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IS1071-mediated recombinational equilibrium in Alcaligenes sp. BR60 carrying the 3-chlorobenzoate catabolic transposon Tn5271
JAMES NG AND R. CAMPBELL WYNDHAM' Ottawa-Carleton Institute of Biology. Carleton University, Ottawa, Ont., Canada KIS 5B6
Received November 27, 1991
Revision received July 30, 1992
Accepted July 31, 1992
NG, J., and WYNDHAM, R. C. 1993. IS1071-mediated recombinational equilibrium in Alcaligenes sp. BR60 carrying the 3-chlorobenzoate catabolic transposon Tn5271. Can. J. Microbiol. 39: 92-100.
In experiments designed to TnS mutagenize the indigenous plasmid pBRC60 of Alcaligenes sp. BR60, kanamycin- resistant mutants were isolated that were cured of this plasmid and that exhibited recombination of the plasmid-located chlorobenzoate catabolic transposon Tn5271 into the chromosome. These events were independent of the location of Tn5 insertions into the genome of strain BR60. The chromosomal recombinants carried at least two novel copies of IS1071, the class I1 insertion sequence flanking Tn5271, compared with the parent strain. Recombination of Tn5271 into the chromosome of Alcaligenes sp. BR60 was also detected following mating in of pBRC60-incompatible (IncP1) plasmids, R68 and pGS65. Chromosomal copies of Tn5271 could be mobilized between Alcaligenes strains via plasmids pBRC40 or R68. Conjugation of the incompatible plasmid pGS65 into Alcaligenes strains in the absence of selection for 3-chlorobenzoate catabolism resulted in the recovery of 85010 of transconjugants in which the entire pBRC60 plasmid had integrated into the chromosome. These transconjugants exhibited complex rearrangements in chromosomal IS1071 copies. A model of recombinational equilibrium involving homologous recombination between plasmid and chromosomal copies of IS1071 is presented. The results are discussed in terms of the IS1071 (class 11) transposition mechanism and the observed products of IS1071-mediated recombination in natural recipients of pBRC60 in aquatic environments.
Key words: transposon, 3-chlorobenzoate catabolism, rearrangement.
NG, J., et WYNDHAM, R. C. 1993. IS1071-mediated recombinational equilibrium in Alcaligenes sp. BR60 carrying the 3-chlorobenzoate catabolic transposon Tn5271. Can. J. Microbiol. 39 : 92-100.
Dans une serie d'experiences avec le transposon TnS visant a la mutagknkse du plasmide pBRC60 indigkne chez Alcaligenes sp. BR60, des mutants resistants a la kanamycine ont Ctk isoles. Ces mutants avaient perdu le plasmide mais ont demontre la recombinaison du transposon Tn5271, responsable du catabolisme du chlorobenzoate et localis6 sur le plasmide pBRC60, au niveau du chromosome. Ces evenements etaient independants de la localisation de l'insertion de Tn5 dans le genome de la souche BR60. Les recombinants chromosomiques portaient au moins deux nouvelles copies d'ISlO71, les sequences d'insertion de classe I1 flanquant Tn5271, par rapport a la souche parente. La recombinaison de Tn5271 au niveau de chromosomes d'Alcaligenes sp. BR60 a aussi Cte detectke par des experiences de conjugaison avec des plasmides d'un autre groupe d'incompatibilite (IncPI), R68 et pGS65. Des copies chromosomiques de Tn5271 pouvaient Ctre mobiliskes entre des souches dlAlcaligenes via le plasmide pBRC40 ou R68. La conjugaison du plasmide incompatible pGS65 dans les souches d'Alcaligenes en absence de selection pour le catabolisme du 3-chlorobenzoate a rCsulte au recouvrement de transconjugants dans lesquels le plasmide pBRC60 a CtC entitrement intCgrC dans le chromosome dans 85% des cas. Ces transconjugants dkmontraient de complexes rearrangements au niveau des copies d'IS1071. Un modkle de recombinaison a l'equilibre impliquant des recombinaisons homologues entre les copies d'IS1071 du plasmide et du chromosome est presente. La discussion des rksultats s'oriente au niveau du mecanisme de transposition d'IS1071 (classe 11) et des produits de la recombinaison mCdiCe par IS1071 qui ont Cte observes chez les porteurs naturels de pBRC60 provenant d'environnements aquatiques.
Mots cl6s : transposon, catabolisme du 3-chlorobenzoate, rearrangements. [Traduit par la redaction]
Introduction
Microorganisms selected for their ability to metabolize unusual organic carbon sources, such as environmental pollutants, often require an adaptation period before optimal growth under laboratory conditions is achieved (Ghosal et al. 1985; Wyndham 1986). Their growth phenotypes, once established, are often unstable and may frequently be lost entirely (Meulien et al. 1981; Eaton and Timmis 1986; Wyndharn et al. 1988; Saint et al. 1990). These observations have prompted many authors to speculate o n the genetic determinants involved in adapta- tion and catabolic instability. In some cases these genetic determinants have been well documented. Pseudomonas putida mt-2 (pWWO) carries the toluene catabolic genes (xyl) on a 39-kb segment of pWWO, which can be excised by
' ~ u t h o r to whom all correspondence should be addressed. Printed in Canada / Imprime au Canada
reciprocal recombination between 1.4-kb directly repeated sequences (Meulien et al. 1981). This 39-kb segment is harboured within the class I1 transposon Tn4651, which is nested within a second class I1 transposon, Tn4653 (Tsuda and Iino 1988). The recombination of the toluene catabolic transposon between chromosomal and plasmid loci has been demonstrated (Sinclair et al. 1986).
A limited number of mobile genetic elements involved in adaptation to chlorinated aromatic pollutants have been characterized. The 3-chlorobenzoate degradative gene cluster, clc, of the 110-kb plasmid pAC27 of P. putida, appears to undergo ready recombination with the chromo- some even in a recA host (Chatterjee and Chakrabarty 1984). Variation in the ability of another Pseudomonas sp., strain B13, to utilize 3-chlorobenzoate was associated with tandem amplification of DNA homologous to the pAC27 clc operon (Rangnekar 1988). These observations have been interpreted
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TABLE 1. Strains and plasmids
Strain Strain phenotype Plasmid Plasmid phenotype Reference or source
E. coli HBlOl HBlOl BW97 MM294 JMlOl JMlOl
pGS9 p15A::TnS suicide donor Selvaraj and Iyer 1983 pJHNl 1 pBR322::TnS J.H. Nash pGS65 RK2 ori/mob Tc', Km' Selvaraj and Iyer 1985 pRK2013 RK2 tra, Km' D. Helinski pBRH2 pUC18 clone of H2 Wyndham et al. 1988 pBRH4 pUC18 clone of H4 Wyndham et al. 1988
Alcaligenes sp. BR60 pBRC60 Tn5271, 3Cba + Wyndham et al. 1988 BR60-1 TnS mutagenized This study BR60-3 Tn5 mutagenized This study BR60-4 Tn5 mutagenized This study BR60-5 Tn5 mutagenized This study BR40 pBRC40 IS1071, 3Cba- Wyndham et al. 1988 BR6020 Cured strain, 3Cba- Wyndham et al. 1988 BR6024 Cm', Trp-, 3Cba- This study BR6024 Cm', Trp - pBRC60 Tn527 1, 3Cba + This study BR6024-12C Cmr, Trp - , 3Cba+ pGS65 Tc', Kmr This study BR6024-25C Cm', Trp - , 3Cba + pGS65 TcT, KmT This study BR6024-225C Cm', Trp - , 3Cba + pGS65 Tc', Km' This study BR6024-225s Cm', Trp - , 3Cba- pGS65 Tc', Kmr This study BR6024-A1/2 CmT, Trp-, Tn5271 (chromosomal), 3Cba+ R68 Tc', Apr, KmT This study BR6024-A1121 Cmr, Trp-, Tn5271 (chromosomal), 3Cba' R68 Tc', Ap', Km' This study BR6024-A2/7 Cm', Trp - , Tn527 1 (chromosomal), 3Cba' R68 Tc', Ap', Km' This study BR605 Tn5271 (chromosomal), 3Cba + R68 Tc', Ap', Kmr Wyndham et al. 1988 BR6053 Tn5271 (chromosomal), 3Cba + Wyndham et al. 1988
NOTE: Ap, ampicillin; 3Cba, 3chlorobenzoate; Cm, chloramphenicol; H2 and H4, Hind111 fragments of pBRC60; Kin, kanamycin; RK2 ori, mob, and tra, origin, mobiliza- tion, and transfer genes, respectively, required for replication and transfer of RK2 derivatives; Tc, tetracycline; Trp, tryptophan.
as indicating gene mobility, although no specific loci involved in transposition or recombination have been characterized.
The 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) catabolic gene cluster chq is flanked by copies of an insertion sequence, IS93 1, in Pseudomonas cepacia ACl 100 (Tomasek et al. 1989). Although transposition of the chq gene cluster has not been demonstrated, the authors speculate that this represents a composite transposon that was selected during the evolution of this strain in continuous culture.
Recently, two groups have reported on transposable elements specifying chloroaromatic catabolic pathways. An Alcaligenes sp. strain BR60 from a chemical landfill in New York State was found to carry a composite transposon, Tn5271, required for the metabolism of 3- and 4-chlorobenzoates (Wyndham and Straus 1988; Wyndham et al. 1988; Nakatsu et al. 1991). Tn5271 is flanked by copies of a unique class I1 (Tn3 family) insertion sequence, IS1071. In another report, a Pseudomonas sp. strain P51 capable of growth on 1,2,4-trichlorobenzene was found to carry chlorobenzene dioxygenase genes within a class I composite transposon, Tn5280 (van der Meer et al. 1991).
Tn5271 was found on the conjugative IncPl plasmid pBRC60 (Wyndham et al. 1988; Burlage et al. 1990). The plasmid is unstable, undergoing deletion of a 14-kb region of DNA at a high frequency. This deletion event and recom- bination of the same region of DNA into the chromosome of this strain were associated with the IS1071 elements flank- ing the catabolic genes. IS1071 is 3.2 kb in length and con-
tains a single 2.91-kb open reading frame for a transposase (TnpA) protein, but lacks a resolvase- or integrase-like func- tion. We predicted that this element should undergo replicative transposition to form co-integrates, but that these transposition products should be stable in a recA host. This was confirmed using a cloned IS1071 element in Escherichia coli HBlOl (Nakatsu et al. 1991). We have yet to determine the nature of transposition and recombination events of Tn5271 or IS1071 in the natural host. In this paper we pre- sent evidence that IS1071 serves as a portable cassette for homologous recombination of Tn5271 between plasmid and chromosomal loci, and of pBRC60 itself with the chromo- some of Alcaligenes sp. BR60. We discuss the evidence that a majority of cells in a BR60 population contain at least one copy of pBRC60 integrated into the chromosome, and that this recombinational equilibrium may be influenced by both cytoplasmic and environmental factors.
Materials and methods Organisms and growth conditions
Alcaligenes sp. isolates and E. coli strains used in this investiga- tion are listed in Table 1. Selective growth conditions for Alcaligenes sp. strains BR60, BR40, BR605, BR6053, and BR6020 were described previously (Wyndham et al. 1988). Alcaligenes sp. BR6024 is a chloramphenicol-resistant, tryptophan-requiring auxotroph isolated by selection for spontaneous resistance to 80 pg chloramphenicol . mL - ', followed by mutagenesis with N-methyl- Nf-nitro-N-nitrosoguanidine (Sigma Chemical Co.; 200 pg -mL -') and counterselection according to the method of Carhart and Hegman (1975). This auxotroph was maintained on minimal medium A (Wyndham 1986), 12 mM succinate, 0.1 mM
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FIG. 1. Restriction enzyme maps of (a) the 3-chlorobenzoate catabolic element Tn5271 on plasmid pBRC60, and ( b ) insertion sequence IS1071. Restriction enzyme abbreviations: Ba, BamHI; Bg, BgnI; E, EcoRI; H, HindIII; Na, NarI; Nr, NruI; Ps, PstI; S, San. Versions of the Tn5271 map have appeared elsewhere (Wyndham et al. 1988; Nakatsu et al. 1991).
tryptophan, and 80 pg . mL - ' chloramphenicol (S-Trp-Cm) agar. The minimum inhibitory concentration of kanamycin sulfate (Sigma) for Alcaligenes sp. BR60 was determined to be 2.5 pg.mL-' for growth in liquid medium A, 12 mM succinate. BR60::TnS mutants were selected immediately following suicide donor mating on medium A, 12 mM succinate, 10 pg . mL -' kanamycin (S-Km) agar. The pool of Km' derivatives was then transferred to minimal medium A, 4 mM 3-chlorobenzoate (Aldrich Chemical Co.), 10 p g . m ~ - ' kanamycin (Cba-Km) broth to select for strains that maintained Tn5271. The pooled 3Cba + , Kmr, Tn5 recipients were then transferred to 1.0% tryp- tone (Difco), 0.5% yeast extract (Difco), 0.5% NaCl (TYE) broth, supplemented with 10 pg.mL-' kanamycin (TYE-Km), and transferred in TYE-Km broth following overnight growth a total of 7 times. The pool was then plated onto TYE-Km agar and Cba- Km agar. Selected isolates from the latter medium, including clones BR60-1, -3, -4, and -5, were maintained on Cba-Km agar.
Alcaligenes sp. BR6024 recipients of plasmid pGS65 were isolated on S-Trp-Cm agar supplemented with 10 pg.mL-' tetracycline (Sigma) (S-Trp-Cm-Tc). Transconjugants were subsequently maintained on S-Trp-Cm-Tc and were replica plated onto 4 mM 3-chlorobenzoate, 0.1 mM tryptophan, 80 pg . mL - ' chloram- phenicol (Cba-Trp-Cm) agar and Cba-Trp-Cm agar supplemented with 10 pg . mL - ' Tc (Cba-Trp-Cm-Tc). Solidified media con- tained 1.8% agar (Difco).
Mating conditions A standard overnight filter mating procedure on
0.2 pm x 22 mm cellulose acetate filters (Millipore Corp.), overly- ing TYE agar and incubated at 32OC, was used for all conjugative transfers. Donors and recipients (and helper plasmid pRK2013 donor in the case of triparental matings) were grown on appro- priate selective media to midexponential phase immediately prior to mating. Dilutions of filter-mating mixtures were carried out in minimal medium A (Wyndham 1986) and plated on selective media for counting recipients and transconjugants. Mating frequencies were expressed as transconjugants per potential recipient.
indicated above. Plasmid DNA was isolated from Alcaligenes isolates using either a large plasmid extraction method of Crosa and Falkow (1981), as modified by Wyndham et al. (1988), or a standard alkaline lysis method (Birnboim and Doly 1979). Total genomic DNA extraction of Alcaligenes strains was based on a version of the Wheatcroft and Watson (1988) method. Restriction enzyme digestion with EcoRI and BgnI (Brl, Bethesda, Md.) and HindIII (Pharmacia, Uppsala, Sweden), and agarose gel elec- trophoresis, were carried out as described (Sambrook et al. 1989). DNA fragment sizes in kilobases, based on electrophoretic mobil- ity relative to HindIII-digested A-phage DNA, were determined with an IBM-compatible program, DNAFRAG (Schaffer and Sederoff 1981).
DNA probes were prepared for the Tn5271 element from the internal 8.8-kb HindIII fragment H2, for the IS1071 element from the internal 1.1-kb HindIII fragment H4, and for Tn5 from the internal 3.2-kb HindIII fragment of this transposon, following HindIII digestion of pBRH2, pBRH4, and pJHN11, respectively, and electrophoresis on low melting temperature agarose (Seaplaque, FMC Corp.). For radioactive probes, the fragment of interest was excised and radiolabeled with 3 2 ~ - d ~ ~ ~ (DuPont Radio- chemicals) using the random primer - Klenow polymerase method (Feinberg and Vogelstein 1984). Separation of labeled probe from unincorporated 3 2 ~ - d ~ ~ ~ was done by Sephadex G-50 (Pharmacia) column chromatography, to give a probe with a spe- cific activity of 5 x 10' cpm.pg DNA-' (1 cpm = 0.0167 Bq).
For nonradioactive probing, the 1.1-kb HindIII fragment H4 was labeled using digoxigenin-11-dUTP via the random primer labeling technique of Boehringer Mannheim, following the manufacturer's instructions.
Transfer of DNA from agarose gels to nylon membranes (Hybond, Amersharn) and hybridization with radiolabeled probes were carried out as described (Sambrook et al. 1989). Hybridiza- tion of digoxigenin-labeled probe, antibody - alkaline phosphatase conjugate binding, and development of the phosphatase colour reaction were carried out as recommended by the supplier (Boehringer Mannheim).
DNA isolation and characterization Plasmid and total genomic DNA isolations were made from Results and discussion
Alcali~enes strains grown on TYE liauid medium at 2S°C and SUD- Tn5 mutanenesis and Tn5271 chromosomal recombination plemeited with theappropriate anGbiotics at the concentratioLs We have localized the 3-chlorobenzoate catabolic genes
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to the 10.5-kb unique region of DNA flanked by IS1071 elements in Fig. 1 (Wyndham et al. 1988; Nakatsu et al. 1991; C. Nakatsu, in preparation). The frequency of loss of these genes by recombination and deletion of the DNA between the IS1071 elements is 1.6 x l o v 3 .cell - ' .gener- ation-' (Nakatsu et al. 1991). The original intent of the Tn5 mutagenesis experiments was to place a selectable antibiotic resistance marker into Tn5271 in preparation for functional mapping of the catabolic region specifying 3-chlorobenzoate degradation. This marker would allow us to select catabolic gene mutants in a stable Tn5271 background.
The frequency of Tn5 transposition in BR60 following introduction of the suicide transposon donor pGS9 was 3.8 x 10 - 5 per recipient. Plasmid DNA isolated from 100 Tn5 mutants selected on Cba-Km agar directly after pGS9 mating revealed the presence of plasmid pBRC60. The plasmid was unchanged, as demonstrated by restriction digestion and by IS1071 fragment H4 probing of BgAI digests of total genomic DNA (data not shown). Mating out of pBRC60 to a plasmid-free recipient, BR6024 (Cmr, Trp -), while selecting for Kmr, resulted in the isolation of many pBRC60: :Tn5 transposition derivatives, but all of the insertions were localized to regions of pBRC60 outside Tn5271 (data not shown).
To select for rare transposition events into Tn5271 in nonessential regions of the transposon, we introduced a selection step applied to the pool of 3Cba' Tn5 recipients that involved seven transfers in TYE-Km broth. We reasoned that Tn5271 instability would result in the loss of this element from cells not being selected for their ability to degrade 3Cba. Eventually, only those cells with Tn5 transposed into Tn5271 would maintain the 3Cba' phenotype. Results showed that following these transfers, only 0.114% (23 out of 20 200) of Kmr colonies remained 3Cba'. Unexpectedly, and unlike the original Tn5-mutagenized BR60 pool, these contained no detectable plasmid when subjected to large plasmid isolation methods in parallel with BR60 (data not presented). To test for linkage between the Kmr and 3Cba' markers in these strains, 12 clones were again carried through a series of seven transfers in TYE-Km broth, followed by plating on TYE agar. The result of replica plating 500 colonies from each of the 12 strains on TYE agar, TYE-Km agar, and Cba agar was that the Kmr marker was present in 100% of colonies, while the 3Cba' marker was present in an average of 87.7% of colonies for all pools, corresponding to a fre- quency of deletion of Tn5271 of 1.1 x 10 - 3 . cell - ' . gene- ration-'. This frequency of loss was not significantly dif- ferent from that in the parent, Alcaligenes sp. BR60.
These results implied that Tn5 and Tn527 1 were unlinked in the Tn5 mutagenized strains, and that both transposons were located in the chromosome of BR60. To confirm that Tn5 and Tn5271 were present in the mutagenized strains, total genomic DNA was isolated from Km', 3Cba' clones, designated BR60- 1, -3, -4, and -5, as well as from the parent BR60 and spontaneous Tn5271 deletion derivatives. Total genomic DNAs of BR60, BR60-4, and BR60-5, digested with EcoRI or HindIII and probed with the internal 3.2-kb HindIII fragment of Tn5, are shown in Fig. 2. The 3.2-kb Tn5 diagnostic fragment is present in the HindIII digests of the Kmr clones. The EcoRI digests demonstrate that Tn5 has integrated at different locations in the genomes of these
FIG. 2. Hybridization of a Tn5 internal 3.2-kb HindIII frag- ment probe to total genomic DNA of lanes 1 and 2. BR60 (KrnS); lanes 3 and 4, BR60-4 (Km'); lanes 5 and 6, BR60-5 (Km'). All strains were recovered from Tn5 mutagenesis experiments. Total genomic DNAs of each strain were digested with EcoRI (E) or HindIII (H) and loaded at equivalent concentrations per lane. Frag- ment sizes in kilobases relative to a A-Hind111 marker (not shown) are indicated.
two clones. Some of the weaker hybridization bands in these digests may be due to partial genomic digestion or to sec- ondary transposition events involving Tn5 or IS50. Similarly variable fragment sizes (other than the 3.2-kb internal HindIII fragment) were observed in hybridizations to other BR60 clones carrying Tn5. There was no hybridization to total genomic DNA fragments from a BR60 KmS clone reisolated from the Tn5 mutagenesis experiment (Fig. 2, lanes 1 and 2). These results show that Kmr in the mutagenized strains correlates with the physical presence of Tn5.
The loss of plasmid pBRC60 from the Tn5 mutants, while they retained the 3Cba' phenotype, implied that the cba genes, or a fragment of the plasmid carrying these genes, had recombined into the chromosome of these mutants. EcoRI and HindIII digests of total genomic DNA of each of four Tn5-labeled derivatives BR60-1, -3, -4, and -5, as well as BR60, BR40, and a chromosomal cba gene recom- binant, BR6053, isolated previously (Wyndham et al. 1988) were probed with the 8.8-kb H2 fragment of Tn5271 (data not shown). Hybridization patterns for the Tn5 mutants and BR6053 were identical, indicating that the Tn5 mutants carried Tn5271 in their chromosomes. BR60 and BR40 hybridizations showed the additional large HindIII fragment (HI) diagnostic for the pBRC60 and pBRC40 plasmids in these strains.
The isolation of this set of Tn5271 chromosomal inser- tion mutants raised the question of whether chromosomal recombination of Tn5271 was a localized or random event.
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FIG. 3. Hybridization of IS1071 internal 1.1-kb Hind111 fragment H4 to total genomic DNA of BR60 and derivatives. DNA of each strain was digested with BglII and resolved by electrophoresis. Gels (not shown) were blotted and hybridized as described in Materials and methods. Fragment sizes indicated on the left are relative to X-Hind111 digest markers marked on the right. Strains BR60, 6053, and 6020 (Wyndham et al. 1988) are shown for reference. BR60-1, -3, -4, and -5 (lanes 4-7) are Tn5-mutagenized strains. Strains BR6024-A1/2, -A1/21, and -A2/7 (lanes 8-10) are recipients of Tn5271 mobilized from Alcaligenes sp. BR605 by plasmid R68.
Total genomic digests of Tn5 mutants BR60-1, -3, -4, and -5 were made using BgnI, which does not cut within the IS1071 sequence (Fig. 1). These digests were resolved and probed with an internal fragment of IS1071 (H4) to deter- mine the number of copies of this insertion sequence in the genome, and to establish whether different chromosomal loci were involved in recombination in each case (Fig. 3). Total genomic DNAs of reference strains were included in this probing experiment. The parent strain BR60 contained seven BgnI fragments carrying copies of IS1071. Two of these, on BgnI fragments of 24.8 and 11.6 kb (Fig. 3, lane l), contained the two IS1071 copies carried on pBRC60. The remaining five IS1071 copies were chromosomal. The cba gene chromosomal recombinant BR6053 (Wyndham et al. 1988) also carried seven copies of IS1071, all of them associated with the chromosome. The two pBRC60 diag- nostic fragments of 11.6 and 24.8 kb were missing from the genomic digest and at least four new IS1071-containing frag- ment sizes were created during recombination (Fig. 3). The 3Cba-, cured strain BR6020 (Wyndham et al. 1988) retained three IS1071 copies. Tn5 mutants BR60-1, -3, -4, and -5 exhibited a unique pattern of IS1071 distribution in the chromosome. There were four IS1071-containing BgAI fragment sizes in these Tn5-mutants, except for BR60-4, which lacked the 5-kb fragment. The novel 8- and 14-kb IS1071-containing fragments (the middle two fragments in lanes 4-7 of Fig. 3) were not present in the parent strain BR60, although the 14-kb fragment corresponds in size to a BR6053 copy of IS1071.
Thus, Tn5 mutagenesis in Alcaligenes sp. BR60 failed to yield an insertion of Tn5 in the internal region of the com- posite transposon Tn5271, but resulted in the loss of
pBRC60 and the recovery of chromosomal recombinants of the cba genes associated with Tn5271 in a portion of the Tn5 recipients. These chromosomal Tn5271 strains were apparently more stable with regard to cba gene maintenance, because they retained these genes following prolonged sub- culturing in the absence of selection. These events were inde- pendent of the location of Tn5 insertion in the genome of BR60. The appearance of novel arrangements of IS1071 in the chromosomes of the Tn5 mutants suggests that IS1071 was involved in the recombination of Tn5271 into the chromosome.
Tn5 mutagenesis has been used effectively in the genetic analysis of many Gram-negative species because it transposes at a high frequency and is relatively stable once established. It can insert into many sites despite occasional hotspots (Berg and Berg 1983; de Bruijn and Lupski 1984; Berg 1989; Lodge et al. 1989). However, in experiments with the hydrogen autotroph Alcaligenes eutrophus HI, attempts to isolate Tn5 mutants of the hydrogen oxidation (hox) genes on the indigenous plasmid pAEl resulted in an unusually high fre- quency of plasrnid curing, or deletion of a 50-kb pAEl DNA segment (Chow etal . 1989). All Tn5 mutants of A. eutrophus H1 exhibiting plasmid deletions had Tn5 inserted at different locations in the genome, distantly located from the plasmid deletion region (Chow et al. 1989).
Similarly, A. eutrophus H16 has been mutagenized with Tn5, resulting in a high percentage of nonrevertible mutants that had lost the autotrophic growth phenotype (Srivastava et al. 1982). These authors did not check for plasmid curing or deletion of hox genes. Alcaligenes eutrophus H16 har- bours the 450-kb plasmid pHGl that carries the hox gene cluster flanked by two structural gene complexes, hox S and
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FIG. 4. Hybridization of IS1071 internal 1.1-kb Hind111 fragment H4 (digoxigenin-labeled) to total genomic DNA of BR6024-derivative strains 12C (3Cba+), 225C (3Cba+), 2253 (3Cba-), and 25C (3Cba+), with the plasmid-cured BR60-derived strain 6020 (3Cba-) shown for comparison. DNA of each strain was digested wtih BgnI and resolved by gel electrophoresis. The gel (not shown) was blotted and hybridized with the IS1071 probe. Fragment sizes in kilobases on the left are relative to X-Hind111 marker frag- ments indicated on the right.
hox P, which encode two hydrogenases (Friedrich et al. 1990). Spontaneous deletions and insertions have occurred in the hox gene cluster and these events have been associated with transposition of the 1.3-kb insertion element IS491, two copies of which have been mapped in the hox gene cluster of pHGl (Friedrich et al. 1990).
The observed events in Alcaligenes sp. BR60 and the autotrophic Alcaligenes species support the hypothesis of Chow et al. (1989), that Tn5 is not directly involved in pro- moting deletion and recombination events. Rather, they pro- pose that transposition and (or) mutagenesis result in increases in homologous recombination activities in these cells (Chow et al. 1989). Other evidence supporting this hypothesis may be found in experiments on Tn5 mutagenesis in Pseudomonasputida RE204 (pRE4) (Eaton and Timmis 1986) and Rhizobium meliloti (Ruvkun et al. 1982), and in experiments on the induction of SOS DNA repair in cells undergoing TnlO transposition (Roberts and Kleckner 1988). Others have found that single transposition events seem to increase the frequencies of transposition of different mobile genetic elements in the same cell line (Read and Jaskunas 1980). Mutation frequencies, including transposition, may be influenced by the physiological status of cells (Cairns et al. 1988; Shapiro and Higgins 1989). Recent studies on the transposition of Tn5 derivatives in E. coli have shown that the time cells spend under quiescent conditions influences the frequency of appearance of chromosomal transposon insertions (Guzzo and DuBow 1991). Similar events may have occurred during the subculturing of Tn5 mutants of BR60 to yield chromosomal IS1071 rearrange- ments and Tn5271 recombinants.
Multiple copies of indigenous insertion sequences such as IS491 and IS1071 will act as substrates for homologous recombination. If these indigenous elements are at the same time transposing to new locations within the genome, the
possibilities for complex rearrangements of DNA are considerable.
Mobilization of Tn5271 from the chromosome To determine whether Tn5271 was intact within the chro-
mosome of the recombinants we had selected, experiments were carried out to mobilize the element using conjugative plasmids. Initial experiments involved triparental matings between Alcaligenes sp. BR40 (pBRC40, the mobilizing plasmid), the Tn5271 chromosomal recombinants BR6053 or BR60-1, -3, -4, and -5, and the recipient BR6024 (Cmr, Trp-). In each case plasmid pBRC40 mobilized Tn5271 into the recipient, at an average frequency of 1.6 x l o p 8 per recipient. The final product of mobilization was iden- tical in restriction digest pattern to pBRC60 (data not shown), indicating that mobilization was due to homolo- gous recombination between IS1071 elements and not to transposition.
Plasmid R68 was then used as the mobilizing vector in matings between Alcaligenes sp. BR605 (chromosomal Tn5271; R68, the mobilizing plasmid) and BR6024 (Cmr, Trp-) . Mobilization occurred at a frequency of 3.0 x per recipient. Plasmid screening and restriction digestion indicated that each recipient contained R68 in its native form, and that following mobilization Tn5271 had become integrated into the chromosome of the recipient. Total genomic DNA was isolated from three of these recip- ients, BR6024-A1/2, -A1/21, and -A2/7, digested with BglII, blotted, and probed with the IS1071 probe H4. The results are shown in Fig. 3, lanes 8-10. Allowing for dif- ferences in the loading of DNA in the lanes, the sizes of ISlO7l-containing BglII fragments were identical to those in the chromosomal Tn527 1 recombinant BR6053.
These results demonstrated that Tn5271 could be mobilized from the chromosome by conjugative plasmids.
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FIG. 5. Recombinational equilibrium in Alcaligenes sp. BR60. Circles represent plasmids and the straight line represents the chro- mosome. Boxes represent plasmid or chromosomal copies of IS1071 showing recombination at unknown points within the elements. Although only one chromosomal copy of IS1071 is shown in A, experimental evidence indicates there may be from three to nine copies that can act as substrates for recombination in this host.
The mechanism in the case of pBRC40 probably involved homologous recombination between IS1071 elements in the plasmid and chromosome. The mechanism in the case of R68 is unknown, but may have involved homologous recom- bination or transposition, since this plasmid contains both class I1 transposon sequences homologous to IS1071, and also the insertion sequence IS21.
Plasmid pBRC60 recombination following incompatible matings
Experiments designed to perturb the indigenous plasmid pBRC60 of Alcaligenes sp. BR60 were carried out to test the hypotheses that Tn5 is not directly involved in the selec- tion of deletion or recombination derivatives. Alcaligenes sp. BR6024 (Cmr, Trp -, carrying pBRC60) was used as a recipient for the introduction of an incompatible plasmid that lacks transposition functions. The broad host range plasmid pGS65 (Selvaraj and Iyer 1985) is derived from RK2, an IncPl group plasmid. It carries only essential sequences for replication (ori and trf), mobilization (mob), and nontransposing resistance determinants. Plasmid pBRC60 has recently been shown to be related, although distantly, to the IncPl plasmids (Burlage et al. 1990), and we have presented evidence that it is incompatible with IncP 1 -group plasmids in Alcaligenes (Wyndharn et al. 1988). Mobilization of pGS65 with the helper plasmid pRK2013 from E. coli BW97 into Alcaligenes sp. BR6024 (pBRC60) occurred at a frequency of 7.8 x l o p 5 per recipient. This is three orders of magnitude lower than the 6.9 x per recipient frequency of mobilization into the plasmid-free host strain BR6024, presumably because of exclusion owing to incompatibility. Transconjugants were selected on S-Trp-
Cm-Tc agar (no selection for Tn5271), and 100 were purified on the same medium. These transconjugants were screened for plasmid content and were found to carry pGS65 only. The catabolic plasmid pBRC60 could not be detected in these transconjugants, using several large plasmid isolation methods that could readily recover this plasmid from the parent strains BR6024 (pBRC60) or BR60 (pBRC60) (data not shown). The 100 transconjugants were then replica plated onto Cba-Trp-Cm-Tc to select for chlorobenzoate metabolism. Of these, 85% exhibited a 3Cba+ phenotype, although many of them required a period of adaptation to recover full 3Cba metabolic activity. Adaptation was characterized by initial slow growth, the accumulation of intermediates of 3-chlorobenzoate metabolism, and the appearance of fast-growing mutant colonies (data not shown). The remaining 15% were nonrevertible 3Cba- mutants. Total genomic DNA was isolated from 12 BR6024 (pGS65, 3CbaP) and 12 BR6024 (pGS65, 3Cba') transconjugants, digested with BglII, and probed with a digoxigenin-labeled H4 fragment from IS1071. The results in Fig. 4 show a selected number of these probed digests. All 3Cba- transconjugants (for example, clone 2258, Fig. 4, lane 3) carried only three copies of IS1071 on BglII fragment sizes identical to those observed in the plasmid- cured derivative BR6020 (Fig. 4, lane 5). The three 3Cba' derivatives shown in Fig. 4, clones 12C, 225C, and 25C, con- tained between six and nine copies of the IS1071 element, not including weak hybridization signals. Several of these copies were found on novel BglII fragment sizes with respect to each other and the original parent strains BR6024 (data not shown) and BR60 (see Fig. 3, which is the genomic hybridization pattern for the same restriction digest and probe). However, note that for each 3Cba+ transcon- jugant, there were hybridizing fragments corresponding in size to at least one of the two pBRC60 BglII fragments car- rying IS1071 (1 1.6 and 24.8 kb). This result indicated that some part or all of the pBRC60 plasmid was present in these 3Cba + transconjugants. Indeed, when the 85% of pGS65 recipients that retained the 3Cba+ phenotype were removed from Tc selection and subcultured on 3Cba-Trp- Cm then screened for plasmid content, pBRC60 was once again detectable in cell lysates, while pGS65 was not (data not shown). After excision of pBRC60 from the chromo- some the cells were found to be TcS.
These results show that as long as selection for the incom- ing IncPl plasmid pGS65 was applied, this plasmid was replicated in the cytoplasm. Plasmid pBRC60 either was eliminated entirely owing to incompatibility, giving rise to the 15% of transconjugants that were 3CbaW, or was maintained in its entirety integrated into the chromosome. Because selection for the 3Cba + phenotype was not applied during selection of the transconjugants, the results indicate that up to 85% of cells in a population may contain a copy of pBRC60 in the integrated state in addition to the form freely replicating in the cytoplasm. The evidence of Fig. 4 supports the conclusion that integration of pBRC60 into the chromosome involved recombination between IS1071 copies in the plasmid and chromosome, resulting in the variation in digest pattern observed. When selection was removed for Tcr and the cells were shifted to chlorobenzoate metabolism, pBRC60 was excised from the chromosome and replicated in the cytoplasm, while pGS65 was eliminated owing to incompatibility.
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Conclusions A simple model for recombinational equilibrium in
Alcaligenes sp. BR60 is shown in Fig. 5. The model pro- poses that IS1071 serves as a mobile cassette for homolo- gous recombination within the genome of BR60, and that plasmid pBRC60 exists in at least three configurations in any given population of cells. The relative frequencies within a population of each plasmid arrangement will depend upon both cytoplasmic factors and the prevailing environmental conditions. For example, under strong selection for chloroaromatic pollutant catabolism in a natural microbial community, pBRC60 replicates in the cytoplasm and is capable of conjugative transfer to a range of Gram-negative recipients (Fulthorpe and Wyndham 1991, 1992). In the absence of selection, the frequency of pBRC40 in the cytoplasm increases, with deletion of Tn5271 occurring from either the plasmid or the chromosome (Figs. 5A, 5B) at a frequency of about 10 - - cell - . generation - ' (Wyndham et al. 1988). The deletion event involves homologous recom- bination between direct repeats of IS1071 (Nakatsu et al. 1991). In this study we have shown that transposon mutagenesis and mating in of an incompatible plasmid can shift the recombinational equilibrium in the host popula- tion toward chromosomal integration of either the transposon alone or the entire pBRC60 plasmid. The fre- quency with which copies of IS1071 appear in new locations in the chromosome suggests that transposition of IS1071 superimposes an additional level of complexity on this equilibrium.
The complete nucleotide sequence of IS 107 1 has recently been reported from our laboratory (Nakatsu et al. 1991). In comparing this element to the Tn3 family of mobile genetic elements, IS1071 is found to be a mobile TnpA transposase gene, lacking other sequences involved in antibiotic resistance, cointegrate resolution (Sherratt 1989), or integrase-like activity (Mahillon and Lereclus 1988). IS1071 carries out only the first step in the classical Tn3-family transposition mechanism, that of cointegrate formation. Cointegrates formed by IS1071 are resolved by the host's endogenous recombination system (Nakatsu et al. 1991). Because IS1071 transposition is replicative, as for other Tn3-family transposons, the tendency will be to increase the number of copies of this element in the genome, thereby increasing the number of potential substrates for homologous recombination. The recombination events we have observed in Alcaligenes sp. BR60 in response to Tn5 mutagenesis and plasmid incompatibility may indeed be a consequence of increased endogenous cellular recombination as proposed by Chow et al. (1989). This increased activity would tend to decrease the number of copies of IS1071 in the genome, at the same time causing deletions of genomic DNA lying between recombining IS1071 elements. Thus, a form of equilibrium may be established that is driven by transposition and plasmid integration on one side and by recombination and deletion on the other.
The nature of the host bacterium will determine to a large extent the nature of the recombinational equilibrium involv- ing IS1071. We have recently shown that in freshwater microbial communities the frequency of Tn5271 increases in response to micromolar concentrations of 3-chlorobenzoate, 4-chloroaniline, and 3-chlorobiphenyl (Fulthorpe and Wyndham 1991, 1992). This increase is largely due to the transfer of pBRC60 into a range of natural
hosts that are better adapted to survival in the aquatic envi- ronment. One set of natural recipients identified as Acidovorax delafieldii, isolated following exposure to chlorobenzoate and chloroaniline, contained only recom- bined fragments of pBRC60 (strain PR63, Fulthorpe and Wyndham 1991) or IS1071 integrated into a plasmid unrelated to pBRC60 (strains T6-11 and T6-13, Fulthorpe and Wyndham 1992). These results can be interpreted in the context of the model proposed here. Some natural recipi- ents will replicate pBRC60 efficiently and will slowly attain recombinational equilibrium with respect to IS1071, as this element transposes into the genome. Others, such as Acidovorax delafieldii, will not maintain pBRC60 without substantial rearrangements, or will maintain the insertion sequence or transposon alone following cointegrate forma- tion and homologous recombination. The recombinational equilibrium with respect to IS1071 in these hosts will be influenced by the level of expression of the TnpA gene, as well as by the activity of the host's recombination systems. We are currently investigating the transpositional activity of IS1071 in its natural host range.
Acknowledgements The authors gratefully acknowledge the support of the
Natural Sciences and Engineering Research Council and the capable assistance of Mrs. Maysoon Salih.
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