E.A.
GREENE
AND
P.C.
ZAMBRYSKI
Agrobacteria
BACTERIAL
CONJUGATION
mate in opine dens
Plant cells transformed by Agrobacteria synthesize opines, high levels of which induce the bacteria to conjugate their Ti plasmid and thus transfer the genes required for opine utilization. Agyobacteria harboring a tumor-inducing (Ti) plasmid genetically transform plants, engineering the development of a crown gall tumor. This tumor provides an ecological niche for the bacteria. Transfer DNA (T-DNA) from the Ti plasmid is stably integrated into the genome of a plant cell. The integrated T-DNA directs the overproduction of phytohormones and the production of an unusual source of carbon and nitrogen, opines. Because the specialized enzymes required for opine transport and catabolism are encoded by the Ti plasmid, the opines support the growth of Agrobacteria to the exclusion of most other soil bacteria. The phytohormones produced at the infection site cause local undifferentiated growth, enlarging the crown gall tumor and thus increasing the production of opines. When opines are abundant, Agrobacteria respond by expressing the genes required for opine utilization and by conjugating their Ti plasmids. Under all other conditions, Ti conjugation is strongly repressed. Whereas opines clrectly stimulate the expression of genes required for their catabolism, they signal the expression of conjugal transfer ( tra) genes indirectly [ 11. Opines from the crown gall begin a signalling cascade that stimulates the bacteria to produce a diffusible second messenger, known as conjugating factor (CF) [2]. The CF signal in turn activates expression of tra genes [3,4]. Central to coordinating the bacteria’s response to the opine signal is the transcriptional repressor, AccR. First, opines de-repress AccR, allowing expression of genes involved in opine utilization [ 11. As a result of increased expression of opine transport proteins, opines are sequestered in the bacteria and subsequently catabolized. Although Agrobacteria remove opines from the crown gall environment, the opines stimulate the production of the second signal, CF, which freely diffuses from the bacteria into the surrounding environment. Through derepressing AccR, opines stimulate the expression both of enzymes that produce CF and of the CF receptor, TraR [2]. By stimulating both the secondary signal and its receptor, opines amplify their effect on conjugation, increasing the frequency of transfer of the Ti plasmid by five orders of magnitude [I]. The secondary signal, CF, and its receptor, TraR, were recently identified [3,4]. Because of their similarity to a well studied signal and receptor pair, one can easily predict their mode of actio,n. CF is a modiied homoserine lactone, similar in structure to the autoinducer (AI) of the LXX operon in vibrio j&+-i [3]. The Ti-plasmid traR gene, which encodes the CF receptor, shows sequence similarity to lux& the K fischeti gene for the AI receptor [4]. By @
Current
Biology
analogy to LuxR, TraR is presumed to be a transcriptional activator that senses CF directly and induces expression of the tra genes (Fig. 1). Like the response in the Zux system, induction of the tra genes shows tremendous speciiicity for the substrate, CF [3,4]. Although the Tiencoded gene(s) required for CF production have not yet been identified, the method used to identify this gene in I/: jiscberi can easily be adapted to Agrobacteria [ 51. First, one must identify Ti-plasmid mutants that are able to respond to CF but can no longer produce the factor. With these mutants one can use complement&ion to identify the gene(s) responsible for CF production. More and more examples are being found of bacterial species that use homoserine lactone derivatives to moniter and respond to cell density, and the best studied of these is I? fischeri. While living in the open ocean I/: jkcberi makes AI, which is freely diffusible and so cannot accumulate in the ocean to concentrations high enough to induce the lux operon. Only after T/: $s&eri colonizes the light organ of the fish Monocentris japonicus and reaches high cell densities does AI accumulate to a critical concentration. When the concentration of AI exceeds a threshold level, its sensor, the transcriptional activator LuxR, induces the lux operon and the bacteria become bioluminescent [5]. Another plant pathogen, Erwinia caratovora, has recently been shown to utilize a modified homoserine lactone derivative identical to AI to monitor its cell density on the plant. When the signal exceeds a threshold concentration, the pathogen produces cell-wall degrading enzymes [6] . The gene responsible for producing the signal shows sequence similarity to the I/: jk-cberi lU3GIgene [7]. In crown gall tumors, do Agrobacteria use CF simply as a secondary messenger to amplify the opine signal or, like I/: ficberi in the light organ of its host fish, could Agrobacteria use the CF concentration to monitor the donor cell density in the developing gall? Recent meth ods developed by Guyon et al. [8] provide the means to pursue these questions. These researchers compared growth of an Agrobacteria inoculum on untransformed lotus plants to growth on lotus plants genetically en@ neered to produce opines. They found that Agrobacteria growing on transformed plants reached concentrations ten-fold higher than those reached by Agrobacteria growing on plants that were not transformed, growing to a density that should allow CF to accumulate above the threshold required for conjugation. A T&like plasmid, the Ri, or root-inducing plasmid, provided an advantage to Agrobacteria growing on opine-producing plants. Plants were inoculated with a mixture of Agrobacteria in which 1993,
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Agrobacferhm
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tissue
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S
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fraR expression _,G, ,,_,,,, I, ,,,,
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B 1993 Current Biology
Fig. 1. During the establishment of a crown in inducing the expression of genes required
gall tumor for opine
on plants utilization
only 10 % of the bacteria carried an Ri plasmid. After three weeks, 80 % of the bacteria isolated from the infected plants carried an Ri p1asmid:A.s the chromosomal backgrounds of the original Agrobacteria inocula with and without the Ri plasmid were identical, the authors could not tell whether bacteria carrying an Ri plasmid simply outgrew bacteria lacking an Ri plasmid, or conjugation of the Ri plasmid into plasmidless recipients contributed to the appearance of so many Ri plasmidcontaining bacteria. Repeating this type of experiment using Agrobacteria with marked chromosomes would allow researchers to quantify the extent of conjugation that occurs in the environment of an opine-producing plant. By reducing the fraction of donor cells in the original inoculum, one could readily test the effect that donor-cell density has on the rate of conjugation. Donor Agrobacteria ready to conjugate should have no trouble finding recipients that lack a Ti plasmid. In the plant-soil environment, the Ti plasmid is lost from Agrobacteria populations, because propagating a Ti plasmid provides a selective disadvantage when opines are not available [B]. Also, there are circumstances in the plant-soil environment where harboring a Ti plasmid is lethal to the bacteria. The Ti plasmid-encoded mechanisms for transporting and catabolizing opines provide a direct target for the toxin, agrocin 84, produced by Agrobacterium radiobacter strain K84 [ 91. Once it has been taken up by an opine-utilizing bacterium, agrocin 84 is cleaved, releasing a modified nucleotide that poisons the cell. The soil bacterium A. radiobacter K84 can utilize opines, but is resistant to the toxin; it presumably makes
infected by Agrobacteria, and Ti-plasmid conjugation
intercellular signalling molecules in the infecting bacteria.
play
a role
agrocin 84 to kill opine-utilizing Agrobacteria at an established crown gall, so that it can colonize the gall itself. It is to the advantage of the Agrobacteria population that only a fraction of the population harbors a Ti plasmid, and that that fraction transfers the plasmid only when opines are available. When opines are available, donor Agrobacteria release CF to the environment. Might CF signal to recipient bacteria, priming them for conjugation? There is a precedent for donor and recipient bacteria using diffusible signals to communicate. In Enterococcus faecalis the recipient secretes a pheromone and the donor secretes a peptide inhibitor of pheromone action [lo]. If the donor senses a concentration of pheromone that overcomes the threshold set by the peptide inhibitor, the donor and recipient produce substances that cause clumping of bacteria and lead to conjugation. When CF was added to Agyobacteria cultures, Zhang and Kerr [Z] could not detect clumping of donor cells, recipient cells or a mixture of donor and recipient cells. Clumping may not be required for Ti conjugation, so this negative result is difficult to interpret. When the same authors preincubated recipient bacteria with opines and CF before mating with an inefficient donor strain, they saw no improvement in transfer efficiency [2]. Either CF cannot prime the recipient for conjugation, or the inefficient donor cannot take advantage of the primed recipient. Recipient bacteria may be passive players in conjugation. Rather than selecting its recipient, the Ti plasmid may conjugate to bacteria indiscriminately but be stably
DISPATCH
maintained only in bacteria that can support its replication. The Ti plasmid would be behaving like ‘selfish DNA, propagating itself in new hosts when the availability of opines would improve the transformed hosts’ chances. of survival. Among soil bacteria, there is good evidence for horizontal transfer of genetic material [I I]. The opine-utilization operon, in particular, may have been transferred horizontally, as Agrobucteria are not &he only soil bacteria that can catabolize this nutrient. h!bizobium spp. and some Pseudomonas strains are able to transport opines against a concentration gradient and can compete with Agrobacteria for this nutrient source [ 121, Conjugation of the Ti plasmid is tightly regulated. The plasmid is transferred only when opines are available and when the concentration of CF, and possibly the density of donor cells, is above a threshold. All of the known elements that regulate Ti transfer are encoded by the Ti plasmid: the T-DNA, which engineers the plant to produce opines; AccR, which responds to opines; TraR and CF, which amplify the opine signal. The Ti plasmid may not limit its transfer to recipient Agrobacteria. In an opine-producing crown gall, we need to consider the question: which genome is actively colonizing the gall, the Agrobacteria or the Ti plasmid? References 1. VON BODMAN SB, HAYMAN GT, FARRAM) SK: Opine
2.
catabolism and conjugal transfer of the nopaline Ti plasmid pTiC58 are coordinately regulated by a single repressor. Proc Nat1 Acad Sci USA 1992, 89643-647. WANG L, KERR A: A diffusible compound can enhance conjugal transfer of the Ti plasmid in Agobacterium tumefaciens. J Bact 1991, 173~1867-1872.
IN THE APRIL 1993 ISSUE OF CURRENT
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BANG gation
L, MURPHY PJ, KERRA, TATE ME: Agrobacterium conjuand gene regulation by N-acyl+homoserine lactones.
Nature 1993, 362:446-448. 4.
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10. 11. 12.
facPIPER KR, BECK VON BODMAN S, FARRAND SK: Conjugation tor of Agrobacterium tumefaciens Ti plasmid regulates Ti plasmid transfer by autoinduction. Nature 1993,. 362:448-450. ENGEBRECHT J, SILVERMAN M: Identification of genes and gene products necessary for bacterial bioluminescence. Proc Nat1 Acud Sci USA 1984, 81:4154-4158. JONES S, Yu B, BAINTON NJ, BIRDSAU M, BYCROFT BW, CHHABRA SR, Cox AJR, GOLBY P, REEVES PJ, STEPHENS S, eT AC.: The Zux autoinducer regulates the production of exoenzyme virnlence determinants in Erwinia cartovora and Pseudomonas aeruginosa. EMBO J 1993, 12:2477-2482. PIRHONEN M, FLFZGO D, HEWNHEIMO R, PALVA ET: A small diffusible signal molecule is responsible for the global control of virulence and exoenzyme production in the plant pathogen Erwinia caratovora. EMBO J 1993, 12:2467-2476. GUYON P, PETIT A, TEMPE J, DESSAUX y: Transformed plants producing opines specifically promote growth of opine-degrading Agrobacteria Mol Plant Microbe Int 1993, 6~92-98. HAYMAN GT, FARRAND SK: Agrobacterium plasmids encode structurally and functionally different loci for catabolism of agrocmopine-type opines. Mel Gen Genet 1990, 2233465473. CLEWELI. DB: Bacterial sex pheromone-induced plasmid transfer. Cell 1993, 73:9-12. WK~TTAM TS: Population genetics: sex in the soil. Curr Biol 1992, 2~676687. BERGERON J, MACLEOD R.4, DION P: Specificity of octopine uptake by Rbizobium and Pseudomonas strains. i?ppl Env Microbial 1990, 56:1453-1458.
E.A. Greene and P.C. Zambryski, Department of Plant Biology, University of California, Berkeley, California 94720, USA
OliNION
IN BIOTECHNOLOGY
Robert Fraley and Jeff Schell edited the following reviews on Plant Biotechnology: Signal transduction and calcium channels in higher plants by Raoul Ranjeva, Patrice Thuleau and Julian I. Schroeder Genome mapping in plants by Andrew H. Paterson and Rod A Wing Production of disease-resistant transgenic plants by Richard Broglie and Karen Broglie Trans-inactivation of gene expression in plants by Jan M. Kooter and Joseph N.M. Mol Control of pigmentation in natural and transgenic plants by Gert Forkmann Plant biotechnology transfer to developing countries by David W. Altman Genetic engineering of commercially useful biosynthetic pathways in transgenic plants by Ganesh M. Kishmore and Christopher R. Somerville Particle gun-mediated transformation by Paul Christou
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