0021-9193/78/0133-0210$02.00/0 JouRNAL OF BACTERIoLOGY, Jan. 1978, p. 210-216 Copyright © 1978 American Society for Microbiology
Vol. 133, No. 1
Printed in U.S.A.
Evolution of Pseudomonas R-Plasmids: Consequences of Tnl Insertion and Resultant Partial Diploidy to Chromosome and Tra- R-Plasmid Mobilization RONALD H. OLSEN Department of Microbiology, University of Michigan Medical School, Ann Arbor, Michigan 48104
Received for publication 29 July 1977 Tnl transposes from pRO161, a Tra- derivative of RP1, to Pseudomonas aeruginosa sex factor FP2. The acquisition of Tnl by FP2 results in its ability to mobilize pRO161 to other bacteria. Genetic evidence presented here suggests two sequential mechanisms. Initially, transposition ofTnl results in trans-diploidy for the Tra+ and Tra- plasmids. This subsequently allows mobilization of the Tra- R-plasmid dependent on a host recombination mechanism. Transconjugants from this mating contain either stable cointegrate R-plasmids or aggregates resulting from dissociation of the cointegrates into a Tra+ and Tra- plasmid.
These aggregates have lost at least part of Tnl from their parent FP2::Tnl component, but now they mobilize the Tra- R-plasmid from a recombinationdeficient (Recd) genetic background as well as from Rec+ donor strains. Transconjugants from these retransfer matings are aggregates. These results suggest a contribution of transposons to R-plasmid evolution and dissemination beyond the mere acquisition of resistance to a given antibiotic.
R-plasmids have been designated class 1 (1) or cointegrate (3) if they exist as a single replicon. Other R-plasmids have been shown to exist as separate components, one bearing the R-determinants specifying resistance to antibiotics and the other encoding for transfer functions. These have been designated class 2 (1) or aggregate (3) R-plasmids. Several reports have focused on the genetic behavior of aggregate Rplasmids to determine the ubiquity of the ability of sex factors to mobilize nonconjugative R-plasmids. Smith and Heller (16) tested F and the transfer factor for colicin I for their ability to mobilize several nonconjugative R-plasmids. These authors observed lack of specificity for mobilization of the nonconjugative plamids by F or the transfer factor for colicin L. However, these sex factors were not equivalent in their plasmid donor ability (Pda). In any case, though, their results clearly indicate possible evolutionary relatedness between conjugative (Tra+) and nonconjugative R-plasmids which extends beyond incompatibility group. A similar study by van Embden and Cohen (17), using a threecomponent mating system, has also demonstrated the ability of different conjugative plasmids to mobilize a small nonconjugative R-plasmid which encodes resistance to tetracycline (Tcr). It is therefore apparent, from such studies and from numerous others, that some conjugative plasmids can mobilize nonconjugative plas-
mids although unrelated to them by present criteria for plaid classification. We noted previously the mobilization of pRO161, a transfer-defective (Tral) variant of the P incompatibility group R-plaid RP1 (9, 15). The mobilization of pRO161 was medi&ted by the unrelated Tra+ R-plasmids FP2 or R388, accompanied by their acquisition of carbenicillin resistance (Cbr) from the Tra- R-plaid pRO161. These Tra+/Tra- doubles were found to be diploid for the R-determinant which specifies Cbr (5). Accordingly, as previously proposed (9, 15), it seems likely that the acquisition of Tnl (Cbr) by either R388, an incompatibility group W R-plasmid, or FP2, a Pseudomonas aeruginosa sex factor described by Holloway (6), reflects transposition of Tnl to these Tra+ R-plasmids. Multicopies of pRO161, then, would allow for the availability of a remaining pRO161 copy for mobilization promoted by shared Tnl homology with the transposed Tra+ companion
plasmid.
In this report, further changes occurring in the Tra+/Tra- aggregates after serial retransfers are described. It appears that Tnl may contribute to the evolution of conjugative R-plasmids beyond the mere acquisition of resistance to ampicillin and related antibiotics. This study focuses on genetic evidence for the sequential changes in aggregates which occur during and after the acquisition of Pda. The results suggest 210
R-PLASMID EVOLUTION Voi.VPSEUDOMONAS 133, 1978
that, initially, intermolecular recombination (following transposition of Cbr) accounts for the mobilization of pRO161. This is followed in transconjugants by either the maintenance of a cointegrate or its dissociation into separate Tra+ and Tra7 components Dissociation, in turn, is accompanied by the loss of the determinant Cb' from the Tra+ component of the aggregate complex. Interestingly, the Tra plasmid component of the aggregate is now mobilized independently of host bacterium recombination activity. MATERIALS AND METHODS Bacterial stri and plmids The relevant properties of the bacterial strains and plasmids used in this study are listed in Table 1. Media. Minimal medium (VBG) and complex medium (TN) were prepared as described previously (9, 10). When complex medium with brain heart infusion was used for the determination of resistance to mercuric chloride (Hgr), it contained (per liter) 36 g of brain heart infusion (Difco Laboratories, Detroit, Mich.) and 30 pg of Hg per mL Brain heart iusion medium was solidified by the addition of 20 g of agar per liter. When nutritional selection against auxotrophic donors was done, amino acid requirements were satisfied by the addition of these components to a final concentration of 20 gtg/mL Antibiotic supplements were as described in the tables. Mating and testng of exconjugants. All mating were done in TN broth medium. For this, TN broth medium was inoculated with overnight growth from TN agar which contained appropriate antibiotic or Hg for the strain containing a plasmid. These broth cultures were incubated for 3 h with agitation at 370C. Inoculation was adjusted to result in approximately TABLE 1. Bacteria and plasmids used Bacterial strain or plasmid-
P.
aeruginosa PAO2 PA038 PAO170
PA02003 Plasmid pRO271 pRO161
Relevant characteristic
ser-3 leu-38 leu-38 (FP2) arg-32 rec-2 (2)
bla mer (9) Cbr Tcr Tra- IncP, derived from RP1 (15)b
mer (6) FP2 R751 IV IncP (8) a The sources of these bacterial stains and plasmids have been given in previous publications (9, 10, 15). b RP1 from our laboratory is indisuishable from RP4 (11). Hence, the transposable genetic element that specifies Cbr is probably the same. In view of this, we have designated the Cbr tansposon used here as Tnl, as suggested by Cohen (4). R-plasmid pRO161 is a deletant of RP1 (15).
21
10' cells per ml of TN broth culture after 3 h of growth. Donor and recipient cells were mixed 1:1 and incubated at 37°C for 2 h. Mating mixtures were centrifuged at ambient temperature, and cell pellets were suspended to 1/10 the original volume of 0.01 M phosphate buffer (pH 7.0). Cell suspensions were diluted and plated onto VBG medium supplemented with the nutrients required by the recipient and with one of the antibiotics to which the plasmid confers resistance in the donor. Plates were incubated for 48 h at 37°C. Transconjugants were purified by picking colonies into liquid suspension, followed by streaking out for single-colony isolation on solid medium identical to that used for their primary isolation. Incompatibility was determined with appropriate selective medium as described previously (10).
RESULTS Mobilization by Pseudomonas sex factor FP2. Of ediate concern in this work and our prior report (9) is the possibility that pRO161 may be mobilized by a cryptic plasmid contained in P. aeruginusa PAO and its auxo-
trophicderivatives,PA02,PA038,andPA02003. Therefore, as a control for this work, pRO161 was transduced to these strains. None of them showed transfer of either CbT or pRO161 (i.e., transfer of Cbr and Tcr) to other P. aeruginosa strains. Furthermore, conjugal recipient bacteria that contained FP2 showed entry exclusion, as measured by lower frequencies of transconjugant formation, towards all the donor strains containing plasmids used in this study. These observations, then, diminish or preclude the likelihood of a cryptic plasmid being responsible for the observations noted below. The strains used are isogenic except for auxotrophic markers or maintenance of FP2. Physical characterization by Pemberton and Clark (13) has also failed to reveal the presence in these strains of a cryptic plamid of sufficient molecular size to encode for transfer functions that might promote pRO161 mobilization to other bacteria. We have previously described the sequence of events leading to the acquisition of the Cb' determinant by Pseudomonas sex factor FP2 (9). Data shown in Table 2 now point up the frequency of this event and confirm previous phenotypic properties reported for the FP2 Cbr plasmid pRO271. A comparison of the FP2+ and FP2+/pRO161+ donors shows that they yield similar numbers of ser-3+ transconjugants and, hence, that pRO161 does not affect the chromosome donor ability (Cda) of FP2. FP2 does not mobilize pRO161 at a detectable frequency (
Vol. 133, No. 1
Printed in U.S.A.
Evolution of Pseudomonas R-Plasmids: Consequences of Tnl Insertion and Resultant Partial Diploidy to Chromosome and Tra- R-Plasmid Mobilization RONALD H. OLSEN Department of Microbiology, University of Michigan Medical School, Ann Arbor, Michigan 48104
Received for publication 29 July 1977 Tnl transposes from pRO161, a Tra- derivative of RP1, to Pseudomonas aeruginosa sex factor FP2. The acquisition of Tnl by FP2 results in its ability to mobilize pRO161 to other bacteria. Genetic evidence presented here suggests two sequential mechanisms. Initially, transposition ofTnl results in trans-diploidy for the Tra+ and Tra- plasmids. This subsequently allows mobilization of the Tra- R-plasmid dependent on a host recombination mechanism. Transconjugants from this mating contain either stable cointegrate R-plasmids or aggregates resulting from dissociation of the cointegrates into a Tra+ and Tra- plasmid.
These aggregates have lost at least part of Tnl from their parent FP2::Tnl component, but now they mobilize the Tra- R-plasmid from a recombinationdeficient (Recd) genetic background as well as from Rec+ donor strains. Transconjugants from these retransfer matings are aggregates. These results suggest a contribution of transposons to R-plasmid evolution and dissemination beyond the mere acquisition of resistance to a given antibiotic.
R-plasmids have been designated class 1 (1) or cointegrate (3) if they exist as a single replicon. Other R-plasmids have been shown to exist as separate components, one bearing the R-determinants specifying resistance to antibiotics and the other encoding for transfer functions. These have been designated class 2 (1) or aggregate (3) R-plasmids. Several reports have focused on the genetic behavior of aggregate Rplasmids to determine the ubiquity of the ability of sex factors to mobilize nonconjugative R-plasmids. Smith and Heller (16) tested F and the transfer factor for colicin I for their ability to mobilize several nonconjugative R-plasmids. These authors observed lack of specificity for mobilization of the nonconjugative plamids by F or the transfer factor for colicin L. However, these sex factors were not equivalent in their plasmid donor ability (Pda). In any case, though, their results clearly indicate possible evolutionary relatedness between conjugative (Tra+) and nonconjugative R-plasmids which extends beyond incompatibility group. A similar study by van Embden and Cohen (17), using a threecomponent mating system, has also demonstrated the ability of different conjugative plasmids to mobilize a small nonconjugative R-plasmid which encodes resistance to tetracycline (Tcr). It is therefore apparent, from such studies and from numerous others, that some conjugative plasmids can mobilize nonconjugative plas-
mids although unrelated to them by present criteria for plaid classification. We noted previously the mobilization of pRO161, a transfer-defective (Tral) variant of the P incompatibility group R-plaid RP1 (9, 15). The mobilization of pRO161 was medi&ted by the unrelated Tra+ R-plasmids FP2 or R388, accompanied by their acquisition of carbenicillin resistance (Cbr) from the Tra- R-plaid pRO161. These Tra+/Tra- doubles were found to be diploid for the R-determinant which specifies Cbr (5). Accordingly, as previously proposed (9, 15), it seems likely that the acquisition of Tnl (Cbr) by either R388, an incompatibility group W R-plasmid, or FP2, a Pseudomonas aeruginosa sex factor described by Holloway (6), reflects transposition of Tnl to these Tra+ R-plasmids. Multicopies of pRO161, then, would allow for the availability of a remaining pRO161 copy for mobilization promoted by shared Tnl homology with the transposed Tra+ companion
plasmid.
In this report, further changes occurring in the Tra+/Tra- aggregates after serial retransfers are described. It appears that Tnl may contribute to the evolution of conjugative R-plasmids beyond the mere acquisition of resistance to ampicillin and related antibiotics. This study focuses on genetic evidence for the sequential changes in aggregates which occur during and after the acquisition of Pda. The results suggest 210
R-PLASMID EVOLUTION Voi.VPSEUDOMONAS 133, 1978
that, initially, intermolecular recombination (following transposition of Cbr) accounts for the mobilization of pRO161. This is followed in transconjugants by either the maintenance of a cointegrate or its dissociation into separate Tra+ and Tra7 components Dissociation, in turn, is accompanied by the loss of the determinant Cb' from the Tra+ component of the aggregate complex. Interestingly, the Tra plasmid component of the aggregate is now mobilized independently of host bacterium recombination activity. MATERIALS AND METHODS Bacterial stri and plmids The relevant properties of the bacterial strains and plasmids used in this study are listed in Table 1. Media. Minimal medium (VBG) and complex medium (TN) were prepared as described previously (9, 10). When complex medium with brain heart infusion was used for the determination of resistance to mercuric chloride (Hgr), it contained (per liter) 36 g of brain heart infusion (Difco Laboratories, Detroit, Mich.) and 30 pg of Hg per mL Brain heart iusion medium was solidified by the addition of 20 g of agar per liter. When nutritional selection against auxotrophic donors was done, amino acid requirements were satisfied by the addition of these components to a final concentration of 20 gtg/mL Antibiotic supplements were as described in the tables. Mating and testng of exconjugants. All mating were done in TN broth medium. For this, TN broth medium was inoculated with overnight growth from TN agar which contained appropriate antibiotic or Hg for the strain containing a plasmid. These broth cultures were incubated for 3 h with agitation at 370C. Inoculation was adjusted to result in approximately TABLE 1. Bacteria and plasmids used Bacterial strain or plasmid-
P.
aeruginosa PAO2 PA038 PAO170
PA02003 Plasmid pRO271 pRO161
Relevant characteristic
ser-3 leu-38 leu-38 (FP2) arg-32 rec-2 (2)
bla mer (9) Cbr Tcr Tra- IncP, derived from RP1 (15)b
mer (6) FP2 R751 IV IncP (8) a The sources of these bacterial stains and plasmids have been given in previous publications (9, 10, 15). b RP1 from our laboratory is indisuishable from RP4 (11). Hence, the transposable genetic element that specifies Cbr is probably the same. In view of this, we have designated the Cbr tansposon used here as Tnl, as suggested by Cohen (4). R-plasmid pRO161 is a deletant of RP1 (15).
21
10' cells per ml of TN broth culture after 3 h of growth. Donor and recipient cells were mixed 1:1 and incubated at 37°C for 2 h. Mating mixtures were centrifuged at ambient temperature, and cell pellets were suspended to 1/10 the original volume of 0.01 M phosphate buffer (pH 7.0). Cell suspensions were diluted and plated onto VBG medium supplemented with the nutrients required by the recipient and with one of the antibiotics to which the plasmid confers resistance in the donor. Plates were incubated for 48 h at 37°C. Transconjugants were purified by picking colonies into liquid suspension, followed by streaking out for single-colony isolation on solid medium identical to that used for their primary isolation. Incompatibility was determined with appropriate selective medium as described previously (10).
RESULTS Mobilization by Pseudomonas sex factor FP2. Of ediate concern in this work and our prior report (9) is the possibility that pRO161 may be mobilized by a cryptic plasmid contained in P. aeruginusa PAO and its auxo-
trophicderivatives,PA02,PA038,andPA02003. Therefore, as a control for this work, pRO161 was transduced to these strains. None of them showed transfer of either CbT or pRO161 (i.e., transfer of Cbr and Tcr) to other P. aeruginosa strains. Furthermore, conjugal recipient bacteria that contained FP2 showed entry exclusion, as measured by lower frequencies of transconjugant formation, towards all the donor strains containing plasmids used in this study. These observations, then, diminish or preclude the likelihood of a cryptic plasmid being responsible for the observations noted below. The strains used are isogenic except for auxotrophic markers or maintenance of FP2. Physical characterization by Pemberton and Clark (13) has also failed to reveal the presence in these strains of a cryptic plamid of sufficient molecular size to encode for transfer functions that might promote pRO161 mobilization to other bacteria. We have previously described the sequence of events leading to the acquisition of the Cb' determinant by Pseudomonas sex factor FP2 (9). Data shown in Table 2 now point up the frequency of this event and confirm previous phenotypic properties reported for the FP2 Cbr plasmid pRO271. A comparison of the FP2+ and FP2+/pRO161+ donors shows that they yield similar numbers of ser-3+ transconjugants and, hence, that pRO161 does not affect the chromosome donor ability (Cda) of FP2. FP2 does not mobilize pRO161 at a detectable frequency (
