relevance. Future improvements and developments in sequencing technologies, expression vectors, alternative host systems, and novel screening assays will help advance the field further by revealing novel taxonomic and genetic diversity. This Research Topic aims to showcase the utility of metagenomics to gain insights on the microbial and genomic diversity in different environments by revealing the breadth of novelty that was in the past, largely untapped. This Research Topic comprises 19 submissions from experts in the field and covers a broad range of themes and article types (Review, Methods, Perspective, Opinion, and Original Research articles). We have broadly grouped the articles under four themes; functional metagenomics, targeted metagenomics, sequence-based metagenomics, and host-associated.

The small-molecule biosynthetic diversity encoded within the genomes of uncultured bacteria is an attractive target for the discovery of natural products using functional metagenomics. Phenotypes commonly associated with the production of small molecules, such as antibiosis, altered pigmentation, or altered colony morphology, are easily identified from screens of arrayed metagenomic library clones. However, functional metagenomic screening methods are limited by their intrinsic dependence on a heterologous expression host. Toward the goal of increasing the small-molecule biosynthetic diversity found in functional metagenomic studies, we report the phenotypic screening of broad-host-range environmental DNA libraries in six different proteobacteria: Agrobacterium tumefaciens, Burkholderia graminis, Caulobacter vibrioides, Escherichia coli, Pseudomonas putida, and Ralstonia metallidurans. Clone-specific small molecules found in culture broth extracts from pigmented and antibacterially active clones, as well as the genetic elements responsible for the biosynthesis of these metabolites, are described. The host strains used in this investigation provided access to unique sets of clones showing minimal overlap, thus demonstrating the potential advantage conferred on functional metagenomics through the use of multiple diverse host species.

Mercury ions (Hg2+), a heavy metal contaminant of strong biotoxicity, pose a serious threat to ecosystems and human health in aquatic environments. Developing highly sensitive and specific detection methods is therefore of great importance. This study presents a novel label-free fluorescent biosensor for Hg2+ by ingeniously coupling target-induced aptamer switching with rolling circle amplification (RCA). Upon Hg2+ binding, the conformational change releases a sequestered primer to initiate RCA, generating G-quadruplex-rich DNA products that produce a strong “turn-on” signal with N-methylmesoporphyrin IX (NMM). Under optimized conditions, the assay exhibits excellent linearity from 10 to 1000 nM with a detection limit of 3.2 nM, along with high selectivity over competing metal ions. Validation using spiked environmental water samples yielded accurate and reproducible recoveries in the range of 93.8% to 106.0%. With its operational simplicity, high sensitivity, and robust performance in complex matrices, this label-free strategy offers a reliable and promising platform for detecting Hg2+ in environmental waters.

Extremophiles provide a one-of-a-kind source of enzymes with properties that allow them to endure the rigorous industrial conversion of lignocellulose biomass into fermentable sugars. However, the fact that most of these organisms fail to grow under typical culture conditions limits the accessibility to these enzymes. In this study, we employed a functional metagenomics approach to identify carbohydrate-degrading enzymes from Ethiopian soda lakes, which are extreme environments harboring a high microbial diversity. Out of 21,000 clones screened for the five carbohydrate hydrolyzing enzymes, 408 clones were found positive. Cellulase and amylase, gave high hit ratio of 1:75 and 1:280, respectively. A total of 378 genes involved in the degradation of complex carbohydrates were identified by combining high-throughput sequencing of 22 selected clones and bioinformatics analysis using a customized workflow. Around 41% of the annotated genes belonged to the Glycoside Hydrolases (GH). Multiple GHs were identified, indicating the potential to discover novel CAZymes useful for the enzymatic degradation of lignocellulose biomass from the Ethiopian soda Lakes. More than 73% of the annotated GH genes were linked to bacterial origins, with Halomonas as the most likely source. Biochemical characterization of the three enzymes from the selected clones (amylase, cellulase, and pectinase) showed that they are active in elevated temperatures, high pH, and high salt concentrations. These properties strongly indicate that the evaluated enzymes have the potential to be used for applications in various industrial processes, particularly in biorefinery for lignocellulose biomass conversion.

Chapter six - Functional Metagenomics of Methylotrophs

Rapid, sensitive, and accurate detection of analytes present in low concentrations in complex matrixes is a critical challenge. One issue that affects many biosensor protocols is the number and nature of the interferences present in complex matrixes such as plasma, urine, stool, and environmental samples, resulting in loss of sensitivity and specificity. We have developed a method for rapid purification, concentration, and detection of target analytes from complex matrixes using antibody-coated superparamagnetic nanobeads (immunomagnetic beads, or IMBs). The surface plasmon resonance (SPR) detection signal from staphylococcal enterotoxin B (SEB) was dramatically increased when the IMBs were used as detection amplifiers. When SEB detection included a 10-fold concentration/purification IMB protocol, a substantial increase in detection sensitivity was observed. This procedure was used to successfully purify and concentrate SEB from serum and stool samples, then amplify the SPR detection signal. SEB at a concentration of 100 pg/mL was easily detected in both buffer and stool samples using this procedure. The IMB protocol also served to verify the analyte detection by using two different anti-SEB antibodies, mouse monoclonal antibodies attached to the magnetic nanobeads and rabbit polyclonal antibodies on the SPR sensor surface. Multiple detections of SEB in stool were performed using the same sensor surface by regenerating the sensor surfaces with a pH 2.2 buffer wash.

BackgroundFunctional metagenomics is a promising strategy for the exploration of the biocatalytic potential of microbiomes in order to uncover novel enzymes for industrial processes (e.g. biorefining or bleaching pulp). Most current methodologies used to screen for enzymes involved in plant biomass degradation are based on the use of single substrates. Moreover, highly diverse environments are used as metagenomic sources. However, such methods suffer from low hit rates of positive clones and hence the discovery of novel enzymatic activities from metagenomes has been hampered.ResultsHere, we constructed fosmid libraries from two wheat straw-degrading microbial consortia, denoted RWS (bred on untreated wheat straw) and TWS (bred on heat-treated wheat straw). Approximately 22,000 clones from each library were screened for (hemi)cellulose-degrading enzymes using a multi-chromogenic substrate approach. The screens yielded 71 positive clones for both libraries, giving hit rates of 1:440 and 1:1,047 for RWS and TWS, respectively. Seven clones (NT2-2, T5-5, NT18-17, T4-1, 10BT, NT18-21 and T17-2) were selected for sequence analyses. Their inserts revealed the presence of 18 genes encoding enzymes belonging to twelve different glycosyl hydrolase families (GH2, GH3, GH13, GH17, GH20, GH27, GH32, GH39, GH53, GH58, GH65 and GH109). These encompassed several carbohydrate-active gene clusters traceable mainly to Klebsiella related species. Detailed functional analyses showed that clone NT2-2 (containing a beta-galactosidase of ~116 kDa) had highest enzymatic activity at 55 °C and pH 9.0. Additionally, clone T5-5 (containing a beta-xylosidase of ~86 kDa) showed > 90 % of enzymatic activity at 55 °C and pH 10.0.ConclusionsThis study employed a high-throughput method for rapid screening of fosmid metagenomic libraries for (hemi)cellulose-degrading enzymes. The approach, consisting of screens on multi-substrates coupled to further analyses, revealed high hit rates, as compared with recent other studies. Two clones, 10BT and T4-1, required the presence of multiple substrates for detectable activity, indicating a new avenue in library activity screening. Finally, clones NT2-2, T5-5 and NT18-17 were found to encode putative novel thermo-alkaline enzymes, which could represent a starting point for further biotechnological applications.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-2404-0) contains supplementary material, which is available to authorized users.

In April 2013, outbreaks of acute gastroenteritis were reported at three schools in Jeonju, South Korea. Epidemiological investigations were performed to characterize the outbreaks and implement appropriate control measures. Retrospective cohort studies were performed at these schools. Stool and environmental samples were collected for bacterial and viral assessment. A food supplier of the schools, food company X, was inspected, and samples of cabbage kimchi and groundwater were tested for norovirus by real-time reverse-transcriptase polymerase chain reaction. The relatedness of the detected norovirus strains was evaluated by phylogenetic analysis. Of the 3347 questionnaires distributed, 631 (attack rate: 18.9%) met the case definition. Among the consumed food items, kimchi products (i.e., cabbage and fresh kimchi) were significantly associated with illness. The kimchi products were supplied by food company X. Among stool samples from 95 students and 34 food handlers at the 3 schools, 39 (41.1%) and 14 (41.2%) samples, respectively, were positive for norovirus. The samples of groundwater and cabbage kimchi at food company X were positive for norovirus. The predominant genotype of norovirus detected in the patient, groundwater, and cabbage kimchi samples, GI.4, shared high nucleotide identity. Kimchi products tainted with norovirus GI.4 from contaminated groundwater were linked to the acute gastroenteritis outbreaks. Therefore, kimchi manufacturers in South Korea should apply chlorine disinfection when using groundwater. Moreover, more stringent sanitation requirements and strict regulations for food companies are recommended.

Colorectal cancer (CRC) is the third most common cancer and leading cause of cancer-related deaths worldwide. However, current CRC screening methods are complex, invasive, and tend to exhibit low sensitivity. Recent evidence has highlighted gut microbiota dysbiosis, especially elevated Fusobacterium nucleatum levels, as a promising biomarker for CRC. In this study, a sensitive and specific detection platform was developed for F. nucleatum by combining a highly specific aptamer with rolling circle amplification (RCA) and the CRISPR/Cas12a technology. The aptamer enables specific target recognition, while RCA amplifies the target signal, and the Cas12a-mediated cleavage of a fluorescence-quenching substrate generates a quantifiable fluorescence or grayscale signal. Using a microplate reader, this assay achieved a limit of detection (LOD) of 3.68 CFU/mL; furthermore, by incorporating smartphone-assisted ImageJ grayscale analysis, it elevated the LOD to 4.30 CFU/mL, thereby enabling a dual-mode output along with on-site applicability. Additionally, the strong correlation between the two signals allowed for mutual validation. Upon application to clinical fecal samples, the developed method sensitively distinguished CRC patients from healthy controls, and its results correlated with the quantitative polymerase chain reaction results. This triple-synergistic platform, integrating aptamer specificity, RCA amplification, and CRISPR/Cas12a sensitivity, enables the noninvasive, ultrasensitive detection of F. nucleatum, supporting early CRC screening, prognosis monitoring, and microbiome-targeted therapy. Moreover, this approach overcomes the challenges of detecting low-abundance bacteria in early stage CRC and advances the precision of microbiome-based diagnostics for CRC.

Microorganisms that thrive in hypersaline environments on the surface of our planet are exposed to the harmful effects of ultraviolet radiation. Therefore, for their protection, they have sunscreen pigments and highly efficient DNA repair and protection systems. The present study aimed to identify new genes involved in UV radiation resistance from these microorganisms, many of which cannot be cultured in the laboratory. Thus, a functional metagenomic approach was used and for this, small-insert libraries were constructed with DNA isolated from microorganisms of high-altitude Andean hypersaline lakes in Argentina (Diamante and Ojo Seco lakes, 4,589 and 3,200 m, respectively) and from the Es Trenc solar saltern in Spain. The libraries were hosted in a UV radiation-sensitive strain of Escherichia coli (recA mutant) and they were exposed to UVB. The resistant colonies were analyzed and as a result, four clones were identified with environmental DNA fragments containing five genes that conferred resistance to UV radiation in E. coli. One gene encoded a RecA-like protein, complementing the mutation in recA that makes the E. coli host strain more sensitive to UV radiation. Two other genes from the same DNA fragment encoded a TATA-box binding protein and an unknown protein, both responsible for UV resistance. Interestingly, two other genes from different and remote environments, the Ojo Seco Andean lake and the Es Trenc saltern, encoded two hypothetical proteins that can be considered homologous based on their significant amino acid similarity (49%). All of these genes also conferred resistance to 4-nitroquinoline 1-oxide (4-NQO), a compound that mimics the effect of UV radiation on DNA, and also to perchlorate, a powerful oxidant that can induce DNA damage. Furthermore, the hypothetical protein from the Es Trenc salterns was localized as discrete foci possibly associated with damaged sites in the DNA in cells treated with 4-NQO, so it could be involved in the repair of damaged DNA. In summary, novel genes involved in resistance to UV radiation, 4-NQO and perchlorate have been identified in this work and two of them encoding hypothetical proteins that could be involved in DNA damage repair activities not previously described.

The microbial communities from the Tinto River, a natural acid mine drainage environment, were explored to search for novel genes involved in arsenic resistance using a functional metagenomic approach. Seven pentavalent arsenate resistance clones were selected and analysed to find the genes responsible for this phenotype. Insights about their possible mechanisms of resistance were obtained from sequence similarities and cellular arsenic concentration. A total of 19 individual open reading frames were analysed, and each one was individually cloned and assayed for its ability to confer arsenic resistance in Escherichia coli cells. A total of 13 functionally active genes involved in arsenic resistance were identified, and they could be classified into different global processes: transport, stress response, DNA damage repair, phospholipids biosynthesis, amino acid biosynthesis and RNA-modifying enzymes. Most genes (11) encode proteins not previously related to heavy metal resistance or hypothetical or unknown proteins. On the other hand, two genes were previously related to heavy metal resistance in microorganisms. In addition, the ClpB chaperone and the RNA-modifying enzymes retrieved in this work were shown to increase the cell survival under different stress conditions (heat shock, acid pH and UV radiation). Thus, these results reveal novel insights about unidentified mechanisms of arsenic resistance.

Wastewater treatment plants (WWTPs) are engineered structures that collect, concentrate, and treat human waste, ultimately releasing treated wastewater into local environments. While WWTPs efficiently remove most biosolids, it has been shown that many antibiotics and antibiotic-resistant bacteria can survive the treatment process. To determine how WWTPs influence the concentration and dissemination of antibiotic-resistant genes into the environment, a functional metagenomic approach was used to identify a novel antibiotic resistance gene within a WWTP, and quantitative PCR (qPCR) was used to determine gene copy numbers within the facility and the local coastal ecosystem. From the WWTP metagenomic library, the fosmid insert contained in one highly resistant clone (MIC, ≈ 416 μg ml(-1) ampicillin) was sequenced and annotated, revealing 33 putative genes, including a 927-bp gene that is 42% identical to a functionally characterized β-lactamase from Staphylococcus aureus PC1. Isolation and subcloning of this gene, referred to as bla(M-1), conferred ampicillin resistance to its Escherichia coli host. When normalized to volume, qPCR showed increased concentrations of bla(M-1) during initial treatment stages but 2-fold-decreased concentrations during the final treatment stage. The concentration ng(-1) DNA increased throughout the WWTP process from influent to effluent, suggesting that bla(M-1) makes up a significant proportion of the overall genetic material being released into the coastal ecosystem. Average discharge was estimated to be 3.9 × 10(14) copies of the bla(M-1) gene released daily into this coastal ecosystem. Furthermore, the gene was observed in all sampled coastal water and sediment samples surrounding the facility. Our results suggest that WWTPs may be a pathway for the dissemination of novel antibiotic resistance genes into the environment.