Nearest-Neighbor Effects Modulate loxP Spacer DNA Chemical Shifts and Guide Oligonucleotide Design for Nuclear Magnetic Resonance Studies.

The Cre-loxP gene editing tool enables site-specific editing of DNA without leaving lesions that must be repaired by error-prone cellular processes. Cre recombines two 34-bp loxP DNA sites that feature a pair of palindromic recombinase-binding elements flanking an asymmetric 8-bp spacer region, via assembly of a tetrameric intasome complex and formation of a Holliday junction intermediate. Recombination proceeds by coordinated nucleophilic attack by pairs of catalytic tyrosine residues on specific phosphodiester bonds in the spacer regions of opposing strands. Despite not making base-specific contacts with the asymmetric spacer region of the DNA, Cre exhibits a preference for initial cleavage on one of the strands, suggesting that intrinsic properties of the uncontacted 8-bp spacer region give rise to this preference. Furthermore, little is known about the structural and dynamic features of the loxP spacer that make it a suitable target for Cre. To enable NMR spectroscopic studies of the spacer, we have aimed to identify a fragment of the 34-bp loxP site that retains the structural features of the spacer while minimizing the spectral crowding and line-broadening seen in longer oligonucleotides. Sequence-specific chemical shift differences between spacer oligos of different lengths, and of a mutant that inverts strand cleavage order, reveal how both nearest-neighbor and next-nearest-neighbor effects dominate the chemical environment experienced by the spacer. We have identified a 16-bp oligonucleotide that preserves the structural environment of the spacer, setting the stage for NMR-based structure determination and dynamics investigations.

Interactions between Viral Regulatory Proteins Ensure an MOI-Independent Probability of Lysogeny during Infection by Bacteriophage P1.

Phage P1 is a temperate phage which makes the lytic or lysogenic decision upon infecting bacteria. During the lytic cycle, progeny phages are produced and the cell lyses, and in the lysogenic cycle, P1 DNA exists as a low-copy-number plasmid and replicates autonomously. Previous studies at the bulk level showed that P1 lysogenization was independent of multiplicity of infection (MOI; the number of phages infecting a cell), whereas lysogenization probability of the paradigmatic phage λ increases with MOI. However, the mechanism underlying the P1 behavior is unclear. In this work, using a fluorescent reporter system, we demonstrated this P1 MOI-independent lysogenic response at the single-cell level. We further observed that the activity of the major repressor of lytic functions (C1) is a determining factor for the final cell fate. Specifically, the repression activity of P1, which arises from a combination of C1, the anti-repressor Coi, and the corepressor Lxc, remains constant for different MOI, which results in the MOI-independent lysogenic response. Additionally, by increasing the distance between phages that infect a single cell, we were able to engineer a λ-like, MOI-dependent lysogenization upon P1 infection. This suggests that the large separation of coinfecting phages attenuates the effective communication between them, allowing them to make decisions independently of each other. Our work establishes a highly quantitative framework to describe P1 lysogeny establishment. This system plays an important role in disseminating antibiotic resistance by P1-like plasmids and provides an alternative to the lifestyle of phage λ. IMPORTANCE Phage P1 has been shown potentially to play an important role in disseminating antibiotic resistance among bacteria during lysogenization, as evidenced by the prevalence of P1 phage-like elements in animal and human pathogens. In contrast to phage λ, a cell fate decision-making paradigm, P1 lysogenization was shown to be independent of MOI. In this work, we built a simple genetic model to elucidate this MOI independency based on the gene-regulatory circuitry of P1. We also proposed that the effective communication between coinfecting phages contributes to the lysis-lysogeny decision-making of P1 and highlighted the significance of spatial organization in the process of cell fate determination in a single-cell environment. Finally, our work provides new insights into different strategies acquired by viruses to interact with their bacterial hosts in different scenarios for their optimal survival.

Nonspecific DNA binding by P1 ParA determines the distribution of plasmid partition and repressor activities.

The faithful segregation, or "partition," of many low-copy number bacterial plasmids is driven by plasmid-encoded ATPases that are represented by the P1 plasmid ParA protein. ParA binds to the bacterial nucleoid via an ATP-dependent nonspecific DNA (nsDNA)-binding activity, which is essential for partition. ParA also has a site-specific DNA-binding activity to the par operator (parOP), which requires either ATP or ADP, and which is essential for it to act as a transcriptional repressor but is dispensable for partition. Here we examine how DNA binding by ParA contributes to the relative distribution of its plasmid partition and repressor activities, using a ParA with an alanine substitution at Arg351, a residue previously predicted to participate in site-specific DNA binding. In vivo, the parAR351A allele is compromised for partition, but its repressor activity is dramatically improved so that it behaves as a "super-repressor." In vitro, ParAR351A binds and hydrolyzes ATP, and undergoes a specific conformational change required for nsDNA binding, but its nsDNA-binding activity is significantly damaged. This defect in turn significantly reduces the assembly and stability of partition complexes formed by the interaction of ParA with ParB, the centromere-binding protein, and DNA. In contrast, the R351A change shows only a mild defect in site-specific DNA binding. We conclude that the partition defect is due to altered nsDNA binding kinetics and affinity for the bacterial chromosome. Furthermore, the super-repressor phenotype is explained by an increased pool of non-nucleoid bound ParA that is competent to bind parOP and repress transcription.

The intrinsically disordered domain of the antitoxin Phd chaperones the toxin Doc against irreversible inactivation and misfolding.

The toxin Doc from the phd/doc toxin-antitoxin module targets the cellular translation machinery and is inhibited by its antitoxin partner Phd. Here we show that Phd also functions as a chaperone, keeping Doc in an active, correctly folded conformation. In the absence of Phd, Doc exists in a relatively expanded state that is prone to dimerization through domain swapping with its active site loop acting as hinge region. The domain-swapped dimer is not capable of arresting protein synthesis in vitro, whereas the Doc monomer is. Upon binding to Phd, Doc becomes more compact and is secured in its monomeric state with a neutralized active site.

Purification and crystallization of Phd, the antitoxin of the phd/doc operon.

The antitoxin Phd from the phd/doc module of bacteriophage P1 was crystallized in two distinct crystal forms. Crystals of His-tagged Phd contain a C-terminally truncated version of the protein and diffract to 2.20 A resolution. Crystals of untagged Phd purified from the Phd-Doc complex diffract to 2.25 A resolution. These crystals are partially merohedrally twinned and contain the full-length version of the protein.

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

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

Bacterial addiction module toxin Doc inhibits translation elongation through its association with the 30S ribosomal subunit.

Bacterial toxin-antitoxin (TA) systems (or "addiction modules") typically facilitate cell survival during intervals of stress by inducing a state of reversible growth arrest. However, upon prolonged stress, TA toxin action leads to cell death. TA systems have also been implicated in several clinically important phenomena: biofilm formation, bacterial persistence during antibiotic treatment, and bacterial pathogenesis. TA systems harbored by pathogens also serve as attractive antibiotic targets. To date, the mechanism of action of the majority of known TA toxins has not yet been elucidated. We determined the mode of action of the Doc toxin of the Phd-Doc TA system. Doc expression resulted in rapid cell growth arrest and marked inhibition of translation without significant perturbation of transcription or replication. However, Doc did not cleave mRNA as do other addiction-module toxins whose activities result in translation inhibition. Instead, Doc induction mimicked the effects of treatment with the aminoglycoside antibiotic hygromycin B (HygB): Both Doc and HygB interacted with 30S ribosomal subunits, stabilized polysomes, and resulted in a significant increase in mRNA half-life. HygB also competed with ribosome-bound Doc, whereas HygB-resistant mutants suppressed Doc toxicity, suggesting that the Doc-binding site includes that of HygB (i.e., helix 44 region of 16S rRNA containing the A, P, and E sites). Overall, our results illuminate an intracellular target and mechanism of TA toxin action drawn from aminoglycoside antibiotics: Doc toxicity is the result of inhibition of translation elongation, possibly at the translocation step, through its interaction with the 30S ribosomal subunit.

Phage like it HOT: solution structure of the bacteriophage P1-encoded HOT protein, a homolog of the theta subunit of E. coli DNA polymerase III.

DNA polymerase III, the main replicative polymerase of E. coli, contains a small subunit, theta, that binds to the epsilon proofreading subunit and appears to enhance the enzyme's proofreading function--especially under extreme conditions. It was recently discovered that E. coli bacteriophage P1 encodes a theta homolog, named HOT. The (1)H-(15)N HSQC spectrum of HOT exhibits more uniform intensities and less evidence of conformational exchange than that of theta; this uniformity facilitates a determination of the HOT solution structure by NMR. The structure contains three alpha helices, as reported previously for theta; however, the folding topology of the two proteins is very different. Residual dipolar coupling measurements on labeled theta support the conclusion that it is structurally homologous with HOT. As judged by CD measurements, the melting temperature of HOT was 62 degrees C, compared to 56 degrees C for theta, consistent with other data suggesting greater thermal stability of the HOT protein.

Genome of bacteriophage P1.

P1 is a bacteriophage of Escherichia coli and other enteric bacteria. It lysogenizes its hosts as a circular, low-copy-number plasmid. We have determined the complete nucleotide sequences of two strains of a P1 thermoinducible mutant, P1 c1-100. The P1 genome (93,601 bp) contains at least 117 genes, of which almost two-thirds had not been sequenced previously and 49 have no homologs in other organisms. Protein-coding genes occupy 92% of the genome and are organized in 45 operons, of which four are decisive for the choice between lysis and lysogeny. Four others ensure plasmid maintenance. The majority of the remaining 37 operons are involved in lytic development. Seventeen operons are transcribed from sigma(70) promoters directly controlled by the master phage repressor C1. Late operons are transcribed from promoters recognized by the E. coli RNA polymerase holoenzyme in the presence of the Lpa protein, the product of a C1-controlled P1 gene. Three species of P1-encoded tRNAs provide differential controls of translation, and a P1-encoded DNA methyltransferase with putative bifunctionality influences transcription, replication, and DNA packaging. The genome is particularly rich in Chi recombinogenic sites. The base content and distribution in P1 DNA indicate that replication of P1 from its plasmid origin had more impact on the base compositional asymmetries of the P1 genome than replication from the lytic origin of replication.

Phylogenetic and functional analysis of the bacteriophage P1 single-stranded DNA-binding protein.

Bacteriophage P1 encodes a single-stranded DNA-binding protein (SSB-P1), which shows 66% amino acid sequence identity to the SSB protein of the host bacterium Escherichia coli. A phylogenetic analysis indicated that the P1 ssb gene coexists with its E. coli counterpart as an independent unit and does not represent a recent acquisition of the phage. The P1 and E. coli SSB proteins are fully functionally interchangeable. SSB-P1 is nonessential for phage growth in an exponentially growing E. coli host, and it is sufficient to promote bacterial growth in the absence of the E. coli SSB protein. Expression studies showed that the P1 ssb gene is transcribed only, in an rpoS-independent fashion, during stationary-phase growth in E. coli. Mixed infection experiments demonstrated that a wild-type phage has a selective advantage over an ssb-null mutant when exposed to a bacterial host in the stationary phase. These results reconciled the observed evolutionary conservation with the seemingly redundant presence of ssb genes in many bacteriophages and conjugative plasmids.

P1 ParB domain structure includes two independent multimerization domains.

ParB is one of two P1-encoded proteins that are required for active partition of the P1 prophage in Escherichia coli. To probe the native domain structure of ParB, we performed limited proteolytic digestions of full-length ParB, as well as of several N-terminal and C-terminal deletion fragments of ParB. The C-terminal 140 amino acids of ParB form a very trypsin-resistant domain. In contrast, the N terminus is more susceptible to proteolysis, suggesting that it forms a less stably folded domain or domains. Because native ParB is a dimer in solution, we analyzed the ability of ParB fragments to dimerize, using both the yeast two-hybrid system and in vitro chemical cross-linking of purified proteins. These studies revealed that the C-terminal 59 amino acids of ParB, a region within the protease-resistant domain, are sufficient for dimerization. Cross-linking and yeast two-hybrid experiments also revealed the presence of a second self-association domain within the N-terminal half of ParB. The cross-linking data also suggest that the C terminus is inhibitory to multimerization through the N-terminal domain in vitro. We propose that the two multimerization domains play distinct roles in partition complex formation.

Headful packaging revisited: the packaging of more than one DNA molecule into a bacteriophage P1 head.

Like a variety of other bacteriophages, such as T4 and P22, bacteriophage P1 packages DNA by a "headful" mechanism in which the capacity of the viral capsid determines the size of the single DNA molecule that is packaged. Because of the long-standing and general acceptance of this packaging mechanism, we were surprised to discover that some of our observations, using the in vitro P1 packaging system, could be explained by the packaging of less than headful-sized (< 110 kb) DNA molecules into a P1 capsid. To account for these observations, we describe results that support a model of in vitro P1 packaging in which multiple less than headful-sized DNA molecules are taken into a P1 head until that head has been filled. The results further suggest that the phage so generated can occasionally inject more than one DNA molecule into a cell upon viral infection. The data that supports these conclusions are: (1) the DNAs of the circular P1 cloning vectors pAd10sacBII (32 kb) and pNS358 (14 kb) are packaged in vitro with an efficiency of about 6 to 12% of that of longer concatemers of these DNAs. (2) The in vitro packaging of two differentially marked, less than 18 kb plasmid DNAs in the same reaction results in the production of a phage that can occasionally inject both DNAs into the same cell upon infection. (3) Virus particles generated by the packaging of either pAd10sacBII plasmid DNA or the two differently marked plasmids have a density in CsCl equilibrium gradients that is the same as P1 plaque-forming phage, suggesting that the former phage contain a headful of DNA. These results cannot be explained by Cre-mediated site-specific recombination between plasmids in the P1 packaging extracts. Finally, we present in vivo experiments that are also consistent with the headful packaging of multiple DNAs into a P1 head.

Purification of the C1 repressor of bacteriophage P1 by fast protein liquid chromatography.

A fast protein liquid chromatographic method is described for the purification of the C1 repressor of bacteriophage P1 and its truncated form C1*. By using one crude extract, both repressor proteins were purified in parallel to homogeneity and were shown to interact specifically with P1 operator DNA in vitro. The method involves an affinity chromatographic step on heparin-Sepharose, followed by a combination of ion-exchange chromatography on Q Sepharose and S Sepharose. The availability of a homogeneous preparation of the phage repressor is a prerequisite for studies on its structure-function relationship.