Identifying Active Phage Lysins through Functional Viral Metagenomics

Recent metagenomic sequencing studies of uncultured viral populations have provided novel insights into the ecology of environmental bacteriophage. At the same time, viral metagenomes could also represent a potential source of recombinant proteins with biotechnological value. In order to identify such proteins, a novel two-step screening technique was devised for cloning phage lytic enzymes from uncultured viral DNA. This plasmid-based approach first involves a primary screen in which transformed Escherichia coli clones that demonstrate colony lysis following exposure to inducing agent are identified. This effect, which can be due to the expression of membrane-permeabilizing phage holins, is discerned by the development a hemolytic effect in surrounding blood agar. In a secondary step, the clones identified in the primary screen are overlaid with autoclaved Gram-negative bacteria (specifically Pseudomonas aeruginosa) to assay directly for recombinant expression of lytic enzymes, which are often encoded proximally to holins in phage genomes. As proof-of-principle, the method was applied to a viral metagenomic library constructed from mixed animal feces, and 26 actively expressed lytic enzymes were cloned. These proteins include both Gram-positive-like and Gram-negative-like enzymes, as well as several atypical lysins whose predicted structures are less common among known phage. Overall, this study represents one of the first functional screens of a viral metagenomic population, and it provides a general approach for characterizing lysins from uncultured phage.The field of metagenomics has expanded rapidly in recent years, providing access to environmental microorganisms that would remain unapproachable by standard, culture-based methods. The foundation of metagenomics lies in the direct extraction of DNA/RNA from environmental samples (e.g., soil, water, or feces) without prior isolation of individual microbial species (reviewed in references 18 and 32). It has been estimated that only a small proportion of naturally occurring microbes—approximately 1% of soil bacteria, for instance—are culturable under standard laboratory conditions (31). In this light, metagenomics has become an increasingly common tool for studying diverse ecosystems, from around the globe to within the human body.Overall, metagenomics research can be divided into two general categories: sequence-based and functional. In the former, environmental DNA is sequenced in mass and compared with genetic databases to address broad questions of ecology, taxonomy, and diversity. Some of the most extensive metagenomic studies to date have been sequence based in nature, benefiting from the development of high-throughput sequencing technologies. Notable examples include a 76-megabase study of an acid mine biofilm (33), a 1-gigabase analysis of the Sargasso Sea (35), and a 6.3-gigabase sampling of global oceanic samples (25). In functional metagenomics, by contrast, environmental genes are recombinantly expressed within a host organism, which is monitored for the acquisition of a desired phenotype. Rather than providing insight into entire ecosystems, functional studies aim to identify individual molecules with biomedical or industrial value. Targeted compounds may be either proteins (usually enzymes) encoded directly by environmental genes or small molecules synthesized by several enzymes of a gene cluster. Numerous classes of molecules have been identified to date, with particular interest in the areas of biosynthesis, biomass degradation, and antibiotic discovery (reviewed in references 2, 34, and 36).While bacteria provide the majority of DNA to most metagenomic pools, recent studies have begun focusing on subsets of total environmental populations. A prominent example is viral metagenomics, in which viral particles (predominately bacteriophage) are purified from cellular material prior to DNA extraction (reviewed in references 10 and 12). Although the yield of DNA from environmental phage isolates is generally low, PCR amplification techniques have been developed to overcome this issue (4, 26). Viral metagenomic analyses have been conducted on a growing number of samples, including ones purified from soil (15), seawater (4, 39), and human feces (3). These studies have revealed a remarkable abundance of novel sequences, supporting the notion that phage represent the largest source of untapped genetic diversity on the planet (19). Despite this wealth of information, viral metagenomic studies to date have remained predominantly sequence based in nature. In this regard, functional screens of viral metagenomes could provide a large source of recombinant molecules.Recently one class of phage-encoded protein has received particular attention from the biotechnology field: phage lytic enzymes (also referred to as endolysins or lysins) (reviewed in references 16 and 17). These peptidoglycan hydrolases are expressed late in the infective cycle of double-stranded DNA phage, and—along with a membrane-permeabilizing protein known as a holin—they are responsible for disrupting the bacterial cell envelope and freeing progeny viral particles. Despite this conserved biological function, phage lysins (especially Gram-positive ones) are a tremendously diverse group of proteins whose enzymatic specificity includes various bonds within the peptidoglycan macromolecule. They include glycosyl hydrolases that target the polysaccharide backbone (muramidases/lysozymes and glucosaminidases), alanine amidases that target the initial l-alanine of the pentapeptide stem, and endopeptidases that target subsequent peptide bonds in the stem or cross bridge. While lysins of Gram-negative phage generally consist of an enzymatic domain alone, Gram-positive lysins are modular and combine an N-terminal lytic domain with a C-terminal binding domain that can recognize various epitopes within the target cell envelope.Although researchers have known of lysins for decades, interest has increased markedly in recent years after it was proposed that they could act as novel anti-infective agents against Gram-positive pathogens, whose peptidoglycan is directly accessible from the extracellular space (8, 23, 28). A growing number of in vitro and in vivo studies have confirmed the ability of recombinantly expressed lysins to kill such organisms, and their appeal lies in both the potency and the specificity they demonstrate toward individual Gram-positive species. This enzybiotic value of phage lysins goes alongside additional proposed applications in the areas of food (11), agricultural (20), veterinary (7), and industrial science (21, 40).Considering this potential, lytic enzymes represent an intriguing functional target for viral metagenomic screens. At the same time, identifying lysins in this manner would present several distinct challenges. Aside from general concerns common to all functional screens (e.g., protein expression and solubility), metagenomic lysin identification would face the following particular issues. (i) Clonal toxicity: recombinant lysin expression is typically well tolerated by host bacteria, since the enzymes are sequestered in the cytoplasm away from the peptidoglycan layer. Holins, on the other hand, interact nonspecifically with plasma membranes and are generally toxic to an Escherichia coli host, inducing bacteriolysis from within (9). Since holins are short (∼100 residues) and are often encoded adjacent to lysins, they can lead to selective toxicity of many of the clones one hopes to identify. In a metagenomic screen, where numerous lysins are present within a single library, this effect could lead to a significant loss of positive hits. (ii) Target bacterial species: in standard phage genomic screens, lysin-encoding clones are selected by their ability to kill the host bacterium of the encoding phage, which generally demonstrates the highest sensitivity (27). In a metagenomic screen, however, numerous host species of unknown origin could be present within a sample, confounding this choice of screening agent.To address these issues, we have devised a novel functional strategy for the general cloning of lytic enzymes from uncultured phage DNA. It utilizes a plasmid-based E. coli expression system and consists of a two-step process. Following induction by arabinose, clones are first screened for holin-mediated lysis by a hemolytic effect they create in the surrounding blood agar. These initial hits are then restreaked as patches and overlaid with Gram-negative cells whose outer membranes have been permeabilized by autoclaving, serving as a general source of peptidoglycan. The clones are observed for surrounding Gram-negative clearing zones to assay directly for the recombinant production of lytic enzymes encoded adjacent to the holins. As proof-of-principle, we applied our methodology to a viral metagenomic library constructed from mixed animal feces, identifying 26 actively expressed lysins of diverse molecular architectures. The first of its kind, this study presents a general model for lysin identification through viral metagenomics, highlighting the potential of this field for cloning of proteins of biotechnological or academic value.