Cos Site Research Articles

L-Cysteine-Tuned the Hierarchical Structure Based on Benzimidazole: Synthesis, Characterization, and Application in Ratiometric Electrochemiluminescence Immunoassay.

Efficient and robust electrochemiluminescence (ECL) emitters are crucial for enhancing the ECL immunosensor sensitivity. This study introduces a novel ECL emitter, CoBIM/Cys, featuring a hierarchical core-shell structure. The core of the structure is created through the swift coordination between the sulfhydryl and carboxyl groups of l-cysteine (l-Cys) and cobalt ions (Co2+), while the shell is constructed by sequentially coordinating benzimidazole (BIM) with Co2+. This design yields a greater specific surface area and a more intricate porous structure compared to CoBIM, markedly enhancing mass transfer and luminophore accessibility. Moreover, the l-Cys and Co2+ core introduces Co-S and Co-O catalytic sites, which improve the catalytic decomposition of H2O2, leading to an increased production of hydroperoxyl radicals (OOH•). This mechanism substantially amplifies the ECL performance. Leveraging the competitive interaction between isoluminol and BIM for OOH• during ECL emission, we developed a ratiometric immunosensor for cardiac troponin I (cTnI) detection. This immunosensor demonstrates a remarkably broad detection range (1 pg mL-1 to 10 ng mL-1), a low detection limit (0.4 pg mL-1), and exceptional reproducibility and specificity.

Two neglected but valuable genetic tools for Escherichia coli and other bacteria: In vivo cosmid packaging and inducible plasmid replication.

In physiology and synthetic biology, it can be advantageous to introduce a gene into a naive bacterial host under conditions in which all cells receive the gene and remain fully functional. This cannot be done by the usual chemical transformation and electroporation methods due to low efficiency and cell death, respectively. However, in vivo packaging of plasmids (called cosmids) that contain the 223 bp cos site of phage λ results in phage particles that contain concatemers of the cosmid that can be transduced into all cells of a culture. An historical shortcoming of in vivo packaging of cosmids was inefficient packaging and contamination of the particles containing cosmid DNA with a great excess of infectious λ phage. Manipulation of the packaging phage and the host has eliminated these shortcomings resulting in particles that contain only cosmid DNA. Plasmids have the drawback that they can be difficult to remove from cells. Plasmids with conditional replication provide a means to "cure" plasmids from cells. The prevalent conditional replication plasmids are temperature-sensitive plasmids, which are cured at high growth temperature. However, inducible replication plasmids are in some cases more useful, especially since this approach has been applied to plasmids having diverse replication and compatibility properties.

Insights into a viral motor: the structure of the HK97 packaging termination assembly.

Double-stranded DNA viruses utilise machinery, made of terminase proteins, to package viral DNA into the capsid. For cos bacteriophage, a defined signal, recognised by small terminase, flanks each genome unit. Here we present the first structural data for a cos virus DNA packaging motor, assembled from the bacteriophage HK97 terminase proteins, procapsids encompassing the portal protein, and DNA containing a cos site. The cryo-EM structure is consistent with the packaging termination state adopted after DNA cleavage, with DNA density within the large terminase assembly ending abruptly at the portal protein entrance. Retention of the large terminase complex after cleavage of the short DNA substrate suggests that motor dissociation from the capsid requires headful pressure, in common with pac viruses. Interestingly, the clip domain of the 12-subunit portal protein does not adhere to C12 symmetry, indicating asymmetry induced by binding of the large terminase/DNA. The motor assembly is also highly asymmetric, showing a ring of 5 large terminase monomers, tilted against the portal. Variable degrees of extension between N- and C-terminal domains of individual subunits suggest a mechanism of DNA translocation driven by inter-domain contraction and relaxation.

Phages from Genus Bruynoghevirus and Phage Therapy: Pseudomonas Phage Delta Case.

The applicability and safety of bacteriophage Delta as a potential anti-Pseudomonas aeruginosa agent belonging to genus Bruynoghevirus (family Podoviridae) was characterised. Phage Delta belongs to the species Pseudomonas virus PaP3, which has been described as a temperate, with cos sites at the end of the genome. The phage Delta possesses a genome of 45,970 bp that encodes tRNA for proline (Pro), aspartic acid (Asp) and asparagine (Asn) and does not encode any known protein involved in lysogeny formation or persistence. Analysis showed that phage Delta has 182 bp direct terminal repeats at the end of genome and lysogeny was confirmed, neither upon infection at low nor at high multiplicity of infection (MOI). The turbid plaques that appear on certain host lawns can result from bacteriophage insensitive mutants that occur with higher frequency (10−4). In silico analysis showed that the genome of Delta phage does not encode any known bacterial toxin or virulence factor, determinants of antibiotic resistance and known human allergens. Based on the broad host range and high lytic activity against planktonic and biofilm cells, phage Delta represents a promising candidate for phage therapy.

HK97 gp74 Possesses an α-Helical Insertion in the ββα Fold That Affects Its Metal Binding, cos Site Digestion, and In Vivo Activities.

The last gene in the genome of the bacteriophage HK97 encodes gp74, an HNH endonuclease. HNH motifs contain two conserved His residues and an invariant Asn residue, and they adopt a ββα structure. gp74 is essential for phage head morphogenesis, likely because gp74 enhances the specific endonuclease activity of the HK97 terminase complex. Notably, the ability of gp74 to enhance the terminase-mediated cleavage of the phage cos site requires an intact HNH motif in gp74. Mutation of H82, the conserved metal-binding His residue in the HNH motif, to Ala abrogates gp74-mediated stimulation of terminase activity. Here, we present nuclear magnetic resonance (NMR) studies demonstrating that gp74 contains an α-helical insertion in the Ω-loop, which connects the two β-strands of the ββα fold, and a disordered C-terminal tail. NMR data indicate that the Ω-loop insert makes contacts to the ββα fold and influences the ability of gp74 to bind divalent metal ions. Further, the Ω-loop insert and C-terminal tail contribute to gp74-mediated DNA digestion and to gp74 activity in phage morphogenesis. The data presented here enrich our molecular-level understanding of how HNH endonucleases enhance terminase-mediated digestion of the cos site and contribute to the phage replication cycle.IMPORTANCE This study demonstrates that residues outside the canonical ββα fold, namely, the Ω-loop α-helical insert and a disordered C-terminal tail, regulate the activity of the HNH endonuclease gp74. The increased divalent metal ion binding when the Ω-loop insert is removed compared to reduced cos site digestion and phage formation indicates that the Ω-loop insert plays multiple regulatory roles. The data presented here provide insights into the molecular basis of the involvement of HNH proteins in phage DNA packing.

Strategies for purification of the bacteriophage HK97 small and large terminase subunits that yield pure and homogeneous samples that are functional

Packaging the viral genome in the head of double-stranded DNA viruses, such as bacteriophages, requires the activity of a terminase. The bacteriophage terminase consists of a small terminase subunit (TerS), which binds the viral DNA, and a large terminase subunit (TerL) that possesses the ATPase and nuclease activities for packaging the DNA in the phage head. Some phages require additional components for DNA packaging, such as the HNH endonuclease gp74 in the bacteriophage HK97. Gp74 enhances the activity of terminase-mediated digestion of the cohesive (cos) site that connects individual genomes in phage concatemeric DNA, a pre-requisite to DNA packaging, and this enhancement requires an intact HNH motif in gp74. Testing of whether gp74 alters the terminase DNA binding or enzymatic activities requires obtaining isolated samples of pure TerS and TerL, which has been challenging owing to the poor solubility of these proteins. To this end, we developed methods to obtain purified TerS and TerL proteins that are active. TerS is expressed solubly in E. coli as a fusion with SUMO, which can be removed during purification to yield a TerS nonamer (TerS9). Homogenous samples of a TerL monomer are also obtained, but the homogeneity of the sample depends on the solution conditions, as seen for other terminases. DNA binding, ATPase, and nuclease assays demonstrate that our preparations of TerS9 and TerL are functional, and that they also function with gp74. Purified TerS9 and TerL enable studies into the molecular basis by which gp74 regulates terminase activity in phage maturation.

The Isolation and Characterization of Kronos, a Novel Caulobacter Rhizosphere Phage that is Similar to Lambdoid Phages.

Despite their ubiquity, relatively few bacteriophages have been characterized. Here, we set out to explore Caulobacter bacteriophages (caulophages) in the rhizosphere and characterized Kronos, the first caulophage isolated from the rhizosphere. Kronos is a member of the Siphoviridae family since it has a long flexible tail. In addition, an analysis of the Kronos genome indicated that many of the predicted proteins were distantly related to those of bacteriophages in the lambdoid family. Consistent with this observation, we were able to demonstrate the presence of cos sites that are similar to those found at the ends of lambdoid phage genomes. Moreover, Kronos displayed a relatively rare head and tail morphology compared to other caulophages but was similar to that of the lambdoid phages. Taken together, these data indicate that Kronos is distantly related to lambdoid phages and may represent a new Siphoviridae genus.

Physical and Functional Characterization of a Viral Genome Maturation Complex

Genome packaging is strongly conserved in the complex double-stranded DNA viruses, including the herpesviruses and many bacteriophages. In these cases, viral DNA is packaged into a procapsid shell by a terminase enzyme. The packaging substrate is typically a concatemer composed of multiple genomes linked in a head-to-tail fashion, and terminase enzymes perform two essential functions: 1) excision of a unit length genome from the concatemer (genome maturation) and 2) translocation of the duplex into a procapsid (genome packaging). While the packaging motors have been described in some detail, the maturation complexes remain ill characterized. Here we describe the assembly, physical characteristics, and catalytic activity of the λ-genome maturation complex. The λ-terminase protomer is composed of one large catalytic subunit tightly associated with two DNA recognition subunits. The isolated protomer binds DNA weakly and does not discriminate between nonspecific DNA and duplexes that contain the packaging initiation sequence, cos. The Escherichia coli integration host factor protein (IHF) is required for efficient λ-development in vivo and a specific IHF recognition sequence is found within cos. We show that IHF and the terminase protomer cooperatively assemble at the cos site and that the small terminase subunit plays the dominant role in complex assembly. Analytical ultracentrifugation analysis reveals that the maturation complex is composed of four protomers and one IHF heterodimer bound at the cos site. Tetramer assembly activates the cos-cleavage nuclease activity of the enzyme, which matures the genome end in preparation for packaging. The stoichiometry and catalytic activity of the complex is reminiscent of the type IIE and IIF restriction endonucleases and the two systems may share mechanistic features. This study, to our knowledge, provides our first detailed glimpse into the structural and functional features of a viral genome maturation complex, an essential intermediate in the development of complex dsDNA viruses.

CRISPR-Cas systems exploit viral DNA injection to establish and maintain adaptive immunity.

CRISPR-Cas systems provide protection against viral1 and plasmid2 infection by capturing short DNA sequences from these invaders and integrating them into the CRISPR locus of the prokaryotic host1. These sequences, known as spacers, are transcribed into short RNA guides3–5 that specify the cleavage site of Cas nucleases in the genome of the invader6–8. When spacer sequences are acquired during viral infection is not known. To investigate this, we followed spacer acquisition in Staphylococcus aureus cells harboring a type II CRISPR-Cas9 system after infection with the staphylococcal bacteriophage ϕ12. We found that new spacers are acquired immediately following infection preferentially from the cos site, the viral free DNA end that is first injected into the cell. Analysis of spacer acquisition after infection with mutant phages demonstrated that most spacers are acquired during DNA injection, but not during other stages of the viral cycle that produce free DNA ends, such as DNA replication or packaging. Finally, we showed that spacers acquired from early-injected genomic regions, which direct Cas9 cleavage of the viral DNA immediately after infection, provide better immunity than spacers acquired from late-injected regions. Our results reveal that CRISPR-Cas systems exploit the phage life cycle to generate a pattern of spacer acquisition that ensures the success of the CRISPR immune response.

Genetics of critical contacts and clashes in the DNA packaging specificities of bacteriophages λ and 21

The cos sites in λ and 21 chromosomes contain binding sites that recruit terminase to initiate DNA packaging. The small subunits of terminase, gpNu1 (λ) and gp1 (21), have winged helix-turn-helix DNA binding domains, where the recognition helixes differ in four of nine residues. To initiate packaging, the small subunit binds three R sequences in the cosB subsite. λ and 21 cannot package each other׳s DNA, due to recognition helix and R sequence differences. In λ and 21 cosBs, two bp, tri1 and tri2, are conserved in the R sequences yet differ between the phages; they are proposed to play a role in phage-specific packaging by λ and 21. Genetic experiments done with mixed and matched terminase and cosB alleles show packaging specificity depends on favorable contacts and clashes. These interactions indicate that the recognition helixes orient with residues 20 and 24 proximal to tri2 and tri1, respectively.

The Binding Parameters of Integration Host Factor to the Lambda Phage Cosi1 Site and Holliday Junction DNA

Integration Host Factor (IHF) is a heterodimeric DNA-binding protein produced by E.coli. IHF interacts with the minor groove and phosphodiester backbone of the DNA to create sharp bends in the DNA and is often referred to as an architectural protein. Although the protein binds sequence-specifically, it also recognizes DNA conformation. Prokaryotic cellular processes involving IHF include viral integration, replication and activation of gene expression. We have recently shown that IHF exhibits high binding affinity for the Holliday or four-way junction DNA (4WJ), an intermediate in homologous recombination where strands from different chromosomes cross over. Förster Resonance Energy Transfer (FRET), where the 5'-ends of DNA strands are labeled with fluorophores, is used to determine the conformational changes of the 4WJ when interacting with DNA-binding proteins. These measurements have shown that the 4WJ opens upon binding with IHF, which is consistent with data promoting the protein's involvement in recombinase action. Subsequent FRET experiments performed confirm this observation, and provide a value for the efficiency of binding and the degree of distortion induced. Interactions between IHF and the cos I1 site of DNA from the lambda bacteriophage will also be explored. It is known that IHF induces bending at cos sites. The affinity of binding to Δ cos DNA is compared to that between IHF and its consensus sequence (H1) in E. coli DNA. Gel mobility shift assay (GMSA) and solutions experiments will measure the binding and affinity of IHF with the cos I1 site. FRET experiments with labeled cosI1 DNA will reveal the distortion induced upon IHF binding and these results will be compared to those with IHF binding to the H1 site.

Improved plasmid-based system for fully regulated off-to-on gene expression in Escherichia coli: Application to production of toxic proteins

In previous work transduction of Escherichia coli with phage λ particles carrying packaged plasmids was shown to provide complete off-to-on expression of plasmid-borne genes (Cronan, J.E., 2003. J. Bacteriol. 185, 6522–6529). The plasmids used contained the phage λcos site (and hence are cosmids) and were very efficiently packaged into λ phage particles in vivo upon induction of λ lysogens having an inactivated cos site. However, a shortcoming was that the stocks contained a variable fraction of infectious λ phage which arose by recombinational repair of the inactive cos site. I report that the construction of E. coli strains that eliminate the background of infectious phage and show that the system can be utilized to express proteins by the phage T7 RNA polymerase/pET vector system.

Functional identification of the DNA packaging terminase from Pseudomonas aeruginosa phage PaP3

Terminase proteins are responsible for DNA recognition and initiation of DNA packaging in phages. We previously reported the genomic sequence of a temperate Pseudomonas aeruginosa phage, PaP3, and determined its precise integration site in the host bacterial chromosome. In this study, we present a detailed functional identification of the DNA packaging terminase for phage PaP3. The purified large subunit p03 was demonstrated to possess ATPase and nuclease activities, as well as the ability to bind to specific DNA when it is unassembled. In addition, a small terminase subunit (p01) of a new type was found and shown to bind specifically to cos-containing DNA and stimulate the cos-cleavage and ATPase activities of p03. The results presented here suggest that PaP3 utilizes a typical cos site mechanism for DNA packaging and provide a first step towards understanding the molecular mechanism of the PaP3 DNA packaging reaction.Electronic supplementary materialThe online version of this article (doi:10.1007/s00705-012-1409-5) contains supplementary material, which is available to authorized users.

Novel broad host range shuttle vectors for expression in Escherichia coli, Bacillus subtilis and Pseudomonas putida

Novel shuttle vectors named pEBP were constructed to allow the gene expression in different bacterial hosts including Escherichia coli, Bacillus subtilis and Pseudomonas putida. These vectors share the inducible promoters P(T7) and P(Xyl) and a cos site to enable packaging of plasmid DNA into phage, and carry different multiple cloning sites and antibiotic resistance genes. Vector pEBP41 generally replicates episomally while pEBP18 replicates episomally in Gram-negative bacteria only, but integrates into the chromosome of B. subtilis. Plasmid copy numbers determined for E. coli and P. putida were in the range of 5-50 per cell. The functionality of pEBP18 and pEBP41 was confirmed by expression of two lipolytic enzymes, namely lipase A from B. subtilis and cutinase from the eukaryotic fungus Fusarium solani pisi in three different host strains. Additionally, we report here the construction of a T7 RNA polymerase-based expression strain of P. putida.

Energy Independent Helicase Activity of a Viral Genome Packaging Motor

Terminase enzymes are integral components of the virus assembly process in many complex double-stranded (ds) DNA viruses, facilitating the packaging of DNA into the procapsid. The bacteriophage λ terminase also possesses a DNA maturation activity, which includes site specific nicking of the duplex at the cohesive end (cos) site and subsequent separation of the annealed strands (“helicase” activity). Previous studies observed that ATP is required for strand separation and it has been presumed that ATP hydrolysis is required for the reaction.

Energy-Independent Helicase Activity of a Viral Genome Packaging Motor

The assembly of complex double-stranded DNA viruses includes a genome packaging step where viral DNA is translocated into the confines of a preformed procapsid shell. In most cases, the preferred packaging substrate is a linear concatemer of viral genomes linked head-to-tail. Viral terminase enzymes are responsible for both excision of an individual genome from the concatemer (DNA maturation) and translocation of the duplex into the capsid (DNA packaging). Bacteriophage λ terminase site-specifically nicks viral DNA at the cos site in a concatemer and then physically separates the nicked, annealed strands to mature the genome in preparation for packaging. Here we present biochemical studies on the so-called helicase activity of λ terminase. Previous studies reported that ATP is required for strand separation, and it has been presumed that ATP hydrolysis is required to drive the reaction. We show that ADP and nonhydrolyzable ATP analogues also support strand separation at low (micromolar) concentrations. In addition, the Escherichia coli integration host factor protein (IHF) strongly stimulates the reaction in a nucleotide-independent manner. Finally, we show that elevated concentrations of nucleotide inhibit both ATP- and IHF-stimulated strand separation by λ terminase. We present a model where nucleotide and IHF interact with the large terminase subunit and viral DNA, respectively, to engender a site-specifically bound, catalytically competent genome maturation complex. In contrast, binding of nucleotide to the low-affinity ATP binding site in the small terminase subunit mediates a conformational switch that down-regulates maturation activities and activates the DNA packaging activity of the enzyme. This affords a motor complex that binds tightly, but nonspecifically, to DNA as it translocates the duplex into the capsid shell. These studies have yielded mechanistic insight into the assembly of the maturation complex on viral DNA and its transition to a mobile packaging motor that may be common to all of the complex double-stranded DNA viruses.

Cooperative Assembly of Host and Viral Proteins into a DNA Packaging Motor Complex Analyzed by Fluorescence-Monitored Analytical Ultracentrifugation

Packaging of viral genomes into procapsids by terminaseenzymes is conserved in complex double-stranded DNA viruses. Terminases bind to linear concatemers of replicated viral genomes linked end-to-end and concomitantly excise (mature) and translocate (package) a single genome per procapsid. While the terminase maturation complex requires site-specific recognition to mature the genome ends at each cos site within the concatemer, the terminase motor complex must bind DNA tightly, but non-specifically, during packaging. The bacteriophage λ protomer is a heterotrimer composed of two different subunits that separately confer catalytic activities and site-specific DNA binding. Although our data demonstrate that the protomer self-associates into a ring-like tetramer, the stoichiometries of terminase in the maturation and packaging complexes remains unknown. We investigated the nature of the terminase-DNA maturation complex and the factors that mediate site-specific vs. non-specific DNA binding modes. Specifically, we utilized electrophoretic mobility shift assays (EMSA) and analytical ultracentrifugation (AUC) to examine assembly of λ terminase and E. coli Integration Host Factor (IHF, required for λ development in vivo) on model DNA substrates. We first demonstrate that terminase protomer does not significantly discriminate between cos-containing and non-specific DNA substrates using EMSA; however, in the presence of IHF the enzyme forms specific complexes with cos-DNA at concentrations much lower than those required to bind non-specific DNA. We next utilize AUC to demonstrate that a binary complex is formed between cos-DNA and IHF. Addition of λ terminase to this binary complex affords two higher-order ternary complexes in a concentration-dependent manner. The data demonstrate that IHF promotes cooperative and site-specific assembly of terminase at the packaging initiation site cos. The nature of the nucleoprotein complexes will be discussed in relation to their role in viral genome packaging.

Plasmid transduction using bacteriophage Phi(adh) for expression of CC chemokines by Lactobacillus gasseri ADH.

Vaginal mucosal microfloras are typically dominated by Gram-positive Lactobacillus species, and colonization of vaginal mucosa by exogenous microbicide-secreting Lactobacillus strains has been proposed as a means of enhancing this natural mucosal barrier against human immunodeficiency virus (HIV) infection. We asked whether an alternative strategy could be utilized whereby anti-HIV molecules are expressed within the cervicovaginal milieu by endogenous vaginal Lactobacillus populations which have been engineered in situ via transduction. In this study, we therefore investigated the feasibility of utilizing transduction for the expression of two HIV coreceptor antagonists, the CC chemokines CCL5 and CCL3, in a predominant vaginal Lactobacillus species, Lactobacillus gasseri. Modifying a previously established transduction model, which utilizes L. gasseri ADH and its prophage Phiadh, we show that mitomycin C induction of L. gasseri ADH transformants containing pGK12-based plasmids with CCL5 and CCL3 expression and secretion cassettes (under the control of promoters P6 and P59, respectively) and a 232-bp Phiadh cos site fragment results in the production of transducing particles which contain 8 to 9 copies of concatemeric plasmid DNA. High-frequency transduction for these particles (almost 6 orders of magnitude greater than that for pGK12 alone) was observed, and transductants were found to contain recircularized expression plasmids upon subsequent culture. Importantly, transductants produced CC chemokines at levels comparable to those produced by electroporation-derived transformants. Our findings therefore lend support to the potential use of transduction in vaginal Lactobacillus species as a novel strategy for the prevention of HIV infection across mucosal membranes.

Sequence Analysis of Leuconostoc mesenteroides Bacteriophage Φ1-A4 Isolated from an Industrial Vegetable Fermentation

Vegetable fermentations rely on the proper succession of a variety of lactic acid bacteria (LAB). Leuconostoc mesenteroides initiates fermentation. As fermentation proceeds, L. mesenteroides dies off and other LAB complete the fermentation. Phages infecting L. mesenteroides may significantly influence the die-off of L. mesenteroides. However, no L. mesenteroides phages have been previously genetically characterized. Knowledge of more phage genome sequences may provide new insights into phage genomics, phage evolution, and phage-host interactions. We have determined the complete genome sequence of L. mesenteroides phage Phi1-A4, isolated from an industrial sauerkraut fermentation. The phage possesses a linear, double-stranded DNA genome consisting of 29,508 bp with a G+C content of 36%. Fifty open reading frames (ORFs) were predicted. Putative functions were assigned to 26 ORFs (52%), including 5 ORFs of structural proteins. The phage genome was modularly organized, containing DNA replication, DNA-packaging, head and tail morphogenesis, cell lysis, and DNA regulation/modification modules. In silico analyses showed that Phi1-A4 is a unique lytic phage with a large-scale genome inversion ( approximately 30% of the genome). The genome inversion encompassed the lysis module, part of the structural protein module, and a cos site. The endolysin gene was flanked by two holin genes. The tail morphogenesis module was interspersed with cell lysis genes and other genes with unknown functions. The predicted amino acid sequences of the phage proteins showed little similarity to other phages, but functional analyses showed that Phi1-A4 clusters with several Lactococcus phages. To our knowledge, Phi1-A4 is the first genetically characterized L. mesenteroides phage.

Genomic Characterization of the Intron-Containing T7-Like Phage phiL7 ofXanthomonas campestris

The lytic phage phiL7, which morphologically belongs to the Siphoviridae family, infects Xanthomonas campestris pv. campestris. Nucleotide sequence analysis has revealed that phiL7 contains a linear double-stranded DNA genome (44,080 bp, 56% G+C) with a 3'-protruding cos site (5'-TTACCGGAC-3') and 59 possible genes. Among the deduced proteins, 32 have homologs with known functions and 18 show no database similarities; moreover, the genes encoding these 18 proteins mostly have varying G+C contents and form clusters dispersed along the genome. Only 39 genes have sequences related (27% to 78%) to those of sequenced genes of X. oryzae pv. oryzae phages, although the genome size and architecture of these Xanthomonas phages are similar. These findings suggest that phiL7 acquired genes by horizontal transfer, followed by evolution via various types of mutations. Major differences were found between phiL7 and the X. oryzae pv. oryzae phages: (i) phiL7 has a group I intron inserted in the DNA polymerase gene, the first such intron observed in Xanthomonas phages; (ii) although infection of phiL7 exerted inhibition to the host RNA polymerase, similar to the situations in X. oryzae pv. oryzae phages Xp10 and Xop411, sequence analysis did not identify a homologue of the Xp10 p7 that controls the shift from host RNA polymerase (RNAP) to viral RNAP during transcription; and (iii) phiL7 lacks the tail fiber protein gene that exhibits domain duplications thought to be important for host range determination in OP1, and sequence analysis suggested that p20 (tail protein III) instead has the potential to play this role.