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Annu. Rev. Genet. 2003. 37:3–29 doi: 10.1146/annurev.genet.37.110801.142807 c 2003 by Annual Reviews. All rights reserved Copyright ° First published online as a Review in Advance on September 8, 2003
TRANSPOSON-BASED STRATEGIES FOR MICROBIAL FUNCTIONAL GENOMICS AND PROTEOMICS
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Finbarr Hayes Department of Biomolecular Sciences, University of Manchester Institute of Science and Technology, PO Box 88, Manchester M60 1QD, England; email:
[email protected]
Key Words transposon, mutagenesis, bacteria, pentapeptide scanning, signature-tagged mutagenesis ■ Abstract Transposons are mobile genetic elements that can relocate from one genomic location to another. As well as modulating gene expression and contributing to genome plasticity and evolution, transposons are remarkably diverse molecular tools for both whole-genome and single-gene studies in bacteria, yeast, and other microorganisms. Efficient but simple in vitro transposition reactions now allow the mutational analysis of previously recalcitrant microorganisms. Transposon-based signaturetagged mutagenesis and genetic footprinting strategies have pinpointed essential genes and genes that are crucial for the infectivity of a variety of human and other pathogens. Individual proteins and protein complexes can be dissected by transposon-mediated scanning linker mutagenesis. These and other transposon-based approaches have reaffirmed the usefulness of these elements as simple yet highly effective mutagens for both functional genomic and proteomic studies of microorganisms.
CONTENTS TRANSPOSONS: A BRIEF PERSPECTIVE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . TRANSPOSON ORGANIZATION AND MOBILITY . . . . . . . . . . . . . . . . . . . . . . . . MAKING TRANSPOSONS JUMP IN VITRO . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . GENE SEQUENCING WITH TRANSPOSONS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . TRANSPOSONS FOR GENE FUSIONS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . SIGNATURE-TAGGED MUTAGENESIS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . GENETIC FOOTPRINTING WITH TRANSPOSONS . . . . . . . . . . . . . . . . . . . . . . . . TRANSPOSONS AND MICROARRAY TECHNOLOGY . . . . . . . . . . . . . . . . . . . . . SCANNING LINKER MUTAGENESIS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . TRANSPOSONS AS GENETIC TOOLS IN HIGHER EUKARYOTES . . . . . . . . . . CONCLUDING REMARKS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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TRANSPOSONS: A BRIEF PERSPECTIVE Transposable elements are discrete segments of DNA that can relocate between genomic sites. The movement of transposable elements is highly regulated and can profoundly influence gene expression, as Barbara McClintock observed in the 1940s and 1950s during her discovery of transposition in maize (27). Transposable elements are ubiquitous; they are present in Eubacteria, Archaea, and Eukarya, including in humans in whom they constitute a significant fraction of the genome. In the 1960s and 1970s, transposable elements were isolated in bacteria whose amenability to genetic manipulation facilitated both detailed molecular studies of the transposition process as well as the development of transposons as molecular tools. In bacteria, transposons were widely employed as random insertion mutagens both at a genomic level and in the analysis of the organization of individual genes. With the advent of high-throughput molecular biology approaches such as rapid nucleotide sequencing and site-directed mutagenesis, transposons fell somewhat out of favor as microbial genetic tools. However, in recent years the value of transposons as malleable and powerful genetic instruments has once again been recognized. In particular, the current expansion in nucleotide sequence data, e.g., ∼2.8 × 1010 bases available in the GenBank database at the time of writing, including many tens of microbial whole-genome sequences, requires experimental as well as bioinformatic strategies for its interpretation. This review focuses on existing and emerging transposon-based methods that can be applied either on a genome-wide scale or at the level of single genes and proteins.
TRANSPOSON ORGANIZATION AND MOBILITY Transposable elements in bacteria range from simple insertion sequence (IS) elements that consist of a gene(s) for transposition bounded by inverted repeat sequences, to composite transposons composed of a pair of IS elements that bracket additional genetic information for antibiotic resistance or other properties, to more complex conjugative transposons that exhibit hybrid properties of transposons, plasmids, and bacteriophages (Figure 1). Other mobile genetic elements, including plasmids, mutator bacteriophage, integrons, mobile introns, shufflons, and pathogenicity islands, also contribute to the genetic plasticity and evolution of bacteria and other microorganisms. As it is beyond the scope of this review to provide a comprehensive description of the diverse genetic organizations and transposition strategies of microbial transposable elements, the reader is referred to recent perceptive discussions of transposon architecture and movement (30, 109), IS classification and distribution (99), selected paradigms of transposition (53, 81, 118, 124), and less well-characterized transposable elements (14, 83, 129). Transposable tools for microbial genomics and proteomics to date have been based principally on a few, well-characterized elements from Gram-negative bacteria that also serve as useful models with which to highlight some salient features
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Figure 1 Genetic organization of well-characterized transposons that have been used extensively as genetic tools. The insertion sequence IS1 is included for comparison. Genes that are involved in the transposition of the elements are indicated by filled arrows. Triangles denote repeat sequences. The hatched box in Tn3 shows the position of the recombination site used during cointegrate resolution. The locations of the insertion sequences IS10 and IS50 in the composite transposons Tn10 and Tn5, respectively, are shown.
of transposon behavior. The transposons most favored as genetic tools are those that insert randomly or near-randomly, or can be manipulated to behave in this way, as is the case with the transposons discussed briefly here. Tn5 is a composite transposon that consists of two inversely oriented copies of the IS50 element that are separated by genes specifying resistance to kanamycin, bleomycin, and streptomycin (Figure 1). The IS50 elements are nonidentical and only one of the elements, IS50R, encodes proteins that are active for transposition; IS50L produces shortened, inactive versions of these proteins (124). Overlapping genes on IS50R produce a transposase (Tnp) that is responsible, along with regulatory host
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factors, for the movement of Tn5 between genomic locations, and a truncated version of Tnp that posttranslationally downregulates Tnp activity. Each IS50 element harbors 19-bp repeat sequences at its termini with which Tnp interacts. Tn5 transposes by a cut-and-paste mechanism in which the element is excised from its resident location and inserted at a new position (Figure 2A). Transposition proceeds by a sequence of steps, of which the first involves the assembly of the ends of the element into a synaptic complex mediated by Tnp. DNA cleavage at the ends of the transposon releases the element from its donor site. This is followed by capture of the target site and strand transfer events, again within a nucleoprotein synapse, that rejoin the transposon ends to the new site. The strand transfer complex subsequently disassembles and host factors repair DNA gaps at the insertion and donor sites (124). A number of other well-characterized transposons, including Tn7 and Tn10, also move by a cut-and-paste mechanism, although these elements have different genetic organizations (Figure 1).
Figure 2 Simplistic representation of cut-and-paste (A) and replicative (B) transposition. In the cut-and-paste mechanism, the transposase protein (ovals) binds to the ends of the transposon (thick arc) and brings these ends together in a synaptic complex (step 1). Strand cleavage releases the transposon (step 2), which captures the new target site (double lines) (step 3) where further strand exchange reactions integrate the transposon at the new location (step 4). In replicative transposition, the transposition process involves cointegration of the donor replicon that harbors the transposon and the target molecule with concomitant duplication of the transposon (step 1). The action of a transposon-specified site-specific recombinase resolves the cointegrate intermediate to regenerate the intact donor replicon and the target molecule that now harbors a copy of the transposon (step 2).
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Transposon Tn3 (Figure 1) exemplifies elements that move by replicative transposition (53). Tn3 contains genes for both a transposase and a site-specific recombinase, and specifies ampicillin resistance. The element is delimited by 38-bp terminal inverted repeats that are recognized by the transposase. During transposition of Tn3 and similar elements, a cointegrate intermediate is formed in which the complete donor molecule that harbors the transposon is inserted into the target replicon with concomitant duplication of the transposon (Figure 2B). Within the cointegrate structure the donor replicon is flanked by copies of the transposon. Resolution of this intermediate is achieved by the action of a transposon-encoded site-specific recombinase of the serine or tyrosine family that regenerates the donor molecule as well as a target molecule that has acquired a copy of the transposon. The mechanisms of cut-and-paste and replicative transposition are both now understood in considerable molecular detail and share many common biochemical features (30). This comprehensive understanding of transposon biology has in part promoted the resurgence in the use of transposons as flexible genetic tools.
MAKING TRANSPOSONS JUMP IN VITRO In vitro transposition reactions were first developed to facilitate detailed biochemical studies of transposition mechanisms (19, 25, 114, 122). In vitro transposition has now been described for bacterial transposons Tn3 (98), Tn5 (47, 49), Tn7 (10), Tn10 (20), Tn552 (93), IS911 (121), bacteriophage Mu (57, 112), the Ty1 transposon of yeast (33), and the mariner transposon of insects (89). In their most uncomplicated format, in vitro transposition reactions require only the transposon terminal inverted repeats, purified transposase, the DNA target substrate, and a simple reaction buffer. Furthermore, the reactions can proceed with high efficiency, for example, producing thousands of transformants of Escherichia coli per microgram of substrate plasmid DNA used in a transposition reaction (52). In addition to revealing details of the transposition process, other important exploitations of in vitro transposition have emerged for genomic studies. In particular, in vitro transposition reactions have been used to generate genomewide insertion mutations in a diversity of bacteria and in the yeast Saccharomyces cerevisiae (Table 1). Typically, purified genomic DNA of the target organism is subjected to in vitro transposition, followed by transformation of the mutated DNA into the host with selection for a marker on the transposon. Homologous recombination within the cell results in replacement of the wild-type gene with the mutated allele. The transposon mutants can be screened as a pool in a model system in vivo if the target microorganism is a pathogen (145) or otherwise can be analyzed for relevant phenotypic alterations in vitro. A variation on this theme involves the construction of DNA transposition complexes (transpososomes) in vitro in the absence of divalent metal ions that are essential for progression of the transposition reaction to completion. The preassembled transpososomes are readily transformable and, once in the metal ion-rich intracellular environment, produce normal chromosomal transposon insertions (37, 47, 54, 70, 87). Other in vitro transposition reactions
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TABLE 1 Microbial genomes mutagenized using in vitro transposition reactions
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Microorganism
Significance
Transposon
Reference
Campylobacter jejuni
Food-borne pathogen
Tn552
26, 66
Erwinia carotovora
Plant pathogen
Mu
87
Escherichia coli
Model bacterium for genetic analysis
Tn5 Mu
47 87
Haemophilus influenzae
Pulmonary infectious agent
mariner Tn7
2, 3 56
Helicobacter pylori
Gastric infections and ulcers
mariner
55
Mycobacterium spp.
Opportunistic pathogen
Tn552
13, 79
Neisseria meningitidis
Meningitis agent
Tn10
145
Proteus vulgaris
Opportunistic pathogen
Tn5
47, 70
Pseudomonas sp.
Opportunistic pathogen
Tn5
70
Rhodococcus sp.
Opportunistic pathogen
Tn5
37
Saccharomyces cerevisiae
Model lower eukaryotic for genetic analysis
Tn5
47
Salmonella typhimirium
Food-borne pathogen
Tn5 Mu
47, 70 87
Streptococcus pneumoniae
Pneumonia agent
mariner
2
Streptomyces coelicolor
Antibiotic producer
Tn5, mariner
44
Synechocystis sp.
Photosynthetic cyanobacterium
NDa
12
Xylella fastidiosa
Plant pathogen
Tn5
54
Yersinia enterocolitica
Systemic infectious agent
Mu
87
a
ND, not described.
have facilitated the tagging and characterization of cryptic plasmids (1, 79), the generation of gene fusions with inducible promoters (50), and the fusion of target proteins with a green fluorescent reporter protein (108). The rapid pace with which microbial genomes are being sequenced far outstrips the rate at which tractable genetic systems are being developed to analyze these genomes. The application of in vitro transposition approaches will assist the dissection of at least some of these sequenced but otherwise largely uncharacterized genomes (38, 60).
GENE SEQUENCING WITH TRANSPOSONS The inherent ability of transposons to insert into novel DNA sites makes them ideal sources of portable priming sites for nucleotide sequence determination of uncharacterized regions. Using sequencing primers that anneal near the end(s) of the transposon, a collection of random transposon insertions into a cloned DNA fragment of interest can be used to generate a set of overlapping sequence contigs
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that can be assembled into the entire sequence of the fragment (e.g., 15, 52, 94, 119, 144). This method has been extended to sequence analysis of cDNA libraries (139) and potentially is of special use in the analysis of regions whose sequences might otherwise be technically difficult to decipher (33). A number of transposable elements, including Tn3, Tn5, and Mu, have been successfully developed as sequencing tools. Recently, sequencing reactions with transposon-specific primers have been used to localize transposon insertion sites directly in genomic DNA of yeast and bacteria without a requirement for prior cloning or amplification of the transposon-target junction region (70, 71). This strategy, although not yet in widespread application, has the potential to accelerate considerably the characterization of transposon disruptions in genes of interest that have been identified by signature-tagged mutagenesis or genetic footprinting, for example. Thus, although high-throughput sequencing strategies that allow the rapid and precise sequencing of whole genomes have been developed in recent years (51), transposon-mediated approaches to nucleotide sequencing remain highly relevant.
TRANSPOSONS FOR GENE FUSIONS The indiscriminate way in which many transposons insert into DNA makes these elements useful to produce random transcriptional or translational fusions between a gene(s) of interest and a reporter gene incorporated into the transposon. The utility of transposons in generating gene fusions was first demonstrated using Tn10-lacZ and Tn5-phoA derivatives to produce transcriptional and translational fusions, respectively (18, 102, 152). The lacZ and phoA genes continue to be the reporter systems of choice for these studies because of the ease with which their respective enzymatic products, β-galactosidase and alkaline phosphatase, can be quantitated chromogenically. Nevertheless, other reporter genes, such as those encoding green fluorescent protein and luciferase, have also been employed (e.g., 42, 108, 149), as have delivery systems other than Tn5 and Tn10 (e.g., 13, 31, 46, 117). In fusion systems, the transposase protein is commonly supplied in trans from a regulatable promoter so as to minimize the size of the delivery vector, to allow controlled expression of the transposase gene, and to reduce the frequency of secondary transposition events from the primary insertion site. Transposon derivatives for use in generating translational fusions to phoA typically are designed so that the phoA gene is truncated of its 50 signal peptide sequence but is fused in-frame to one end of the transposon (Figure 3). An in-frame insertion of the phoA transposon into a target gene produces a hybrid protein product composed of a portion of the N terminus of the target protein, a short peptide specified by the transposon end, and the bulk of PhoA. As PhoA is active mainly in the bacterial periplasm, alkaline phosphatase activity is detected in the transposon mutant only if the target protein is exported across the cytoplasmic membrane and if the site of the fusion is within a periplasmic region of the target. Thus, PhoA fusion technology is widely used to identify exported and cell envelope–associated
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Figure 3 The use of a transposon harboring a truncated phoA gene to produce protein translational fusions. A. The full-length phoA gene includes a 50 sequence (hatched box) that specifies a signal peptide in PhoA. This 50 sequence is absent in Tn-phoA. The transposon also usually contains an antibiotic resistance selective marker (abr). B. Inframe insertion of Tn-phoA in a target gene (cylinder) will produce a fusion protein whose N terminus is derived from the target protein. If this region includes signals for periplasmic localization, the fusion protein is transported to the membrane (stippled box) and the alkaline phosphatase moeity will be activated.
polytopic proteins and to explore the membrane topology of these proteins (100). Moreover, single proteins or multiprotein complexes of interest can be dissected using PhoA fusions, or genomewide screens can be performed (for recent examples, see 9, 13, 88, 146, 155). In contrast to transposons designed to produce translational fusions with phoA, transposons that artificially harbor the lacZ gene have been used principally to produce random transcriptional fusions of this gene throughout a genome (6, 32, 117, 141). These fusions can provide preliminary information on the strength of the promoter that controls expression of the disrupted gene. More important, by monitoring lacZ expression from this promoter using different physiological, environmental, or genetic parameters, it is possible to deduce the signals involved in negative and/or positive regulation of the promoter. If the analysis of the lacZ fusions is designed appropriately, the information obtained can be remarkably subtle (6, 80, 130, 153).
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In a combination of phoA and lacZ fusion approaches, novel transposon derivatives have been developed that facilitate the combined analysis of periplasmic and cytoplasmic domains of membrane proteins (4). Furthermore, a number of artificial elements have been designed in which an outward-reading, regulatable promoter is positioned near the transposon end. If transposition occurs between the promoter region and start codon of a gene, transcription of the gene is switched from the natural to the regulatable promoter, from which expression can be either induced or repressed (24, 76, 123, 148). In this way, genome-wide screens can be designed that assess the contribution of individual genes to cell viability, for example (77). Reporter fusion technology and other emerging transposon-based methods for generating random fusions between genes (113) will continue to be invaluable tools for both whole-genome and single-gene studies.
SIGNATURE-TAGGED MUTAGENESIS The identification of microbial virulence genes implicated in animal disease is a technical challenge because it usually requires negative selection of attenuated mutants. The development by Holden and coworkers of transposon-based signature-tagged mutagenesis (STM) partly surmounted this obstacle by allowing the large-scale mutagenesis, in vivo screening, and recovery of a spectrum of avirulent mutants (67, 137). STM has now been applied to a diversity of pathogenic bacteria and has provided invaluable signposts to their modes of virulence (reviewed in 92, 106, 138). The procedure has also suggested potential strategies for the development of novel antimicrobial agents and vaccines. In STM, a pool of synthetic oligonucleotides with random internal sequences (∼40 bp) but with invariable 30 and 50 ends are cloned in a transposon delivery vehicle (Figure 4). The pool of oligonucleotides originally comprised as many as 1017 molecules but is usually now refined to <100 unique tags that do not cross-hybridize in subsequent steps and that reproducibly amplify by PCR. The tagged transposon pool is used to randomly mutagenize the genome of the pathogen of interest. Transposon mutants are arrayed on plates for later screening and recovery. Pools of mutants are used to infect an animal or cell culture model system, and the infection is allowed to proceed for an appropriate period. Bacteria are recovered from selected tissues and genomic DNAs of the recovered bacteria (output pool) and the inoculum (input pool) are prepared. The tags present in both genomic preparations are amplified by PCR using the invariable ends of the tags as priming sites, radioactively labeled, and hybridized against the colony arrays or in DNA dot blots of the arrays. Transposon mutants that do not hybridize with the output pool probe are candidates that have failed to establish in the animal model and thus potentially are impaired in virulence. These mutants are then retested individually and relevant techniques are used to pinpoint the site of the transposon insertion and to characterize the isolates in more detail. STM was first developed using a mini-Tn5 transposon as the tag delivery system and has been applied successfully to a variety of pathogenic Proteobacteria in
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which this transposon is most active, including Brucella spp., E. coli, Klebsiella pneumoniae, Legionella pneumophila, Salmonella spp., Vibrio cholerae, and Yersinia spp. (8, 35, 40, 67, 78, 104, 110, 137). Furthermore, the STM technique has been extended to Gram-positive pathogens, in which Tn5 transposes poorly, if at all, by employing transposons Tn917, Tn1545, and IS1096 that originate from these bacteria (5, 16, 29, 75, 107), or the mariner transposon from insects (62). Thus, the choice of transposon in STM is dictated in part by the pathogen under scrutiny. The transposon additionally should insert randomly or near-randomly to ensure that a wide breadth of mutants is generated. Nevertheless, some degree of insertion specificity can be tolerated (5), and even Tn5, which has been used most extensively and successfully for STM, does not insert entirely without site-specificity (95). Furthermore, by using either homologous recombination or in vitro transposition followed by recombination to generate tagged mutants, STM has been adapted for use in certain bacteria and single-celled eukaryotes, where amenable transposition systems are less readily available (68, 82, 90, 96, 115, 145). Although STM has often illustrated the importance of housekeeping genes in infectivity, pathogen establishment, and disease progression, the technique has proven particularly valuable both in the identification of novel virulence loci and in the confirmation of the roles of previously identified virulence genes (106). Among the hundreds of virulence genes identified in STM screens, those that specify factors associated with the bacterial cell wall or cell membrane are among the most commonly found, thereby emphasizing the significance of the physical interaction between pathogen and host in disease. As examples, mutations that affected genes for integral membrane and membrane-associated proteins, adherence, and cell wall decoration led to attenuation of the foodborne pathogen Listeria monocytogenes in a murine infection model (5). Similarly, both genes for polysaccharide biosynthesis and other potential virulence genes were identified in STM of Yersinia pseudotuberculosis in mice and of Streptococcus agalactiae in a neonatal rat sepsis ←−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−− − Figure 4 Signature-tagged mutagenesis. A collection of transposon derivatives each of which contains a unique oligonucleotide tag (filled box labeled N) bracketed by common primer binding sites (hatched boxes) is used to mutagenize a genome of interest. A segment of this genome is represented with three essential loci A–C. Three distinct transposon insertions in this region are shown and the different tags in these insertions are denoted 1–3. The complete pool of insertions, which might number several thousand clones, is arrayed for later recovery (input pool). Pools of mutants are also used to inoculate the animal model. After an appropriate infection period, genomic DNA is recovered from the bacteria (output pool), the tags are amplified using the common primer binding sites, and the PCR products are end-labeled. These probes are hybridized against the input arrays. Those colonies in the input pool that fail to hybridize with the output pool DNA are candidates that have been depleted during infection and thus are likely to contain transposon insertions in genes that are important for some aspect of the infection process.
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infection model (75, 78). In a classical example, a new pathogenicity island encoding a type III secretion system was identified by STM of the enteric pathogen Salmonella typhimurium (137). This type of system is commonly employed by pathogenic bacteria for the secretion of virulence effector molecules into the cytosol of the host cell (28). Although transposon-based STM is a remarkably powerful method to identify virulence factors and has been extended to the use of attenuated mutants as live vaccines (43), a number of considerations concerning the pathogen-host interaction and other aspects of the infection process need to be evaluated before embarking on a mutant screen (92, 106, 138). Nevertheless, when used in parallel with microarray and other whole-scale genome approaches, STM has specially formidable potential not only in pathogenicity studies but also in the analysis of bacterial establishment and persistence in diverse ecological niches.
GENETIC FOOTPRINTING WITH TRANSPOSONS Like STM, genetic footprinting involves the production of a large pool of transposon insertion mutants and the subsequent identification of individuals or genes that become depleted when this pool is propagated under certain environmental conditions (142, 143). The first step in genetic footprinting is the generation of a comprehensive transposon random insertion pool that optimally will include mutants with insertions at different locations in the gene of interest. The second step involves outgrowth of the mutagenized pool under the relevant test conditions for a specific number of generations. Finally, genomic DNA is retrieved from the pool before and after growth. These DNAs are subjected to PCR using a pair of primers, one that anneals to the transposon ends and the other that primes from within or nearby the gene of interest (Figure 5). The depletion of PCR products −−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−− → Figure 5 Genetic footprinting with transposons. A transposon (open box) is used to comprehensively mutagenize the microorganism of interest. Four transposon insertions are shown: Three of these insertions (M1–M3) are at different locations in an essential gene A, whereas the fourth insertion (M4) is located in an inessential gene B. Genomic DNA is prepared from the entire mutant pool (T0 genomic sample). The mutant pool also is grown under appropriate experimental conditions, e.g., in minimal growth medium, for a specific number of generations, after which genomic DNA is prepared (T1 genomic sample). Primers P1 and P2, which anneal upstream of gene A and within the transposon, respectively, are used in separate amplification reactions with the T0 and T1 DNAs. The products are analyzed on a sequencing gel. As A is an essential gene under the test conditions, the amplification products M1–M3 that are present in the T0 sample are absent from the T1 sample owing to depletion of the corresponding inviable mutants during growth. In contrast, the M4 amplification product is still evident at T1 because the corresponding transposon mutation is in a dispensable gene.
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following growth under the experimental conditions is indicative of a reduction of the number of cells in the population that harbor transposon insertions in the test gene, which thus is important for viability under these conditions. If the growth of mutants containing a disruption of the gene of interest is unimpaired under the test conditions, no significant difference in the pattern of PCR products before and after outgrowth will be observed (142, 143). Individual genes of interest can be tested by genetic footprinting, or a spectrum of genes can be analyzed using DNA aliquots derived from a single outgrowth experiment. Genetic footprinting first was used to identify genes that were essential for the viability of S. cerevisiae under different growth conditions (142, 143). Using the Ty1 transposon a systematic genetic footprinting analysis of chromosome V of S. cerevisiae indicated that an unexpectedly large proportion (>50%) of the genes on this chromosome was required for proper growth under test conditions that included growth in rich and minimal media, in a high-salt medium, at elevated temperature, and under mating conditions. These important genes included a number of loci of hitherto unknown function or that previous mutational analysis had indicated were dispensable. Genetic footprinting compares the behavior of cells with the mutations of interest in cocultivation with wild-type cells and cells that contain a breadth of other insertion mutations. In this situation, subtle competitive disadvantages might be detectable for insertion mutants that are not evident when strains with null or other mutations in the gene of interest are tested in isolation. The genetic footprinting strategy has been extended to bacteria using marinerfamily transposons for mutagenesis in Pseudomonas aeruginosa and Haemophilus influenzae (3, 154), and with modified derivatives of Tn5 and Tn10 that were delivered to the E. coli genome in vitro and in vivo, respectively (45, 61). These studies demonstrated that genetic footprinting can be as powerful an experimental approach for whole-genome mutagenesis studies in bacteria as it is in yeast. Furthermore, these bacterial trials also revealed the essentiality of certain genes of previously indeterminate significance. For example, the first gene in a three-gene operon of unknown function was shown by genetic footprinting to be required for the viability of E. coli not only in minimal growth medium but also in rich medium (61). Simultaneously, this cryptic gene was identified by more traditional approaches as a gene for dephosphocoenzyme A kinase, a protein required for synthesis of the essential Coenzyme A cofactor (111).
TRANSPOSONS AND MICROARRAY TECHNOLOGY Since the technique’s inception in 1995 (133), microarray technology has been extensively employed for monitoring genome-wide gene expression in a multitude of prokaryotic and eukaryotic systems (134, 140). By combining microarray technology with the simplicity and power of transposon insertion mutagenesis, Church, Rubin, and coworkers identified genes that contributed to the fitness of cells growing in minimal medium compared to those growing in rich medium. The method involved using subgenic-resolution microarrays of mini-Tn10 transposon mutants of E. coli or mariner-based transposon mutants of Mycobacterium spp.
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(7, 131, 132). As expected, genes required for the biosynthesis of numerous amino acids, nucleotides, and lipopolysaccharides were particularly prevalent among those required for optimal growth in minimal media but a number of putative metabolic enzymes and transcriptional repressors that might be involved in growth in nutrient-limiting conditions were also identified by this method (7). Although this strategy has some limitations, including the inability to identify essential genes, possible polar effects of transposon insertions and cross-feeding of mutants by nutrients released by nearby cells, it can potentially be applied to a variety of bacteria and other microorganisms in which it is possible to construct a comprehensive library of transposon insertions. As mentioned previously, the use of in vitro transposition technology should broaden the scope of microorganisms that are amenable to combined transposon and microarray analyses. Furthermore, it should be feasible to perform microarray studies using a variety of conditions in which the growth of the transposon mutant pool is modulated, e.g., different ionic concentrations, temperatures, or pH, to deduce more about the factors that contribute to microbial fitness and evolution. To identify previously overlooked genes in S. cerevisiae, shuttle transposon mutagenesis (135) using a Tn3 derivative was used to construct random genomic transcriptional and translational fusions to the lacZ gene of E. coli (86). Almost 200 previously unannotated but transcriptionally active genes, corresponding to approximately 10% of all of the tagged genes, were identified by sequencing across the junction of each lacZ fusion that produced significant β-galactosidase activity. The expression of each new transposon-tagged gene was verified in a microarray format consisting of oligonucleotides representing each gene. Combined with bioinformatics and other in silico approaches, the number of new genes in this study was refined to 137 genes, which represents a significant proportion of the yeast genome (86). In a parallel study, transposon-based tagging of proteins with a 93-amino acid epitope that is detectable by immunofluorescence allowed the subcellular localization of the majority of the S. cerevisiae proteome (84). Although novel combinations of transposon mutagenesis and microarray technology have also been applied in other microbial systems (21, 41), the joint impact of these strategies will only be realized in the next few years.
SCANNING LINKER MUTAGENESIS The construction and analysis of proteins with insertions of short peptides [scanning linker mutagenesis (SLM)] is a powerful strategy for dissecting protein structure-function relationships. The insertion and imprecise excision of a transposon from the corresponding cloned gene is now the preferred method for the random introduction of peptides into a target protein (64, 103). The transposon excision event is designed so as to embed an in-frame fingerprint at the insertion point. The excision process is most often achieved by digestion of the transposon-containing plasmid with a restriction enzyme that cleaves near to both ends of the element but that does not have recognition sites within the remainder of the recombinant plasmid. Religation of the restriction fragment bearing the target gene reconstitutes
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Figure 6 Scanning linker mutagenesis. The transposon (open box) is used to mutagenize the cloned target gene (filled arrow) (step 1). The bulk of the transposon is deleted from the disrupted gene by restriction digestion with an enzyme (A) that has recognition sites near both ends of the transposon. The plasmid backbone is religated to produce a gene containing an in-frame insertion (step 2). This insertion will produce a peptide insertion in the corresponding protein (step 3).
the preexisting plasmid but with an in-frame insertion in the gene (Figure 6). A variety of restriction enzymes have been used so that even if the target gene harbors a restriction site that complicates the use of one transposon-enzyme combination, another combination is likely to be suitable (Table 2). Site-specific recombination reactions have also been employed in the formation of the insertion. Depending on the transposon selected, the corresponding peptide fingerprint in the target protein is between 4 and 93 amino acids. A variety of transposons have been adapted for use in SLM, and in vivo and/or in vitro delivery of the transposon to the cloned target gene is feasible depending on the transposon selected (Table 2). Furthermore, although it is possible to construct and analyze single transposon insertions in a target gene, it is often more convenient and efficient to process large pools of mutants before progressing to the systematic characterization of individual mutants from this pool. In vitro transposition reactions have an inherent capacity to generate extensive mutant arrays, but SLM approaches that employ in vivo transposition can also be adapted easily to produce comprehensive mutant pools (64).
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TABLE 2 Scanning linker mutagenesis using transposons
Transposona
In vivo or in vitro transposition
Strategy for deleting transposon bulk
Peptide size (amino acids)
Reference
Tn552
In vitro
BsrGI cleavage and religate
4–7
52
IS21
In vivo
BglII or SalI cleavage and religate
4 or 11
136
Tn4430
In vivo
KpnI cleavage and religate
5
58
Tn7
In vitro
PmeI cleavage and religate
5
11
Mu
In vitro
NotI cleavage and religate
5
91, 147
Tn5
In vivo
NotI cleavage and religate
24
36
Tn5
In vitro
SfiI or NcoI cleavage and religate
25
4
Tn5
In vivo
BamHI cleavage and religate
31
101
Tn5
In vitro
Cre-loxP site-specific recombination
42
4
Tn3
In vivo
Cre-loxP site-specific recombination
45
69
Tn5
In vivo
Cre-loxP site-specific recombination
63
9
mariner
In vitro
FLP-FRT site-specific recombination
67
22
Tn3
In vivo
Cre-loxP site-specific recombination
89 or 93
128
a
The transposons used often are modified versions of the parental transposons shown.
Pentapeptide scanning mutagenesis (PSM) is an SLM approach based on transposon Tn4430 (58, 64). This element was identified in the Gram-positive bacterium Bacillus thuringiensis, but it also transposes efficiently in E. coli. Recognition sites for the KpnI restriction enzyme fortuitously are located 5 bp from either end of Tn4430. The transposon also duplicates 5 bp of target site sequence during transposition. Thus, KpnI digestion of a plasmid that contains Tn4430 liberates the bulk of the transposon. Religation of the plasmid backbone reproducibly produces a 15-bp insertion that will result in a pentapeptide tag when the insertion is within a protein-encoding sequence. Because of the comparatively modest size of the insertion, and the fact that the procedure very rarely introduces a termination codon at the insertion point and is easy to use, PSM has proven to be specially valuable in analyzing structure-function relationships in a variety of prokaryotic and eukaryotic proteins (17, 58, 63–65, 127, 159). A few examples of PSM emphasize its potential in dissecting protein function. First, BRCA1 is a human tumor suppressor protein that, among other functions, is a transcriptional activator. PSM
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of the carboxyl terminus of BRCA1 revealed an extensive region of the protein that is highly tolerant of insertions with regard to transcription activation, probably because this region is nonglobular and highly flexible. A distinct region of BRCA1 that might directly interact with host transcription factors was also demonstrated in this study (63). Second, PSM analysis was used recently to dissect protein-protein interactions within the multiprotein secretion complex that transports exoproteins in Pseudomonas aeruginosa (127). Protein-protein interactions in toxin-antitoxin complexes involved in bacterial programmed cell death, cell cycle arrest, and genome maintenance are among other associations that have been dissected by PSM (unpublished data). Transposon-based SLM techniques are specially useful in the rapid identification of protein regions that are highly tolerant of peptide insertions and of sites at which insertions entirely abolish protein activity. SLM can be an efficient method by which to introduce epitope tags in proteins (101), as well as revealing unexpected facets of protein function and organization (64).
TRANSPOSONS AS GENETIC TOOLS IN HIGHER EUKARYOTES It is beyond the remit of this discussion to describe in detail the uses of transposons as tools for the genome-wide manipulation of multicellular eukaryotic organisms. However, sophisticated transposon-based strategies have been developed for insertion mutagenesis in a number of animal and plant models. In particular, mutagenesis with P elements in Drosophila (126), the Tc1/mariner family of transposons in Caenorhabditis elegans (120), and a variety of transposons of the hAT and CACTA superfamilies in Arabidopsis, maize, and other plants (116, 151) is a well-established method for gene disruption and tagging. The use of heterologous transposons in mammalian systems also has been described recently, which expands the range of options available for transposon mutagenesis studies in these systems (34, 39, 72, 74, 97, 158), as well as suggesting novel approaches to gene therapy (125, 156). As is the case with microbial genomes, the expanse of data emerging from genome-sequencing projects of higher eukaryotes requires novel methods by which to interpret this information. Although transposon mutagenesis in these systems has lagged behind more notable advances in bacteria and lower eukaryotes, transposable elements are also being rediscovered as malleable but effective tools with which to undertake functional genomics studies of multicellular organisms (59).
CONCLUDING REMARKS Spectacular strides have been made in recent years in deciphering the entire genome sequences of a variety of organisms. Although these remarkable advances have ushered in a new era in the biosciences, broadly applicable experimental strategies are needed to unravel how these diverse genomes direct the organization
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of cells that are capable of flourishing in disparate environments, for example. Transposons have reemerged in recent years as simple yet sophisticated tools for this purpose. Transposons can now be used for insertion mutagenesis in vivo and in vitro, which potentially allows genome-wide earmarking even of bacteria that do not yet have extensive genetic toolkits (e.g., 73, 160). Signature-tagged mutagenesis has proven to be an invaluable resource in understanding the infectivity, virulence, and establishment properties of a variety of important bacterial pathogens (106). Transposons continue to find regular use as tools in gene sequencing and protein fusion technologies. Individual proteins and entire proteomes can now be marked and dissected with transposon-delivered peptide tags (64, 103). Strategies for the rational manipulation of whole genomes using transposons have also been developed (48, 157). Moreover, transposons are finding ever more subtle uses in multicellular organisms (e.g., 105). Undoubtedly, transposable elements will bolster other advances in functional and structural genomics in the near future and will remain essential and versatile instruments in the molecular microbiologist’s repertoire (23, 38, 59, 60, 77, 85, 150). ACKNOWLEDGMENTS Work in the author’s laboratory is supported by the Wellcome Trust and the Biotechnology and Biological Sciences Research Council. I thank Bernard Hallet for collaborative work on pentapeptide scanning mutagenesis. The Annual Review of Genetics is online at http://genet.annualreviews.org
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Annual Review of Genetics Volume 37, 2003
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CONTENTS IRA HERSKOWITZ (1946–2003), The Editors TRANSPOSON-BASED STRATEGIES FOR MICROBIAL FUNCTIONAL GENOMICS AND PROTEOMICS, Finbarr Hayes ERROR-PRONE DNA POLYMERASES: WHEN MAKING A MISTAKE IS THE ONLY WAY TO GET AHEAD, Alison J. Rattray and Jeffrey N. Strathern
GENETICS OF HAIR AND SKIN COLOR, Jonathan L. Rees THIOL-BASED REGULATORY SWITCHES, Mark S.B. Paget and Mark J. Buttner
1 3
31 67 91
PSEUDOGENES: ARE THEY “JUNK” OR FUNCTIONAL DNA? Evgeniy S. Balakirev and Francisco J. Ayala
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UNUSUAL LIFE STYLE OF GIANT CHLORELLA VIRUSES, James L. Van Etten
153
GENETICS OF LACTASE PERSISTENCE AND LACTOSE INTOLERANCE, Dallas M. Swallow
CELL POLARITY AND THE CYTOSKELETON IN THE CAENORHABDITIS ELEGANS ZYGOTE, Stephan Q. Schneider and Bruce Bowerman THE SPINDLE ASSEMBLY AND SPINDLE POSITION CHECKPOINTS, Daniel J. Lew and Daniel J. Burke
197 221 251
LATERAL GENE TRANSFER AND THE ORIGINS OF PROKARYOTIC GROUPS, Yan Boucher, Christophe J. Douady, R. Thane Papke, David A. Walsh, Mary Ellen R. Boudreau, Camilla L. Nesbø, Rebecca J. Case, and W. Ford Doolittle
283
GENETICS OF AGING IN THE FRUIT FLY, DROSOPHILA MELANOGASTER, Stephen L. Helfand and Blanka Rogina
329
NATURAL SELECTION AND THE EVOLUTION OF GENOME IMPRINTING, Elena de la Casa-Esper´on and Carmen Sapienza
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THE NEED FOR WINTER IN THE SWITCH TO FLOWERING, Ian R. Henderson, Chikako Shindo, and Caroline Dean
TRANSMISSION RATIO DISTORTION IN MICE, Mary F. Lyon
371 393
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CONTENTS
STRUCTURE, DIVERSITY, AND EVOLUTION OF PROTEIN TOXINS FROM SPORE-FORMING ENTOMOPATHOGENIC BACTERIA, Ruud A. de Maagd, Alejandra Bravo, Colin Berry, Neil Crickmore, and H. Ernst Schnepf
YEAST VACUOLE INHERITANCE AND DYNAMICS, Lois S. Weisman HEPARAN SULFATE CORE PROTEINS IN CELL-CELL SIGNALING, Kenneth L. Kramer and H. Joseph Yost
409 435 461
RETROTRANPOSONS PROVIDE AN EVOLUTIONARILY ROBUST NON-TELOMERASE MECHANISM TO MAINTAIN TELOMERES, Annu. Rev. Genet. 2003.37:3-29. Downloaded from arjournals.annualreviews.org by Universidad Nacional de la Plata on 09/29/08. For personal use only.
Mary-Lou Pardue and P.G. DeBaryshe
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A CYANOBACTERIAL CIRCADIAN TIMING MECHANISM, S.S. Golden, J.L. Ditty, and S.B. Williams
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REGULATION OF CELL CYCLES IN DROSOPHILA DEVELOPMENT: INTRINSIC AND EXTRINSIC CUES, Laura A. Lee and Terry L. Orr-Weaver RECOGNITION AND RESPONSE IN THE PLANT IMMUNE SYSTEM,
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Zachary Nimchuk, Thomas Eulgem, Ben F. Holt III, and Jeffrey L. Dangl
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RECA-DEPENDENT RECOVERY OF ARRESTED DNA REPLICATION FORKS, Justin Courcelle and Phillip C. Hanawalt
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INDEXES Subject Index Cumulative Index of Contributing Authors, Volumes 33–37 Cumulative Index of Chapter Titles, Volumes 33–37
ERRATA An online log of corrections to Annual Review of Genetics chapters may be found at http://genet.annualreviews.org/errata.shtml
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