JouRNAL OF BACTERIOLOGY, Feb. 1977, p. 809-814 Copyright © 1977 American Society for Microbiology
Vol. 129, No. 2 Printed in U.S.A.
Transposition of a Beta-Lactamase Locus from RP1 into Pseudomonas putida Degradative Plasmids MICHAEL BENEDIK, MICHAEL FENNEWALD, AND JAMES SHAPIRO* Department of Microbiology, University of Chicago, Chicago, Illinois 60637
Received for publication 23 September 1976
The beta-lactamase gene from the RP1 plasmid transposes into at least two Pseudomonas putida degradative plasmids. Donor strains that carry RP1 (bla+ tet+ aphA +) and a degradative plasmid yield transconjugants that have only the bla+ marker of RP1. This occurs in up to 80% of all bla+ transconjugants. Segregation of the bla+ marker requires the presence of a degradative plasmid in the donor and is only observed in transconjugants that have received degradative markers. The bla+ tet aphA transconjugants show 100%o linkage of bla+ to degradative markers in conjugation, transduction, and transformation crosses. A transduction cross of an (RP1), (SAL) donor shows that 8% ofall SAL plasmids also carry the transposed bla+ marker. Tn401 is the name we assign to the bla+ transposon from RP1 observed in Pseudomonas. Its identity with the RP1 bla+ transposon observed in Escherichia coli is not known. In four cases, Tn401 has inserted into the camphor genes of the CAM-OCT plasmid. A number of antibiotic resistance loci have the important property of transposing themselves from one replicon to another (A. Campbell, D. Berg, D. Botstein, R. Novick, and P. Starlinger, in A. I. Bukhari, J. Shapiro, and S. Adhya [ed.], DNA Insertion Elements, Episomes and Plasmids, in press). In Escherichia coli, the TEM-1 and TEM-2 beta-lactamase genes of several plasmids, including RP1, exhibit this property and can insert into the bacterial chromosome (1), other plasmids (14, 16, 23), and temperate bacteriophages (G. Weinstock and D. Botstein, personal communication; Shapiro, unpublished observation). We have been studying a group of plasmids that encode the ability to degrade different groups of hydrocarbons in Pseudomonas putida and Psuedomonas aeruginosa. These include CAM, SAL, and OCT plasmids, which determine growth on camphor, salicylate, or alkanes (5, 7, 22). Such plasmids have been termed "degradative plasmids." However, they do not appear to be a unique set of genetic elements because they belong to several incompatibility groups, one of which (IncP-2) contains a number of drug resistance plasmids as well as CAM and OCT (G. A. Jacoby and J. Shapiro, in A. I. Bukhari, J. Shapiro, and S. Adhya [ed.], DNA Insertion Elements, Episomes and Plasmids, in press). In this paper, we report the transposition of the bla+ locus from RP1 into the SAL and CAMOCT plasmids in P. putida. (In this paper we adopt the nomenclature proposed by Novick et
al. [20]. The relevant phenotypic symbols are Cb = resistance to carbenicillin, Nm = resistance to neomycin, and Tc = resistance to tetracycline. The relevant genotypic symbols are tet = tetracycline resistance determinant, bla = beta-lactamase [Cb] determinant, aphA = aminoglycoside phosphotransferase A [Nm] determinant, cam = determinants for camphor-oxidizing enzymes, and alk = determinants for alkane-oxidizing enzymes.) We name this transposon Tn401, until such time as it may be shown to be the same as the RP1 bla+ transposon observed in E. coli (1, 23). MATERIALS AND METHODS Bacterial strains. The bacterial strains used in this research are listed in Table 1. The P. aeruginosa strains are all derivatives of the PAC line, and the P. putida strains are all derivatives of the PpG1 line. The RP1 plasmid in E. coli MRPO comes from a Salmonella (RP1) strain from the laboratory of R. Olsen. Drug resistance plasmids can be crossed from prototrophic P. putida strains into PAC1 or PAC5, because only the latter strains will grow on alkanes longer than dodecane (cf. 19). Bacteriophages. We have used two transducing phages: pfl6 for P. putida (8), obtained from I. C. Gunsalus, and F116L for P. aeruginosa (17), obtained from B. W. Holloway. The lines of pfl6 used are two poorer-growing mutants induced by nitrosoguanidine mutagenesis, called pfl6 no. 1 and pfl6 no. 4. The size of the deoxyribonucleic acid (DNA) molecule encapsidated by pfl6 is 97.7 x 106 daltons (L. MacHattie, personal communication). F116L is a subline of phage F116, which shows higher cotransduction frequencies (17). The size of the DNA mole809
810
BENEDIK, FENNEWALD, AND SHAPIRO
J. BACTERIOL.
TABLE 1. Bacterial strains" Species
P. putida
Strain
Genotype
PpG1 PpS5 PpS81 PpS102 PpS108 PpS145 PpS208 PpS236 PpS239 PpS284 PpS288 PpS338 PpS345-348 PpS352 PpS353-354 PpS366 PpS369 PpS370 PpS376 PAC5 PAS65 PAS71
Prototroph met-5 (OCT) alcA81 trp-102 ilv-108 met-145 (CAM-OCT) met-145 (CAM-OCT alk208) trp-236 (RP1, SAL, CAM) met-5 (OCT, SAI, 239::Tn401) trp-102 (RP1) met-145 (RP1, CAM-OCT) alcA81 trp-338 alcA81 (CAM-OCT::Tn401) alcA81 (CAM-OCT cam-352::Tn401) alcA81 trp-338 (CAM-OCT::Tn401) trp-102 str (CAM-OCT::Tn401) trp-102 (SAL 239::Tn401) ilv-108 (SAL 239::Tn401) trp-102 (SAL 376::Tn401) his-5 his-5 (CAM-OCT cam-352::Tn401) his-5 (CAM-OCT)
Source
I. C. Gunsalus Ac75 of A. M. Chakrabarty NTG mutant of PpG1 NTG mutant of PpG1 NTG mutant of PpG1 References 6, 10, 12 NTG mutant of PpS145 Ac26 of A. M. Chakrabarty PpS236 x PpS5 E. coli (RP1) x PpS102 E. coli (RP1) x PpS145 HNO2 mutant of PpS81 PpS288 x PpS81 PpS288 x PpS338 PpS288 x PpS338 PpS345 x PpS102str PpS239 x PpS102 pfl6 * PpS369 x PpS108 pfl6 - PpS236 x PpS102 P. aeruginosa P. H. Clark-e PpS352 x PAC5 PpS145 x PAC5 "Genotypic symbols either use the standard nomenclature symbols or have been previously defined (2, 12, 20); Jacoby and Shapiro; in press. The allele numbers correspond to the strains of original isolation. Tn401 is defined in the text. NTG, N-methyl-N'-nitro-N-nitrosoguanidine.
cule encapsidated by F116L is 38 x 106 daltons (15). All phage lysates were prepared by the confluent lysis method on the appropriate donor strain. Phage pfl6 lysates were sterilized by storage in broth over chloroform. Phage F116 is inactivated by chloroform, and lysates were therefore sterilized by filtration. Both phages were stable in our hands at 4°C in broth. Media, chemicals, and culture methods. Media, chemicals, and culture methods have generally been described previously (2, 12, 19). Antibiotics were added to either nutrient or minimal agar at the following concentrations (micrograms per milliliter): carbenicillin, 2,000 for P. putida and 500 forP. aeruginosa; neomycin, 50; tetracycline, 50. Bacterial crosses. In general, bacterial crosses were performed as previously described (2). For crosses in which antibiotic-resistant exconjugants were selected, the donor-recipient mixtures were incubated for 30 min at 32°C, diluted with an equal volume of fresh broth, and incubated for a further 120 to 240 min at 32°C with aeration to allow expression of the resistant phenotype before plating. When plasmids were crossed into P. aeruginosa strains, the recipients were sometimes grown at 43°C to eliminate host restriction. Transduction. For phage pfl6, transductions were carried out as previously described (8), using ultraviolet inactivation and antiserum to prevent killing of transductants. We have found transduction with this virulent phage to be very unpredictable and highly dependent on the marker transduced. For phage F116L, we used the procedure of Betz et al. (3) with the following improvement. Phage F116L is sensitive to ethylene-bis-(oxyethyleneni-
trilo)tetraacetic acid (EGTA; Eastman); consequently, spreading selective plates with 0.1 ml of a 1 M EGTA solution results in a 10- to 100-fold increase in the yield of transductants, and the colonies are of more uniform size. The same effect can be achieved using recipients lysogenic for F116L. Extraction of plasmid DNA. Extraction of plasmid DNA was done by either of two methods, with comparable results. One was the method of Palchaudhuri and Chakrabarty (21). The other was the method of Clewell and Helinski (11), with the substitution of Triton X-100 for Brij in the detergent mixture. CsCl gradients were adjusted to a refractive index of 1.399 to 1.400 before adding 1/20 volume of ethidium bromide (10 mg/ml in water), mixing, and centrifuging. Transformation by plasmid DNA. We used the method of Lederberg and Cohen (18).
RESULTS Segregation of the bla ' marker from RP1. We first observed transposition of the bla+ marker during strain construction. The cross PpS236 (RP1, SAL, CAM) x PpS5 (OCT) gave rise to a sal+ recombinant that was Cbr but Nms and T&. Since we had not previously observed segregation in RP1 crosses, we examined this in more detail. In crosses with PpS236 as donor, 30 to 80% of the Cbr transconjugants were Nms and T&' (Table 2, donor PpS236). Selection for either Nmr or Tcr yielded only a low level of segregation, and the absence of the Nmr Tcr Cbs class of transconjugants indicated that PpS236
P. PUTIDA bla+ TRANSPOSON
VOL. 129, 1977
does not harbor two drug resistance plasmids (Cb and Nm Tc). For segregation of the bla+ marker to occur, other plasmids must be present in the donor strain. This conclusion comes from the data in the Tables 2 and 3. The bla+ marker did not segregate from PpS284 with only the RP1 plasmid, but it did segregate from PpS288 with both RP1 and the CAM-OCT plasmid. Moreover, segregation only occurred when the RP1 donor had also transferred the second plasmid. In the four crosses with PpS288 as donor, only 12 to 30% of all Cbr transconjugants had received CAM-OCT, but when we examined the Nrin T&l among these, all of them had recieved CAMOCT. In addition to the four crosses shown in Table 3, we repeated this experiment another 24 times. We always obtained the same result: all Cbr Nms Tcs transconjugants contain CAMOCT. In four cases, we found bla+ alk+ camtransconjugants. These resulted from insertion of Tn401 into the camphor genes (see below). Incorporation of the bla+ marker into the degradative plasmids. Transposition of the bla+ determinant into the degradative plasmids would give linkage between the Cbr and degradative phenotyes in conjugation crosses. Table 4 shows that all markers transferred jointly regardless of the marker selected. Based on these results, we designated the hybrid plasmids SAL::Tn401 and CAM-OCT::Tn401. (To
811
distinguish independent isolates of SAL::Tn401 where we did not know the site of insertion, we put an isolation number before the double colon; otherwise, we followed the recommendation of Campbell et al. [in press].) To eliminate one possible artefact in our results, i.e., a very high incidence of cotransfer of separate plasmids, we performed the transduction and transformation experiments summarized in Table 5. Since the probability is very low that physically separate plasmids will either be incorporated in a single phage particle or jointly taken up from a DNA solution, these experiments distinguish between separate or covalently joined plasmids for Cb resistance and degradative markers. Our experiments with the CAM-OCT::Tn401 plasmids were limited to CAM-OCT cam-352::Tn401 for two technical reasons: (i) we have not yet isolated CAMOCT plasmid DNA, and (ii) CAM-OCT is not transduced intact by either pfl6 or F116L. In we cases three investigated, the SAL239::Tn401, SAL376::Tn401, and CAM-OCT cam-352::Tn401, it is clear that bla+ and degradative markers are physically present on a single DNA molecule. In particular, the 100% linkage between bla+ and cam on CAM-OCT cam-352::Tn401 demonstrates that both changes in the plasmid resulted from a single genetic event, the insertion of Tn401 into one of the camphor genes.
TABLE 2. Segregation of antibiotic markers in crosses with RP1 donorsa Recombinant classes (Cb Nm Tc) Donor
Recipient
Selected markers +++
PpG1
PpS236trp- (RP1, SAL, CAM)
PpS103
trp+ bla+ trp+ tet+ trp+ aphA+
14 62 59
trp+ bla+ trp+ tet+ trp+ aphA+
28 34 44
-++ +-+ ++0 0 0
+
0 0
1 0
0 5
56 0 0
0 0 0
1 8 0
0 0 0
14 0 0
.----+ 0 0 2
0 0 0
0 0 0
0 0 0
0 0 120 0 0 0 0 trp+ bla+ PpS284 trp- (RP1) PpS103 a Recombinants were selected on appropriately supplemented minimal glucose agar containing antibiotics. After colonies appeared, they were picked into the same medium, incubated overnight, and replicaplated to the nutrient agar with antibiotics. + = Growth, resistance; - = no growth, sensitivity.
TABLE 3. Segregation of antibiotics and degradative markers in crosses with RP1 and CAM-OCT plasmidsa Recombinant classes among bla+ tranconjugants tested (Cb Nm Tc Cam Alk)
Donor cul-
+++++-
ture
+-+++ +-+---
----
+--++
++-++
++---
Others
3 0 4 2 4 1 0 26 0 4 0 0 0 0 6 0 0 0 10 0 0 0 C 22 0 0 0 0 10 22 2 0 0 D aIndependent cultures of strain PpS288 met-(RP1) (CAM-OCT) were crossed to PpS81, and met+ bla+ recombinants were selected. These were picked into selective medium and replica-plated to score RP1 and CAM-OCT markers.
A B
106 80 81 94
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BENEDIK, FENNEWALD, AND SHAPIRO
812
TABLE 4. Joint transfer of carbenicillin resistance and degradative markers from transposition strains by conjugation" Recipient
Donor strain
Selected markers
Colonies tested
Exconjugants found
PpS347 strs (CAM-OCT::Tn4Ol)
PpS81 strr
bla+ strr alk+ strr
82 95
82 bla+ cam+ alk+ strr 95 bla+ cam+ alk+ strr
PpS348 strs (CAM-OCT::Tn4Ol)
PpS81 strr
bla+ strr alk+ strr
92 105
92 bla+ cam+ alk+ strr 105 bla+ cam+ alk+ trpr
PpS352 trp-(CAM-OCT cam-352::Tn4Ol)
PpS81
bla+ trp+ alk+ trp+
88 110
88 bla+ cam-- alk+ trp+ 110 bla+ cam- alk+ trpr
PpS353
PpS81
bla+ trp+ alk+ trp+ cam+ trp+
101 98 117
101 bla+ cam+ alk+ trpr 98 bla+ cam+ alk+ trpr 117 bla+ cam+ alk+ trpr
PpS354 trp-(CAM-OCT::Tn4Ol)
PpS81
bla+ trp+ alk+ trp+ cam+ trp+
43 121 109
43 bla+ cam+ alk+ trpr 121 bla+ cam+ alk+ trpr 109 bla+ cam+ alk+ strr
PpS366 trp-(CAM-OCT::Tn4Ol)
PpS81
bla+ trp+ alk+ trp+ cam+ trp+
91 136 133
91 bla+ cam+ alk+ trpr 136 bla+ cam+ alk+ trpr 133 bla+ cam+ alk+ trpr
PpS239 met(OCT, SAL239::Tn4Ol)
PpS102 trp--
sal+ met+
103
103 bla+ sal+ met+ trp-
trp-(CAM-OCT::Tn4Ol)
65 65 bla+ sal+ ilv+ trpsal+ ilv+ PpS102trpPpS370 92 bla+ sal+ ilv+ trp 92 bla+ ilv+ ilv-(SAL239::Tn4O1) " Recombinants were selected on appropriately supplemented minimal glucose carbenicillin agar, minimal streptomycin agar (125 /.L/ml) with octane added in the vapor phase, minimal agar with either octane or camphor added in the vapor phase, or minimal tryptophan agar containing 0.01 M sodium salicylate. Colonies were picked into the same selective medium and replica-plated after overnight incubation. TABLE 5. Joint transfer of Cb' and degradation ability by transduction and transformation" Donor Recipient
Vector
Selected Colonies markers tested
Recombinants found
PpS108
pf 16
bla+
60
60 bla+ sal+
PpS369(SAL239::Tn401)
PpS102
pfl6
bla+
81
81 bla+ sal+
PpS370(SAL239::Tn401)
PpS102 PpS102 PpS102
pf16 Plasmid DNA Plasmid DNA
bla+ bla+ sal+
147 170 50
147 bla+ sal+ 170 bla+ sal+ 50 bla+ sal+
PpS376(SAL376::Tn401)
PpS102 PpS102
Plasmid DNA Plasmid DNA
bla+ sal+
25 25
25 bla+ sal+ 25 bla+ sal+
PpS352(CAM-OCT cam352::Tn401)
PpS208(CAM-OCT alk-208)
pf 16
bla+
22
22 bla+ cam
PpS239-
(OCT, SAL239::Tn401)
328 328 bla- cam+ cam+ F116L PAS65(CAM-OCT cam-352::Tn401) "Transduction and transformation were performed as described in Materials and Methods. Colonies were selected on nutrient carbenicillin agar or appropriately supplemented minimal salicylate and minimal camphor agar. Recombinant colonies were picked into the selective medium and replica-plated for nonselected markers. PAS71(CAM-OCT)
VOL. 129, 1977
Origin of the SAL::Tn401 plasmids. One of the most striking results in Tables 2 and 3 is the high frequency with which we find transposition events in P. putida. We wanted to know if these events occur in the absence of conjugation. Phage pf16 grown on PpS236 was used to transduce PpS102 to sal+. Eight percent of the sal+ transductions (9/114 tested) had received SAL::Tn401. DNA from one of these (PpS376) carries both sal+ and bla+ on-a single molecule (Table 5). Thus, SAL::Tn401 plasmids exist prior to conjugation and compose a high proportion of all SAL plasmids in the culture. The even higher proportion of SAL::Tn401 strains among the bla+ transconjugants is due to selective effects, because SAL fertility inhibits RP1 (Jacoby and Shapiro, in press). Reversion of Tn401 insertion mutations. We examined strains carrying four independent CAM-OCT cam::Tn401 plasmids for reversion to cam+. No cam+ revertants were found in more than 109 cells of any cam::Tn401 strain. DISCUSSION Our results show that a blat transposon in the RP1 plasmid can insert itself into the P. putida SAL and CAM-OCT plasmids. This conclusion comes from the observation that crosses with (RP1, SAL) and (RP1, CAM-OCT)donors yield hybrid CIf-degradative plasmids, which behave as single entities in conjugation, transduction, and transformation crosses. We call this P. putida transposon Tn401. Our data do not permit a detailed comparison of transposition events in P. putida and E. coli. We do not know if transposition in Pseudomonas is independent of the host general recombination system (cf. 1, 14, 23), although insertion of Tn401 into the camphor genes indicates that gross genetic homology is not necessary. We also do not know if Tn401 inserts itself into the Pseudomonas genome at many or a few sites. On the one hand, we were unable to isolate Tn401 inserted into the chromosome ofeitherP. putida or P. aeruginosa or into the alk genes of CAM-OCT. This would suggest that we are observing frequent insertion into a few plasmid hot spots. On the other hand, the four independent nonreverting insertions into the cam loci recombine with each other in transductional crosses; since they do not "self" to give cam+, these results indicate that each insertion is at a different site. We do not yet know if Tn401 has the same short terminal repeats observed in the Tn2 and Th3 bla+ transposons (14, 18) or whether it has the same 4.0 x 106 molecular weight as the RP1 bla+ transposon measured in E. coli (23). Preliminary measure-
P. PUTIDA bla+ TRANSPOSON
813
ments of isolated SAL239::TnA suggest that it may be as much as 4.7 to 6.2 negadaltons larger than SAL. Despite these limitations, two points of comparison are clear. First, as in our cam::Tn401 insertions, mutations caused by a bla+ transposon in E. coli do not revert (G. Weinstock and D. Botstein, personal communication). Second, Tn401 transposes from RP1 into SAL at very high frequencies. Transduction shows that 8% of all SAL plasmids from an (RP1, SAL) strain have Tn401 insertions. This figure is about two orders of magnitude greater than the frequency of Tn2 transpositions in comparable experiments in E. coli (14). ACKNOWLEDGMENTS We thank A. M. Chakrabarty, P. H. Clarke, I. C. Gunsalus, and B. W. Holloway for bacterial strains and phage stocks and Chia-Chung Che and Ruth Timmons for technical assistance. This research was supported in part by a grant from the Louis Block Fund of the University of Chicago, the National Science Foundation (GB 43352 and BMS75-08591), and the Public Health Service. M. F. was the recipient of a Public Health Service predoctoral traineeship (GM 603) from the National Institute of General Medical Sciences.
LITERATURE CITED 1. Bennett, P. M., and M. H. Richmond. 1976. Translocation of a discrete piece of deoxyribonucleic acid carrying an amp gene between replicons in Escherichia coli. J. Bacteriol. 125:1-6. 2. Benson, S., and J. Shapiro. 1976. Plasmid-determined alcohol dehydrogenase activity in alkane-utilizing strains ofPseudomonas putida. J. Bacteriol. 126:794798. 3. Betz, J. L., J. E. Brown, P. H. Clarke, and M. Day. 1974. Genetic analysis of amidase mutants of Pseudomonas aeruginosa. Genet. Res. 23:235-359. 4. Bryan, L. E., and M. S. Shahrabadi. 1975. Properties of P-2 R factors of Pseudomonas aeruginosa, some of which mediate gentamycin resistance, p. 53-55. In D. Schlessinger (ed.), Microbiology- 1974. American Society for Microbiology, Washington, D.C. 5. Chakrabarty, A. M. 1972. Genetic basis of the biodegradation of salicylate in Pseudomonas. J. Bacteriol. 112:815-823. 6. Chakrabarty, A. M. 1973. Genetic fusion of incompatible plasmids in Pseudomonas. Proc. Natl. Acad. Sci. U.S.A. 70:1641-1644. 7. Chakrabarty, A. M., G. Chou, and I. C. Gunsalus. 1973. Genetic regulation of octane dissimilation plasmid in Pseudomonas. Proc. Natl. Acad. Sci. U.S.A. 70:11371140. 8. Chakrabarty, A. M., C. F. Gunsalus and I. C. Gunsalus. 1967. Transduction and the clustering of genes in the fluorescent pseudomonads. Proc. Natl. Acad. Sci. 60:168-175. 9. Chakrabarty, A. M., and I. C. Gunsalus. 1971. Degradative pathways specified by extrachromosomal gene clusters in Pseudomonas. Genetics 68:s10. 10. Chou, G., D. Katz, and I. C. Gunsalus. 1974. Fusion and compatibility of camphor and octane plasmids in Pseudomonas. Proc. Natl. Acad. Sci. U.S.A. 71:26752678. 11. Clewell, D. B., and D. R. Helinski. 1967. Supercoiled
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circular DNA-protein complexes in E. coli: purification and induced conversion to open circular DNA form. Proc. Natl. Acad. Sci. U.S.A. 62:1159-1166. Grund, A., J. Shapiro, M. Fennewald, P. Bacha, J. Leahy, K. Markbreiter, M. Neider, and M. Toepfer. 1975. Regulation of alkane oxidation in Psuedomonas p4tida. J. Bacteriol. 123:546-556. Hedges, R. W., and A. Jacob. 1974. Transposition of ampicillin resistance from RP4 to other replicons. Mol. Gen. Genet. 132:31-40. Heffron, F., C. Rubens, and S. Falkow. 1975. Translocation of a plasmid DNA sequence which mediates ampicillin resistance: molecular nature and specificity of insertion. Proc. Natl. Acad. Sci. U.S.A. 72:3623-3627. Holloway, B. W., and V. Krishnapillai. 1975. Bacteriophages and bacteriocins, p. 99-132. In P. H. Clarke and M. H. Richmond (ed.), Genetics and Biochemistry of Pseudomonas. John Wiley & Sons, London. Kopecko, D. J., and S. N. Cohen. 1975. Site specific recA-independent recombination between bacterial plasmids: involvement of palindromes at the recombination level. Proc. Natl. Acad. Sci. U.S.A. 72:13731377. Krishnapillai, V. 1971. A novel transducing phage. Its
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role in recognition of a possible new host-controlled modification system in Pseudomona aerugi.iosa. Mol. Gen. Genet. 114:134-143. Lederberg, E. M., and S. N. Cohen. 1974. Transformation of Salmonella typhimurium by plasmid deoxyribonucleic acid. J. Bacteriol. 119:1072-1074. Nieder, M., and J. Shapiro. 1975. Physiological function of the Pseudomonas putida PpG6 (Pseudomonas oleovorans) alkane hydroxylase: monoterminal oxidation of alkanes and fatty acids. J. Bacteriol. 211:93-98. Novick, R. P., R. C. Clowes, S. N. Cohen, R. Curtiss III, N. Datta, and S. Falkow. 1976. Uniform nomenclature for bacterial plasmids: a proposal. Bacteriol. Rev. 40:168-189. Palchaudhuri, S., and A. M. Chakrabarty. 1976. Isolation of plasmid DNA from Pseudomonas putida. J. Bacteriol. 126:410-416. Rheinwald, J. G., A. M. Chakrabarty, and I. C. Gunsalus. 1973. A transmissible plasmid controlling camphor oxidation in Pseudomonas putida. Proc. Natl. Acad. Sci. U.S.A. 70:885-889. Richmond, M. H., and R. B. Sykes. 1972. The chromosomal integration of a /3-lactamase gene derived from the P-type R factor RP1 in E. coli. Genet. Res. 20:231237.
Vol. 129, No. 2 Printed in U.S.A.
Transposition of a Beta-Lactamase Locus from RP1 into Pseudomonas putida Degradative Plasmids MICHAEL BENEDIK, MICHAEL FENNEWALD, AND JAMES SHAPIRO* Department of Microbiology, University of Chicago, Chicago, Illinois 60637
Received for publication 23 September 1976
The beta-lactamase gene from the RP1 plasmid transposes into at least two Pseudomonas putida degradative plasmids. Donor strains that carry RP1 (bla+ tet+ aphA +) and a degradative plasmid yield transconjugants that have only the bla+ marker of RP1. This occurs in up to 80% of all bla+ transconjugants. Segregation of the bla+ marker requires the presence of a degradative plasmid in the donor and is only observed in transconjugants that have received degradative markers. The bla+ tet aphA transconjugants show 100%o linkage of bla+ to degradative markers in conjugation, transduction, and transformation crosses. A transduction cross of an (RP1), (SAL) donor shows that 8% ofall SAL plasmids also carry the transposed bla+ marker. Tn401 is the name we assign to the bla+ transposon from RP1 observed in Pseudomonas. Its identity with the RP1 bla+ transposon observed in Escherichia coli is not known. In four cases, Tn401 has inserted into the camphor genes of the CAM-OCT plasmid. A number of antibiotic resistance loci have the important property of transposing themselves from one replicon to another (A. Campbell, D. Berg, D. Botstein, R. Novick, and P. Starlinger, in A. I. Bukhari, J. Shapiro, and S. Adhya [ed.], DNA Insertion Elements, Episomes and Plasmids, in press). In Escherichia coli, the TEM-1 and TEM-2 beta-lactamase genes of several plasmids, including RP1, exhibit this property and can insert into the bacterial chromosome (1), other plasmids (14, 16, 23), and temperate bacteriophages (G. Weinstock and D. Botstein, personal communication; Shapiro, unpublished observation). We have been studying a group of plasmids that encode the ability to degrade different groups of hydrocarbons in Pseudomonas putida and Psuedomonas aeruginosa. These include CAM, SAL, and OCT plasmids, which determine growth on camphor, salicylate, or alkanes (5, 7, 22). Such plasmids have been termed "degradative plasmids." However, they do not appear to be a unique set of genetic elements because they belong to several incompatibility groups, one of which (IncP-2) contains a number of drug resistance plasmids as well as CAM and OCT (G. A. Jacoby and J. Shapiro, in A. I. Bukhari, J. Shapiro, and S. Adhya [ed.], DNA Insertion Elements, Episomes and Plasmids, in press). In this paper, we report the transposition of the bla+ locus from RP1 into the SAL and CAMOCT plasmids in P. putida. (In this paper we adopt the nomenclature proposed by Novick et
al. [20]. The relevant phenotypic symbols are Cb = resistance to carbenicillin, Nm = resistance to neomycin, and Tc = resistance to tetracycline. The relevant genotypic symbols are tet = tetracycline resistance determinant, bla = beta-lactamase [Cb] determinant, aphA = aminoglycoside phosphotransferase A [Nm] determinant, cam = determinants for camphor-oxidizing enzymes, and alk = determinants for alkane-oxidizing enzymes.) We name this transposon Tn401, until such time as it may be shown to be the same as the RP1 bla+ transposon observed in E. coli (1, 23). MATERIALS AND METHODS Bacterial strains. The bacterial strains used in this research are listed in Table 1. The P. aeruginosa strains are all derivatives of the PAC line, and the P. putida strains are all derivatives of the PpG1 line. The RP1 plasmid in E. coli MRPO comes from a Salmonella (RP1) strain from the laboratory of R. Olsen. Drug resistance plasmids can be crossed from prototrophic P. putida strains into PAC1 or PAC5, because only the latter strains will grow on alkanes longer than dodecane (cf. 19). Bacteriophages. We have used two transducing phages: pfl6 for P. putida (8), obtained from I. C. Gunsalus, and F116L for P. aeruginosa (17), obtained from B. W. Holloway. The lines of pfl6 used are two poorer-growing mutants induced by nitrosoguanidine mutagenesis, called pfl6 no. 1 and pfl6 no. 4. The size of the deoxyribonucleic acid (DNA) molecule encapsidated by pfl6 is 97.7 x 106 daltons (L. MacHattie, personal communication). F116L is a subline of phage F116, which shows higher cotransduction frequencies (17). The size of the DNA mole809
810
BENEDIK, FENNEWALD, AND SHAPIRO
J. BACTERIOL.
TABLE 1. Bacterial strains" Species
P. putida
Strain
Genotype
PpG1 PpS5 PpS81 PpS102 PpS108 PpS145 PpS208 PpS236 PpS239 PpS284 PpS288 PpS338 PpS345-348 PpS352 PpS353-354 PpS366 PpS369 PpS370 PpS376 PAC5 PAS65 PAS71
Prototroph met-5 (OCT) alcA81 trp-102 ilv-108 met-145 (CAM-OCT) met-145 (CAM-OCT alk208) trp-236 (RP1, SAL, CAM) met-5 (OCT, SAI, 239::Tn401) trp-102 (RP1) met-145 (RP1, CAM-OCT) alcA81 trp-338 alcA81 (CAM-OCT::Tn401) alcA81 (CAM-OCT cam-352::Tn401) alcA81 trp-338 (CAM-OCT::Tn401) trp-102 str (CAM-OCT::Tn401) trp-102 (SAL 239::Tn401) ilv-108 (SAL 239::Tn401) trp-102 (SAL 376::Tn401) his-5 his-5 (CAM-OCT cam-352::Tn401) his-5 (CAM-OCT)
Source
I. C. Gunsalus Ac75 of A. M. Chakrabarty NTG mutant of PpG1 NTG mutant of PpG1 NTG mutant of PpG1 References 6, 10, 12 NTG mutant of PpS145 Ac26 of A. M. Chakrabarty PpS236 x PpS5 E. coli (RP1) x PpS102 E. coli (RP1) x PpS145 HNO2 mutant of PpS81 PpS288 x PpS81 PpS288 x PpS338 PpS288 x PpS338 PpS345 x PpS102str PpS239 x PpS102 pfl6 * PpS369 x PpS108 pfl6 - PpS236 x PpS102 P. aeruginosa P. H. Clark-e PpS352 x PAC5 PpS145 x PAC5 "Genotypic symbols either use the standard nomenclature symbols or have been previously defined (2, 12, 20); Jacoby and Shapiro; in press. The allele numbers correspond to the strains of original isolation. Tn401 is defined in the text. NTG, N-methyl-N'-nitro-N-nitrosoguanidine.
cule encapsidated by F116L is 38 x 106 daltons (15). All phage lysates were prepared by the confluent lysis method on the appropriate donor strain. Phage pfl6 lysates were sterilized by storage in broth over chloroform. Phage F116 is inactivated by chloroform, and lysates were therefore sterilized by filtration. Both phages were stable in our hands at 4°C in broth. Media, chemicals, and culture methods. Media, chemicals, and culture methods have generally been described previously (2, 12, 19). Antibiotics were added to either nutrient or minimal agar at the following concentrations (micrograms per milliliter): carbenicillin, 2,000 for P. putida and 500 forP. aeruginosa; neomycin, 50; tetracycline, 50. Bacterial crosses. In general, bacterial crosses were performed as previously described (2). For crosses in which antibiotic-resistant exconjugants were selected, the donor-recipient mixtures were incubated for 30 min at 32°C, diluted with an equal volume of fresh broth, and incubated for a further 120 to 240 min at 32°C with aeration to allow expression of the resistant phenotype before plating. When plasmids were crossed into P. aeruginosa strains, the recipients were sometimes grown at 43°C to eliminate host restriction. Transduction. For phage pfl6, transductions were carried out as previously described (8), using ultraviolet inactivation and antiserum to prevent killing of transductants. We have found transduction with this virulent phage to be very unpredictable and highly dependent on the marker transduced. For phage F116L, we used the procedure of Betz et al. (3) with the following improvement. Phage F116L is sensitive to ethylene-bis-(oxyethyleneni-
trilo)tetraacetic acid (EGTA; Eastman); consequently, spreading selective plates with 0.1 ml of a 1 M EGTA solution results in a 10- to 100-fold increase in the yield of transductants, and the colonies are of more uniform size. The same effect can be achieved using recipients lysogenic for F116L. Extraction of plasmid DNA. Extraction of plasmid DNA was done by either of two methods, with comparable results. One was the method of Palchaudhuri and Chakrabarty (21). The other was the method of Clewell and Helinski (11), with the substitution of Triton X-100 for Brij in the detergent mixture. CsCl gradients were adjusted to a refractive index of 1.399 to 1.400 before adding 1/20 volume of ethidium bromide (10 mg/ml in water), mixing, and centrifuging. Transformation by plasmid DNA. We used the method of Lederberg and Cohen (18).
RESULTS Segregation of the bla ' marker from RP1. We first observed transposition of the bla+ marker during strain construction. The cross PpS236 (RP1, SAL, CAM) x PpS5 (OCT) gave rise to a sal+ recombinant that was Cbr but Nms and T&. Since we had not previously observed segregation in RP1 crosses, we examined this in more detail. In crosses with PpS236 as donor, 30 to 80% of the Cbr transconjugants were Nms and T&' (Table 2, donor PpS236). Selection for either Nmr or Tcr yielded only a low level of segregation, and the absence of the Nmr Tcr Cbs class of transconjugants indicated that PpS236
P. PUTIDA bla+ TRANSPOSON
VOL. 129, 1977
does not harbor two drug resistance plasmids (Cb and Nm Tc). For segregation of the bla+ marker to occur, other plasmids must be present in the donor strain. This conclusion comes from the data in the Tables 2 and 3. The bla+ marker did not segregate from PpS284 with only the RP1 plasmid, but it did segregate from PpS288 with both RP1 and the CAM-OCT plasmid. Moreover, segregation only occurred when the RP1 donor had also transferred the second plasmid. In the four crosses with PpS288 as donor, only 12 to 30% of all Cbr transconjugants had received CAM-OCT, but when we examined the Nrin T&l among these, all of them had recieved CAMOCT. In addition to the four crosses shown in Table 3, we repeated this experiment another 24 times. We always obtained the same result: all Cbr Nms Tcs transconjugants contain CAMOCT. In four cases, we found bla+ alk+ camtransconjugants. These resulted from insertion of Tn401 into the camphor genes (see below). Incorporation of the bla+ marker into the degradative plasmids. Transposition of the bla+ determinant into the degradative plasmids would give linkage between the Cbr and degradative phenotyes in conjugation crosses. Table 4 shows that all markers transferred jointly regardless of the marker selected. Based on these results, we designated the hybrid plasmids SAL::Tn401 and CAM-OCT::Tn401. (To
811
distinguish independent isolates of SAL::Tn401 where we did not know the site of insertion, we put an isolation number before the double colon; otherwise, we followed the recommendation of Campbell et al. [in press].) To eliminate one possible artefact in our results, i.e., a very high incidence of cotransfer of separate plasmids, we performed the transduction and transformation experiments summarized in Table 5. Since the probability is very low that physically separate plasmids will either be incorporated in a single phage particle or jointly taken up from a DNA solution, these experiments distinguish between separate or covalently joined plasmids for Cb resistance and degradative markers. Our experiments with the CAM-OCT::Tn401 plasmids were limited to CAM-OCT cam-352::Tn401 for two technical reasons: (i) we have not yet isolated CAMOCT plasmid DNA, and (ii) CAM-OCT is not transduced intact by either pfl6 or F116L. In we cases three investigated, the SAL239::Tn401, SAL376::Tn401, and CAM-OCT cam-352::Tn401, it is clear that bla+ and degradative markers are physically present on a single DNA molecule. In particular, the 100% linkage between bla+ and cam on CAM-OCT cam-352::Tn401 demonstrates that both changes in the plasmid resulted from a single genetic event, the insertion of Tn401 into one of the camphor genes.
TABLE 2. Segregation of antibiotic markers in crosses with RP1 donorsa Recombinant classes (Cb Nm Tc) Donor
Recipient
Selected markers +++
PpG1
PpS236trp- (RP1, SAL, CAM)
PpS103
trp+ bla+ trp+ tet+ trp+ aphA+
14 62 59
trp+ bla+ trp+ tet+ trp+ aphA+
28 34 44
-++ +-+ ++0 0 0
+
0 0
1 0
0 5
56 0 0
0 0 0
1 8 0
0 0 0
14 0 0
.----+ 0 0 2
0 0 0
0 0 0
0 0 0
0 0 120 0 0 0 0 trp+ bla+ PpS284 trp- (RP1) PpS103 a Recombinants were selected on appropriately supplemented minimal glucose agar containing antibiotics. After colonies appeared, they were picked into the same medium, incubated overnight, and replicaplated to the nutrient agar with antibiotics. + = Growth, resistance; - = no growth, sensitivity.
TABLE 3. Segregation of antibiotics and degradative markers in crosses with RP1 and CAM-OCT plasmidsa Recombinant classes among bla+ tranconjugants tested (Cb Nm Tc Cam Alk)
Donor cul-
+++++-
ture
+-+++ +-+---
----
+--++
++-++
++---
Others
3 0 4 2 4 1 0 26 0 4 0 0 0 0 6 0 0 0 10 0 0 0 C 22 0 0 0 0 10 22 2 0 0 D aIndependent cultures of strain PpS288 met-(RP1) (CAM-OCT) were crossed to PpS81, and met+ bla+ recombinants were selected. These were picked into selective medium and replica-plated to score RP1 and CAM-OCT markers.
A B
106 80 81 94
J. BACTERIOL.
BENEDIK, FENNEWALD, AND SHAPIRO
812
TABLE 4. Joint transfer of carbenicillin resistance and degradative markers from transposition strains by conjugation" Recipient
Donor strain
Selected markers
Colonies tested
Exconjugants found
PpS347 strs (CAM-OCT::Tn4Ol)
PpS81 strr
bla+ strr alk+ strr
82 95
82 bla+ cam+ alk+ strr 95 bla+ cam+ alk+ strr
PpS348 strs (CAM-OCT::Tn4Ol)
PpS81 strr
bla+ strr alk+ strr
92 105
92 bla+ cam+ alk+ strr 105 bla+ cam+ alk+ trpr
PpS352 trp-(CAM-OCT cam-352::Tn4Ol)
PpS81
bla+ trp+ alk+ trp+
88 110
88 bla+ cam-- alk+ trp+ 110 bla+ cam- alk+ trpr
PpS353
PpS81
bla+ trp+ alk+ trp+ cam+ trp+
101 98 117
101 bla+ cam+ alk+ trpr 98 bla+ cam+ alk+ trpr 117 bla+ cam+ alk+ trpr
PpS354 trp-(CAM-OCT::Tn4Ol)
PpS81
bla+ trp+ alk+ trp+ cam+ trp+
43 121 109
43 bla+ cam+ alk+ trpr 121 bla+ cam+ alk+ trpr 109 bla+ cam+ alk+ strr
PpS366 trp-(CAM-OCT::Tn4Ol)
PpS81
bla+ trp+ alk+ trp+ cam+ trp+
91 136 133
91 bla+ cam+ alk+ trpr 136 bla+ cam+ alk+ trpr 133 bla+ cam+ alk+ trpr
PpS239 met(OCT, SAL239::Tn4Ol)
PpS102 trp--
sal+ met+
103
103 bla+ sal+ met+ trp-
trp-(CAM-OCT::Tn4Ol)
65 65 bla+ sal+ ilv+ trpsal+ ilv+ PpS102trpPpS370 92 bla+ sal+ ilv+ trp 92 bla+ ilv+ ilv-(SAL239::Tn4O1) " Recombinants were selected on appropriately supplemented minimal glucose carbenicillin agar, minimal streptomycin agar (125 /.L/ml) with octane added in the vapor phase, minimal agar with either octane or camphor added in the vapor phase, or minimal tryptophan agar containing 0.01 M sodium salicylate. Colonies were picked into the same selective medium and replica-plated after overnight incubation. TABLE 5. Joint transfer of Cb' and degradation ability by transduction and transformation" Donor Recipient
Vector
Selected Colonies markers tested
Recombinants found
PpS108
pf 16
bla+
60
60 bla+ sal+
PpS369(SAL239::Tn401)
PpS102
pfl6
bla+
81
81 bla+ sal+
PpS370(SAL239::Tn401)
PpS102 PpS102 PpS102
pf16 Plasmid DNA Plasmid DNA
bla+ bla+ sal+
147 170 50
147 bla+ sal+ 170 bla+ sal+ 50 bla+ sal+
PpS376(SAL376::Tn401)
PpS102 PpS102
Plasmid DNA Plasmid DNA
bla+ sal+
25 25
25 bla+ sal+ 25 bla+ sal+
PpS352(CAM-OCT cam352::Tn401)
PpS208(CAM-OCT alk-208)
pf 16
bla+
22
22 bla+ cam
PpS239-
(OCT, SAL239::Tn401)
328 328 bla- cam+ cam+ F116L PAS65(CAM-OCT cam-352::Tn401) "Transduction and transformation were performed as described in Materials and Methods. Colonies were selected on nutrient carbenicillin agar or appropriately supplemented minimal salicylate and minimal camphor agar. Recombinant colonies were picked into the selective medium and replica-plated for nonselected markers. PAS71(CAM-OCT)
VOL. 129, 1977
Origin of the SAL::Tn401 plasmids. One of the most striking results in Tables 2 and 3 is the high frequency with which we find transposition events in P. putida. We wanted to know if these events occur in the absence of conjugation. Phage pf16 grown on PpS236 was used to transduce PpS102 to sal+. Eight percent of the sal+ transductions (9/114 tested) had received SAL::Tn401. DNA from one of these (PpS376) carries both sal+ and bla+ on-a single molecule (Table 5). Thus, SAL::Tn401 plasmids exist prior to conjugation and compose a high proportion of all SAL plasmids in the culture. The even higher proportion of SAL::Tn401 strains among the bla+ transconjugants is due to selective effects, because SAL fertility inhibits RP1 (Jacoby and Shapiro, in press). Reversion of Tn401 insertion mutations. We examined strains carrying four independent CAM-OCT cam::Tn401 plasmids for reversion to cam+. No cam+ revertants were found in more than 109 cells of any cam::Tn401 strain. DISCUSSION Our results show that a blat transposon in the RP1 plasmid can insert itself into the P. putida SAL and CAM-OCT plasmids. This conclusion comes from the observation that crosses with (RP1, SAL) and (RP1, CAM-OCT)donors yield hybrid CIf-degradative plasmids, which behave as single entities in conjugation, transduction, and transformation crosses. We call this P. putida transposon Tn401. Our data do not permit a detailed comparison of transposition events in P. putida and E. coli. We do not know if transposition in Pseudomonas is independent of the host general recombination system (cf. 1, 14, 23), although insertion of Tn401 into the camphor genes indicates that gross genetic homology is not necessary. We also do not know if Tn401 inserts itself into the Pseudomonas genome at many or a few sites. On the one hand, we were unable to isolate Tn401 inserted into the chromosome ofeitherP. putida or P. aeruginosa or into the alk genes of CAM-OCT. This would suggest that we are observing frequent insertion into a few plasmid hot spots. On the other hand, the four independent nonreverting insertions into the cam loci recombine with each other in transductional crosses; since they do not "self" to give cam+, these results indicate that each insertion is at a different site. We do not yet know if Tn401 has the same short terminal repeats observed in the Tn2 and Th3 bla+ transposons (14, 18) or whether it has the same 4.0 x 106 molecular weight as the RP1 bla+ transposon measured in E. coli (23). Preliminary measure-
P. PUTIDA bla+ TRANSPOSON
813
ments of isolated SAL239::TnA suggest that it may be as much as 4.7 to 6.2 negadaltons larger than SAL. Despite these limitations, two points of comparison are clear. First, as in our cam::Tn401 insertions, mutations caused by a bla+ transposon in E. coli do not revert (G. Weinstock and D. Botstein, personal communication). Second, Tn401 transposes from RP1 into SAL at very high frequencies. Transduction shows that 8% of all SAL plasmids from an (RP1, SAL) strain have Tn401 insertions. This figure is about two orders of magnitude greater than the frequency of Tn2 transpositions in comparable experiments in E. coli (14). ACKNOWLEDGMENTS We thank A. M. Chakrabarty, P. H. Clarke, I. C. Gunsalus, and B. W. Holloway for bacterial strains and phage stocks and Chia-Chung Che and Ruth Timmons for technical assistance. This research was supported in part by a grant from the Louis Block Fund of the University of Chicago, the National Science Foundation (GB 43352 and BMS75-08591), and the Public Health Service. M. F. was the recipient of a Public Health Service predoctoral traineeship (GM 603) from the National Institute of General Medical Sciences.
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