The Role of O-antigen in P1 Transduction of Shigella flexneri and Escherichia coli with its Alternative S' Tail Fibre.

Enterobacteria phage P1 expresses two types of tail fibre, S and S'. Despite the wide usage of phage P1 for transduction, the host range and the receptor for its alternative S' tail fibre was never determined. Here, a ΔS-cin Δpac E. coli P1 lysogenic strain was generated to allow packaging of phagemid DNA into P1 phage having either S or S' tail fibre. P1(S') could transduce phagemid DNA into Shigella flexneri 2a 2457O, Shigella flexneri 5a M90T and Escherichia coli O3 efficiently. Mutational analysis of the O-antigen assembly genes and LPS inhibition assays indicated that P1(S') transduction requires at least one O-antigen unit. E. coli O111:B4 LPS produced a high neutralising effect against P1(S') transduction, indicating that this E. coli strain could be susceptible to P1(S')-mediated transduction. Mutations in the O-antigen modification genes of S. flexneri 2a 2457O and S. flexneri 5a M90T did not cause significant changes to P1(S') transduction efficiency. A higher transduction efficiency of P1(S') improved the delivery of a cas9 antimicrobial phagemid into both S. flexneri 2457O and M90T. These findings provide novel insights into P1 tropism-switching, by identifying the bacterial strains which are susceptible to P1(S')-mediated transduction, as well as demonstrating its potential for delivering a DNA sequence-specific Cas9 antimicrobial into clinically relevant S. flexneri.

Bacteriophage P1 does not show spatial preference when infecting Escherichia coli.

To begin its infection, a bacteriophage first needs to adsorb to cells. The adsorption site on the cell surface may influence viral DNA injection, gene expression and cell-fate development. Here, we study the early steps of the infection cycle of coliphage P1, focusing on their correlation with spatial locations at the single-cell level. By fluorescently labeling P1 virions, we found that P1 shows no spatial preference on cell surface adsorption. In addition, live-cell phage DNA imaging revealed that adsorption sites do not affect the success rate for P1 in injecting its DNA into the cell. Furthermore, the lysis-lysogeny decision of P1 does not depend on the adsorption site, based on fluorescence reporters for the lytic and lysogenic pathways. These findings highlight the different infection strategies used by the two paradigmatic coliphages differ from those found in the paradigmatic phage lambda, highlighting that different infection strategies are used by phages.

The Legacy of Nat Sternberg: The Genesis of Cre-lox Technology.

Cre-lox of bacteriophage P1 has become one of the most widely used tools for genetic engineering in eukaryotes. The origins of this tool date to more than 30 years ago when Nat L. Sternberg discovered the recombinase, Cre, and its specific locus of crossover, lox, while studying the maintenance of bacteriophage P1 as a stable plasmid. Recombinations mediated by Cre assist in cyclization of the DNA of infecting phage and in resolution of prophage multimers created by generalized recombination. Early in vitro work demonstrated that, although it shares similarities with the well-characterized bacteriophage λ integration, Cre-lox is in many ways far simpler in its requirements for carrying out recombination. These features would prove critical for its development as a powerful and versatile tool in genetic engineering. We review the history of the discovery and characterization of Cre-lox and touch upon the present direction of Cre-lox research.

Bacteriophage P1 pac sites inserted into the chromosome greatly increase packaging and transduction of Escherichia coli genomic DNA.

The Escherichia coli bacteriophage P1 packages host chromosome separately from phage DNA, and transfers it to recipient cells at low frequency in a process called generalized transduction. Phage genomes are packaged from concatemers beginning at a specific site, pac. To increase transduction rate, we have inserted pac into the chromosome at up to five equally spaced positions; at least this many are fully tolerated in the absence of P1 infection. A single chromosomal pac greatly increases transduction of downstream markers without decreasing phage yields; 3.5 × as much total chromosomal DNA is packaged. Additional insertions decrease phage yield by > 90% and also decrease phage DNA synthesis, although less dramatically. Packaging of chromosomal markers near to and downstream of each inserted pac site is, at the same time, increased by greater than 10 fold. Transduction of markers near an inserted pac site can be increased by over 1000-fold, potentially allowing identification of such transductants by screening.

CRP represses the CRISPR/Cas system in Escherichia coli: evidence that endogenous CRISPR spacers impede phage P1 replication.

The CRISPR/Cas system is an important aspect in bacterial immunology. The anti-phage activity of the CRISPR system has been established using synthetic CRISPR spacers, but in vivo studies of endogenous CRISPR spacers are relatively scarce. Here, we showed that bacteriophage P1 titre in Escherichia coli decreased in the glucose-containing medium compared with that in the absence of glucose. This glucose effect of E. coli against phage P1 infection disappeared in cse3 deletion mutants. The effect on the susceptibility to phage P1 was associated with cAMP receptor protein (CRP)-mediated repression of cas genes transcription and crRNA maturation. Analysis of the regulatory element in the cse1 promoter region revealed a novel CRP binding site, which overlapped with a LeuO binding site. Furthermore, the limited sequence identity between endogenous spacers and the phage P1 genome was necessary and sufficient for CRISPR-mediated repression of phage P1 replication. Trans-expression of the third and seventh spacers in the CRISPR I region or third and sixth spacers in the CRISPR II region effectively reduced phage P1 titres in the CRISPR deletion mutants. These results demonstrate a novel regulatory mechanism for cas repression by CRP and provide evidence that endogenous spacers can repress phage P1 replication in E. coli.

Natural lysogenization and transduction in Salmonella enterica serovar Choleraesuis by bacteriophage P1.

It has been reported that bacteriophage P1 injects DNA into serovar Choleraesuis without evidence of productive infection. However, we found that P1 generates progeny and is capable of transduction in serovar Choleraesuis. This is not the case with other serovars of Salmonella enterica we tested. Therefore, P1 could play a role in serovar Choleraesuis evolution and contribute to its genetic manipulation and analysis.

Assembling new Escherichia coli strains by transduction using phage P1.

A protocol is described that allows the transfer of genetic material from one Escherichia coli strain to another using bacteriophage P1. P1 transduction can be used to construct new bacterial strains containing multiple alleles, to restore a locus to wild type, to move specific genetic markers from one strain to another, to relocate different mutant genes to a common genetic background, and to evaluate second-site suppression of a mutant allele. Because of these abilities, P1 transduction remains a staple in the arsenal of genetic tools that have kept E. coli at the forefront of model bacterial systems. The protocol incorporates some updated steps and discusses general principles of bacteriophage handling and the infection process.

Visualization of bacteriophage P1 infection by cryo-electron tomography of tiny Escherichia coli.

Bacteriophage P1 has a contractile tail that targets the conserved lipopolysaccharide on the outer membrane surface of the host for initial adsorption. The mechanism by which P1 DNA enters the host cell is not well understood, mainly because the transient molecular interactions between bacteriophage and bacteria have been difficult to study by conventional approaches. Here, we engineered tiny E. coli host cells so that the initial stages of P1-host interactions could be captured in unprecedented detail by cryo-electron tomography. Analysis of three-dimensional reconstructions of frozen-hydrated specimens revealed three predominant configurations: an extended tail stage with DNA present in the phage head, a contracted tail stage with DNA, and a contracted tail stage without DNA. Comparative analysis of various conformations indicated that there is uniform penetration of the inner tail tube into the E. coli periplasm and a significant movement of the baseplate away from the outer membrane during tail contraction.

Dissipation of proton motive force is not sufficient to induce the phage shock protein response in Escherichia coli.

Phage shock proteins (Psp) and their homologues are found in species from the three domains of life: Bacteria, Archaea and Eukarya (e.g. higher plants). In enterobacteria, the Psp response helps to maintain the proton motive force (PMF) of the cell when the inner membrane integrity is impaired. The presumed ability of ArcB to sense redox changes in the cellular quinone pool and the strong decrease of psp induction in ΔubiG or ΔarcAB backgrounds suggest a link between the Psp response and the quinone pool. The authors now provide evidence indicating that the physiological signal for inducing psp by secretin-induced stress is neither the quinone redox state nor a drop in PMF. Neither the loss of the H(+)-gradient nor the dissipation of the electrical potential alone is sufficient to induce the Psp response. A set of electron transport mutants differing in their redox states due to the lack of a NADH dehydrogenase and a quinol oxidase, but retaining a normal PMF displayed low levels of psp induction inversely related to oxidised ubiquinone levels under microaerobic growth and independent of PMF. In contrast, cells displaying higher secretin induced psp expression showed increased levels of ubiquinone. Taken together, this study suggests that not a single but likely multiple signals are needed to be integrated to induce the Psp response.

Identification of a peptide sequence that improves transport of macromolecules across the intestinal mucosal barrier targeting goblet cells.

In this study, we demonstrated that the CSKSSDYQC-peptide ligand which was identified from a random phage-peptide library through an in vivo phage display technique with rats could prominently improve the transport efficiency of macromolecules, such as large filamentous phage particles (M13 bacteriophage), across the intestinal mucosal barrier. Synthetic CSKSSDYQC-peptide ligands significantly inhibited the binding of phage P1 encoding CSKSSDYQC-peptide ligands to the intestinal mucosal tissue and immunohistochemical analysis showed that the CSKSSDYQC-peptide ligands could be transported across the intestinal mucosal barrier via goblet cells as their specific gateway. Thus, we inferred that CSKSSDYQC-peptide ligand might have a specific receptor on the goblet cells and transported from intestinal lumen to systemic circulation by transcytosis mechanism. These results suggest that CSKSSDYQC-ligand could be a promising tool for development of an efficient oral delivery system for macromolecular therapeutics in the carrier-drug conjugate strategy.

The bacteriophage P1 hot gene, encoding a homolog of the E. coli DNA polymerase III theta subunit, is expressed during both lysogenic and lytic growth stages.

The bacteriophage P1 hot gene product is a homolog of the theta subunit of E. coli DNA polymerase III. Previous studies with hot cloned on a plasmid have shown that Hot protein can substitute for theta, as evidenced by its stabilizing effect on certain dnaQ mutator mutants carrying an unstable pol III proofreading subunit (epsilon subunit). These results are consistent with Hot, like theta, being a replication protein involved in stabilizing the intrinsically unstable epsilon proofreading function. However, the function of hot for the viral life cycle is less clear. In the present study, we show that the hot gene is not essential. Based on its promoter structure, hot has been previously classified as a "late" phage gene, a property that is not easily reconciled with a presumed replication function. Here, we clarify this issue by demonstrating that P1 hot is actively expressed both during the lysogenic state and in the early stages of a lytic induction, in addition to its expression in the late stage of phage development. The results indicate that P1 hot has a complex expression pattern, compatible with a model in which Hot may affect the host replication machinery to benefit overall phage replication.

High-frequency phage-mediated gene transfer among Escherichia coli cells, determined at the single-cell level.

Recent whole-genome analysis suggests that lateral gene transfer by bacteriophages has contributed significantly to the genetic diversity of bacteria. To accurately determine the frequency of phage-mediated gene transfer, we employed cycling primed in situ amplification-fluorescent in situ hybridization (CPRINS-FISH) and investigated the movement of the ampicillin resistance gene among Escherichia coli cells mediated by phage at the single-cell level. Phages P1 and T4 and the newly isolated E. coli phage EC10 were used as vectors. The transduction frequencies determined by conventional plating were 3x10(-8) to 2x10(-6), 1x10(-8) to 4x10(-8), and <4x10(-9) to 4x10(-8) per PFU for phages P1, T4, and EC10, respectively. The frequencies of DNA transfer determined by CPRINS-FISH were 7x10(-4) to 1x10(-3), 9x10(-4) to 3x10(-3), and 5x10(-4) to 4x10(-3) for phages P1, T4, and EC10, respectively. Direct viable counting combined with CPRINS-FISH revealed that more than 20% of the cells carrying the transferred gene retained their viabilities. These results revealed that the difference in the number of viable cells carrying the transferred gene and the number of cells capable of growth on the selective medium was 3 to 4 orders of magnitude, indicating that phage-mediated exchange of DNA sequences among bacteria occurs with unexpectedly high frequency.

Escherichia coli mazEF-mediated cell death as a defense mechanism that inhibits the spread of phage P1.

The Escherichia coli gene pair mazEF is a regulatable chromosomal toxin-antitoxin module: mazF encodes a stable toxin and mazE encodes for a labile antitoxin that overcomes the lethal effect of MazF. Because MazE is labile, inhibition of mazE expression results in cell death. We studied the effect of mazEF on the development of bacteriophage P1 upon thermoinduction of the prophage P1CM c1ts and upon infection with virulent phage particles (P1vir). In several E. coli strains, we showed that the Delta mazEF derivative strains produced significantly more phages than did the parent strain. In addition, upon induction of K38(P1CM c1ts), nearly all of the Delta mazEF mutant cells lysed; in contrast, very few of the parental mazEF + K38 cells underwent lysis. However, most of these cells did not remain viable. Thus, while the Delta mazEF cells die as a result of the lytic action of the phage, most of the mazEF+ cells are killed by a different mechanism, apparently through the action of the chromosomal mazEF system itself. Furthermore, the introduction of lysogens into a growing non-lysogenic culture is lethal to Delta mazEF but not for mazEF+ cultures. Thus, although mazEF action causes individual cells to die, upon phage growth this is generally beneficial to the bacterial culture because it causes P1 phage exclusion from the bacterial population. These results provide additional support for the view that bacterial cultures may share some of the characteristics of multicellular organisms.

Plasmid partitioning and the spreading of P1 partition protein ParB.

Bacterial plasmids of low copy number, P1 prophage among them, are actively partitioned to nascent daughter cells. The process is typically mediated by a pair of plasmid-encoded proteins and a cis-acting DNA site or cluster of sites, referred to as the plasmid centromere. P1 ParB protein, which binds to the P1 centromere (parS), can spread for several kilobases along flanking DNA. We argue that studies of mutant ParB that demonstrated a strong correlation between spreading capacity and the ability to engage in partitioning may be misleading, and describe here a critical test of the dependence of partitioning on the spreading of the wild-type protein. Physical constraints imposed on the spreading of P1 ParB were found to have only a minor, but reproducible, effect on partitioning. We conclude that, whereas extensive ParB spreading is not required for partitioning, spreading may have an auxiliary role in the process.

Escherichia coli SspA is a transcription activator for bacteriophage P1 late genes.

The stringent starvation protein A (SspA), an Escherichia coli RNA polymerase (RNAP)-associated protein, has been reported to be essential for lytic growth of bacteriophage P1. Unlike P1 early promoters, P1 late promoters are not recognized by RNAP alone. A phage-encoded early protein, Lpa (late promoter activator protein, formerly called gp10), has been shown to be required for P1 late transcription in vivo. Here, we demonstrate that SspA is a transcription activator for P1 late genes. Our results indicated that Lpa is not limiting in an sspA mutant. However, the transcription of P1 late genes was deficient in an sspA mutant in vivo. We demonstrated that SspA/Lpa are required for transcription activation of the P1 late promoter Ps in vitro. In addition, SspA and Lpa were shown to facilitate the binding of RNAP to Ps late promoter DNA. Activation of late transcription by SspA/Lpa was dependent on holoenzyme containing sigma70 but not sigmaS, indicating that the two activators discriminate between the two forms of the holoenzyme. Furthermore, P1 early gene expression was downregulated in the wild-type background, whereas it persisted in the sspA mutant background, indicating that SspA/Lpa mediate the transcriptional switch from the early to the late genes during P1 lytic growth. Thus, this work provides the first evidence for a function of the E. coli RNAP-associated protein SspA.

The P1 plasmid in action: time-lapse photomicroscopy reveals some unexpected aspects of plasmid partition.

The prophage of bacteriophage P1 is a low copy number plasmid in Escherichia coli and is segregated to daughter cells by an active partition system. The dynamics of the partition process have now been successfully followed by time-lapse photomicroscopy. The process appears to be fundamentally different from that previously inferred from statistical analysis of fixed cells. A focus containing several plasmid copies is captured at the cell center. Immediately before cell division, the copies eject bi-directionally along the long axis of the cell. Cell division traps one or more plasmid copies in each daughter cell. These copies are free to move, associate, and disassociate. Later, they are captured to the new cell center to re-start the cycle. Studies with mutants suggest that the ability to segregate accurately at a very late stage in the cell cycle is dependent on a novel ability of the plasmid to control cell division. Should segregation be delayed, cell division is also delayed until segregation is successfully completed.

Overcoming the phage replication threshold: a mathematical model with implications for phage therapy.

Prior observations of phage-host systems in vitro have led to the conclusion that susceptible host cell populations must reach a critical density before phage replication can occur. Such a replication threshold density would have broad implications for the therapeutic use of phage. In this report, we demonstrate experimentally that no such replication threshold exists and explain the previous data used to support the existence of the threshold in terms of a classical model of the kinetics of colloidal particle interactions in solution. This result leads us to conclude that the frequently used measure of multiplicity of infection (MOI), computed as the ratio of the number of phage to the number of cells, is generally inappropriate for situations in which cell concentrations are less than 10(7)/ml. In its place, we propose an alternative measure, MOI(actual), that takes into account the cell concentration and adsorption time. Properties of this function are elucidated that explain the demonstrated usefulness of MOI at high cell densities, as well as some unexpected consequences at low concentrations. In addition, the concept of MOI(actual) allows us to write simple formulas for computing practical quantities, such as the number of phage sufficient to infect 99.99% of host cells at arbitrary concentrations.

Postsegregational killing mediated by the P1 phage "addiction module" phd-doc requires the Escherichia coli programmed cell death system mazEF.

"Addiction modules" consist of two genes; the product of the second is long lived and toxic, while the product of the first is short lived and antagonizes the lethal action of the toxin. The extrachromosomal addiction module phd-doc, located on the P1 prophage, is responsible for the postsegregational killing effect (death of plasmid-free cells). The Escherichia coli chromosomal addiction module analogue, mazEF, is responsible for the induction of programmed cell death. Here we show that the postsegregational killing mediated by the P1 phd-doc module depends on the presence of the E. coli mazEF system. In addition, we demonstrate that under conditions of postsegregational killing, mediated by phd-doc, protein synthesis of E. coli is inhibited. Based on our findings, we suggest the existence of a coupling between the phd-doc and mazEF systems.