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In vivo expression technology strategies: valuable tools for biotechnology
440
In vivo expression technology strategies: valuable tools for biotechnology Paul B Rainey* and Gail M Preston† Whole genome sequences have shown that bacteria possess a significant number of genes that have no known function. It is probable that many of these are required for survival in environments other than the agar plate. In vivo selection strategies provide a means of obtaining genes active in complex natural environments. Direct access to these genes is essential for understanding ecological performance and provides novel opportunities for biotechnology. Addresses Department of Plant Sciences, University of Oxford, South Parks Road, Oxford OX1 3RB, UK *e-mail: [email protected] † e-mail: [email protected] Current Opinion in Biotechnology 2000, 11:440–444 0958-1669/00/$ — see front matter © 2000 Elsevier Science Ltd. All rights reserved. Abbreviations DAP diaminopimelate IVET in vivo expression technology RIVET recombinase-based IVET
Introduction For most of the past century, microbiology has been devoted to studying the physiology and genetics of bacteria in vitro. This means that we now understand a great deal about the lives of bacteria on agar plates, but have little understanding of the determinants of ecological success in the wild. Without an understanding of the causes of ecological success our understanding of the biology of bacteria will remain incomplete and the full potential of these organisms in biotechnology will remain unrealised. Many strategies have been used to study ecological performance [1–3]. Bottom-up (genes to population) and top-down (population to genes) approaches are both useful. The bottom-up approach is commonly employed for studies of bacteria, although is rarely pursued to the population level. The typical genes-to-phenotype strategy involves identification of traits on the basis of gene inactivation. This is a powerful approach that has been fundamental to the majority of advances in molecular microbiology, but despite its power, insertional mutagenesis is not always appropriate for the analysis of phenotypes as complex as ecological performance. For most organisms, in most environments, there is no primary determinant of ecological performance; this is because it is determined by complex epistatic interactions among many different gene products that each has a long evolutionary history. Traits having greatest effect on ecological performance are likely to be those that show subtle quantitative variation and such traits are unlikely to produce ‘defective’ phenotypes when inactivated [3].
Recent advances in gene fusion technologies provide an alternative way to study complex phenotypes. Rather than identifying genes on the basis of function loss, ecologically significant genes can be identified on the basis of their positive contribution toward a specific phenotype. A study wishing to understand the mechanistic basis of ecological performance in a leaf colonising biocontrol isolate might therefore begin by identifying those genes that are specifically induced in the leaf environment. One advantage of this approach is that it considers bacteria as integrated organisms rather than as a toolbox of independent genes and phenotypes. A second advantage, of particular relevance to biotechnology, is the opportunities to access novel traits. Most significantly, bottom-up, positive selection approaches open the door to understanding ecological performance in bacteria at a mechanistic level in the wild. In vivo expression technology (IVET) strategies, despite their tremendous power, have been little used, except in studies of animal pathogenicity. This brief review is intended to draw attention to IVET strategies, their development, and potential in biotechnology. Length prevents discussion of biological insights gained from these strategies and we refer the reader to primary sources and to two recent reviews [4–6].
IVET: the basic strategy The first gene fusion technology involving positive selection was developed to study pathogenicity in Xanthomonas campestris on turnip seedlings [7]. The strategy involved selection of environment-specific genes on the basis of their ability to drive expression of a gene essential for survival. In 1993, this strategy was modified and extended to the study of Salmonella pathogenicity in a mammalian host where the term ‘in vivo expression technology’ was coined [8]. IVET has since been applied extensively to the study of Salmonella virulence and has resulted in the discovery of many novel genes. It has provided valuable insights into the ecology of the host environment [4,9,10] and has recently been used to explore regulation and temporal expression patterns of Vibrio cholerae virulence genes during infection [11,12••]. The basic IVET strategy is described in Figure 1 — modifications are discussed below. The strategy is based upon a bacterial strain with a conditional mutation in a biosynthetic gene that is involved in the synthesis of an essential growth factor. Provided that exogenous levels of the growth factor are negligible, then growth in the wild cannot occur unless the missing gene is introduced into the bacterial cell. Introduction into the mutant strain of a promoterless but otherwise functional copy of the biosynthetic gene, to which random fragments of wild-type DNA have been cloned (in place of the promoter) to facilitate
IVET strategies Rainey and Preston
incorporation of the gene into the host genome, provides a means of selecting for promoters that are active in the environment. Selection of such promoters (and corresponding genes) is performed simply by recovery of bacteria that have grown in the wild — growth in the wild can only occur if the function of the mutated biosynthetic gene has been restored. This can only occur where transcription of the promoterless biosynthetic gene is initiated. A subsequent screening step using a second promoterless reporter gene (e.g. ‘lacZ) facilitates differentiation of those promoters active solely in the wild from those that are active both in the wild and in vitro (Figure 1).
Development of IVET strategies There are two essential phases in the development of an IVET strategy. The first requires a decision as to the strength and specificity of the selective regime and involves construction of a conditionally compromised strain. The second phase involves construction of a vector for trapping environment-induced promoter–gene fusions. Both phases are essential, but the first is critical and determines ultimate success and utility. The strength and specificity of selection determines the breadth of environmentally responsive fusions recovered. A study seeking to identify all genes activated in a particular environment is likely to use a general strategy, based, for example, on a strain deficient in the biosynthesis of an essential growth factor (and ideally would use two complementary strategies that use different selective regimes). A weak selective regime has the advantage of isolating a wide range of environment-response genes, but risks false positives. A strong selective regime is likely to recover only highly induced promoters, risking, if selection is too severe, identifying only constitutively expressed fusions and missing transiently expressed genes. Identification of genes induced under specific environmental conditions requires a selection strategy based on a gene that is essential for survival in that specific environment. This can be difficult to achieve and is usually limited to well-studied systems where genes essential for specific environmental challenges are already known, but novel selective regimes are being developed (see below). Development of the selective ‘vehicle’ (phase 2) is relatively straightforward. At its simplest it is a suicide plasmid with multiple cloning sites, selectable antibiotic resistance marker and a promoterless, but otherwise functional, copy of the gene that forms the basis of the selective regime. This is typically fused to a second (and sometimes third) promoterless reporter gene, such as lacZ (β-galactosidase), uidA (β-glucuronidase), gfp (green fluorescent protein), or a combination (see Figure 1) to enable differentiation between constitutive and environment-induced promoters. A final consideration is the recovery of chromosomally integrated fusions following selection — a stage that can be problematic and partly responsible for the slow application
441
Figure 1
Clone fragments of genomic DNA into pIVET
pIVET
+
‘egf ‘lacZY MCS Integrate into genome of strain carrying a deletion of egf ‘egf ‘lacZY ∆egf
pIVET Inoculate environment with library of fusions
Harvest bacteria from environment after 14 days
E N V I R O N M E N T
Re-inoculate environment with putative environmentinduced fusions to confirm induction
Screen for β-gal activity on agar plates. White colonies contain fusions to putative environmentspecific genes
Selective medium containing X-gal and essential growth factor Current Opinion in Biotechnology
Selection for genes showing environment-specific elevation in expression. IVET is a promoter-trapping strategy that selects for genes displaying elevated levels of expression on the basis of their ability to drive the expression of a gene that is essential for survival in a given environment. The basic strategy is based upon random integration of a promoterless ‘essential growth factor’ (‘egf) gene into the chromosome of a bacterial strain carrying a chromosomal deletion of egf. The egf deletion strain cannot grow in the environment because the essential growth factor is absent or severely limiting; growth can only occur if the promoterless ‘egf gene is inserted downstream of an active promoter. Recovery of strains from the environment following selection for the ability to synthesise the essential growth factor results in the isolation of promoters that are either constitutive or environment-specific. In order to distinguish between these two, a promoterless marker operon, for example, lacZY (β-galactosidase [β-gal]), is fused to ‘egf, thus enabling the lactose phenotype of the recovered cells to be determined. Lac+ strains contain fusions to constitutive promoters (dark colonies), whereas Lac– strains (white colonies) contain fusions to promoters active in response to environmental signals.
and development of IVET strategies in biotechnology. For a thorough analysis it is necessary to screen large numbers of environmentally induced fusions by DNA sequencing.
442
Expression vectors and delivery systems
This relies upon an efficient means of recovering IVET fusion plasmids from the host chromosome. In Salmonella, recovery of fusions is readily achieved by transduction using bacteriophage P22 [13], but for many bacteria transducing phage are not available. Fusions can be cloned by standard molecular biology, but these techniques are cumbersome and not suitable for the recovery of large numbers of fusions. An alternative strategy is conjugative cloning, which uses a helper plasmid to provide transfer functions that enable the rescue and transfer of any integrated IVET plasmid into a replication-permissive Escherichia coli host [14]. This is a simple technique that has been shown to work in enteric bacteria, Pseudomonas and Rhizobium. There is no reason for it not to be generally applicable.
General selective strategies Knowledge of the nutritional status of the targeted environment is an important aspect in the development of strategies using nutritionally compromised mutants. Without knowledge of limiting nutrients it is difficult to chose suitable selective strategies (although see RIVET strategies below). In the mammalian host environment, general selective strategies for Salmonella [8] and Pseudomonas aeruginosa [15] have employed purine auxotrophy (purA); thymine auxotrophy has also proved useful [8]. Recent work on Actinobacillus pleuropneumoniae has used a riboflavin-requiring mutant to study patterns of gene expression in the pig lung [16•], and uracil selection has been used to isolate genes expressed by the fungal pathogen Histoplasma capsulatum during infection of a mouse model [17•]. For use in the plant–soil environment, three general strategies have been developed. Two were developed for Pseudomonas fluorescens following a screen of a range of nutritionally compromised transposon mutants for lack of growth in a model sugar beet rhizosphere. One is based on the water-soluble vitamin pantothenate (panB) [18••] (which has since been extended to Rhizobium leguminosarum [D Allaway, N Schofield, PS Poole, personal communication]). The other is based on a combined diaminopimelate (DAP)- and lysine-requiring auxotroph caused by a deletion of dapB [19] (M Gal, G Preston, P Rainey, unpublished data). The third strategy was developed for leaf-colonising P. syringae and is based upon methionine auxotrophy (metXW) (M Marco, S Lindow, personal communication). Of these three strategies, DAP-based selection is applicable to an extensive range of environments — it is a solely bacterial product, absent from soil, water, plant and animal surfaces and tissues and is an essential component of the peptidoglycan of all Gram-negative and some Gram-positive bacteria. DAP auxotrophy is lethal and causes lysis of rapidly growing cells, although non-growing cells remain viable for long periods, which ensures that selection is not too stringent [18]. A selective regime with similar broad applicability has been developed for P. aeruginosa and is based on auxotrophy for DAP, threonine, lysine and methionine, caused by
deletion of the gene encoding aspartate beta-semialdehyde dehydrogenase (asd) [20,21]. Two general selection strategies that largely eliminate the need for conditional lethal mutants have also been developed. One employs antibiotic-based selection and simply requires transcriptional fusions to a promoterless copy of an antibiotic-resistance gene and selection in an environment replete in the antibiotic. This has been successful in mammalian models of infection [9,22–24], but less so in other environments where the presence of large amounts of antibiotic is either impractical or likely to radically alter the environment [7]. The second strategy is a recombinase-based IVET (RIVET), developed originally to identify infectioninduced genes in Vibrio cholerae [11,25]. RIVET is based on a transcriptional reporter that encodes a site-specific DNA recombinase, which catalyses excision of a selectable substrate gene cassette (e.g. tetracycline resistance) from the bacterial genome. Evidence of promoter activity in the wild is inferred from the products of genetic recombination (loss of tetracycline resistance), which acts as a heritable reporter. The elegance of this strategy is twofold. Firstly, it overcomes a criticism often levelled at IVET strategies, namely, that they fail to detect transiently expressed genes. Secondly, the strategy has considerable general utility (requiring only magnesium ions and negatively supercoiled DNA) and is applicable to fastidious organisms and to viruses and eukaryotes. Indeed, development of such a technology has recently been reported for Candida albicans [26••], based upon FLP-mediated recombination with the enzyme inosine monophosphate (IMP) dehydrogenase as a deletable marker.
Specific selective strategies An important goal of many studies, particularly of microbe–host interactions, is to identify and characterise genes that contribute to specific processes, for example, colonisation or parasitism of hosts. This has been elegantly achieved using specific selective regimes that are exemplified in studies of plant–microbe interactions. To identify Rhizobium meliloti genes induced within root nodules during the infection and differentiation stages of symbiosis Oke and Long [27••] used a R. meliloti strain defective in the expression of bacA (transcriptionally fused to uidA), a gene required for bacteroid differentiation. Selection for active promoters was readily achieved by harvesting bacteria from nodules because only strains containing fusions to active promoters could cause nodule development. Those fusions specific to infection and differentiation were distinguished from constitutive promoters by determining β-glucuronidase activity under free-living conditions. Conceptually similar strategies have been developed to study parasitism of plants by Xanthomonas campestris (D Nennstiel, U Bonas, personal communication) and P. syringae, (J Boch, V Joardar, L Gao, T Robertson, B Kunkel, personal communication). The
IVET strategies Rainey and Preston
selective regime, in both cases, is a result of a defect in the hrp cluster (hrpB1 and hrcC, respectively) which is required for pathogenicity. A similar strategy was recently reported for the intracellular pathogen Listeria monocytogenes, based on a listeriolysin (toxin) negative mutant [28•]. A particularly innovative example has been described for isolating genes specific for colonisation of surfaces by P. aeruginosa [29••]. The strategy is based upon a promoterless reporter operon composed of sacB (levan sucrase), aacC1 (gentamicin resistance) and gfp. A library of strains containing transcriptional fusions to this synthetic operon are selected in shaken broth culture in the presence of sucrose, which, because of the toxicity of sacB in a sucrose-rich environment, eliminates strains containing fusions to genes active in the liquid-phase. The remaining cells are then selected on a solid surface in the presence of gentamycin to select genes specific to surface colonisation. The potential of this and similar selective strategies is considerable, and limited only by imagination.
Monitoring activity of bacteria in the environment IVET strategies generate a chromosomal fusion between a tagged (or trapped) gene and a selectable maker that is typically fused to a second reporter — part of the IVET plasmid. Generation of a transcriptional fusion to the primary selectable marker is essential for IVET selection, whereas the second reporter enables constitutive fusions to be distinguished from those that are environment-specific. The second reporter can also be used, however, to monitor spatial and temporal activity of any given fusion. In the case of standard IVET strategies, reporters such as lacZ or uidA can generate quantitative data on precise levels of transcription in different environments and at different times [8,18,30]. These reporters can also be used for in situ studies, although of more use are reporters such as gfp or lux, which allow detection of activity at the level of individual bacterial cells. The recombinase reporter at the heart of RIVET also enables spatial and temporal information on patterns of gene expression to be determined simply by scoring the frequency of tetracycline-sensitive cells isolated from different environments. Such an approach was recently used by Lee et al. [12••] to determine temporal expression patterns of V. cholerae virulence genes during infection of a host.
Conclusions IVET strategies enable a direct insight into the lives of bacteria in natural environments. Such knowledge stands to benefit biotechnology in multiple ways. The discovery of new compounds and processes and enzymes is one immediate benefit, but at a more fundamental level, knowledge of patterns of gene expression in the wild can ultimately provide insights into the causes of ecological success. Knowledge of these causes is as important for the design of efficient biocontrol strains, as it is for the construction of strains for industrial fermentations.
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Update Recently, a further novel extension of IVET has been described, termed IVIAT (in vivo induced antigen technology), that allows identification of in vivo induced genes in human infections without the use of animal models [31]. The strategy takes sera from patients currently experiencing infection from the pathogen of interest and absorbs this against the pathogen that has been grown in vitro, thus leaving in the serum antibodies against antigens that are expressed during infection but not during in vitro cultivation. To obtain bacterial genes expressing the antibodies of interest, the absorbed serum is challenged with an expression library of the pathogen’s DNA. The strategy is simple to use and readily adapted to a variety of different purposes. For a recent review of IVET strategies and their application in pathogenic bacteria, see the comprehensive account by Heithoff et al. [6].
Acknowledgements This work is supported by grants from the BBSRC, NERC and the European Union.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as:
• of special interest •• of outstanding interest 1.
Dykhuizen DE: Natural selection and the single gene. In Population Genetics of Bacteria. Edited by Baumberg S, Young JPW, Wellington EMH, Saunders JR. Cambridge: Cambridge University Press; 1995:161-173.
2.
Dawkins R: The Extended Phenotype. Oxford: Oxford University Press; 1982.
3.
Lenski RE: Molecules are more than markers: new directions in molecular microbial ecology. Mol Ecol 1995, 4:643-651.
4.
Heithoff DM, Conner CP, Mahan MJ: Dissecting the biology of a pathogen during infection. Trends Microbiol 1997, 5:509-513.
5.
Lee SH, Camilli A: Novel approaches to monitor bacterial gene expression in infected tissue and host. Curr Opin Microbiol 2000, 3:97-101.
6.
Heithoff DM, Sinsheimer RL, Low DA, Mahan MJ: In vitro gene expression and the adaptive response: from pathogenesis to vaccines and antimicrobials. Phil Trans R Soc Lond B Biol Sci 2000, 355:633-642.
7.
Osbourn AE, Barber CE, Daniels MJ: Identification of plant-induced genes of the bacterial pathogen Xanthomonas campestris pathovar campestris using a promoter-probe plasmid. EMBO J 1987, 6:23-28.
8.
Mahan MJ, Slauch JM, Mekalanos JJ: Selection of bacterial virulence genes that are specifically induced in host tissues. Science 1993, 259:686-688.
9.
Mahan MJ, Tobias JW, Slauch JM, Hanna PC, Collier RJ, Mekalanos JJ: Antibiotic-based selection for bacterial genes that are specifically induced during infection of a host. Proc Natl Acad Sci USA 1995, 92:669-673.
10. Conner CP, Heithoff DM, Julio SM, Sinsheimer RL, Mahan MJ: Differential patterns of acquired virulence genes distinguish Salmonella strains. Proc Natl Acad Sci USA 1998, 95:4641-4645. 11. Camilli A, Mekalanos JJ: Use of recombinase gene fusions to identify Vibrio cholerae genes induced during infection. Mol Microbiol 1995, 18:671-683.
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12. Lee SH, Hava DL, Waldor MK, Camilli A: Regulation and temporal •• expression patterns of Vibrio cholerae virulence genes during infection. Cell 1999, 99:625-634. An important objective of studies of bacteria in vivo is to understand when and where specific genes are expressed. This paper shows how the RIVET strategy can be used to monitor patterns of gene regulation within host tissue.
22. Young GM, Miller VL: Identification of novel chromosomal loci affecting Yersinia enterocolitica pathogenesis. Mol Microbiol 1997, 25:319-328. 23. Kilic AO, Herzberg MC, Meyer MW, Zhao XM, Tao L: Streptococcal reporter gene-fusion vector for identification of in vivo expressed genes. Plasmid 1999, 42:67-72.
13. Mahan MJ, Slauch JM, Mekalanos JJ: Bacteriophage P22 transduction of integrated plasmids: single-step cloning of Salmonella typhimurium gene fusions. J Bacteriol 1993, 175:7086-7091.
24. Janakiraman A, Slauch JM: The putative iron trapsort system SitABCD encoded on SPI1 is required for virulence of Salmonella typhimurium. Mol Microbiol 2000, 35:1146-1155.
14. Rainey PB, Heithoff DM, Mahan MJ: Single-step conjugative cloning of bacterial gene fusions involved in microbe–host interactions. Mol Gen Genet 1997, 256:84-87.
25. Camilli A, Beattie DT, Mekalanos JJ: Use of genetic recombination as a reporter of gene expression. Proc Natl Acad Sci USA 1994, 91:2634-2638.
15. Wang J, Mushegian A, Lory S, Jin S: Large-scale isolation of candidate virulence genes of Pseudomonas aeruginosa by in vivo selection. Proc Natl Acad Sci USA 1996, 93:10434-10439.
26. Staib P, Kretschmar M, Nichterlein T, Kohler G, Michel S, Hof H, •• Hacker J, Morschhauser J: Host-induced, stage-specific virulence gene activation in Candida albicans during infection. Mol Microbiol 1999, 32:533-546. Reports the development of a recombinase-based IVET strategy for use in a fungus and its application to the study of pathogenesis.
16. Fuller TE, Shea RJ, Thacker BJ, Mulks MH: Identification of in vivo • induced genes in Actinobacillus pleuropneumoniae. Microb Pathog 1999, 27:311-27. This paper describes a riboflavin-based IVET strategy for selecting genes actively transcribed in the pig lung. 17. •
Retallack DM, Deepe GSJ, Woods JP: Applying in vivo expression technology (IVET) to the fungal pathogen Histoplasma capsulatum. Microb Pathog 2000, 28:169-182. The authors describe a uracil-based IVET strategy for a fungal pathogen to enable selection of virulence genes expressed in a mouse model. 18. Rainey PB: Adaptation of Pseudomonas fluorescens to the plant •• rhizosphere. Environ Microbiol 1999, 1:243-257. This paper describes the development and application of an IVET strategy from first principles for studying genes induced in the plant rhizosphere. 19. Gal M: Development of in vivo expression technology (IVET) and its use to isolate Pseudomonas fluorescens genes induced in the plant rhizosphere. [D. Phil. Thesis]. Oxford: University of Oxford; 1999. 20. Hoang TT, Williams S, Schweizer HP, Lam JS: Molecular genetic analysis of the region containing the essential Pseudomonas aeruginosa asd gene encoding aspartate-beta-semialdehyde dehydrogenase. Microbiol 1997, 143:899-907. 21. Handfield M, Lehoux DE, Sanschagrin F, Mahan MJ, Woods DE, Levesque RC: In vivo-induced genes in Pseudomonas aeruginosa. Infect Immun 2000, 68:2359-2362.
27. ••
Oke V, Long SR: Bacterial genes induced within the nodule during the Rhizobium-legume symbiosis. Mol Microbiol 1999, 32:837-849. The authors describe an elegant extension of the basic IVET strategy to enable isolation of enes induced during infection and differentiation of legume nodules.
28. Gahan CG, Hill C: The use of listeriolysin to identify in vivo • induced genes in the Gram positive intracellular pathogen Listeria monocytogenes. Mol Microbiol 2000, 36:498-507. The first IVET strategy that uses toxin production as a selectable phenotype. 29. Jarrett C, Franklin MJ: Development of a selection strategy to •• identify Pseudomonas aeruginosa genes induced during surfaceassociated growth. American Society for Microbiology General Meeting: 1999 May 30–June 3; Chicago. Washington: American Society for Microbiology; 1999. An elegant IVET strategy for the isolation of surface-active genes. 30. Slauch JM, Mahan MJ, Mekalanos JJ: Measurement of transcriptional activity on pathogenic bacteria recovered directly from infected host tissue. BioTechniques 1994, 16:641-644. 31. Handfield M, Brady LJ, Progulske-Fox A, Hillman JD: IVIAT: a novel method to identify microbial genes expressed specifically during human infections. Trends Microbiol 2000, 8:336-339.
In vivo expression technology strategies: valuable tools for biotechnology Paul B Rainey* and Gail M Preston† Whole genome sequences have shown that bacteria possess a significant number of genes that have no known function. It is probable that many of these are required for survival in environments other than the agar plate. In vivo selection strategies provide a means of obtaining genes active in complex natural environments. Direct access to these genes is essential for understanding ecological performance and provides novel opportunities for biotechnology. Addresses Department of Plant Sciences, University of Oxford, South Parks Road, Oxford OX1 3RB, UK *e-mail: [email protected] † e-mail: [email protected] Current Opinion in Biotechnology 2000, 11:440–444 0958-1669/00/$ — see front matter © 2000 Elsevier Science Ltd. All rights reserved. Abbreviations DAP diaminopimelate IVET in vivo expression technology RIVET recombinase-based IVET
Introduction For most of the past century, microbiology has been devoted to studying the physiology and genetics of bacteria in vitro. This means that we now understand a great deal about the lives of bacteria on agar plates, but have little understanding of the determinants of ecological success in the wild. Without an understanding of the causes of ecological success our understanding of the biology of bacteria will remain incomplete and the full potential of these organisms in biotechnology will remain unrealised. Many strategies have been used to study ecological performance [1–3]. Bottom-up (genes to population) and top-down (population to genes) approaches are both useful. The bottom-up approach is commonly employed for studies of bacteria, although is rarely pursued to the population level. The typical genes-to-phenotype strategy involves identification of traits on the basis of gene inactivation. This is a powerful approach that has been fundamental to the majority of advances in molecular microbiology, but despite its power, insertional mutagenesis is not always appropriate for the analysis of phenotypes as complex as ecological performance. For most organisms, in most environments, there is no primary determinant of ecological performance; this is because it is determined by complex epistatic interactions among many different gene products that each has a long evolutionary history. Traits having greatest effect on ecological performance are likely to be those that show subtle quantitative variation and such traits are unlikely to produce ‘defective’ phenotypes when inactivated [3].
Recent advances in gene fusion technologies provide an alternative way to study complex phenotypes. Rather than identifying genes on the basis of function loss, ecologically significant genes can be identified on the basis of their positive contribution toward a specific phenotype. A study wishing to understand the mechanistic basis of ecological performance in a leaf colonising biocontrol isolate might therefore begin by identifying those genes that are specifically induced in the leaf environment. One advantage of this approach is that it considers bacteria as integrated organisms rather than as a toolbox of independent genes and phenotypes. A second advantage, of particular relevance to biotechnology, is the opportunities to access novel traits. Most significantly, bottom-up, positive selection approaches open the door to understanding ecological performance in bacteria at a mechanistic level in the wild. In vivo expression technology (IVET) strategies, despite their tremendous power, have been little used, except in studies of animal pathogenicity. This brief review is intended to draw attention to IVET strategies, their development, and potential in biotechnology. Length prevents discussion of biological insights gained from these strategies and we refer the reader to primary sources and to two recent reviews [4–6].
IVET: the basic strategy The first gene fusion technology involving positive selection was developed to study pathogenicity in Xanthomonas campestris on turnip seedlings [7]. The strategy involved selection of environment-specific genes on the basis of their ability to drive expression of a gene essential for survival. In 1993, this strategy was modified and extended to the study of Salmonella pathogenicity in a mammalian host where the term ‘in vivo expression technology’ was coined [8]. IVET has since been applied extensively to the study of Salmonella virulence and has resulted in the discovery of many novel genes. It has provided valuable insights into the ecology of the host environment [4,9,10] and has recently been used to explore regulation and temporal expression patterns of Vibrio cholerae virulence genes during infection [11,12••]. The basic IVET strategy is described in Figure 1 — modifications are discussed below. The strategy is based upon a bacterial strain with a conditional mutation in a biosynthetic gene that is involved in the synthesis of an essential growth factor. Provided that exogenous levels of the growth factor are negligible, then growth in the wild cannot occur unless the missing gene is introduced into the bacterial cell. Introduction into the mutant strain of a promoterless but otherwise functional copy of the biosynthetic gene, to which random fragments of wild-type DNA have been cloned (in place of the promoter) to facilitate
IVET strategies Rainey and Preston
incorporation of the gene into the host genome, provides a means of selecting for promoters that are active in the environment. Selection of such promoters (and corresponding genes) is performed simply by recovery of bacteria that have grown in the wild — growth in the wild can only occur if the function of the mutated biosynthetic gene has been restored. This can only occur where transcription of the promoterless biosynthetic gene is initiated. A subsequent screening step using a second promoterless reporter gene (e.g. ‘lacZ) facilitates differentiation of those promoters active solely in the wild from those that are active both in the wild and in vitro (Figure 1).
Development of IVET strategies There are two essential phases in the development of an IVET strategy. The first requires a decision as to the strength and specificity of the selective regime and involves construction of a conditionally compromised strain. The second phase involves construction of a vector for trapping environment-induced promoter–gene fusions. Both phases are essential, but the first is critical and determines ultimate success and utility. The strength and specificity of selection determines the breadth of environmentally responsive fusions recovered. A study seeking to identify all genes activated in a particular environment is likely to use a general strategy, based, for example, on a strain deficient in the biosynthesis of an essential growth factor (and ideally would use two complementary strategies that use different selective regimes). A weak selective regime has the advantage of isolating a wide range of environment-response genes, but risks false positives. A strong selective regime is likely to recover only highly induced promoters, risking, if selection is too severe, identifying only constitutively expressed fusions and missing transiently expressed genes. Identification of genes induced under specific environmental conditions requires a selection strategy based on a gene that is essential for survival in that specific environment. This can be difficult to achieve and is usually limited to well-studied systems where genes essential for specific environmental challenges are already known, but novel selective regimes are being developed (see below). Development of the selective ‘vehicle’ (phase 2) is relatively straightforward. At its simplest it is a suicide plasmid with multiple cloning sites, selectable antibiotic resistance marker and a promoterless, but otherwise functional, copy of the gene that forms the basis of the selective regime. This is typically fused to a second (and sometimes third) promoterless reporter gene, such as lacZ (β-galactosidase), uidA (β-glucuronidase), gfp (green fluorescent protein), or a combination (see Figure 1) to enable differentiation between constitutive and environment-induced promoters. A final consideration is the recovery of chromosomally integrated fusions following selection — a stage that can be problematic and partly responsible for the slow application
441
Figure 1
Clone fragments of genomic DNA into pIVET
pIVET
+
‘egf ‘lacZY MCS Integrate into genome of strain carrying a deletion of egf ‘egf ‘lacZY ∆egf
pIVET Inoculate environment with library of fusions
Harvest bacteria from environment after 14 days
E N V I R O N M E N T
Re-inoculate environment with putative environmentinduced fusions to confirm induction
Screen for β-gal activity on agar plates. White colonies contain fusions to putative environmentspecific genes
Selective medium containing X-gal and essential growth factor Current Opinion in Biotechnology
Selection for genes showing environment-specific elevation in expression. IVET is a promoter-trapping strategy that selects for genes displaying elevated levels of expression on the basis of their ability to drive the expression of a gene that is essential for survival in a given environment. The basic strategy is based upon random integration of a promoterless ‘essential growth factor’ (‘egf) gene into the chromosome of a bacterial strain carrying a chromosomal deletion of egf. The egf deletion strain cannot grow in the environment because the essential growth factor is absent or severely limiting; growth can only occur if the promoterless ‘egf gene is inserted downstream of an active promoter. Recovery of strains from the environment following selection for the ability to synthesise the essential growth factor results in the isolation of promoters that are either constitutive or environment-specific. In order to distinguish between these two, a promoterless marker operon, for example, lacZY (β-galactosidase [β-gal]), is fused to ‘egf, thus enabling the lactose phenotype of the recovered cells to be determined. Lac+ strains contain fusions to constitutive promoters (dark colonies), whereas Lac– strains (white colonies) contain fusions to promoters active in response to environmental signals.
and development of IVET strategies in biotechnology. For a thorough analysis it is necessary to screen large numbers of environmentally induced fusions by DNA sequencing.
442
Expression vectors and delivery systems
This relies upon an efficient means of recovering IVET fusion plasmids from the host chromosome. In Salmonella, recovery of fusions is readily achieved by transduction using bacteriophage P22 [13], but for many bacteria transducing phage are not available. Fusions can be cloned by standard molecular biology, but these techniques are cumbersome and not suitable for the recovery of large numbers of fusions. An alternative strategy is conjugative cloning, which uses a helper plasmid to provide transfer functions that enable the rescue and transfer of any integrated IVET plasmid into a replication-permissive Escherichia coli host [14]. This is a simple technique that has been shown to work in enteric bacteria, Pseudomonas and Rhizobium. There is no reason for it not to be generally applicable.
General selective strategies Knowledge of the nutritional status of the targeted environment is an important aspect in the development of strategies using nutritionally compromised mutants. Without knowledge of limiting nutrients it is difficult to chose suitable selective strategies (although see RIVET strategies below). In the mammalian host environment, general selective strategies for Salmonella [8] and Pseudomonas aeruginosa [15] have employed purine auxotrophy (purA); thymine auxotrophy has also proved useful [8]. Recent work on Actinobacillus pleuropneumoniae has used a riboflavin-requiring mutant to study patterns of gene expression in the pig lung [16•], and uracil selection has been used to isolate genes expressed by the fungal pathogen Histoplasma capsulatum during infection of a mouse model [17•]. For use in the plant–soil environment, three general strategies have been developed. Two were developed for Pseudomonas fluorescens following a screen of a range of nutritionally compromised transposon mutants for lack of growth in a model sugar beet rhizosphere. One is based on the water-soluble vitamin pantothenate (panB) [18••] (which has since been extended to Rhizobium leguminosarum [D Allaway, N Schofield, PS Poole, personal communication]). The other is based on a combined diaminopimelate (DAP)- and lysine-requiring auxotroph caused by a deletion of dapB [19] (M Gal, G Preston, P Rainey, unpublished data). The third strategy was developed for leaf-colonising P. syringae and is based upon methionine auxotrophy (metXW) (M Marco, S Lindow, personal communication). Of these three strategies, DAP-based selection is applicable to an extensive range of environments — it is a solely bacterial product, absent from soil, water, plant and animal surfaces and tissues and is an essential component of the peptidoglycan of all Gram-negative and some Gram-positive bacteria. DAP auxotrophy is lethal and causes lysis of rapidly growing cells, although non-growing cells remain viable for long periods, which ensures that selection is not too stringent [18]. A selective regime with similar broad applicability has been developed for P. aeruginosa and is based on auxotrophy for DAP, threonine, lysine and methionine, caused by
deletion of the gene encoding aspartate beta-semialdehyde dehydrogenase (asd) [20,21]. Two general selection strategies that largely eliminate the need for conditional lethal mutants have also been developed. One employs antibiotic-based selection and simply requires transcriptional fusions to a promoterless copy of an antibiotic-resistance gene and selection in an environment replete in the antibiotic. This has been successful in mammalian models of infection [9,22–24], but less so in other environments where the presence of large amounts of antibiotic is either impractical or likely to radically alter the environment [7]. The second strategy is a recombinase-based IVET (RIVET), developed originally to identify infectioninduced genes in Vibrio cholerae [11,25]. RIVET is based on a transcriptional reporter that encodes a site-specific DNA recombinase, which catalyses excision of a selectable substrate gene cassette (e.g. tetracycline resistance) from the bacterial genome. Evidence of promoter activity in the wild is inferred from the products of genetic recombination (loss of tetracycline resistance), which acts as a heritable reporter. The elegance of this strategy is twofold. Firstly, it overcomes a criticism often levelled at IVET strategies, namely, that they fail to detect transiently expressed genes. Secondly, the strategy has considerable general utility (requiring only magnesium ions and negatively supercoiled DNA) and is applicable to fastidious organisms and to viruses and eukaryotes. Indeed, development of such a technology has recently been reported for Candida albicans [26••], based upon FLP-mediated recombination with the enzyme inosine monophosphate (IMP) dehydrogenase as a deletable marker.
Specific selective strategies An important goal of many studies, particularly of microbe–host interactions, is to identify and characterise genes that contribute to specific processes, for example, colonisation or parasitism of hosts. This has been elegantly achieved using specific selective regimes that are exemplified in studies of plant–microbe interactions. To identify Rhizobium meliloti genes induced within root nodules during the infection and differentiation stages of symbiosis Oke and Long [27••] used a R. meliloti strain defective in the expression of bacA (transcriptionally fused to uidA), a gene required for bacteroid differentiation. Selection for active promoters was readily achieved by harvesting bacteria from nodules because only strains containing fusions to active promoters could cause nodule development. Those fusions specific to infection and differentiation were distinguished from constitutive promoters by determining β-glucuronidase activity under free-living conditions. Conceptually similar strategies have been developed to study parasitism of plants by Xanthomonas campestris (D Nennstiel, U Bonas, personal communication) and P. syringae, (J Boch, V Joardar, L Gao, T Robertson, B Kunkel, personal communication). The
IVET strategies Rainey and Preston
selective regime, in both cases, is a result of a defect in the hrp cluster (hrpB1 and hrcC, respectively) which is required for pathogenicity. A similar strategy was recently reported for the intracellular pathogen Listeria monocytogenes, based on a listeriolysin (toxin) negative mutant [28•]. A particularly innovative example has been described for isolating genes specific for colonisation of surfaces by P. aeruginosa [29••]. The strategy is based upon a promoterless reporter operon composed of sacB (levan sucrase), aacC1 (gentamicin resistance) and gfp. A library of strains containing transcriptional fusions to this synthetic operon are selected in shaken broth culture in the presence of sucrose, which, because of the toxicity of sacB in a sucrose-rich environment, eliminates strains containing fusions to genes active in the liquid-phase. The remaining cells are then selected on a solid surface in the presence of gentamycin to select genes specific to surface colonisation. The potential of this and similar selective strategies is considerable, and limited only by imagination.
Monitoring activity of bacteria in the environment IVET strategies generate a chromosomal fusion between a tagged (or trapped) gene and a selectable maker that is typically fused to a second reporter — part of the IVET plasmid. Generation of a transcriptional fusion to the primary selectable marker is essential for IVET selection, whereas the second reporter enables constitutive fusions to be distinguished from those that are environment-specific. The second reporter can also be used, however, to monitor spatial and temporal activity of any given fusion. In the case of standard IVET strategies, reporters such as lacZ or uidA can generate quantitative data on precise levels of transcription in different environments and at different times [8,18,30]. These reporters can also be used for in situ studies, although of more use are reporters such as gfp or lux, which allow detection of activity at the level of individual bacterial cells. The recombinase reporter at the heart of RIVET also enables spatial and temporal information on patterns of gene expression to be determined simply by scoring the frequency of tetracycline-sensitive cells isolated from different environments. Such an approach was recently used by Lee et al. [12••] to determine temporal expression patterns of V. cholerae virulence genes during infection of a host.
Conclusions IVET strategies enable a direct insight into the lives of bacteria in natural environments. Such knowledge stands to benefit biotechnology in multiple ways. The discovery of new compounds and processes and enzymes is one immediate benefit, but at a more fundamental level, knowledge of patterns of gene expression in the wild can ultimately provide insights into the causes of ecological success. Knowledge of these causes is as important for the design of efficient biocontrol strains, as it is for the construction of strains for industrial fermentations.
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Update Recently, a further novel extension of IVET has been described, termed IVIAT (in vivo induced antigen technology), that allows identification of in vivo induced genes in human infections without the use of animal models [31]. The strategy takes sera from patients currently experiencing infection from the pathogen of interest and absorbs this against the pathogen that has been grown in vitro, thus leaving in the serum antibodies against antigens that are expressed during infection but not during in vitro cultivation. To obtain bacterial genes expressing the antibodies of interest, the absorbed serum is challenged with an expression library of the pathogen’s DNA. The strategy is simple to use and readily adapted to a variety of different purposes. For a recent review of IVET strategies and their application in pathogenic bacteria, see the comprehensive account by Heithoff et al. [6].
Acknowledgements This work is supported by grants from the BBSRC, NERC and the European Union.
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