Molecular analysis of a cytotoxin-converting phage, φCTX, of Pseudomonas aeruginosa: structure of the attP–cos–ctx region and integration into the serine tRNA gene

The Pseudomonas aeruginosa ctx gene encoding cytotoxin is carried by a temperate phage φCTX. The genome of φCTX is a 35.5 kb double-stranded DNA with cohesive ends (cos). It is unique in that the ctx gene and attP site of φCTX exist very close to the respective cohesive ends. In this study, we determined the structure of this attP–cos–ctx region. The termini of φCTX are 21-base 5′ extended-single-stranded DNAs. The ctxgene is located 361 bp downstream of the left end (cosL). The attP core sequence of 30 bp exists only 647 bp apart from the right end (cosR). The attP–cos–ctx region contains six kinds of repeats and integration host factor-binding sequences and showed sequence-directed static bends, suggesting its potential to form a highly ordered structure. In addition, φCTX was found to integrate into the serine tRNA gene which was mapped to the 43–45 min region on the P. aeruginosachromosome.     

Plastid marker gene excision by the phiC31 phage site-specific recombinase

Marker genes are essential for selective amplification of rare transformed plastid genome copies to obtain genetically stable transplastomic plants. However, the marker gene becomes dispensable when homoplastomic plants are obtained. Here we report excision of plastid marker genes by the phiC31 phage site-specific integrase (Int) that mediates recombination between bacterial (attB) and phage (attP) attachment sites. We tested marker gene excision in a two-step process. First we transformed the tobacco plastid genome with the pCK2 vector in which the spectinomycin resistance (aadA) marker gene is flanked with suitably oriented attB and attP sites. The transformed plastid genomes were stable in the absence of Int. We then transformed the nucleus with a gene encoding a plastid-targeted Int that led to efficient marker gene excision. The aadA marker free Nt-pCK2-Int plants were resistant to phosphinothricin herbicides since the pCK2 plastid vector also carried a bar herbicide resistance gene that, due to the choice of its promoter, causes a yellowish-golden (aurea) phenotype. Int-mediated marker excision reported here is an alternative to the currently used CRE/loxP plastid marker excision system and expands the repertoire of the tools available for the manipulation of the plastid genome.     

Control of Staphylococcus aureus pathogenicity island excision

Staphylococcus aureus pathogenicity islands (SaPIs) are a group of related 15–17 kb mobile genetic elements that commonly carry genes for superantigen toxins and other virulence factors. The key feature of their mobility is the induction of SaPI excision and replication by certain phages and their efficient encapsidation into specific small‐headed phage‐like infectious particles. Previous work demonstrated that chromosomal integration depends on the SaPI‐encoded recombinase, Int. However, although involved in the process, Int alone was not sufficient to mediate efficient SaPI excision from chromosomal sites, and we expected that SaPI excision would involve an Xis function, which could be encoded by a helper phage or by the SaPI, itself. Here we report that the latter is the case. In vivo recombination assays with plasmids in Escherichia coli demonstrate that SaPI‐coded Xis is absolutely required for recombination between the SaPI att_L and att_R sites, and that both sites, as well as their flanking SaPI sequences, are required for SaPI excision. Mutational analysis reveals that Xis is essential for efficient horizontal SaPI transfer to a recipient strain. Finally, we show that the master regulator of the SaPI life cycle, Stl, blocks expression of int and xis by binding to inverted repeats present in the promoter region, thus controlling SaPI excision.     

Site-specific recombination in <Emphasis Type="Italic">Arabidopsis</Emphasis> plants promoted by the Integrase protein of coliphage HK022

The gene encoding the wild type Integrase protein of coliphage HK022 was integrated chromosomally and expressed in Arabidopsis thaliana plants. Double-transgenic plants cloned with the int gene as well as with a T-DNA fragment carrying the proper att sites in a tandem orientation showed that Int catalyzed a site-specific integration reaction (attP × attB) as well as a site-specific excision reaction (attL × attR). The reactions took place without the need to provide any of the accessory proteins that are required by Int in the bacterial host. When expressed in tobacco plants a GFP-Int fusion exhibits a predominant nuclear localization.These authors contributed equally to this work     

Prevalence and distribution of bacteriophage φAa DNA in strains of Actinobacillus actinomycetemcomitans

Abstract φAa is a bacteriophage that was originally isolated by induction of a lysogenic strain of the oral bacterium Actinobacillus actinomycetemcomitans . Since the discovery of phage φAa , additional phages infecting several other strains of A. actinomycetemcomitans have been identified. To determine the prevalence of φAa or φAa -related temperate phages in this species, a φAa -specific DNA probe was prepared to screen for homologous sequences among 42 strains of A. actinomycetemcomitans . Fourteen (33%) of the 42 strains examined contained DNA sequences that hybridized with the phage φAa probe. A bacteriophage designated φAa ₃₃₃₈₄ was isolated by induction from one of the strains (ATCC 33384) that contained a sequence that hybridized with the φAa probe. The φAa probe hybridized with the DNA extracted from bacteriophage φAa ₃₃₃₈₄. The distribution of the phage φAa sequence among A. actinomycetemcomitans serotypes was 5/13 (38%) of the serotype a strains, 0/16 (0%) of the serotype b strains, and 9/13 (69%) of the serotype c strains. The results of this investigation suggest that the target sequence prepared from the phage φAa genome is fairly common in the A. actinomycetemcomitans chromosome, and that the sequence is distributed among the A. actinomycetemcomitans serotypes in a seemingly nonrandom manner.     

A diversity of serine phage integrases mediate site-specific recombination in mammalian cells

This study evaluated the ability of five serine phage integrases, from phages A118, U153, Bxb1, φFC1, and φRV1, to mediate recombination in mammalian cells. Two types of recombination were investigated, including the ability of an integrase to mediate recombination between its own phage att sites in the context of a mammalian cell and the ability of an integrase to perform genomic integration pairing a phage att site with an endogenous mammalian sequence. We demonstrated that the A118 integrase mediated precise intra-molecular recombination of a plasmid containing its attB and attP sites at a frequency of ∼ 50% in human cells. The closely related U153 integrase also performed efficient recombination in human cells on a plasmid containing the attB and attP sites of A118. The integrases from phages Bxb1, φFC1, and φRV1 carried out such recombination at their attB and attP sites at frequencies ranging from 11 to 75%. Furthermore, the A118 integrase mediated recombination between its attP site on a plasmid and pseudo attB sites in the human genome, i.e. native sequences with partial identity to attB. Fifteen such A118 pseudo att sites were analyzed, and a consensus recognition site was identified. The other integrases did not mediate integration at genomic sequences at a frequency above background. These site-specific integrases represent valuable new tools for manipulating eukaryotic genomes.     

Control of lytic development in the Streptomyces temperate phage φC31

The repressor gene. c, is required for maintenance of lysogeny in the Streptomyces phage φC31. The c gene expresses three in-frame N-terminally different protein isoforms at least one of which is thought to bind to a 17bp highly conserved inverted repeat (CIR) sequence found at 18 (or more) loci throughout the φC31 genome. Here we present evidence that one of these loci, CIR6, and its interaction with the products of the repressor gene are critical in the control of the lytic pathway in φC31. To the right of CIR6, according to the standard map of φC31, an ‘immediate-early’ promoter. ap1, was discovered after insertion of a fragment containing CIR6 upstream of a promoterless kanamycin-resistance gene. aphll, to form pCIA2. pCIA2 conferred kanamycin resistance upon Streptomyces coelicolor A3(2) but not upon a φC31 lysogen of S. coelicolor. Operator-constitutive (O^c) mutants of pCIA2 were isolated and the mutations lay in CIR6, i.e. CIR6:G14T and CIR6:C2A. Primer extension analysis of RNA prepared from an induced, temperature-sensitive lysogen of S. coelicolor localized a mRNA 5′ endpoint 21 bp to the right of CIR6. The importance of the ap1/CIR6 region in the regulation of lytic growth was demonstrated by the analysis of a virulent mutant, φC31 vir1, capable of forming plaques on an S. coelicolorφC31 lysogen, φC31 vir1 contained a DNA inversion with the breakpoints lying within the integrase gene (which lies approximately 7kbp to the right of CIR6) and in the essential early region between CIR6 and the -10 sequence for ap1. The separation of ap1 from its operator was thought to be the basis for the virulent phenotype in φC31 vir1. Band-shift assays and DNase I footprinting experiments using purified 42kDa repressor isoform confirmed that CIRs 5 and 6 were indeed the targets for binding of this protein. The 42 kDa repressor bound to CIR6 with higher affinity than to CIR5 in spite of their identical core sequences. Repressor bound at CIR6 facilitated binding at CIR5. The high-affinity binding to CIR6 was abolished with the O^c mutant, CIR6:G14T. Hydroxyl radical footprinting and dimethyl sulphate methylation protection of the 42 kDa repressor–CIR6 interaction suggested that the protein bound in the major groove and to one face of the DNA.     

Site-specific integration of the phage ΦCTX genome into the Pseudomonas aeruginosa chromosome: characterization of the functional integrase gene located close to and upstream of attP

The site-specific integration of the phage CTX genome, which carries the gene for a pore-forming cytotoxin, into the Pseudomonas aeruginosa chromosome was analysed. The 1,167 by integrase gene, int, located immediately upstream of the attachment site, attP, was characterized using plasmid constructs, harbouring the integration functions, and serving as an integration probe in both P. aeruginosa and Escherichia coli. The attP plasmids p1000/p400 in the presence of the int plasmid pIBH and attP-int plasmids pINT/pINTS can be stably integrated into the P. aeruginosa chromosome. Successful recombination between the attP plasmid p1000 and the attB plasmid p5.1, in the presence of the int plasmid pIBH in E. coli HB101 showed that the int gene is active in trans in E. coli. The int gene product was detected as a 43 kDa protein in E. coli maxicells harbouring pINT. Proposed integration arm regions downstream of attP are not necessary for the integration process. pINT and phage CTX could be integrated together into P. aeruginosa chromosomal DNA, yielding double integrates.     

Complete nucleotide sequence and molecular characterization of the temperate staphylococcal bacteriophage φPVL carrying Panton–Valentine leukocidin genes

The staphylococcal Panton–Valentine leukocidin (PVL) genes, [lukS-PV–lukF-PV], exist on the genome of a temperate bacteriophage φPVL isolated from mitomycin C-induced Staphylococcus aureus V8 (ATCC 49775) (Kaneko, J., Kimura, T., Kawakami, Y., Tomita, T., Kamio, Y., 1997b. Panton–Valentine leukocidin genes in phage-like particle isolated from mitomycin C-treated Staphylococcus aureus V8 (ATCC 49775). Biosci. Biotechnol. Biochem. 61, 1960–1962). In this study, the complete nucleotide sequence of the φPVL genome was analyzed, and the att sites (attL, attR, and attB) required for site-specific integration of φPVL into the host chromosome were also determined. The linear double-stranded φPVL genome comprised 41 401 bp with 3′ staggered cohesive ends (cos) of nine bases and contained 63 ORFs, among which the regulatory proteins involved in DNA replication, structural proteins, a holin, a lysin, an integrase, and dUTPase, were tentatively identified by the comparison of the deduced amino acid sequences and by the analysis of the proteins isolated from φPVL particles. The [lukS-PV–lukF-PV], attP, and int (integrase gene) of φPVL were all located very close to one another within a 4.0-kb segment on the genome in the order given, and this segment was located at the center from the left and the right cos sites. In addition, the attP region contained five direct repeat sequences that showed a high degree of homology with the recombinase-binding sites of some other S. aureus bacteriophages. The φPVL genome was found to integrate into an ORF encoding an unknown protein comprising 725 amino acid residues with two leucine zipper-like motifs.     

Diversity,evolution and life strategies of CbK-like phages

Caulobacter phage CbK has been extensively studied as a model system in virology and bacteriology. Lysogeny-related genes have been found in each CbK-like isolate, suggesting a life strategy of both lytic and lysogenic cycles. However, whether CbK-related phages can enter lysogeny is still undetermined. This study identified new CbK-like sequences and expanded the collection of CbK-related phages. A common ancestry with a temperate lifestyle was predicted for the group, however, which subsequently evolved into two clades of different genome sizes and host associations. Through the examination of phage recombinase genes, alignment of attachment sites on the phage and bacterial genomes (attP–attB pairing), and the experimental validation, different lifestyles were found among the different members. A majority of clade II members retain a lysogenic lifestyle, whereas all clade I members have evolved into an obligate lytic lifestyle via a loss of the gene encoding Cre-like recombinase and the coupled attP fragment. We postulated that the loss of lysogeny may be a by-product of the increase in phage genome size, and vice versa. Clade I is likely to overcome the costs through maintaining more auxiliary metabolic genes (AMGs), particularly for those involved in protein metabolism, to strengthen host takeover and further benefit virion production.     

Electron microscope heteroduplex studies of sequence relations among plasmids of Escherichia coli. VI. Mapping of F14 sequences homologous to phi 80dmetBJF and phi 80dargECBH bacteriophages

The positions of the metBJF and the argECBH sequences on F14 have been mapped by studying heteroduplexes of F14 with φ80dmet and φ80darg transducing phage DNAs. The structures of the DNAs of the transducing phage φ80d-metB isolated by Konrad (1969), of two φ80dmetB phages isolated by Press et al. (1971), and of some derived φ80darg phages, have been determined. They all have complex structures. In addition to the bacterial chromosome sequences corresponding to the met and arg genes, they contain certain F sequences, which have been recognized as active in F-related recombination events. Plausible models for the integration and excision events leading to the formation of the phage DNA molecules are proposed.     

Prophage, φPV83-pro,Carrying Panton-Valentine Leukocidin Genes,on the Staphylococcus aureus P83 Chromosome: Comparative Analysis of the Genome Structures of φPV83-pro, φPVL, φ11, and Other Phages

Staphylococcus aureus P83 has Panton-Valentine leukocidin (PVL)-like genes, lukM and lukF-PV. Here, lukM and lukF-PV genes were found on the genome of a prophage, which was designated as φPV83-pro. The precise genome size was 45,636 bp with att core sequences of 10 base pairs. Sixty-four ORFs were identified on the φPV83-pro genome, including two extra operons, lukM-lukF-PV and orfs63-64. The lukM-lukF-PV cluster was located 2.1 kb upstream of the attL site. The most striking feature of the φPV83-pro genome was a constituent of at least 4 regions from φ11, φPVL, and other phages, i.e., (i) att sites identical with those of φ11, (ii) a cos sequence and the genes encoding packaging and head proteins of φPVL (occupied half region of φPV83-pro), and (iii) the other two regions which showed no significant similarity with known phages (occupied about 40% of φPV83-pro). Furthermore, two insertion sequences, ISSA1 and ISSA2 were integrated into attL site and orf44, respectively. φPV83-pro was not induced as phage particles from S. aureus P83 regardless of its treatment with mitomycin C. The insertion of ISSA1 into the attL site was one of the reasons of the failure of the induction of the phage particles by mitomycin C treatment of the strain P83.     

Site-specific integration of the phage ΦCTX genome into the Pseudomonas aeruginosa chromosome: characterization of the functional integrase gene located close to and upstream of attP

The site-specific integration of the phage ?CTX genome, which carries the gene for a pore-forming cytotoxin, into the Pseudomonas aeruginosa chromosome was analysed. The 1,167 by integrase gene, int, located immediately upstream of the attachment site, attP, was characterized using plasmid constructs, harbouring the integration functions, and serving as an integration probe in both P. aeruginosa and Escherichia coli. The attP plasmids p1000/p400 in the presence of the int plasmid pIBH and attP-int plasmids pINT/pINTS can be stably integrated into the P. aeruginosa chromosome. Successful recombination between the attP plasmid p1000 and the attB plasmid p5.1, in the presence of the int plasmid pIBH in E. coli HB101 showed that the int gene is active in trans in E. coli. The int gene product was detected as a 43 kDa protein in E. coli maxicells harbouring pINT. Proposed integration arm regions downstream of attP are not necessary for the integration process. pINT and phage ?CTX could be integrated together into P. aeruginosa chromosomal DNA, yielding double integrates.     

Genetic interaction between the nip1 mutation and genes affecting integration and excision in phage P2

Summary The excision of prophage P2 is controlled by two genes, int and cox. (The cox gene discussed in this report is defined by the cox class II mutants, defined by Six and Lindqvist, 1978). The combined activity of these two genes is rather inefficient, however, since only about 1% of the lysogens carrying an int ⁺ cox ⁺ prophage actually produce phage when derepressed. The efficiency of phage production (and presumably excision) can be increased 100-fold by an additional mutation called nip1 (Calendar et al., 1972), which is dominant and is located in or near the int gene.The nip1 mutation was mapped between c5, a mutation in the C gene, and an amber int mutation, int150. Phages carrying nip1 and either int150 or a cox mutation, cox3, were prepared by recombination. The nip1 mutation was found to increase excision only when it was located on the same chromosome as an active int ⁺ gene and only if cox ⁺ gene product was also available. The cox gene, known to be located between genes B and C (Lindahl and Sunshine, 1972), was further localized to a region between 77.2 to 78.1% from the conventional left end of the P2 chromosome by comparing the ability of phages with overlapping deletions to promote excision of the prophage in a P2 nip1 c5 cox3 lysogen.Other features of the integration-excision system in P2 are discussed.     

Cutting out the φC31 prophage

To establish a lysogenic lifestyle, the temperate bacteriophage φC31 integrates its genome into the chromosome of its Streptomyces host, by site-specific recombination between attP (the attachment site in the phage DNA) and attB (the chromosomal attachment site). This reaction is promoted by a phage-encoded serine recombinase Int. To return to the lytic lifestyle, the prophage excises its DNA by a similar Int-mediated reaction between the recombinant sites flanking the prophage, attL and attR. φC31 Int has been developed into a popular experimental tool for integration of transgenic DNA into the genomes of eukaryotic organisms. However, until now it has not been possible to use Int to promote the reverse reaction, excision. In many other phages, the presence of a recombination directionality factor (RDF) protein biases the phage-encoded integrase towards prophage excision, whereas absence of the RDF favours integration; but the φC31 RDF had proved elusive. In this issue of Molecular Microbiology, Khaleel et al. (2011) report the identification and purification of the φC31 RDF, and show that it both promotes excision and inhibits integration by direct protein-protein interactions with Int itself.     

Streptomyces lividans 66 contains a gene for phage resistance which is similar to the phage λea59 endonuclease gene

The DNA of wild-type Streptomyces lividans 66 is degraded during electrophoresis in buffers containing traces of ferrous iron. S. lividans ZX1, a mutant selected for resistance to DNA degradation, simuiltaneously became sensitive to φHAU3, a wide-host-range temperate bacteriophage. A DNA fragment conferring φHAU3 resistance was cloned; it contains a phage resistance gene whose deduced amino acid sequence is similar to the phage λ Ea59 endonuclease. The S. lividansφHAU3 resistance does not seem to be a classical restriction-modification system, because no host-modified phages able to propagate on the wild-type strain could be isolated. The cloned fragment did not make the host DNA prone to degradation during electrophoresis, indicating that the two phenotypes are controlled by different genes which were deleted together from the chromosome of ZX1.     

Insights into the diversity of φRSM phages infecting strains of the phytopathogen Ralstonia solanacearum complex: regulation and evolution

The filamentous φRSM phages (φRSM1 and φRSM3) have integration/excision capabilities in the phytopathogenic bacterium Ralstonia solanacearum. In the present study, we further investigated φRSM-like sequences present in the genomes of R. solanacearum strains belonging to the four major evolutionary lineages (phylotypes I–IV). Based on bioinformatics and comparative genomic analyses, we found that φRSM homologs are highly diverse in R. solanacearum complex strains. We detected an open reading frame (ORF)15 located upstream of the gene for φRSM integrase, which exhibited amino acid sequence similarity to phage repressor proteins. ORF15-encoded protein (a putative repressor) was found to encode a 104-residue polypeptide containing a DNA-binding (helix-turn-helix) domain and was expressed in R. solanacearum lysogenic strains. This suggested that φRSM3-ORF15 might be involved in the establishment and maintenance of a lysogenic state, as well as in phage immunity. Comparison of the putative repressor proteins and their binding sites within φRSM-related prophages provides insights into how these regulatory systems of filamentous phages have evolved and diverged in the R. solanacearum complex. In conclusion, φRSM phages represent a unique group of filamentous phages that are equipped with innate integration/excision (ORF14) and regulatory systems (ORF15).     

Identification of Site-Specific Recombination Genes int and xis of the Rhizobium Temperate Phage 16-3

Phage 16-3 is a temperate phage of Rhizobium meliloti 41 which integrates its genome with high efficiency into the host chromosome by site-specific recombination through DNA sequences of attB and attP. Here we report the identification of two phage-encoded genes required for recombinations at these sites: int (phage integration) and xis (prophage excision). We concluded that Int protein of phage 16-3 belongs to the integrase family of tyrosine recombinases. Despite similarities to the cognate systems of the lambdoid phages, the 16-3 int xis att system is not active in Escherichia coli, probably due to requirements for host factors that differ in Rhizobium meliloti and E. coli. The application of the 16-3 site-specific recombination system in biotechnology is discussed.     

Properties and products of the cloned int gene of bacteriophage P2

Summary Fragments of DNA of the temperate phage P2, generated by treatment with the restriction enzyme PstI, have been cloned into the plasmid pBR322. One such fragment, which has its endpoints within phage genes T and C, carries the structural P2 int gene as well as its promoter and the phage att site. When introduced into a suitable bacterial host, the cloned fragment mediates the integration and excision of int ^- mutants of P2 and recombination within the phage att site in mixed infection. All these activities are independent of the orientation of the fragment within the plasmid.When introduced into minicells, the fragment produces, in addition to the products of genes D and U, a protein of 35–37,000 daltons identified as the int protein. A study of the map location of two amber int mutants, together with the sizes of the polypeptides they produce, indicates that the P2 int gene is transcribed from right to left on the P2 map, i.e. starting near gene C and proceeding toward att.