Structural and Functional Analysis of Viral siRNAs

A large amount of short interfering RNA (vsiRNA) is generated from plant viruses during infection, but the function, structure and biogenesis of these is not understood. We profiled vsiRNAs using two different high-throughput sequencing platforms and also developed a hybridisation based array approach. The profiles obtained through the Solexa platform and by hybridisation were very similar to each other but different from the 454 profile. Both deep sequencing techniques revealed a strong bias in vsiRNAs for the positive strand of the virus and identified regions on the viral genome that produced vsiRNA in much higher abundance than other regions. The hybridisation approach also showed that the position of highly abundant vsiRNAs was the same in different plant species and in the absence of RDR6. We used the Terminator 5′-Phosphate-Dependent Exonuclease to study the 5′ end of vsiRNAs and showed that a perfect control duplex was not digested by the enzyme without denaturation and that the efficiency of the Terminator was strongly affected by the concentration of the substrate. We found that most vsiRNAs have 5′ monophosphates, which was also confirmed by profiling short RNA libraries following either direct ligation of adapters to the 5′ end of short RNAs or after replacing any potential 5′ ends with monophosphates. The Terminator experiments also showed that vsiRNAs were not perfect duplexes. Using a sensor construct we also found that regions from the viral genome that were complementary to non-abundant vsiRNAs were targeted in planta just as efficiently as regions recognised by abundant vsiRNAs. Different high-throughput sequencing techniques have different reproducible sequence bias and generate different profiles of short RNAs. The Terminator exonuclease does not process double stranded RNA, and because short RNAs can quickly re-anneal at high concentration, this assay can be misleading if the substrate is not denatured and not analysed in a dilution series. The sequence profiles and Terminator digests suggest that CymRSV siRNAs are produced from the structured positive strand rather than from perfect double stranded RNA or by RNA dependent RNA polymerase.     

A Compact Viral Processing Proteinase/Ubiquitin Hydrolase from the OTU Family

Turnip yellow mosaic virus (TYMV) - a member of the alphavirus-like supergroup of viruses - serves as a model system for positive-stranded RNA virus membrane-bound replication. TYMV encodes a precursor replication polyprotein that is processed by the endoproteolytic activity of its internal cysteine proteinase domain (PRO). We recently reported that PRO is actually a multifunctional enzyme with a specific ubiquitin hydrolase (DUB) activity that contributes to viral infectivity. Here, we report the crystal structure of the 150-residue PRO. Strikingly, PRO displays no homology to other processing proteinases from positive-stranded RNA viruses, including that of alphaviruses. Instead, the closest structural homologs of PRO are DUBs from the Ovarian tumor (OTU) family. In the crystal, one molecule''s C-terminus inserts into the catalytic cleft of the next, providing a view of the N-terminal product complex in replication polyprotein processing. This allows us to locate the specificity determinants of PRO for its proteinase substrates. In addition to the catalytic cleft, at the exit of which the active site is unusually pared down and solvent-exposed, a key element in molecular recognition by PRO is a lobe N-terminal to the catalytic domain. Docking models and the activities of PRO and PRO mutants in a deubiquitylating assay suggest that this N-terminal lobe is also likely involved in PRO''s DUB function. Our data thus establish that DUBs can evolve to specifically hydrolyze both iso- and endopeptide bonds with different sequences. This is achieved by the use of multiple specificity determinants, as recognition of substrate patches distant from the cleavage sites allows a relaxed specificity of PRO at the sites themselves. Our results thus shed light on how such a compact protein achieves a diversity of key functions in viral genome replication and host-pathogen interaction.     

Structural and Functional Comparison of Polysaccharide-Degrading Enzymes

Sugar molecules as well as enzymes degrading them are ubiquitously present in physiological systems, especially for vertebrates. Polysaccharides have at least two aspects to their function, one due to their mechanical properties and the second one involves multiple regulatory processes or interactions between molecules, cells, or extracellular space. Various bacteria exert exogenous pressures on their host organism to diversity glycans and their structures in order for the host organism to evade the destructive function of such microbes. Many bacterial organism produce glycan-degrading enzymes in order to facilitate their invasion of host tissues. Such polysaccharide degrading enzymes utilize mainly two modes of polysaccharide-degradation, a hydrolysis and a β-elimination process. The three-dimensional structures of several of these enzymes have been elucidated recently using X-ray crystallography. There are many common structural motifs among these enzymes, mainly the presence of an elongated cleft transversing these molecules which functions as a polysaccharide substrate binding site as well as the catalytic site for these enzymes. The detailed structural information obtained about these enzymes allowed formulation of proposed mechanisms of their action. The polysaccharide lyases utilize a proton acceptance and donation mechanism (PAD), whereas polysaccharide hydrolases use a direct double displacement (DD) mechanism to degrade their substrates.     

A Structural and Functional Comparison Between Infectious and Non-Infectious Autocatalytic Recombinant PrP Conformers

Infectious prions contain a self-propagating, misfolded conformer of the prion protein termed PrP^Sc. A critical prediction of the protein-only hypothesis is that autocatalytic PrP^Sc molecules should be infectious. However, some autocatalytic recombinant PrP^Sc molecules have low or undetectable levels of specific infectivity in bioassays, and the essential determinants of recombinant prion infectivity remain obscure. To identify structural and functional features specifically associated with infectivity, we compared the properties of two autocatalytic recombinant PrP conformers derived from the same original template, which differ by >10⁵-fold in specific infectivity for wild-type mice. Structurally, hydrogen/deuterium exchange mass spectrometry (DXMS) studies revealed that solvent accessibility profiles of infectious and non-infectious autocatalytic recombinant PrP conformers are remarkably similar throughout their protease-resistant cores, except for two domains encompassing residues 91-115 and 144-163. Raman spectroscopy and immunoprecipitation studies confirm that these domains adopt distinct conformations within infectious versus non-infectious autocatalytic recombinant PrP conformers. Functionally, in vitro prion propagation experiments show that the non-infectious conformer is unable to seed mouse PrP^C substrates containing a glycosylphosphatidylinositol (GPI) anchor, including native PrP^C. Taken together, these results indicate that having a conformation that can be specifically adopted by post-translationally modified PrP^C molecules is an essential determinant of biological infectivity for recombinant prions, and suggest that this ability is associated with discrete features of PrP^Sc structure.     

Structure of the A20 OTU domain and mechanistic insights into deubiquitination

RACK1 (receptor for activated C kinase 1) is an abundant scaffolding protein, which binds active PKCbetaII (protein kinase C betaII) increasing its activity in vitro. RACK1 has also been described as a component of the small ribosomal subunit, in proximity to the mRNA exit channel. In the present study we tested the hypothesis that PKCbetaII plays a specific role in translational control and verified whether it may associate with the ribosomal machinery. We find that specific inhibition of PKCbetaI/II reduces translation as well as global PKC inhibition, but without affecting phosphorylation of mTOR (mammalian target of rapamycin) targets. These results suggest that PKCbetaII acts as a specific PKC isoform affecting translation in an mTOR-independent fashion, possibly close to the ribosomal machinery. Using far-Western analysis, we found that PKCbetaII binds ribosomes in vitro. Co-immunoprecipitation studies indicate that a small but reproducible pool of PKCbetaII is associated with membranes containing ribosomes, suggesting that in vivo PKCbetaII may also physically interact with the ribosomal machinery. Polysomal profiles show that stimulation of PKC results in an increased polysomes/80S ratio, associated with a shift of PKCbetaII to the heavier part of the gradient. A RACK1-derived peptide that inhibits the binding of active PKCbetaII to RACK1 reduces the polysomes/80S ratio and methionine incorporation, suggesting that binding of PKCbetaII to RACK1 is important for PKC-mediated translational control. Finally, down-regulation of RACK1 by siRNA (small interfering RNA) impairs the PKC-mediated increase of translation. Taken together the results of the present study show that PKCbetaII can act as a specific PKC isoform regulating translation, in an mTOR-independent fashion, possibly close to the ribosomal machinery.     

Structural Alphabets for Protein Structure Classification: A Comparison Study

Finding structural similarities between proteins often helps reveal shared functionality, which otherwise might not be detected by native sequence information alone. Such similarity is usually detected and quantified by protein structure alignment. Determining the optimal alignment between two protein structures, however, remains a hard problem. An alternative approach is to approximate each three-dimensional protein structure using a sequence of motifs derived from a structural alphabet. Using this approach, structure comparison is performed by comparing the corresponding motif sequences or structural sequences. In this article, we measure the performance of such alphabets in the context of the protein structure classification problem. We consider both local and global structural sequences. Each letter of a local structural sequence corresponds to the best matching fragment to the corresponding local segment of the protein structure. The global structural sequence is designed to generate the best possible complete chain that matches the full protein structure. We use an alphabet of 20 letters, corresponding to a library of 20 motifs or protein fragments having four residues. We show that the global structural sequences approximate well the native structures of proteins, with an average coordinate root mean square of 0.69 Å over 2225 test proteins. The approximation is best for all α-proteins, while relatively poorer for all β-proteins. We then test the performance of four different sequence representations of proteins (their native sequence, the sequence of their secondary-structure elements, and the local and global structural sequences based on our fragment library) with different classifiers in their ability to classify proteins that belong to five distinct folds of CATH. Without surprise, the primary sequence alone performs poorly as a structure classifier. We show that addition of either secondary-structure information or local information from the structural sequence considerably improves the classification accuracy. The two fragment-based sequences perform better than the secondary-structure sequence but not well enough at this stage to be a viable alternative to more computationally intensive methods based on protein structure alignment.     

Death and Kinship in Hinduism: Structural and Functional Interpretations

This paper is, first, a structural analysis demonstrating that in Hindu sacred law death rites reflect a conception of kinship wherein intimacy is affected not only by such matters as genealogical distance, but also by differences in spiritual purity among kinsmen. Second, the paper shows that structural analysis of particular civilizations have more than intrinsic value, that they are also a necessary adjunct to highly general functional theories.     

Neural Correlates of Emotional Personality: A Structural and Functional Magnetic Resonance Imaging Study

Studies addressing brain correlates of emotional personality have remained sparse, despite the involvement of emotional personality in health and well-being. This study investigates structural and functional brain correlates of psychological and physiological measures related to emotional personality. Psychological measures included neuroticism, extraversion, and agreeableness scores, as assessed using a standard personality questionnaire. As a physiological measure we used a cardiac amplitude signature, the so-called E_κ value (computed from the electrocardiogram) which has previously been related to tender emotionality. Questionnaire scores and E_κ values were related to both functional (eigenvector centrality mapping, ECM) and structural (voxel-based morphometry, VBM) neuroimaging data. Functional magnetic resonance imaging (fMRI) data were obtained from 22 individuals (12 females) while listening to music (joy, fear, or neutral music). ECM results showed that agreeableness scores correlated with centrality values in the dorsolateral prefrontal cortex, the anterior cingulate cortex, and the ventral striatum (nucleus accumbens). Individuals with higher E_κ values (indexing higher tender emotionality) showed higher centrality values in the subiculum of the right hippocampal formation. Structural MRI data from an independent sample of 59 individuals (34 females) showed that neuroticism scores correlated with volume of the left amygdaloid complex. In addition, individuals with higher E_κ showed larger gray matter volume in the same portion of the subiculum in which individuals with higher E_κ showed higher centrality values. Our results highlight a role of the amygdala in neuroticism. Moreover, they indicate that a cardiac signature related to emotionality (E_κ) correlates with both function (increased network centrality) and structure (grey matter volume) of the subiculum of the hippocampal formation, suggesting a role of the hippocampal formation for emotional personality. Results are the first to show personality-related differences using eigenvector centrality mapping, and the first to show structural brain differences for a physiological measure associated with personality.     

桩蛋白的结构与功能

桩蛋白(paxillin)是近年来发现的一种信号蛋白,主要定位于黏着斑,包含LD模体、LIM结构域、SH2和SH3结合结构域,在整个分子中还散在着多种磷酸化位点,共同构成了桩蛋白的多结构域性结构。桩蛋白分子本身的酶活性尚不清楚,但很可能作为细胞内的一种接头蛋白与多种功能蛋白质结合。另外．作为黏着斑的重要组成部分,桩蛋白不仅参与了整合蛋白介导的信号转导和黏着斑的组装,在细胞黏附和迁移过程中也发挥了重要作用。     

Functional and Structural Properties of Ovomucin

Relationships between the structural (hydrophobicity and viscosity) and functional (foaming and emulsifying) properties of proteins were investigated by using a polymeric form of ovomucin (soluble type), and dissociated ovomucins which were treated with sonication (sonicated type) and reduction (reduced type). The soluble, sonicated and reduced ovomucins were ascertained to have excellent foaming and emulsifying properties. The foaming properties of the ovomucins decreased in proportion to decreases in the viscosity as the dissociation proceeded, in the order of the soluble, sonicated and reduced types. On the other hand, the emulsifying properties of ovomucins increased in proportion to increases in the surface hydrophobicity as the dissociation proceeded.

Thus, it was suggested that the foaming properties of ovomucins were dependent upon viscosity, and that the emulsifying properties of ovomucins were dependent upon surface hydrophobicity.     

Structural and Functional Diversity of Desmosomes

Abstract

Desmosomes anchor intermediate filaments at sites of cell contact established by the interaction of cadherins extending from opposing cells. The incorporation of cadherins, catenin adaptors, and cytoskeletal elements resembles the closely related adherens junction. However, the recruitment of intermediate filaments distinguishes desmosomes and imparts a unique function. By linking the load-bearing intermediate filaments of neighboring cells, desmosomes create mechanically contiguous cell sheets and, in so doing, confer structural integrity to the tissues they populate. This trait and a well-established role in human disease have long captured the attention of cell biologists, as evidenced by a publication record dating back to the mid-1860s. Likewise, emerging data implicating the desmosome in signaling events pertinent to organismal development, carcinogenesis, and genetic disorders will secure a prominent role for desmosomes in future biological and biomedical investigations.     

Structural and Functional Studies of Truncated Hemolysin A from Proteus mirabilis

In this study we analyzed the structure and function of a truncated form of hemolysin A (HpmA265) from Proteus mirabilis using a series of functional and structural studies. Hemolysin A belongs to the two-partner secretion pathway. The two-partner secretion pathway has been identified as the most common protein secretion pathway among Gram-negative bacteria. Currently, the mechanism of action for the two-partner hemolysin members is not fully understood. In this study, hemolysis experiments revealed a unidirectional, cooperative, biphasic activity profile after full-length, inactive hemolysin A was seeded with truncated hemolysin A. We also solved the first x-ray structure of a TpsA hemolysin. The truncated hemolysin A formed a right-handed parallel β-helix with three adjoining segments of anti-parallel β-sheet. A CXXC disulfide bond, four buried solvent molecules, and a carboxyamide ladder were all located at the third complete β-helix coil. Replacement of the CXXC motif led to decreased activity and stability according to hemolysis and CD studies. Furthermore, the crystal structure revealed a sterically compatible, dry dimeric interface formed via anti-parallel β-sheet interactions between neighboring β-helix monomers. Laser scanning confocal microscopy further supported the unidirectional interconversion of full-length hemolysin A. From these results, a model has been proposed, where cooperative, β-strand interactions between HpmA265 and neighboring full-length hemolysin A molecules, facilitated in part by the highly conserved CXXC pattern, account for the template-assisted hemolysis.Hemolysin A (HpmA)² and B (HpmB) from Proteus mirabilis belong to the Type V_b or two-partner secretion pathway (1), the most widespread of the five porin-type protein translocating systems found within bacterial, fungal, plant, and animal kingdoms (2). Cell surface adhesions, iron-acquisition proteins, and cytolysins/hemolysins all use two-partner secretion pathways (3–5). The A-component of the two-partner secretion in P. mirabilis is a 166-kDa virulence factor capable of mammalian blood cell lysis upon secretion from the cell. This is accomplished by Sec-dependent transport to the periplasm followed by N-terminal proteolytic processing. Extracellular secretion occurs by transport through the B-component, HpmB, which is a 16-stranded β-barrel transmembrane channel (6). In addition to its role in efficient secretion, HpmB is also necessary for activation of the larger exoprotein A-component (HpmA) (7–10).Studies on hemolytic TpsA members report that: 1) a truncated TpsA containing the N-terminal secretion cap (11) complements and restores hemolytic activity within a non-secreted/inactive pool of full-length TpsA (12), 2) the conserved cysteine residues within a CXXC motif are not required for secretion (12), and 3) the first asparagine within a NPNG hemagglutinin motif is required for efficient secretion (13). Other investigations demonstrate significant conformational change within TpsA members during B-component dependent secretion (8, 14–16).Recent x-ray crystal structures for two TpsA adhesion orthologs, hemagglutinin from Bordetella pertussis (FHA) and high molecular weight protein from Haemophilus influenzae (HMW1) adopt a right-handed parallel β-helix similar to pectate lyase (11, 18, 19). The 301-residue N-terminal FHA fragment (Fha30) contains a 37 parallel stranded β-helix. Stabilization of type I β-turns at two highly conserved regions: ⁶⁶NPNL and ¹⁰⁵NPNG is proposed to play a large role in the ability of this N-terminal fragment to rapidly adopt β-helix architecture (11, 20). Despite 21% sequence identity, the 371-residue HMW1 structure (HMW1-PP) is a similar 47 parallel stranded β-helix. A hypothesis arose from these β-helix structures that suggests extracellular secretion through TpsB channels, and the progressive folding of TpsA members is energetically coupled. Full-length TpsA adhesion members have been proposed to have a filamentous appearance built from a right-handed β-helix fold (21). To date, there is little known about the full-length HpmA domain architecture. However, there are two filamentous hemagglutinin type domains. The N-terminal domain is positioned between residues 30 and 167, whereas the C-terminal domain lies between residues 1200 and 1264 and has been proposed to facilitate cellular aggregation.In this work, we investigated the functional and structural role of truncated hemolysin A (HpmA265) during the template-assisted activation of hemolysis. A previous investigation with ShlA, a homologous TpsA member from Serratia marcescens, has shown similar complementation using a 255-amino acid fragment (12). Here, we demonstrate that HmpA265 can cooperatively cross seed an inactive pool of full-length hemolysin A (HpmA*) to form an exotoxin measured by our template-assisted hemolytic assay (TAHA). We also report that the CXXC motif provides structural stability and facilitates reversible re-folding. The structure reveals a right-handed β-helix, similar to those of FHA and HMW1. A number of conserved features found at the putative subunit interface suggest a mechanism by which activation of inactive HpmA* occurs.     

Sleep-Inducing Properties of DSIP Analogs: Structural and Functional Relationships

The sleep-inducing activity of Delta Sleep-Inducing Peptide (DSIP) and its 13 synthetic analogs has been studied on rabbits with preliminary implanted electrodes. The peptides were injected into the lateral ventricle of cerebrum. Polygraphic computer monitoring of sleep–wake states was carried out at daytime for 7–12 h. DSIP and most analogs had no statistically significant effect on sleep compared to the control administration of saline to the same animals. [NMeAla²]DSIP and [Pro²]DSIP had a pronounced sleep-inducing effect and reliably increased the proportion of slow-wave sleep by 10–15% on average compared to the control. Several other analogs had a week sleep-inducing effect, increasing the proportion of slow-wave sleep during specific recording time only. [-Ala²]DSIP significantly suppressed sleep. In addition, this analog, as well as parent DSIP and four proline-containing nonapeptides, slightly increased the body temperature. The revealed differences may be due to both conformation properties and proteolytic resistance of the studied molecules, and it may reflect their indirect involvement in the control sleep–wake hormonal processes.     

Structural and Functional Characterization of Paramecium Dynein: Initial Studies

ABSTRACT Dynein arms and isolated dynein from Paramecium tetraurelia ciliary axonemes are comparable in structure, direction of force generation, and microtubule translocation ability to other dyneins. In situ arms have dimensions and substructure similar to those of Tetrahymena. Based on spoke arrangement in intact axonemes, arms translocate axonemal microtubules in sliding such that active dynein arms are (-) end directed motors and the doublet to which the body and cape of the arms binds (N) translocates the adjacent doublet (N+1) upward. After salt extraction, based on ATPase activity, paramecium dynein is found as a 22S and a 14S species. the 22S dynein is a three-headed molecule that has unfolded from the in situ dimensions; the 14S dynein is single headed. Both dyneins can be photocleaved by UV light (350 nm) in the presence of Mg^2-, ATP and vanadate; the photocleavage pattern of 22S dynein differs from that seen with Tetrahymena. Both isolated dyneins translocate taxol-stabilized, bovine brain microtubules in vitro. Under standard conditions, 22S dynein, like comparable dyneins from other organisms, translocates at velocities that are about three times faster than 14S dynein.     

Peptide Toxins in Sea Anemones: Structural and Functional Aspects

Sea anemones are a rich source of two classes of peptide toxins, sodium channel toxins and potassium channel toxins, which have been or will be useful tools for studying the structure and function of specific ion channels. Most of the known sodium channel toxins delay channel inactivation by binding to the receptor site 3 and most of the known potassium channel toxins selectively inhibit Kv1 channels. The following peptide toxins are functionally unique among the known sodium or potassium channel toxins: APETx2, which inhibits acid-sensing ion channels in sensory neurons; BDS-I and II, which show selectivity for Kv3.4 channels and APETx1, which inhibits human ether-a-go-go-related gene potassium channels. In addition, structurally novel peptide toxins, such as an epidermal growth factor (EGF)-like toxin (gigantoxin I), have also been isolated from some sea anemones although their functions remain to be clarified.     

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.