Department of Microbiology and Immunology, Medical University of South Carolina, 173 Ashley Avenue BSB-201, Charleston, SC 29403, USA1
Author for correspondence: Caroline Westwater. Tel: +1 843 792 7703. Fax: +1 843 792 2464. e-mail: westwatc{at}musc.edu
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ABSTRACT |
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Keywords: delivery system, bacteriophage, gene transfer, transduction
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INTRODUCTION |
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Recombinant DNA manipulations in bacteria typically involve initial cloning and molecular analyses in E. coli, followed by reintroduction of the cloned DNA into the original host genetic background for studies of gene expression and reverse genetics. Some species are recalcitrant to standard transformation techniques. Therefore, genetic analysis of these species is largely impaired. Most bacterial species possess restriction/modification systems that have evolved to protect the cell from foreign DNA (Bickle & Krüger, 1993 ). Modification of DNA can differ between species and among strains of the same species, creating barriers to gene transfer. To facilitate the movement of DNA some transformation protocols are limited to specific strains that are defective in one or more restriction systems (Novick, 1990
; Takagi & Kisumi, 1985
). Non-specific barriers such as high intra- or extracellular nuclease activity can also have profound effects on transformation efficiency (Omenn & Friedman, 1970
; Shireen et al., 1990
; Wu et al., 2001
). Genetic exchange between mutated laboratory strains and clinical or environmental isolates can be hampered by the lack of alternative methods for the delivery of genes.
The ability to electroporate protoplasts, spheroplasts and intact cells has advanced microbiological studies in organisms where other transformation procedures have failed (Chassy et al., 1988 ). However, the generation of cells lacking cell walls can be labour-intensive, time-consuming and difficult to reproduce. In addition, these methods normally require optimization of numerous strain-dependent parameters for efficient transformation and regeneration. Transformation efficiencies of intact cells can be highly variable depending on the growth media, growth phase and final concentration of cells, composition of the electroporation medium, electric parameters and conditions used to select for transformants.
The bacteriophage P1 has been widely used in the construction of recombinant bacteria because of its ability to transduce chromosomal markers as well as episomes such as F and R plasmids (Arber, 1960 ; Lennox, 1955
). Besides E. coli B, C and K, P1 can adsorb to and inject its DNA into 25 Gram-negative species (Yarmolinsky & Sternberg, 1988
). Such a lifestyle has resulted in the evolution of an antirestriction function. P1 DNA is only weakly restricted when it infects a cell carrying type I restriction and modification systems even though DNA purified from P1 phage particles is a good substrate for type I restriction enzymes in vitro (Iida et al., 1987
). This protection has been attributed to the presence of the defence against restriction proteins, DarA and DarB, within the phage head. The Dar proteins protect any DNA, including transduced DNA, from restriction (Iida et al., 1987
).
In this communication, we describe the construction of a phagemid vector, P1pBHR-T, which can be used for cloning in E. coli or several Gram-negative hosts. We also describe the development of a P1 phage delivery system that will have great use for the movement of P1pBHR-T between a variety of clinically relevant Gram-negative species.
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METHODS |
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Phagemid delivery and analysis.
An overnight culture of the host strain was diluted in LB and grown to mid-exponential phase (OD600 0·4). The cells were centrifuged at 2500 g for 10 min at 4 °C and concentrated to OD600 2·0 (108 c.f.u. ml-1) with LC medium. Phage (100 µl) was added at various multiplicity of infection (m.o.i.) and allowed to adsorb to the cells (100 µl) for 15 min at 32 °C. LC medium containing 10 mM sodium citrate was added (800 µl), and cells were incubated at 32 °C for 45 min or 90 min to allow expression of antibiotic-resistance genes (kanamycin and carbenicillin, respectively). The infected cells were centrifuged at 7000 g for 5 min and resuspended in 100 µl LC medium containing 10 mM sodium citrate. Transductants were detected by spotting 7·5 µl samples of a 10-fold serial dilution of the mixture onto LB agar plates containing appropriate antibiotic selection. Plates were scored following overnight incubation at 32 °C. No transductants were observed when 107 viable bacteria were assayed on selective media in the absence of phage lysate. P1pBHR-T was recovered from transduced cells by the alkaline lysis method (QIAprep miniprep kit, Qiagen).
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RESULTS |
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The elements necessary for packaging into P1 phage capsids were inserted into pBBR122. These elements included the P1 lytic replicon and minimal pac site. The lytic replicon consists of the C1 repressor-controlled P53 promoter, the promoter P53 antisense and the kilA and repL genes. The KilA protein is not essential for replicon function but is lethal to the bacterial cell. The kilA gene was therefore inactivated by an in-frame deletion, resulting in a protein 52% of the original size. During the late stages of the phage life cycle the lytic replicon initiates a rolling-circle mode of replication that generates concatemeric DNA, which is the substrate for packaging. Packaging is initiated when phage-encoded proteins recognize and cleave the unique pac site. The DNA is then brought into the empty P1 phage head, and packaging proceeds unidirectionally until the head is full. Since the P1 phage head can package approximately 110115 kb of DNA (Sternberg, 1990 ), fragments as large as 100 kb could potentially be cloned and delivered by this system. The ability to clone high-molecular-mass DNA fragments is important in the analysis of large genes or gene clusters that encode components of biochemical or signalling pathways.
Production of phagemid-containing virions
The phagemid was maintained in a P1 lysogen which provided all the replication factors needed to activate the lytic cycle and all the structural components to form mature viral particles. The P1 prophage carried a chloramphenicol-resistance marker and the c1.100 temperature-sensitive repressor mutation. This mutation enabled the P1 lytic cycle to be rapidly induced when the temperature of the exponential-phase lysogenic culture was shifted from 32 °C to 42 °C. Lysates typically contained approximately 80% P1 and 20% phagemid particles as determined by infecting E. coli C600 and C600(P1), respectively (data not shown).
Delivery to multiple strains of E. coli
The ability to deliver the phagemid to multiple strains of bacteria was tested first with laboratory strains and clinical isolates of E. coli. Since the host recombination system may affect concatemer resolution and plasmid rearrangement, RecA+ (C600 and JM101) and RecA- (DH5 and JM109) strains were included. Increasing titres of phage were added to fixed numbers of bacterial cells and limited to a single round of infection by the addition of 10 mM sodium citrate. After infection, phagemid-containing transductants were selected by virtue of their ability to grow in the presence of antibiotics. As shown in Fig. 2(a)
, the total number of transductants increased progressively as the m.o.i. increased. Antibiotic-resistant colonies were not recovered if the phage lysate or cells were tested alone. Since the P1 prophage carried the c1.100 mutation, cells infected with this phage were rendered temperature sensitive. Therefore, to minimize induction of the P1 lytic cycle and maximize the number of transductants recovered all procedures were performed at 32 °C.
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Successful delivery of P1pBHR-T was confirmed by extraction of this plasmid from representative isolates. Antibiotic-resistant transductants harboured plasmid DNA whose migration was identical to that originally seen in the parent strain (Fig. 2b). Restriction enzyme digestion demonstrated that gross deletions or genetic rearrangements in P1pBHR-T did not occur as a consequence of packaging or recircularization. Acquisition of P1pBHR-T did not result in displacement (incompatibility) of native plasmids in clinical isolates (data not shown).
The delivery vehicle can transduce many Gram-negative species
To demonstrate the utility of the delivery system, transduction of the phagemid was tested in various Gram-negative bacteria including Pseudomonas aeruginosa, Klebsiella pneumoniae, Citrobacter freundii, Shigella flexneri and Shigella dysenteriae. All the bacteria tested were successfully transduced by the P1 delivery system (Figs 3a and 4a
). The P. aeruginosa clinical isolate PA-1 was transduced at a lower efficiency than the laboratory strain PAO1 (Fig. 3a
). It is noteworthy that a similar effect has been reported for electroporation of P. aeruginosa isolates from lung sputum of cystic fibrosis patients and wild-type strains isolated from different sources for other Gram-negative species (Diver et al., 1990
; Wirth et al., 1989
). Functionality of the pBBR122 origin of replication among the Gram-negative species was confirmed by extraction and analysis of P1pBHR-T from representative transductants (Figs 3b
, 4b
and 4c
).
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DISCUSSION |
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Since the various Gram-negative bacteria accepted DNA packaged from another bacterial genus (E. coli), this suggested protection of the DNA by the P1 Dar proteins. Both the DarA and DarB proteins were provided by the P1 prophage and are thought to be incorporated into the phagemid-carrying P1 capsids during lysate production. These internal head proteins have been shown to bind phage DNA and are injected along with the infecting DNA into the recipient cell. Moreover, DNA binding is not specific for the P1 phage genome because any DNA packaged into a P1 phage head has been shown to be protected against restriction (Iida et al., 1987 ). Alternatively, the species tested may simply not possess an effective restriction/modification system or the transduced phagemid DNA may lack a restriction endonuclease recognition sequence recognized by these systems. The results obtained suggest that the delivery system may be applicable to the transduction of many different Gram-negative bacteria. There are additional bacterial species to which it should be possible to extend this P1 delivery system. The host range of P1 includes, for example, Yersina pestis, Yersina pseudotuberculosis and Salmonella typhimurium (Lawton & Molnar, 1972
; Murooka & Harada, 1979
; Okada & Watanabe, 1968
).
This procedure could be used for many genetic approaches, including the construction of strains, heterologous expression of genes and proteins, and analysis of endogenous gene expression. One important advantage of a phage delivery system is that, in contrast to transformation, phage infection normally occurs at high frequency in hosts competent for that phage. Low transformation efficiency of many bacteria has prevented the introduction of a gene library into these bacteria for direct complementation. In addition to using this procedure for the generation of recombinant bacteria, it should also be possible to construct genomic libraries in the phagemid vector. After obtaining transformants in E. coli the library could be pooled and infected en masse with P1 phage, generating an entire packaged library. This could be used to transduce any P1-sensitive host in vitro and in vivo. Bacterial transduction has been reported in environmental settings such as marine and freshwater aquatic habitats and in soil (Miller, 1998 ; Zeph et al., 1988
). In this regard, the P1 delivery system could be helpful in addressing questions concerning the fate of genetically engineered vectors released into these environments and the transfer by transduction of DNA to indigenous organisms.
One of the challenges of current molecular therapy is how to deliver the therapeutic agent to the offending bacterium. Because of their importance as pathogens, effort was directed towards the development of delivery vectors and improvements of methods to introduce recombinant DNA molecules into Gram-negative bacteria that are not naturally competent. Clinically important micro-organisms that are rapidly developing resistance to available antimicrobials include Gram-negative bacteria that cause urinary tract infections (Gupta et al., 1999 ), foodborne infections (Glynn et al., 1998
), bloodstream infections (Pittet & Wenzel, 1995
) and infections transmitted in health-care settings (Richard et al., 1994
; Wiener et al., 1999
). Besides being a valuable tool for delivering DNA in vitro this technology provides the opportunity for targeting bacterial cells in vivo. This system could be used as a delivery vehicle for oral vaccines if the natural enteric flora of the gastrointestinal tract was targeted. In this approach P1 phage would deliver phagemids engineered to express pathogen-specific immunogenic epitopes on the surface of the bacteria (Zuercher et al., 2000
). Alternatively, phage-delivered vectors could direct oral bacteria to secrete salivary histatin or other antimicrobial peptides (Hancock & Chapple, 1999
). This approach may be useful in the management of mucosal candidiasis and development of antimicrobial therapies.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 7 August 2001;
revised 16 November 2001;
accepted 21 November 2001.
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