Temperate Coliphage Research Articles

Equine Intestinal O-Seroconverting Temperate Coliphage Hf4s: Genomic and Biological Characterization.

Tailed bacteriophages constitute the bulk of the intestinal viromes of vertebrate animals. However, the relationships between lytic and lysogenic lifestyles of phages in these ecosystems are not always clear and may vary between the species or even between the individuals. The human intestinal (fecal) viromes are dominated mostly by temperate phages, while in horse feces virulent phages are more prevalent. To our knowledge, all the previously reported isolates of horse fecal coliphages are virulent. Temperate coliphage Hf4s was isolated from horse feces, from the indigenous equine Escherichia coli 4s strain. It is a podovirus related to the Lederbergvirus genus (including the well-characterized Salmonella bacteriophage P22). Hf4s recognizes the host O antigen as its primary receptor and possesses a functional O antigen seroconversion cluster that renders the lysogens protected from superinfection by the same bacteriophage and also abolishes the adsorption of some indigenous equine virulent coliphages, such as DT57C, while other phages, such as G7C or phiKT, retain the ability to infect E. coli 4s (Hf4s) lysogens. IMPORTANCE The relationships between virulent and temperate bacteriophages and their impact on high-density symbiotic microbial ecosystems of animals are not always clear and may vary between species or even between individuals. The horse intestinal virome is dominated by virulent phages, and Hf4s is the first temperate equine intestinal coliphage characterized. It recognizes the host O antigen as its primary receptor and possesses a functional O antigen seroconversion cluster that renders the lysogens protected from superinfection by some indigenous equine virulent coliphages, such as DT57C, while other phages, such as G7C or phiKT, retain the ability to infect E. coli 4s (Hf4s) lysogens. These findings raise questions on the significance of bacteriophage-bacteriophage interactions within the ecology of microbial viruses in mammal intestinal ecosystems.

Instability of CII is needed for efficient switching between lytic and lysogenic development in bacteriophage 186.

The CII protein of temperate coliphage 186, like the unrelated CII protein of phage λ, is a transcriptional activator that primes expression of the CI immunity repressor and is critical for efficient establishment of lysogeny. 186-CII is also highly unstable, and we show that in vivo degradation is mediated by both FtsH and RseP. We investigated the role of CII instability by constructing a 186 phage encoding a protease resistant CII. The stabilised-CII phage was defective in the lysis-lysogeny decision: choosing lysogeny with close to 100% frequency after infection, and forming prophages that were defective in entering lytic development after UV treatment. While lysogenic CI concentration was unaffected by CII stabilisation, lysogenic transcription and CI expression was elevated after UV. A stochastic model of the 186 network after infection indicated that an unstable CII allowed a rapid increase in CI expression without a large overshoot of the lysogenic level, suggesting that instability enables a decisive commitment to lysogeny with a rapid attainment of sensitivity to prophage induction.

Studies on Escherichia coliHflKC suggest the presence of an unidentified λ factor that influences the lysis-lysogeny switch

BackgroundThe lysis-lysogeny decision in the temperate coliphage λ is influenced by a number of phage proteins (CII and CIII) as well as host factors, viz. Escherichia coli HflB, HflKC and HflD. Prominent among these are the transcription factor CII and HflB, an ATP-dependent protease that degrades CII. Stabilization of CII promotes lysogeny, while its destabilization induces the lytic mode of development. All other factors that influence the lytic/lysogenic decision are known to act by their effects on the stability of CII. Deletion of hflKC has no effect on the stability of CII. However, when λ infects ΔhflKC cells, turbid plaques are produced, indicating stabilization of CII under these conditions.ResultsWe find that CII is stabilized in ΔhflKC cells even without infection by λ, if CIII is present. Nevertheless, we also obtained turbid plaques when a ΔhflKC host was infected by a cIII-defective phage (λcIII67). This observation raises a fundamental question: does lysogeny necessarily correlate with the stabilization of CII? Our experiments indicate that CII is indeed stabilized under these conditions, implying that stabilization of CII is possible in ΔhflKC cells even in the absence of CIII, leading to lysogeny.ConclusionWe propose that a yet unidentified CII-stabilizing factor in λ may influence the lysis-lysogeny decision in ΔhflKC cells.

Identification of bases required for P2 integrase core binding and recombination

Temperate coliphage P2 integrates its genome into the host chromosome upon lysogenization via a site-specific recombination event mediated by an integrase belonging to the complex family of tyrosine recombinases. The host integration site attB (BOB′) is localized in the end of the cyaR gene and shares 27 nucleotides with the core of attP (COC′). In the present study we determine the minimal attB site using an in vivo recombination assay. Ten nt on the left side (B) are found to be nonessential for recombination. We show that the integrase has higher affinity for the right side (B′) compared to B and that artificial B′OB′ and an attP site with a matching core (C′OC′) are efficient substrates for recombination in vitro. We have analyzed single nucleotides in attB and find that sequence homology within a non-centrally located quadruplet in the hypothetical overlap region is essential for efficient recombination in vivo.

Conversion of Linear DNA with Hairpin Telomeres into a Circular Molecule in the Course of Phage N15 Lytic Replication

The prophage of temperate coliphage N15 is not integrated into the bacterial chromosome but exists as a linear plasmid molecule with covalently closed ends. Upon infection of an Escherichia coli cell, the phage DNA circularises via cohesive ends. A phage-encoded enzyme, protelomerase, then cuts at another site, telRL, and forms hairpin ends (telomeres) of linear plasmid prophage. Bidirectional replication of a linear plasmid produces a molecule with duplicated telomeres that is subsequently resolved into unit-length plasmids by protelomerase. Here, we analysed the structures of N15 DNA in the course of phage lytic development and found that, upon circularisation of infecting phage DNA, the circular molecule is not used to initiate lambda-like lytic replication but is converted into a linear plasmid. Replication of N15 DNA then follows the plasmid mode, and only at late steps did the circular unit-length molecules that could start lambda-like late replication and DNA encapsidation appear. Consistently, we found that protelomerase is required not only for plasmid replication but also for phage N15 lytic development. We found that conversion of linear plasmid with hairpin telomeres into a circular molecule with unresolved telomeres in the course of lytic development depends on N15 DNA replication, suggesting that the circular monomer is generated not directly from a linear plasmid in reverse "telomere fusion" reaction but results from resolution of a circular dimer intermediate of plasmid replication into two circular molecules with joined telRL telomeres. Mutation H415A in protelomerase results in accumulation of monomeric circular molecules in the course of replication of linear plasmid. The switch to circular plasmid formation in the course of lytic replication of N15 may result from depletion of protelomerase or modification of its activity by some phage-encoded factor at late steps of lytic growth.

Properties of HflX, an Enigmatic Protein from Escherichia coli

The Escherichia coli gene hflX was first identified as part of the hflA operon, mutations in which led to an increased frequency of lysogenization upon infection of the bacterium by the temperate coliphage lambda. Independent mutational studies have also indicated that the HflX protein has a role in transposition. Based on the sequence of its gene, HflX is predicted to be a GTP-binding protein, very likely a GTPase. We report here purification and characterization of the HflX protein. We also specifically examined its suggested functional roles mentioned above. Our results show that HflX is a monomeric protein with a high (30% to 40%) content of helices. It exhibits GTPase as well as ATPase activities, but it has no role in lambda lysogeny or in transposition.

Probing the Antiprotease Activity of λCIII, an Inhibitor of theEscherichia coliMetalloprotease HflB (FtsH)

The CIII protein encoded by the temperate coliphage lambda acts as an inhibitor of the ubiquitous Escherichia coli metalloprotease HflB (FtsH). This inhibition results in the stabilization of transcription factor lambdaCII, thereby helping the phage to lysogenize the host bacterium. LambdaCIII, a small (54-residue) protein of unknown structure, also protects sigma(32), another specific substrate of HflB. In order to understand the details of the inhibitory mechanism of CIII, we cloned and expressed the protein with an N-terminal six-histidine tag. We also synthesized and studied a 28-amino-acid peptide, CIIIC, encompassing the central 14 to 41 residues of CIII that exhibited antiproteolytic activity. Our studies show that CIII exists as a dimer under native conditions, aided by an intersubunit disulfide bond, which is dispensable for dimerization. Unlike CIII, CIIIC resists digestion by HflB. While CIII binds to HflB, it does not bind to CII. On the basis of these results, we discuss various mechanisms for the antiproteolytic activity of CIII.

Role of C-Terminal Residues in Oligomerization and Stability of λ CII: Implications for Lysis-Lysogeny Decision of the Phage

A crucial element in the lysis-lysogeny decision of the temperate coliphage lambda is the phage protein CII, which has several interesting properties. It promotes lysogeny through activation of three phage promoters p(E), p(I) and p(aQ), recognizing a direct repeat sequence TTGCN6TTGC at each. The three-dimensional structure of CII, a homo-tetramer of 97 residue subunits, is unknown. It is an unstable protein in vivo, being rapidly degraded by the host protease HflB (FtsH). This instability is essential for the function of CII in the lysis-lysogeny switch. From NMR and limited proteolysis we show that about 15 C-terminal residues of CII are highly flexible, and may act as a target for proteolysis in vivo. From in vitro transcription, isothermal calorimetry and gel chromatography of CII (1-97) and its truncated fragments CIIA (4-81/82) and CIIB (4-69), we find that residues 70-81/82 are essential for (a) tetramer formation, (b) operator binding and (c) transcription activation. Presumably, tetramerization is necessary for the latter functions. Based on these results, we propose a model for CII structure, in which protein-protein contacts for dimer and tetramer formation are different. The implications of tetrameric organization, essential for CII activity, on the recognition of the direct repeat sequence is discussed.

The genome and proteome of coliphage T1

The genome of enterobacterial phage T1 has been sequenced, revealing that its 50.7-kb terminally redundant, circularly permuted sequence contains 48,836 bp of nonredundant nucleotides. Seventy-seven open reading frames (ORFs) were identified, with a high percentage of small genes located at the termini of the genomes displaying no homology to existing phage or prophage proteins. Of the genes showing homologs (47%), we identified those involved in host DNA degradation (three endonucleases) and T1 replication (DNA helicase, primase, and single-stranded DNA-binding proteins) and recombination (RecE and Erf homologs). While the tail genes showed homology to those from temperate coliphage N15, the capsid biosynthetic genes were unique. Phage proteins were resolved by 2D gel electrophoresis, and mass spectrometry was used to identify several of the spots including the major head, portal, and tail proteins, thus verifying the annotation.

Partition operon expression in the linear plasmid prophage N15 is controlled by both Sop proteins and protelomerase.

The temperate coliphage N15, unlike most low copy-number prokaryotic replicons, is maintained as a linear DNA molecule with covalently closed ends. Accurate partitioning of the plasmid prophage is assured by a close homologue of the sop locus of the F plasmid. However, the region upstream of the N15 sopAB genes contains multiple putative promoters, in contrast to F sop whose expression is driven by one negatively autoregulated promoter. In addition, the centromere of N15 is represented by four inverted repeats located at widely separated sites within the region essential for replication and control of lytic functions. We have analysed expression of N15 sop genes. We find that transcription of N15 sop is driven by two major promoters. The first, P1, is similar in sequence and function to the F sop promoter; it is repressed by Sop proteins. The second promoter, P2, is upstream of P1 and is several times stronger. It is insensitive to regulation by Sop proteins but is tightly repressed by protelomerase, the N15 enzyme that completes prophage replication by generating hairpin telomeres. These results establish a regulatory link between the partition system and other processes of N15 maintenance.

Establishing lysogenic transcription in the temperate coliphage 186.

A single-copy chromosomal reporter system was used to measure the intrinsic strengths and interactions between the three promoters involved in the establishment of lysogeny by coliphage 186. The maintenance lysogenic promoter p(L) for the immunity repressor gene cI is intrinsically approximately 20-fold weaker than the lytic promoter p(R). These promoters are arranged face-to-face, and transcription from p(L) is further weakened some 14-fold by the activity of p(R). Efficient establishment of lysogeny requires the p(E) promoter, which lies upstream of p(L) and is activated by the phage CII protein to a level comparable to that of p(R). Transcription of p(E) is less sensitive to converging p(R) transcription and raises cI transcription at least 55-fold. The p(E) promoter does not occlude p(L) but inhibits lytic transcription by 50%. This interference is not due to bound CII preventing elongation of the lytic transcript. The p(E) RNA is antisense to the anti-immune repressor gene apl, but any role of this in the establishment of lysogeny appears to be minimal.

Use of a linear multicopy vector based on the mini-replicon of temperate coliphage N15 for cloning DNA with abnormal secondary structures

A new cloning vector pN15L is described. It is a linear 13.8 kb plasmid based on the coliphage N15 mini-replicon. The vector capacity exceeds 50 kb and the copy number is 250 per Escherichia coli chromosome. We show that some artificial and natural palindromes and approximately 5% of human DNA Bgl II fragments can be cloned effectively in linear vector pN15L, whereas they either sharply reduce the copy number of circular vector pUC19 or cannot be cloned at all. We conclude that pN15L may be usefully employed to clone large imperfect palindromes and some abnormal sequences of human DNA.

Use of a linear multicopy vector based on the mini-replicon of temperate coliphage N15 for cloning DNA with abnormal secondary structures.

A new cloning vector pN15L is described. It is a linear 13.8 kb plasmid based on the coliphage N15 mini-replicon. The vector capacity exceeds 50 kb and the copy number is 250 per Escherichia coli chromosome. We show that some artificial and natural palindromes and approximately 5% of human DNA Bgl II fragments can be cloned effectively in linear vector pN15L, whereas they either sharply reduce the copy number of circular vector pUC19 or cannot be cloned at all. We conclude that pN15L may be usefully employed to clone large imperfect palindromes and some abnormal sequences of human DNA.

The Late-Expressed Region of the Temperate Coliphage 186 Genome

The late-lytic region of the genome of bacteriophage 186 encodes the phage proteins that synthesize the complex viral particle and lyse the bacterial host. We report the completion of the DNA sequence of the late region and the assignment of 18 previously identified genes to open reading frames in the sequence. The 186 late region is similar to the late region of phage P2, sharing 26 genes of known function: the single gene for activation of late gene transcription, 6 genes for construction of DNA-containing heads, 16 for tail morphogenesis, and 3 for cell lysis. We identified two 186 late genes with unknown function; one is homologous to previously unrecognised genes in P2, HP1, and φCTX, and the other may modulate DNA packaging. The 186 late region, like the rest of the genome, lacks the lysogenic conversion genes that are carried by P2, allowing the 186 late region to be transcribed from only three late promoters rather than four. The relative absence of lysogenic conversion genes in 186 suggests that the two phages have evolved to use the lytic and lysogenic reproductive modes to different extents.

The dual role of Apl in prophage induction of coliphage 186.

In the present study we show that the Apl protein of the temperate coliphage 186 combines, in one protein, the activities of the coliphage lambda proteins Cro and Xis. We have shown previously that Apl represses both the lysogenic promoter, pL, and the major lytic promoter, pR, and is required for excision of the prophage. Apl binds at two locations on the phage chromosome, i.e. between pR and pL and at the phage-attachment site. Using an in vivo recombination assay, we now show that the role of Apl in excision is in the process itself and is not simply a consequence of repression of pR or pL. To study the repressive role of Apl at the switch promoters we isolated Apl-resistant operator mutants and used them to demonstrate a requirement for Apl in the efficient derepression of the lysogenic promoter during prophage induction. We conclude that Apl is both an excisionase and transcriptional repressor.

Phage HK022 Roi protein inhibits phage lytic growth in Escherichia coli integration host factor mutants.

Temperate coliphage HK022 requires integration host factor (IHF) for lytic growth. The determinant responsible for this requirement was identified as a new gene (roi) located between genes P and Q. This gene encodes a DNA-binding protein (Roi) containing a helix-turn-helix motif. We have shown that Roi binds a site within its own gene that is closely linked to an IHF binding site. By gel retardation experiments, we have found that IHF binding stabilizes the interaction of Roi with its gene. We have isolated three independent phage mutants that are able to grow on an IHF- host. They carry different mutations scattered in the roi gene and specifying single amino-acid changes. The interactions of all three Roi mutant proteins with the Roi binding site differed from that of the wild type. Roi displays strong similarities, in its C-terminal half, to two putative DNA-binding proteins of bacteriophage P1: Ant1 and KilA. The mode of action of the Roi protein and the possibility that IHF is modulating the expression and/or the action of Roi are discussed.

Tail Sheath and Tail Tube Genes of the Temperate Coliphage 186

The nucleotide sequence of the phage 186 genome from 46.3-52.5% was determined and was found to contain two open reading frames highly similar to the tail sheath gene FI and tail tube gene FII of phage P2. The reading frames were identified as genes J and I by marker rescue experiments. A late promoter p J was identified by galK reporter and primer extension studies. Northern analysis suggested that the p J transcript predominantly terminated immediately after gene I , although some transcripts could extend to the terminator t B at 67.3%.

Mechanisms of genome propagation and helper exploitation by satellite phage P4

Temperate coliphage P2 and satellite phage P4 have icosahedral capsids and contractile tails with side tail fibers. Because P4 requires all the capsid, tail, and lysis genes (late genes) of P2, the genomes of these phages are in constant communication during P4 development. The P4 genome (11,624 bp) and the P2 genome (33.8 kb) share homologous cos sites of 55 bp which are essential for generating 19-bp cohesive ends but are otherwise dissimilar. P4 turns on the expression of helper phage late genes by two mechanisms: derepression of P2 prophage and transactivation of P2 late-gene promoters. P4 also exploits the morphopoietic pathway of P2 by controlling the capsid size to fit its smaller genome. The P4 sid gene product is responsible for capsid size determination, and the P2 capsid gene product, gpN, is used to build both sizes. The P2 capsid contains 420 capsid protein subunits, and P4 contains 240 subunits. The size reduction appears to involve a major change of the whole hexamer complex. The P4 particles are less stable to heat inactivation, unless their capsids are coated with a P4-encoded decoration protein (the psu gene product). P4 uses a small RNA molecule as its immunity factor. Expression of P4 replication functions is prevented by premature transcription termination effected by this small RNA molecule, which contains a sequence that is complementary to a sequence in the transcript that it terminates.

Genes for the establishment and maintenance of lysogeny by the temperate coliphage 186

To identify the genes in coliphage 186 that are required for lysogeny, we isolated clear-plaque mutants. Complementation studies and DNA sequencing identified two genes, the cI gene for the immunity maintenance repressor and the cII gene, which is required only for the establishment of lysogeny. One mutant carried a change in the LexA-binding site controlling expression of the antirepression protein Tum.

Xis and Fis proteins prevent site-specific DNA inversion in lysogens of phage HK022

HK022, a temperate coliphage related to lambda, forms lysogens by inserting its DNA into the bacterial chromosome through site-specific recombination. The Escherichia coli Fis and phage Xis proteins promote excision of HK022 DNA from the bacterial chromosome. These two proteins also act during lysogenization to prevent a prophage rearrangement: lysogens formed in the absence of either Fis or Xis frequently carried a prophage that had suffered a site-specific internal DNA inversion. The inversion is a product of recombination between the phage attachment site and a secondary attachment site located within the HK022 left operon. In the absence of both Fis and Xis, the majority of lysogens carried a prophage with an inversion. Inversion occurs during lysogenization at about the same time as prophage insertion but is rare during lytic phage growth. Phages carrying the inverted segment are viable but have a defect in lysogenization, and we therefore suggest that prevention of this rearrangement is an important biological role of Xis and Fis for HK022. Although Fis and Xis are known to promote excision of lambda prophage, they had no detectable effect on lambda recombination at secondary attachment sites. HK022 cIts lysogens that were blocked in excisive recombination because of mutation in fis or xis typically produced high yields of phage after thermal induction, regardless of whether they carried an inverted prophage. The usual requirement for prophage excision was bypassed in these lysogens because they carried two or more prophages inserted in tandem at the bacterial attachment site; in such lysogens, viable phage particles can be formed by in situ packaging of unexcised chromosomes.