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1.  Complete Genomic Sequence and Mass Spectrometric Analysis of Highly Diverse, Atypical Bacillus thuringiensis phage 0305φ8-36 
Virology  2007;368(2):405-421.
To investigate the apparent genomic complexity of long-genome bacteriophages, we have sequenced the 218,948-bp genome (6479 bp terminal repeat), and identified the virion proteins (55), of Bacillus thuringiensis bacteriophage 0305φ8-36. Phage 0305φ8-36 is an atypical myovirus with three large curly tail fibers. An accurate mode of DNA pyrosequencing was used to sequence the genome and mass spectrometry was used to accomplish the comprehensive virion protein survey. Advanced informatic techniques were used to identify classical morphogenesis genes. The 0305φ8-36 genes were highly diverged; 19% of 247 closely spaced genes have similarity to proteins with known functions. Genes for virion-associated, apparently fibrous proteins in a new class were found, in addition to strong candidates for the curly fiber genes. Phage 0305φ8-36 has twice the virion protein coding sequence of T4. Based on its genomic isolation, 0305φ8-36 is a resource for future studies of vertical gene transmission.
PMCID: PMC2171028  PMID: 17673272
myovirus; Bacillus thuringiensis; pyrosequencing; virion protein; mass spectrometry
2.  Propagating the missing bacteriophages: a large bacteriophage in a new class 
Virology Journal  2007;4:21.
The number of successful propagations/isolations of soil-borne bacteriophages is small in comparison to the number of bacteriophages observed by microscopy (great plaque count anomaly). As one resolution of the great plaque count anomaly, we use propagation in ultra-dilute agarose gels to isolate a Bacillus thuringiensis bacteriophage with a large head (95 nm in diameter), tail (486 × 26 nm), corkscrew-like tail fibers (187 × 10 nm) and genome (221 Kb) that cannot be detected by the usual procedures of microbiology. This new bacteriophage, called 0305φ8-36 (first number is month/year of isolation; remaining two numbers identify the host and bacteriophage), has a high dependence of plaque size on the concentration of a supporting agarose gel. Bacteriophage 0305φ8-36 does not propagate in the traditional gels used for bacteriophage plaque formation and also does not produce visible lysis of liquid cultures. Bacteriophage 0305φ8-36 aggregates and, during de novo isolation from the environment, is likely to be invisible to procedures of physical detection that use either filtration or centrifugal pelleting to remove bacteria. Bacteriophage 0305φ8-36 is in a new genomic class, based on genes for both structural components and DNA packaging ATPase. Thus, knowledge of environmental virus diversity is expanded with prospect of greater future expansion.
PMCID: PMC1817643  PMID: 17324288
3.  Comparative genomics of Bacillus thuringiensis phage 0305φ8-36: defining patterns of descent in a novel ancient phage lineage 
Virology Journal  2007;4:97.
The recently sequenced 218 kb genome of morphologically atypical Bacillus thuringiensis phage 0305φ8-36 exhibited only limited detectable homology to known bacteriophages. The only known relative of this phage is a string of phage-like genes called BtI1 in the chromosome of B. thuringiensis israelensis. The high degree of divergence and novelty of phage genomes pose challenges in how to describe the phage from its genomic sequences.
Phage 0305φ8-36 and BtI1 are estimated to have diverged 2.0 – 2.5 billion years ago. Positionally biased Blast searches aligned 30 homologous structure or morphogenesis genes between 0305φ8-36 and BtI1 that have maintained the same gene order. Functional clustering of the genes helped identify additional gene functions. A conserved long tape measure gene indicates that a long tail is an evolutionarily stable property of this phage lineage. An unusual form of the tail chaperonin system split to two genes was characterized, as was a hyperplastic homologue of the T4gp27 hub gene. Within this region some segments were best described as encoding a conservative array of structure domains fused with a variable component of exchangeable domains. Other segments were best described as multigene units engaged in modular horizontal exchange. The non-structure genes of 0305φ8-36 appear to include the remnants of two replicative systems leading to the hypothesis that the genome plan was created by fusion of two ancestral viruses. The case for a member of the RNAi RNA-directed RNA polymerase family residing in 0305φ8-36 was strengthened by extending the hidden Markov model of this family. Finally, it was noted that prospective transcriptional promoters were distributed in a gradient of small to large transcripts starting from a fixed end of the genome.
Genomic organization at a level higher than individual gene sequence comparison can be analyzed to aid in understanding large phage genomes. Methods of analysis include 1) applying a time scale, 2) augmenting blast scores with positional information, 3) categorizing genomic rearrangements into one of several processes with characteristic rates and outcomes, and 4) correlating apparent transcript sizes with genomic position, gene content, and promoter motifs.
PMCID: PMC2147016  PMID: 17919320
4.  Aggregates of bacteriophage 0305φ8-36 seed future growth 
Virology Journal  2007;4:131.
Lytic bacteriophage 0305φ8-36 forms visually observed aggregates during plaque formation. Aggregates intrinsically lower propagation potential. In the present study, the following observations indicate that lost propagation potential is regained with time: (1) Aggregates sometimes concentrate at the edge of clear plaques. (2) A semi-clear ring sometimes forms beyond the plaques. (3) Formation of a ring is completely correlated with the presence of aggregates at the same angular displacement along the plaque edge. To explain this aggregate-derived lowering/raising of propagation potential, the following hypothesis is presented: Aggregation/dissociation of bacteriophage of 0305φ8-36 is a selected phenomenon that evolved to maintain high host finding rate in a trade-off with maintaining high rate of bacteriophage progeny production. This hypothesis explains ringed plaque morphology observed for other bacteriophages and predicts that aggregates will undergo time-dependent change in structure as propagation potential increases. In support, fluorescence microscopy reveals time-dependent change in the distance between resolution-limited particles in aggregates.
PMCID: PMC2222632  PMID: 18053210
5.  Activity, specificity and structure of I-Bth0305I: a representative of a new homing endonuclease family 
Nucleic Acids Research  2011;39(22):9705-9719.
Novel family of putative homing endonuclease genes was recently discovered during analyses of metagenomic and genomic sequence data. One such protein is encoded within a group I intron that resides in the recA gene of the Bacillus thuringiensis 0305ϕ8–36 bacteriophage. Named I-Bth0305I, the endonuclease cleaves a DNA target in the uninterrupted recA gene at a position immediately adjacent to the intron insertion site. The enzyme displays a multidomain, homodimeric architecture and footprints a DNA region of ∼60 bp. Its highest specificity corresponds to a 14-bp pseudopalindromic sequence that is directly centered across the DNA cleavage site. Unlike many homing endonucleases, the specificity profile of the enzyme is evenly distributed across much of its target site, such that few single base pair substitutions cause a significant decrease in cleavage activity. A crystal structure of its C-terminal domain confirms a nuclease fold that is homologous to very short patch repair (Vsr) endonucleases. The domain architecture and DNA recognition profile displayed by I-Bth0305I, which is the prototype of a homing lineage that we term the ‘EDxHD’ family, are distinct from previously characterized homing endonucleases.
PMCID: PMC3239194  PMID: 21890897
6.  Genomic, Proteomic and Physiological Characterization of a T5-like Bacteriophage for Control of Shiga Toxin-Producing Escherichia coli O157:H7 
PLoS ONE  2012;7(4):e34585.
Despite multiple control measures, Escherichia coli O157:H7 (STEC O157:H7) continues to be responsible for many food borne outbreaks in North America and elsewhere. Bacteriophage therapy may prove useful for controlling this pathogen in the host, their environment and food. Bacteriophage vB_EcoS_AKFV33 (AKFV33), a T5-like phage of Siphoviridae lysed common phage types of STEC O157:H7 and not non-O157 E. coli. Moreover, STEC O157:H7 isolated from the same feedlot pen from which the phage was obtained, were highly susceptible to AKFV33. Adsorption rate constant and burst size were estimated to be 9.31×10−9 ml/min and 350 PFU/infected cell, respectively. The genome of AKVF33 was 108,853 bp (38.95% G+C), containing 160 open reading frames (ORFs), 22 tRNA genes and 32 strong promoters recognized by host RNA polymerase. Of 12 ORFs without homologues to T5-like phages, 7 predicted novel proteins while others exhibited low identity (<60%) to proteins in the National Centre for Biotechnology Information database. AKVF33 also lacked the L-shaped tail fiber protein typical of T5, but was predicted to have tail fibers comprised of 2 novel proteins with low identity (37–41%) to tail fibers of E. coli phage phiEco32 of Podoviridae, a putative side tail fiber protein of a prophage from E. coli IAI39 and a conserved domain protein of E. coli MS196-1. The receptor-binding tail protein (pb5) shared an overall identify of 29–72% to that of other T5-like phages, with no region coding for more than 6 amino acids in common. Proteomic analysis identified 4 structural proteins corresponding to the capsid, major tail, tail fiber and pore-forming tail tip (pb2). The genome of AKFV33 lacked regions coding for known virulence factors, integration-related proteins or antibiotic resistance determinants. Phage AKFV33 is a unique, highly lytic STEC O157:H7-specific T5-like phage that may have considerable potential as a pre- and post-harvest biocontrol agent.
PMCID: PMC3326045  PMID: 22514640
7.  Evidence for bacteriophage T7 tail extension during DNA injection 
BMC Research Notes  2008;1:36.
Electron micrographs of bacteriophage T7 reveal a tail shorter than needed to reach host cytoplasm during infection-initiating injection of a T7 DNA molecule through the tail and cell envelope. However, recent data indicate that internal T7 proteins are injected before the DNA molecule is injected. Thus, bacteriophage/host adsorption potentially causes internal proteins to become external and lengthen the tail for DNA injection. But, the proposed adsorption-induced tail lengthening has never been visualized.
In the present study, electron microscopy of particles in T7 lysates reveals a needle-like capsid extension that attaches partially emptied bacteriophage T7 capsids to non-capsid vesicles and sometimes enters an attached vesicle. This extension is 40–55 nm long, 1.7–2.4× longer than the T7 tail and likely to be the proposed lengthened tail. The extension is 8–11 nm in diameter, thinner than most of the tail, with an axial hole 3–4 nm in diameter. Though the bound vesicles are not identified by microscopy, these vesicles resemble the major vesicles in T7 lysates, found to be E. coli outer membrane vesicles by non-denaturing agarose gel electrophoresis, followed by mass spectrometry.
The observed lengthened tail is long enough to reach host cytoplasm during DNA injection. Its channel is wide enough to be a conduit for DNA injection and narrow enough to clamp DNA during a previously observed stalling/re-starting of injection. However, its outer diameter is too large to explain formation by passing of an intact assembly through any known capsid hole unless the hole is widened.
PMCID: PMC2525648  PMID: 18710489
8.  Multifunctional Roles of a Bacteriophage ϕ29 Morphogenetic Factor in Assembly and Infection 
Journal of molecular biology  2008;378(4):802-815.
Low copy number proteins within macromolecular complexes, such as viruses, can be critical to biological function while comprising a minimal mass fraction of the complex. The Bacillus subtilis dsDNA bacteriophage ϕ29 gene 13 product (gp13), previously undetected in the virion, was identified and localized to the distal tip of the tail knob. Western blots and immuno-electron microscopy detected a few copies of gp13 in ϕ29, DNA-free particles, purified tails, and defective particles produced in suppressor-sensitive (sus) mutant sus13(330) infections. Particles assembled in the absence of intact gp13 (sus13(342) and sus13(330)) had the gross morphology of ϕ29 but were noninfectious. gp13 has predicted structural homology and sequence similarity to the M23 metalloprotease LytM. Poised at the tip of the ϕ29 tail knob, gp13 may serve as a plug to help restrain the highly pressurized packaged genome. Also, in this position, gp13 may be the first virion protein to contact the cell wall in infection, acting as a pilot protein to depolymerize the cell wall. gp13 may facilitate juxtaposing of the tail knob onto the cytoplasmic membrane and the triggering of genome injection.
PMCID: PMC2443984  PMID: 18394643
bacteriophage ϕ29; morphogenetic factor; immuno-electron microscopy; tail protein; endopeptidase
9.  Characterization of the Genome, Proteome, and Structure of Yersiniophage ϕR1-37 
Journal of Virology  2012;86(23):12625-12642.
The bacteriophage vB_YecM-ϕR1-37 (ϕR1-37) is a lytic yersiniophage that can propagate naturally in different Yersinia species carrying the correct lipopolysaccharide receptor. This large-tailed phage has deoxyuridine (dU) instead of thymidine in its DNA. In this study, we determined the genomic sequence of phage ϕR1-37, mapped parts of the phage transcriptome, characterized the phage particle proteome, and characterized the virion structure by cryo-electron microscopy and image reconstruction. The 262,391-bp genome of ϕR1-37 is one of the largest sequenced phage genomes, and it contains 367 putative open reading frames (ORFs) and 5 tRNA genes. Mass-spectrometric analysis identified 69 phage particle structural proteins with the genes scattered throughout the genome. A total of 269 of the ORFs (73%) lack homologues in sequence databases. Based on terminator and promoter sequences identified from the intergenic regions, the phage genome was predicted to consist of 40 to 60 transcriptional units. Image reconstruction revealed that the ϕR1-37 capsid consists of hexameric capsomers arranged on a T=27 lattice similar to the bacteriophage ϕKZ. The tail of ϕR1-37 has a contractile sheath. We conclude that phage ϕR1-37 is a representative of a novel phage type that carries the dU-containing genome in a ϕKZ-like head.
PMCID: PMC3497697  PMID: 22973030
10.  Cytotoxic Chromosomal Targeting by CRISPR/Cas Systems Can Reshape Bacterial Genomes and Expel or Remodel Pathogenicity Islands 
PLoS Genetics  2013;9(4):e1003454.
In prokaryotes, clustered regularly interspaced short palindromic repeats (CRISPRs) and their associated (Cas) proteins constitute a defence system against bacteriophages and plasmids. CRISPR/Cas systems acquire short spacer sequences from foreign genetic elements and incorporate these into their CRISPR arrays, generating a memory of past invaders. Defence is provided by short non-coding RNAs that guide Cas proteins to cleave complementary nucleic acids. While most spacers are acquired from phages and plasmids, there are examples of spacers that match genes elsewhere in the host bacterial chromosome. In Pectobacterium atrosepticum the type I-F CRISPR/Cas system has acquired a self-complementary spacer that perfectly matches a protospacer target in a horizontally acquired island (HAI2) involved in plant pathogenicity. Given the paucity of experimental data about CRISPR/Cas–mediated chromosomal targeting, we examined this process by developing a tightly controlled system. Chromosomal targeting was highly toxic via targeting of DNA and resulted in growth inhibition and cellular filamentation. The toxic phenotype was avoided by mutations in the cas operon, the CRISPR repeats, the protospacer target, and protospacer-adjacent motif (PAM) beside the target. Indeed, the natural self-targeting spacer was non-toxic due to a single nucleotide mutation adjacent to the target in the PAM sequence. Furthermore, we show that chromosomal targeting can result in large-scale genomic alterations, including the remodelling or deletion of entire pre-existing pathogenicity islands. These features can be engineered for the targeted deletion of large regions of bacterial chromosomes. In conclusion, in DNA–targeting CRISPR/Cas systems, chromosomal interference is deleterious by causing DNA damage and providing a strong selective pressure for genome alterations, which may have consequences for bacterial evolution and pathogenicity.
Author Summary
Bacteria have evolved mechanisms that provide protection from continual invasion by viruses and other foreign elements. Resistance systems, known as CRISPR/Cas, were recently discovered and equip bacteria and archaea with an “adaptive immune system.” This adaptive immunity provides a highly evolvable sequence-specific small RNA–based memory of past invasions by viruses and foreign genetic elements. There are many cases where these systems appear to target regions within the bacterial host's own genome (a possible autoimmunity), but the evolutionary rationale for this is unclear. Here, we demonstrate that CRISPR/Cas targeting of the host chromosome is highly toxic but that cells survive through mutations that alleviate the immune mechanism. We have used this phenotype to gain insight into how these systems function and show that large changes in the bacterial genome can occur. For example, targeting of a chromosomal pathogenicity island, important for virulence of the potato pathogen Pectobacterium atrosepticum, resulted in deletion of the island, which constituted ∼2% of the bacterial genome. These results have broad significance for the role of CRISPR/Cas systems and their impact on the evolution of bacterial genomes and virulence. In addition, this study demonstrates their potential as a tool for the targeted deletion of specific regions of bacterial chromosomes.
PMCID: PMC3630108  PMID: 23637624
11.  The essential genome of a bacterium 
This study reports the essential Caulobacter genome at 8 bp resolution determined by saturated transposon mutagenesis and high-throughput sequencing. This strategy is applicable to full genome essentiality studies in a broad class of bacterial species.
The essential Caulobacter genome was determined at 8 bp resolution using hyper-saturated transposon mutagenesis coupled with high-throughput sequencing.Essential protein-coding sequences comprise 90% of the essential genome; the remaining 10% comprising essential non-coding RNA sequences, gene regulatory elements and essential genome replication features.Of the 3876 annotated open reading frames (ORFs), 480 (12.4%) were essential ORFs, 3240 (83.6%) were non-essential ORFs and 156 (4.0%) were ORFs that severely impacted fitness when mutated.The essential elements are preferentially positioned near the origin and terminus of the Caulobacter chromosome.This high-resolution strategy is applicable to high-throughput, full genome essentiality studies and large-scale genetic perturbation experiments in a broad class of bacterial species.
The regulatory events that control polar differentiation and cell-cycle progression in the bacterium Caulobacter crescentus are highly integrated, and they have to occur in the proper order (McAdams and Shapiro, 2011). Components of the core regulatory circuit are largely known. Full discovery of its essential genome, including non-coding, regulatory and coding elements, is a prerequisite for understanding the complete regulatory network of this bacterial cell. We have identified all the essential coding and non-coding elements of the Caulobacter chromosome using a hyper-saturated transposon mutagenesis strategy that is scalable and can be readily extended to obtain rapid and accurate identification of the essential genome elements of any sequenced bacterial species at a resolution of a few base pairs.
We engineered a Tn5 derivative transposon (Tn5Pxyl) that carries at one end an inducible outward pointing Pxyl promoter (Christen et al, 2010). We showed that this transposon construct inserts into the genome randomly where it can activate or disrupt transcription at the site of integration, depending on the insertion orientation. DNA from hundred of thousands of transposon insertion sites reading outward into flanking genomic regions was parallel PCR amplified and sequenced by Illumina paired-end sequencing to locate the insertion site in each mutant strain (Figure 1). A single sequencing run on DNA from a mutagenized cell population yielded 118 million raw sequencing reads. Of these, >90 million (>80%) read outward from the transposon element into adjacent genomic DNA regions and the insertion site could be mapped with single nucleotide resolution. This yielded the location and orientation of 428 735 independent transposon insertions in the 4-Mbp Caulobacter genome.
Within non-coding sequences of the Caulobacter genome, we detected 130 non-disruptable DNA segments between 90 and 393 bp long in addition to all essential promoter elements. Among 27 previously identified and validated sRNAs (Landt et al, 2008), three were contained within non-disruptable DNA segments and another three were partially disruptable, that is, insertions caused a notable growth defect. Two additional small RNAs found to be essential are the transfer-messenger RNA (tmRNA) and the ribozyme RNAseP (Landt et al, 2008). In addition to the 8 non-disruptable sRNAs, 29 out of the 130 intergenic essential non-coding sequences contained non-redundant tRNA genes; duplicated tRNA genes were non-essential. We also identified two non-disruptable DNA segments within the chromosomal origin of replication. Thus, we resolved essential non-coding RNAs, tRNAs and essential replication elements within the origin region of the chromosome. An additional 90 non-disruptable small genome elements of currently unknown function were identified. Eighteen of these are conserved in at least one closely related species. Only 2 could encode a protein of over 50 amino acids.
For each of the 3876 annotated open reading frames (ORFs), we analyzed the distribution, orientation, and genetic context of transposon insertions. There are 480 essential ORFs and 3240 non-essential ORFs. In addition, there were 156 ORFs that severely impacted fitness when mutated. The 8-bp resolution allowed a dissection of the essential and non-essential regions of the coding sequences. Sixty ORFs had transposon insertions within a significant portion of their 3′ region but lacked insertions in the essential 5′ coding region, allowing the identification of non-essential protein segments. For example, transposon insertions in the essential cell-cycle regulatory gene divL, a tyrosine kinase, showed that the last 204 C-terminal amino acids did not impact viability, confirming previous reports that the C-terminal ATPase domain of DivL is dispensable for viability (Reisinger et al, 2007; Iniesta et al, 2010). In addition, we found that 30 out of 480 (6.3%) of the essential ORFs appear to be shorter than the annotated ORF, suggesting that these are probably mis-annotated.
Among the 480 ORFs essential for growth on rich media, there were 10 essential transcriptional regulatory proteins, including 5 previously identified cell-cycle regulators (McAdams and Shapiro, 2003; Holtzendorff et al, 2004; Collier and Shapiro, 2007; Gora et al, 2010; Tan et al, 2010) and 5 uncharacterized predicted transcription factors. In addition, two RNA polymerase sigma factors RpoH and RpoD, as well as the anti-sigma factor ChrR, which mitigates rpoE-dependent stress response under physiological growth conditions (Lourenco and Gomes, 2009), were also found to be essential. Thus, a set of 10 transcription factors, 2 RNA polymerase sigma factors and 1 anti-sigma factor are the core essential transcriptional regulators for growth on rich media. To further characterize the core components of the Caulobacter cell-cycle control network, we identified all essential regulatory sequences and operon transcripts. Altogether, the 480 essential protein-coding and 37 essential RNA-coding Caulobacter genes are organized into operons such that 402 individual promoter regions are sufficient to regulate their expression. Of these 402 essential promoters, the transcription start sites (TSSs) of 105 were previously identified (McGrath et al, 2007).
The essential genome features are non-uniformly distributed on the Caulobacter genome and enriched near the origin and the terminus regions. In contrast, the chromosomal positions of the published E. coli essential coding sequences (Rocha, 2004) are preferentially located at either side of the origin (Figure 4A). This indicates that there are selective pressures on chromosomal positioning of some essential elements (Figure 4A).
The strategy described in this report could be readily extended to quickly determine the essential genome for a large class of bacterial species.
Caulobacter crescentus is a model organism for the integrated circuitry that runs a bacterial cell cycle. Full discovery of its essential genome, including non-coding, regulatory and coding elements, is a prerequisite for understanding the complete regulatory network of a bacterial cell. Using hyper-saturated transposon mutagenesis coupled with high-throughput sequencing, we determined the essential Caulobacter genome at 8 bp resolution, including 1012 essential genome features: 480 ORFs, 402 regulatory sequences and 130 non-coding elements, including 90 intergenic segments of unknown function. The essential transcriptional circuitry for growth on rich media includes 10 transcription factors, 2 RNA polymerase sigma factors and 1 anti-sigma factor. We identified all essential promoter elements for the cell cycle-regulated genes. The essential elements are preferentially positioned near the origin and terminus of the chromosome. The high-resolution strategy used here is applicable to high-throughput, full genome essentiality studies and large-scale genetic perturbation experiments in a broad class of bacterial species.
PMCID: PMC3202797  PMID: 21878915
functional genomics; next-generation sequencing; systems biology; transposon mutagenesis
12.  Mechanism of Membranous Tunnelling Nanotube Formation in Viral Genome Delivery 
PLoS Biology  2013;11(9):e1001667.
Abrescia and colleagues demonstrate how the bacteriophage PRD1, a model membrane-containing virus, generates a self-polymerizing protein-lipid nanotube to deliver its viral genome to a host cell.
In internal membrane-containing viruses, a lipid vesicle enclosed by the icosahedral capsid protects the genome. It has been postulated that this internal membrane is the genome delivery device of the virus. Viruses built with this architectural principle infect hosts in all three domains of cellular life. Here, using a combination of electron microscopy techniques, we investigate bacteriophage PRD1, the best understood model for such viruses, to unveil the mechanism behind the genome translocation across the cell envelope. To deliver its double-stranded DNA, the icosahedral protein-rich virus membrane transforms into a tubular structure protruding from one of the 12 vertices of the capsid. We suggest that this viral nanotube exits from the same vertex used for DNA packaging, which is biochemically distinct from the other 11. The tube crosses the capsid through an aperture corresponding to the loss of the peripentonal P3 major capsid protein trimers, penton protein P31 and membrane protein P16. The remodeling of the internal viral membrane is nucleated by changes in osmolarity and loss of capsid-membrane interactions as consequence of the de-capping of the vertices. This engages the polymerization of the tail tube, which is structured by membrane-associated proteins. We have observed that the proteo-lipidic tube in vivo can pierce the gram-negative bacterial cell envelope allowing the viral genome to be shuttled to the host cell. The internal diameter of the tube allows one double-stranded DNA chain to be translocated. We conclude that the assembly principles of the viral tunneling nanotube take advantage of proteo-lipid interactions that confer to the tail tube elastic, mechanical and functional properties employed also in other protein-membrane systems.
Author Summary
Viral survival and propagation depend on the ability of the viruses to transfer their genetic material to a host cell. Viral genome delivery has been described for viruses that directly enclose their genome in a capsid or nucleocapsid, but not for internal membrane-containing viruses in which the genome is protected by a lipid vesicle enclosed by the icosahedral capsid. The latter infect organisms across the three domains of life. We use a range of electron microscopy techniques to reveal how one such virus, the bacteriophage PRD1, which uses gram negative bacteria as its host, delivers its double-stranded DNA to the bacteria across the cell envelope. The PRD1 bacteriophage is special in that it doesn't carry a rigid tail; rather it creates a tube tail when needed at the time of infection to pass its DNA through to the host. We now show that this tube formation is accomplished via concerted restructuring of the icosahedral capsid and remodeling of the internal icosahedral protein-rich virus membrane. We also find that this tail tube is studded with membrane-associated proteins and its internal diameter allows one double-stranded DNA chain to be injected. Finally, we capture PRD1 in 3-D with the proteo-lipidic tail piercing the gram-negative bacterial cell and shuttling its viral genome in vivo. These results provide insights into a new mechanism of viral genome delivery.
PMCID: PMC3782422  PMID: 24086111
13.  First genome sequences of Achromobacter phages reveal new members of the N4 family 
Virology Journal  2014;11:14.
Multi-resistant Achromobacter xylosoxidans has been recognized as an emerging pathogen causing nosocomially acquired infections during the last years. Phages as natural opponents could be an alternative to fight such infections. Bacteriophages against this opportunistic pathogen were isolated in a recent study. This study shows a molecular analysis of two podoviruses and reveals first insights into the genomic structure of Achromobacter phages so far.
Growth curve experiments and adsorption kinetics were performed for both phages. Adsorption and propagation in cells were visualized by electron microscopy. Both phage genomes were sequenced with the PacBio RS II system based on single molecule, real-time (SMRT) technology and annotated with several bioinformatic tools. To further elucidate the evolutionary relationships between the phage genomes, a phylogenomic analysis was conducted using the genome Blast Distance Phylogeny approach (GBDP).
In this study, we present the first detailed analysis of genome sequences of two Achromobacter phages so far. Phages JWAlpha and JWDelta were isolated from two different waste water treatment plants in Germany. Both phages belong to the Podoviridae and contain linear, double-stranded DNA with a length of 72329 bp and 73659 bp, respectively. 92 and 89 putative open reading frames were identified for JWAlpha and JWDelta, respectively, by bioinformatic analysis with several tools. The genomes have nearly the same organization and could be divided into different clusters for transcription, replication, host interaction, head and tail structure and lysis. Detailed annotation via protein comparisons with BLASTP revealed strong similarities to N4-like phages.
Analysis of the genomes of Achromobacter phages JWAlpha and JWDelta and comparisons of different gene clusters with other phages revealed that they might be strongly related to other N4-like phages, especially of the Escherichia group. Although all these phages show a highly conserved genomic structure and partially strong similarities at the amino acid level, some differences could be identified. Those differences, e.g. the existence of specific genes for replication or host interaction in some N4-like phages, seem to be interesting targets for further examination of function and specific mechanisms, which might enlighten the mechanism of phage establishment in the host cell after infection.
PMCID: PMC3915230  PMID: 24468270
Achromobacter xylosoxidans; N4-like phage; Genome; Lar-like protein; N4likevirus; Podoviridae; GBDP
14.  Characterization of the dsDNA prophage sequences in the genome of Neisseria gonorrhoeae and visualization of productive bacteriophage 
BMC Microbiology  2007;7:66.
Bioinformatic analysis of the genome sequence of Neisseria gonorrhoeae revealed the presence of nine probable prophage islands. The distribution, conservation and function of many of these sequences, and their ability to produce bacteriophage particles are unknown.
Our analysis of the genomic sequence of FA1090 identified five genomic regions (NgoΦ1 – 5) that are related to dsDNA lysogenic phage. The genetic content of the dsDNA prophage sequences were examined in detail and found to contain blocks of genes encoding for proteins homologous to proteins responsible for phage DNA replication, structural proteins and proteins responsible for phage assembly. The DNA sequences from NgoΦ1, NgoΦ2 and NgoΦ3 contain some significant regions of identity. A unique region of NgoΦ2 showed very high similarity with the Pseudomonas aeruginosa generalized transducing phage F116. Comparative analysis at the nucleotide and protein levels suggests that the sequences of NgoΦ1 and NgoΦ2 encode functionally active phages, while NgoΦ3, NgoΦ4 and NgoΦ5 encode incomplete genomes. Expression of the NgoΦ1 and NgoΦ2 repressors in Escherichia coli inhibit the growth of E. coli and the propagation of phage λ. The NgoΦ2 repressor was able to inhibit transcription of N. gonorrhoeae genes and Haemophilus influenzae HP1 phage promoters. The holin gene of NgoΦ1 (identical to that encoded by NgoΦ2), when expressed in E. coli, could serve as substitute for the phage λ s gene. We were able to detect the presence of the DNA derived from NgoΦ1 in the cultures of N. gonorrhoeae. Electron microscopy analysis of culture supernatants revealed the presence of multiple forms of bacteriophage particles.
These data suggest that the genes similar to dsDNA lysogenic phage present in the gonococcus are generally conserved in this pathogen and that they are able to regulate the expression of other neisserial genes. Since phage particles were only present in culture supernatants after induction with mitomycin C, it indicates that the gonococcus also regulates the expression of bacteriophage genes.
PMCID: PMC1931599  PMID: 17615066
15.  Insights into Bacteriophage T5 Structure from Analysis of Its Morphogenesis Genes and Protein Components 
Journal of Virology  2014;88(2):1162-1174.
Bacteriophage T5 represents a large family of lytic Siphoviridae infecting Gram-negative bacteria. The low-resolution structure of T5 showed the T=13 geometry of the capsid and the unusual trimeric organization of the tail tube, and the assembly pathway of the capsid was established. Although major structural proteins of T5 have been identified in these studies, most of the genes encoding the morphogenesis proteins remained to be identified. Here, we combine a proteomic analysis of T5 particles with a bioinformatic study and electron microscopic immunolocalization to assign function to the genes encoding the structural proteins, the packaging proteins, and other nonstructural components required for T5 assembly. A head maturation protease that likely accounts for the cleavage of the different capsid proteins is identified. Two other proteins involved in capsid maturation add originality to the T5 capsid assembly mechanism: the single head-to-tail joining protein, which closes the T5 capsid after DNA packaging, and the nicking endonuclease responsible for the single-strand interruptions in the T5 genome. We localize most of the tail proteins that were hitherto uncharacterized and provide a detailed description of the tail tip composition. Our findings highlight novel variations of viral assembly strategies and of virion particle architecture. They further recommend T5 for exploring phage structure and assembly and for deciphering conformational rearrangements that accompany DNA transfer from the capsid to the host cytoplasm.
PMCID: PMC3911642  PMID: 24198424
16.  A Tail-like Assembly at the Portal Vertex in Intact Herpes Simplex Type-1 Virions 
PLoS Pathogens  2012;8(10):e1002961.
Herpes viruses are prevalent and well characterized human pathogens. Despite extensive study, much remains to be learned about the structure of the genome packaging and release machinery in the capsids of these large and complex double-stranded DNA viruses. However, such machinery is well characterized in tailed bacteriophage, which share a common evolutionary origin with herpesvirus. In tailed bacteriophage, the genome exits from the virus particle through a portal and is transferred into the host cell by a complex apparatus (i.e. the tail) located at the portal vertex. Here we use electron cryo-tomography of human herpes simplex type-1 (HSV-1) virions to reveal a previously unsuspected feature at the portal vertex, which extends across the HSV-1 tegument layer to form a connection between the capsid and the viral membrane. The location of this assembly suggests that it plays a role in genome release into the nucleus and is also important for virion architecture.
Author Summary
It is estimated that 90% of the world's adult population is infected with Herpes Simplex Virus (HSV), which is responsible for causing cold sores, blindness, encephalitis, even death. One problem faced by all viruses is how to ensure that their genetic material enters the host cell. In viruses that infect bacteria (bacteriophage), genome transfer from the virus into the cell is carried out by a specialized structure known as the tail. Studies have shown that bacteriophage and herpesviruses share a common evolutionary origin. However, until now, no tail-like structure has been seen in herpes. We have used newly developed electron microscopy based techniques to identify a structure in HSV, which recapitulates features of the bacteriophage tail and may also be involved in release of DNA from the virus particle at the nuclear pore. Such advances in understanding the structures that drive virus replication may suggest new therapeutic targets to counter viral infection.
PMCID: PMC3464221  PMID: 23055933
17.  Visualization of Bacteriophage T3 Capsids with DNA Incompletely Packaged In Vivo 
Journal of molecular biology  2008;384(5):1384-1399.
The tightly packaged dsDNA genome in the mature particles of many tailed bacteriophages has been shown to form multiple concentric rings when reconstructed from cryo-electron micrographs. However, recent single-particle DNA packaging force measurements have suggested that incompletely packaged DNA (ipDNA) is less ordered when it is shorter than ∼25% of the full genome length. The study presented here initially achieves both the isolation and the ipDNA length-based fractionation of ipDNA-containing T3 phage capsids (ipDNA-capsids) produced by DNA packaging in vivo; some ipDNA has quantized lengths, as judged by high-resolution gel electrophoresis of expelled DNA. This is the first isolation of such particles among the tailed dsDNA bacteriophages. The ipDNA-capsids are a minor component (containing ∼10-4 of packaged DNA in all particles) and are initially detected by non-denaturing gel electrophoresis after partial purification by buoyant density centrifugation. The primary contaminants are aggregates of phage particles and empty capsids. This study then investigates ipDNA conformations by the first cryo-electron microscopy (cryo-EM) of ipDNA-capsids produced in vivo. The 3-D structures of DNA-free capsids, ipDNA-capsids with various lengths of ipDNA, and mature bacteriophage are reconstructed, which reveals the typical T=7l icosahedral shell of many tailed dsDNA bacteriophages. Though the icosahedral shell structures of these capsids are indistinguishable at the current resolution for the protein shell (∼15 Å), the conformations of the DNA inside the shell are drastically different. T3 ipDNA-capsids with 10.6 kb or shorter dsDNA (<28% of total genome) have an ipDNA conformation indistinguishable from random. However, T3 ipDNA-capsids with 22 kb DNA (58% of total genome) forms a single DNA ring next to the inner surface of the capsid shell. In contrast, dsDNA fully packaged (38.2 kb) in mature T3 phage particles forms multiple concentric rings like those seen in other tailed dsDNA bacteriophages. The distance between the icosahedral shell and the outermost DNA ring decreases in the mature, fully packaged phage structure. These results suggest that, in the early stage of DNA packaging, the dsDNA genome is randomly distributed inside the capsid, not preferentially packaged against the inner surface of the capsid shell, and that the multiple concentric dsDNA rings seen later are the results of pressure-driven close-packing.
PMCID: PMC2628292  PMID: 18952096
Agarose gel electrophoresis; Buoyant density centrifugation; Cryo-EM; 3-D reconstruction; Mass spectrometry; DNA packaging
18.  CpG Island Mapping by Epigenome Prediction 
PLoS Computational Biology  2007;3(6):e110.
CpG islands were originally identified by epigenetic and functional properties, namely, absence of DNA methylation and frequent promoter association. However, this concept was quickly replaced by simple DNA sequence criteria, which allowed for genome-wide annotation of CpG islands in the absence of large-scale epigenetic datasets. Although widely used, the current CpG island criteria incur significant disadvantages: (1) reliance on arbitrary threshold parameters that bear little biological justification, (2) failure to account for widespread heterogeneity among CpG islands, and (3) apparent lack of specificity when applied to the human genome. This study is driven by the idea that a quantitative score of “CpG island strength” that incorporates epigenetic and functional aspects can help resolve these issues. We construct an epigenome prediction pipeline that links the DNA sequence of CpG islands to their epigenetic states, including DNA methylation, histone modifications, and chromatin accessibility. By training support vector machines on epigenetic data for CpG islands on human Chromosomes 21 and 22, we identify informative DNA attributes that correlate with open versus compact chromatin structures. These DNA attributes are used to predict the epigenetic states of all CpG islands genome-wide. Combining predictions for multiple epigenetic features, we estimate the inherent CpG island strength for each CpG island in the human genome, i.e., its inherent tendency to exhibit an open and transcriptionally competent chromatin structure. We extensively validate our results on independent datasets, showing that the CpG island strength predictions are applicable and informative across different tissues and cell types, and we derive improved maps of predicted “bona fide” CpG islands. The mapping of CpG islands by epigenome prediction is conceptually superior to identifying CpG islands by widely used sequence criteria since it links CpG island detection to their characteristic epigenetic and functional states. And it is superior to purely experimental epigenome mapping for CpG island detection since it abstracts from specific properties that are limited to a single cell type or tissue. In addition, using computational epigenetics methods we could identify high correlation between the epigenome and characteristics of the DNA sequence, a finding which emphasizes the need for a better understanding of the mechanistic links between genome and epigenome.
Author Summary
A key challenge for bioinformatic research is the identification of regulatory regions in the human genome. Regulatory regions are DNA elements that control gene expression and thereby contribute to the organism's phenotype. An important class of regulatory regions consists of so-called CpG islands, which are characterized by frequent occurrence of the CG sequence pattern. CpG islands are strongly associated with open and transcriptionally competent chromatin structure, they play a critical role in gene regulation, and they are involved in the epigenetic causes of cancer. In this article we make several conceptual improvements to the definition and mapping of CpG islands. First, we show that the traditional distinction between CpG islands and non-CpG islands is too harsh, and instead we propose a quantitative measure of CpG island strength to gradually distinguish between stronger and weaker regulatory regions. Second, by genome-wide comparison of multiple epigenome datasets we identify high correlation between features of the genome's DNA sequence and the epigenome, indicating strong functional interdependence. Third, we develop and apply a novel method for predicting the strength of all CpG islands in the human genome, giving rise to an improved and more accurate CpG island mapping.
PMCID: PMC1892605  PMID: 17559301
19.  Functions involved in bacteriophage P2-induced host cell lysis and identification of a new tail gene. 
Journal of Bacteriology  1994;176(16):4974-4984.
Successful completion of the bacteriophage P2 lytic cycle requires phage-induced lysis of its Escherichia coli host, a process that is poorly understood. Genetic analysis of lysis-deficient mutants defined a single locus, gene K, which lies within the largest late transcription unit of P2 and maps between head gene L and tail gene R. We determined and analyzed the DNA sequence of a ca. 2.1-kb EcoRV fragment that spans the entire region from L to R, thus completing the sequence of this operon. This region contains all of the functions necessary for host cell lysis. Sequence analysis revealed five open reading frames, initially designated orf19 through orf23. All of the existing lysis mutants--ts60, am12, am76, and am218--were located in orf21, which must therefore correspond to gene K. The K gene product has extensive amino acid sequence similarity to the product of gene R of bacteriophage lambda, and its exhibits endolysin function. Site-directed mutagenesis and reverse genetics were used to create P2 amber mutants in each of the four other newly identified open reading frames. Both orf19 (gene X) and orf20 (gene Y) encode essential functions, whereas orf22 (lysA) and orf23 (lysB) are nonessential. Gene Y encodes a polypeptide with striking similarities to the family of holin proteins exemplified by gpS of phage lambda, and the Yam mutant displayed the expected properties of a holin mutant. The gene products of lysA and lysB, although nonessential, appear to play a role in the correct timing of lysis, since a lysA amber mutant caused slightly accelerated lysis and a lysB amber mutant slightly delayed lysis of nonpermissive strains. Gene X must encode a tail protein, since lysates from nonpermissive cells infected with the X amber mutant were complemented in vitro by similar lysates of cells infected with P2 head mutants but not with tail mutants.
PMCID: PMC196335  PMID: 8051010
20.  Genomic and proteomic characterization of SuMu, a Mu-like bacteriophage infecting Haemophilus parasuis 
BMC Genomics  2012;13:331.
Haemophilus parasuis, the causative agent of Glässer’s disease, is prevalent in swine herds and clinical signs associated with this disease are meningitis, polyserositis, polyarthritis, and bacterial pneumonia. Six to eight week old pigs in segregated early weaning herds are particularly susceptible to the disease. Insufficient colostral antibody at weaning or the mixing of pigs with heterologous virulent H. parasuis strains from other farm sources in the nursery or grower-finisher stage are considered to be factors for the outbreak of Glässer’s disease. Previously, a Mu-like bacteriophage portal gene was detected in a virulent swine isolate of H. parasuis by nested polymerase chain reaction. Mu-like bacteriophages are related phyologenetically to enterobacteriophage Mu and are thought to carry virulence genes or to induce host expression of virulence genes. This study characterizes the Mu-like bacteriophage, named SuMu, isolated from a virulent H. parasuis isolate.
Characterization was done by genomic comparison to enterobacteriophage Mu and proteomic identification of various homologs by mass spectrometry. This is the first report of isolation and characterization of this bacteriophage from the Myoviridae family, a double-stranded DNA bacteriophage with a contractile tail, from a virulent field isolate of H. parasuis. The genome size of bacteriophage SuMu was 37,151 bp. DNA sequencing revealed fifty five open reading frames, including twenty five homologs to Mu-like bacteriophage proteins: Nlp, phage transposase-C-terminal, COG2842, Gam-like protein, gp16, Mor, peptidoglycan recognition protein, gp29, gp30, gpG, gp32, gp34, gp36, gp37, gpL, phage tail tube protein, DNA circulation protein, gpP, gp45, gp46, gp47, COG3778, tail fiber protein gp37-C terminal, tail fiber assembly protein, and Com. The last open reading frame was homologous to IS1414. The G + C content of bacteriophage SuMu was 41.87% while its H. parasuis host genome’s G + C content was 39.93%. Twenty protein homologs to bacteriophage proteins, including 15 structural proteins, one lysogeny-related and one lysis-related protein, and three DNA replication proteins were identified by mass spectrometry. One of the tail proteins, gp36, may be a virulence-related protein.
Bacteriophage SuMu was characterized by genomic and proteomic methods and compared to enterobacteriophage Mu.
PMCID: PMC3447690  PMID: 22823751
Haemophilus parasuis; Bacteriophage; Virulence
21.  Characterization and Comparative Genomic Analysis of a Novel Bacteriophage, SFP10, Simultaneously Inhibiting both Salmonella enterica and Escherichia coli O157:H7 
Salmonella enterica and Escherichia coli O157:H7 are major food-borne pathogens causing serious illness. Phage SFP10, which revealed effective infection of both S. enterica and E. coli O157:H7, was isolated and characterized. SFP10 contains a 158-kb double-stranded DNA genome belonging to the Vi01 phage-like family Myoviridae. In vitro adsorption assays showed that the adsorption constant rates to both Salmonella enterica serovar Typhimurium and E. coli O157:H7 were 2.50 × 10−8 ml/min and 1.91 × 10−8 ml/min, respectively. One-step growth analysis revealed that SFP10 has a shorter latent period (25 min) and a larger burst size (>200 PFU) than ordinary Myoviridae phages, suggesting effective host infection and lytic activity. However, differential development of resistance to SFP10 in S. Typhimurium and E. coli O157:H7 was observed; bacteriophage-insensitive mutant (BIM) frequencies of 1.19 × 10−2 CFU/ml for S. Typhimurium and 4.58 × 10−5 CFU/ml for E. coli O157:H7 were found, indicating that SFP10 should be active and stable for control of E. coli O157:H7 with minimal emergence of SFP10-resistant pathogens but may not be for S. Typhimurium. Specific mutation of rfaL in S. Typhimurium and E. coli O157:H7 revealed the O antigen as an SFP10 receptor for both bacteria. Genome sequence analysis of SFP10 and its comparative analysis with homologous Salmonella Vi01 and Shigella phiSboM-AG3 phages revealed that their tail fiber and tail spike genes share low sequence identity, implying that the genes are major host specificity determinants. This is the first report identifying specific infection and inhibition of Salmonella Typhimurium and E. coli O157:H7 by a single bacteriophage.
PMCID: PMC3255626  PMID: 22020516
22.  Genetic analysis of structural proteins in the adsorption apparatus of bacteriophage epsilon 15 
World Journal of Virology  2013;2(4):152-159.
AIM: To probe the organizational structure of the adsorption apparatus of bacteriophage epsilon 15 (E15) using genetic and biochemical methodology
METHODS: Hydroxylamine was used to create nonsense mutants of bacteriophage E15. The mutants were then screened for defects in their adsorption apparatus proteins, initially by measuring the concentrations of free tail spike proteins in lysates of cells that had been infected by the phage mutants under non-permissive growth conditions. Phage strains whose infected cell lysates contained above-average levels of free tail spike protein under non-permissive growth conditions were assumed to contain nonsense mutations in genes coding for adsorption apparatus proteins. These mutants were characterized by classical genetic mapping methods as well as automated sequencing of several of their genes. Finally, sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography were used to examine the protein compositions of the radioactive particles produced when the various mutants were grown on a non-permissive host cell in the presence of 35S-methionine and co-purified along with E15wt phage on CsCl block gradients.
RESULTS: Our results are consistent with gp4 forming the portal ring structure of E15. In addition, they show that proteins gp15 and gp17 likely comprise the central tube portion of the E15 adsorption apparatus, with gp17 being more distally positioned than gp15 and dependent upon both gp15 and gp16 for its attachment. Finally, our data indicates that tail spike proteins comprised of gp20 can assemble onto nascent virions that contain gp7, gp10, gp4 and packaged DNA, but which lack both gp15 and gp17, thereby forming particles that are of sufficient stability to survive CsCl buoyant density centrifugation.
CONCLUSION: The portal ring (gp4) of E15 is bound to tail spikes (gp20) and the tail tube (gp15 and gp17); gp17’s attachment requires both gp15 and gp16.
PMCID: PMC3832910  PMID: 24286036
Epsilon15; Virion structure; Salmonella phages
23.  The complete genome sequence of EC1-UPM, a novel N4-like bacteriophage that infects Escherichia coli O78:K80 
Virology Journal  2013;10:308.
Bacteriophage EC1-UPM is an N4-like bacteriophage which specifically infects Escherichia coli O78:K80, an avian pathogenic strain that causes colibacillosis in poultry. The complete genome sequence of bacteriophage EC1-UPM was analysed and compared with other closely related N4-like phage groups to assess their genetic similarities and differences.
Bacteriophage EC1-UPM displays a very similar codon usage profile with its host and does not contain any tRNA gene. Comparative genomics analysis reveals close resemblance of bacteriophage EC1-UPM to three N4-like bacteriophages namely vB_EcoP_G7C, IME11 and KBNP21 with a total of 44 protein coding genes shared at 70% identity threshold. The genomic region coding for the tail fiber protein was found to be unique in bacteriophage EC1-UPM. Further annotation of the tail fiber protein using HHpred, a highly sensitive homology detection tool, reveals the presence of protein structure homologous to various polysaccharide processing proteins in its C-terminus. Leveraging on the availability of multiple N4-like bacteriophage genome sequences, the core genes of N4-like bacteriophages were identified and used to perform a multilocus phylogenetic analysis which enabled the construction of a phylogenetic tree with higher confidence than phylogenetic trees based on single genes.
We report for the first time the complete genome sequence of a N4-like bacteriophage which is lytic against avian pathogenic Escherichia coli O78:K80. A novel 928 amino acid residues tail fiber protein was identified in EC1-UPM which may be useful to further the understanding of phage-host specificity. Multilocus phylogenetic analysis using core genes of sequenced N4-like phages showed that the evolutionary relationship correlated well with the pattern of host specificity.
PMCID: PMC3853248  PMID: 24134834
Bacteriophage EC1-UPM; Tail fiber protein; Complete genome; Multilocus phylogenetic analysis
24.  Clostridium perfringens bacteriophages ΦCP39O and ΦCP26F: genomic organization and proteomic analysis of the virions 
Archives of virology  2010;156(1):25-35.
Poultry intestinal material, sewage and poultry processing drainage water were screened for virulent Clostridium perfringens bacteriophages. Viruses isolated from broiler chicken offal washes (O) and poultry feces (F), designated ΦCP39O and ΦCP26F, respectively, produced clear plaques on host strains. Both bacteriophages had isometric heads of 57 nm in diameter with 100-nm non-contractile tails characteristic of members of the family Siphoviridae in the order Caudovirales. The double-strand DNA genome of bacteriophage ΦCP39O was 38,753 base pairs (bp), while the ΦCP26F genome was 39,188 bp, with an average GC content of 30.3%. Both viral genomes contained 62 potential open reading frames (ORFs) predicted to be encoded on one strand. Among the ORFs, 29 predicted proteins had no known similarity while others encoded putative bacteriophage capsid components such as a pre-neck/appendage, tail, tape measure and portal proteins. Other genes encoded a predicted DNA primase, single-strand DNA-binding protein, terminase, thymidylate synthase and a transcription factor. Potential lytic enzymes such as a fibronectin-binding autolysin, an amidase/hydrolase and a holin were encoded in the viral genomes. Several ORFs encoded proteins that gave BLASTP matches with proteins from Clostridium spp. and other Gram-positive bacterial and bacteriophage genomes as well as unknown putative Collinsella aerofaciens proteins. Proteomics analysis of the purified viruses resulted in the identification of the putative pre-neck/appendage protein and a minor structural protein encoded by large open reading frames. Variants of the portal protein were identified, and several mycobacteriophage gp6-like protein variants were detected in large amounts relative to other virion proteins. The predicted amino acid sequences of the pre-neck/appendage proteins had major differences in the central portion of the protein between the two phage gene products. Based on phylogenetic analysis of the large terminase protein, these phages are predicted to be pac-type, using a head-full DNA packaging strategy.
PMCID: PMC4127328  PMID: 20963614
25.  A new gene of bacteriophage P4 that controls DNA replication. 
Journal of Bacteriology  1994;176(19):6059-6065.
Bacteriophage P4 replication may result in either a lytic cycle or plasmid maintenance, depending on the presence or absence, respectively, of helper phase P2 genome. Bacteriophage P4 DNA replication depends on the product of gene alpha, which has origin recognition, primase, and helicase activities. An open reading frame with the coding capacity for a protein of 106 amino acids (orf106) is located upstream of the alpha gene. Genes orf106 and alpha are transcriptionally coregulated. Three amber mutations and an internal deletion (del51) were introduced into orf106. All of the amber mutations exhibited a polar effect on transcription of the downstream alpha gene. The P4 del51 mutant was slightly defective in lytic growth and could not be propagated in the plasmid state. In this latter condition, P4 DNA overreplication was observed. Overexpression of Orf106 severely inhibited P4 DNA replication, preventing P4 lytic growth and plasmid maintenance. The inhibitory effect of Orf106 on P4 replication was not observed when both orf106 and alpha were overexpressed. We suggest that orf106 is involved in P4 replication and that a balanced expression of orf106 relative to alpha may be necessary for proper P4 DNA replication. In particular, orf106 appears to be essential for the control of P4 genome replication in the plasmid state. We propose that orf106 be named cnr, for copy number regulation.
PMCID: PMC196825  PMID: 7928967

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