Plasmid promiscuity and chastity and its uses

Biotech. Adv. Vol. 8, pp. 515-537,1990

0734-9750/90 $0.00 + .50 © 1990 Pergamon Press ptc

Printed in Great Britain, All Rights Reserved.

P L A S M I D P R O M I S C U I T Y A N D C H A S T I T Y A N D ITS USES V. N. IYER Department of B i o l o g y and Institute of Biochemistry, Curleton University, Ottawa, Ontario, Canada

ABSTRACT

Bacterial plasmids are obligate and l n t r a c e l l u l a r genetic elements that replicate and are maintained autonomously from the chromosome. They are ubiquitous. Someof them are r e l a t i v e l y more promiscuous than others. Plasmld genetic systems that contribute to r e l a t i v e promiscuity or chastity In naturally occurring plasmlds are described and discussed. Both the promiscuity and the chastity of plasmld-based genetlc systems have applications In bacterial molecular genetics, In the production of recombinant DNA products and In the breedlng and use of desirable bacteria.

The role of

these systems In such applications ls considered. KEYM)ROS

Vectors, broad-host-range, narrow-host-range, bacterial plasmlds, transfer, cloning, environmental-release. INTB(X}UOTIONAND SCOPE The discovery and exploitation of microorganisms wlth useful metabolic properties from t h e l r natural habltats has been the corner-stone of Industrial microbiology. In many Instances, this approach was supplemented by microbial mutatlon and selection programmes and this remains a useful enterprise. However, with the advent of recombinant DNA technology, we are no longer constrained to the specles In whlch the product or the a c t l v l t y was f i r s t discovered. Thls Is w e l l - I l l u s t r a t e d by t i e change of our view of EscherfchYa coZY, an organlsm whlch unt11 recently was not considered as belng I n d u s t r i a l l y Important.

As a consequence of the fact that E. col1 ls the

515

516

V . N . IYER

best-understood organism (57) and that many of the concepts and technology of recombinant ONA were developed with t h l s bacterium, several d i f f e r e n t genes from d i s t a n t l y related organisms have been introduced Into t h l s species to produce commercially useful products (e.g. 8).

Thls ls l l k e l y to continue.

However, although E. coll w111 In many cases f u l f i l

a role as an intermediate

host, there are circumstances when I t w111 not be the preferred host for the engineering of a bacterium or a product.

I t is therefore necessary to

consider technology for the t r a n s f e r , maintenance and expression of genes that may on occasion be cloned and characterized In E. t o l l but w i l l then need to be t r a n s f e r r e d , maintained and expressed In the bacterium of choice. Promiscuity associated with plasmlds or plasmld gene vectors can make t h i s possible.

Promiscuity ls however not always desirable and there are

circumstances In blotechnology where I t w i l l be useful to r e s t r a i n or control promiscuity.

Seeking an understanding of the basis of plasmld promiscuity or

of plasmld c h a s t l t y Is therefore important.

The i n t e n t of t h i s c o n t r i b u t i o n

ls to review s e l e c t i v e l y p e r t i n e n t Information t h a t may lead to such an understanding, to expose areas of r e l a t l v e ignorance and to lead the reader to a vast l l t e r a t u r e on the subject.

In thls way, the potential user could make

a judlclous constructlon or selection of a plasmld vector wlth propertles deslrable for a particular purpose. The examples used w i l l be i l l u s t r a t i v e rather than comprehenslve, and except In special cases, the references that are provided w l l l not be complete but rather those that w l l l lead the reader to access appropriate l i t e r a t u r e .

PLASMID PROMISCUITY IN NATURE; WHEN IS A PLASMID CONSIDERED PROMISCUOUS? N a t u r a l l y - o c c u r r i n g plasmids that have been considered to be promiscuous (14, 48, 59, 71, 81) must, as a r u l e , have at t h e i r dtsposal mechanisms f o r t h e i r t r a n s f e r from one b a c t e r i a l species to a broad range of other species.

They

must also have mechanisms t h a t promote t h e l r ab111ty to r e p l i c a t e and be maintained In a stable manner In these c e l l s or c e l l populations a f t e r they acquire the plasmld.

Plasmlds t h a t have l i m i t e d host-ranges for e i t h e r of

these two groups of processes would not as a rule be ca]led promiscuous.

At

present, there is I n s u f f i c i e n t Information to e s t a b l i s h absolute c r i t e r i a for promiscuity because of the f o l l o w i n g reasons: (a) the s e l e c t i o n of b a c t e r i a l strains and species for tests on host-range have often been a r b i t r a r y rather than systematic; t h i s ls understandable because attempts to r e s t r u c t u r e b a c t e r i a l taxonomy to r e f l e c t natural r e l a t i o n s h i p s are In t h e i r infancy (89) and spe~les and genera as defined a t present may be p h y l o g e n e t i c a l l y intermixed, (b) the c r i t e r i a used by d i f f e r e n t i n v e s t i g a t o r s to draw

PLASMID PROMISCUITY AND CHASTITY

inferences about host-range have not been uniform; for example, some Investigators have drawn inferences on the host-range of a plasmld following the successful selection of a recipient carrying the plasmld or plasmld marker; others have drawn a posltlve Inference about host-range only I f the plasmld ls maintained in a recipient population for a number of generations without deliberate selection for plasmld maintenance, (c) inferences about a genus have sometimes been made from examining only one or two species in the genus, (d) naturally-occurring plasmlds can be co-Integrates of more than one plasmld; unless the plasmld Is genetically or physically characterized, this w i l l not be known and Inferences w l l l be llmlted. Whlle these are a l l limitations in defining promiscuity In naturally occurring plasmlds, such plasmlds are seldom used d i r e c t l y for biotechnologlcal applications. Plasmld vectors used In blotechnology are usually engineered and since t h e i r construction and characterization no longer poses serious limitations, they ought to be better-characterized than the natural plasmlds from whlch they derive. Whenused for commercial or environmentalapplications, the hostrange of these vectors Is best determinedby experlment rather than being assumed from the propertles of the naturally-occurrlng plasmids from which they derive. Detalled structural and functional studles on representative members of naturally-occurrlng plasmld groups is important becausean understanding of the various strategies that plasmlds have evolved to malntain themselves in one or more hosts can be very useful in designing and constructing plasmid vectors that are best-sulted for a particular purpose. For practical and historical reasons, the best-known plasmids are those that include EscherlchYa colY K-12 as one of t h e i r hosts. Several but not a l l such plasmlds have been conveniently classified lnto l n t r a c e l l u l a r lncompatabtllty groups and in E. c o i l about t h i r t y such groups have been i d e n t i f i e d . Among these, the P group of plasmlds (examples RK2, R751) has long been heralded as being extremely promiscuous and recent reviews (71, 81) have included the groups C (example R40-a), N (examples pCUl, R46), O (examples RSFIOIO) and W (example pSa) as being promiscuous among Gram negative eubacterla. These reviews and an earller review (39) w111 be useful to consult for access to the extensive primary l i t e r a t u r e on thls group of plasmlds. The following are some b r l e f comments on them. (a) These plasmlds must by d e f i n i t i o n have promiscuous systems for Interbacterial transfer and also promiscuous systems for l n t r a - b a c t e r l a l maintenance. (b) Plasmlds of a l l five groups are self-transmissible by bacterial mating or can be mobilized for e f f i c i e n t transfer by other self-transmissible

517

518

Table 1.

V. N. IYER

Examples of less-well-known naturally 0ccurrlng plasmlds for which there ls some evtdence of promiscuity.

Plasmld

Hosts

References

pLS1

Bacillus s u b t l l l s Escherfchla co11 Streptococcus pneumonlae

49

pAl~l

Lactobaclllus case! Staphylococcus aureus Streptococcus pneumonfae Clostrfdfum acetobutylfcum

14, 31, 62

pU8110

8aclllus s u b t l / l s Staphylococcus aureus

28, 64

TOL

Escherlchla co11 Pseudomonas aeruglnosa Pseudomonas putlda

4

pFA3 (pJD4)

Escherlchfa col! Haemophllus Nelsserla gonorrhoeae

26, 91

pNG2

Corynebacterlum Escherlchla co11

75

pC194

Staphylococcus aureus Escherfchla colf

27

PLASMID PROMISCUITY AND CHASTITY

plasmlds. This is indicative of the relative Importance of bacterial mating systems in determining plasmld promlsculty among Gram negative eubacterla in nature. (c) Any plasmld, whether promiscuous or not, is composed of several genetic systems each of which could have different l i m i t s to promiscuity. For a thoughtful use of such systems, we need to f i r s t establish the host-range of each component system and the genetic or physiological circumstances that l i m i t or expand i t s host-range. I t is by adjusting these circumstances or/and by a r t i f i c i a l l y recomblnlng the different component systems that i t may be possible to exploit f u l l y t h e l r promiscuity or chastity. At present, the molecular basis of promiscuity or of chastity is not completely understood for any plasmld. Though obvious, I t is also useful to emphasize that transfer to and maintenance in E. coll is not a pre-condltlon for a plasmld to quallfy as promiscuous. As plasmlds and plasmld promiscuity are explored in other bacteria, i t is l i k e l y that examples of promiscuous p]asmlds that may not or may include E. coll as one of t h e i r hosts w l l l be uncovered. Table 1 l i s t s examples of some less well-known naturally-occurring plasmlds that may be rewarding to examine. GENETIC SYSTEMSTHAT PROMOTEOR LIMIT PROMISCUITY A plasmld can be viewed as an assemblage of different genetic systems each of which may have had a separate evolutionary hlstory and at present is r e l a t i v e l y chaste or promiscuous. Fig. 1 Indicates possible mechanisms by which a bacterial plasmld may galn entry lnto a new host and eventually be maintained In such a host. Genetlc systems could exist to l l m l t or promote each of these steps. I t w l l l be useful to consider them separately. Systems for intercellular transfer.

Bacterial matlnq and cell fusion. The mating system of the classical plasmid F Is the best-described and has served as a paradigm for other mating systems of Gram negative eubacterla (87, 88). Although thls plasmid Is not promiscuous, there is evidence to indicate that l t s mating system ls operative In matlngs between E. coll and Gram posltlve bacteria (29) and even between E. coll and yeast (Saccharom¥ces cerevlslae) at a low frequency (32}. There are also other conjugatlve plasmlds of enteric bacteria such as ColIb-P9 or R64drdll which themselves have a narrow-host-range but the mating systems of

519

V.N.IYER

520

BACTERIAL MATING NATURAL ONA TRANSFORMATION GENERAL TRANSDUCTION

INTERCELLULAR TRANSFER

TRANSFER OF DNA COMPLEXED WITH PROTEIN

AVOIDANCE OF RESTRICTION

ESTABLISHMENT

CIRCULARIZATION

ATTACHMENT TO CELL

MEMBRANE?

MAINTENANCE

REPLICATION

~

DISTRIBUTION RESCUE BY RECOMBINATION

FIg.1. Possible modes of plasmid entry, establishment and maintenance.

PLASMID PROMISCUITY AND CHASTITY

which have a broader host-range (e.g. 6). Potentially therefore, this system may be amenable to exploitation for gene transfer from E. coll to distantly related bacteria. At present however i t ls the promiscuous matlng systems of N, P, W and G groups of plasmlds that have been most u t i l i z e d . A common feature of these mating systems that ls relevant t o t h e i r use Is that the efficiency of mating can be very high when matlngs are conducted on solid or semi-solid surfaces. For example, using plasmtds that specify the N matlng system and a donor to recipient ratio of 10:1, i t has been possible In our laboratory to detect transconJugants easily wlthout the use of selectable markers. The host-range of these mating systems tend to be broader than t h e i r respective systems for plasmld establishment and maintenance. ConJugatlve plasmlds have also been Identified in several Gram posltlve eubacterla and some of them medlate e f f i c i e n t matlng only on solid surfaces and they are promiscuous (14). A sub-class of these Gram positive conJugatlve plasmlds are those found so far only In Enterococcus faecalfs and which make t h e i r donors responsive to matlng pheromones excreted by potential recipients. Genetlc and functional analysis has been undertaken on the mating systems of two of this class of E. faecalls plasmlds, pAD1 and pCF10 (17, 85, 12). The mating systems of these pheromone-responsive plasmlds are apparently chaste. Chastity In these cases could be determined by the I n a b i l i t y of other recipient bacterial species to produce and transmit the relevant pheromone. The behavlour of these plasmlds and t h e i r hosts underscores the role of recipients In the s p e c i f i c i t y of bacterial mating reactions, a role which ls not s u f f i c i e n t l y evident in matlngs Involving Gram negative eubacterla. Conjugatlve plasmlds have been reported in the i n d u s t r i a l l y important group of bacteria, the $treptom¥ces and also In StreptovertYclillum and Streptosoranglum. There is evldence Indicating that the mating systems of these plasmlds have a degree of promiscuity. For a detailed consideration of $treptomyces plasmlds, the attention of the reader Is drawn to a recent review by Hopwood and to references therein (36). I t ls not clear that the mating systems of these plasmlds are any more promiscuous than t h e i r systems for establishment and maintenance. However, there do exist non-conJugatlve plasmlds that can be transmitted to and maintained in several species of

$treptom¥ces. The consideration of the I n t e r c e l l u l a r transfer of $treptom¥ces plasmlds and of the pheromone-responsive Enterococcus plasmlds along with the mating systems of the Gram negative eubacteria should not be taken to imply a s i m i l a r i t y In details of gene transfer mechanism. The presence and functlon of protelnaceous plasmld-coded surface organelles called sex p i l l Is

52~

522

V . N . 1YER

characteristic of most mating systems of Gram negative eubacterla whlle these are not observed In Streptom¥ces or Enterococcus. $treptom¥ces plasmlds are Imagined to be transferred by a process of hyphal fusion that ls presumably determined by host-chromoson~l genes wlth the plasmld providing only an orlgln of transfer and possibly, functions related to the u t i l i z a t i o n of this origin. Natural DNA transformation.

When cultures of certaln bacterial specles (e.g.

Bacillus subtllls) are subjected to particular growth regimens, a fraction of the popu|atlon achieves a temporary state called 'competence' when thls fraction is ab]e to take up extrace]lular DNA and Integrate I t Into Its genome. The phenomenonls known to occur In diverse genera of both Gram posltlve and Gram negative eubacterla (e.g. Baclllus, Haemophllus) and has been referred to as 'natura| transformation' to distinguish I t from such nonphysiological procedures of transformation such as those now used routlne]y for E. coll. Even though natural transformation with plasmld DNA 1s a low frequency event, t t is possible that I t has contributed to the dispersal, in nature, of certain plasmlds. Natural transformation wlth plasmld DNA has been important for the development and use of cloning vectors In Bacillus species

(28, 51). Generalized transductlono Generalized transductlon Is the a b i l i t y of a virus particle to accidentally encapsldate any part of host DNA and transmit thls DNA to other hosts through a viral Infection process. I t Is a widespread phenomenonamong eubacterla and where studied, has lnvolved the encapsldatlon and transmission of a continuous fragment of DNA of a size that approximates viral DNA and whlch is determined by the volume of the capsld. Hence, the term 'head f u l l packaging' (52).

Plasmld DNA can be packaged and therefore

can be presumably dispersed by transductlon but only wlthln narrow boundaries determined by the host cell-surface-attachment s p e c if ic it y of the vlrus. In the phototrophlc bacterium Rhodabacter capsulatus, a process slmllar to general transductlon and called 'capsductlon' exfsts (53). In this process, fragments of host DNA are packaged In particles and the DNA is transmitted to recipients but these particles called 'gene transfer agents' are not a subclass of viral particles. These agents can however be used In the same way as transducing particles. Transfer of DNA complexed wlth proteln.

A well-known example of a promiscuous

transfer system is associated with the plant tumor-inducing plasmlds of AgFobacterlum species. In this case, a system contained in a region of the plasmld called the vtr region promotes the transfer of plasmld DNA Into the

PLASMID PROMISCUITY AND CHASTITY

cells of a broad range of plants.

523

There Is substantial circumstantial

evidence Indicating a resemblance of thls transfer process to that of transfer occurring during bacterial mating (reviewed In 93). However, certain aspects of the process also begin to suggest an analogy to the extrusion of singlestranded DNA bacteriophages. For Instance, there ls recent evldence suggesting that large amounts of one of the vtr reglon protelns (VIrE2) can p o t e n t i a l l y complex with the transferring slngle strand of DNA (11, 13, 25, 73).

The v i r system does not however mediate Inter-bacterial gene transfer

between Agrobacterlum species for whlch AgrobacterYum plasmlds have evolved a separate mating system. However, I t has been reported (7) that the leading region of the promiscuous transfer system of a plasmld of Gram negative eubacterla can promote plasmld DNA transfer to plant cells. Mechanisms l l m l t l n q or promotlnq plasmld establishment. In several of the examples of plasmld DNA transfer that were considered, the available evldence Is consistent with a b e l i e f that the DNA that is transferred Is 11near and that the transfer is polarized. This DNA is now In a p o t e n t i a l l y hostile environment. Furthermore, most bacterial plasmlds exist In vegetative cells as clrcular molecules that are covalently closed. The establishment In a recipient cell of a transferred llnear plasmld DNA molecule prior to Its replication must thus Involve both Its ab111ty to override the potential h o s t i l i t y of the new environment and to reclrcularlze. Some circumstances that deter plasmld establishment are known but mechanisms of plasmid establishment are not well-understood. DNA r e s t r i c t i o n endonucleases which are widespread In bacterla constitute an effective barrier to plasmld establishment. Several r e s t r i c t i o n endonucleases are coded by plasmlds Indigenous to a bacterial host and which w l l l therefore r e s t r i c t other lncomlng plasmlds. The effects of r e s t r i c t i o n endonucleases can usually be overcome by selecting for the small fraction of transferred plasmlds that escape thls r e s t r i c t i o n and become modlfled by the modification system of the host. Alternatively, mutants of hosts that are defective In r e s t r i c t i o n may be Isolated and used. In natural transformation systems with llnear DNA, the DNA Is detectably single-stranded during or soon after entry. Since almost a l l r e s t r i c t i o n endonucleases use double-stranded DNA as substrates, the transferred single-stranded DNA In such systems may be temporarily protected until they are consumed by recombination In a successful transformation reaction. The reason why the transformation of competent B. s u b t l l i s cells wlth plasmld DNA ls I n e f f i c i e n t r e l a t i v e to chromosomal DNA ls

524

V . N . IYER

not understood (15). The mechanism by which l i n e a r l y transferred plasmld DNA molecules reclrcularlze In recipients is unknown. I t occurs e f f i c i e n t l y In cells that are deficient in general homologous recombination (RecA" E. coll). In bacterial conjugation, one p o s s i b i l i t y Is that i t occurs by a reversal of the reactions that I n i t i a t e s strand nicking at the region of the orlgln of transfer when the plasmld was In the donor. Site-specific conservative recombination would be another p o s s i b i l i t y . Irrespective of the mechanism of clr c u l a r l z a t l o n of transferred plasmld DNA, I n a b i l i t y to do so tn a particular host w l l l l i k e l y prevent plasmld establishment. I t ls also possible that plasmld establishment lnvolves the attachment of certaln of lt s regions to the cell membrane (21). Mechanisms l l m l t l n q or promotlnq maintenance. The maintenance of a plasmld in host cells after it s establishment, can be determined by i t s a b i l i t y to replicate and by the a b i l i t y of the resultant plasmld copies to be distributed to daughter cells. In addition, plasmld maintenance may be affected indirectly by host-mediated alterations in plasmld copy number control or by structural i n s t a b i l i t y of the plasmld. Replication. Plasmlds dlsplay different modes of DNA replication as well as diff e re n t mechanisms of replication control. They have been reviewed recently and extensively for different groups of plasmlds (59, 65, 81, 94). However, the f u l l range of mechanisms that plasmlds can exploit ls probably s t l l l not known. For example, for one of the best-studied plasmlds, ColE1, i t is known that replication normally Involves f i r s t the production of a plasmld RNA transcript by the E. co11RNA polymerase and that this transcript is then processed by the host enzyme RNAse H to provide the primer for the synthesis of the leading plasmld DNA strand (65). However, I t has been found recently that In a host that ls deficient in RNAse H, replication s t i l l proceeds by an alternative means (61). In another example, I t has been found that replication of the baslc repllcon of the plasmld pCU1 is independent of the host polymerase, PolI and depends on a plasmld-determlned replication protein. Inactivation of this replication protein did not however abolish plasmld DNA replication but instead uncovered an a b l l l t y of the plasmld to now replicate by a PolI-dependent mode (45, 46). I t is not yet known whether these two replication modes have different host-ranges. I f so, this would provide one means by which a plasmld repllcon acquires replication promiscuity. A second is by simply'combining two plasmlds of different groups and d if f e r e n t hostranges Into a slngle colntegrate plasmld. There are several examples of

PLASMID PROMISCUITY AND CHASTITY

525

naturally occurring colntegrate plasmlds (9, 44, 63, 78, 90). A t h i r d mechanism for a plasmtd to achieve replication promiscuity ls for l t s repllcon to encode and produce products In a host that make I t r e l a t i v e l y independent of components that may otherwise be needed to be supplied by the host. Recent studies indicate that the IncQ group repllcon of plasmlds such as RSFIOIO and Rl162 may employ this strategy (20, 30). For replication In at least nine different species of Gram negative bacteria, the origin of vegetative replication and the replication proteln called TrfA of the IncP group plasmld RK2 ls s u f f i c i e n t (71). The mechanism by whlch this promiscuity ls achieved is not known. However, for stable maintenance of the plasmld In other bacterial species, additional and variable regions on the plasmld appear to be necessary (71). These l a t t e r regions are functionally organized In a complex way (82) and some of them like t r f B are co-regulated with the essential t r f A gene that determines TrfA. However I t is not clear that such a complex network ls causally related to plasmld promiscuity. Recent studles on the small promiscuous plasmid pLS1 which replicates In some Gram positive bacteria and In E. c o / l suggests that one of Its proteins which can repress the synthesis of i t s replication protein has s i g n i f i c a n t homology to the TrfB protein of RK2 at l t s N termlnal reglon (16). The repllcon of the promiscuous IncW plasmld pSa has been delineated to a reglon of about 4 kllobase palrs that functions In E. c o l i (80). The host-range of the repllcon region ls not known. However, a mutation in i t s replication gene was reported to be complemented by the otherwise unrelated plasmld R6K which is not known to be promiscuous (80). $eqreqatlonal stab111ty. In the literature Involvlng plasmlds, especlally promiscuous plasmids, there are instances In which a plasmld can be introduced into a host and be maintained In thls host but only provlded selection for the plasmid is malntalned (e.g. 47).

In the absence of selection, the population

tends to lose the plasmld. Thls suggests that the plasmld has the capacity to replicate In the host but that I t is either segregationally unstable or that the hosts carrying I t are at a growth dlsadvantage relative to hosts that do not carry the plasmid. PlasmidDNA sequences determining such s t a b i l i t y of inheritance have been studied extensively In some plasmlds such as CoIEI, F, RI and PI. These studies suggest that most naturally-occurrlng plasmids have more than one klnd of genetic system to ensure stable plasmld inheritance In populations. I t Is only recently that these systems have been uncovered by mutational and s t a b i l i t y studles in E. coli. One such system involves either the prevention of formation or the resolution by recombination of plasmld multlmers that interfere with their partitioning or random distribution to

5z6

V . N . IYER

daughter c e l l s (2, 78). A second system ls the selective k1111ng from w l t h l n the cell of those c e l l s that lose the plasmtd (41, 60, 88). A t h l r d system Is one whlch a c t l v e l y p a r t i t i o n s plasmld copies In a non-random fashlon (1,56). I t Is also emphasized that In Individual c e l l s , a l l plasmlds have a certain d i s t r i b u t i o n In copy number that ls determined by Its r e p l i c a t i o n control system(s).

Modulation of these control systems which can be Imposed by such

factors as the bacterial host ( I . e . host chromosome-determined factors) or growth conditions can a f f e c t this copy-number d i s t r i b u t i o n and therefore plasmtd s t a b i l i t y or apparent host-range (10, 33). These recent observations suggest that s t a b i l i t y of a plasmld In a bacterial population Is not as a rule determined unlquely by any one mechanism but rather by the cumulative e f f e c t of several mechanisms. Rescue by recombination. Classical studles on the plasmld F (40) I l l u s t r a t e d that an e n t i r e plasmld can lnsert I t s e l f Into the bacterial chromosome In a reversible manner to consume and then restore the plasmld state. There are other reports of slmllar situations (e.g. 42). In bacterial populations, they could be medlated by one or more of several recombination pathways and are often mediated by transposable elements. This phenomenon Is mentioned here b r l e f l y only to draw a t t e n t i o n to one other potential of bacterial plasmlds that a f f e c t t h e | r maintenance In bacterial hosts. ARTIFICIALLY EXPANDING OR LIMITING PLASMIDMAINTENANCE Potential applications are the emphasis of this concluding sectlon.

There are

many bacterla of practical Importance for which vectors for gene c]onlng, expression and manipulation would prove to be an asset. For several of the promiscuous plasmlds that were described In the prevlous sections, there ls now s u f f i c i e n t Information to enable the construction of clonlng vectors. One way of securing broad-host-range Is to construct and use vectors that are colntegrates of two baslc repllcons each of whlch has a d i f f e r e n t host-range (e.g. 9, 44, 78, 90). A d i f f e r e n t but not mutually exclusive way Is to use vectors that are based on slngle basic repllcons that have a broad-host-range such as those of the ZncN, P, Q or W plasmlds of Gram negatlve bacteria (81). For d e l i v e r y of clones Into various Gram negative bacteria, cassettes containing the o r l g l n of conjugattve transfer and related cls-actlng m o b i l i z a t i o n functions (mob cassettes} are conveniently Incorporated Into vectors, a l l or most trans-actlng functlon being provlded by the chromosome or a non-self-transmissible plasmtd I . e . one with l t s own mo___bbcassette removed

Tn5

Tn5

Tnl

Tn9

Tnt0

Tn5

pSUP2021

pGS9

pGS18

pGS27

pGS16

pRK340

Tc

Cm, Tra(pcm)

KmlNm, Sm, 61e

Tc

Cm

KalNm, Sm, Die, Ap

KmlNm, Sm, Ble

Tn5

pSUPI021

Cm, Tc, Mob( ) Ap. '~l, Mob(~)

Transposon ~electable amrkertpl on the: Plasmid excluding Transposon transposon

Lead Reference

The suicldal effect is based on the heatsensitivity of the vector. This Illposes a limitation.

The TnS-carrying pGS9 has proved to be the most generally useful. Derivatives of Tn5 containing mrkers other than the native ones my extend i t s usefulness. 43

74

These two vectors are 76 used when resident in the E. coil host 517-1 that provides promiscuous transfer functions from Its chromosome.

Co~oeents I f any

Illustrative examples of SUicide plasmd-host Systems for transposon Insertions*.

Plasmtd

Table 2.

Leglonella

Agrobacterlom Azosptrtllue Azotobacter Br~yrhfzoblum Plant-associated Pseudomnas Rhlzobl~l

Erwlnla Plant-associated Pseudomonas Pseudolonas aeruglnosa Rhlzoblom Xanfhomonas

Has proved useful in:

~4

Z

©

[-"

Tn917

pE194Ts(PTV1Ts)

Tra(Kz)

Cm

Tra(Col[b-Pg)

Tra(lncP-1O) Cb

TracKz)

Erm

Die, Tc

KmlNm.Sm,

KmlNm, Sm, 81e

KmlNm

Tp/Sm

Tc

KmlNm

Under non-permissive conditions, the plasmid may insert into the chromosome.

Range of auxotrophs obtained was limited.

35

92

86

18

50

Bacillus

Soil Pseudomonas

Methylobacterlum

Aclnetobacter calcoacetlcus

Caulobacter crescen£us

Plant-assoc|ated Pseudomonas

The i l l u s t r a t i v e exmples were chosen only to lndlcate the range of species In which such vectors have proved to be useful. For a historical or comprehensive treatment of such vectors, see references 38 and 76. Abbreviations: Ap, ampictllin; 81e, hleomyctn; Cb, carbenic1111n; Cm, chloramphenlcol; Erm. erythromycln (mecro]ldes); Km, kanamycln; Mob, meblllzatlon with source of region in parenthesis; Nm. neomycin; Sa, streptomycin; TO, tetracycline and Tra, con]ugative transfer.

Tn5

Tn5

pMO75

TnlO

Tn5-132

pRK2013::RnS-132

pLG223

Tn7

pRK2013::Tn7

pLG221

Tn903

TnlOHH

pRK2013::Tn903

pRK2013::Tn10HH

PLASMID PROMISCUITY AND CHASTITY

(38, 76). For a l l vectors whether promiscuous or not, s t a b i l i t y and the factors that may influence I t are important considerations. For most practlca| applications, this ts best determined experimentally under 6ondlttons whlch wl]1 simulate l ts intended use. A very large number of plasmtd vectors suitable for clonlng In a variety of both Gram posltlve and Gram negative bacterla now exist. A compendiumof vectors ls beyond the lntended scope of thls a r t l c l e .

The following references are however

recommended as those that w111 provlde access to most general and special purpose vectors suitable for cloning and gene expression In a varlety of eubacterla: 9, 19, 22, 23, 67, 69, 90. There can be Important applications to l l m l t l n g the expression of plasmld systems that promote promiscuous establishment or maintenance while not affecting the promiscuity or efficiency of transfer. For example, the a b l l l t y to conduct mutagenesls and other useful manipulations with bacterial transposons has often depended on the development and use of so-called plasmld sulclde vector-host systems; that Is, creating conditions whlch w i l l ensure e f f i c i e n t dellvery of a transposon-carrytng plasmld lnto a stratn (e.g. by uslng e f f i c i e n t and promiscuous mating systems) but preventing the establishment or maintenance of the plasmld In the stratn. In thls circumstance, selecting for the transposon marker Is a convenient way of obtaining Insertions. Examples of such dellvery systems are shown In Table 2. The current a v a i l a b i l i t y of several such systems enables an Investigator to select a system that Is best sulted to a particular species, strain or purpose.

I t ls emphasized that the potential usefulness of transposons and

transposon Insertions goes well beyond merely obtaining Insertion mutants (e.g. 5). A major problem In the use of plasmlds In Industrial bloreactors Is the segregatlonal loss of plasmids from a population in the absence of selection for the plasmld (37). Such segregatlonal loss could occur because of a deficiency In any of the mechanisms considered e a r l i e r . The deficiencies could arise as an inadvertent consequence of the fn v i t r o construction of vectors. For Gram negative eubacterla, various schemes have been devlsed to reduce the accumulation of plasmld-less segregants.

These lnclude the

Insertion lnto the vector of a p a r t i t i o n locus (54, 77), the use of the hok/sok (parB) k t l l e r or addiction system of plasmld R1 (24) and the transplacement In E. colY of the essential ssb gene from the chromosometo the plasmld vector (66). In other situations such as the deliberate release of a bacterium carrylng an engineered plasmld Into a natural environment,

529

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V . N . IYER

segregatlonal i n s t a b i l i t y may be advantageous. I t would be desirable to secure such I n s t a b i l i t y In a predictable way. Molln et a l (55) recently constructed a derivative composed of a 300 base-pair lnverton that contains a promoter controlling the hokA gene of plasmld R1 (a gene controlling a sulclde function). Using this plasmld construct, they were able to secure, in laboratory experiments, a loss of v i a b i l i t y of a fraction of the cells per unit time. These and slmllar other schemes suggest that i t may be possible to deslgn and use 'containment cassettes' engineered into bacteria for environmental release and that such bacteria w l l l have a progressively lower probability of surviving after environmental release. However, i t is emphasized that there are a large number of potential ways by whlch a gene in a released bacterial population could theoretically be rescued by i n vivo mechanisms. There are Increasing scenarios where the environmental release of a bacterium carrying an engineered plasmid can be seen to be p o t e n t i a l l y beneficial or corrective. Potential risks can also be imagined. In using plasmld-based systems for environmental release, those that provide the a b i l i t y to monitor the object of potential concern and, i f possible, allow a degree of p r e d i c t a b i l i t y of l t s survival In a glven environment, are generally to be preferred. REFERENCES 1.

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