Metagenomic insights into the prokaryotic communities of heavy metal-contaminated hypersaline soils.

Saline soils and their microbial communities have recently been studied in response to ongoing desertification of agricultural soils caused by anthropogenic impacts and climate change. Here we describe the prokaryotic microbiota of hypersaline soils in the Odiel Saltmarshes Natural Area of Southwest Spain. This region has been strongly affected by mining and industrial activity and feature high levels of certain heavy metals. We sequenced 18 shotgun metagenomes through Illumina NovaSeq from samples obtained from three different areas in 2020 and 2021. Taxogenomic analyses demonstrate that these soils harbored equal proportions of archaea and bacteria, with Methanobacteriota, Pseudomonadota, Bacteroidota, Gemmatimonadota, and Balneolota as most abundant phyla. Functions related to the transport of heavy metal outside the cytoplasm are among the most relevant features of the community (i.e., ZntA and CopA enzymes). They seem to be indispensable to avoid the increase of zinc and copper concentration inside the cell. Besides, the archaeal phylum Methanobacteriota is the main arsenic detoxifier within the microbiota although arsenic related genes are widely distributed in the community. Regarding the osmoregulation strategies, "salt-out" mechanism was identified in part of the bacterial population, whereas "salt-in" mechanism was present in both domains, Bacteria and Archaea. De novo biosynthesis of two of the most universal compatible solutes was detected, with predominance of glycine betaine biosynthesis (betAB genes) over ectoine (ectABC genes). Furthermore, doeABCD gene cluster related to the use of ectoine as carbon and energy source was solely identified in Pseudomonadota and Methanobacteriota.

Integrating functional metagenomics to decipher microbiome-immune interactions.

Microbial metabolites can be viewed as the cytokines of the microbiome, transmitting information about the microbial and metabolic environment of the gut to orchestrate and modulate local and systemic immune responses. Still, many immunology studies focus solely on the taxonomy and community structure of the gut microbiota rather than its functions. Early sequencing-based microbiota profiling approaches relied on PCR amplification of small regions of bacterial and fungal genomes to facilitate identification of the microbes present. However, recent microbiome analysis methods, particularly shotgun metagenomic sequencing, now enable culture-independent profiling of microbiome functions and metabolites in addition to taxonomic characterization. In this review, we showcase recent advances in functional metagenomics methods and applications and discuss the current limitations and potential avenues for future development. Importantly, we highlight a few examples of key areas of opportunity in immunology research where integrating functional metagenomic analyses of the microbiome can substantially enhance a mechanistic understanding of microbiome-immune interactions and their contributions to health and disease states.

Cat and dog feces as reservoirs of diverse novel antibiotic resistance genes.

Companion animals have the potential to greatly enhance the physical and mental health of humans, thus leading to an increased focus on the interactions between humans and pets. Currently, the inappropriate and excessive utilization of antimicrobial agents has become prevalent in veterinary clinical practice for pets. This antibiotic contamination phenomenon has a profound impact on the enrichment of antibiotic resistance bacteria (ARB) and antibiotic resistance genes (ARGs) in pets. However, the pet-associated resistome, especially the novel ARGs in pets, represents a relatively neglected area. In this study, we successfully constructed a total of 12 libraries using the functional metagenomics approach to assess the diversity of ARGs in pet cats and dogs from four pet hospitals. Through the integration of functional screening and high-throughput sequencing, a total of 122 antibiotic resistance determinants were identified, of which 15 were classified as putative novel ARGs originating from five classes. Functional assessment demonstrated that 6 novel ARGs including one ß-lactam, two macrolides, two aminoglycosides, and one rifamycin (RIF), namely blaPF, ermPF, msrPF, aac(6')PF, aph(3')PF, and arrPF, exhibited functionally activity in conferring bacterial phenotypic resistance by increasing the minimum inhibitory concentrations (MICs) with a 4- to 128-fold. Genetic context analysis demonstrated that, with the exception of aac(6')PF and arrPF, the remaining four novel ARGs were found adjacent to mobile genetic elements (MGEs) including IS elements or transposases, which provided a prerequisite for horizontal transfer of these novel ARGs, thereby offering an explanation for their detection in diverse samples collected from various sampling sites. The current study has unveiled the significant role of cat and dog feces as one source of reservoirs of diverse novel ARGs, while also highlighting the potential adverse consequences of their further spread to medically significant pathogens and human commensal organisms.

Intraspecific variation in antibiotic resistance potential within E. coli.

Intraspecific genomic diversity brings the potential for an unreported and diverse reservoir of cryptic antibiotic resistance genes in pathogens, as cryptic resistance can occur without major mutations and horizontal transmission. Here, we predicted the differences in the types of antibiotics and genes that induce cryptic and latent resistance between micro-diverse Escherichia coli strains. For example, we hypothesize that known resistance genes will be the culprit of latent resistance within clinical strains. We used a modified functional metagenomics method to induce expression in eight E. coli strains. We found a total of 66 individual genes conferring phenotypic resistance to 11 out of 16 antibiotics. A total of 14 known antibiotic resistance genes comprised 21% of total identified genes, whereas the majority (52 genes) were unclassified cryptic resistance genes. Between the eight strains, 1.2% of core orthologous genes were positive (conferred resistance in at least one strain). Sixty-four percent of positive orthologous genes conferred resistance to only one strain, demonstrating high intraspecific variability of latent resistance genes. Cryptic resistance genes comprised most resistance genes among laboratory and clinical strains as well as natural, semisynthetic, and synthetic antibiotics. Known antibiotic resistance genes primarily conferred resistance to multiple antibiotics from varying origins and within multiple strains. Hence, it is uncommon for E. coli to develop cross-cryptic resistance to antibiotics from multiple origins or within multiple strains. We have uncovered prospective and previously unknown resistance genes as well as antibiotics that have the potential to trigger latent antibiotic resistance in E. coli strains from varying origins.IMPORTANCEIntraspecific genomic diversity may be a driving force in the emergence of adaptive antibiotic resistance. Adaptive antibiotic resistance enables sensitive bacterial cells to acquire temporary antibiotic resistance, creating an optimal window for the development of permanent mutational resistance. In this study, we investigate cryptic resistance, an adaptive resistance mechanism, and unveil novel (cryptic) antibiotic resistance genes that confer resistance when amplified within eight E. coli strains derived from clinical and laboratory origins. We identify the potential of cryptic resistance genes to confer cross-resistance to antibiotics from varying origins and within multiple strains. We discern antibiotic characteristics that promote latent resistance in multiple strains, considering intraspecific diversity. This study may help detect novel resistance genes and functional genes that could become responsible for cryptic resistance among diverse strains and antibiotics, thus also identifying potential novel antibiotic targets and mechanisms.

A novel family of sugar-specific phosphodiesterases that remove zwitterionic modifications of GlcNAc.

The zwitterions phosphorylcholine (PC) and phosphoethanolamine (PE) are often found esterified to certain sugars in polysaccharides and glycoconjugates in a wide range of biological species. One such modification involves PC attachment to the 6-carbon of N-acetylglucosamine (GlcNAc-6-PC) in N-glycans and glycosphingolipids (GSLs) of parasitic nematodes, a modification that helps the parasite evade host immunity. Knowledge of enzymes involved in the synthesis and degradation of PC and PE modifications is limited. More detailed studies on such enzymes would contribute to a better understanding of the function of PC modifications and have potential application in the structural analysis of zwitterion-modified glycans. In this study, we used functional metagenomic screening to identify phosphodiesterases encoded in a human fecal DNA fosmid library that remove PC from GlcNAc-6-PC. A novel bacterial phosphodiesterase was identified and biochemically characterized. This enzyme (termed GlcNAc-PDase) shows remarkable substrate preference for GlcNAc-6-PC and GlcNAc-6-PE, with little or no activity on other zwitterion-modified hexoses. The identified GlcNAc-PDase protein sequence is a member of the large endonuclease/exonuclease/phosphatase superfamily where it defines a distinct subfamily of related sequences of previously unknown function, mostly from Clostridium bacteria species. Finally, we demonstrate use of GlcNAc-PDase to confirm the presence of GlcNAc-6-PC in N-glycans and GSLs of the parasitic nematode Brugia malayi in a glycoanalytical workflow.

Discovery of two bifunctional/multifunctional cellulases by functional metagenomics.

Cellulases are widely used in industry, and the usage in bioconversion of biofuels makes cellulases more valuable. In this study, two tandem genes that encoded cellulases ZF994-1 and ZF994-2, respectively, were identified on a cosmid from a soil metagenomic library. Phylogenetic analysis indicated that ZF994-1 and ZF994-2 belonged to glycoside hydrolase family 12 (GH12), and GH3, respectively. Based on the substrate specificity analysis, the recombinant ZF994-1 exhibited weak endoglucanase activity, moderate ß-1,3-glucanase and ß-1,4-mannanase activities, and strong ß-glucosidase activity, while the recombinant ZF994-2 exhibited moderate endoglucanase activity and strong ß-glucosidase activity. More than 45% ß-glucosidase activity of the recombinant ZF994-1 retained in the buffer containing 3 M glucose, indicating the good tolerance against glucose. The recombinant ZF994-2 showed high activity in the presence of metal ions and organic reagents, exhibiting potential industrial applications.

Interactive Web-Based Services for Metagenomic Data Analysis and Comparisons.

Recently, sequencing technologies have become readily available, and scientists are more motivated to conduct metagenomic research to unveil the potential of a myriad of ecosystems and biomes. Metagenomics studies the composition and functions of microbial communities and paves the way to multiple applications in medicine, industry, and ecology. Nonetheless, the immense amount of sequencing data of metagenomics research and the few user-friendly analysis tools and pipelines carry a new challenge to the data analysis.Web-based bioinformatics tools are now being developed to facilitate the analysis of complex metagenomic data without prior knowledge of any programming languages or special installation. Specialized web tools help answer researchers' main questions on the taxonomic classification, functional capabilities, discrepancies between two ecosystems, and the probable functional correlations between the members of a specific microbial community. With an Internet connection and a few clicks, researchers can conveniently and efficiently analyze the metagenomic datasets, summarize results, and visualize key information on the composition and the functional potential of metagenomic samples under study. This chapter provides a simple guide to a few of the fundamental web-based services used for metagenomic data analyses, such as BV-BRC, RDP, MG-RAST, MicrobiomeAnalyst, METAGENassist, and MGnify.

Functional metagenomics uncovers nitrile-hydrolysing enzymes in a coal metagenome.

Introduction: Nitriles are the most toxic compounds that can lead to serious human illness through inhalation and consumption due to environmental pollution. Nitrilases can highly degrade nitriles isolated from the natural ecosystem. In the current study, we focused on the discovery of novel nitrilases from a coal metagenome using in silico mining. Methods: Coal metagenomic DNA was isolated and sequenced on the Illumina platform. Quality reads were assembled using MEGAHIT, and statistics were checked using QUAST. Annotation was performed using the automated tool SqueezeMeta. The annotated amino acid sequences were mined for nitrilase from the unclassified organism. Sequence alignment and phylogenetic analyses were carried out using ClustalW and MEGA11. Conserved regions of the amino acid sequences were identified using InterProScan and NCBI-CDD servers. The physicochemical properties of the amino acids were measured using ExPASy's ProtParam. Furthermore, NetSurfP was used for 2D structure prediction, while AlphaFold2 in Chimera X 1.4 was used for 3D structure prediction. To check the solvation of the predicted protein, a dynamic simulation was conducted on the WebGRO server. Ligands were extracted from the Protein Data Bank (PDB) for molecular docking upon active site prediction using the CASTp server. Results and discussion: In silico mining of annotated metagenomic data revealed nitrilase from unclassified Alphaproteobacteria. By using the artificial intelligence program AlphaFold2, the 3D structure was predicted with a per-residue confidence statistic score of about 95.8%, and the stability of the predicted model was verified with molecular dynamics for a 100-ns simulation. Molecular docking analysis determined the binding affinity of a novel nitrilase with nitriles. The binding scores produced by the novel nitrilase were approximately similar to those of the other prokaryotic nitrilase crystal structures, with a deviation of ±0.5.

Synthetic Biology Toolbox for Antarctic Pseudomonas sp. Strains: Toward a Psychrophilic Nonmodel Chassis for Function-Driven Metagenomics.

One major limitation of function-driven metagenomics is the ability of the host to express the metagenomic DNA correctly. Differences in the transcriptional, translational, and post-translational machinery between the organism to which the DNA belongs and the host strain are all factors that influence the success of a functional screening. For this reason, the use of alternative hosts is an appropriate approach to favor the identification of enzymatic activities in function-driven metagenomics. To be implemented, appropriate tools should be designed to build the metagenomic libraries in those hosts. Moreover, discovery of new chassis and characterization of synthetic biology toolbox in nonmodel bacteria is an active field of research to expand the potential of these organisms in processes of industrial interest. Here, we assessed the suitability of two Antarctic psychrotolerant Pseudomonas strains as putative alternative hosts for function-driven metagenomics using pSEVA modular vectors as scaffold. We determined a set of synthetic biology tools suitable for these hosts and, as a proof of concept, we demonstrated their fitness for heterologous protein expression. These hosts represent a step forward for the prospection and identification of psychrophilic enzymes of biotechnological interest.

Functional metagenomic libraries generated from anthropogenically impacted environments reveal importance of metabolic genes in biocide and antibiotic resistance.

Anthropogenic activities result in the release of antimicrobial resistant bacteria and a cocktail of antimicrobial compounds into the environment that may directly select or indirectly co-select for antimicrobial resistance (AMR). Many studies use metagenome sequencing or qPCR-based approaches to study the environmental resistome but these methods are limited by a priori knowledge. In this study, a functional metagenomic approach was used to explore biocide resistance mechanisms in two contaminated environments and a pristine site, and to identify whether potentially novel genes conferring biocide resistance also conferred resistance or reduced susceptibility to antibiotics. Resistance was predominately mediated through novel mechanisms exclusive of the well-known qac efflux genes. UDP-galactose 4-epimerase (galE) -like genes were identified in both contaminated environments and were shown to confer cross-resistance to biocides and clinically important antibiotics for the first time (to our knowledge), compared to knockout mutants. GalE -like genes were also co-located with transposons, suggesting mobilisation potential. These results show that housekeeping genes may play a significant yet underappreciated role in AMR in environmental microbiomes.

Functional metagenomics reveals wildlife as natural reservoirs of novel ß-lactamases.

The antibiotic resistances in bacteria are believed to rapidly evolve over time in the anthropogenic environments which enriched with selection pressures. However, the knowledge regarding the development of antibiotic resistance in wildlife and their habitats is scarce. It is, therefore, of great interest and significance to unveil the yet-unknown antibiotic resistances in wildlife in accordance with One Health concept. To this end, we analyzed the samples taken from wildlife and surrounding environments using a functional metagenomics approach. By functional screening in combination with Illumina sequencing, a total of 32 candidate genes which encoding putative novel ß-lactamase were identified. These putative ß-lactamase were taxonomically assigned into bacteria of 23 genera from 7 phyla, where Proteobacteria, Actinobacteria and Firmicutes were dominant. The following functional assessment demonstrated that 4 novel ß-lactamases, namely blaSSA, blaSSB1, blaSSB2 and blaSSD, were functionally active to confer the phenotypical resistance to bacteria by increasing MICs up to 128-fold. Further analysis indicated that the novel ß-lactamases identified in the current study were able to hydrolyze a broad spectrum of ß-lactams including cephalosporins, and they were genetically unique comparing with known ß-lactamases. The plausible transmission of some novel ß-lactamase genes was supported by our results as the same gene was detected in different samples from different sites. This study shed the light on the active role of wildlife and associated environments as natural reservoirs of novel ß-lactamases, implying that the antibiotic resistances might evolve in absence of selection pressure and threaten public health once spread into clinically important pathogens.

Differential response to prolonged amoxicillin treatment: long-term resilience of the microbiome versus long-lasting perturbations in the gut resistome.

The collateral impact of antibiotics on the microbiome has attained increasing attention. However, the ecological consequences of long-term antibiotic exposure on the gut microbiome, including antibiotic resistance, are still limited. Here, we investigated long-term exposure effects to amoxicillin on the human gut microbiome and resistome. Fecal samples were collected from 20 patients receiving 3-months of amoxicillin or placebo treatment as part of a Norwegian multicenter clinical trial on chronic low back pain (AIM study). Samples were collected at baseline, last day of treatment, and 9 months after antibiotic cessation. The abundance and diversity of microbial and resistome composition were characterized using whole shotgun and functional metagenomic sequencing data. While the microbiome profiles of placebo subjects were stable over time, discernible changes in diversity and overall microbiome composition were observed after amoxicillin treatment. In particular, health-associated short-chain fatty acid producing species significantly decreased in proportion. However, these changes were short-lived as the microbiome showed overall recovery 9 months post-treatment. On the other hand, exposure to long-term amoxicillin was associated with an increase in total antimicrobial resistance gene load and diversity of antimicrobial resistance genes, with persistent changes even at 9 months post-treatment. Additionally, beta-lactam resistance was the most affected antibiotic class, suggesting a targeted response to amoxicillin, although changes at the gene level varied across individuals. Overall, our results suggest that the impact of prolonged amoxicillin exposure was more explicit and long-lasting in the fecal resistome than in microbiome composition. Such information is relevant for designing rational administration guidelines for antibiotic therapies.

Functional Metagenomics to Study Antibiotic Resistance.

The construction and screening of metagenomic expression libraries have a great potential to identify novel genes with desired functions. Here, we describe metagenomic library preparation from fecal DNA, screening of libraries for antibiotic resistance genes (ARGs), massively parallel DNA sequencing of the enriched DNA fragments, and a computational pipeline for high-throughput assembly and annotation of functionally selected DNA.

Functional Metagenomics as a Tool to Tap into Natural Diversity of Valuable Biotechnological Compounds.

The marine ecosystem covers more than 70% of the world's surface, and oceans represent a source of varied types of organisms due to the diversified environment. Consequently, the marine environment is an exceptional depot of novel bioactive natural products, with structural and chemical features generally not found in terrestrial habitats. Here, in particular, microbes represent a vast source of unknown and probably new physiological characteristics. They have evolved during extended evolutionary processes of physiological adaptations under various environmental conditions and selection pressures. However, to date, the biodiversity of marine microbes and the versatility of their bioactive compounds and metabolites have not been fully explored. Thus, metagenomic tools are required to exploit the untapped marine microbial diversity and their bioactive compounds. This chapter focuses on function-based marine metagenomics to screen for bioactive molecules of value for biotechnology. Functional metagenomic strategies are described, including sampling in the marine environment, constructing marine metagenomic large-insert libraries, and examples on function-based screens for quorum quenching and anti-biofilm activities.

Activity-Based Screening of Metagenomic Fosmid Libraries for Hydrogen-Uptake Enzymes.

Here, we outline how to identify hydrogenase enzymes from metagenomic fosmid libraries through an activity-based screening approach. A metagenomic fosmid library is constructed in E. coli and the fosmids are transferred into a hydrogenase deletion mutant of Shewanella oneidensis MR-1 (ΔhyaB) via triparental mating. If a fosmid clone exhibits hydrogen-uptake activity, S. oneidensis' phenotype is restored and hydrogenase activity is indicated by a color change of the medium from yellow to colorless. The screen enables screening of 48 metagenomic fosmid clones in parallel.

Identification of PKS Gene Clusters from Metagenomic Libraries Using a Next-Generation Sequencing Approach.

Microbial secondary metabolites have been an important source of bioactive compounds with diverse applications from medicine to agriculture, noticeably those encoded by polyketide synthase (PKS) clusters due to their astounding chemical diversity. While most discovered compounds originate from culturable microorganisms, yet-to-be cultured microbes represent a reservoir of previously inaccessible compounds. The advent and development of metagenomics have allowed not only the characterization of these microorganisms but also their metabolic potential, making viable the prospection of environmental PKS for natural product discovery.Study of environmental PKSs often relies on the construction of metagenomic libraries and their mining, with clones containing PKS clusters identified via amplification of conserved domains and then screened for an activity of interest. Compounds produced by clones exhibiting the desired bioactivity can be isolated and characterized. However, these approaches can be less sensitive and biased against more divergent clusters, in addition to precluding the use of bioinformatics for cluster characterization prior to expression. While direct shotgun sequencing of metagenomes has identified and profiled a great number of PKSs from different environments and yet-to-be cultured microorganisms, it does not lend itself well to heterologous expression, the cruxes of natural product discovery.Here, we describe a strategy for sequencing entire metagenomic libraries while maintaining correspondence between sequence and clone, allowing the full characterization and annotation of all clusters present in a library using bioinformatic tools and then seamlessly passing clones of interest for activity screening through heterologous expression. Once a library is sequenced, the methods herein can be adapted for the mining of any biosynthetic gene cluster of interest within a metagenomic library.

Discovery of novel carbohydrate degrading enzymes from soda lakes through functional metagenomics.

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.

Isolation of novel cold-tolerance genes from rhizosphere microorganisms of Antarctic plants by functional metagenomics.

The microorganisms that thrive in Antarctica, one of the coldest environments on the planet, have developed diverse adaptation mechanisms to survive in these extreme conditions. Through functional metagenomics, in this work, 29 new genes related to cold tolerance have been isolated and characterized from metagenomic libraries of microorganisms from the rhizosphere of two Antarctic plants. Both libraries were hosted in two cold-sensitive strains of Escherichia coli: DH10B ΔcsdA and DH10B ΔcsdA Δrnr. The csdA gene encodes a DEAD-box RNA helicase and rnr gene encodes an exoribonuclease, both essential for cold-adaptation. Cold-tolerance tests have been carried out in solid and liquid media at 15°C. Among the cold-tolerance genes identified, 12 encode hypothetical and unknown proteins, and 17 encode a wide variety of different proteins previously related to other well-characterized ones involved in metabolism reactions, transport and membrane processes, or genetic information processes. Most of them have been connected to cold-tolerance mechanisms. Interestingly, 13 genes had no homologs in E. coli, thus potentially providing entirely new adaptation strategies for this bacterium. Moreover, ten genes also conferred resistance to UV-B radiation, another extreme condition in Antarctica.

Functional mining of novel terpene synthases from metagenomes.

BACKGROUND: Terpenes are one of the most diverse and abundant classes of natural biomolecules, collectively enabling a variety of therapeutic, energy, and cosmetic applications. Recent genomics investigations have predicted a large untapped reservoir of bacterial terpene synthases residing in the genomes of uncultivated organisms living in the soil, indicating a vast array of putative terpenoids waiting to be discovered. RESULTS: We aimed to develop a high-throughput functional metagenomic screening system for identifying novel terpene synthases from bacterial metagenomes by relieving the toxicity of terpene biosynthesis precursors to the Escherichia coli host. The precursor toxicity was achieved using an inducible operon encoding the prenyl pyrophosphate synthetic pathway and supplementation of the mevalonate precursor. Host strain and screening procedures were finely optimized to minimize false positives arising from spontaneous mutations, which avoid the precursor toxicity. Our functional metagenomic screening of human fecal metagenomes yielded a novel ß-farnesene synthase, which does not show amino acid sequence similarity to known ß-farnesene synthases. Engineered S. cerevisiae expressing the screened ß-farnesene synthase produced 120 mg/L ß-farnesene from glucose (2.86 mg/g glucose) with a productivity of 0.721 g/L∙h. CONCLUSIONS: A unique functional metagenomic screening procedure was established for screening terpene synthases from metagenomic libraries. This research proves the potential of functional metagenomics as a sequence-independent avenue for isolating targeted enzymes from uncultivated organisms in various environmental habitats.