Control of the Serine Integrase Reaction: Roles of the Coiled-Coil and Helix E Regions in DNA Site Synapsis and Recombination.

Bacteriophage serine integrases catalyze highly specific recombination reactions between defined DNA segments called att sites. These reactions are reversible depending upon the presence of a second phage-encoded directionality factor. The bipartite C-terminal DNA-binding region of integrases includes a recombinase domain (RD) connected to a zinc-binding domain (ZD), which contains a long flexible coiled-coil (CC) motif that extends away from the bound DNA. We directly show that the identities of the phage A118 integrase att sites are specified by the DNA spacing between the RD and ZD DNA recognition determinants, which in turn directs the relative trajectories of the CC motifs on each subunit of the att-bound integrase dimer. Recombination between compatible dimer-bound att sites requires minimal-length CC motifs and 14 residues surrounding the tip where the pairing of CC motifs between synapsing dimers occurs. Our alanine-scanning data suggest that molecular interactions between CC motif tips may differ in integrative (attP × attB) and excisive (attL × attR) recombination reactions. We identify mutations in 5 residues within the integrase oligomerization helix that control the remodeling of dimers into tetramers during synaptic complex formation. Whereas most of these gain-of-function mutants still require the CC motifs for synapsis, one mutant efficiently, but indiscriminately, forms synaptic complexes without the CC motifs. However, the CC motifs are still required for recombination, suggesting a function for the CC motifs after the initial assembly of the integrase synaptic tetramer. IMPORTANCE The robust and exquisitely regulated site-specific recombination reactions promoted by serine integrases are integral to the life cycle of temperate bacteriophage and, in the case of the A118 prophage, are an important virulence factor of Listeria monocytogenes. The properties of these recombinases have led to their repurposing into tools for genetic engineering and synthetic biology. In this report, we identify determinants regulating synaptic complex formation between correct DNA sites, including the DNA architecture responsible for specifying the identity of recombination sites, features of the unique coiled-coil structure on the integrase that are required to initiate synapsis, and amino acid residues on the integrase oligomerization helix that control the remodeling of synapsing dimers into a tetramer active for DNA strand exchange.

Atomic structures of an entire contractile injection system in both the extended and contracted states.

Contractile injection systems are sophisticated multiprotein nanomachines that puncture target cell membranes. Although the number of atomic-resolution insights into contractile bacteriophage tails, bacterial type six secretion systems and R-pyocins is rapidly increasing, structural information on the contraction of bacterial phage-like protein-translocation structures directed towards eukaryotic hosts is scarce. Here, we characterize the antifeeding prophage AFP from Serratia entomophila by cryo-electron microscopy. We present the high-resolution structure of the entire AFP particle in the extended state, trace 11 protein chains de novo from the apical cap to the needle tip, describe localization variants and perform specific structural comparisons with related systems. We analyse inter-subunit interactions and highlight their universal conservation within contractile injection systems while revealing the specificities of AFP. Furthermore, we provide the structure of the AFP sheath-baseplate complex in a contracted state. This study reveals atomic details of interaction networks that accompany and define the contraction mechanism of toxin-delivery tailocins, offering a comprehensive framework for understanding their mode of action and for their possible adaptation as biocontrol agents.

Characterization of two glycosyl hydrolases, putative prophage endolysins, that target Clostridium perfringens.

Clostridium perfringens, a spore-forming anaerobic bacterium, causes food poisoning and gas gangrene in humans and is an agent of necrotizing enteritis in poultry, swine and cattle. Endolysins are peptidoglycan hydrolases from bacteriophage that degrade the bacterial host cell wall causing lysis and thus harbor antimicrobial therapy potential. The genes for the PlyCP10 and PlyCP41 endolysins were found in prophage regions of the genomes from C. perfringens strains Cp10 and Cp41, respectively. The gene for PlyCP10 encodes a protein of 351 amino acids, while the gene for PlyCP41 encodes a protein of 335 amino acids. Both proteins harbor predicted glycosyl hydrolase domains. Recombinant PlyCP10 and PlyCP41 were expressed in E. coli with C-terminal His-tags, purified by nickel chromatography and characterized in vitro. PlyCP10 activity was greatest at pH 6.0, and between 50 and 100 mM NaCl. PlyCP41 activity was greatest between pH 6.5 and 7.0, and at 50 mM NaCl, with retention of activity as high as 600 mM NaCl. PlyCP10 lost most of its activity above 42°C, whereas PlyCP41 survived at 50°C for 30 min and still retained >60% activity. Both enzymes had lytic activity against 75 C. perfringens strains (isolates from poultry, swine and cattle) suggesting therapeutic potential.

The structure of DLP12 endolysin exhibiting alternate loop conformation and comparative analysis with other endolysins.

The lytic enzyme, endolysin, is encoded by bacteriophages (phages) to destroy the peptidoglycan layer of host bacterial cells. The release of phage progenies to start the new infection cycle is dependent on the cell lysis event. Endolysin encoded by DLP12 cryptic prophage is a SAR endolysin which is retained by the bacterium presumably due to the benefit it confers. The structure of DLP12 endolysin (Id: 4ZPU) determined at 2.4 Å resolution is presented here. The DLP12 endolysin structure shows a modular nature and is organized into distinct structural regions. One of the monomers has the loops at the active site in a different conformation. This has led to a suggestion of depicting possibly active and inactive state of DLP12 endolysin. Comparison of DLP12 endolysin structure and sequence with those of related endolysins shows the core three-dimensional fold is similar and the catalytic triad geometry is highly conserved despite the sequence differences. Features essential for T4 lysozyme structure and function such as the distance between catalytic groups, salt bridge and presence of nucleophilic water are conserved in DLP12 endolysin and other endolysins analyzed.

Dynamic interactions between prophages induce lysis in Propionibacterium acnes.

Progress in next-generation sequencing technologies has facilitated investigations into microbial dynamics. An important bacterium in the dairy industry is Propionibacterium freudenreichii, which is exploited to manufacture Swiss cheeses. A healthy culture of these bacteria ensures a consistent cheese with formed 'eyes' and pleasant flavour profile, and the investigation of prophages and their interactions with these bacteria could assist in the maintenance of the standard of this food product. Two bacteriophages, termed PFR1 and PFR2, were chemically induced using mitomycin C from two different dairy strains of P. freudenreichii. Both phages have identical genomes; however, PFR2 was found to contain an insertion sequence, IS204. Host range characterisation showed that PFR1 was able to form plaques on a wild type Propionibacterium acnes strain, whereas PFR2 could not. The lytic plaques observed on P. acnes were a result of PFR1 inducing the lytic cycle of a pseudolysogenic phage in P. acnes. Further investigation revealed that both PFR1 and PFR2 could infect P. acnes but not replicate. This study demonstrates the dynamic interactions between phages, which may alter their lytic capacity under certain conditions. To our knowledge, this is the first report of two phages interacting to kill their host.

Two novel temperate bacteriophages co-existing in Aeromonas sp. ARM81 - characterization of their genomes, proteomes and DNA methyltransferases.

Aeromonas species are causative agents of a wide spectrum of diseases in animals and humans. Although these bacteria are commonly found in various environments, little is known about their phages. Thus far, only one temperate Aeromonas phage has been characterized. Whole-genome sequencing of an Aeromonas sp. strain ARM81 revealed the presence of two prophage clusters. One of them is integrated into the chromosome and the other was maintained as an extrachromosomal, linear plasmid-like prophage encoding a protelomerase. Both prophages were artificially and spontaneously inducible. We separately isolated both phages and compared their genomes with other known viruses. The novel phages show no similarity to the previously characterized Aeromonas phages and might represent new evolutionary lineages of viruses infecting Aeromonadaceae. Apart from the comparative genomic analyses of these phages, complemented with their structural and molecular characterization, a functional analysis of four DNA methyltransferases encoded by these viruses was conducted. One of the investigated N6-adenine-modifying enzymes shares sequence specificity with a Dam-like methyltransferase of its bacterial host, while another one is non-specific, as it catalyzes adenine methylation in various sequence contexts. The presented results shed new light on the diversity of Aeromonas temperate phages.

The solution structures of two prophage homologues of the bacteriophage λ Ea8.5 protein reveal a newly discovered hybrid homeodomain/zinc-finger fold.

A cluster of genes in the exoxis region of bacteriophage λ are capable of inhibiting the initiation of DNA synthesis in Escherichia coli. The most indispensible gene in this region is ea8.5. Here, we report the nuclear magnetic resonance structures of two ea8.5 orthologs from enteropathogenic E. coli and Pseudomonas putida prophages. Both proteins are characterized by a fused homeodomain/zinc-finger fold that escaped detection by primary sequence search methods. While these folds are both associated with a nucleic acid binding function, the amino acid composition suggests otherwise, leading to the possibility that Ea8.5 associates with other viral and host proteins.

X-ray structure of a superinfection exclusion lipoprotein from phage TP-J34 and identification of the tape measure protein as its target.

Lipoproteins of temperate phage are a broad family of membrane proteins encoded in the lysogeny module of temperate phages. Expression of the ltp(TP-J34) gene of temperate Streptococcus thermophilus phage TP-J34 interferes with phage infection at the stage of triggering DNA release and injection into the cell. Here, we report the first structure of a superinfection exclusion protein. We have expressed and determined the X-ray structure of Ltp(TP-J34). The soluble domain of Ltp(TP-J34) is composed of a tandem of three-helix helix-turn-helix (HTH) domains exhibiting a highly negatively charged surface. By isolating mutants of lactococcal phage P008wt with reduced sensitivities to Ltp(TP-J34) and by genome sequencing of such mutants we obtained evidence supporting the notion that Ltp(TP-J34) targets the phage's tape measure protein (TMP) and blocks its insertion into the cytoplasmic membrane.

Spontaneous release of bacteriophage particles by Lactobacillus rhamnosus pen.

The identification of bacteriophage proteins on the surface of Lactobacillus rhamnosus Pen was performed by LC-MS/MS analysis. Among the identified proteins, we found a phage-derived major tail protein, two major head proteins, a portal protein, and a host specificity protein. Electron microscopy of a cell surface extract revealed the presence of phage particles in the analyzed samples. The partial sequence of genes encoding the major tail protein for all tested L. rhamnosus strains was determined with specific primers designed in this study. Next, RT-PCR analysis allowed detection of the expression of the major tail protein gene in L. rhamnosus strain Pen at all stages of bacterial growth. The transcription of genes encoding the major tail protein was also proved for other L. rhamnosus strains used in this study. The present work demonstrates the spontanous release of prophage-encoded particles by a commercial probiotic L. rhamnosus strain, which did not significantly affect the bacterial growth of the analyzed strain.

Solution NMR structure of hypothetical protein CV_2116 encoded by a viral prophage element in Chromobacterium violaceum.

CV_2116 is a small hypothetical protein of 82 amino acids from the Gram-negative coccobacillus Chromobacterium violaceum. A PSI-BLAST search using the CV_2116 sequence as a query identified only one hit (E = 2e(-07)) corresponding to a hypothetical protein OR16_04617 from Cupriavidus basilensis OR16, which failed to provide insight into the function of CV_2116. The CV_2116 gene was cloned into the p15TvLic expression plasmid, transformed into E. coli, and (13)C- and (15)N-labeled NMR samples of CV_2116 were overexpressed in E. coli and purified for structure determination using NMR spectroscopy. The resulting high-quality solution NMR structure of CV_2116 revealed a novel α + ß fold containing two anti-parallel ß-sheets in the N-terminal two-thirds of the protein and one α-helix in the C-terminal third of the protein. CV_2116 does not belong to any known protein sequence family and a Dali search indicated that no similar structures exist in the protein data bank. Although no function of CV_2116 could be derived from either sequence or structural similarity searches, the neighboring genes of CV_2116 encode various proteins annotated as similar to bacteriophage tail assembly proteins. Interestingly, C. violaceum exhibits an extensive network of bacteriophage tail-like structures that likely result from lateral gene transfer by incorporation of viral DNA into its genome (prophages) due to bacteriophage infection. Indeed, C. violaceum has been shown to contain four prophage elements and CV_2116 resides in the fourth of these elements. Analysis of the putative operon in which CV_2116 resides indicates that CV_2116 might be a component of the bacteriophage tail-like assembly that occurs in C. violaceum.

The structure of Bacillus subtilis SPß prophage dUTPase and its complexes with two nucleotides.

dUTPases are housekeeping enzymes which catalyse the hydrolysis of dUTP to dUMP in an ion-dependent manner. Bacillus subtilis has both a genomic and an SPß prophage homotrimeric dUTPase. Here, structure determination of the prophage apoenzyme and of its complexes with dUDP and dUpNHpp-Mg(2+) is described at 1.75, 1.9 and 2.55 Šresolution, respectively. The C-terminal extension, which carries the conserved motif V, is disordered in all three structures. Unlike all other trimeric dUTPases for which structures are available, with the exception of the Bacillus genomic enzyme, the aromatic residue covering the uridine and acting as the Phe-lid is close to motif III in the sequence rather than in motif V. This is in spite of the presence of an aromatic amino acid at the usual Phe-lid position in motif V. The alternative position of the Phe-lid requires a reconsideration of its role in the catalytic cycle of the enzyme. In the dUpNHpp-Mg(2+) complex a water can be seen at the position expected for nucleophilic attack on the α-phosphate, in spite of motif V being disordered. Differences in the active site between the free enzyme and the dUDP and dUpNHpp-Mg(2+) complexes shows that the triphosphate moiety needs to be in the gauche conformation to trigger the conformational changes that can be seen in both B. subtilis dUTPases.

Microarray analysis and draft genomes of two Escherichia coli O157:H7 lineage II cattle isolates FRIK966 and FRIK2000 investigating lack of Shiga toxin expression.

The existence of two separate genetic lineages of Escherichia coli O157:H7 has previously been reported, and research indicates that lineage I could be more pathogenic toward human hosts than lineage II. We have previously shown that lineage I as a group expresses higher levels of Shiga toxin 2 (Stx2) than lineage II. To help evaluate why lineage II strains do not express appreciable levels of this toxin, whole-genome microarrays were performed using Agilent custom microarrays. Gene expression of the two representative bovine lineage II strains (FRIK966 and FRIK2000) were compared with gene expression of E. coli O157:H7 EDL933 (lineage I clinical type strain). Missing or differentially expressed genes and pathways were identified. Quantitative reverse transcription-polymerase chain reaction was performed to validate the microarray data. Draft genomes of FRIK966 and FRIK2000 were sequenced using Roche Applied Science/454 GS-FLX technology shotgun and paired-end approaches followed by de novo assembly. These assemblies were compared with the lineage I genome sequences from E. coli O157:H7 EDL933. The bacteriophage 933W, which encodes the Stx2 genes, showed a notable repression in gene expression. Polymerase chain reaction primers, based upon EDL933 genomic information, were also designed against all of the potentially missing genes of this bacteriophage. Most of the structural genes associated with the bacteriophage were found to be absent from the genome of the two bovine strains. These analyses, combined with evaluation of the genomic information, suggest that transposon (IS629) rearrangements may be associated with disruption of the bacteriophage genome in the FRIK strains. The results support the hypothesis that lineage II strains may be less of a risk as human foodborne pathogens. The microarray and genome data have been made available to the scientific community to allow continuing analysis of these cattle-isolated lineage II genomes and their gene expression.

Doc of prophage P1 is inhibited by its antitoxin partner Phd through fold complementation.

Prokaryotic toxin-antitoxin modules are involved in major physiological events set in motion under stress conditions. The toxin Doc (death on curing) from the phd/doc module on phage P1 hosts the C-terminal domain of its antitoxin partner Phd (prevents host death) through fold complementation. This Phd domain is intrinsically disordered in solution and folds into an alpha-helix upon binding to Doc. The details of the interactions reveal the molecular basis for the inhibitory action of the antitoxin. The complex resembles the Fic (filamentation induced by cAMP) proteins and suggests a possible evolutionary origin for the phd/doc operon. Doc induces growth arrest of Escherichia coli cells in a reversible manner, by targeting the protein synthesis machinery. Moreover, Doc activates the endogenous E. coli RelE mRNA interferase but does not require this or any other known chromosomal toxin-antitoxin locus for its action in vivo.

Subclassification and targeted characterization of prophage-encoded two-component cell lysis cassette.

Bacteriophage induced lysis of host bacterial cell is mediated by a two component cell lysis cassette comprised of holin and lysozyme. Prophages are integrated forms of bacteriophages in bacterial genomes providing a repertoire for bacterial evolution. Analysis using the prophage database (http://bicmku.in:8082) constructed by us showed 47 prophages were associated with putative two component cell lysis genes. These proteins cluster into four different subgroups. In this process, a putative holin (essd) and endolysin (ybcS), encoded by the defective lambdoid prophage DLP12 was found to be similar to two component cell lysis genes in functional bacteriophages like p21 and P1. The holin essd was found to have a characteristic dual start motif with two transmembrane regions and C-terminal charged residues as in class II holins. Expression of a fusion construct of essd in Escherichia coli showed slow growth. However, under appropriate conditions, this protein could be over expressed and purified for structure function studies. The second component of the cell lysis cassette, ybcS, was found to have an N-terminal SAR (Signal Arrest Release) transmembrane domain. The construct of ybcS has been over expressed in E.coli and the purified protein was functional, exhibiting lytic activity against E.coli and Salmonella typhi cell wall substrate. Such targeted sequence- structure-function characterization of proteins encoded by cryptic prophages will help understand the contribution of prophage proteins to bacterial evolution.

Structure and lytic activity of a Bacillus anthracis prophage endolysin.

We report a structural and functional analysis of the lambda prophage Ba02 endolysin (PlyL) encoded by the Bacillus anthracis genome. We show that PlyL comprises two autonomously folded domains, an N-terminal catalytic domain and a C-terminal cell wall-binding domain. We determined the crystal structure of the catalytic domain; its three-dimensional fold is related to that of the cell wall amidase, T7 lysozyme, and contains a conserved zinc coordination site and other components of the catalytic machinery. We demonstrate that PlyL is an N-acetylmuramoyl-L-alanine amidase that cleaves the cell wall of several Bacillus species when applied exogenously. We show, unexpectedly, that the catalytic domain of PlyL cleaves more efficiently than the full-length protein, except in the case of Bacillus cereus, and using GFP-tagged cell wall-binding domain, we detected strong binding of the cell wall-binding domain to B. cereus but not to other species tested. We further show that a related endolysin (Ply21) from the B. cereus phage, TP21, shows a similar pattern of behavior. To explain these data, and the species specificity of PlyL, we propose that the C-terminal domain inhibits the activity of the catalytic domain through intramolecular interactions that are relieved upon binding of the C-terminal domain to the cell wall. Furthermore, our data show that (when applied exogenously) targeting of the enzyme to the cell wall is not a prerequisite of its lytic activity, which is inherently high. These results may have broad implications for the design of endolysins as therapeutic agents.

Genetic structure and chromosomal integration site of the cryptic prophage CP-1639 encoding Shiga toxin 1.

The sequence of 50 625 bp of chromosomal DNA derived from Shiga-toxin (Stx)-producing Escherichia coli (STEC) O111: H- strain 1639/77 was determined. This DNA fragment contains the cryptic Stx1-encoding prophage CP-1639 and its flanking chromosomal regions. The genome of CP-1639 basically resembles that of lambdoid phages in structure, but contains three IS629 elements, one of which disrupts the gene of a tail fibre component. The prophage genome lacks parts of the recombination region including integrase and excisionase genes. Moreover, a capsid protein gene is absent. CP-1639 is closely associated with an integrase gene of an ancient integrative element. This element consists of three ORFs of unknown origin and a truncated integrase gene homologous to intA of CP4-57. By PCR analysis and sequencing, it was shown that this integrative element is present in a number of non-O157 STEC serotypes and in non-STEC strains, where it is located at the 3'-end of the chromosomal ssrA gene. Whereas in most E. coli O111: H- strains, prophages are inserted in this site, E. coli O26 strains contain the integrative element not connected to a prophage. In E. coli O103 strains, the genetic structure of this region is variable. Comparison of DNA sequences of this particular site in E. coli O157: H7 strain EDL933, E. coli O111: H- strain 1639/77 and E. coli K-12 strain MG1655 showed that the ssrA gene is associated in all cases with the presence of foreign DNA. The results of this study have shown that the cryptic prophage CP-1639 is associated with an integrative element at a particular site in the E. coli chromosome that possesses high genetic variability.

Functional comparison of the transposition core machineries of phage Mu and Haemophilus influenzae Mu-like prophage Hin-Mu reveals interchangeable components.

Bacteriophage Mu uses DNA transposition for propagation and is a model for transposition studies in general. Recent identification of Mu-like prophages within bacterial genomes offers new material for evolutionary and comparative functional studies. One such prophage, Hin-Mu of Haemophilus influenzae Rd, was studied for its transpositional properties. The components of its transposition core machinery, the encoded transposase (MuA(Hin)) and the transposase binding sites, were evaluated for functional properties by sequence comparisons and DNase I footprinting. Transpositional activity of Hin-Mu was examined by in vitro assays directly assessing the assembly and catalytic function of the transposition core machinery. The Hin-Mu components readily assembled catalytically competent protein-DNA complexes, transpososomes. Thus, Hin-Mu encodes a functional transposase and contains critical transposase binding sites. Despite marked sequence differences, components of the Hin-Mu and Mu transposition core machineries are partially interchangeable, reflecting both conservation and flexibility in the functionally important regions within the transpososome structure.

The role of prophage-like elements in the diversity of Salmonella enterica serovars.

The Salmonella enterica serovar Typhi CT18 (S.Typhi) chromosome harbours seven distinct prophage-like elements, some of which may encode functional bacteriophages. In silico analyses were used to investigate these regions in S.Typhi CT18, and ultimately compare these integrated bacteriophages against 40 other Salmonella isolates using DNA microarray technology. S.Typhi CT18 contains prophages that show similarity to the lambda, Mu, P2 and P4 bacteriophage families. When compared to other S.Typhi isolates, these elements were generally conserved, supporting a clonal origin of this serovar. However, distinct variation was detected within a broad range of Salmonella serovars; many of the prophage regions are predicted to be specific to S.Typhi. Some of the P2 family prophage analysed have the potential to carry non-essential "cargo" genes within the hyper-variable tail region, an observation that suggests that these bacteriophage may confer a level of specialisation on their host. Lysogenic bacteriophages therefore play a crucial role in the generation of genetic diversity within S.enterica.