selection of independent plasmids determining phenol degradation inpseudomonas putida and the...

PLASMID 24,25-36 (1990)

Selection of Independent Plasmids Determining Phenol Degradation in Pseudomonas putida and the Cloning and Expression of Genes Encoding

Phenol Monooxygenase and Catechol 1,2-Dioxygenase

MAIA KIVISAAR, RITA H~RAK, LAGLE KASAK, AIN HEINARU, AND JAAN HABICHT

Laboratov of Plasmid Biology, Estonian Biocenter, 202400 Tartu, Tiihetorn Toomel, Estonian SSR, USSR

Received September 8, 1989; revised February 20, 1990

Long-term cultivation of the Pseudomonas putida multiplasmid strain EST1020 on phenol resulted in the formation of individual PHE plasmids determining phenol degradation. Four types of PHE plasmids, pESTl024, pEST1026, pEST1028, and pESTl029, are characterized. They all contain a transferrable replicon similar to pWWO-8 with a partly duplicated DNA sequence of the 17-kb transposable element of this plasmid and include various amounts of DNA that carry genes encoding phenol degradation (phe genes). We cloned the genes determining phenol monooxygenase and catechol 1,2dioxygenase from the Pseudomonas sp. parent strain plasmid DNA into the broad host range vector pAYC32 and studied the expression of the cloned DNA. The formation of a new hybrid metabolic plasmid, pEST1354, was demonstrated in P. putida Paw85 as the result of transposition of the 17-kb genetic element from the chromosome of Paw85 into the plasmid carrying cloned phe genes. The target site for the 17-kb transposon was localized in the vector DNA, just near the cloning site. In subcloning experiments we found two regions in the I7-kb DNA stretch that are involved in the expression of the cloned phe genes. o 1990 Academic

RN, Inc.

The TOL plasmid pWW0 is the most studied among the degradative plasmids specific for aromatic hydrocarbons and provides a good illustration of recombination events between different DNA regions (Williams et al., 1983). The genetic and molecular organization of the catabolic operons of the TOL plasmid have been characterized in detail (Nakazawa et al., 1986; Timmis et al., 1986; Williams et al., 1983), while knowledge about the other parts of this large 115kb plasmid is limited. All genes encoding the degradation of toluene and related aromatic compounds (xyl genes) have been localized in a 39-kb segment on the pWW0 genome (Williams et al., 1983). The loss of toluene degradative function in pWWO-8 has been correlated with the excision of this 39-kb segment from pWW0 by reciprocal recombination between the 1.4-kb direct repeats (Meulien et al., 198 1). The 56- kb pWW0 region including xyl genes is transposable and so is its 17-kb derivative with the deletion of the internal catabolic 39-kb segment (Cane and Williams, 1982; Jeenes and

Williams, 1982; Tsuda and Iino, 1987) (Fig. 4).

Pseudomonas putida strains Paw85 and Paw340 are plasmid-free strains which are derived from the pWWO-harboring strain Paw 1 (Williams and Murray, 1974; Bayley et al., 1977; Franklin and Williams, 1980). These strains contain the 17-kb transposon of pWW0 in their chromosomes (Meulien and Broda, 1982; Williams et al., 1983). The insertion of the 17-kb transposon from the host chromosome into introduced plasmids in Paw340 has been described by Cane and Wil- liams ( 1982).

Although certain phenotypic characters of soil pseudomonads such as m-toluate, naphthalene, and salicylate degradation are usually plasmid encoded (Boronin et al., 1980; Cane and Williams, 1982; Connors and Bamsley, 1982; Duggleby et al., 1977; Dunn and Gun- salus, 1973; Williams and Murray, 1974; Wil- liams and Worsey, 1976), it has been generally assumed that phenol degradation in bacteria is chromosomally encoded. We found that the

25 0147-619X/90 $3.00 Copyright 0 1990 by Academic Press, Inc. All tights of reproduction in any form reserved.

26 KIVISAAR ET AL.

TABLE 1

BACTERIALSTRAINSANDPLASMIDS

Strain designation Characteristics’ Plasmid Reference

P. putida Paw1 Paw8 Paw85 Paw340 EST3036

E. coli DHl

C600

mTol+Phe- mTol-Phe- mTol-Phe- mTol-Phe-TrpSm’ mTol-Phe-Cb’(Tn 401)Km’Tc’Tra

F- recA1 endA 1 gyrA96 thi-1 hsdRl7 (rk; ml) supE44 recA1

thr leu thi lac4 Ap’Sm’

pwwo pWWO-8

pEST3036

pAYC32

Williams and Murray ( 1974) Bayley et al. ( 1977) Bayley et al. (1977) Franklin and Williams (1980)

Hanahan (1983)

Chistoserdov and Tsygankov (1984)

a Phenotype designations: mTol+ and Phe+ denote the ability of bacteria to grow on m-toluate and phenol, as sole sources ofcarbon. Trp, requirement for tryptophan; Sm’, Cb’, Km’, Tc’, and Ap’, resistance to streptomycin, carbenicillin, kanamycin, tetracycline, and ampicillin, respectively; Tra-, nonconjugative plasmid.

genes specifying the degradation of phenol and m-toluate in Pseudomonas sp. multiplasmid strain EST1001 were transferrable to P. putida Paw340 (Kivisaar et al., 1989) and in this paper we show the isolation of independent degradative plasmids encoding phenol catabolism (PHE plasmids) which have evolved from the EST 100 1 multiplasmid system in the Paw340 background. We cloned the genes encoding phenol monooxygenase and catechol 1,2- dioxygenase, the two first enzymes in the phenol degradation pathway, from the parent strain plasmid DNA into the broad host range vector pAYC32. This paper presents evidence for the control of the expression of the cloned genes in P. putida Paw strains by the insertion of the 17-kb transposon from the host chromosome into the plasmid carrying these genes.

MATERIALS AND METHODS

Bacterial Strains and Plasmids

The standard strains and plasmids and their sources are listed in Table 1. The isolation of EST 100 1 and EST 1020 has been described by us previously (Kivisaar et al., 1989). Pseudo- monas sp. EST 100 1 contains three plasmids of different molecular sizes and has the ability to grow on phenol (Phe+) and on m-toluate

(mTol+). EST1020 derives from the cross of EST 100 1 with the P. putida plasmid-free strain PaW340. EST1020 is able to utilize phenol and m-toluate as growth substrates and har- bors several plasmids. The genealogy of strains and plasmids constructed in this study is shown in Fig. 1. pEST3036 (in host strain P. putida EST3036) was constructed in our laboratory by A. Mae. pEST3036 is a tra derivative of RP 1 which contains the 7.2-kb insertion of the TOL plasmid pWW0 DNA in its tru region. The broad host range vector pAYC32, a derivative of RSFlOlO, was obtained from Y. D. Tsygankov. pAYC32 is 9.7 kb in size and provides resistance to ampicillin and streptomycin. Insertional inactivation of the Sm’ gene provides selection of hybrid plasmids which carry HindIII, ClaI, EcoRI, SacI, or Sac11 fragments (Chistoserdov and Tsygan- kov, 1984).

Media and Culture Conditions

Luria broth (LB)’ was used as the complete medium and M9 as the minimal medium. Antibiotics were added to the following final concentrations: streptomycin, 30 pg/ml for

’ Abbreviations used: LB, Luria broth; C 120, catechol 1,2-dioxygenase.

CLONING OF GENES ENCODING PHENOL DEGRADATION 27

ultiplasmid system

selective plating

EST1021 (Phe+mTol+) rcultiplasmid system

I selective plating

I

transfer into

on phenol P.pltida PaW85

I

EST1021 plasmid DNA P.p&& PaY340 transformation

EST& (Phe+mTol-1 ESTlOZO-85 (Phe+mTol+J ESTlOZJ'fPhe+mTol-1 pESTlOO nultiplasmid system EST1024 (Phe+mTol-)

I

cloning of 5.5-kb CM fragnent into pAVC32, transformation of P.p&&fa Paw85

EST1332 (Phe-Cb') I

continuous culture EST1025 (Phe+mTol‘)

fermentation pESTlO

on Phenol EST1028 (Phe+nTol-) pESTlO

EST1029 (Phe+mTol-1 pEST1332 pESTlOZ9

EST1027 (Phe:mTol:) T"401

I insertion of 17-kb trans-

ESTl$",l;;; mTo1 ) nutagenesis EST;;fMi;:'Cbr'

poson into pEST1332

ESTl35; (Phe+Cb') pEST1354

I

deletions occur frti 17-kb DNA, transformation of P.&i& PaW85

EST1454 (Phe'Cb') cloning of 0.2 kb SacIKYaI pEST1454 fragment from 17 kb DNA

EST1411 (Phe+'-Cb') Phe+ se'ectfon EST1412 (Phe+Cb') i"to pEST1332 EST1415 (Phe*Cb? pEST1411 pEST1412 pEST1415

FIG. 1. Genealogy of the strains and plasmids constructed in this study.

Escherichia coli and 1000 pg/ml for P. putida; ampicillin, 50 pg/ml for E. coli; carbenicillin, 3000 g/ml; tetracycline, 25 pg/ml; and kanamycin 50 &ml for P. putida. Auxotrophic requirements were added to a final concentration of 20 pg/mI; m-toluate was added to a final concentration of 5 mM; glycose was added to a final concentration of 10 mM; and phenol was added to a final concentration of 2.5 mM. P. putida strains were incubated at 30°C and E. coli strains at 37°C respectively.

Elimination of the Ability of Bacteria to Utilize Phenol

After lo-40 generations of growth on L- broth the cultures of P. putida Phe+ strains were spread onto L-plates, and 100-400 individual colonies were tested for their ability to grow on minimal plates containing phenol.

Mating Experiments and Transposon Mutagenesis of PHE Plasmid pESTlO

Conjugal matings of bacteria were carried out as described by Williams and Murray (1974). Transfer of the Phe+ character was tested by the selection of transconjugants on phenol containing minimal selective plates.

pEST3036 (Cb’Sm’Tc’), a Tra- derivative of RPl, was used as the source of the Cb’- determining transposon TnBO1. pEST1026 was conjugally transferred into a SmS proto- trophic strain harboring pEST3036. While pEST3036 itself is nonconjugative, the mating of clones carrying both pEST3036 and pESTlO with Paw340 (Sm’Trp) and the selection of Cb’ transconjugants enabled us to select mutants of pESTlO with Tn401 in their DNA. In order to be sure that there was no PEST 1026-mediated cotransfer of pEST3036, we tested transconjugants for their resistance to tetracycline and analyzed plasmid DNA of Cb’Tc” clones. Clones containing a single plasmid were checked as above and were tested for their ability to utilize phenol as a carbon source.

DNA Manipulations

Plasmid DNA was isolated by the procedures of Hansen and Olsen (I 978) and Con- nors and Bamsley ( 1982). The DNA cleavage by restriction endonucleases, the analysis by agarose gel electrophoresis, the transfer to nitrocellulose filters, the preparation of 32P-labeled DNA, the DNA-DNA hybridization, and the cloning procedures were performed as described by Maniatis et al. ( 1982). Transfor-

28 KIVISAAR ET AL.

mation of P. putidu cells with plasmid DNA was done as described by Bagdasarian and Timmis ( 1982) and for E. coli transformation the CaCl* treatment of cells as described by Maniatis et al. (1982) was used. The size of restriction fragments was calculated by com- paring their mobilities with those of fragments of pWW0 of known sizes.

Enzyme Assays

The cultures of P. putidu to be used for enzyme assays were grown in 30 ml M9 liquid medium with phenol as the sole source of carbon and energy or in LB in the absence or presence of phenol to the midexponential phase of growth. Cells of E. coli were cultivated in 30 ml of LB or LB + phenol medium. The centrifuged cells were washed twice with 0.1 M K-Na-phosphate buffer (pH 7.5) and re- suspended in 5 ml of the same buffer. For cell-free extracts the cells were broken by son- ication and cell debris was removed by cen- trifugation at 50,OOOg for 30 min. Catechol 1,2-dioxygenase was determined by the procedure of Hegeman ( 1966). Protein was measured by the method of Lowry et al. (195 1).

The uptake of oxygen by whole-cell suspen- sions was determined at 30°C with the Clark oxygen electrode according to the procedure of Sala-Trepat and Evans (197 1). The reaction vessel of the oxygen electrode contained 1.4 ml of 0.1 M K-Na-phosphate buffer, pH 7.5, saturated with air and the cell suspension in a total volume of 1.5 ml. After the determi- nation of endogenous uptake of oxygen, 5 pmol of phenol was added to the reaction vessel and the uptake of oxygen was measured. The rate of phenol oxidation was calculated as the difference of the two measurements.

RESULTS

Selection of Independent PHE Plasmids from the Multiplasmid Strain P. putida EST1020

Pseudomonas sp. EST 100 1 and its transconjugant with P. putidu PaW340, EST1020, are able to utilize phenol and m-toluate as

sources of carbon and energy. Both strains contain plasmids of different molecular sizes. Transformation of P. putida Paw340 with the TOL plasmid pWW0 enabled us to select mTol+ transformants at the frequency of 1 Oe5/ pg of TOL DNA, but we failed to isolate mTol+ or Phe+ transformants using plasmid DNA that was isolated from either EST 100 1 or EST 1020 cells grown on phenol or m-toluate. From these data we concluded that there is no independent plasmid determining phenol or m-toluate degradation in EST 100 1 and EST 1020.

The XhoI restriction pattern of plasmid DNA from EST1020 demonstrated the presence of restriction fragments of sizes similar to those of pWW0 fragments in this digest (Fig. 2A). After long-term growth of EST 1020 on minimal plates containing phenol we detected rear-

A A B C

H

I

.I

FIG. 2. Agarose gel electrophoresis of XhoI restriction endonuclease digests of plasmid DNA. (A) Lane 1, Paw 1 (pWW0); lane 2, EST1020, cells grown on phenol. (B) Lane 1, EST102 1, cells grown on phenol; lane 2, Paw1 (pWW0). Letters A to J on the sides of the gels show pWW0 XhoI restriction fragments, as designated by Downing and Broda (1979), from top to bottom, respectively: X/&A, 5 1 kb; XhoI-B, 23 kb; XhoI-C, 16 kb; XhoI- D, 6.5 kb; Xhol-E, 5.3 kb; XhoI-F, 4.7 kb; XhoI-G, 4.2 kb; XhoI-H, 2.7 kb; XhoI-I, 2.3 kb; XhoI-J, 1.6 kb.


rangements in its plasmid DNA. For example, XhoI restriction fragments with mobilities similar to those of XhoI-D, XhuI-E, XhoI-G, XhoI- I, and XhoI-J, that are known to belong to the TOL catabolic region, disappeared from the digest of plasmid DNA (Fig. 2B). The changed strain was designated EST1021 (Fig. 1). The m-toluate degradation genes of EST 102 1 cells grown on phenol probably were located chromosomally, since the bacteria retained the ability to grow on m-toluate.

The P. putida strain Paw340 was transformed with the EST102 1 plasmid DNA. Phe+mToll transformants appeared at a frequency of 10V5/ pg of plasmid DNA on phenol selective plates and 20 clones that were investigated contained single plasmids. Agarose gel electrophoresis of XhoI and EcoRIdigested plasmid DNA from 6 clones revealed differences in their plasmids: all digests contained restriction fragments of the same size as those generated by similar digests of pWWO-8, but there were some differences in other fragments. Three types of plasmids, PEST 1024, PEST 1026, and PEST 1028, were characterized (Table 2).

Another way to select phenol-utilizing Pseu- domonas strains containing single plasmids was the continuo&low fermentation of bacteria on phenol as the sole source of carbon and energy. The Phe+mTol- clone EST1029 was isolated (Fig. 1) by the cultivation of the Phe+mTol+ transconjugant EST1020-85 (the starting strain was obtained by mating of EST1020 with Paw85 and harbored plasmids of different molecular sizes; data not shown) in a continuous culture on phenol for 80 generations. EST 1029 contained only the single plasmid pEST1029, which, according to the sum of lengths of hag- ments generated by XhoI digest, was approxi- mately 100 kb in size (Table 2).

In the following studies we used PEST 1026 as the prototype plasmid. The recipient strains Paw85 and Paw340 could be transformed with PEST 1026 to obtain transformants of the Phef phenotype. pESTlO was also transmissible by conjugation among strains derived from P. putida PaWl: transconjugants arose at a frequency of 1 OV3 per donor cell, exhibited the Phe+ phenotype, and contained a plasmid

identical to pEST1026. These results showed that PEST 1026 is the individual PHE plasmid; i.e., it is responsible for the phenol degradation in our P. putida strains.

Molecular Organization of PHE Plasmids

After the growth of the Phe+mTol+ parent strain Pseudomonas sp. EST 100 1 on phenol- containing minimal media we isolated the Phe+mTol- clone EST 1005, which contained only one 44-kb plasmid pESTlOO (Fig. 1). No homology was detected between PEST 1005 and TOL plasmid pWW0 (Figs. 3A and 3B, lanes 3). The hybridization pattern of EcoRI restriction fragments of pESTlO with the PEST 1005 radioactive probe revealed that a part of DNA in pESTlO is homologous with pESTlO (Figs. 3A and 3B, lanes 1, see PEST 1026 EcoRI restriction fragments in Table 2). PEST 1026 carries the information for the phenol degradation in P. putida strains. We supposed that the DNA homologous between pESTlO and pESTlO contained phenol degradation genes and therefore we designated this DNA phe DNA.

A radioactive probe of PEST 1026 was used to reveal the homology between this plasmid and TOL plasmid pWW0. Xhol restriction fragments XhuI-A and XhoI-H of pWW0 that hybridized with pESTlO in Fig. 3D were present in the XhoI digest of pWWO-8 too (see pWWO-8 restriction map in Fig. 4). XhoI- b of pWWO-8 was derived from the joining of pWW0 fragments XhoI-B and Xho-F after the deletion of the internal 39-kb TOL catabolic segment from pWW0 (Downing et al., 1979). These two fragments of pWW0 hybridize with pESTlO as shown in Fig. 3D. No hybridization was detected between pESTlO and pWW0 fragments XhoI-C, XhoI-D, XhoI-E, XhoI-G, XhoI-I, and XhoI-J, that belong to the 39-kb catabolic region of pwwo.

After the growth of EST1026 for 40 generations in nutrient broth we isolated Phe- derivatives that contained a plasmid with restriction pattern identical to that of pWWO- 8. The hybridization pattern of nitrocellulose-

30 KIVISAAR ET AL.

TABLE 2

MOLECULAR SIZES (kb)o~XhoI AND EcoRI RESTRICTION FFUGMENTSOFTHE PHE FTASMIDSAND pWWO-8”

EcoRl XhoI

pWWO-8 pESTlO pESTlO pESTlO pWWO-8 pESTlO pESTlO pESTlO pESTlO

b 10.2

C 7.8

5.9 5.5 5.5 5.1 4.9 4.9 4.3

M 3.7 N 3.2

0 2.8

R 2.4

S T U W X

1.9 1.7 1.5 1.35 1.35

Z 0.95 Z 0.8

z”

Total

W)

0.3

76.0 106.9 108.5 110.0 76.7 107.5 108.5 109.9 99.6

10.8 10.2

7.8 7.06 5.9 5.5 5.5 5.1 4.9 4.9 4.3

10.8 10.8 10.2 10.2 8.6’ 8.66 7.8 7.8

5.9 5.5 5.5 5.1 4.9 4.9 4.3

3.7 3.7 3.2 3.2 2.9 2.9 2.8 2.8 2.8 2.8 2.5b 2.5’ 2.4 2.4 2.4 2.4 1.9 1.9 1.7 1.7 1.5 1.5 1.35 1.35 1.35 1.35 I.0 1.0 0.95 0.95 0.8 0.8 0.8 0.8 0.6 0.6 0.3 0.3

5.9 5.5 5.5 5.1 4.9 4.9 4.3 4.06 3.7 3.2 2.9 2.8 2.8

2.4 2.4 1.9 1.7 1.5 1.35 1.35 1.0 0.95 0.8 0.8 0.6 0.3

A 49 49 49 49 49 b 25 25 25 25 25

106 lob 96

8’ 6.9’ 6.9’ 6.9’

4.8’ 4.6 4.6 4.6 4.6 3.4b 3.4b 3.4b 3.4 3.4 3.4 3.4

H 2.7 2.7 2.7 2.7 2.7 2.7 2.7 2.7 2.7 0.8 0.8 0.8 0.8

’ Letters designate the restriction fragments of pWWO-8 derived from the TOL plasmid pWW0. b Fragments which differed in pEST1024, pEST1026, pEST1028, and pESTlO digests.

blotted X/z01 fragments of pESTlO with inated from EST1021 DNA. The physical cloned fragments of the 17-kb transposon re- structure of these plasmids is depicted in Fig. 4. vealed that more XhoI fragments than in the pWWO-8 digest hybridized with the 17-kb sequence in the pESTlO digest (data not

Cloning of phe Genes from pESTlOO

shown). This result suggested that part of the and the Expression of Cloned DNA

17-kb DNA might be duplicated in PEST 1026. in P. putida

We constructed the restriction map of Matings that were performed to insert PEST 1026 and other PHE plasmids that orig- Tn401 into PEST 1026 lead to the isolation of


f

f

10.8

2.8-

2.5 2.4

FIG. 3. (A) Agarose gel electrophoresis of restriction fragments of various plasmids. Lane 1, pESTlO (EcoRI); lane 2, pEST1226 (EcoRI); lane 3, pWW0 (EcoRI/SacI). Numbers indicate the molecular size of EcoRI fragments of pESTlO (in kb) that differ from EcoRI restriction fragments of pWWO-8. (B) The autoradiograph of the nitrocellulose-blotted DNA after hybridization with 32P-labeled pEST1005. Numbers indicate the molecular size of restriction fragments (in kb) which hybridize with the labeled PEST 1005 probe. (C) Agarose gel electrophoresis of XhoI-digested pWW0. (D) Autoradiograph of the nitrocellulose-blotted DNA after hybridization with 32P-labeled pESTlO as a probe. Letters A to J between panels C and D show pWW0 X/z01 restriction fragments.

two types of clones. Six investigated PheCb’ clones had lost all phe DNA from their plasmids. These plasmids differed from pWWO- 8 only by the Tn401 insertion into the EcoRI- b fragment (see Fig. 4A). Four PheCb’ clones, that contained Tn40I in their plasmids, were also obtained and one such isolate, EST1 226, was used for a more detailed study. EcoRI digestion of the EST 1226 plasmid, PEST 1226, showed a large deletion in the phe DNA (Figs. 3A and 3B, compare lanes 1 and 2), but this deletion did not affect the genes responsible for the expression of the Phe+ phenotype. PEST 1226 was transfer-r-able by conjugation and by transformation and conferred the PheCb’ phenotype in P. putida hosts. The part of the phe DNA which remained after the

Tn40Z insertion into PEST 1026 in PEST 1226 is shown in Fig. 4B.

The 5.5-kb PEST 1005 ClaI restriction fragment covers nearly all of the sequence com- mon in PEST 1005 and PEST 1226. We cloned this fragment from pESTlO DNA into the ClaI site of pAYC32, a derivative of the broad host range plasmid RSFlO 10. The recombi- nant plasmid pEST1332 in E. coli DH I was used for restriction mapping of the cloned DNA segment. The restriction map of the cloned phe DNA in PEST 1332 is presented in Fig. 5.

We transformed the plasmid-free strain P. putida PaW85 with pESTI 332 and selected clones on phenol and on glucose plus carbenicillin selective plates. Transformants arose

32 KIVISAAR ET AL.

A o 10 20 30 40 50 60 70 kb I I I I I I I I

W I 2 Z’ z**’

ECURI L 1 J 1x1 M 1 C ]ISl G 1 I IlJl b IINIIIK I F IIoITIH IllI b I

ECURI XhoI

pEST1226 -

FIG. 4. (A) EcoRI and XhoI cleavage map of pWWO-8. The insertion site for Tn401 in EST1026 Phe- derivatives is shown by an arrow. (B) Schematic representation of the structures of PHE plasmids selected from the multiplasmid strain EST1020. The black box below the map of pWWO-8 indicates the 17-kb transposable element. The 17-kb segment of pWWO-8 is formed from the two regions localized to the left and to the right of pWW0 catabolic genes which are designated as LH and RH, respectively. The lower part of the figure shows the EcoRI and X/r01 cleavage map of phe DNA and its flanking regions in pEST1026. A different amount of phe DNA (thin lines) is located between the stretches of DNA originating from the 17-kb LH segment (black boxes) in different PHE plasmids. The thick lines indicate the phe DNA (with its flanks originating from the 17-kb element) which remained in pEST1226 after the insertion of Tn401 into pEST1026.

only on glucose plus carbenicillin plates. After transformants gave a few Phe+ colonies at a 5-6 days of cultivation on phenol-containing frequency of 10e7 per cell. Six colonies that selective media the P. putida PaW85 Cb’ were investigated contained a single plasmid,

S 1 nESTI :P

-c pEST1411 c-

-c pEST1412 c-

-c pEST1415 w "I-*

PIG. 5. Cloning of the phe genes into the broad host range vector pAYC32 and the insertion of the 17- kb transposable element into the vector DNA just near the cloned DNA. Cleavage sites for EcoRI (E), X/z01 (X), Hind111 (H), ClaI (C), and Sac1 are shown. The black box indicates the sequence from the 17-kb element and the open box shows phe DNA. At the lower part of the figure the lines indicate the DNA regions present in pESTI deletants and in pEST1415.


designated pESTl354. Digestion of PEST 1354 with XhoI, EcoRI, HindIII, and ClaI showed insertion of the 17-kb DNA segment into the vector DNA near the cloning site (Fig. 5). Re- striction analysis of PEST 1354 DNA and hybridization of PEST 1354 with the TOL plasmid pWW0 (data not shown) demonstrated the identity of the 17-kb insertion in PEST 1354 with the 17-kb transposon from the TOL plasmid pWW0, which is localized chromosomally in P. putida strains Paw85 and Paw340 (Meulien and Broda, 1982; Wil- liams et al., 1983).

We studied the stability of the PheCb’ phenotype in the strain P. putida EST1354 harboring PEST 1354. After 10 generations of growth on nutrient broth, derivatives of EST1 354 which were unable to utilize phenol arose at a frequency of 80% of 400 single colonies investigated, while less than 1% of them lost the carbenicillin resistance marker and plasmid DNA as well. The analysis of the plasmid DNA from PheCb’ derivatives demonstrated that all of them had lost the 17-kb tmnsposon. These clones gave Phe+ revertants and in this case the 17-kb element reinserted back into the plasmid. To summarize, although the 17-kb transposon locates in the chromosome of P. putida PaW85, a cis loca- tion of this element with cloned DNA was ob- viously necessary for the expression of the phenol degradative function.

We found two regions in the 17-kb element that step up the expression of cloned phenol degradation genes (phe genes). To localize these sequences, deletions from the 17-kb transposon were made in pESTl354. We transformed the P. putida strain Paw85 with different deletants, selected clones for Cb’, and tested for their ability to grow on phenol-containing minimal media. The Sac1 deletant pEST1454 had the deletion in the right-hand part of the 17-kb transposon (see Fig. 5) and this deletion did not affect the expression of the phe genes. The deletant PEST 14 11 contained the cloned phe genes and only 1.7 kb of DNA from the right hand of the 17-kb transposon, (Fig. 5). Paw85 transformants with PEST 14 11 grew slowly on phenol. These

clones gave some large colonies on phenol- containing media. One of these mutants was designated EST 14 12. Subsequent subclonings showed that the DNA region that was involved in the expression of the cloned genes in PEST 14 12 is localized in the 0.2-kb SacI/CZaI fragment in the right hand of the 17-kb element. Cloning this fragment from PEST 14 12 into Sac1 + ClaI-digested pEST1332 resulted in pEST14 15 (see Fig. 5). Transformation of P. putida strains Paw85 and Paw340 with pEST1415 and also with pEST1354, PEST 14 12, and pEST1454 followed by selection for the Phe+ phenotype of bacteria oc- curred at a frequency of 10p5/pg of plasmid DNA.

Phenol monooxygenase catalyzes the degradation of phenol to catechol. Catechol is cleaved by catechol 1 ,Zdioxygenase (C 120) or by catechol2,3-dioxygenase via the ortho- and meta-pathway.

Oxidation of phenol by whole-cell suspen- sions of P. putida strains harboring PEST 1354 or PEST 14 12 indicated the presence of active phenol monooxygenase in the cells. The ortho- pathway enzymes were used for phenol degradation in these strains (Table 3). The high basal level of C 120 was established in LB-cultivated cells of P. putida Paw85 harboring pEST1354 or pEST1412. In principle, since P, putida Paw85 contains functional ortho- pathway genes in its chromosome (Bayley et al., 1977), the expression of the Cl20 gene in these strains could be due to the transcription of the chromosomal Cl20 gene. However, since P. putida Paw85 itself did not express Cl20 at detectable levels when grown in LB, this enzyme appeared to be encoded by the cloned phe DNA, too. Measurements of C 120 activities in crude extracts of P. putida cells carrying pEST1354 or pEST1412, grown either in LB or in LB in the presence of phenol, suggested that phenol itself does not induce Cl 20. Interestingly, a lo-fold lower level of C 120 was observed in P. putida cells carrying pEST1332.

Enzyme assays from E. coli DHl clones carrying either pEST1412 or pEST1354 con- firmed that the cloned phe DNA carries in-

34 KIVISAAR ET AL.

TABLE 3

ENZYME ASSAYS OF P. putida AND E. coli STRAINS CONTAINING CLONED phe DNA

Bacterial strain Growth and plasmid substrate

O2 uptake by whole-cell

suspension” c120*

P. putida Paw85 pEST1332 PEST 1332 pEST1354 pEST1354 pEST1354 pEST1412 pESTl412 pEST1412

E. co/i DHI pEST1332 pESTI pEST1354 pEST1354 pEST1412 pEST1412

LB LB LB + phenol LB LB + phenol phenol LB LB + phenol phenol LB LB LB + phenol LB LB + phenol LB LB + phenol

<5 <0.005 <5 0.02 <5 0.02 10 0.20 30 0.32

250 1.60 60 0.32

120 0.38 160 1.90 t5 <0.005 <5 -co.005 <5 <0.005 <5 0.06 10 0.03 20 0.24 40 0.40

4 Expressed as microliters of oxygen taken up per hour per milligram (dry wt) of cells. Values are calculated as differences from endogenous uptake.

* Expressed as micromoles of substrate used per minute per milligram of protein.

formation for phenol monooxygenase and catechol 1,2-dioxygenase. These two enzymes were not detectable in E. coli cells containing PEST 1332 (Table 3).

DISCUSSION

Plasmids of different molecular sizes were isolated from the parent strain Pseudomonas sp. EST1001 and from its P. putidu transconjugant EST1020. There was no individual plasmid determining phenol or m-toluate degradation in these multiplasmid strains. We showed that the multiplasmid system present in these Phe+ pseudomonads could be con- verted into a much simpler system containing just one transformable plasmid, responsible for the degradation of phenol. Two approaches were used to select single-plasmid-containing Phe+ clones from our multiplasmid strain EST1020. (i) We selected EST102 1, in which structural rearrangements in plasmid DNA and supposedly recombinations between plasmid and chromosomal DNA had taken place

following serial growth of EST 1020 on phenol- containing minimal media. Plasmid DNA that was isolated from this multiplasmid strain made it possible to obtain single-plasmid-containing Phe+ transformants in PaW340. (ii) We selected Phe+mTol- clones that harbored a single IOO-kb plasmid during continuous- flow cultivation of the Phe+mTol+ strain EST 1020-85 on phenol.

The structure of PHE plasmids is presented in Fig. 4. A duplicated region of the left-hand part of the 17-kb DNA flanks both ends of the phe DNA in PHE plasmids. Furthermore, all the phe DNA with the duplicated region originating from the 17-kb DNA is located in a pWWO-8-like transfer replicon.

PHE plasmids that were isolated from the multiplasmid strain EST1020 are of interest in the context of similar structural rearrangements which take place either in PHE plasmids or in TOL plasmid pWW0. For example, the 39-kb DNA segment that corresponds to the xyl catabolic operons of pWW0 is .deleted in the mTol- P. putida strain Paw8 carrying


pWWO-8 (Bayley et al., 1977; Downing and Broda, 1979). Excision of the 39-kb segment from pWW0 involves 1.4-kb direct repeats (Meulien et al., 1981). In Phe- clones originating from the strain EST 1026 the phe DNA was deleted from pESTlO and these clones contained pWWO-8. We cannot exclude the possibility that the excision of the phe DNA from PEST 1026 is mediated by the duplicated regions originating from the 17-kb DNA. The exact localization of these regions is in progress.

The Cl20 gene has been cloned from the chromosome of Acinetobacter calcoaceticus and P. aeruginosa (Neidle and Ornston, 1986; Kukor et al., 1988). Plasmid-encoded Cl 20 has been cloned by Ghosal et al. (1985). Our construct pEST1332 contains 5.5 kb of DNA cloned from PEST 1005 DNA into a broad host range vector plasmid based on RSF 10 IO. The structural genes for phenol monooxygenase and C 120 were established in the cloned DNA. For the expression of the phenol degradative function in P. putida Paw85 cells that were transformed with pEST1332, the host chromosome contributed to the formation of the functional PHE plasmid by inserting into it the 17-kb transposon. In subcloning experiments we found that two regions of the 17-kb DNA can be used in the expression of the cloned phe genes. One of these is localized in the 0.2-kb ClaI/SacI fragment of the right hand of 17-kb transposon (see PEST 14 15 in Fig. 5) and another to the left from the above- mentioned 0.2-kb sequence (pEST1454 in Fig. 5).

Although the growth phenotype of P. putida cells carrying PEST 1332 demonstrated that the cloned phe genes were not expressed in this plasmid and the chromosomally encoded Cl20 of P. putida Paw85 was not detectable in cells grown in LB, a low basal level of C 120 was established in P. putida cells carrying pEST1332. The level of Cl20 was lo-fold higher in cells carrying pESTl354 and PEST 14 12 grown in LB (Table 3), thereby in- dicating that the 17-kb DNA sequences acting in cis with the cloned phe DNA enhance the expression of C 120.

The expression of the phe genes in PEST 1354 and in pESTl4 12 are controlled by different sequences of the 17-kb transposon. The level of C 120 in E. coli cells grown in LB that carried PEST 1354 was lower than that in P. putida cells harboring that plasmid. In the case of PEST 14 12 it was observed that the levels of expression of C 120 of this plasmid in P. putida and E. coli grown in LB were equal. The low expression of the xyl operons in E. coli was a reflection of the inefficient transcription of the xyl operons by E. coli poly- merase (Nakazawa et al., 1986). We cannot exclude the possibility that due to different sequences of the 17-kb DNA, which control the expression of the phe genes in PEST 1354 and PEST 1412, the efficiency of transcription of the phe genes in pEST1354 and pEST1412 is also different. Further studies are in progress to resolve this problem.

ACKNOWLEDGMENTS

We thank Y. D. Tsygankov for vector pAYC32. We also acknowledge Richard Villems and Andres Raukas for critical reading this manuscript.

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Communicated by Kurt Nordstriim

selection of independent plasmids determining phenol degradation inpseudomonas putida and the...

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