[]~EVIEWS

Genetics of gene transfer between species

F a i t h f u l transmission of traits from parent to offspring is vital to the survival of a species. The need to reproduce offspring accurately is complicated by the demands of a changing environment, requiring compensatory changes in organisms. Further complicating JACK A. HEINEMANN the strategy for survival are assaults on the g e n o m e that alter its informational content, by damage or in- Bacteria transfer genetic information to members of at least three of tbe flve biological kingdoms. Gene transfer fidelities in replication. The fundamental roles of sex are to supply an between species may play the same role as sex between members of a single species, providing genetic diversity organism with genetic variation and to provide the means for repairing DNA damageL2. If genetic in- and material for repair of genomic damage. formation were to flow b e t w e e n neighbors, or horizontally, as it does'vertically between parent and off- resistances in bacteria'S. Resistance determinant~ ar~: often found on plasmids that direct conjugation bespring, then all organisms might have access to the tween bacteria and are often grouped with other rc evolutionary advantages of sex. Indeed one such sistance determinants. The dissemination of antibiotic mechanism of DNA transmission - bacterial conjuresistance factors through populations of pathogens gation - may be able to provide the benefits of sex to and nonpathogens attests to the horizontal transmission possibly all species. of traits over geographical distances in a relatively For horizontal transmission to occur, genetic mashort time. terial must be routed around at least two hypothetical Plasmid-mediated conjugation is the most thorbarriers: transfer barriers that prevent the delivery of oughly studied mechanism of contact-dependent DNA genetic information from a donor cell or virus, and transferS2 ~5. Conjugal plasmids are assigned to incomestablishment barriers that block inheritance of n e w patibility (inc) groups, which reflect the fact that closely molecules. Transfer and establishment barriers could related plasmids fail to cohabit stably in a single cell 16. discourage contact between genetic donors and recipiA self-transferable plasmid encodes both cis- and transents, degrade the genetic material, exclude the molecule from replication or segregation processes, or acting functions necessary for its mobilization and transfer between cells. For descriptive purposes, it is useful preclude the expression of genes necessary for the inheritance of transferred molecules. Nevertheless, mechanisms of DNA transmission do bridge large evolutionary distances and these are probably underestimates of the extent of DNA transfer (Fig. 1). Gene transmission by conjugation essentially links all Gram-negative bacteria, while studies using Escherichia coli have established that DNA is transmitted to and from Gram-positive bacteria as well >7. As described below, both E. coli and the plant pathogen Agrobacterium tumefaciens can transEubacteria mit DNA to eukaryotic species 8-m These findings provide the basis for hypothesizing the existence of a network of genetic exchange among organisms, implying that establishment barriers are: (1) the most effective defense against organisms that move nucleic acids for predation, such as viruses or bacteria, and (2) the means FIGI[ll by which species remain distinct in Known extent of the DNA exchange network. Illustration of potential DNA the midst of genetic promiscuity. distribution routes between biological kingdoms. Conjugation occurs between most. if not all, eubacteria, even between Gram-negative and Gram-positive species Bacterial sex (indicated by bidirectional solid arrows). Conjugation can also occur between Bacterial conjugation provides a prokaryotes and eukaryotes (indicated by unidirectional solid arrows) - including mechanism by which species could many plants and the fungi S. cerevisiae and Schizosaccbaromrcespombe. The achieve rapid adaptation to environremarkable range of species known to be capable of participating in DNA exchange_. mental changes, even if adaptation suggests that members of the remaining two kingdoms may be equally susceptible requires that the organism acquire to the transfer of DNA from bacteria (illustrated by unidirectional outlined arrows~ multiple traits simultaneously. ConjuThe existence of bacteria naturally amenable to the uptake of free DNA from the gation has probably played this environment suggests a possible mechanism for the transfer of I)NA back to prokaryotes from eukauotes (represented by unidirectional dashed arrows) role in the evolution of antibiotic "riG .IUNF19t)1 VOL. 7 NO. 6 ~'I~)9I Els~_'xk~S~icmv Publishers LtdtUK)0168 9'iV9 91'$0200

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[]~EVIEWS to organize this information on the basis of function into three loci - tra, for plasmid transfer, mob, for plasmid mobilization, and oriT, for origin of transfer. The tra and mob loci encode trans-acting functions and oriT acts in cis (Fig. 2a, b). The mob functions are plasmid specific; their specificities probably arise from a necessity to interact with oriZ The products of mob genes from different incompatibility groups usually will not recognize a heterologous oriT. In contrast, the tra genes are not plasmid specific; they encode functions that can interact with the mob functions of plasmids belonging to different incompatibility groups. For example, the plasmid colE1 supports its own mobilization (i.e. has mob and oriTfunctions) but requires a self-transferable plasmid, such as F, to supply tra functions for transmission. For the discussion that follows, general statements about the DNA transmission process will be based on findings involving both the transfer and establishment of the most extensively studied conjugal elements, plasmids of the incP group and the F sex factor of E. coll, Conjugation initiates with contact between donor and recipient cells. Surface structures of donor cells, called pill, mediate cell-cell contact. A pilus-cell interaction may provide the donor cell with sufficient information to recognize a potential recipient, effect more extensive surface contacts, and generate a signal whose reception results in the preparation of DNA for transfer. A strand- and site-specific cleavage at oriT is the first event in the mob-encoded metabolic pathway that culminates in DNA transfer. Helix unwinding always proceeds in the same direction from oriT after nicking, generating the identical molecular species each time. The displaced linear strand is probably the intermediate structure directed to the recipient cell. Meanwhile, the remaining strand serves as a template for the synthesis of a replacement strand in the donor. Some conjugal plasmids direct the transfer of proteins between cells, probably as DNA escorts (Ref. 17 and references therein). Little is known about how the DNA or DNA-protein complex leaves the donor and enters the recipient cell. However, the particular protein functions that a plasmid encodes to accompany the DNA molecule may affect the range of species in which the transferred molecule can establish.

Pathogenic DNA transmission So far one microbe has been shown to use DNA transmission in pathogenesis, in this case transmitting DNA to organisms of a different biological kingdom ls,~9. A. tumefaciens causes crown gall, a tumorous proliferation of plant cells released from normal metabolic and reproductive controls. Not only does the bacterium cause disease in a broad range of dicots, it can transfer DNA to even more species than display symptoms, including such monocots as Zea mays and Asparagus officinali,s19. Crown galls synthesize and secrete a member of a class of compounds called opines. Although only disease-causing strains of A. tumefaciens can utilize opines, genes determining both a virulence and opine-consuming phenotype are linked together on a plasmid that is transmissible to non,virulent strains. The transmissible element of A. tumefaciens that is necessary for virulence and tumorigenesis is called Ti

because of its association with tumor induction. A segment of the Ti plasmid, dubbed T-DNA for transfer DNA, is liberated from the whole of Ti during pathogenesis and transferred to the plant (Fig. 2c). T-DNA is responsible for tumorigenesis, but does not encode functions necessary for DNA transfer. DNA mobilization and transfer are dependent upon a trans-acting locus of Ti called vir, so-called because inactivation results in attenuation of virulence. Transfer is followed by integration of T-DNA sequences into the plant genome. Once these sequences are integrated, plant cellular functions ensure replication of T-DNA, segregation of T-DNA to daughter cells and expression of T-DNA genes, which encode three biosynthetic pathways. Two pathways provide alternative routes for the synthesis of the growth stimulators auxin and cytokinin, releasing the host cell from the normal plant regulation of cell division. The third pathway directs production of an opine. Not only does T-DNA impart reproductive autonomy to cells harboring it, the now renegade cells serve as factories producing a wealth of carbon and nitrogen in the form of opines.

Pathogenicity and sex DNA transfer to plants occurs by a mechanism similar to the conjugal transfer of DNA between bacteria (Fig. 2d). Loci on Ti are analogous to the functional loci found on conjugal plasmids. The trans-acting vir functions are analogous to tra and mob. Sequences immediately bordering the T-DNA on the left and right are, like oriT, required in cis. DNA mobilization is similar in both systems. Both oriT and the T-DNA border sequences delineate sites at which endonucleases introduce single-strand breaks and mobilize adjacent sequences unidirectionally. In vivo studies of T-DNA intermediates generated both in A. tumefaciens and heterologously in E. coli indicate that T-DNA separation from Ti is polar, from the right to the left border. Moreover, some of these intermediates resemble putative DNA intermediates in conjugation 20. The most compelling evidence that agrobacteria have subverted conjugation to effect pathogenesis is the demonstration that certain pathogenic functions can be replaced by sexual functions. Buchanan-Wollaston and colleagues have shown that the mob--oriTfunctions of a mobilizable plasmid can substitute for the T-DNA border sequences during pathogenesis9. Whereas the right border sequence is essential for tumorigenesis when the T-DNA resides in the Ti plasmid, a bacterial cloning vector lacking border sequences but encoding mob, oriT and a marker selectable in plants was efficiently transmitted by AgrobacWrium. Disruption of the plasmid mob region or oriTsequence abolished mobility of the plant markers. In this case, a conjugally mobilized DNA intermediate could be recognized by vir functions, which acted like tra to effect DNA transfer between organisms. Likewise, vir functions also appear to promote conjugal transmission of the Ti plasmid and chromosomal markers between strains of agrobacteria 21,22.

lnterkingdom sex Prokaryotic and eukaryotic cells look different and, a priori, transmission of DNA to a eukaryote would seem more difficult than transmission of DNA to

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D~EVIEWS (c)

(a)

viy CdS/

Functions

Functions (b)

(d) PlasmidGeneral

Trans-acting

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Conjugal

Virulence vir

mob

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FIG~ Genetics of sexual and pathogenic plasmids. {a) Schematic description of an ind: conjugal plasmid (black ellipsei and the bNA transfer functions; ira, mob and oriT represent approximately 30 conjugation-specific functions encoded by self-transferable plasmids. Arrows indicate the site of action of trans-acting functions, mob at oriT and tra at the cell surface (black squarel. (b) Characteristics of the three types of functions encoded by conjugal elements. (c) Schematic outline of the Ti plasmid (outlined and black arcs) and DNA transfer functions. The outlined sector of the Ti plasmid is probably not transferred to the plant. The vir functions reside in this sector and, as indicated by the arrow, act in trans at the T-DNA (black sector), first by introducing single-strand cleavages at the T-DNA borders (thin black sectors) then perhaps by association with the T-DNA during transfer across the cell surface (black square) during pathogenesis. One strand of the T-DNA region is transferred to plants during an infection. (d) Genetically analogous functions involved in conjugation and pathogenicity.

another prokaryote. To determine whether the Agrobacterium Ti plasmid, or its bacterial host, might be unique in possessing functions required for the transmission of DNA to eukaryotes, we attempted to cross a different prokaryote--eukaryotc pair (Ref. 8; J.A. Heinenmnn, PhD Thesis, University of Oregon, 1989). E. colt and the yeast Saccbaromvces cerevisiae suited us well because they are distantly related and enjoy no known ecological relationship, making it unlikely that they have evolved a specific interaction like Agrobacterium and plants. Two plasmids were introduced into E. colt cells. One, a yeast-bacteria shuttle plasmid, could replicate in both S. cerevisiae and E. coli, had selectable markers for both species, and had an oriZ The other was a self-transferable plasmid that could supply the necessary trans-acting functions to transfer both plasmids between strains of E. coil Bacterial donors were mixed with veast on minimal agar that allowed the growth of only those yeast cells that had inherited the shuttle plasmid resident in E. coli.

These donors, but not donors lacking either the selftransferable plasmid or the shuttle plasmiU, clkitcd the: growth of yeast on the selective medium. Yeast that grew contained the shuttle plasmid, since plasmid DNA with the same genetic markers and restriction fragmenl profiles as the shuttle plasmid could be isolated from the yeast. Therefore, the transfer of DNA to eukaryotes is not an exclusive property of either the Ti plasmM or Agrobacterium. Moreover, DNA transmission to },cast is ph}'sicallx and genetically like conjugal DNA transmission between two bacteria. Physical nmnipulations that per turb DNA transmission between conjugating bacteria similarly perturb the transmission of DNA to yeast from bacteria. Treatments that prohibitect cell contact or disrupted donor cell integrity interrupted transmission. As in conjugation, the bacteria-yeast relationship was insensitive to exogenous DNasc and could not be rescued by the exogenous addition of plasmid I)NA when donors lacked a shuttle plasmid.

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H~EVIEWS A series of genetic experiments provides the most compelling evidence that bacteria conjugate with yeast (Fig. 3). Three bacterial donors were constructed, each missing a different plasmid locus necessary for conjugation - tra, mob or orfT. Bacteria that contained any two of the three functional loci could not promote the growth of yeast on selective medium. Control donors containing all three conjugal loci could always transmit the shuttle plasmid to yeast. Therefore, conjugation was necessary for DNA transmission, and both necessary and sufficient to achieve DNA transfer, between a prokaryote and a eukaryote. The frequency at which DNA is transmitted to yeast by bacterial conjugation is similar to the frequency of transmission observed in crosses between bacteria. Under conditions in which the population of yeast cells remains in vast excess over the bacterial population throughout the course of the experiment, and a marker on a small self-replicating molecule of DNA is monitored, the ratio of the number of yeast cells inheriting plasmids to the number of bacterial donors approaches 0.1 (compared to 1.0 for crosses involving two strains of E. coli) (Ref. 8; J.A. Heinemann, unpublished). Nearly one in every ten bacteria successfully transmits a ,molecule of DNA to yeast, and possibly more accomplish DNA transfer. We then attempted to determine whether plasmids with different bacterial host ranges also varied in their abilities to transmit to yeast. Two different conjugal plasmids, a member of the incP and a member of the incF group, which, in bacterial crosses, display a broad and a narrow host range, respectively, were engineered to carry a marker gene selectable in yeast and a replication sequence active in yeast. Consistent with the hypothesis that plasmids are limited by their ability to establish rather than transfer, the transmission of each from bacteria [as well as DNA transferred by incI plasmids (B. Wilkins, pers. commun.)] to yeast was detected at comparable frequencies. Agrobacterium has overcome replication barriers by allowing the integration of transferred DNA into a host replicon - a plant chromosome. T-DNA integration does not appear to be guided into the plant genome by DNA

sequence homology between the T-DNA and its site of integration. This feature of the DNA transfer system of A. tumefaciens is unlike that observed in conjugation between bacteria. Like Agrobacterium, a type of donor E. coli, called Hfr because it is associated with a high frequency of recombination, relies on the recombination of transferred and resident DNA molecules for successful gene transmission z3. F plasmid integration- into the chromosome of its host causes the Hfr phenotype. When the plasmid mobilizes, the attached chromosome is also conducted into the recipient. If mating is interrupted before the transmission of the entire chromosome and plasmid, the transferred sequences must integrate into resident sequences to survive. However, in E. coli, integration is governed by the extent to which two molecules are alike 24. Recombination functions encoded by the chromosome or conjugal elements could actively suppress a natural tendency of DNA molecules to recombine, tolerating this tendency only when the molecules are homologous. Do conjugal elements allow transferred molecules to integrate into replicons in a eukaryotic recipient? DNA transferred from bacteria to yeast by conjugal plasmids can establish by integrating into resident yeast replicons (Ref. 25; J.A. Heinemann, unpublished). Integration into resident replicons occurs when both molecules share regions of homology. However, integration of this sort is rare, probably because, like E. coli, S. cerevisiae demands extensive sequence homology between molecules before it permits their recombination. It remains unclear whether homology requirements are relaxed in plants or whether the Agrobacterium system differs from conjugation by promoting recombination between foreign and resident molecules in an environment otherwise hostile to nonhomologous recombination.

The network, individuality and disease

Conjugation may provide a mechanism through which a community of organisms can achieve rapid, punctuated strides in evolution by scattering mixtures of traits. Promiscuous DNA transmission may have allowed bacterial species threatened by both medical and agricultural applications of antibiotics to survive. If, instead, their subscriptions to the network of DNA exchange had lapsed, the result might have been extinction. No single conjugal element has an unlimited host range. However, there is a significant overlap of host ranges among the conjugal elements, allowing the sequential transmission of markers between most, if not all, bacterial species. Sharing may involve mob, oriT tra, mob, oriT tra, oriT more than just conjugal plasmids. HeW Plasmids can accumulate and deliver The genetics of interkingdom conjugation. Genetic demonstration that tra and mob, transposable elements and some required for the conjugal transmission of DNA between bacteria, are similarly plasmids transfer other self-mobilized required for DNA transmission between bacteria and yeast. As many as three plasmids (such as colE1 by F), plasmids were introduced into bacteria. Each encodes one of the three sets of markers contained on regions of the functions necessary for conjugation. Each of these bacteria was combined with yeast chromosomes of their hosts 26, or on a rhedium permissive for the growth only of yeast that inherit a shuttle plasmid the entire host chromosome (such as (supplying or/T). As indicated by the white colonies, only bacteria expressing all in Hfr strains). Moreover, the transfer three functions promote yeast growth. "ri~;l~:~v: 1991 voi.. 7 NO. 6

[]~EVIEWS ranges of the conjugal elements are not as restricted as their host ranges. There are, however, barriers that discourage DNA transmission. Transfer barriers inhibit DNA transfer and establishment barriers block inheritance. Barriers to establishment rather than transfer appear to limit transmission by conjugation. Establishment barriers can be overcome through the acquisition of replication sequences, by integration into a resident replicon, and by mechanisms that provide immunity to such host defenses as endonuclease restriction. Over evolutionary time, it is conceivable that sequences could occasionally evade the establishment barriers. For example, a recent 'study' found that compensatory mutations restoring the ability of a mutant bacteriayeast shuttle plasmid to replicate in yeast occurred more commonly in the sequences of bacterial origin than in the sequences of yeast origin, suggesting that minor changes in DNA sequence can dramatically alter the host range of a transferable element 27. Several instances of putative horizontal inheritance of genes have also been reported 2~-3t. The extensive transfer range of conjugal elements leads to the suggestion that recipient cells of all kingdoms have in c o m m o n a feature that can be adapted to allow the import of DNA. This conserved 'port-ofentry' for nucleic acids.would probably be the site at which divergent mechanisms of transfer converge. Viruses and conjugal elements may promote DNA transfer by moving genetic information through the same portal. If all cells are equally susceptible to the entry of nucleic acids, then establishment barriers are the last best defense species have to maintain their identity and guard against pathogens that prey u p o n all species. Why is the promiscuous transfer of DNA tolerated by nature? First, there may not be a selection against transfer, but against expressing the genes on transferred molecules of DNA. Natural barriers to DNA transmission are presented by a diversity of speciesspecific replication requirements and c/s-acting signals required for gene expression. Such barriers to the establishment of vectors that might otherwise exploit the network for predation, such as viruses or the Ti plasmid, limit pathogenesis without preventing the transfer of genetic information between unrelated organisms. Second, there may be selective advantage to acquiring traits developed and refined by other organisms ('variation hypothesis%~-'. Third, access to potentially useful sequences of genetic information may provide individuals with sequences from which to repair damage to their genomes and consequently extend their lives until they can reproduce ('repair hypothesis') 1,-'. The day-to-day utility of using transferred DNA to salvage damaged genomes may promote the survival of individuals without resulting in the transmission of any particular trait. Therefore, not only the extent but also the impact of horizontal transmission may be difficult to determine by analysis of gene products and nucleotide sequences alone.

all of whom have contributed to this presentation. I am grateful to Tammy Walsh, Ga W Hettrick and Bob Evans for help in preparing the manuscript.

References 1 Bernstein, H., Hopf, F. and Michod, R. (1987) Adv. Genet. 24, 323-370 2 Michod, R.E., Wojciechowski, M.F. and Hoclzer. MA. (19881 Genetics 118, 31-39 3 Trieu-Cuot, P., Carlier, C. and Courvalin, P. (19881 J. Bacteriol. 170, 4388-4391 4 Trieu-Cuot, P., Carlier, C., Martin, P. and Courvalin, P. (1987) FEMSMicrobiol. Lett. 48, 289-294 5 Mazodier, R, Petter, R. and Thompson, C. (19891 J. Bacteriol. 171, 3583-3585 6 Guiney, D.G. (19821.L Biol. Biol. 162, 699-703 7 Thiel, T. and Wolk, C.R (19871 MethodsEnzl,moL 153, 232-243 8 Heinemann, J.A. and Sprague, G.F., Jr (19891 Nature 340, 205-209 9 Buchanan-Wollaston, V., Passiatore, J.E. and Cannon. t-' (1987) Nature 328, 172-175 10 Sikorski, R.S. el al. (19901 Nature 345, 581-582 11 Wright, K. (1990) Science249, 22-24 12 Willetts, N. and Skurray, R. (1987) in Eschericbia coli and Salmonella typhimurium (Neidhardt, EC. et al., eds), pp. 1110-1133, American Society for Microbiology 13 lppen-Ihler, K.A. and Minkely, E.G., Jr (1986) Annu. Rev. Genet. 20, 5934524 14 Willetts, N. and Wilkins, P,. (19841 MicrobioI. Rev. i8, 2441 15 Thomas, C.M and Smith, C.A. (1987) Atom. Rel'. Microbiol. 41, 77-101 16 Kues, U. and Stahl, U. (1989) Microbiol. Rev. 53, q91-510 17 Rees, C.E.D. and Wilkins, B.M. (19901 Mol. Microbiol..¢, 1199-1205 18 Zambryski, P., Tempe, J. and Schell, J. (1989) (,ell "~(~, 193-201 19 Binns, A.N. and Thomashow, MF. (19881 Amttl Ret' Microbiol. 42, 5754506 20 Veluthambi, K.,Jayaswal, R.K. and Gelvin, S.B. (19871 Proc. Natl Acad. Sci. USA 84, 1881-1885 21 Steck, T.R. and Kado, C.I. (19901J. BacterioL 1"72, 2191-2193 22 Dessaux, Y. et al. (1989)J. Bacleriol. 171,031~3q~306 23 Low, K.B. (1987) in Escbericbia coli and Salmo~wlla tlphimurium (Neidhardt, F.C. et al., eds), pp. 113i 113-. American Society for Microbiology 24 Rayssiguier, C., Thaler, D.S. and Radman, M. ( 19891 Nature 342, 390-401 25 Heinemann, J.A. and Sprague, G.F., Jr lletbod.~ K~IZl'~)I(~[ (in press) 26 Holloway, B. and Love, K.B. ( 19871 in k~cbericbia col~ and Salmo~wlla O'phimurillm (Neidhardt. F.C. el al.. cds), pp. 1145-1153, American Socict3 for Micr~)biol{)g3 27 Kipling, D. and Kearsey, S.E. (19901 Mol. Cell 13iol. 10, 265-272 28 Carlson, T.A. and Chelm, B.K. (19861 Nature 322. 568-570 29 Lewin, R. (19821 &ience 217, 42--43 30 Holmgren, A. and Branden, C. (19891 ,\~mre 3-~2. 248-251 31 Weigel, B.J. et al. (19881J. Bacteriol. 1"0. 381- 3820 32 Syvanen. M. (1987) 1. Mol. F~t'ol. 26. 1(>23

J.A HE, VEMaXX *S *.~' TnL" LaBORATORr O~ MldRomat

Acknowledgements 1 thank Drs Kit Tilly, Patti Rosa, Seth Pincus. John Swanson. William Sistrom, Roderick Capaldi and Frank Sta 11,

STRUCIZ;RE AND F~WCTIO~%NIAID, NIH, ROCKY MOI'~'E41.x" LaBORArOmE&HaMlzmN, M T 5 9 8 4 0 , USA.

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rlc, iU×E 1991 VOL..7 XO. 6