Genetic engineering strategies for environmental applications Vfctor de Lorenzo Centro de Investigaciones Biol6gicas-CSIC, Madrid, Spain Environmental applications of genetically engineered microorganisms are currently hampered not only by legal regulations restricting their release, but also by the frequent dearth of adequate genetic tools for their construction in the laboratory. Recent approaches to strain development include the use of non-antibiotic markers as selection determinants, the use of transposon-vectors for the permanent acquisition of recombinant genes, and the utilization of expression devices based on promoters from promiscuous plasmids and biodegradative pathway genes. Current Opinion in Biotechnology 1992, 3:227-231
Introduction In spite of the tremendous potential of genetically engineered microorganisms (GEMs) in environmental, agricultural and mining applications [1], the fact is that very few examples of actual utilization in the field have b e e n recorded in recent years. Restrictive legal regulations, raised by the unpredictability of GEMs in natural environments [2"q, are to be blamed in part for the lack of progress, but gaps in the genetic technology available for the construction of GEMs in the laboratory have b e c o m e major bottlenecks as well. GEM predictability requires at least three genetic factors: recombinant gene stability, gene expression and gene containment, all of which are essential for the realistic design of strains capable of growth in conditions which are totally unlike those of the laboratory. Moreover, GEMs with properties such as sensitivity to conventional antibiotics and tractable phenotypes are attractive, as they facilitate monitoring in natural habitats and the avoidance of undue spread of resistance genes. Although numerous bacterial species show p h e n o types of environmental and agricultural interest, state of the art technologies permit genetic engineering of only a few of them. Consequently, applications in the field, apart from the enterobacteria, are limited to Pseudomonas and related genera such as Alcaligenes, Rhizobium, Agrobacterium, Acinetobacter and Rhodobacter. Other genera with tremendous metabolic potential such as the filamentous bacteria (e.g. Streptomyces), the 'extremophilic' bacteria (e.g. Thiobacillus and Sulfolobus) and fungi, must await further advances in genetic technology before recombinant strains with engineered traits can be devised for release into the environment.
This review will discuss recent a p p r o a c h e s to the construction of GEMs for uncontained applications, focusing mainly on vector d e v e l o p m e n t for Pseudomonas and related species. Several designs for recombinant live vaccines have potential as tools for devising GEMs for release into the environment and will also be discussed here. Monitoring of GEMs in the environment has been reviewed elsewhere [3,4"] and is also discussed by Stahl and Kane ( p p 244-252).
Unconventional selection markers for GEMs The most widely used selection markers for genetic manipulations are antibiotics of therapeutic value, but the use of such markers is undesirable where bacteria are to be released in large quantities. Some alternatives have therefore b e e n developed. One involves the use of herbicide resistance genes like the bargene of Streptomyces hygroscopicus which, w h e n expressed with a functional promoter, affords resistance to the herbicide bialaphos in a n u m b e r of Gram-negative bacteria [5"q. The bar gene has b e e n used as a selection marker in transposon vectors (see below). In addition, it forms active 3'-terminal gene fusions [6], which may further increase its environmental applications if combined with other markers or reporter genes. Other possible markers involve resistance to heavy metal ions. Genes conferring resistance to both arsenite and mercuric salts or organomercurials have b e e n used as selection markers in Tn5- and Tnl0-derived transposon vectors
[5"]. An alternative to selection of resistance genes involves the use of specialized host-vector systems, in which an essential gene that has b e e n precisely deleted from
Abbreviation
GEM--genetically engineeredmicroorganism. © Current Biology Ltd ISSN 0958-1669
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Environmental biotechnology the host c h r o m o s o m e is supplied by a recombinant plasmid [7,8"',9,10"]. This permits direct selection of recombinant clones without the addition of selective agents. This concept has b e e n successfully tested in the construction of live vaccine candidates [7] and in devising systems to eliminate plasmid-less ceils from a reactor [9], but has not yet b e e n used for environmental applications. Broad host range plasmids carrying the thyA+/thymidylate synthase autoselective marker have b e e n constructed which employ a thyA- mutant strain as the host [8"']. Another approach used to obtain engineered strains devoid of antibiotic resistance is the insertion of the gene(s) of interest into the c h r o m o s o m e of the target bacteria b y means of h o m o l o g o u s flanking sequences [11,12"]. The cloning vectors used may carry an antibiotic resistance gene, which is eventually lost after the double recombination event. This approach, although very simple, has not b e e n widely used in Pseudomonas, perhaps because of the difficulty in selecting for double crossing-over events.
Plasmid vectors An impressive collection of broad host-range plasmid vectors, based on the IncQ, IncP-1 and IncW replicons, is available [13]. The best d e v e l o p e d are those derived from the IncQ group (RSF1010 and related), which include cloning, p r o m o t e > p r o b i n g and regulated expression vectors [14",15"]. These plasmids may prove to b e inadequate, however, w h e n the time comes to maintain and express a recombinant p h e n o t y p e in the field. Virtually all available vectors carry resistances to antibiotics and only a f e w plasmids with alternative markers have been d e v e l o p e d [8"']. More importantly, recombinant plasmids tend to be unstable in the absence of selective pressure. This p r o b l e m may be overcome by including cassettes with the parB (hok/sok) system in the constructions [16], which eliminates plasmid-less segregants. This stabilization system has b e e n successfully used in Pseudomonas [16] and in recombinant intestinal colonizers [17]. Alternatively, autoselection plasmids of the type mentioned above [7,8"',9] could be used. New replicons with the potential for further development as plasmid vectors tailored for applications in the field have been isolated. Among t h e m is pPS10, a plasmid originally isolated from a strain of P. savastanoi which colonizes the olive tree [18"]. This plasmid replicates in Pseudomonas, and its basic replicon consists of only a short or/.Vand the gene for the trans-acting and auto-regulated replication protein RepA [19]. In spite of the large number of n e w natural plasmids which have b e c o m e available recently, none are yet able to contest RSF1010 as the favourite replicon for broad hostrange vector development. This plasmid, which was first found nearly 20 years ago, was last year shown to possess the astonishing ability to replicate not only in
a variety of Gram-negative bacteria, but also in Grampositives, including Streptomyces and Mycobacterium species [20"q. This will surely change s o m e current ideas o n intergeneric barriers to genetic interchange, and will raise some concern over the risks of dissemination of recombinant genes cloned in RSF1010 derivatives and other promiscuous replicons. Additionally, this plasmid is b o u n d to be an excellent source not only of a broad host-range replication machinery, but also of promiscuous promoters and translation initiation regions (derived from the plasmid's replication protein genes and resistance genes) of remarkable potential for vector development.
Transposon vectors The use of transposons as cloning vectors has received notable attention during the last few years, as they are e x p e c t e d to increase the stability of cloned genes once they are inserted into the c h r o m o s o m e s of GEMs. Archetypal natural transposons, such as Tn5, Tnl0, Tn7 and Tn9, are large and limited in the n u m b e r of permissive sites available for cloning. Moreover they contain antibiotic resistance genes and m a y re-transpose themselves. Some attempts at modification have therefore b e e n m a d e to adapt t h e m for engineering purposes. Early attempts to use transposons as vectors w e r e based o n Tn7 [21], but more recently, several types of transposon vectors for use in Gram-negative bacteria have b e c o m e available. Among them is a collection of vectors b a s e d on the properties of IS1 [22]. In this system, a mobile pBR322-derived plasmid was used to assemble a defective c o p y of the IS1 transposase gene acting in cis, but external to two ends of IS1 flanking an antibiotic resistance gene, along with several restriction sites, which w e r e included for cloning purposes. Non-enteric exconjugants, which carry chromosomal insertions of the region flanked by the IS1 termini, can then be isolated. The same concept, although in a m o r e elaborate form, is present in the series of Tn5- and Tnl0-derived mini-transposon vectors containing non-antibiotic selection determinants [5",23"]. These w e r e specifically designed to construct GEMs for environmental applications, and they provide a straightforward procedure for cloning and stably inserting foreign genes into the c h r o m o s o m e s of various Gram-negative bacteria. The modular nature of the constructions facilitates the design of m a n y types of elements in which n e w selective markers m a y b e introduced, and in which promoter probes and a w h o l e range of expression devices may be engineered. Furthermore, these mini-transposons permit target ceils to undergo several rounds of transposition, and therefore the n u m b e r of insertions in the same strain is limited only b y the availability of distinct selection markers. By a less sophisticated approach, Tn5-derived constructions with several extra restriction sites have b e e n p r o d u c e d which facilitate cloning of heterologous DNA fragments [24"].
Genetic engineering strategies for environmental applications de Lorenzo 229 Gene expression 'in the field' GEMs destined for release into the environment should express their recombinant genes under the control of signals present in the location where the bacteria are expected to grow. This is also important for the design of built-in genetic circuits of containment to impede strain proliferation and/or horizontal gene transfer (see below). The utilization of promoters which have b e e n widely used in Esherichia coli genetics (e.g. Ptac, Ptrp, PL/R etc.) in released GEMs is unrealistic owing to the high cost of the inducer and the complications experienced in activating the system. A possible alternative is the use of regulated promoters from catabolic plasmids of Pseudomonas. Broad host-range plasmids are available which contain the Pm promoter of the TOL plasmid, along with its native regulator xylS [25], or mutant varieties like xylS2 [26], and which will cause transcription of heterologous genes in response to the addition of various benzoates. Yen [27" ] has constructed an expression cassette based on the nahR-regulated PG promoter of the NAH7 plasmid, which directs salicylateinducible transcription of downstream heterologous genes. This type of expresion device permits the design of recombinant strains with p h e n o t y p e s that are responsive to chemical signals present in the contaminated location even, in s o m e cases, to the pollutants to be degraded. Transposon vectors [23"] allow the construction of mobile elements in which a promoter-less gene of interest is inserted at random in front of chromosomal promoters so that a specific level of expression m a y b e chosen. This procedure has b e e n used successfully to produce pertussis toxin in Bordetella bronchiseptica [28"] and should also be of use for environmental GEMs. Promoters and expression cassettes derived from natural catabolic and promiscuous plasmids, phages [29,30] and transposons [31] should be further explored for use in expression systems. The T7-polymerase system, which has a remarkable potential for soil bacteria, has b e e n used very rarely and only for expression in laboratory conditions [30]. Combination of the T7 polymerase gene with catabolic promoters or growth-phase d e p e n d e n t promoters should afford the engineering of complex phenotypes to be manifested as a response to the presence of specific chemicals, or during a certain stage of growth.
rate schemes which e m p l o y the regulatory network of the TOL plasmid to generate a model containment circuit for biodegradative strains, based on the toxic hoklike, gefgene. Unfortunately, the hok-related systems are not 100 % efficient [33"',34,35"]. The high frequency of surviving cells demonstrates the need for more complex circuits to ensure effective containment in the environment.
Perspectives Although m a n y broad host-range vectors are available for the genetic engineering of Gram-negative bacteria, procedures to ensure the ecological predictability of GEMs are still in the very early stages of development. Efforts to engineer n e w characteristics must be c o m p l e m e n t e d by novel genetic approaches to ensure that the recombinant genes involved perform properly in the field and do not cause undesirable side effects. Surprisingly, this aspect has received very little attention, and virtually no patents addressing this issue, and only a small n u m b e r of papers describing purpose-specific tools, have been published in the last few years. Transposon vectors, promoters and translation initiation regions derived from promiscuous plasmids will probably be the preferred elements for genetic engineering of strains p r o g r a m m e d to benefit the environment safely.
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.
LINDOWSE, PANOPOULOS NJ, MCFARLAND BL: G e n e t i c Engin e e r i n g o f Bacteria f r o m M a n a g e d a n d N a t u r a l Habit a t s . Science 1989, 244:1300-1307.
2. COLWELLR: R i s k A s s e s s m e n t i n E n v i r o n m e n t a l B i o t e c h •. n o l o g y . Curr Opin Btotechnol 1991, 2:470475. A short review on issues, both legal and scientific, related to the ecological risks of biotechnology. This article points out the need to know more about microbial ecology before the impact of GEMs in natural ecosystems can be realistically evaluated. 3.
PICKUPRW, SAUNDERSJR: D e t e c t i o n o f Genetically Eng i n e e r e d Traits a m o n g Bacteria i n t h e E n v i r o n m e n t . Trends Biotechnol 1990, 8:329-334.
4.
Strain and gene containment D e v e l o p m e n t of genetic circuits to i m p e d e proliferation of recombinant bacteria in conditions other than those in which they have b e e n designed to grow is an important, but challenging aspect o f GEM construction. Endowing strains with mutations to prevent undesirable spread was the only a p p r o a c h available until Molin [32] developed a conditional suicide device b a s e d on the properties of the lethal hok gene. Contreras et al. [33"'] have recently d e v e l o p e d elabo-
PICKUPRW: D e v e l o p m e n t o f M o l e c u l a r M e t h o d s f o r t h e D e t e c t i o n o f S p e c i f i c Bacteria in t h e E n v i r o n m e n t . J Gen Microbiol 1991, 137:1009-1019. A review of n e w procedures for following the fate of bacteria and recombinant genes can'ied by GEMs in natural habitats. HERREROM, DE LORENZO V, TIMMIS KN: Transposon Vectors Containing Non-antibiotic Selection Markers for C l o n i n g a n d Stable C h r o m o s o m a l I n s e r t i o n o f Fore i g n DNA i n G r a m - n e g a t i v e Bacteria. J Bacteriol 1990, 172:6557-6567. Description of a collection of n e w transposon-based tools tailored for constructing GEMs for environmental applications and live vaccine development. Enormous possibilities of further development for specific applications in the field. 5. •.
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BO~q'eP, MAN J, GOSSELg V, THOEN C, LAUWEREYSM: C h a r a c terization of Phosphinothricin Acetyltransferase and C - t e r m i n a l E n z y m a t i c a l l y Active F u s i o n P r o t e i n s . Gene 1991, 102:33-37.
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NAKAYAMAK, KELLY SM, CURTISS R III: C o n s t r u c t i o n o f a n ASD + E x p r e s s i o n - c l o n i n g Vector: Stable M a i n t e n a n c e a n d H i g h Level o f E x p r e s s i o n o f C l o n e d G e n e s i n a Salmonella V a c c i n e Strain. Biotechnology 1988, 6:693~597
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Ross P, O'GARA F, CONDON S: T h y m i d y l a t e S y n t h a s e G e n e f r o m Lactococcus lactis as a G e n e t i c M a r k e r : Art Alt e r n a t i v e to A n t i b i o t i c R e s i s t a n c e Genes. Appl Environ Microbiol 1990, 56:2164-2169. This paper reports one of the few attempts to develop plasmid vectors for environmental/agricultural applications without the u s e of antibiotics of clinical importance. The constructions described can be u s e d in s o m e soil bacteria, but the performance of the system in microcosms is u n k n o w n . 9.
PORTER RE), BLACK S, PANNURI S, CARLSON A: U s e o f Escherichia coil ssb G e n e to P r e v e n t B i o r e a c t o r T a k e o v e r b y P l a s m i d l e s s Cells. Biotechnology 1990, 8:47-50.
MORONAR, YEADON J, CONSIDINE A, MORONA J, MANNING, PA: C o n s t r u c t i o n o f P l a s m i d s V e c t o r s w i t h N o n - a n t i b i o t i c S e l e c t i o n S y s t e m B a s e d o n t h e Escherichia coil thyA G e n e : A p p l i c a t i o n to C h o l e r a V a c c i n e D e v e l o p m e n t . Gene 1991, 107:139-144. Describes a n auto-selection vector for enterobacteria similar in concept to that described in [8"']. Even t h o u g h the scenario for the release of GEMs is very different, the problem to solve (i.e. maintenance of recombinant g e n e s in the absence of antibiotic selection) is identical to that posed by recombinant bacteria for environmental applications.
18.
NIETO C; FERNANDEZ-TRESGUERRES E, SANCHEZ N, VICENTE M, DLAZ R: C l o n i n g Vectors f r o m a N a t u r a l l y O c c u r r i n g P l a s m i d o f Pseudomonas savastanoi, S p e c i f i c a l l y T a i l o r e d f o r G e n e t i c M a n i p u l a t i o n s i n Pseudomonas. Gene 1990, 87:145-149. Description of a collection of plasmid vectors derived from a n e w replicon w h i c h seems to be specific for Pseudomonas species. G o o d possibilities of future developments for environmental applications. 19. 20. •.
GORMLEYE, DAVIES J: T r a n s f e r o f P l a s m i d RSF1010 b y C o n j u g a t i o n f r o m Escherichia coli to Streptomyces lividans and Mycobacterium smegmatis, J Bacte~ol 1991, 173:6705-6708. This p a p e r will probably b e c o m e a landmark for studies o n genetic promiscuity. T h e authors s h o w that RSF1010 can not only be transfered conjugatively into two Gram-positive species, but can also replicate stably and express its antibiotic resistances in the n e w hosts. The promiscuity of functional elements of RSF1010 is a source of building blocks for broad host-range vector development. 21.
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STRUGNELLRA, MASKELL D, FAIRWEATHER M, PICKARD D, COCKAYNE A, PENN C, DOUGAN G: Stable E x p r e s s i o n of Foreign Antigens from the Chromosomes of Salmonella t y p h i m u r i u m V a c c i n e Strains. Gene 1990, 88:57-63. Gives a strategy for c h r o m o s o m a l insertion of heterologous g e n e s based o n h o m o l o g o u s recombination of flanking sequences. 13.
SCHMIDHAUSERTJ, DITrA G, HELINSK] DR: B r o a d H o s t R a n g e P l a s m i d C l o n i n g Vectors f o r G r a m - n e g a t i v e Bact e r i m In Vectors: A Survey of Molecular Cloning Vectors and their Uses. Edited by Rodriguez LR, Denhardt DT. Boston: Butterworths; 1987:287-332.
MORALESV, BAGDASARIAN MM, BAGDASARIAN M: P r o l n i s c u o u s P l a s m i d s o f t h e I n c Q G r o u p : M o d e o f Replicat i o n a n d Use f o r G e n e C l o n i n g i n G r a m - n e g a t i v e Bacteria. In Pseudomonas: Biotransformations, Pathogenesis and Evolving Biotechnology. Edited by Silver S. Washington DC: American Society for Microbiology; 1990:229-241. An excellent review of the history a n d recent developments of RSP1010-based plasmid vectors.
23.
DE LORENZO V, HERRERO M, JACUBZIK U, TIMMIS KN: MiniTrt5 T r a n s p o s o n D e r i v a t i v e s f o r I n s e r t i o n M u t a g e n e sis, P r o m o t e r P r o b i n g a n d C h r o m o s o m a l I n s e r t i o n o f C l o n e d DNA i n G r a m - n e g a t i v e E u b a c t e r i a . J Bacteriol 1990, 172:6568-6572. T r a n s p o s o n vectors containing a variety of selection markers a n d reporter g e n e s which further e x p a n d the u s e s of constructions described in [5"']. 24.
KANIGA K, DAVISON J: T r a n s p o s o n V e c t o r s f o r Stable Chromosomal Integration of Cloned Genes in Rhizos p h e r e Bacteria. Gene 1991, 100:201-205. Describes Tn5-based transposon vectors w h i c h are used to stably introduce a xylE marker g e n e to track the fate of Pseudomonas in the environment. 25.
MERMOD N, RAMOS JL, LEHRBACH PR, TIMMIS K_N: Vector for Regulated Expression of Cloned Genes in a W i d e R a n g e o f G r a m - n e g a t i v e B a c t e r i a . J Bacterlol 1986, 167:447-454.
26.
RAMOSJL, GONZ~LEZ-CARRER6 M, TIMMIS KN: B r o a d H o s t R a n g e E x p r e s s i o n Vectors C o n t a i n i n g M a n i p u l a t e d Meta-cleavage Pathway Regulatory Elements of the TOL P l a s m i d . FEBS Lett 1988, 226:241-246.
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MORALESV, B~CKMANA, BAGDASARIANM: A H o s t R a n g e L o w C o p y N u m b e r Vectors r e c t S c r e e n i n g f o r R e c o m b i n a n t s . Gene Description of the most up-to-date RSF1010-based tors.
Series o f W i d e t h a t A l l o w Di1991, 9 7 : 3 9 4 7 . expression vec-
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GEe,DES K: T h e p a r B (hok-sok) Locus o f t h e P l a s m i d RI: a G e n e r a l P u r p o s e P l a s m i d S t a b i l i z a t i o n S y s t e m . Biotechnology 1988, 6:1402-1405.
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BURGHOFFRL, LAUX DC, COHEN PS: C o n s t r u c t i o n o f Stable C l o n i n g Vectors t h a t d o n o t S e g r e g a t e f r o m a H u m a n F e c a l Escherichia coU S t r a i n i n t h e S t r e p t o m y c i n t r e a t e d M o u s e L a r g e I n t e s t i n e . Infect Immun 1990, 58:1141-1145.
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WALKERM, ROHDE M, WEHLANDJ, TIMMIS KN: C o n s t r u c t i o n o f M i n i - t r a n s p o s o n s f o r C o n s t i t u t i v e a n d I n d u c i b l e Exp r e s s i o n o f P e r t u s s i s T o x i n i n bvg-negative Bordeteila bronchiseptica. Infect Imm 1991, 59:4238-4248. Attractive u s e of mini-transposons to select a certain level of expression of a heterologous gene in a strain, the genetics of w h i c h are not well k n o w n . This approach has great promise for the design of environmental GEMs. 29.
FREY J, MUDD EA, K~ISCH H: A B a c t e r i o p h a g e T4 Expression Cassette that Functions Efficiently In a W i d e R a n g e o f G r a m - n e g a t i v e Bacteria. Gene 1988, 62:237-247.
30.
DAVISON J, CHEVALIER M, BRUNEL F. B a c t e r i o p h a g e T'7 RNA p o l y m e r a s e - c o n t r o l l e d Specific G e n e E x p r e s s i o n i n pseudomonas. Gene 1989, 83:371-375.
Genetic engineering strategies for environmental applications de Lorenzo 31.
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32.
MOLIN S, KLEMM P, POULSEN LK, BIEHL H, GERDES K, ANDERSSON P: C o n d i t i o n a l Suicide S y s t e m f o r Cont a i n m e n t o f B a c t e r i a a n d P l a s m i d s . Biotechnology 1987, 5:1315-1318.
33. •.
CONTRERASA, MOLIN S, RAMOS JL: C o n d i t i o n a l S u i c i d e Containment System for Bacteria which Mineralize A r o m a t i c s . Appl Environ Microbiol 1991, 57:1504-1508. First attempt to design a containment system adapted to biodegradarive strains. The goal is to destroy GEMs after the pollutant present in the m e d i u m has been degraded. The model system is set u p with E. coli a n d it works within certain limits. Adaptation to Pseudomonas is awaited.
34.
BEJ AK, PERLIN MH, ATLAS RM: M o d e l Suicide Vector for Containment of GeneticaUy Engineered Microorg a n i s m s . Appl Environ Microbiol 1988, 54:2472-2477.
35.
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Victor de Lorenzo, Centro de Investigaciones Bioi6gicas, Vel~zquez 144, 28006 Madrid, Spain.
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Introduction In spite of the tremendous potential of genetically engineered microorganisms (GEMs) in environmental, agricultural and mining applications [1], the fact is that very few examples of actual utilization in the field have b e e n recorded in recent years. Restrictive legal regulations, raised by the unpredictability of GEMs in natural environments [2"q, are to be blamed in part for the lack of progress, but gaps in the genetic technology available for the construction of GEMs in the laboratory have b e c o m e major bottlenecks as well. GEM predictability requires at least three genetic factors: recombinant gene stability, gene expression and gene containment, all of which are essential for the realistic design of strains capable of growth in conditions which are totally unlike those of the laboratory. Moreover, GEMs with properties such as sensitivity to conventional antibiotics and tractable phenotypes are attractive, as they facilitate monitoring in natural habitats and the avoidance of undue spread of resistance genes. Although numerous bacterial species show p h e n o types of environmental and agricultural interest, state of the art technologies permit genetic engineering of only a few of them. Consequently, applications in the field, apart from the enterobacteria, are limited to Pseudomonas and related genera such as Alcaligenes, Rhizobium, Agrobacterium, Acinetobacter and Rhodobacter. Other genera with tremendous metabolic potential such as the filamentous bacteria (e.g. Streptomyces), the 'extremophilic' bacteria (e.g. Thiobacillus and Sulfolobus) and fungi, must await further advances in genetic technology before recombinant strains with engineered traits can be devised for release into the environment.
This review will discuss recent a p p r o a c h e s to the construction of GEMs for uncontained applications, focusing mainly on vector d e v e l o p m e n t for Pseudomonas and related species. Several designs for recombinant live vaccines have potential as tools for devising GEMs for release into the environment and will also be discussed here. Monitoring of GEMs in the environment has been reviewed elsewhere [3,4"] and is also discussed by Stahl and Kane ( p p 244-252).
Unconventional selection markers for GEMs The most widely used selection markers for genetic manipulations are antibiotics of therapeutic value, but the use of such markers is undesirable where bacteria are to be released in large quantities. Some alternatives have therefore b e e n developed. One involves the use of herbicide resistance genes like the bargene of Streptomyces hygroscopicus which, w h e n expressed with a functional promoter, affords resistance to the herbicide bialaphos in a n u m b e r of Gram-negative bacteria [5"q. The bar gene has b e e n used as a selection marker in transposon vectors (see below). In addition, it forms active 3'-terminal gene fusions [6], which may further increase its environmental applications if combined with other markers or reporter genes. Other possible markers involve resistance to heavy metal ions. Genes conferring resistance to both arsenite and mercuric salts or organomercurials have b e e n used as selection markers in Tn5- and Tnl0-derived transposon vectors
[5"]. An alternative to selection of resistance genes involves the use of specialized host-vector systems, in which an essential gene that has b e e n precisely deleted from
Abbreviation
GEM--genetically engineeredmicroorganism. © Current Biology Ltd ISSN 0958-1669
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228
Environmental biotechnology the host c h r o m o s o m e is supplied by a recombinant plasmid [7,8"',9,10"]. This permits direct selection of recombinant clones without the addition of selective agents. This concept has b e e n successfully tested in the construction of live vaccine candidates [7] and in devising systems to eliminate plasmid-less ceils from a reactor [9], but has not yet b e e n used for environmental applications. Broad host range plasmids carrying the thyA+/thymidylate synthase autoselective marker have b e e n constructed which employ a thyA- mutant strain as the host [8"']. Another approach used to obtain engineered strains devoid of antibiotic resistance is the insertion of the gene(s) of interest into the c h r o m o s o m e of the target bacteria b y means of h o m o l o g o u s flanking sequences [11,12"]. The cloning vectors used may carry an antibiotic resistance gene, which is eventually lost after the double recombination event. This approach, although very simple, has not b e e n widely used in Pseudomonas, perhaps because of the difficulty in selecting for double crossing-over events.
Plasmid vectors An impressive collection of broad host-range plasmid vectors, based on the IncQ, IncP-1 and IncW replicons, is available [13]. The best d e v e l o p e d are those derived from the IncQ group (RSF1010 and related), which include cloning, p r o m o t e > p r o b i n g and regulated expression vectors [14",15"]. These plasmids may prove to b e inadequate, however, w h e n the time comes to maintain and express a recombinant p h e n o t y p e in the field. Virtually all available vectors carry resistances to antibiotics and only a f e w plasmids with alternative markers have been d e v e l o p e d [8"']. More importantly, recombinant plasmids tend to be unstable in the absence of selective pressure. This p r o b l e m may be overcome by including cassettes with the parB (hok/sok) system in the constructions [16], which eliminates plasmid-less segregants. This stabilization system has b e e n successfully used in Pseudomonas [16] and in recombinant intestinal colonizers [17]. Alternatively, autoselection plasmids of the type mentioned above [7,8"',9] could be used. New replicons with the potential for further development as plasmid vectors tailored for applications in the field have been isolated. Among t h e m is pPS10, a plasmid originally isolated from a strain of P. savastanoi which colonizes the olive tree [18"]. This plasmid replicates in Pseudomonas, and its basic replicon consists of only a short or/.Vand the gene for the trans-acting and auto-regulated replication protein RepA [19]. In spite of the large number of n e w natural plasmids which have b e c o m e available recently, none are yet able to contest RSF1010 as the favourite replicon for broad hostrange vector development. This plasmid, which was first found nearly 20 years ago, was last year shown to possess the astonishing ability to replicate not only in
a variety of Gram-negative bacteria, but also in Grampositives, including Streptomyces and Mycobacterium species [20"q. This will surely change s o m e current ideas o n intergeneric barriers to genetic interchange, and will raise some concern over the risks of dissemination of recombinant genes cloned in RSF1010 derivatives and other promiscuous replicons. Additionally, this plasmid is b o u n d to be an excellent source not only of a broad host-range replication machinery, but also of promiscuous promoters and translation initiation regions (derived from the plasmid's replication protein genes and resistance genes) of remarkable potential for vector development.
Transposon vectors The use of transposons as cloning vectors has received notable attention during the last few years, as they are e x p e c t e d to increase the stability of cloned genes once they are inserted into the c h r o m o s o m e s of GEMs. Archetypal natural transposons, such as Tn5, Tnl0, Tn7 and Tn9, are large and limited in the n u m b e r of permissive sites available for cloning. Moreover they contain antibiotic resistance genes and m a y re-transpose themselves. Some attempts at modification have therefore b e e n m a d e to adapt t h e m for engineering purposes. Early attempts to use transposons as vectors w e r e based o n Tn7 [21], but more recently, several types of transposon vectors for use in Gram-negative bacteria have b e c o m e available. Among them is a collection of vectors b a s e d on the properties of IS1 [22]. In this system, a mobile pBR322-derived plasmid was used to assemble a defective c o p y of the IS1 transposase gene acting in cis, but external to two ends of IS1 flanking an antibiotic resistance gene, along with several restriction sites, which w e r e included for cloning purposes. Non-enteric exconjugants, which carry chromosomal insertions of the region flanked by the IS1 termini, can then be isolated. The same concept, although in a m o r e elaborate form, is present in the series of Tn5- and Tnl0-derived mini-transposon vectors containing non-antibiotic selection determinants [5",23"]. These w e r e specifically designed to construct GEMs for environmental applications, and they provide a straightforward procedure for cloning and stably inserting foreign genes into the c h r o m o s o m e s of various Gram-negative bacteria. The modular nature of the constructions facilitates the design of m a n y types of elements in which n e w selective markers m a y b e introduced, and in which promoter probes and a w h o l e range of expression devices may be engineered. Furthermore, these mini-transposons permit target ceils to undergo several rounds of transposition, and therefore the n u m b e r of insertions in the same strain is limited only b y the availability of distinct selection markers. By a less sophisticated approach, Tn5-derived constructions with several extra restriction sites have b e e n p r o d u c e d which facilitate cloning of heterologous DNA fragments [24"].
Genetic engineering strategies for environmental applications de Lorenzo 229 Gene expression 'in the field' GEMs destined for release into the environment should express their recombinant genes under the control of signals present in the location where the bacteria are expected to grow. This is also important for the design of built-in genetic circuits of containment to impede strain proliferation and/or horizontal gene transfer (see below). The utilization of promoters which have b e e n widely used in Esherichia coli genetics (e.g. Ptac, Ptrp, PL/R etc.) in released GEMs is unrealistic owing to the high cost of the inducer and the complications experienced in activating the system. A possible alternative is the use of regulated promoters from catabolic plasmids of Pseudomonas. Broad host-range plasmids are available which contain the Pm promoter of the TOL plasmid, along with its native regulator xylS [25], or mutant varieties like xylS2 [26], and which will cause transcription of heterologous genes in response to the addition of various benzoates. Yen [27" ] has constructed an expression cassette based on the nahR-regulated PG promoter of the NAH7 plasmid, which directs salicylateinducible transcription of downstream heterologous genes. This type of expresion device permits the design of recombinant strains with p h e n o t y p e s that are responsive to chemical signals present in the contaminated location even, in s o m e cases, to the pollutants to be degraded. Transposon vectors [23"] allow the construction of mobile elements in which a promoter-less gene of interest is inserted at random in front of chromosomal promoters so that a specific level of expression m a y b e chosen. This procedure has b e e n used successfully to produce pertussis toxin in Bordetella bronchiseptica [28"] and should also be of use for environmental GEMs. Promoters and expression cassettes derived from natural catabolic and promiscuous plasmids, phages [29,30] and transposons [31] should be further explored for use in expression systems. The T7-polymerase system, which has a remarkable potential for soil bacteria, has b e e n used very rarely and only for expression in laboratory conditions [30]. Combination of the T7 polymerase gene with catabolic promoters or growth-phase d e p e n d e n t promoters should afford the engineering of complex phenotypes to be manifested as a response to the presence of specific chemicals, or during a certain stage of growth.
rate schemes which e m p l o y the regulatory network of the TOL plasmid to generate a model containment circuit for biodegradative strains, based on the toxic hoklike, gefgene. Unfortunately, the hok-related systems are not 100 % efficient [33"',34,35"]. The high frequency of surviving cells demonstrates the need for more complex circuits to ensure effective containment in the environment.
Perspectives Although m a n y broad host-range vectors are available for the genetic engineering of Gram-negative bacteria, procedures to ensure the ecological predictability of GEMs are still in the very early stages of development. Efforts to engineer n e w characteristics must be c o m p l e m e n t e d by novel genetic approaches to ensure that the recombinant genes involved perform properly in the field and do not cause undesirable side effects. Surprisingly, this aspect has received very little attention, and virtually no patents addressing this issue, and only a small n u m b e r of papers describing purpose-specific tools, have been published in the last few years. Transposon vectors, promoters and translation initiation regions derived from promiscuous plasmids will probably be the preferred elements for genetic engineering of strains p r o g r a m m e d to benefit the environment safely.
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.
LINDOWSE, PANOPOULOS NJ, MCFARLAND BL: G e n e t i c Engin e e r i n g o f Bacteria f r o m M a n a g e d a n d N a t u r a l Habit a t s . Science 1989, 244:1300-1307.
2. COLWELLR: R i s k A s s e s s m e n t i n E n v i r o n m e n t a l B i o t e c h •. n o l o g y . Curr Opin Btotechnol 1991, 2:470475. A short review on issues, both legal and scientific, related to the ecological risks of biotechnology. This article points out the need to know more about microbial ecology before the impact of GEMs in natural ecosystems can be realistically evaluated. 3.
PICKUPRW, SAUNDERSJR: D e t e c t i o n o f Genetically Eng i n e e r e d Traits a m o n g Bacteria i n t h e E n v i r o n m e n t . Trends Biotechnol 1990, 8:329-334.
4.
Strain and gene containment D e v e l o p m e n t of genetic circuits to i m p e d e proliferation of recombinant bacteria in conditions other than those in which they have b e e n designed to grow is an important, but challenging aspect o f GEM construction. Endowing strains with mutations to prevent undesirable spread was the only a p p r o a c h available until Molin [32] developed a conditional suicide device b a s e d on the properties of the lethal hok gene. Contreras et al. [33"'] have recently d e v e l o p e d elabo-
PICKUPRW: D e v e l o p m e n t o f M o l e c u l a r M e t h o d s f o r t h e D e t e c t i o n o f S p e c i f i c Bacteria in t h e E n v i r o n m e n t . J Gen Microbiol 1991, 137:1009-1019. A review of n e w procedures for following the fate of bacteria and recombinant genes can'ied by GEMs in natural habitats. HERREROM, DE LORENZO V, TIMMIS KN: Transposon Vectors Containing Non-antibiotic Selection Markers for C l o n i n g a n d Stable C h r o m o s o m a l I n s e r t i o n o f Fore i g n DNA i n G r a m - n e g a t i v e Bacteria. J Bacteriol 1990, 172:6557-6567. Description of a collection of n e w transposon-based tools tailored for constructing GEMs for environmental applications and live vaccine development. Enormous possibilities of further development for specific applications in the field. 5. •.
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NAKAYAMAK, KELLY SM, CURTISS R III: C o n s t r u c t i o n o f a n ASD + E x p r e s s i o n - c l o n i n g Vector: Stable M a i n t e n a n c e a n d H i g h Level o f E x p r e s s i o n o f C l o n e d G e n e s i n a Salmonella V a c c i n e Strain. Biotechnology 1988, 6:693~597
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Ross P, O'GARA F, CONDON S: T h y m i d y l a t e S y n t h a s e G e n e f r o m Lactococcus lactis as a G e n e t i c M a r k e r : Art Alt e r n a t i v e to A n t i b i o t i c R e s i s t a n c e Genes. Appl Environ Microbiol 1990, 56:2164-2169. This paper reports one of the few attempts to develop plasmid vectors for environmental/agricultural applications without the u s e of antibiotics of clinical importance. The constructions described can be u s e d in s o m e soil bacteria, but the performance of the system in microcosms is u n k n o w n . 9.
PORTER RE), BLACK S, PANNURI S, CARLSON A: U s e o f Escherichia coil ssb G e n e to P r e v e n t B i o r e a c t o r T a k e o v e r b y P l a s m i d l e s s Cells. Biotechnology 1990, 8:47-50.
MORONAR, YEADON J, CONSIDINE A, MORONA J, MANNING, PA: C o n s t r u c t i o n o f P l a s m i d s V e c t o r s w i t h N o n - a n t i b i o t i c S e l e c t i o n S y s t e m B a s e d o n t h e Escherichia coil thyA G e n e : A p p l i c a t i o n to C h o l e r a V a c c i n e D e v e l o p m e n t . Gene 1991, 107:139-144. Describes a n auto-selection vector for enterobacteria similar in concept to that described in [8"']. Even t h o u g h the scenario for the release of GEMs is very different, the problem to solve (i.e. maintenance of recombinant g e n e s in the absence of antibiotic selection) is identical to that posed by recombinant bacteria for environmental applications.
18.
NIETO C; FERNANDEZ-TRESGUERRES E, SANCHEZ N, VICENTE M, DLAZ R: C l o n i n g Vectors f r o m a N a t u r a l l y O c c u r r i n g P l a s m i d o f Pseudomonas savastanoi, S p e c i f i c a l l y T a i l o r e d f o r G e n e t i c M a n i p u l a t i o n s i n Pseudomonas. Gene 1990, 87:145-149. Description of a collection of plasmid vectors derived from a n e w replicon w h i c h seems to be specific for Pseudomonas species. G o o d possibilities of future developments for environmental applications. 19. 20. •.
GORMLEYE, DAVIES J: T r a n s f e r o f P l a s m i d RSF1010 b y C o n j u g a t i o n f r o m Escherichia coli to Streptomyces lividans and Mycobacterium smegmatis, J Bacte~ol 1991, 173:6705-6708. This p a p e r will probably b e c o m e a landmark for studies o n genetic promiscuity. T h e authors s h o w that RSF1010 can not only be transfered conjugatively into two Gram-positive species, but can also replicate stably and express its antibiotic resistances in the n e w hosts. The promiscuity of functional elements of RSF1010 is a source of building blocks for broad host-range vector development. 21.
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SCHMIDHAUSERTJ, DITrA G, HELINSK] DR: B r o a d H o s t R a n g e P l a s m i d C l o n i n g Vectors f o r G r a m - n e g a t i v e Bact e r i m In Vectors: A Survey of Molecular Cloning Vectors and their Uses. Edited by Rodriguez LR, Denhardt DT. Boston: Butterworths; 1987:287-332.
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23.
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30.
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32.
MOLIN S, KLEMM P, POULSEN LK, BIEHL H, GERDES K, ANDERSSON P: C o n d i t i o n a l Suicide S y s t e m f o r Cont a i n m e n t o f B a c t e r i a a n d P l a s m i d s . Biotechnology 1987, 5:1315-1318.
33. •.
CONTRERASA, MOLIN S, RAMOS JL: C o n d i t i o n a l S u i c i d e Containment System for Bacteria which Mineralize A r o m a t i c s . Appl Environ Microbiol 1991, 57:1504-1508. First attempt to design a containment system adapted to biodegradarive strains. The goal is to destroy GEMs after the pollutant present in the m e d i u m has been degraded. The model system is set u p with E. coli a n d it works within certain limits. Adaptation to Pseudomonas is awaited.
34.
BEJ AK, PERLIN MH, ATLAS RM: M o d e l Suicide Vector for Containment of GeneticaUy Engineered Microorg a n i s m s . Appl Environ Microbiol 1988, 54:2472-2477.
35.
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Victor de Lorenzo, Centro de Investigaciones Bioi6gicas, Vel~zquez 144, 28006 Madrid, Spain.
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