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Bacteriophage recombination systems have been widely used in biotechnology for modifying prokaryotic species, for creating transgenic animals and plants, and more recently, for human cell gene manipulation. In contrast to homologous recombination, which benefits from the endogenous recombination machinery of the cell, site-specific recombination requires an exogenous source of recombinase in mammalian cells. The mechanism of bacteriophage evolution and their coexistence with bacterial cells has become a point of interest ever since bacterial viruses’ life cycles were first explored. Phage recombinases have already been exploited as valuable genetic tools and new phage enzymes, and their potential application to genetic engineering and genome manipulation, vectorology, and generation of new transgene delivery vectors, and cell therapy are attractive areas of research that continue to be investigated. The significance and role of phage recombination systems in biotechnology is reviewed in this paper, with specific focus on homologous and site-specific recombination conferred by the coli phages, λ, and N15, the integrase from the Streptomyces phage, ΦC31, the recombination system of phage P1, and the recently characterized recombination functions of Yersinia phage, PY54. Key steps of the molecular mechanisms involving phage recombination functions and their application to molecular engineering, our novel exploitations of the PY54-derived recombination system, and its application to the development of new DNA vectors are discussed.

Bacteriophage P1 is among the best described bacterial viruses used in molecular biology. Here, we report that deficiency in the host cell DksA protein, an E. coli global transcription regulator, improves P1 lytic development. Using genetic and microbiological approaches, we investigated several aspects of P1vir biology in an attempt to understand the basis of this phenomenon. We found several minor improvements in phage development in the dksA mutant host, including more efficient adsorption to bacterial cell and phage DNA replication. In addition, gene expression of the main repressor of lysogeny C1, the late promoter activator Lpa, and lysozyme are downregulated in the dksA mutant. We also found nucleotide substitutions located in the phage immunity region immI, which may be responsible for permanent virulence of phage P1vir. We suggest that downregulation of C1 may lead to a less effective repression of lysogeny maintaining genes and that P1vir may be balancing between lysis and lysogeny, although finally it is able to enter the lytic pathway only. The mentioned improvements, such as more efficient replication and more “gentle” cell lysis, while considered minor individually, together may account for the phenomenon of a more efficient P1 phage development in a DksA-deficient host.

Phage P1 was isolated from the abnormal fermented liquid using Lactobacillus plantarum (L. plantarum) IMAU10120. To date, genetic knowledge regarding L. plantarum phage diversity is still limited, and further in-depth sequencing analysis of isolated L. plantarum phages can fill this gap. Here, we investigated the whole genome sequence of L. plantarum phage P1, sequenced by Illumina HiSeq platform, to decipher its genomic characteristics and putative DNA packaging mechanism. It was revealed that phage P1 was 73,787 bp in length, which was composed of linear double-stranded DNA (dsDNA), and the GC content was 39.17%. Its genome contained 86 coding sequences for various functions, such as adsorption, injection, replication, assembly, and release. Moreover, it was observed that L. plantarum phage P1 utilized the ‘cohesive ends’ DNA packaging mechanism. This study furthered the genomic knowledge of L. plantarum phages and provided some basis for the control of L. plantarum phages in the dairy fermentation industry.

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.

Lateral gene transfer by phages has contributed significantly to the genetic diversity of bacteria. To accurately determine the frequency and range of phage-mediated gene transfer, it is important to understand the movement of DNA among microbes. Using an in situ DNA amplification technique (cycling primed in situ amplification-fluorescent in situ hybridization; CPRINS-FISH), we examined the propensity for phage-mediated gene transfer in freshwater environments at the single-cell level. Phage P1, T4 and isolated Escherichia coli phage EC10 were used as vectors. All E. coli phages mediated gene transfer from E. coli to both plaque-forming and non-plaque-forming Enterobacteriaceae strains at frequencies of 0.3–8 × 10 −3 per plaque-forming unit (PFU), whereas culture methods using selective agar media could not detect transductants in non-plaque-forming strains. The DNA transfer frequencies through phage EC10 ranged from undetectable to 9 × 10 −2 per PFU (undetectable to 2 × 10 −3 per total direct count) when natural bacterial communities were recipients. Direct viable counting combined with CPRINS-FISH revealed that more than 20% of the cells carrying the transferred gene retained their viability in most cases. These results indicate that the exchange of DNA sequences among bacteria occurs frequently and in a wide range of bacteria, and may promote rapid evolution of the prokaryotic genome in freshwater environments.

Key Points Temporal and spatial control of gene expression in the mouse can be achieved using binary transgenic systems, in which gene expression is controlled by the interaction of an effector protein product on a target transgene. These interactions are controlled by crossing mouse lines, or by adding or removing an exogenous inducer. Binary transgenic systems fall into two categories. One is based on transcriptional transactivation and is well suited for activating transgenes in gain-of-function experiments. The other is based on site-specific DNA recombination and can be used to activate transgenes or to generate tissue-specific gene knockouts and cell-lineage markers. The most commonly used transcriptional systems are based on the tetracycline resistance operon of Escherichia coli . The effectors of these systems fall into two categories defined by whether transcription activation occurs upon the administration or depletion of a tetracycline compound (usually doxycycline). The Gal4-based system is a transactivation system that does not require an inducer, but Gal4 transcriptional activation can be controlled by synthetic steroids when a mutated ligand-binding domain is incorporated into a Gal4 chimeric transactivator. The most widely used site-specific DNA recombination system uses the Cre recombinase from bacteriophage P1. The Flp recombinase from Saccharomyces cerevisiae has also been adapted for use in mice. By using gene-targeting techniques to produce mice with modified endogenous genes that can be acted on by Cre or Flp recombinases expressed under the control of tissue-specific promoters, site-specific recombination can be used to inactivate endogenous genes in a spatially controlled manner. Cre/Flp activity can also be controlled temporally by delivering cre / FLP -encoding transgenes in viral vectors, by administering exogenous steroids to mice that carry a chimeric transgene consisting of the cre gene fused to a mutated ligand-binding domain, or by using transcriptional transactivation to control cre / FLP expression. The irreversibility of site-specific recombination makes this technique uniquely suited for a new type of analysis in which the transient tissue-specific expression of cre / FLP is used to permanently activate a reporter target gene for cell-lineage studies. One of the most powerful tools that the molecular biology revolution has given us is the ability to turn genes on and off at our discretion. In the mouse, this has been accomplished by using binary systems in which gene expression is dependent on the interaction of two components, resulting in either transcriptional transactivation or DNA recombination. During recent years, these systems have been used to analyse complex and multi-staged biological processes, such as embryogenesis and cancer, with unprecedented precision. Here, I review these systems and discuss certain studies that exemplify the advantages and limitations of each system.

Ref is an HNH superfamily endonuclease that only cleaves DNA to which RecA protein is bound. The enigmatic physiological function of this unusual enzyme is defined here. Lysogenization by bacteriophage P1 renders E. coli more sensitive to the DNA-damaging antibiotic ciprofloxacin, an example of a phenomenon termed phage-antibiotic synergy (PAS). The complementary effect of phage P1 is uniquely traced to the P1-encoded gene ref. Ref is a P1 function that amplifies the lytic cycle under conditions when the bacterial SOS response is induced due to DNA damage. The effect of Ref is multifaceted. DNA binding by Ref interferes with normal DNA metabolism, and the nuclease activity of Ref enhances genome degradation. Ref also inhibits cell division independently of the SOS response. Ref gene expression is toxic to E. coli in the absence of other P1 functions, both alone and in combination with antibiotics. The RecA proteins of human pathogens Neisseria gonorrhoeae and Staphylococcus aureus serve as cofactors for Ref-mediated DNA cleavage. Ref is especially toxic during the bacterial SOS response and the limited growth of stationary phase cultures, targeting aspects of bacterial physiology that are closely associated with the development of bacterial pathogen persistence.

The Lyz endolysin of bacteriophage P1 was found to cause lysis of the host without a holin. Induction of a plasmid-cloned lyz resulted in lysis, and the lytic event could be triggered prematurely by treatments that dissipate the proton-motive force. Instead of requiring a holin, export was mediated by an N-terminal transmembrane domain (TMD) and required host sec function. Exported Lyz of identical SDS/PAGE mobility was found in both the membrane and periplasmic compartments, indicating that periplasmic Lyz was not generated by the proteolytic cleavage of the membrane-associated form. In gene fusion experiments, the Lyz TMD directed PhoA to both the membrane and periplasmic compartments, whereas the TMD of the integral membrane protein Ftsl restricts Lyz to the membrane. Thus, the N-terminal domain of Lyz is both necessary and sufficient not only for export of this endo-lysin to the membrane but also for its release into the periplasm. The unusual N-terminal domain, rich in residues that are weakly hydrophobic, thus functions as a signal-arrest-release sequence, which first acts as a normal signal-arrest domain to direct the endolysin to the periplasm in membrane-tethered form and then allows it to be released as a soluble active enzyme in the periplasm. Examination of the protein sequences of related bacteriophage endolysins suggests that the presence of an N-terminal signal-arrest-release sequence is not unique to Lyz. These observations are discussed in relation to the role of holins in the control of host lysis by bacteriophage encoding a secretory endolysin.

Cre recombinase from bacteriophage P1 recognizes a 34-bp recombination site, loxP, with exquisite sequence specificity and catalyzes the site-specific insertion, excision, or rearrangement of DNA. To better understand the molecular basis of protein-DNA recognition and generate recombinases with altered specificities, we have developed a directed evolution strategy that can be used to identify recombinases that recognize variant loxP sites. To be selected, members of a library of Cre variants produced by targeted random mutagenesis must rapidly catalyze recombination, in vivo, between two variant loxP sites that are located on a reporter plasmid. Recombination results in an altered pattern of fluorescent protein expression that can be identified by flow cytometry. Fluorescence-activated cell sorting can be used either to screen positively for recombinase variants that recognize a novel loxP site, or negatively for variants that cannot recognize the wild-type loxP site. The use of positive screening alone resulted in a relaxation of recombination site specificity, whereas a combination of positive and negative screening resulted in a switching of specificity. One of the identified recombinases selectively recombines a novel recombination site and operates at a rate identical to that of wild-type Cre. Analysis of the sequences of the resulting Cre variants provides insight into the evolution of these altered specificities. This and other systems should contribute to our understanding of protein-DNA recognition and may eventually be used to evolve custom-tailored recombinases that can be used for gene study and inactivation.

Background Chromosome engineering encompasses a collection of homologous recombination-based techniques that are employed to modify the genome of a model organism in a controlled fashion. Such techniques are widely used in both fundamental and industrial research to introduce multiple insertions in the same Escherichia coli strain. To date, λ-Red recombination (also known as recombineering) and P1 phage transduction are the most successfully implemented chromosome engineering techniques in E. coli . However, due to errors that can occur during the strain creation process, reliable validation methods are essential upon alteration of a strain’s chromosome. Results and discussion Polymerase chain reaction (PCR)-based methods and DNA sequence analysis are rapid and powerful methods to verify successful integration of DNA sequences into a chromosome. Even though these verification methods are necessary, they may not be sufficient in detecting all errors, imposing the requirement of additional validation methods. For example, as extraneous insertions may occur during recombineering, we highlight the use of Southern blotting to detect their presence. These unwanted mutations can be removed via transducing the region of interest into the wild type chromosome using P1 phages. However, in doing so one must verify that both the P1 lysate and the strains utilized are free from contamination with temperate phages, as these can lysogenize inside a cell as a large plasmid. Thus, we illustrate various methods to probe for temperate phage contamination, including cross-streak agar and Evans Blue-Uranine (EBU) plate assays, whereby the latter is a newly reported technique for this purpose in E. coli . Lastly, we discuss methodologies for detecting defects in cell growth and shape characteristics, which should be employed as an additional check. Conclusion The simple, yet crucial validation techniques discussed here can be used to reliably verify any chromosomally engineered E. coli strains for errors such as non-specific insertions in the chromosome, temperate phage contamination, and defects in growth and cell shape. While techniques such as PCR and DNA sequence verification should standardly be performed, we illustrate the necessity of performing these additional assays. The discussed techniques are highly generic and can be easily applied to any type of chromosome engineering.