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Environmental potential of suicide genes
Environmental potential of suicide genes S ren Molin Technical University of Denmark, Lyngby, Denmark Environmental applications of genetically engineered microorganisms have caused concern among scientists, the authorities and the general public. Despite the many potential benefits offered by gene technology to agriculture, environment protection and the medical sector, only a few cases of released engineered bacteria have been permitted. In this review, the design of safer organisms for release purposes is discussed with specific emphasis on the use of suicide systems to limit the survival of bacteria in the environment. Current Opinion in Biotechnology 1993, 4:299-305 Introduction At the beginning of the gene technology debate, it was argued b y many scientists that bacteria used by industry are no longer capable of surviving in the environment, because the necessary functions for this have been lost during cultivation in laboratories, and after genetic manipulation they would be even further disabled. Several experimental observations have cast doubt on such statements [1], and in addition we have no easy way of interpreting reduced numbers of colony formers from environmental samples. Therefore, the ability to predict and control bacterial survival may be very difficult or impossible to attain. We therefore have only two options in the present predicament, assuming that we agree that engineered organisms are beneficial to society and should eventually b e c o m e accepted tools in our strife for improving the quality of life for man and his surroundings. The first option is to accept the potential risk associated with engineered organisms and let the experience gained from practical use of them be the starting point for further work. The second option is to recognize that when we introduce man-made products (either chemicals or living organisms) into the environment, we have an obligation to ensure that such products are 'degradable' and will disappear after a limited period of time; if total elimination is not possible, w e should at least make the utmost effort to reduce concentrations to insignificant levels. This review is based on the opinion that the two options are perhaps compatible; the clean-up strategy is sound and advisable in the absence of experience, and experience may at a later point let us revise some of the precautions. The first practical applications of genetic engineering were the microbial production of h u m a n and animal peptides and proteins. Our broad knowledge and ex-
perience of laboratory organisms such as Escherichia coli, Bacillus subtilis and Saccharomyces cerevisiae made them obvious candidates as host organisms for introduced genes encoding peptide hormones or growth factors. As many of the strains used for these genetic experiments had several mutations rendering the ceils dependent on special nutritional supplies in the growth media, it was accepted that the organisms themselves would not be able to survive for a long time in the environment, let alone out-compete the natural organisms. In contrast, deliberate release of genetically engineered microorganisms (GEMs) to the environment leaves them in competition with the natural population under conditions that change constantly. If bacteria are released to perform a task (plant protection, environmental clean-up, live vaccines, etc.) it is normally essential that they survive for some time, maintaining a reasonable population size during this period. The bacterial species used for environmental releases are most often not very well k n o w n and consequently analogous libraries of mutant strains with markers or disabling traits do not exist. There are, therefore, two arguments against a traditional disablement approach in the case of bacterial releases: first, many mutations would render the strain handicapped in the performance of its actual task in the environment; and second, the proper mutations would often be difficult to obtain. An alternative method to control bacterial establishment and survival is the introduction into competitive wild-type organisms of controllable suicide functions, that do not interfere with normal growth but are expressed under specific conditions defined by the physical or chemical composition of the environment [2]. Thereby, it may be possible to limit survival of the strain to the exact environment where it performs its desired function, in such a w a y that any cell escaping this environment will be killed by induction of
Abbreviations
GEM--genetically engineeredmicroorganism;MB--methylbenzoate. © Current Biology Ltd ISSN 0958-1669
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Environmental biotechnology the suicide system. An even more specific method would be to eliminate gene transfer by coupling a suicide gene to the environmentally relevant gene(s) (e.g. on a plasmid) and thereby control the suicide gene in the released organism b y a chromosomal gene that is not found in the natural population of potential recipients. Several strategies for controlled suicide will be described here. The basic idea behind them all is to couple potent killing genes to environmentally relevant expression control circuits, which will allow the released strain to establish itself in a confined w a y (limited to a certain defined environment or limited in time) with severely reduced possibilities of survival.
Killing genes In the course of studies on the maintenance functions of plasmid R1 in E. coli, it was found that a locus on the plasmid, responsible in part for the high level of plasmid stability during cell growth in the absence of selection pressures, encodes a small polypeptide (52 amino acids) which is highly toxic to the cells [3]. The plasmid stability phenotype of this locus is connected to antisense RNA regulation of the expression of this polypeptide, which ensures tight repression of the gene in cells harbouring the plasmid, because of constitutive synthesis of the antisense RNA. However, in ceils to which the plasmid has not segregated, the antisense RNA decays rapidly leaving a very stable mRNA, which is then translated into the toxic protein resulting in eradication of the plasmid-free cells. Separation of the two genes (regulator and effector) allowed subsequent analysis of the molecular mechanisms involved in both control of expression and the method of cell killing. The gene encoding the small toxic polypeptide was termed hok, for host killing, and the antisense RNA gene was called sok, for suppression of killing. Killing of the cells after induction of the Hok protein was typically followed b y a morphology change in the bacteria observed under the microscope. The cells became almost totally transparent except for cell material accumulating at the poles [3]. The occurrence of such 'ghost' cells indicated that one effect of the induction of Hok could be membrane damage. This was later confirmed by showing that, immediately after induction of the protein, the transmembrane potential collapses, respiration is terminated, and the membrane contains a new small polypeptide the size of H o k [3]. It was therefore concluded that the cellular membrane of E. cob is the target for the Hok protein, and s o m e h o w introduction of the protein i~i'to the membrane leads to cell death by interference with central components of the respiratory system. The position of the hok/sok locus in plasmid R1 stimulated a search for similar genes in other plasmids, and homologous sequences were detected by hybridization in several plasmids of the enteric bacteria. Similarly, previously described genes from a number of plas-
mids turned out to be homologous both genetically and functionally [3]. In some of the hybridization experiments performed to detect plasmid homologues, more than one signal was detected, and these extra hybridization signals were detected even in plasmidfree cells [3]. One of these was rapidly identified by comparison between hok and the so-called relF gene. The relF gene is located in the relB gene cluster at 37 rain on the chromosome map of E. coli. The functional similarity between the two genes is obvious, although no biological significance of the relF gene has so far been indicated. A second hok homologous locus in the E. coli chromosome was isolated by cloning and characterized. This gefgene is located at 1 min on the E. coli map, the protein is functionally identical to the Hok and RelF proteins in terms of the mode of killing and, as in the case of hok expression, gefis regulated by an antisense RNA mechanism (not the case for relF but similar for most other members of this family). A number of properties make the g e f g e n e family an interesting source of suicide functions. The small proteins are all highly toxic in the cell even in small amounts, and the effect of their synthesis is a cascade of reactions resulting from the incorporation of the polypeptides into the cytoplasmic membrane. This central target for the toxic action makes it likely that most, if not all, bacteria are susceptible to the killing activity provided sufficient protein is expressed, and results obtained so far confirm this. It is an extra benefit that the genes are much smaller than average prokaryotic genes (less than 200 base pairs) as this will facilitate genetic manipulation and sequence analysis. It must be emphasized, however, that other types of suicide genes may possess similar advantages, and in the following no specific attention is given to the gef-like genes.
Expression control circuits The key to the design of functional suicide containment systems lies in the regulation of suicide gene expression. As long as the cells are used under defined conditions in the laboratory or in a fermentation plant, there are many possible strategies that have already been developed. However, in situations where the microorganisms are to function in the environment, little information is available about gene expression, and even though some tests may be performed in model ecosystems, the permissible level of extrapolation to the natural environment is limiting. The identification of an optimal expression system is a complex process. First, the promoter for eventual transcription of the killing gene must be sufficiently active under the prevailing conditions in the environment, leading to expression of the protein in actively growing cells, as well as in stationary cells. Cells may be in a state of dormancy most of the time, and ideally the killing should happen even in this state. It can be argued that the dormant cells do not contribute to the
Environmental potential of suicide genes Molin ecosystem activities, and therefore they constitute no potential risk; it is thus sufficient for the suicide system to function if the ceils later become reactivated. The second problem is control. Under conditions of constant variation it is difficult to predict which external signals are relevant to the bacteria, and therefore the choice of expression system for a specific environmental composition or condition is not obvious. In general, the optimal solution will be to design the expression control system in direct combination with the task of the organism, such that spread of bacteria to other localities will induce their suicide system. In some cases, where there is a high level of molecular information about the particular desired function of the bacteria, it is actually possible to achieve this (see below), but in many cases it is far from possible, and other regulatory alternatives have to be invented. Finally, it is important to k n o w whether the suicide function is active under the environmental conditions and the various physiological states of the cells. What will be the effect of a membrane porin in cells that have no membrane growth, and h o w will a toxic protein survive in ceils with increased proteolytic activity? These questions may be answered by monitoring released bacteria in which bioluminescence genes (lux, luc) have b e e n inserted together with the containment systems. The methods available to detect the light emitted from such strains with extremely high sensitivity [4%5,6"'], and the dependence on metabolic activity for these systems to function, should provide an excellent basis for determining the efficacy of the suicide systems in situ.
Degradation of xenobiotics Degradation of man-made compounds in the environment is one of the obvious beneficial applications of released GEMs. Many types of organisms have been isolated from nature that are capable of the mineralization of a large number of organic molecules, and in many cases such natural isolates are quite effective in their clean-up function. In other cases, however, the degradative pathways are either very inefficient, too c o m p o u n d specific, or incomplete resulting in conversion of one problematic c o m p o u n d to another which may be even more problematic from an environmental point of view. In some cases, it may be useful to introduce genes encoding thermoresistant degrading enzymes derived from other organisms [7]. Therefore, genetic manipulation offers the potential of optimization, expansion of specificity, and fusion of pathways to allow efficient and complete mineralization. Deliberate release of engineered microorganisms to the environment in the context of bioremediation is still at the experimental stage, and much scepticism is often expressed about the efficacy of such organisms. In a series of studies on the survival, gene transfer and function of engineered strains of Pseudomonas spp. capa-
ble of degrading substituted aromatics [8"',9"'], it was demonstrated that the organisms survived very well for four weeks in aquatic sediments whether or not the aromatics were present. In addition, it was found that the presence of the GEMs led to an accelerated degradation of the aromatics compared to situations where no engineered bacteria had been introduced. Finally, a significant rate of plasmid transfer was observed in the microcosm. Thus, data from this study show that engineered strains with improved degradative properties (in this case simultaneous mineralization of chloroand methylbenzoates) may survive well and express the genes involved in degradation such that enhanced degradative activity is observed in situ. Bacterial degradation of aromatic compounds such as the benzoates has been studied in great detail for a number of years, and much is n o w k n o w n about the specific degradative pathways and their regulation [10]. The TOL genes found in P. putida are induced in the presence of very low amounts of the substrates (toluene and different benzoates), and the spectrum of inducers and substrates has been further expanded by mutation [11,12]. The control system responsible for the regulation of these genes is quite complex, but in the present context, the important feature is the substrate-dependent transcription from the Pm promoter, for which the regulatory protein is the xylS product. The XylS protein has no activity in the absence of substrate, e.g. methylbenzoate (MB), but in its presence the XylS protein is activated, and the activated regulator acts positively on the Pm promoter to stimulate initiation of transcription, which continues as long as MB is present. In an attempt to design a strain that is fully viable in an environment comprising 3-MB, and which, because of the insertion of a regulated suicide system, is killed in the absence of the compound, a regulatory double loop has been constructed (Fig. 1). The Pm promoter was placed upstream of the lacI gene from E. coli as a substitute for the normal promoter [13,14]. In a cell harbouring this fusion and the xylS gene, a lacpgef fusion was inserted. The resulting strain was viable in substrates containing MB, because in this environment the LacI repressor is synthesized, which ensures repression of the suicide gene. When MB was removed from the substrate the cells were killed after a short period of growth, as no Lac repressor was expressed from the MB-regulated promoter under this condition. In the first demonstration of this system the organism was E. coli, and no degradation of the substrate took place. The same type of construction was later inserted into P. putida harbouring the entire TOL degradative pathway, and in this case it was possible to follow simultaneously the degradation of MB and the elimination of the bacteria after complete mineralization of the aromatic. From experiments performed in soil with and without MB it became clear that the introduction of a suicide system, as the one described here, is an efficient containment strategy even in a complex environment such as soil (LB Jensen, JL Ramos, S Molin, unpublished data).
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Survival
T'+'I "l'-' 0
3MB
I x×,s I I Km
[
0
,e
Yes
Z~
I
I Ap
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Fig. 1. Elements of conditional containment systems based on the TOL plasmid regulator XylS, Lacl and a killing protein. The operation of the killing system is described in the text. (a) Conditions under which survival of the contained bacteria is predicted. (b) Conditions under which it is predicted that the bacteria bearing the containment system will die. 3MB, 3-methylbenzoate. Symbols © and @, inactive and active forms of XylS protein respectively; Z_ and L , Lacl and Gef proteins, respectively.
The major point raised here is that, in connection with bioremediation, the engineered bacteria of interest most often harbor genes from pathways that have been characterized in great detail, and it is therefore possible to design the regulatory loops necessary for the described mode of site-conditioned survival. Even though it may seem difficult to imagine environmental risks in relation to bioremediation, the activity of the microorganism is only predictable in the context for which it was designed, and if it is possible to restrict their survival to the execution of this 'job', there is basically no reason not to do so.
Biopesticides The use of chemicals to combat pests is viewed with increasing concern because of the adverse environmental side effects. Many of the pesticides are recalcitrant and they are toxic to many organisms outside the target group. Although the strategy of biodegradation may also be applied to chemical pesticides it seems irrational to employ compounds that are recognized as harmful to the environment and then try to remove them later. The alternative insect toxins produced by B. thuringiensis offer the following advantages: they are highly specific, they are degradable in the environment, and they are highly toxic to the target organisms. In addition, many different types of toxins have n o w been identified that are active against a broad spectrum of insects [15,16]. It is therefore not surprising that these toxins constitute 90-95% of commercial biopesticides. When B. thuringiensis toxins are used as biopesticides they are sprayed on fields as protein crystals. The crystals are relatively easy to purify and formulate as they are released from the bacteria during cellular lysis w h e n the ceils enter the stationary phase. However,
the crystal proteins are only active in the field for a relatively short period of time, and attempts have therefore been made to design biopesticides based on these toxins with a longer life span in nature. The finding that the toxins may be expressed in other bacteria and maintain their toxicity allows for transfer of the appropriate genes to bacteria that are k n o w n to live in close association with plants (in contrast to B. thuringiensis itself). A particular application of these toxin biopesticides concerns the combatting of mosquitoes b y killing the larvae as they develop in fresh water. As the larvae feed on microorganisms in the water, an ideal formulation of the toxins would be to express them in live cyanobacteria. A recent report [17"] on the successful transfer of a toxin gene from B. thuringiensis subsp. israelensis, which is active against mosquitoes, to a strain of the cyanobacterium Agmenellum quadruplicatum, and the expression in this organism of a stable and active toxin, holds promise for the design of biopesticides to be used in connection with elimination of insect vectors of various diseases. This type of genetically engineered bacteria have not been commercialized yet, mainly because of the lack of knowledge about the potential risks of such organisms to ecosystems. A serious question is whether the organism and its toxin gene will spread outside the target area. For plant-associated bacteria it may be possible to limit the survival of the bacteria to a particular growth zone. Bacterial genes responding to plant c o m p o u n d s like root exudates have been identified [18], and even before the actual regulatory details have been w o r k e d out it may be possible to insert suicide genes in a control circuit involving the plant signals such that cells escaping from the plant zone will be killed. The availability of bacterial gene regulators in combination with other niche-specific regulators, makes it possible to design site-directed induction systems for the expression of suicide functions that will interfere with a totally random and uncontrolled spread of man-made microorganisms from the original site of release. The increasing interest in microbial ecology, and in symbiotic relationships in particular, gives promise for the identification of many such useful control systems from a broad spectrum of bacteria. A different approach to biological containment in the design of biopesticides is stress-induced limitation of survival. In contrast to the life of bacteria in nutrientrich laboratory media, microbial life in the natural environment is probably usually a state of starvation and other types of stress (pH, extreme temperatures, toxic compounds etc.). It is therefore to be expected that any organism released to the environment will meet severe stress conditions sooner or later. Bacterial responses to different forms of stress have been studied for some time and, especially for bacteria like E. coli [19,20"], S. typhimurium [21], and Vibrio app. [221, much is k n o w n about the genetic programmes responsible for coping with these extreme growth conditions. The picture emerging from these studies is that bacteria have differentiation programmes that are in operation w h e n conditions become harsh. The sporulation cycle
Environmental potential of suicide genes Molin of Gram-positive bacteria is an excellent example of gross changes in cellular morphology and metabolism, but many non-sporulating bacteria also possess genetic programmes for stress-induced morphology, and resistance to various factors and metabolic changes. Specific genes are activated in an ordered sequence as a response to stress, as revealed clearly by two-dimensional gel electrophoresis, and analogously certain recovery genes are activated u p o n the return of normal growth conditions after a stress period [22].
mal growth; however, w h e n placed under starvation conditions during which the wild-type organism normally survives well, the double insertion mutant strain s h o w e d decreasing viability resulting in almost total elimination after 10-15 days.
Such studies of differentiation are also being carried out in different plant-associated Pseudomonas strains, and the observations accumulated so far clearly indicate the presence of similar genetic programmes in these bacteria (M Givskov, L Eberl and S Molin, unpublished data). The possibility of fusing stress gene promoters with a killing gene is an obvious containment strategy. Promoters active at late times after induction of a stress programme are especially useful, as killing will not take place until after an extended period of stress. In this way, an undesired rapid elimination of the population after short transient stress periods can be avoided. Alternatively, fusions to specific recovery gene promoters will allow a significant period of survival followed by a subsequent reduction in the population. Among the stress genes of particular interest in the context of suicide containment are those involved in starvation survival. Feast and famine will unquestionably be experienced by bacteria in the environment, and starvation is a condition that is very easy to reproduce in the laboratory. Naturally, the more that is k n o w n about such pathways and their regulation, the better are the possibilities for interfering with and controlling survival in the environment.
Live bacterial vaccines are not often mentioned in connection with the deliberate release of GEMs, but as engineered strains of Salmonella spp. or E. coli are being considered as interesting vectors of antigen presentation, the lack of confinement in this type of application must be taken into consideration.
Limited survival may also be accomplished by mutations in genes whose products are essential under stress conditions. Interesting results have been obtained for Vibrio $14, whose stress-induced differentiation programme has been studied for some years [22]. If cells of this organism are starved for a carbon source they become very small, and the metabolism changes drastically. Specific proteins are synthesized at different times after the onset of starvation, and the pattern of macromolecular synthesis strongly indicates the existence of a regulatory network responsible for the changes and the sequence of events. In an attempt to expose such control circuits, fusions were constructed by the insertion of ~-galactosidase reporter genes into genes that are specifically induced under conditions of starvation (.J ()stling and S Kjelleberg, personal communication). Subsequently, mutagenic transposons were inserted at random in the chromosome, followed by screening for clones that showed a lack of starvationinduced gene expression. After showing that such insertions were not interfering directly with the reporter gene, it was concluded that the second site insertion had inactivated a regulatory gene responsible for activation of one or more genes which are normally turned on after starvation. The two insertion mutations described in genes of importance for starvation-induced gene expression had no significant effect during n o r -
Live vaccines
One promising strategy for designing live vaccines is to make fusions between antigen-coding sequences and genes encoding surface-bound proteins like flagellar proteins, outer membrane proteins or fimbriae [23]. In such cases, the antigenic determinants will be presented on the outside of bacterial cells, and it is believed that the proliferative properties of such strains will make them more effective than traditional vaccines. Obviously, the host organisms must not be pathogens, and therefore biological containment approaches are not supposed to compensate for any pathogenicity exerted by the bacteria inside the animal or human body. Containment is only relevant for the purpose of limiting the spread of the engineered genes and the survival of the strains after their excretion to the environment. Although some of the containment designs mentioned above are also relevant in this context, the most suitable approach for live vaccines based on enteric bacteria is stochastic induction of killing based on recombinational switching of gene expression. Recombinational switches are found in several operons in bacteria and bacteriophages. The switch promoter directing the synthesis of Type 1 fimbriae in E. coli (fimAp) has been used as a model system for stochastic induction of suicide [24]. The promoter is located within a 314 base pair DNA sequence that inverts its orientation approximately 2 x 10-3 times per cell per hour (measured in fast growing ceils) [24]. This invertible sequence comprises a promoter that directs transcription into one of the flanking sequences, so that any gene controlled by the promoter will be switched from on to off with this frequency. The insertion of a suicide gene next to the switch promoter, fimAp, was the first approach used to obtain time-dependent induction of suicide [2,25"], and in a series of subsequent investigations both the hok and the gef genes have been employed (S Molin, unpublished data). E. coli cells harbouring such inserts on plasmids or integrated into the chromosome behave according to expectations: in cell suspensions where no net growth is possible because there is no external carbon source, viable cell counts constantly drop in contrast to the non-contained control strain, indicating that switching occurs in the absence of growth. In fast growing
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Environmentalbiotechnology cultures (doubling times of approximately 30 rain) no effect of the suicide system on the growth rate of the culture is observed, which suggests that the killing rate is insignificant relative to the growth rate. However, if the growth condition is changed to a p o o r carbon source, the consequence of the presence of this type of stochastic suicide system is apparent growth inhibition of the cell population: the slower the cells grow, the more severe is growth inhibition by the fimAp-geffusion, indicating that the killing rate becomes increasingly significant relative to the decreasing growth rates under the chosen conditions. In principle, it seems to be possible to design functional stochastic induction systems for bacterial suicide. The particular system based on the fimA switch promoter unfortunately only works in the enterobacteria, and even in some of these the switch frequency is too low to be of any use. So far the best results have been obtained in E. coli and S. typhimurium, whereas for example no switch takes place at all in P. putida (LB Jensen and S Molin, unpublished data). For live vaccines, this type of containment would allow their proliferation and competition in the gut of the vaccinated individual, whereas progeny excreted to the environment from the animal or human host would be eliminated eventually because of the poor growth conditions in the outside environment combined with the time-dependent stochastic induction of killing.
Conclusion
Acknowledgements Parts of this review are published in Annual Review of Mzcrobiology 1993 [25"], and I am grateful for the permission given by Annual Reviews Inc to u s e these parts here. The work performed in the author's laboratory has been supported by European Commission contracts.
References and recommended reading Papers of particular interest, published within the annual period of review, have b e e n highhghted as: of special interest •o of outstanding interest 1
SOBECKYPA, SCHELLMA, MORAN M.A, HODSON RE: Adaptation o f Model Genetically Engineered M i c r o o r g a n i s m s to Lake Water: G r o w t h Rate E n h a n c e m e n t s and Plasmid L o s s . Appl Environ Microbtol 1992, 58:3630-3637.
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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 for Cont a i n n a e n t o f Bacteria a n d P l a s m i d s . Biotechnology 1987, 5:1315-1318
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GERDESK, POULSEN LK, TI-~STED T, NIELSEN AK, MARTINUSSEN J, ANDREASENPH. The hok Killer G e n e F a m i l y i n GratnNegative Bacteria. New Biol 1990, 2 946-956
4.
DE WEGER LA, DUNBAR P, MAHAFEE WE, LUGTENBERG BJJ, SAYLER GS: Use of B i o l n m i n e s e e n e e Markers to Detect P s e u d o m o n a s s p p in t h e R h i z o s h e r e . Appl Environ Microbiol 1991, 57:3641-3644. A fine paper describing the use of lux g e n e s in relation to detailed studies of plant-microbe interactions. Bioluminescence is s h o w n to be an adequate tool in monitoring specific bacteria relative to their position on plant roots. 5
The predictable argument against the suicide systems described here relates to the failure of systems resulting in total elimination of a population. Even if optimized combinations of killing genes and gene expression sequences are constructed that will be 100% effective u p o n induction, the occurrence of mutations in a cell population will never be eliminated. Therefore, if an organism is considered a serious risk to the environment in connection with applications, the containment of such strains will not be sufficient to remove the potential risk. Even if no direct risks are foreseen, there are still concerns regarding the release of engineered organisms to the environment, and we suggest in this context that, as long as such concerns prevail, attempts should be made to minimize putative problems by reducing the exposure time and persistence of the introduced organisms as much as possible. If this is the purpose of biological containment, a reduction of the population size over a limited time by a factor of a million or even less seems to be of value. There have been many bad':experiences with man-made chemicals accumulating in the environment, extensive use of antibiotics etc., and measures are n o w being taken against these problems by encouraging the use of degradable rather than recalcitrant compounds. In the same way, we find it prudent to use survival-deficient engineered organisms rather that very persistent strains, even in cases where there are no obvious risks.
SHAW JJ, DANE F, m i n e s c e n e e for Microorganisms Environ Microbtol
GEIGER D, KLOEPPER J'W: U s e o f BioluD e t e c t i o n o f Genetically Engineered Released into the Environment. Appl 1992, 58:267-273.
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SILCOCK DJ, WATERHOUSE RN, GLOVER LA, PROSSER JI, KILLHAM K Detection o f a Single G e n e t i c a l l y Modified Bacterial Cell in Soil b y Using Charge Coupled Device-Enhanced M i c r o s c o p y Appl Environ Microbiol 1992, 58:2444-2448. This paper describes the microscopy techniques combined with ultra-sensitive charge-coupled device cameras that have b e e n developed for monitoring single bacterial cells in very complex enviromnents. The results presented here allow previously impossible analyses of bacterial life in the environment, a n d it is certain that this approach will create highly interesting insights into microbial ecology in the coming years. • .
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DONG FM, WANG LL, WANG CM, CHENG JP, HE ZQ, SHENG ZJ, SHEN RQ. M o l e c u l a r C l o n i n g a n d Mapping o f P h e n o l Degradation Genes f r o m Bacillus s t e a r o t h e r m o p h i l u s FDTP-3 a n d Their E x p r e s s i o n in Escherichia c o i l Appl Environ Microbiol 1992, 58:2531-2535
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PIPKE R, WAGNERDOBLER I, TIMMIS KN, DWYER DF. Surviva1 a n d Function o f a Genetically E n g i n e e r e d P s e u d o m o n a d i n A q u a t i c Sediment Microcosms. Appl Environ Microbiol 1992, 58:1259-1265. Shows the potential benefits of redesigning degradative pathways by genetic engineering. The results clearly demonstrate the fitness of the engineered organisms, and the effect on the rate of degradation of the aromatic c o m p o u n d is significant. The data argue against simplified statements o n the lack of positive results in bioremediatlon after applying GEMs. 9. •,
NUSSLEINK, MARIS D, TIMMIS KN, DWYER DF: Express i o n a n d Transfer of Engineered Catabolic Pathways
Environmental potential of suicide genes/violin Harbored by P s e u d o m o n a s s p p I n t r o d u c e d i n t o Activ a t e d S l u d g e M i c r o c o s m s . Appl Environ Mzcrobiol 1992, 58:3380-3386. This is a follow-up to the previous paper, which extends the observations on the advantages of using GEMs in bioremediation contexts. The strains designed are effective, a n d their activities with respect to degradation o f various aromatics could not easily have b e e n encountered from the indigenous microflora.
successful insertion of one of the toxin g e n e s into a cyanobacterium a n d its demonstrated insecticidal activity in this n e w host point to other potential apphcatlons in mosquito control and their role in a n u m b e r of infectious diseases. 18.
CLARKE HRG, LEIGH JA, DOUGLAS CJ M o l e c u l a r S i g n a l s i n t h e I n t e r a c t i o n s B e t w e e n P l a n t s a n d M i c r o b e s . Cell 1992, 71:191-199.
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MATINA. M o l e c u l a r A n a l y s i s o f t h e S t a r v a t i o n Stress i n E s c h e r i c h i a coli. Microb*ol Ecol 1990, 74:185-196
RAMOS JL, MICH~N C, ROJO F, DWYER DF, TIMMIS KN. Signal-Regulator Interactions. Genetic Analysis of the Effector B i n d i n g Site o f XylS, t h e B e n z o a t e - A c t i v a t e d Positive R e g u l a t o r o f P s e u d o m o n a s TOL P i a s m i d MetaC l e a v a g e P a t h w a y O p e r o n . J Mol Biol 1990, 211:373-382.
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RAMOSJL, STOLZ MA, REINEKE W, TIMMIS KN A l t e r e d Eff e c t o r Specificities i n R e g u l a t o r s o f G e n e Expression: TOL P i a s m i d x y l S M u t a n t s a n d T h e i r U s e t o E n g i n e e r E x p a n s i o n o f t h e R a n g e o f A r o m a t i c s D e g r a d e d by Bacteria. Proc Natl A c a d Sci USA 1986, 83:8467-8471.
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DELGADOA, WUBBOLTSMG, ABmL MA, RAMOSJL: N i t r o a r o m a t i c s A r e S u b s t r a t e s f o r t h e TOL P i a s m i d U p p e r - P a t h w a y E n z y m e s . Appl Environ Microbiol 1992, 58.415-417.
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CONTReRAS A, 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
14.
DUQUE E, RAMOS-GONZALEZ MI, DELGADO A, CONTRERAS A, MOLIN S, RAMOS JL. G e n e t i c a l l y E n g i n e e r e d P s e u d o m o n a s S t r a i n s f o r M i n e r a l i T ~ t i o n o f Aromatics: Survivial, P e r f o r m a n c e , Gene Transfer, a n d Biologic a l C o n t a i n m e n t . In Pseudomonas: Molecular Biology a n d Biotechnology. Edited by Galli E, Silver S, Witholt B. Washington DC: American Society for Microbiology, 1992:429-437.
15.
FEITELSONJS, PAYNE J, KIM L: B a c i l l u s t b u r i n g i e n s i s : Ins e c t s a n d B e y o n d . Biotechnology 1992, 10:271-276.
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LECADET MM, CHAUFAUX J, RIMER J, LERECLUS D: C o n s t r u c t i o n o f N o v e l Bacillus t h u r i n g i e n s i s S t r a i n s w i t h D i f f e r e n t I n s e c t i c i d a l Activities b y T r a n s d u c t i o n a n d T r a n s f o r m a t i o n . Appl Environ Microb*ol 1992, 58:840-849
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MURPHYRC, STEVENS SE: C l o n i n g a n d E x p r e s s i o n o f t h e c r y l V D G e n e o f Bacillus t h u r i n g i e n s i s S u b s p . i s r a e lensis i n t h e C y a n o b a c t e r i t t t n A g m e n e l l u m q u a d r u p l i c a t u m PR-6 a n d Its R e s u l t i n g Larvicidal Activity. Appl Environ Microbiol 1992, 58:1650-1655. The Bt toxins have mainly been discussed m the context of agricultural applications as biopestlcldes directed against plant pests. The
20.
NYSTROM T, NEIDHARDT FC. Clotting, M a p p i n g a n d Nucleotide Sequencing of a Gene Encoding a Universal S t r e s s P r o t e i n i n E $ c h e r i c h a coll. Mol Microbiol 1992, 6:3187-3198. This is a very interesting and detailed molecular analysis of a n e w g e n e involved in the stress response of E. colt. It should be considered as a model example of m o d e r n molecular genetics applied to environmental issues. 21.
SPECTORMP: G e n e E x p r e s s i o n i n R e s p o n s e to Multip l e N u t r i e n t - S t a r v a t i o n C o n d i t i o n s i n S a l m o n e l l a typ h i m u r i u m . Microbiol Ecol 1990, 74:175-184
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NYSTROMT, ALBERTSON NH, FLARDH K, KJELLEBERG S: P h y s i o l o g i c a l a n d M o l e c u l a r A d a p t a t i o n to S t a r v a t i o n a n d R e c o v e r y f r o m S t a r v a t i o n b y t h e M a r i n e Vibrio. Microbtol Ecol 1990, 74:129-140.
23.
STOCKERBAD: A r o m a t i c - D e p e n d e n t S a l m o n e l l a as Live V a c c i n e P r e s e n t e r s o f F o r e i g n I n s e r t s i n Fiagellin. Res Microbiol 1990, 141.787-796.
24.
EISENSTEINBI: P h a s e V a r i a t i o n o f T y p e 1 F i m b r i a e i n E $ c h e r i c h t a colt is U n d e r T r a n s c r i p t i o n a l C o n t r o l . Science 1981, 214:337-338.
25. •.
MOLIN S, BOE L, JENSEN LB, KRISTENSEN CS, GIVSKOV M, RAMOSJL, BEJ AK: Suicidal G e n e t i c E l e m e n t s a n d T h e i r U s e i n Biological C o n t a i n m e n t o f Bacteria. A n n Rev Microbiol 1993, 47:139-166. T h e first review d e s c n b m g the potential a n d strategies behind designs of biological containment systems. The review covers all the major attempts m a d e to construct disabled bacteria for a n u m b e r of applications, and the reference list covers most of this field
Soren Molin, Department of Microbiology, Technical University of Denmark, Building 221, DK-2800 Lyngby, Denmark.
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perience of laboratory organisms such as Escherichia coli, Bacillus subtilis and Saccharomyces cerevisiae made them obvious candidates as host organisms for introduced genes encoding peptide hormones or growth factors. As many of the strains used for these genetic experiments had several mutations rendering the ceils dependent on special nutritional supplies in the growth media, it was accepted that the organisms themselves would not be able to survive for a long time in the environment, let alone out-compete the natural organisms. In contrast, deliberate release of genetically engineered microorganisms (GEMs) to the environment leaves them in competition with the natural population under conditions that change constantly. If bacteria are released to perform a task (plant protection, environmental clean-up, live vaccines, etc.) it is normally essential that they survive for some time, maintaining a reasonable population size during this period. The bacterial species used for environmental releases are most often not very well k n o w n and consequently analogous libraries of mutant strains with markers or disabling traits do not exist. There are, therefore, two arguments against a traditional disablement approach in the case of bacterial releases: first, many mutations would render the strain handicapped in the performance of its actual task in the environment; and second, the proper mutations would often be difficult to obtain. An alternative method to control bacterial establishment and survival is the introduction into competitive wild-type organisms of controllable suicide functions, that do not interfere with normal growth but are expressed under specific conditions defined by the physical or chemical composition of the environment [2]. Thereby, it may be possible to limit survival of the strain to the exact environment where it performs its desired function, in such a w a y that any cell escaping this environment will be killed by induction of
Abbreviations
GEM--genetically engineeredmicroorganism;MB--methylbenzoate. © Current Biology Ltd ISSN 0958-1669
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Environmental biotechnology the suicide system. An even more specific method would be to eliminate gene transfer by coupling a suicide gene to the environmentally relevant gene(s) (e.g. on a plasmid) and thereby control the suicide gene in the released organism b y a chromosomal gene that is not found in the natural population of potential recipients. Several strategies for controlled suicide will be described here. The basic idea behind them all is to couple potent killing genes to environmentally relevant expression control circuits, which will allow the released strain to establish itself in a confined w a y (limited to a certain defined environment or limited in time) with severely reduced possibilities of survival.
Killing genes In the course of studies on the maintenance functions of plasmid R1 in E. coli, it was found that a locus on the plasmid, responsible in part for the high level of plasmid stability during cell growth in the absence of selection pressures, encodes a small polypeptide (52 amino acids) which is highly toxic to the cells [3]. The plasmid stability phenotype of this locus is connected to antisense RNA regulation of the expression of this polypeptide, which ensures tight repression of the gene in cells harbouring the plasmid, because of constitutive synthesis of the antisense RNA. However, in ceils to which the plasmid has not segregated, the antisense RNA decays rapidly leaving a very stable mRNA, which is then translated into the toxic protein resulting in eradication of the plasmid-free cells. Separation of the two genes (regulator and effector) allowed subsequent analysis of the molecular mechanisms involved in both control of expression and the method of cell killing. The gene encoding the small toxic polypeptide was termed hok, for host killing, and the antisense RNA gene was called sok, for suppression of killing. Killing of the cells after induction of the Hok protein was typically followed b y a morphology change in the bacteria observed under the microscope. The cells became almost totally transparent except for cell material accumulating at the poles [3]. The occurrence of such 'ghost' cells indicated that one effect of the induction of Hok could be membrane damage. This was later confirmed by showing that, immediately after induction of the protein, the transmembrane potential collapses, respiration is terminated, and the membrane contains a new small polypeptide the size of H o k [3]. It was therefore concluded that the cellular membrane of E. cob is the target for the Hok protein, and s o m e h o w introduction of the protein i~i'to the membrane leads to cell death by interference with central components of the respiratory system. The position of the hok/sok locus in plasmid R1 stimulated a search for similar genes in other plasmids, and homologous sequences were detected by hybridization in several plasmids of the enteric bacteria. Similarly, previously described genes from a number of plas-
mids turned out to be homologous both genetically and functionally [3]. In some of the hybridization experiments performed to detect plasmid homologues, more than one signal was detected, and these extra hybridization signals were detected even in plasmidfree cells [3]. One of these was rapidly identified by comparison between hok and the so-called relF gene. The relF gene is located in the relB gene cluster at 37 rain on the chromosome map of E. coli. The functional similarity between the two genes is obvious, although no biological significance of the relF gene has so far been indicated. A second hok homologous locus in the E. coli chromosome was isolated by cloning and characterized. This gefgene is located at 1 min on the E. coli map, the protein is functionally identical to the Hok and RelF proteins in terms of the mode of killing and, as in the case of hok expression, gefis regulated by an antisense RNA mechanism (not the case for relF but similar for most other members of this family). A number of properties make the g e f g e n e family an interesting source of suicide functions. The small proteins are all highly toxic in the cell even in small amounts, and the effect of their synthesis is a cascade of reactions resulting from the incorporation of the polypeptides into the cytoplasmic membrane. This central target for the toxic action makes it likely that most, if not all, bacteria are susceptible to the killing activity provided sufficient protein is expressed, and results obtained so far confirm this. It is an extra benefit that the genes are much smaller than average prokaryotic genes (less than 200 base pairs) as this will facilitate genetic manipulation and sequence analysis. It must be emphasized, however, that other types of suicide genes may possess similar advantages, and in the following no specific attention is given to the gef-like genes.
Expression control circuits The key to the design of functional suicide containment systems lies in the regulation of suicide gene expression. As long as the cells are used under defined conditions in the laboratory or in a fermentation plant, there are many possible strategies that have already been developed. However, in situations where the microorganisms are to function in the environment, little information is available about gene expression, and even though some tests may be performed in model ecosystems, the permissible level of extrapolation to the natural environment is limiting. The identification of an optimal expression system is a complex process. First, the promoter for eventual transcription of the killing gene must be sufficiently active under the prevailing conditions in the environment, leading to expression of the protein in actively growing cells, as well as in stationary cells. Cells may be in a state of dormancy most of the time, and ideally the killing should happen even in this state. It can be argued that the dormant cells do not contribute to the
Environmental potential of suicide genes Molin ecosystem activities, and therefore they constitute no potential risk; it is thus sufficient for the suicide system to function if the ceils later become reactivated. The second problem is control. Under conditions of constant variation it is difficult to predict which external signals are relevant to the bacteria, and therefore the choice of expression system for a specific environmental composition or condition is not obvious. In general, the optimal solution will be to design the expression control system in direct combination with the task of the organism, such that spread of bacteria to other localities will induce their suicide system. In some cases, where there is a high level of molecular information about the particular desired function of the bacteria, it is actually possible to achieve this (see below), but in many cases it is far from possible, and other regulatory alternatives have to be invented. Finally, it is important to k n o w whether the suicide function is active under the environmental conditions and the various physiological states of the cells. What will be the effect of a membrane porin in cells that have no membrane growth, and h o w will a toxic protein survive in ceils with increased proteolytic activity? These questions may be answered by monitoring released bacteria in which bioluminescence genes (lux, luc) have b e e n inserted together with the containment systems. The methods available to detect the light emitted from such strains with extremely high sensitivity [4%5,6"'], and the dependence on metabolic activity for these systems to function, should provide an excellent basis for determining the efficacy of the suicide systems in situ.
Degradation of xenobiotics Degradation of man-made compounds in the environment is one of the obvious beneficial applications of released GEMs. Many types of organisms have been isolated from nature that are capable of the mineralization of a large number of organic molecules, and in many cases such natural isolates are quite effective in their clean-up function. In other cases, however, the degradative pathways are either very inefficient, too c o m p o u n d specific, or incomplete resulting in conversion of one problematic c o m p o u n d to another which may be even more problematic from an environmental point of view. In some cases, it may be useful to introduce genes encoding thermoresistant degrading enzymes derived from other organisms [7]. Therefore, genetic manipulation offers the potential of optimization, expansion of specificity, and fusion of pathways to allow efficient and complete mineralization. Deliberate release of engineered microorganisms to the environment in the context of bioremediation is still at the experimental stage, and much scepticism is often expressed about the efficacy of such organisms. In a series of studies on the survival, gene transfer and function of engineered strains of Pseudomonas spp. capa-
ble of degrading substituted aromatics [8"',9"'], it was demonstrated that the organisms survived very well for four weeks in aquatic sediments whether or not the aromatics were present. In addition, it was found that the presence of the GEMs led to an accelerated degradation of the aromatics compared to situations where no engineered bacteria had been introduced. Finally, a significant rate of plasmid transfer was observed in the microcosm. Thus, data from this study show that engineered strains with improved degradative properties (in this case simultaneous mineralization of chloroand methylbenzoates) may survive well and express the genes involved in degradation such that enhanced degradative activity is observed in situ. Bacterial degradation of aromatic compounds such as the benzoates has been studied in great detail for a number of years, and much is n o w k n o w n about the specific degradative pathways and their regulation [10]. The TOL genes found in P. putida are induced in the presence of very low amounts of the substrates (toluene and different benzoates), and the spectrum of inducers and substrates has been further expanded by mutation [11,12]. The control system responsible for the regulation of these genes is quite complex, but in the present context, the important feature is the substrate-dependent transcription from the Pm promoter, for which the regulatory protein is the xylS product. The XylS protein has no activity in the absence of substrate, e.g. methylbenzoate (MB), but in its presence the XylS protein is activated, and the activated regulator acts positively on the Pm promoter to stimulate initiation of transcription, which continues as long as MB is present. In an attempt to design a strain that is fully viable in an environment comprising 3-MB, and which, because of the insertion of a regulated suicide system, is killed in the absence of the compound, a regulatory double loop has been constructed (Fig. 1). The Pm promoter was placed upstream of the lacI gene from E. coli as a substitute for the normal promoter [13,14]. In a cell harbouring this fusion and the xylS gene, a lacpgef fusion was inserted. The resulting strain was viable in substrates containing MB, because in this environment the LacI repressor is synthesized, which ensures repression of the suicide gene. When MB was removed from the substrate the cells were killed after a short period of growth, as no Lac repressor was expressed from the MB-regulated promoter under this condition. In the first demonstration of this system the organism was E. coli, and no degradation of the substrate took place. The same type of construction was later inserted into P. putida harbouring the entire TOL degradative pathway, and in this case it was possible to follow simultaneously the degradation of MB and the elimination of the bacteria after complete mineralization of the aromatic. From experiments performed in soil with and without MB it became clear that the introduction of a suicide system, as the one described here, is an efficient containment strategy even in a complex environment such as soil (LB Jensen, JL Ramos, S Molin, unpublished data).
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Survival
T'+'I "l'-' 0
3MB
I x×,s I I Km
[
0
,e
Yes
Z~
I
I Ap
ee
I
No
1
[]
Fig. 1. Elements of conditional containment systems based on the TOL plasmid regulator XylS, Lacl and a killing protein. The operation of the killing system is described in the text. (a) Conditions under which survival of the contained bacteria is predicted. (b) Conditions under which it is predicted that the bacteria bearing the containment system will die. 3MB, 3-methylbenzoate. Symbols © and @, inactive and active forms of XylS protein respectively; Z_ and L , Lacl and Gef proteins, respectively.
The major point raised here is that, in connection with bioremediation, the engineered bacteria of interest most often harbor genes from pathways that have been characterized in great detail, and it is therefore possible to design the regulatory loops necessary for the described mode of site-conditioned survival. Even though it may seem difficult to imagine environmental risks in relation to bioremediation, the activity of the microorganism is only predictable in the context for which it was designed, and if it is possible to restrict their survival to the execution of this 'job', there is basically no reason not to do so.
Biopesticides The use of chemicals to combat pests is viewed with increasing concern because of the adverse environmental side effects. Many of the pesticides are recalcitrant and they are toxic to many organisms outside the target group. Although the strategy of biodegradation may also be applied to chemical pesticides it seems irrational to employ compounds that are recognized as harmful to the environment and then try to remove them later. The alternative insect toxins produced by B. thuringiensis offer the following advantages: they are highly specific, they are degradable in the environment, and they are highly toxic to the target organisms. In addition, many different types of toxins have n o w been identified that are active against a broad spectrum of insects [15,16]. It is therefore not surprising that these toxins constitute 90-95% of commercial biopesticides. When B. thuringiensis toxins are used as biopesticides they are sprayed on fields as protein crystals. The crystals are relatively easy to purify and formulate as they are released from the bacteria during cellular lysis w h e n the ceils enter the stationary phase. However,
the crystal proteins are only active in the field for a relatively short period of time, and attempts have therefore been made to design biopesticides based on these toxins with a longer life span in nature. The finding that the toxins may be expressed in other bacteria and maintain their toxicity allows for transfer of the appropriate genes to bacteria that are k n o w n to live in close association with plants (in contrast to B. thuringiensis itself). A particular application of these toxin biopesticides concerns the combatting of mosquitoes b y killing the larvae as they develop in fresh water. As the larvae feed on microorganisms in the water, an ideal formulation of the toxins would be to express them in live cyanobacteria. A recent report [17"] on the successful transfer of a toxin gene from B. thuringiensis subsp. israelensis, which is active against mosquitoes, to a strain of the cyanobacterium Agmenellum quadruplicatum, and the expression in this organism of a stable and active toxin, holds promise for the design of biopesticides to be used in connection with elimination of insect vectors of various diseases. This type of genetically engineered bacteria have not been commercialized yet, mainly because of the lack of knowledge about the potential risks of such organisms to ecosystems. A serious question is whether the organism and its toxin gene will spread outside the target area. For plant-associated bacteria it may be possible to limit the survival of the bacteria to a particular growth zone. Bacterial genes responding to plant c o m p o u n d s like root exudates have been identified [18], and even before the actual regulatory details have been w o r k e d out it may be possible to insert suicide genes in a control circuit involving the plant signals such that cells escaping from the plant zone will be killed. The availability of bacterial gene regulators in combination with other niche-specific regulators, makes it possible to design site-directed induction systems for the expression of suicide functions that will interfere with a totally random and uncontrolled spread of man-made microorganisms from the original site of release. The increasing interest in microbial ecology, and in symbiotic relationships in particular, gives promise for the identification of many such useful control systems from a broad spectrum of bacteria. A different approach to biological containment in the design of biopesticides is stress-induced limitation of survival. In contrast to the life of bacteria in nutrientrich laboratory media, microbial life in the natural environment is probably usually a state of starvation and other types of stress (pH, extreme temperatures, toxic compounds etc.). It is therefore to be expected that any organism released to the environment will meet severe stress conditions sooner or later. Bacterial responses to different forms of stress have been studied for some time and, especially for bacteria like E. coli [19,20"], S. typhimurium [21], and Vibrio app. [221, much is k n o w n about the genetic programmes responsible for coping with these extreme growth conditions. The picture emerging from these studies is that bacteria have differentiation programmes that are in operation w h e n conditions become harsh. The sporulation cycle
Environmental potential of suicide genes Molin of Gram-positive bacteria is an excellent example of gross changes in cellular morphology and metabolism, but many non-sporulating bacteria also possess genetic programmes for stress-induced morphology, and resistance to various factors and metabolic changes. Specific genes are activated in an ordered sequence as a response to stress, as revealed clearly by two-dimensional gel electrophoresis, and analogously certain recovery genes are activated u p o n the return of normal growth conditions after a stress period [22].
mal growth; however, w h e n placed under starvation conditions during which the wild-type organism normally survives well, the double insertion mutant strain s h o w e d decreasing viability resulting in almost total elimination after 10-15 days.
Such studies of differentiation are also being carried out in different plant-associated Pseudomonas strains, and the observations accumulated so far clearly indicate the presence of similar genetic programmes in these bacteria (M Givskov, L Eberl and S Molin, unpublished data). The possibility of fusing stress gene promoters with a killing gene is an obvious containment strategy. Promoters active at late times after induction of a stress programme are especially useful, as killing will not take place until after an extended period of stress. In this way, an undesired rapid elimination of the population after short transient stress periods can be avoided. Alternatively, fusions to specific recovery gene promoters will allow a significant period of survival followed by a subsequent reduction in the population. Among the stress genes of particular interest in the context of suicide containment are those involved in starvation survival. Feast and famine will unquestionably be experienced by bacteria in the environment, and starvation is a condition that is very easy to reproduce in the laboratory. Naturally, the more that is k n o w n about such pathways and their regulation, the better are the possibilities for interfering with and controlling survival in the environment.
Live bacterial vaccines are not often mentioned in connection with the deliberate release of GEMs, but as engineered strains of Salmonella spp. or E. coli are being considered as interesting vectors of antigen presentation, the lack of confinement in this type of application must be taken into consideration.
Limited survival may also be accomplished by mutations in genes whose products are essential under stress conditions. Interesting results have been obtained for Vibrio $14, whose stress-induced differentiation programme has been studied for some years [22]. If cells of this organism are starved for a carbon source they become very small, and the metabolism changes drastically. Specific proteins are synthesized at different times after the onset of starvation, and the pattern of macromolecular synthesis strongly indicates the existence of a regulatory network responsible for the changes and the sequence of events. In an attempt to expose such control circuits, fusions were constructed by the insertion of ~-galactosidase reporter genes into genes that are specifically induced under conditions of starvation (.J ()stling and S Kjelleberg, personal communication). Subsequently, mutagenic transposons were inserted at random in the chromosome, followed by screening for clones that showed a lack of starvationinduced gene expression. After showing that such insertions were not interfering directly with the reporter gene, it was concluded that the second site insertion had inactivated a regulatory gene responsible for activation of one or more genes which are normally turned on after starvation. The two insertion mutations described in genes of importance for starvation-induced gene expression had no significant effect during n o r -
Live vaccines
One promising strategy for designing live vaccines is to make fusions between antigen-coding sequences and genes encoding surface-bound proteins like flagellar proteins, outer membrane proteins or fimbriae [23]. In such cases, the antigenic determinants will be presented on the outside of bacterial cells, and it is believed that the proliferative properties of such strains will make them more effective than traditional vaccines. Obviously, the host organisms must not be pathogens, and therefore biological containment approaches are not supposed to compensate for any pathogenicity exerted by the bacteria inside the animal or human body. Containment is only relevant for the purpose of limiting the spread of the engineered genes and the survival of the strains after their excretion to the environment. Although some of the containment designs mentioned above are also relevant in this context, the most suitable approach for live vaccines based on enteric bacteria is stochastic induction of killing based on recombinational switching of gene expression. Recombinational switches are found in several operons in bacteria and bacteriophages. The switch promoter directing the synthesis of Type 1 fimbriae in E. coli (fimAp) has been used as a model system for stochastic induction of suicide [24]. The promoter is located within a 314 base pair DNA sequence that inverts its orientation approximately 2 x 10-3 times per cell per hour (measured in fast growing ceils) [24]. This invertible sequence comprises a promoter that directs transcription into one of the flanking sequences, so that any gene controlled by the promoter will be switched from on to off with this frequency. The insertion of a suicide gene next to the switch promoter, fimAp, was the first approach used to obtain time-dependent induction of suicide [2,25"], and in a series of subsequent investigations both the hok and the gef genes have been employed (S Molin, unpublished data). E. coli cells harbouring such inserts on plasmids or integrated into the chromosome behave according to expectations: in cell suspensions where no net growth is possible because there is no external carbon source, viable cell counts constantly drop in contrast to the non-contained control strain, indicating that switching occurs in the absence of growth. In fast growing
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Environmentalbiotechnology cultures (doubling times of approximately 30 rain) no effect of the suicide system on the growth rate of the culture is observed, which suggests that the killing rate is insignificant relative to the growth rate. However, if the growth condition is changed to a p o o r carbon source, the consequence of the presence of this type of stochastic suicide system is apparent growth inhibition of the cell population: the slower the cells grow, the more severe is growth inhibition by the fimAp-geffusion, indicating that the killing rate becomes increasingly significant relative to the decreasing growth rates under the chosen conditions. In principle, it seems to be possible to design functional stochastic induction systems for bacterial suicide. The particular system based on the fimA switch promoter unfortunately only works in the enterobacteria, and even in some of these the switch frequency is too low to be of any use. So far the best results have been obtained in E. coli and S. typhimurium, whereas for example no switch takes place at all in P. putida (LB Jensen and S Molin, unpublished data). For live vaccines, this type of containment would allow their proliferation and competition in the gut of the vaccinated individual, whereas progeny excreted to the environment from the animal or human host would be eliminated eventually because of the poor growth conditions in the outside environment combined with the time-dependent stochastic induction of killing.
Conclusion
Acknowledgements Parts of this review are published in Annual Review of Mzcrobiology 1993 [25"], and I am grateful for the permission given by Annual Reviews Inc to u s e these parts here. The work performed in the author's laboratory has been supported by European Commission contracts.
References and recommended reading Papers of particular interest, published within the annual period of review, have b e e n highhghted as: of special interest •o of outstanding interest 1
SOBECKYPA, SCHELLMA, MORAN M.A, HODSON RE: Adaptation o f Model Genetically Engineered M i c r o o r g a n i s m s to Lake Water: G r o w t h Rate E n h a n c e m e n t s and Plasmid L o s s . Appl Environ Microbtol 1992, 58:3630-3637.
2.
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 for Cont a i n n a e n t o f Bacteria a n d P l a s m i d s . Biotechnology 1987, 5:1315-1318
3.
GERDESK, POULSEN LK, TI-~STED T, NIELSEN AK, MARTINUSSEN J, ANDREASENPH. The hok Killer G e n e F a m i l y i n GratnNegative Bacteria. New Biol 1990, 2 946-956
4.
DE WEGER LA, DUNBAR P, MAHAFEE WE, LUGTENBERG BJJ, SAYLER GS: Use of B i o l n m i n e s e e n e e Markers to Detect P s e u d o m o n a s s p p in t h e R h i z o s h e r e . Appl Environ Microbiol 1991, 57:3641-3644. A fine paper describing the use of lux g e n e s in relation to detailed studies of plant-microbe interactions. Bioluminescence is s h o w n to be an adequate tool in monitoring specific bacteria relative to their position on plant roots. 5
The predictable argument against the suicide systems described here relates to the failure of systems resulting in total elimination of a population. Even if optimized combinations of killing genes and gene expression sequences are constructed that will be 100% effective u p o n induction, the occurrence of mutations in a cell population will never be eliminated. Therefore, if an organism is considered a serious risk to the environment in connection with applications, the containment of such strains will not be sufficient to remove the potential risk. Even if no direct risks are foreseen, there are still concerns regarding the release of engineered organisms to the environment, and we suggest in this context that, as long as such concerns prevail, attempts should be made to minimize putative problems by reducing the exposure time and persistence of the introduced organisms as much as possible. If this is the purpose of biological containment, a reduction of the population size over a limited time by a factor of a million or even less seems to be of value. There have been many bad':experiences with man-made chemicals accumulating in the environment, extensive use of antibiotics etc., and measures are n o w being taken against these problems by encouraging the use of degradable rather than recalcitrant compounds. In the same way, we find it prudent to use survival-deficient engineered organisms rather that very persistent strains, even in cases where there are no obvious risks.
SHAW JJ, DANE F, m i n e s c e n e e for Microorganisms Environ Microbtol
GEIGER D, KLOEPPER J'W: U s e o f BioluD e t e c t i o n o f Genetically Engineered Released into the Environment. Appl 1992, 58:267-273.
6.
SILCOCK DJ, WATERHOUSE RN, GLOVER LA, PROSSER JI, KILLHAM K Detection o f a Single G e n e t i c a l l y Modified Bacterial Cell in Soil b y Using Charge Coupled Device-Enhanced M i c r o s c o p y Appl Environ Microbiol 1992, 58:2444-2448. This paper describes the microscopy techniques combined with ultra-sensitive charge-coupled device cameras that have b e e n developed for monitoring single bacterial cells in very complex enviromnents. The results presented here allow previously impossible analyses of bacterial life in the environment, a n d it is certain that this approach will create highly interesting insights into microbial ecology in the coming years. • .
7.
DONG FM, WANG LL, WANG CM, CHENG JP, HE ZQ, SHENG ZJ, SHEN RQ. M o l e c u l a r C l o n i n g a n d Mapping o f P h e n o l Degradation Genes f r o m Bacillus s t e a r o t h e r m o p h i l u s FDTP-3 a n d Their E x p r e s s i o n in Escherichia c o i l Appl Environ Microbiol 1992, 58:2531-2535
8. •.
PIPKE R, WAGNERDOBLER I, TIMMIS KN, DWYER DF. Surviva1 a n d Function o f a Genetically E n g i n e e r e d P s e u d o m o n a d i n A q u a t i c Sediment Microcosms. Appl Environ Microbiol 1992, 58:1259-1265. Shows the potential benefits of redesigning degradative pathways by genetic engineering. The results clearly demonstrate the fitness of the engineered organisms, and the effect on the rate of degradation of the aromatic c o m p o u n d is significant. The data argue against simplified statements o n the lack of positive results in bioremediatlon after applying GEMs. 9. •,
NUSSLEINK, MARIS D, TIMMIS KN, DWYER DF: Express i o n a n d Transfer of Engineered Catabolic Pathways
Environmental potential of suicide genes/violin Harbored by P s e u d o m o n a s s p p I n t r o d u c e d i n t o Activ a t e d S l u d g e M i c r o c o s m s . Appl Environ Mzcrobiol 1992, 58:3380-3386. This is a follow-up to the previous paper, which extends the observations on the advantages of using GEMs in bioremediation contexts. The strains designed are effective, a n d their activities with respect to degradation o f various aromatics could not easily have b e e n encountered from the indigenous microflora.
successful insertion of one of the toxin g e n e s into a cyanobacterium a n d its demonstrated insecticidal activity in this n e w host point to other potential apphcatlons in mosquito control and their role in a n u m b e r of infectious diseases. 18.
CLARKE HRG, LEIGH JA, DOUGLAS CJ M o l e c u l a r S i g n a l s i n t h e I n t e r a c t i o n s B e t w e e n P l a n t s a n d M i c r o b e s . Cell 1992, 71:191-199.
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MATINA. M o l e c u l a r A n a l y s i s o f t h e S t a r v a t i o n Stress i n E s c h e r i c h i a coli. Microb*ol Ecol 1990, 74:185-196
RAMOS JL, MICH~N C, ROJO F, DWYER DF, TIMMIS KN. Signal-Regulator Interactions. Genetic Analysis of the Effector B i n d i n g Site o f XylS, t h e B e n z o a t e - A c t i v a t e d Positive R e g u l a t o r o f P s e u d o m o n a s TOL P i a s m i d MetaC l e a v a g e P a t h w a y O p e r o n . J Mol Biol 1990, 211:373-382.
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RAMOSJL, STOLZ MA, REINEKE W, TIMMIS KN A l t e r e d Eff e c t o r Specificities i n R e g u l a t o r s o f G e n e Expression: TOL P i a s m i d x y l S M u t a n t s a n d T h e i r U s e t o E n g i n e e r E x p a n s i o n o f t h e R a n g e o f A r o m a t i c s D e g r a d e d by Bacteria. Proc Natl A c a d Sci USA 1986, 83:8467-8471.
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DELGADOA, WUBBOLTSMG, ABmL MA, RAMOSJL: N i t r o a r o m a t i c s A r e S u b s t r a t e s f o r t h e TOL P i a s m i d U p p e r - P a t h w a y E n z y m e s . Appl Environ Microbiol 1992, 58.415-417.
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CONTReRAS A, 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
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FEITELSONJS, PAYNE J, KIM L: B a c i l l u s t b u r i n g i e n s i s : Ins e c t s a n d B e y o n d . Biotechnology 1992, 10:271-276.
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LECADET MM, CHAUFAUX J, RIMER J, LERECLUS D: C o n s t r u c t i o n o f N o v e l Bacillus t h u r i n g i e n s i s S t r a i n s w i t h D i f f e r e n t I n s e c t i c i d a l Activities b y T r a n s d u c t i o n a n d T r a n s f o r m a t i o n . Appl Environ Microb*ol 1992, 58:840-849
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MURPHYRC, STEVENS SE: C l o n i n g a n d E x p r e s s i o n o f t h e c r y l V D G e n e o f Bacillus t h u r i n g i e n s i s S u b s p . i s r a e lensis i n t h e C y a n o b a c t e r i t t t n A g m e n e l l u m q u a d r u p l i c a t u m PR-6 a n d Its R e s u l t i n g Larvicidal Activity. Appl Environ Microbiol 1992, 58:1650-1655. The Bt toxins have mainly been discussed m the context of agricultural applications as biopestlcldes directed against plant pests. The
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NYSTROM T, NEIDHARDT FC. Clotting, M a p p i n g a n d Nucleotide Sequencing of a Gene Encoding a Universal S t r e s s P r o t e i n i n E $ c h e r i c h a coll. Mol Microbiol 1992, 6:3187-3198. This is a very interesting and detailed molecular analysis of a n e w g e n e involved in the stress response of E. colt. It should be considered as a model example of m o d e r n molecular genetics applied to environmental issues. 21.
SPECTORMP: G e n e E x p r e s s i o n i n R e s p o n s e to Multip l e N u t r i e n t - S t a r v a t i o n C o n d i t i o n s i n S a l m o n e l l a typ h i m u r i u m . Microbiol Ecol 1990, 74:175-184
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NYSTROMT, ALBERTSON NH, FLARDH K, KJELLEBERG S: P h y s i o l o g i c a l a n d M o l e c u l a r A d a p t a t i o n to S t a r v a t i o n a n d R e c o v e r y f r o m S t a r v a t i o n b y t h e M a r i n e Vibrio. Microbtol Ecol 1990, 74:129-140.
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STOCKERBAD: A r o m a t i c - D e p e n d e n t S a l m o n e l l a as Live V a c c i n e P r e s e n t e r s o f F o r e i g n I n s e r t s i n Fiagellin. Res Microbiol 1990, 141.787-796.
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EISENSTEINBI: P h a s e V a r i a t i o n o f T y p e 1 F i m b r i a e i n E $ c h e r i c h t a colt is U n d e r T r a n s c r i p t i o n a l C o n t r o l . Science 1981, 214:337-338.
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MOLIN S, BOE L, JENSEN LB, KRISTENSEN CS, GIVSKOV M, RAMOSJL, BEJ AK: Suicidal G e n e t i c E l e m e n t s a n d T h e i r U s e i n Biological C o n t a i n m e n t o f Bacteria. A n n Rev Microbiol 1993, 47:139-166. T h e first review d e s c n b m g the potential a n d strategies behind designs of biological containment systems. The review covers all the major attempts m a d e to construct disabled bacteria for a n u m b e r of applications, and the reference list covers most of this field
Soren Molin, Department of Microbiology, Technical University of Denmark, Building 221, DK-2800 Lyngby, Denmark.
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