Quorum Sensing but Not Autoinduction of Ti Plasmid Conjugal Transfer Requires Control by the Opine Regulon and the Antiactivator TraM

Abstract

Conjugal transfer of the Ti plasmids from Agrobacterium tumefaciens is controlled by autoinduction via the transcriptional activator TraR and the acyl-homoserine lactone ligand, Agrobacterium autoinducer (AAI). This control process is itself regulated by opines, which are small carbon compounds produced by the crown gall tumors that are induced by the bacteria. Opines control autoinduction by regulating the expression of traR. Transfer of pTiC58 from donors grown with agrocinopines A and B, the conjugal opines for this Ti plasmid, was detected only after the donors had reached a population level of 10 7 cells per cm 2 . Donors incubated with the opines and AAI transferred their Ti plasmids at population levels about 10-fold lower than those incubated with opines only. Transcription of the tra regulon, as assessed by monitoring a traA::lacZ reporter, showed a similar dependence on the density of the donor population. However, even in cultures at low population densities that were induced with opines and AAI, there was a temporal lag of between 15 and 20 h in the development of conjugal competence. Moreover, even after this latent period, maximal transfer frequencies required several hours to develop. This lag period was independent of the population density of the donors but could be reduced somewhat by addition of exogenous AAI. Quorum-dependent development of conjugal competence required control by the opine regulon; donors harboring a mutant of pTiC58 deleted for the master opine responsive repressor accR transferred the Ti plasmid at maximum frequencies at very low population densities. Similarly, an otherwise wild-type derivative of pTiC58 lacking traM, which codes for an antiactivator that inhibits TraR activity, transferred at high frequency in a population-independent manner in the absence of the conjugal opines. Thus, while quorum sensing is dependent upon autoinduction, the two phenomena are not synonymous. We conclude that conjugal transfer of pTiC58 is regulated in a quorum-dependent fashion but that supercontrol of the TraR-AAI system by opines and by TraM results in a complex control process that requires not only the accumulation of AAI but also the expression of TraR and the synthesis of this protein at levels that overcome the inhibitory activity of TraM.Conjugal transfer of the Ti plasmids from Agrobacterium tumefaciens is regulated directly by the transcriptional activator TraR and its acyl-homoserine lactone (acyl-HSL) ligand, Agrobacterium autoinducer [AAI; N-(3-oxo-octanoyl)-L-homoserine lactone] (16, 34, 40; reviewed in reference 12). TraR, in its interaction with AAI, controls conjugation by autoinduction, a process by which the bacteria induce gene sets in response to signals they themselves produce. This regulatory strategy is believed to tie plasmid transfer to the population density of the donor in what has come to be called the quorumsensing effect (reviewed in reference 18). The acyl-HSL autoinducers, which are produced by the bacteria themselves, are released into the environment, w...

Induction and Loss of Ti Plasmid Conjugative Competence in Response to the Acyl-Homoserine Lactone Quorum-Sensing Signal

“…In previous studies, we showed that responsiveness to the quormone is delayed for several hours following opine induction (25). While we postulated that this delay is due to TraM, we could not test the hypothesis, since traM mutants are constitutive for transfer.…”

“…Finally, these results strongly suggest that TraM in its interaction with TraR establishes the minimum concentration of the acyl-HSL required to fully induce the system. That donors harboring pCMA1⌬traI (accR ϩ traM), which expresses only basal levels of TraR (25), are essentially as sensitive to quormone as those harboring pKMA1⌬traI (accR traM), which constitutively expresses traR (compare Fig. 2A and C), suggests that sensitivity to signal is not a function of the amount of TraR produced but rather a function of the amount of available TraR, that is, the amount of activator not complexed with TraM.…”

“…Donors harboring pCMA1, in which traR remains repressed but TraM is absent, are constitutive for transfer, demonstrating that in the absence of the antiactivator, basal-level expression of traR is sufficient to activate transcription of the tra regulon at all population sizes tested (25). However, the ⌬traI mutation allowed us to examine the efficiency by which low levels of TraR can activate the tra regulon.…”

“…Since, in the absence of other factors, full activation of TraR by the acyl-HSL signal takes between 10 and 15 min (20), expression and construction of the conjugative transfer apparatus itself require an additional 15 to 20 min. However, other factors modulate the timing and kinetics of the induction of conjugative transfer (25). In this regard, clearly TraM serves to delay development of conjugative competence; transfer of pTiC58⌬accR⌬traIKm, which differs from pKMA1⌬traI only by expressing the antiactivator, does not initiate transfer until 60 min after exposure to the quormone (Fig.…”

“…This observation suggested that even in the absence of the conjugative opine, traR is expressed at a basal level that is sufficient to activate transfer. Based on this observation, we proposed that one role of TraM is to prevent premature conjugation attendant to this low level of expression of the activator gene (14,25). In addition, Ti plasmids with mutations in both traM and accR are hyperconjugative, suggesting that the antiactivator also serves to govern the maximum level of expression of the tra regulon under opine-inducing conditions (14).…”