APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Jan. 2001, p. 42–50 0099-2240/01/$04.00⫹0 DOI: 10.1128/AEM.67.1.42–50.2001 Copyright © 2001, American Society for Microbiology. All Rights Reserved.
Vol. 67, No. 1
Occurrence of Tn4371-Related Mobile Elements and Sequences in (Chloro)biphenyl-Degrading Bacteria DIRK SPRINGAEL,1* ANNEMIE RYNGAERT,1 CHRISTOPHE MERLIN,2† ARIANE TOUSSAINT,2,3‡ 1,4 AND MAX MERGEAY Environmental Technology, Flemish Institute for Technological Research (Vito),1 and Laboratory Microbiology, Radioactive Waste and Clean-up Division, SCK/CEN,4 Boeretang 200, B-2400 Mol, Belgium; Laboratoire de Microbiologie, Universite´ Joseph Fourier, F38041 Grenoble Cedex 9, France2; and Laboratoire de Ge´ne´tique des Procaryotes, Universite´ Libre de Bruxelles, B-6041 Gosselies, Belgium3 Received 17 April 2000/Accepted 19 September 2000
Tn4371, a 55-kb transposable element involved in the degradation and biphenyl or 4-chlorobiphenyl identified in Ralstonia eutropha A5, displays a modular structure including a phage-like integrase gene (int), a Pseudomonas-like (chloro)biphenyl catabolic gene cluster (bph), and RP4- and Ti-plasmid-like transfer genes (trb) (C. Merlin, D. Springael, and A. Toussaint, Plasmid 41:40–54, 1999). Southern blot hybridization was used to examine the presence of different regions of Tn4371 in a collection of (chloro)biphenyl-degrading bacteria originating from different habitats and belonging to different bacterial genera. Tn4371-related sequences were never detected on endogenous plasmids. Although the gene probes containing only bph sequences hybridized to genomic DNA from most strains tested, a limited selection of strains, all -proteobacteria, displayed hybridization patterns similar to the Tn4371 bph cluster. Homology between Tn4371 and DNA of two of those strains, originating from the same area as strain A5, extended outside the catabolic genes and covered the putative transfer region of Tn4371. On the other hand, none of the (chloro)biphenyl degraders hybridized with the outer left part of Tn4371 containing the int gene. The bph catabolic determinant of the two strains displaying homology to the Tn4371 transfer genes and a third strain isolated from the A5 area could be mobilized to a R. eutropha recipient, after insertion into an endogenous or introduced IncP1 plasmid. The mobilized DNA of those strains included all Tn4371 homologous sequences previously identified in their genome. Our observations show that the bph genes present on Tn4371 are highly conserved between different (chloro)biphenyl-degrading hosts, isolated globally but belonging mainly to the -proteobacteria. On the other hand, Tn4371-related mobile elements carrying bph genes are apparently only found in isolates from the environment that provided the Tn4371-bearing isolate A5. and only in some cases has horizontal gene transfer been demonstrated (48). We have described a chromosomally located transposable element, Tn4371, carrying bph genes in Ralstonia eutropha A5, a strain isolated from PCB-contaminated lake sediment of the Fort Loundoun Reservoir, Knoxville, Tenn. (50, 52). Tn4371 could be transferred to other bacteria by the A5 endogenous plasmid pSS50 and by other related plasmids belonging to the incompatibility P (Pseudomonas group P-1) group of plasmids. Moreover, Tn4371 was shown to easily integrate into the chromosome of the recipient bacteria (40, 52). The bph genes present on Tn4371 are most similar in nucleotide sequence (up to 94%) and gene organization to the corresponding bph genes of Achromobacter georgiopolitanum KKS102 (formerly Pseudomonas sp. strain KKS102) isolated in Japan (39). Recent observations on the structure and transposition characteristics of Tn4371 show that the mobile element displays features of a conjugative transposon (40). The left border of the element encodes a product similar to integrases, while its right border contains genes with significant similarity to the trbIG genes involved in transfer of the antibiotic resistance IncP1 plasmid RP4 and the Agrobacterium tumefaciens Ti plasmid (40). However, transfer of Tn4371 between bacteria by means of conjugative transposition has not been demonstrated. The similarity between the bph gene cluster of strain A5 and strain KKS102 suggests a common ancestor and its horizontal
Polychlorinated biphenyl (PCB) degradation genes (encoding enzymes for the conversion of biphenyl and PCBs into benzoate and chlorobenzoates), generally referred to as bph, have been cloned and analyzed from both gram-negative and gram-positive bacteria (1, 2, 3, 12, 17, 20, 21, 26–31, 35, 36, 39, 43, 47, 53, 54). All of these bacteria have in common the ability to utilize biphenyl (BP) and some monochlorinated biphenyls (CBPs) as the sole carbon source following a meta-cleavage pathway which proceeds in four steps producing (chloro)benzoate and a five-carbon fragment (9, 14). Comparison of bph gene sequences indicate that the bph gene clusters of different bacteria share a common ancestor and are spread by horizontal gene transfer (3, 15, 53, 57). Additional gene reshuffling, gene incorporation and gene exchange might explain further the differences observed in bph gene cluster organization between different isolates. On the other hand, although plasmid-encoded bph genes have been reported (8, 34, 48), they are mostly chromosomally encoded
* Corresponding author. Mailing address: Environmental Technology, Flemish Institute for Technological Research (Vito), Boeretang 200, B-2400 Mol, Belgium. Phone: 32-14-335176. Fax: 32-14-580523. E-mail:
[email protected]. † Present address: Institute of Cell and Molecular Biology, Edinburgh University, Edinburgh EH9 3JR, Scotland, United Kingdom. ‡ Present address: Unite de Conformation des Macromolecules Biologiques, Universite Libre de Bruxelles, B-1050, Brussels, Belgium. 42
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43
TABLE 1. Bacterial strains and plasmids used Characteristicsa
Strain or plasmid
Strains Ralstonia eutropha A5
pSS50, Tn4371 (Bph⫹)
Ralstonia eutropha H850 Alcaligenes faecalis Pi434 Acidovorax sp. strain 1C3
Bph⫹ Bph⫹ Bph⫹
Acidovorax sp. strain 4A4
pSS50, Bph⫹
Burkholderia cepacia LB400 Burkholderia cepacia H201 Burkholderia cepacia P159 Burkholderia sp. strain JB1 Comamonas testosteroni LPS10A
Bph⫹ pH201, Bph⫹ Bph⫹ Bph⫹ Bph⫹
Comamonas testosteroni MPN1 Comamonas testosteroni B356 Comamonas testosteroni H336 Comamonas acidovorans H1130 Comamonas acidovorans M3GY Achromobacter sp. strain LBS1C1
Bph⫹ Bph⫹ Bph⫹ pH1130, Bph⫹ Bph⫹ pSS60 (Fcb⫹), Bph⫹
Achromobacter georgiopolitanum strain KKS102 Unidentified strain 4C1
Bph⫹
Pseudomonas sp. strain CB406 Pseudomonas putida JHR Pseudomonas putida BN10 Pseudomonas sp. strain B4 Pseudomonas fluorescens GS15 Pseudomonas vesicularis KDW3 Pseudomonas vesicularis KDW9 Pseudomonas maltophyla Dan Pseudomonas sp. strain CP15 Pseudomonas sp. strain UCR2 Pseudomonas stutzeri GS1 Xanthobacter sp. strain P129 Arthrobacter sp. strain SK19 Corynebacterium sp. strain P109 Ralstonia eutropha CH34 Ralstonia eutropha AE815 Escherichia coli CM140
pWW100 (Bph⫹) Three plasmids, Bph⫹ pWR10 (Bph⫹), pWR11 (Nah⫹) Bph⫹ pGS15, Bph⫹ Bph⫹ Bph⫹ pDan, Bph⫹ Bph⫹ Bph⫹ pGS1, Bph⫹ Bph⫹ Bph⫹ Bph⫹ pMOL30 (Czc⫹), pMOL28 (Cnr⫹) Rifr RP4 (Tcr Knr Ampr)
Plasmids RP4 RP4::Tn4371 RP4::Tn4372 RP4::Tn4373-1 RP4::Tn4373-2
⫹
Bph
Tcr Tcr Tcr Tcr Tcr
Knr Knr Knr Knr Knr
Ampr Ampr; Ampr; Ampr; Ampr;
Origin
Source or reference
Sediment, Fort Loundoun Lake, Knoxville, Tenn. Dredge spoil, Hudson River, New York, N.Y. Soil, New York, N.Y. Lake water, Fort Loundoun Lake, Knoxville, Tenn. Lake water, Fort Loundoun Lake, Knoxville, Tenn. Soil, New York, N.Y. Soil, New York, N.Y. Industrial sewage, Panama City, Fla. Garden soil, Amsterdam, The Netherlands Lake water, Fort Loundoun Lake, Knoxville, Tenn. Soil, Knoxville, Tenn. Activated sludge, Canada Soil, New York, N.Y. Soil, New York, N.Y. In vivo recombinant strain, United States Lake water, Fort Loundoun Lake, Knoxville, Tenn. Soil near oil refinery, Tokyo, Japan
(50)
Lake water, Fort Loundoun Lake, Knoxville, Tenn. Industrial soil, United Kingdom Rhine river sediments, Germany Rhine river sediments, Germany Seine river sediments, France Soil pinch, Germany Sludge, Hoboken, Belgium Sludge, Hoboken, Belgium Rhine river sediments, Germany River sediments, Italy In vivo recombinant strain, United States Soil Pinch, Germany Industrial sewage, Panama City, Fla. River sediment, Belgium Industrial sewage, Panama City, Fla. Zinc decantation tank, Luik, Belgium Plasmid-free derivative of CH34
(44)
(4) (56) (44) (44) (6) (56) (18) (45) (46) (44) (1) (56) (56) (37) (32, 46) (30)
(34) (16) (42) (11) (51) D. Springael D. Springael W. Reineke (13) (19) (51) (18) (51) (18) (38) (52) (33) (33) (52) This study This study This study
Bph⫹ Bph⫹ Bph⫹ Bph⫹
a Tcr, Kmr, Ampr, and Rifr, resistance to tetracycline, kanamycin, ampicillin, and rifampin, respectively; Bph⫹ and Nah⫹, ability to utilize BP and naphthalene as the sole carbon source, respectively; Fcb⫹, ability to dehalogenate 4CBA; Czc⫹, resistance to cadmium, zinc, and cobalt; Cnr⫹, resistance to cobalt and nickel.
dissimination by elements like Tn4371. We therefore examined whether other (chloro)biphenyl-degrading strains carry similar mobile DNA elements. We hybridized plasmid DNA and genomic DNA of more than 35 different (chloro)biphenyl degraders of different origins and belonging to different bacterial genera with gene probes constructed from Tn4371. These gene probes included probes carrying the bph genes and probes carrying DNA outside the catabolic gene cluster such as the int and trbIG genes. In addition, we examined the strains for conjugal transfer and/or transposition of their catabolic determinants.
MATERIALS AND METHODS Bacterial strains, plasmids, and media. Table 1 lists the strains and plasmids used in this study. Strain 1C3 is identical to strain 4A4 except that 1C3 lacks plasmid pSS50 (44). All strains were grown at 30°C in Luria-Bertani (LB) medium or in the chloride-free minimal medium of Dorn et al. (10) containing BP or 4-chlorobiphenyl (4CBP) crystals, 2 mM 4-chlorobenzoate (4CBA), or 0.2% (wt/vol) gluconate. Escherichia coli strains containing recombinant plasmids or RP4-derived plasmids were grown at 37°C in LB medium (49) supplied with the appropriate antibiotic (tetracycline, 20 g/ml; kanamycin, 50 g/ml; ampicillin, 50 g/ml). Gene probes and DNA-DNA hybridization. The different gene probes used in this study and their locations on Tn4371 are depicted in Fig. 1 and are described in Table 2. All gene probes except for gene probe C were cloned from
44
SPRINGAEL ET AL.
APPL. ENVIRON. MICROBIOL.
FIG. 1. Location and specification of fragments of Tn4371 used as gene probes in this study. (A) Restriction map for the enzymes PstI (P), EcoRI (E), and SalI (S) of Tn4371 in RP4::Tn4371 and location of the bph region. (B) Location of the used gene probes on Tn4371 with the indicated cloning sites (P, PstI; S, SalI; B, BamHI; Bg, BglII; Xb, XbaI; Sp, SphI). (C) Enlarged map of the bph regions of Tn4371 and A. georgiopolitanum KKS102 indicating restriction sites PstI (P), SalI (S), EcoRI (E), KpnI (K), XhoI (X), and SmaI (Sm). Restriction sites with an asterisks indicate sites in the KKS102 bph region which are not present in the Tn4371 bph region. The restriction map of the KKS102 bph operon was deduced from its published nucleotide sequence and restriction map (26, 27, 30).
RP4::Tn4371 in pBluescript SKII(⫹). Gene probes C, D and E cover only sequences involved in BP and 4CBP catabolism, i.e., bphR, bphCD, and bphEGForf4bphBA1A2A3B, respectively (39). Gene probe C, cloned in pUC18, contains bphR of A. georgiopolitanum KKS102. Sequencing data demonstrated that Tn4371 carries a similar gene (90% amino acid identity) (41). Gene probe B contains bphA4 and bphR. Gene probe F contains bphS, an open reading frame transcribed in the opposite direction upstream of the structural bph genes (41). Both probes B and F contain additional DNA with unknown function, as shown by DNA sequencing data (D. Springael, unpublished results). Gene probes A and H include the Tn4371 extremities and are believed to be involved in transposition of the element (40). Gene probe H contains the int gene of Tn4371, whereas gene probe A contains the trbIG genes. In most cases, the recombinant pBluescript SKII(⫹) or pUC18 plasmids containing the probes were digested with the appropriate restriction endonucleases and, after gel electrophoresis, the DNA fragments used as probes were recovered from the agarose using a GeneClean kit (La Jolla, Calif.). Alternatively, complete plasmid DNA was used as a probe. Plasmid DNA and PstI-digested genomic DNA were transferred from the agarose gel to a nylon Hybond N⫹ membrane (Amersham International, Buckinghamshire, England) by Southern blotting. Labeling of the gene probes and
detection of hybridization signals were performed using the fluorescein gene image labeling and detection kits of Amersham International. Hybridization was performed at 60°C under high-stringency conditions (leading to detection of ca. 75% identity) according to the manufacturers. After hybridization, the filters were washed twice at 65°C in 0.1⫻ SSC (1⫻ SSC is 0.15 M NaCl plus 0.015 M sodium citrate) containing 0.1% sodium dodecyl sulfate for 30 min. Other molecular biology techniques. Crude preparations of plasmid DNA were obtained as described by Kado and Liu (24). Plasmid DNA for restriction analysis from E. coli was obtained as described by Ish-Horowicz and Burke (23). Total genomic DNA was prepared by a standard small-scale procedure described by Ho ¨fte et al. (22). Restriction endonuclease analysis (Bethesda Research Laboratories) was performed according to the recommendations of the manufacturer. Bacterial matings. Donor and recipients cells were grown at 30°C in nutrient broth. Plate matings occurred as described by Lejeune et al. (33). Transconjugants were seected as described in Results. Chemicals. BP and 4CBA were purchased from Janssen Chimica, Beerse, Belgium, and 4CBP was obtained from Ventron, Karlsruhe, Germany.
TABLE 2. Gene probes used Probe
Size (bp)
Origin
Recombinant plasmid
Contenta
A B C D E F G H
2,350 10,500 1,000 1,400 6,000 6,800 4,300 7,100
BglII-PstI fragment of Tn4371 PstI-PstI fragment of Tn4371 BamHI-SphI fragment of KKS102 PstI-XbaI fragment of Tn4371 SalI-SalI fragment of Tn4371 PstI-PstI fragment of Tn4371 PstI-PstI fragment of Tn4371 PstI-PstI fragment of Tn4371
pECG322 pECG317 pKH211 pMOL993 pECG333 pECG328 pECG316 pECG312
Right extremity of Tn4371, trbIG genes bphA4, bphR, plus unknown genes BphR bphCD⬘ bphEGForf4bphA1A2B⬘ bphS plus unknown genes Unknown genes Left extremity of Tn4371, int gene
a
A prime symbol indicates truncated genes.
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Tn4371 SEQUENCES IN (CHLORO)BIPHENYL DEGRADERS
RESULTS DNA-DNA similarity between plasmid DNA of (chloro)biphenyl-degrading isolates and Tn4371-derived gene probes. Not yet described plasmids were detected in strains H1130, H201, GS1, GS15, Dan, M3GY, and UCR2 (Table 3). Most of the plasmids were ca. 60 kb. Strains A5, 4A4, and LBS1C1 displayed the expected plasmid sizes of pSS50, pSS50, and pSS60, respectively. None of the visible plasmids hybridized with any of the Tn4371 gene probes tested (data not shown). DNA-DNA similarity between genomic DNAs of (chloro)biphenyl-degrading isolates and Tn4371-derived gene probes. The genomic DNAs of the examined PCB-degrading isolates were digested with PstI and hybridized with the Tn4371 gene probes. The results are compiled and presented in Table 3. A limited selection of organisms displayed strong hybridization with the gene probes containing only bph-related sequences, i.e., probes C, D, and E and could be classified into two main groups according to the obtained pattern of hybridization (Fig. 2). Group I strains A5, 1C3, and 4A4 hybridized with probes C, D, and E with hybridization patterns identical to those of Tn4371. Group II strains displayed for probes E and D hybridization patterns identical to the patterns obtained for A. georgiopolitanum KKS102. Group II included strains LPS10A, LBS1C1, KKS102, Pi434, H336, and H201. Most of these strains, except strain KKS102, displayed the same hybridization pattern for probe C. Strain MPN1 showed strong hybridization with probes D and E but did not hybridize with probe C. The hybridization pattern for probe E showed some similarity with the pattern obtained for the group II strains (Fig. 2). Other strains like B356, H1130 and JB1 hybridized moderately strongly with the catabolic gene probes D and E but displayed a different signal pattern than group I and II strains and did not hybridize with probe C (Fig. 2 and data not shown). Another set of PCB degraders, i.e., strains CB406, LB400, H850, GS1, JHR, KDW3, P153, Dan, B4, CP15, and UCR2, hybridized weakly with the catabolic probes D and E, displayed a completely different hybridization pattern, and did not hybridize with probe C (Fig. 2 and data not shown). Strains KDW9, P129, M3GY, and 4C1 and the gram-positive isolates SK19 and P109 did not hybridize with any of the tested catabolic probes (Fig. 2 and data not shown). Probes B and F containing bphA4-bphR and bphS, respectively, hybridized strongly with all of the above-mentioned group I and group II strains (data not shown). However, probe F did not hybridize with the expected 3.5-kb PstI fragment of KKS102 located at the left side of its bph cluster (Fig. 1) and of other group II strains (except for strain LBS1C1), indicating either that those strains do not contain bphS (hybridization would then be due to noncatabolic DNA present on probe F) or that bphS in those strains is located elsewhere in the chromosome. This observation is in agreement with the published sequence of the bph gene cluster of KKS102, which starts to differ substantially from the Tn4371 bph cluster 50 bp upstream of bphE (26, 27, 30). For both probes B and F, strain 4A4, its pSS50 lacking counterpart 1C3, and the Tn4371-bearing strain A5 showed an identical-sized fragment, suggesting that the similarity between Tn4371 and strains 4A4 and 1C3 extends on both sides of the bph gene cluster. Both probes B and F hybridized with some other fragments in strains 4A4 and 1C3. Probes B and F also hybridized with DNA from strains H1130 and JB1 (data not shown). However, H1130 and
45
JB1 did not hybridize with the bphR gene (probe C) which is included in probe B (data not shown), indicating that for probe B similarity was due to bphA4 or non-bph DNA. Strain M3GY hybridized only with probes B and F, and strain KDW9 only hybridized with probe B (data not shown). Probe A containing the trbIG genes located at the Tn4371 right extremity hybridized strongly with genomic DNA of strains 4A4 and 1C3, confirming the existence of Tn4371-related sequences outside of the bph genes in those strains (Fig. 3). Like strain A5, strains 4A4 and 1C3 displayed two bands hybridizing with probe A, indicating either the presence of two copies of the trbIG genes or the presence of related genes in the genome. On the other hand, no hybridization signal was found with strains 1C3 and 4A4 using probe G and probe H containing Tn4371 sequences located at the left extremity, including the int gene (data not shown). Mobilization of BP-degradative genes. Plasmid RP4 was introduced into a selection of the (chloro)biphenyl-degrading isolates (Table 3). The RP4-containing strains were then examined as donors of the Bph⫹ phenotype in matings with the nickel-resistant R. eutropha-like CH34 as a recipient. Strains 4A4, LBS1CI, H201, and H1130 were also tested for transfer and/or mobilization of the Bph⫹ phenotype by means of their endogenous plasmids. Tris minimal medium containing 2 mM Ni to counterselect the donor strain and BP as sole carbon source was used as the selection medium. Transfer of Bph⫹ was only observed from strains 4A4, 1C3 (RP4), and LBS1C1 at frequencies of 10⫺6 per recipient. Using LBS1C1 and 1C3 (RP4) as donors, all CH34 transconjugants contained enlarged pSS60 and RP4 plasmids, respectively (data not shown). Using 4A4 as a donor strain, the Bph⫹ CH34 transconjugants contained apparently unchanged pSS50 plasmids, suggesting integration of the bph genes into the recipient chromosome (data not shown). Plasmid RP4 was introduced into a CH34 Bph⫹ transconjugant obtained from the matings with LBS1C1 and 4A4 to cure the strains from the received plasmids. Selection was performed on BP Tc minimal medium. Exconjugants from these matings still displayed the Bph⫹ phenotype and contained RP4 instead of the enlarged pSS60 or pSS50, as demonstrated by gel electrophoresis of plasmid extracts (data not shown). They were able to further transfer Bph⫹ by means of the introduced RP4 to the R. eutropha-like strain AE815 (Rifr). In both cases, Bph⫹ Rifr transconjugants were obtained at a frequency of 10⫺6 per recipient and contained enlarged RP4 plasmids with sizes similar to RP4::Tn4371 (data not shown). These data mirror the transposition observations made with Tn4371 (52). Therefore, the mobilized elements from LBS1C1 and 4A4 were tentatively designated Tn4372 and Tn4373, respectively. Comparison of Bphⴙ enlarged RP4 plasmids. The PstI restriction patterns of four independently obtained RP4::Tn4372 plasmids, two RP4::Tn4373 plasmids, and RP4::Tn4371 were compared. The four examined RP4::Tn4372 plasmids exhibited slightly different restriction patterns with calculated sizes of the acquired DNA ranging from 62 to 71.4 kb (Fig. 4A). However, the restriction patterns were significantly different from the pattern obtained for RP4::Tn4371. Moreover, two PstI fragments of RP4 instead of the expected one were lost in the RP4::Tn4372 plasmids. One of them was the PstI fragment known to contain the preferential target site of Tn4371 in RP4 (40, 52). The two examined RP4::Tn4373 plasmids, designated as
pWR10, pWR11
pDan
pGS1 PGS15
pH1130
pH201
pSS60
pSS50
pSS50
⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺
Probing plasmid DNA (probes A to H) ⫹⫹⫹ ⫹⫹⫹ ⫹⫹⫹ ⫺ ⫺ ⫹ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ND ⫺ ⫺
A (right extremity, trbIG) ⫹⫹⫹⫹ ⫹⫹⫹⫹ ⫹⫹⫹⫹ ⫹⫹⫹ ⫹⫹⫹ ⫹⫹⫹ ⫹⫹⫹ ⫹⫹⫹ ⫹⫹⫹ ⫺ ⫹⫹⫹ ⫺ ⫹⫹⫹ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫹⫹⫹ ⫺ ⫹⫹⫹ ⫺ ⫹⫹⫹ ⫺ ⫺ ND ⫹⫹⫹ ⫺
B (bphA4R plus unknown) ⫹⫹⫹⫹ ⫹⫹⫹⫹ ⫹⫹⫹⫹ ⫹⫹⫹ ⫹⫹⫹ ⫹⫹⫹ ⫹⫹⫹ ⫹⫹⫹ ⫹⫹⫹ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺
C (bphR) ⫹⫹⫹⫹ ⫹⫹⫹⫹ ⫹⫹⫹⫹ ⫹⫹⫹ ⫹⫹⫹ ⫹⫹⫹ ⫹⫹⫹ ⫹⫹⫹ ⫹⫹⫹ ⫹ ⫹ ⫹⫹⫹ ⫹⫹ ⫹ ⫹ ⫹ ⫹ ⫹⫹ ⫹ ⫹⫹ ⫹⫹ ⫹ ⫹ ⫹ ⫹⫹ ⫺ ⫹ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺
D (bphCD⬘) ⫹⫹⫹⫹ ⫹⫹⫹⫹ ⫹⫹⫹⫹ ⫹⫹⫹ ⫹⫹⫹ ⫹⫹⫹ ⫹⫹⫹ ⫹⫹⫹ ⫹⫹⫹ ⫹⫹⫹ ⫹⫹⫹ ⫹⫹⫹ ⫹⫹⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹⫹ ⫹⫹ ⫹⫹ ⫹ ⫹⫹ ⫹ ⫺ ⫺ ⫺ ⫺ ⫺ ND ⫺ ⫺
E (bphEGForf4bphA1 A2A3B⬘)
Probing genomic DNAa
⫹⫹⫹⫹ ⫹⫹⫹⫹ ⫹⫹⫹⫹ ⫹⫹⫹ ⫹⫹ ⫹⫹⫹ ⫹⫹ ⫹⫹ ⫹⫹ ⫺ ⫹⫹⫹ ⫺ ⫹⫹ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫹⫹ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫹⫹ ⫺
F (bphS plus unknown) ⫹⫹⫹⫹ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺
G (unknown)
⫹⫹⫹⫹ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺
H (left extrimity, int)
⫹ ⫹* ⫹ ⫺* ⫹ ⫺* ⫺* ⫺* ⫺* ⫺* ⫺ ND ⫺* ⫺* ⫺* ⫺* ND ⫺* ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND
Transfer (Bph⫹)*
SPRINGAEL ET AL.
a ⫹⫹⫹⫹, strong hybridization signal with a pattern identical to that obtained for Tn4371; ⫹⫹⫹, strong hybridization signal with a different pattern compared with Tn4371; ⫹⫹, medium hybridization signal with a different pattern compared with Tn4371; ⫹, weak hybridization signal with a different pattern compared with Tn4371; ⫺, no signal determined; ND, not determined; *, based on transfer via an introduced RP4.
R. eutropha A5 Acidovorax sp. strain 1C3 Acidovorax sp. strain 4A4 C. testosteroni LPS10A Achromobacter sp. strain LBS1C1 A. georgiopolitanum KKS102 A. faecalis Pi434 C. testosteroni H336 B. cepacia H201 C. testosteroni B356 C. acidovorans H1130 C. testosteroni MPN1 Burkholderia sp. strain JB1 Pseudomonas sp. strain CB406 B. cepacia LB400 R. eutropha H850 P. stutzeri GS1 P. fluorescens GS15 P. putida JHR B. cepacia P159 P. maltophyla Dan Pseudomonas sp. strain CP15 P. putida BN10 Pseudomonas sp. strain UCR2 Pseudomonas sp. strain B4 Unidentified strain 4C1 P. vesicularis KDW3 P. vesicularis KDW9 Moraxella sp. strain SK12 Corynebacterium sp. strain P109 Xanthobacter sp. strain P129 C. acidovorans M3GY Arthrobacter sp. strain SK19
Strain
Detected plasmid
TABLE 3. Homology between the tested Tn4371-derived gene probes and DNA of the examined (chloro)biphenyl-degrading bacterial strains and the ability of the tested strains to transfer their bph marker to R. eutropha
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FIG. 3. DNA-DNA hybridization analysis of PstI-digested genomic DNAs of relevant chlorobiphenyl-degrading isolates using Tn4371 gene probe A. For probe A, complete plasmid recombinant DNA probe was used as a probe, leading to hybridization with RP4 DNA due to the presence of the bla (Apr) gene, present on both RP4 and pBluescript SKII(⫹) (the arrows indicate the fragments of RP4::Tn4371 hybridizing with vector DNA).
FIG. 2. DNA-DNA hybridization analysis of PstI-digested genomic DNAs of relevant chlorobiphenyl-degrading isolates using Tn4371 fragments containing only catabolic sequences, i.e., probe C (bphR) (A), probe D (bphCD) (B), or probe E (bphEGForf4bphA1A2A3B) (C) as gene probes.
RP4::Tn4373-1 and RP4::Tn4373-2, demonstrated slightly different restriction profiles (Fig. 4B). Moreover, the majority of the bands were common with Tn4371. For both RP4::Tn4373 plasmids, Tn4373 seems to be inserted in RP4 into the preferential target PstI fragment of Tn4371. The sizes of Tn4373 in both RP4-Tn4373-1 and RP4-Tn4373-2 were calculated to ca. 48 kb, indicating insertion of the same DNA segment at two different positions in the RP4 PstI fragment. PstI-restricted RP4::Tn4371, RP4::Tn4373-1, and RP4::Tn4373-2 and one of the RP4::Tn4372 plasmids were Southern blotted and hybridized with the Tn4371-derived probes (Fig. 5). For most probes (probes B, C, D, E, and F), the fragments of RP4::Tn4372 and both RP4::Tn4373 plasmids which hybridized were identical in size to the fragments which previously hybridized in the genomic DNAs of the parental strains LBS1C1 and 4A4, respectively. This showed that all genomic DNA sequences which hybridized previously with the Tn4371 probes also make part of the mobilized bph bearing segments in strains 4A4 and LBS1C1. Only with probe A was a different hybridization pattern ob-
served for the RP4::Tn4373 plasmids in comparison with the 4A4 genomic DNA (Fig. 5A, lanes 3, 6, and 7, see arrows). Moreover, for both plasmids, a different-sized fragment hybridized. In both cases, this fragment contained a junction with RP4. Furthermore, the fragment of RP4::Tn4373-2 hybridizing with probe A was identical in size to the hybridizing fragment of RP4::Tn4371 (Fig. 5A, lanes 4 and 5). These results indicate that insertion of Tn4373 indeed occurred at two different locations in the same PstI fragment of RP4 and that the Tn4373 and Tn4371 right extremities are identical and can become combined with the same sequences in RP4. Probes B and F were shown in the previous experiments to hybridize with more than one PstI fragment of genomic DNA of strain 4A4, including a fragment with a size similar to the 10.5-kb PstI fragment of Tn4371. Only this 10.5-kb fragment seem to make part of Tn4373 and is thus associated with the transferred bph genes of strain 4A4 (Fig. 5B and F, lanes 4, 6, and 7).
FIG. 4. Comparison of PstI restriction patterns of RP4, RP4::Tn4371, and RP4 plasmids containing Tn4372 (A) and Tn4373 (B) of strains LBS1C1 and 4A4, respectively. RJ and LJ indicate the RP4::Tn4371 fragments containing the right and left junctions, respectively, of Tn4371 with RP4.
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FIG. 5. DNA-DNA hybridization analysis of PstI-digested genomic DNAs of chlorobiphenyl-degrading isolates A5, LBS1C1, and 4A4 and PstI-digested enlarged RP4 plasmids carrying bph using Tn4371 gene probes A, B, C, D, E, and F. For probe A, the single and double arrow(s) indicate the different fragments of RP4::Tn4373-1 and RP4::Tn4373-2 hybridizing with probe A. The double arrow additionally indicates the common-sized fragment of RP4::Tn4371 and RP4::Tn4373-2 hybridizing with probe A. For probes A, B, and E, complete plasmid recombinant DNA was used, leading to hybridization with RP4 DNA due to the presence of the bla (Apr) gene, which is present on both RP4 and pBluescript SKII(⫹).
DISCUSSION In this study, we examined the natural host range of Tn4371 and the occurrence of its modular structure encoding a phage-like integrase, a catabolic bph pathway, and RP4/Ti-like transfer functions in a wide variety of (chloro)biphenyl degraders by means of Southern blot hybridization using different parts of Tn4371 as probes. Our observations show that the bph genes present on Tn4371 are highly conserved between different (chloro)biphenyldegrading hosts, isolated globally. Sequence data previously demonstrated that the strains R. eutropha A5 and A. georgiopolitanum KKS102 contain closely related bph gene clusters (39). Both bph gene clusters have the same gene organization and the corresponding genes show ca. 81 to 94% identity at the nucleotide level. The typical bph cluster hybridization patterns of strains designated above as group I and group II strains show that the Tn4371 and KKS102 bph gene clusters are representatives of two
strongly related families of bph gene clusters with a very similar bph gene organization. Both seem to have been and possibly are currently being spread as genetic segments specifying all of the enzymes necessary for the utilization of BP as a carbon source. A possible difference between the group I and group II bph clusters might be that in group II strains bphS is not present or is at least not linked with the other bph genes. Interestingly, all group I and II bacteria belong to the -proteobacteria. This is reminiscent of the fact that the bph genes of Tn4371 only expressed well in Ralstonia and closely related species (52). Therefore, the KKS102 and A5 bph family gene clusters seem to be developed in and for -proteobacterial hosts. Other strains hybridized less strongly with the bph genes of Tn4371 and showed completely different hybridization patterns. The bph gene clusters of some of those strains (i.e., strains B356 and LB400) have been characterized at the molecular level and indeed show a different gene organization
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and a less-conserved DNA sequence of the bph genes in comparison with the corresponding genes in Tn4371 or KKS102 (1, 12, 20, 21, 43, 53). Interestingly, all group I and group II strains also hybridized with probes B and F. This could be due to the presence on those probes of bphA4-bphR and bphS, respectively. On the other hand, strain 4A4 and its pSS50 lacking derivative 1C3 clearly contain Tn4371 sequences from outside the catabolic bph gene cluster. Indeed, the hybridization patterns using those probes were identical to the patterns obtained for Tn4371 itself. Moreover, strong hybridization was observed for probe A. On the other hand, no hybridization was found with the int part of Tn4371. In strain 4A4 and most probably also in strain 1C3, the DNA fragments which show homology to Tn4371 seem to make themselves part of a mobile element, i.e., Tn4373. We might conclude that Tn4371 and Tn4373 contain identical right sides which have been combined with different left sides. Tn4373 may thus contain a different int gene on the left side. Up to now, no functions were assigned to the DNA flanking the catabolic genes at both sides in Tn4371, but sequence data indicate that it represents noncatabolic DNA (Springael, unpublished). Merlin et al. (39) demonstrated the transfer of the right part of Tn4371 without the left part and suggested that Tn4371 consists of different mobile modules. This part of Tn4371 was called Tn-bph, and its physical map fits well with the part of Tn4371 which showed homology to Tn4373. This would indicate that Tn-bph became integrated in two different larger mobile elements. Such module combinations, which create new mobile elements, may be an efficient alternative for enhancing the dissemination power of a catabolic phenotype. Interestingly, both mobile elements Tn4371 and Tn4373 were identified in (chloro)biphenyl-degrading bacteria originating from the same environment. This might indicate that in these cases the bph genes became integrated into these molecular structures in that environment. Mobilization of the Bph⫹ phenotype was not only observed from strains carrying a group I bph gene cluster but also from strain LBS1C1 carrying a group II bph gene cluster. The mobilized element carrying the bph genes in LBS1C1, i.e., Tn4372, seems to differ substantially from Tn4371 and Tn4373 since it did not display, outside the bph genes, sequence homology to Tn4371. Its mechanism of transposition might be therefore different from the suspected excision/integration mechanism of Tn4371. Molecular characterization of Tn4372 is currently going on. In contrast to Tn4371 and Tn4373, Tn4372 seems not to have moved as a discrete DNA fragment, since inserts in RP4 seem to vary in size. Therefore, Tn4372 cannot be classified as a conventional transposon. Similar observations were described by Thomas et al. (55) for the so-called DEH mobile element which carries the Deh1 dehalogenase gene (deh1) responsible for dehalogenation of alkanoic acids in Pseudomonas putida PP3. Alternatively, as indicated by the fact that more than one PstI fragment of RP4 is lost after receiving the bph genes, these variations in size might be due to rearrangements of the RP4 backbone after insertion rather than to transposition of different-sized DNA fragments. With most of the other (chloro)biphenyl-degrading strains, neither conjugal transfer nor mobilization of the bph genes could be observed, indicating that the catabolic deterimant is not located on mobile elements. However, we used only one shuttle system, which is based on transposition of putative bph transposons into
49
RP4 and transfer and expression in R. eutropha. RP4 may not contain suitable target sequences for transposition of bph genes from other strains such as from bacteria related to strain A5, for example, A. eutrophus H850. Alternatively, as for the bph genes of Tn4371 (52), the expression of bph gene clusters from other strains may be host dependent, and we may not have detected expression in the recipient strain CH34. Lloyd-Jones et al. (34) described transposition-like features of the bph cluster of strain Pseudomonas sp. strain CB406, which recombined with RP4 and was transferred and expressed in P. putida. We could not observe translocation of the CB406 bph genes using our system, and the hypothesis of host-dependent expression may provide an explanation for this. Although Tn4371, Tn4372, and Tn4373 seem to differ substantially, they display interesting common features. They have similar sizes, carry a highly similar bph gene cluster, and coexist in the cell with a similar plasmid, which in laboratory matings can be used as a transport vehicle of the transposon. Two possible explanations come to mind to explain the connection of these mobile elements with pSS50 or pSS50-like plasmids in the environment. First, pSS50 may be an efficient vehicle for the acquisition of the transposons in the reservoir environment and may have been used to deliver the transposon(s) to the various cells. Indeed, although Tn4371 shows a structure related to the conjugative transposons, effective transfer of the entire element without the help of a plasmid has not been shown. Second, pSS50-like plasmids may carry genes encoding the dehalogenation steps for complete mineralization of 4CBP as is found in pSS60 (7, 32). Coexistence in the same cell of both sets of genes may be important for avoiding the transformation of 4CBA to toxic by-products (5). ACKNOWLEDGMENTS We thank V. de Lorenzo, F. Fava, D. D. Focht, A. Layton, E. Kim, F. Mondello, Y. Nagata, J. Parsons, W. Reineke, G. S. Sayler, M. Sylvestre, N. Truffaut, P. Williams, and G. Zylstra for providing PCBdegrading isolates and Y. Nagata for providing the bphR gene probe. We are grateful to A. Layton, J. Packard, C. Wyndham, and two anonymous reviewers for discussion and suggestions. This work was partially supported by the EC program ENVIRONMENT (EVSV-CT92-0192), by the EC Concerted Action MECBAD (BIO4-CT-0039), by Tournesol grants from the Flemish and French governments, and by a grant from the MENESR (UPRES 2023). REFERENCES 1. Ahmad, D., R. Masse´, and M. Sylvestre. 1990. Cloning and expression of genes involved in 4-chlorobiphenyl transformation by Pseudomonas testosteroni: homology to polychlorobiphenyl-degrading genes in other bacteria. Gene 86:53–61. 2. Asturias, J. A., and K. N. Timmis. 1993. Three different 2,3-dihydroxybiphenyl-1,2-dioxygenase genes in the gram-positive polychlorobiphenyl-degrading bacterium Rhodococcus globerulus P6. J. Bacteriol. 175:4631–4640. 3. Asturias, J. A., E. Diaz, and K. N. Timmis. 1995. The evolutionary relationship of biphenyl dioxygenase from gram-positive Rhodococcus globerulus P6 to multicomponent dioxygenases from gram-negative bacteria. Gene 156:11– 18. 4. Bedard, D. L., R. E. Wagner, M. J. Brennan, M. L. Haberl, and J. F. Brown, Jr. 1987. Extensive degradation of Arochlors and environmentally transformed polychlorinated biphenyls by Alcaligenes eutrophus H850. Appl. Environ. Microbiol. 53:1094–1102. 5. Blasco, R., M. Mallavarapu, R. M. Wittich, K. N. Timmis, and D. H. Pieper. 1997. Evidence that formation of protoanemonin from metabolites of 4-chlorobiphenyl degradation negatively affects the survival of 4-chlorobiphenyl cometabolizing microorganisms. Appl. Environ. Microbiol. 63:427–434. 6. Bopp, L. H. 1986. Degradation of highly chlorinated PCBs by Pseudomonas strain LB400. J. Ind. Microbiol. 1:23–29. 7. Burlage, R. S., L. A. Bemis, A. C. Layton, G. S. Sayler, and F. Larimer. 1990. Comparative genetic organization of incompatibility group P degradative plasmids. J. Bacteriol. 172:6818–6825.
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