Gene, 127 (1993) 23-29 0 1993 Elsevier Science Publishers B.V. All rights reserved. 0378-I I ~9/93/$06.00

GENE 07025

In-vivo-generated fusion promoters in Pseudomonas putida

(Transposon Tn4652; insertional activation of transcription; adaptive mutations; 0” promoters of Escherichiu co&

phenol monooxygenase)

Allan Nurk, Anu Tamm, Rita Hhak and Maia Kivisaar

Institute of Molecular and Cell Biology, Estonian Biocentre. EE2400 Tam. Estonia

Received by T.K. Misra: 17 June 1992; Accepted: 24 November 1992; Received at publishers: 6 January 1993

23

SUMMARY

Plasmid pESTI carrying the promoterless pheBA operon was cloned into Pseudomonas putidu PaW85, and phenol- utilizing colonies were isolated on minimal plates containing phenol as the only carbon and energy source. In these clones, chromosomally located Tn4652 was transposed upstream from the coding sequence of pheA (encoding phenol monooxygenase). Sequence analysis together with mapping of the transcription start point of the pheBA operon in the recombinant plasmids revealed that fusions of the -10 sequences present in the pheBA operon and -35 sequence located in the terminal inverted repeats of Tn4652 had generated functional promoters under selective pressure in P.

putida ceils. These promoter sequences show similarity to the Escherichia coli RNA polymerase 07’ promoter consensus sequence. In three of the six fusion promoters studied, the generation combined two distinct events: transposition of Tn46.52 into DNA containing potential - 10 sequences and point mutations in these sequences. These mutations made the -10 sequences more like the 07’ promoter consensus sequences.

INTRODUCTION

Activation of downstream genes due to readthrough transcription from a transposable element by its outward- reading promoter sequences is described in several works (Pilacinski et al., 1977; Iida et al., 1983; Wang and Roth, 1988; Lers et al., 1989; Haughland et al., 1990). In am& ~-lactamase-hy~rproducing mutants of E. coli K-12, the insertion of IS2 created a novel 070 promoter in which

Correspondence to: Dr. M. Kivisaar, Institute of Molecular and Cell Biology, Estonian Biocentre, 2 Jakobi Street EE2400 Tartu, Estonia. Tel. (01434) 35376; Fax (01434) 35430; e-mail: maiak@ebc.ee

Abbreviations: A, absorbance (1 cm); Ap, ampicillin; bp, base pair(s); BSA, bovine serum albumin; Cb, carbenicilhn; C120, catechol I,Zdiox- ygenase; DR, direct repeat; IR, inverted repeat; IRL, IR at the left end of Tn; IRR, IR at the right end of Tn; kb, kilobase or 1000 bp; LB, Luria-Bertani (m~ium); nt, nuc~eotid~s); oligo, oligodeoxy~~nucleo- tide; P, promoter; P., Ps~do~o~s~ pheA and phefl, genes encoding PM0 and C120, respectively; PMO, phenol monooxygenase; Tn, transposon; tsp, transcription start point(s); [ 1, denotes plasmid- carrier state.

the - 35 region and 17-bp spacing sequence between the two consensus sequences are present in IS2 DNA, whereas the -10 region from the E. coli ampC promoter

is retained (Jaurin and Normark, 1983). Tn4652 (Tsuda and Iino, 1987), a 17-kb transposon of

P. p~t~da, originates from a TOL plasmid pWW0 that contains genes for the degradation of toluene and xylenes (Worsey and Williams, 1975). The plasmid-free strain P.

putida Paw85 is derived from the pWWO-harbouring strain PaW 1 and has Tn4652 inserted into its chromo- some (Meulien and Broda, 1982). Sequence analysis demonstrated that Tn4652 has 46-bp terminal IRS and that this element generates 5-bp duplication of the target sequence upon insertion (Tsuda et al., 1989). Although Tn4652 was placed into the Tn3-related (class II) Tns, its IRS show considerable sequence divergence from these well-studied Tns (Tsuda et al., 1989).

Phenol monooxygenase (PMO) catalyzes the hydroxyl- ation of phenol to catechol. Catechol 1,Zdioxygenase (C120) is the first enzyme involved in the oxidation of

24

catechol via the ortho pathway. We have cloned the genes pheA encoding PM0 and pheB encoding Cl20 from the plasmid DNA of Pseudomonas sp. EST1001 into the broad host range vector pAYC32 (Kivisaar et al., 1990). These genes are organized into the operon where the gene order is pheBA (Kivisaar et al., 1991). P. putida PaW85 contains the ortho-pathway genes for catechol degrada- tion in its chromosome (Bayley et al., 1977). We have shown that the transfer of the functional pheA into P.

putida PaW85 enables the bacteria to use phenol as a growth substrate (Nurk et al., 1991).

Recombinant plasmid pEST1332 contains the pheBA operon without its natural promoter. Therefore, the genes were expressed only weakly in P. putida PaW85[pEST1332], and this was insufficient to allow the growth of bacteria on minimal medium containing phenol as the only source of carbon and energy. However, we isolated the mutant clones, which exhibited high con- stitutive levels of PM0 and Cl20 activities, when the cells of P. putida PaW85[pEST1332] were selected for their growth on phenol-containing minimal media. Tn4652 was inserted into the single site upstream from pheBA in all six recombinant plasmids investigated (Kivisaar et al., 1990). In this study we show that after 4-6 days of cultivation of bacteria on phenol-containing minimal plates, pheA was activated in P. putida PaW85 cells due to the generation of fusion promoters at the junction between different target DNA sequences upstream from pheA and the IRS of Tn4652. The aim of present study was to determine the molecular mecha- nisms that generate these fusions in response to environ- mental pressures.

RESULTS AND DISCUSSION

(a) Activation of the pheA gene by the transposition of Tn4652

We have shown previously the activation of promoter- less pheBA in pEST1332 by the insertion of Tn46.52 from the chromosome of P. putida PaW85 into the single site upstream from these genes (Kivisaar et al., 1990). In order to study whether the transposition of Tn4652 to other sites could activate the cloned pheBA, we deleted the above-mentioned target DNA sequence from pEST1332 between the EcoRI and ClaI sites in vector plasmid pAYC32 (Fig. 1) and introduced the resultant plasmid pEST1463 into P. putida PaW85. Phenol-utilizing colo- nies were studied for the transposition of Tn4652. Of the clones analyzed 75% had Tn4652 inserted into pEST1463. Transcriptional activation of the phe&l operon in the other 25% was achieved (demonstrated by DNA sequencing and transcription mapping experi-

A

L AK p*cs

pEST1463 , 500 bp ,

Fig. 1. Plasmid maps. (A) Circular map of pEST1332 (Kivisaar et al., 1990) is shown in the middle. Arrow shows the direction of transcription of pheB and pheA. Expanded map of the SacI-Hind111 fragment and of the restriction sites are shown above. DNA of the vector plasmid pAYC32 is indicated by the line and the cloned fragment, by the box. The black boxes show the location of the pheBA operon in the 5.5kb CIaI fragment, and the hatched boxes signify intergenic regions. The ClaI site marked by Cm is methylated in E. co& thus, only the CM site upstream from the phenol degradation genes is cleaved by CM. (B) The linear map of pEST1463. pEST1463 is obtained by deleting the vector DNA upstream from pheBA between EcoRI and CM sites which is the target for Tn4652. The 3’ termini, produced by EcoRI and CM, were filled in with Klenow fragment of E. coli DNA polymerase I. The ligation of blunt ends restored the EcoRI site in the resultant plasmid pEST1463. The 2.1-kb Hind111 fragment, which is localized downstream from the pheBA operon in pEST1332, is also deleted in pEST1463. Five different target sites for Tn4652, indicated by vertical arrows, are shown in the map of pEST1463. LA1 designates the insertion of Tn4652, being oriented by its left arm towards pheBA. RA2, RA3, RA4, and RA7 designate the different insertion sites of Tn4652 in pEST1463, where the Tn is inserted with its right arm towards the transcription of pheA.

RAI is shown by the vertical arrow in A in the map of pEST1332. The hybrid plasmid pESTl354, which was created in this recombination, has been described by us previously (Kivisaar et al., 1990). Only relevant restriction sites are shown: S, SacI; E, EcoRI; C, CM; H, HindIII; AC,

AccI; N, NlaIV; P, PstI; E47, Eco47111.

ments) as the result of a point mutation upstream from the coding sequence of pheB, which led to the creation of the constitutively expressing promoter of the operon (not shown in this paper).

Different insertions of Tn4652 into pEST1463 are shown in Fig. 1 and Table I. The target site 140 bp upstream from the PstI site inside of pheB (see RA2 in Fig. 1) was strongly preferred over the others: 15 hybrid plasmids of the 19 investigated contained the same inser- tion. The other four targets were unique. Thus, although RA2 was strongly preferred and in the case of pEST1332 we identified only one type of insertion, RAI, Tn4652 could activate pheA from different distances. Except for LAI, the Tn was situated with its right end oriented in the transcriptional direction of pheA.

25

TABLE I

Sequence analysis of the insertions of Tn46.52 in the hybrid plasmids

-35 -10

TTGACA TATAAT

Insertione Position of insertion (bp)b

Target The 5-bp flanking sequence region in hybrid

(5 bp) plasmidsd

RAl 1259-1255 TATCA TATCA

RA2 312-308 TATGA TATGA RA3 728-724 TGTAT TATI-A RA4 70-66 TAAAC TAAAC RA7 85-81 TCTAA TATAA LA1 1175-1171 TACTT TATAC

a See Fig. 1. b The distance from the ATG start codon of pheA is shown. The nt sequence of the 3482-bp DNA region carrying pheB and pheA have the GenBank accession No. M57500. The nt sequences of the double- stranded templates (4 ug) were determined by the dideoxy chain- termination method @anger et al., 1977) with Sequenase, using synthetic oligos (20 ng). Primer 5’-TACAATAGCTTAG and primer 5’- ATCATATATTTAC, complementary to the sequences 43-55 bp inwards of the left and right end of Tn4652, respectively, were used to determine the junction sequences of Tn4652 in the hybrid plasmids described in Fig. 1. ’ Tn46.52 generates 5-bp duplication of the target sequence upon insertion. d Sequences flanking the IRR of Tn4652 are shown for insertions RAI, RA2, RA3, RA4, and RA7, and the sequence contiguous with the IRL of the Tn is presented in the case of LAI. The mutant nt are in bold- face type.

At 17 bp inward of the element, both IRS of Tn4652 contain a TTGCCT sequence that resembles the -35 hexamer TTGACA of the 07e-recognized promoters of E. coli. Sequencing of the junction DNAs of Tn46.52 in plasmids carrying the activated pheA revealed the -10 hexamer-resembling sequences adjacent to the right arm of Tn4652 in the case of RAl, RA2, RA3, RA4 and RA7, and to the left arm of the Tn in the case of LA1 (Fig. 2). These sequences matched the -10 consensus TATAAT in 4-5 positions. We did not find the -10 hexamer- resembling sequences flanking the insertion of Tn4652 when there was no selection for gene activation (data not shown). These findings led us to the hypothesis that under selective conditions, fusions between the - 35 hexamer of the IRS of Tn4652 and the -IO hexamer found in the target DNA created promoters for the transcription of pheA.

(b) Effect of cloning of the Tn4652 ends upstream from the pheBA operon on its expression

In some cases bacterial transposable elements have been found to activate or enhance the expression of adja- cent genes due to readthrou~ transcription from the ele- ment outside (see, e.g., Pilacinski et al., 1977). In order to determine whether the ends of Tn4652 contain a func- tional promoter for the transcription of adjacent genes,

IBL TAAlW@%@gAlWCGGtXTGACCCC

IRB TAA~ATCXGGCATAWCC

WA2

8 PBA3 TAA~ATfXGrXXTAACCCC@A!lTA’@WXWTG

PBA4 TAA~ATCl’UWATAAtXCX@ACl@-lWA&CC

Pl?A7 TAA!W#KZi@TCKGGCA TAACtXt$~@ZATA~T

PU1 TAATY@i%$PATClYXGCATtXCXX+AT&~&

Fig. 2. Sequence characterization of the individual insertions of Tn4652. The 29 terminal nt of the right- and the left-end IRS of the Tn, IRR and IRL, respectively, and 16 nt adjacent to the insertions are shown. Two hexamers homologous to the E. co& a70 -35 and -10 consensus sequences are boxed. Formation of the fusion promoters PRAI, PRAZ, PRA3, PRA4, PRA7, and PLAI by the fusion of the IRS of T&652 with the target DNA (see the 5-bp flanking regions in Table I) in the different insertions RAl, RA2, RA3, RA4, RA7, and LAI, respectively, is represented. Asterisks mark tsg, determined by primer- extension experiments (Fig. 4).

we cloned the ends of the Tn into pEST1332 upstream from the promoterless pheBA operon. The expression of the pheBA operon was studied by measuring the activity of Ct20 in P. p~~j~~ cells carrying these constructs.

We used RAI and LA1 as the source of the Tn46.52 ends. Cloning procedures are described in Fig. 3. Only the right end of Tn4652, cloned from RAl, activated the pheBA operon expression (pRA1 in Fig. 3), whereas the left end did not. In the case of LAl, Tn4652 was oriented by its left arm towards the direction of the transcription of pheBA, and the genes were also activated. Cloning of the 86-bp DNA fragment, which contained the left end of the Tn, from this recombinant piasmid into pEST1332 (see pLA1 in Fig. 3) led to the activation of pheBA. On the other hand, we did not observe the activation of the phe genes by the sequences at the right end of Tn4652. These data clearly demonstrate that not only sequences of Tn4652, but also sequences adjacent to the inserted DNA, are important for gene expression and that the fusions of the ends of Tn4652 with the sequences of target DNA created the functional promoters for transcription of the downstream genes.

(c) Activation of the tran~ription of the piteBA operon by cloned fusion promoters

In addition to the plasmids pRA1 and pLA1, which include the fusion promoters designated as PRAI and

26

A , 2kb ,

DHdE 5HC H E E XH XXI

1” ” I I I II I

IRL inpT T”np5 mp* IRR

B , 200 bp ( Actlvlty of Cl20

DRAM 1” P.p”tida

0.08

pLA1 IRL

IAL

PLAR1 SD HC” AC

IRA

0.48

0.012

Fig. 3. Maps and promoter activity of Tn4652. (A) Physical and genetic map of Tn4652 (Tsuda et al., 1989). The 46-bp terminal inverted repeats of the left and right ends of the Tn are designated as IRL and IRR, respectively. Genes tnpA, tnpT and tnpS encode the cointegration and resolution functions of Tn4652. (B) Maps and promoter activity of the ends of Tn4652. Methods: The Tn ends were cloned into pBluescript KS(+) (Stratagene, La Jolla, CA). Polylinker multicloning sites Sac1 and CIaI were used in order to reclone them in proper orientation into pEST1332, upstream from the pheEA operon. The 63-bp D&-C/a1 fragment, which contains the right-end DNA of the Tn up to the DraI site, was cloned from the hybrid plasmid pEST1354 (see RAl in Fig. 1A) into pEST1332 to obtain pRA1. Plasmid pRAL1 contains in pEST1332 the 79-bp DraI-EcoRI fragment, which covers the left end of the inserted Tn. The hybrid plasmid carrying the insertion LA1 (Fig. 1) served as the source of the ends of Tn46.52; it is inserted upstream from the pheBA operon in the direction opposite to the insertion RAl. Plasmid pLA1 contains the 86-bp DraI-AccI fragment, including the left end, and pLAR1 carries the 135-bp DraI-EcoRI fragment, including the right end of the Tn in pEST1332, respectively. Open boxes indicate the DNA of the ends of Tn4652, hatched boxes denote the DNA upstream from pheB up to the ClaI site, and the black boxes represent the DNA of pheB. Lines represent the DNA of the vector pAYC32. The activities of Cl20 (determined by the procedure of Hegeman, 1966), measured in the cell-free extracts of P. putida Paw85 carrying these plasmids, are listed to the right of the plasmid maps. The values for Cl20 were calculated as umol of product (cis-cis muconate) formed/min/mg of protein. Cl20 activities are means of four independent assays. Protein concentrations were determined by the method of Bradford (1976), with BSA as standard. Clonings were performed in E. coli strains HBlOl (Boyer and Roulland-Dussoix, 1969) and TGl (Gibson, 1984). The Pseudomonas host strain was P. putida Paw85 (Bayley et al., 1977). Transformation of E. coli cells with plasmid DNA was done as described by Hanahan (1983). For P. putida the same protocol was used, except that MgClz solution was added to RF1 at a final concen- tration of 100 mM and to RF2 at a final concentration of 10 mM. LB was used as the complete medium. Antibiotics Ap (100 ug/ml) for E. co/i or Cb (1500 ug/ml) for P. putida were added to media as required for the selection of plasmid-containing strains. Abbreviations for restric- tion sites are the same as those in Fig. 1 plus:D, DraI; Ha, HaeIII; X, XhoI; A, AluI.

PLAl, respectively, the plasmids pRA2, pRA3, pRA4, and pRA7 carrying the other four fusion promoters were con- structed (Table II). Measurement of the Cl20 and PM0 activities in P. putida Paw85 cells carrying pEST1332

TABLE II

Activities of Cl20 and PM0 in P. putida Paw85 clones harbouring pESTl332 with different fusion promoters’

Plasmid C120b PM0

pEST1332 0.005 7 pRA1 0.08 65 pRA2 0.05 30 pRA3 0.29 165 pRA4 0.22 135 pRA7 0.21 125 pLA1 0.48 190

’ The construction of the plasmids pRA1 and pLA1 carrying the fusion promoters PRAl and PLAI, respectively, is described in Fig. 3. The same strategy was used for the construction of the other plasmids pRA2, pRA3, pRA4 and pRA7 by cloning of the junction DNA as the 198-bp DraI-PstI fragment from RA2, the 182-bp DraI-NlaIV fragment from RA3, the 80-bp DraI-Eco47111 fragment from RA4, and the 95-bp DraI- Eco47111 fragment from RA7 upstream from pheB into pEST1332 using the restriction sites of the polylinker of pBluescript KS(+). The values of Cl20 and PM0 are means of four independent assays. b Specific activities of Cl20 in cell-free extract of bacteria, expressed as umol of product (cis-cis muconate) formed/min/mg of total protein. ’ Specific activities of PM0 in cell-free extract of bacteria, calculated as nmol of NADPH decreased at A smo,,/min/mg of total protein. PM0 was measured as described by Beadle and Smith (1982).

with different fusions (Table II) showed that the level of expression of pheB and pheA in these recombinant plas- mids depended on the cloned sequence.

Primer extension (using RNA extracted from P. putida Paw85 cells carrying plasmids where the fusion pro- moters were cloned upstream from the pheBA operon) revealed that the transcript initiated 6-8 bp downstream from the - 10 hexamer of these promoters (Figs. 2 and 4).

(d) Possible mechanisms for the in vivo generation of the fusion promoters

Tn46.52 generates the duplication of 5 bp of the target sequence upon insertion (Tsuda et al., 1989). Sequencing of the junctions between Tn4652 and the @e&4 operon in hybrid plasmids revealed that the 5-bp DRs generated in this way contained mutations (in the case of RA3 and RA7) adjacent to the right arm of Tn4652 (Table I). On RA3 the right-hand IR (IRR) of the Tn was flanked by the sequence TATTA instead of the target sequence TGTAT (AT replacing the original G). RA7 contained A instead of C in its 5-bp DR 84 bp upstream from the ATG start codon of pheA. In the case of LAl, when the Tn was oriented by its left arm towards the transcription of pheBA, TA were inserted between the left terminus of Tn4652 and target DNA. Hence, mutations appeared only in these repeats which served as - 10 sequences of the fusion promoters, and in this context they were revealed as up-promoter mutations: sequences TATAAT instead of TCTAAT in the case of RA7 and TATTAT

27

3 C-G C-G T-A

;I; I C-G A-T T-A

ii2 A-T T-A A-T

*C-G

\

0 A T C P

G-C T-A T-A G-C C-G A-T

*C-G A-T

i C-G C-G

G A T C P

A-T 1 C-G T-A ;

i

G A T C P

PRAl PRA2

G A T C P G A T C P G A T C P

1; C-G A-T T-A A-T

*G-G A-T A-T A- i T-A

T-A

C-G

;I; T-A

WC A-T A-T G-C I

PRA7

Fig.4. Mapping of the rsp (5’ ends) of transcripts from the fusion promoters, as determined by primer extension of the RNA samples with reverse transcriptase (lanes P, transcripts are shown by arrows). Sequence ladders were generated by using the same oligos that were used for transcript mapping (lanes G, A, T, C). The sequences of 20 bp in the tsp region, including four terminal C of Tn465.2 (see Fig. 2), are shown at the left of sequencing reactions, and the positions on the sequences corresponding to the tsp are shown by asterisks. Vertical lines mark -IO regions of the fusion promoters. Oligo S-GTATGCTTGGCAGTCGT, complementary to the nt sequences -I20 to -136 relative to the start codon of phel, was used in p~mer-extension analysis. Total RNA (20 up), purified from P. put~d# cells according to Blomberg et al. (1990), was used as the template. The RNA samples were brought to the final volume of 50 pi and incubated for 30 min at 37°C with 10 units of avian reverse transcriptase (Promega, ‘Madison, WI) in the presence of dCTP, dGTP and dTTP (0.15 pM each), 50 mM Tris pH 8.3/75 mM KCljlO mM MgCIJO.5 mM s~rmidine/S pCi of [a-32P]dATP and 20 units of placental RNase inhibitor (Promega, Madison, WI). After the addition of dNTPs to the final concentration of 0.5 mM each, the incubation was prolonged 30 min for strand extension. The reactions were stopped by phenol extraction, and then the samples were denatured with 0.2 M NaOH for 5 min, neutralized with 0.3 M Na-acetate pH 4.5, and ethanol-precipitate.

instead of TGTATG in the case of RA3 flanked the end of the Tn in the fusion promoters PRA7 and PRA3 (Fig. 2). Insertion of TA between the left end of Tn46.52 and the 5-bp direct repeat TACTT of LA1 generated the -10 sequence TATACT in the fusion promoter PLAl

(Table I and Fig. 2). Thus, two distinct events, transposi- tion of Tn46.52 into DNA containing the potential -10 sequence and up-promoter mutation in this sequence, were required for the generation of the fusion promoters PRA3, PRA7 and PLAI.

28

Previous studies have shown that after a long delay the rates of certain kinds of mutations, i.e., just those which adapt the cell to an available energy source, increase in stationary-phase cells under selective condi- tions (Shapiro, 1984; Cairns et al., 1988; Hall, 1988; Stahl, 1988; Boe, 1990; Boe and Marinus, 1991).

Several models for the generation of the adaptive mut- ations have been proposed. Stahl (1988) suggests that the starved cells maintain some metabolism by the digestion of macromolecules for energy and building blocks. The replication of the DNA segments, occasionally nicked and single-stranded, may introduce some mistakes pro- ducing heteroduplex. If the transcribed strand of the het- eroduplex carries a desirable mutation, it can be transcribed and translated into a mutant gene product allowing cell growth and the stabilization of the mutation by replication of the intermediate DNA and segregation of it into one of the daughter cells. Boe (1990) supports the mechanism originally proposed by Stahl (1988) and also proposes that transcription of heteroduplex DNA may inhibit the DNA proofreading enzymes and thereby facilitate the fixation of an adaptive mutation.

Several experiments show that transposition is increased in stationary-phase cells (reviewed by Fennewald et al., 1981). It is also known that transposi- tion produces single-stranded DNA regions. We suppose that the more mutable single-stranded regions of DNA created during transposition increased the frequency of up-promoter mutations in potential -10 regions in the target DNA. The potential -35 hexamer TTGCCT of the fusion promoters, which is located at both ends of Tn4652, could provide the signal for recognition by RNA polymerase. In the event that the DNA flanking the end of the inserted Tn contains a functional -10 sequence, RNA polymerase initiates transcription of the PM0 gene, and the cells utilize phenol for growth on phenol- containing minimal media and fix the up-promoter muta- tion during replication and cell division.

We wish to thank Dr. Mikael Skurnik and Prof. Richard Villems for their helpful comments on this paper.

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