Department of Genetics, Institute of Molecular and Cell Biology, Estonian Biocentre and Tartu University, Riia 23, 51010 Tartu, Estonia1
Author for correspondence: Maia Kivisaar. Tel: +372 7 375015. Fax: +372 7 420286. e-mail: maiak{at}ebc.ee
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ABSTRACT |
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Keywords: pheBA, catBCA, operons, exponential silencing of transcription
Abbreviations: CCM, cis,cis-muconate; ß-Gal, ß-galactosidase
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INTRODUCTION |
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In Pseudomonas species, phenolic compounds are transformed by different enzymes to central intermediates, such as protocatechuate and (substituted) catechols (Harayama & Timmis, 1989 ). Typically, unsubstituted compounds, such as benzoate, are metabolized by an ortho-cleavage pathway (Fig. 1
). The genes for benzoate metabolism, including ortho-pathway genes, are chromosomally encoded in P. putida (Harwood & Parales, 1996
). The catBCA operon encodes three enzymes of the ortho-pathway required for benzoate catabolism, namely muconate lactonizing enzyme I, muconolactone isomerase and catechol 1,2-dioxygenase, respectively (Houghton et al., 1995
). The induction of this operon, which is
54-independent, requires a LysR family transcriptional activator, CatR, and an inducer molecule, cis,cis-muconate (CCM), an intermediate of the ortho-pathway (Rothmel et al., 1990
, 1991
). The pheB and pheA genes originating from plasmid DNA of Pseudomonas sp. EST1001 encode catechol 1,2-dioxygenase and phenol monooxygenase, respectively (Kivisaar et al., 1990
). When the pheBA operon is introduced into P. putida, the bacteria acquire the ability to degrade phenol (Kivisaar et al., 1991
, 1990
). The pheBA promoter resembles the catBCA promoter and is also activated by CatR (Kasak et al., 1993
; Parsek et al., 1995
). Comparative studies of the interaction of CatR with the promoters of the pheBA and catBCA operons have revealed that the CatR-mediated activation mechanism is well conserved, despite the different origins of these operons (Parsek et al., 1995
; Tover et al., 2000
).
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METHODS |
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To investigate the regulation of transcription from the pheBA and catBCA promoters in minimal medium, P. putida cells were grown overnight in MOPS medium (Neidhardt et al., 1974 ) either with 0·5% CAA or amino acids at a final concentration of 200 ng each amino acid ml-1. The overnight bacterial culture was diluted with fresh medium (1:20, v/v) grown to exponential phase and then diluted again in minimal medium containing 0·2% glucose. The OD580 of the diluted culture was 0·1. Sodium benzoate was added at a final concentration of 10 mM at the beginning of the experiment and 1 ml samples were taken at different time points during growth to assay the ß-Gal activity in cell suspensions.
To investigate the possible effect of overexpression of the CatR protein on the transcription from the pheBA and catBCA promoters, the culture was grown overnight in LB medium and thereafter diluted into fresh LB medium as indicated above. To induce the expression of CatR, IPTG (at a final concentration of 0·5 mM) was added to the growth medium. When the culture reached the exponential growth phase, it was diluted again into fresh LB medium containing 0·5 mM IPTG. At this step, 5 mM sodium benzoate was added for the induction of transcription from the pheBA and catBCA promoters.
A ß-Gal assay with the cell lysates was carried out as specified by Miller (1992) . In all cases at least three independent measurements were made. Protein concentration in crude lysates was measured by the method of Bradford (1976)
. ß-Gal measurement in cell suspensions was performed by modification of the standard protocol of Miller (1992)
. The amount of Z buffer (60 mM Na2HPO4, 40 mM NaH2PO4, 10 mM KCl, 1 mM MgSO4 and 50 mM ß-mercaptoethanol) in a test tube was 1·6 ml. ONGP (2-nitrophenyl-ß-D-galactopyranoside; 400 µl of a 50 mg ml-1 solution), 100 µl 0·002% SDS and 100 µl chloroform were added into the reaction mixture. Finally, bacteria were added into the test tube and mixed well. The ß-Gal reaction was stopped by the addition of 1 ml 1 M Na2CO3. Data of at least three independent experiments are presented in all figures.
Cloning procedures.
Construction of the pheBA promoterlacZ transcriptional fusion (plasmid pZ-pheBA) was described previously (Tover et al., 2000 ). For the cloning of the catBCA promoter into the promoterprobe plasmid pKTlacZ (Hõrak & Kivisaar, 1998
), the 207 bp catBCA promoter region from the P. putida chromosome was amplified by using oligonucleotides cat35 (5'-GGGCTGCCAGCCGCGGGCCC-3') and AFC62 [5'-AGCGCGGCGGCTCGACGACGCTG(PstI)CAGAGC-3'], complementary to the upstream and downstream regions of the catBCA promoter, respectively. The PCR amplification product was cleaved with PstI and cloned into pBluescript SK(+) cleaved with PstI and EcoRV. The sequence of the catBCA promoter was verified by DNA sequencing. Subsequently, the catBCA promoter was inserted using BamHI- and XhoI-generated ends into the promoterprobe vector pKTlacZ to obtain plasmid pZ-catBCA. For the construction of P. putida CatR overexpression strain PaWCatR+, the catR gene was cloned from plasmid pKR
HF (Rothmel et al., 1991
) by using HindIII- and EcoRI-generated ends to the vector pBRlacItac (Ojangu et al., 2000
) cleaved with same enzymes (pBRlacItac-catR in Table 1
). The CatR expression cassette lacIq-Ptac-catR was inserted into pUC18Not (Herrero et al., 1990
) using EcoRI- and BamHI-generated ends. Thereafter, the lacIq-Ptac-catR sequence was inserted into the NotI site of pUTmini-Tn5 Km2 (de Lorenzo et al., 1990
) and pUTlacItac-catR was selected in E. coli C118
pir (Herrero et al., 1990
). The lacIq-Ptac-catR expression cassette was inserted into the chromosome of P. putida strain PaW85 (Bayley et al., 1977
) by the method of random insertion using E. coli S17-1
pir (Miller & Mekalanos, 1988
) as donor strain. P. putida PaW85CatR+ was selected at 30 °C on glucose/kanamycin plates. The expression of CatR in PaW85CatR+ was verified by Western blot analysis using polyclonal antibodies against P. putida CatR protein.
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RESULTS AND DISCUSSION |
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We have previously constructed a P. putida S-deficient strain, PKS54, that is a derivative of the wild-type strain PaW85 (Ojangu et al., 2000
). Plasmids pZ-pheBA and pZ-catBCA, containing the pheBAlacZ and catBCAlacZ transcriptional fusions, respectively, were introduced into the rpoS-deficient strain, PKS54, and into the wild-type strain, PaW85. The level of expression of ß-Gal activity measured in exponential- and stationary-phase cells of PKS54 was compared with that estimated in PaW85. The results presented in Fig. 2
show that in the case of the pheBA promoter, ß-Gal activity remained approximately threefold lower in the
S-deficient strain than in the wild-type strain in stationary-phase cultures (Fig. 2a
). At the same time, lack of expression of
S in P. putida cells did not affect transcription from the catBCA promoter (Fig. 2b
). The sequence of the -10 region of the catBCA promoter (CAATCCT) shows more similarity to the consensus CTATACT proposed for the promoters recognized by E
S than that of the pheBA promoter (CTAGCTT). Based on in vivo experiments presented in this paper we cannot state that the pheBA promoter is recognized by
S. However, the nucleotide sequence of the -10 region of the promoter is only one component that determines
S-dependent transcription. There is increasing evidence that additional regulators play a crucial role in establishing sigma factor specificity at stress-inducible promoters (Hengge-Aronis, 1999
). Moreover, coming back to the sequence determinants, we have recently shown on fusion promoters generated in a starving population of P. putida that not only the -10 hexameric sequence, but also sequence downstream from the -10 hexamer is important for
S-dependent transcription (Ojangu et al., 2000
).
The positive effect of S observed in the case of the pheBA promoter can give P. putida cells a little advantage to use phenol as a single source of carbon and energy under stressful conditions. However, this effect is insufficient to account for the inhibition of the pheBA promoter during exponential growth. Moreover, transcription from the catBCA promoter was not influenced by the presence of
S in P. putida cells. It is obvious, therefore, that stationary-phase-specific transcription from the pheBA and catBCA promoters must be regulated by some other mechanism than
S-mediated control.
Modulation of pheBA and catBCA promoter activity by growth phase does not operate through the amount of regulator protein CatR
Transcriptional activation from the pheBA and catBCA promoters requires the presence of CatR and an inducer molecule CCM (Kasak et al., 1993 ; Parsek et al., 1995
). Therefore, we examined whether the amount of the regulator protein CatR would be limiting in transcriptional activation from the pheBA and catBCA promoters in exponentially growing cells. To study the expression of the catR gene during the growth cycle, the promoter of the catR gene was cloned into plasmid pKTlacZ to generate a catRlacZ transcriptional fusion. The activity of this fusion was compared in the wild-type P. putida strain PaW85 and in its rpoS-deficient mutant PKS54. In both strains, the level of ß-Gal expression increased in stationary-phase cells when compared to that observed in exponentially growing cells (Fig. 3
). Therefore, although catR promoter activity remained at a very low level during all growth phases of the bacteria, the intracellular amount of CatR (undetectable by Western blot analysis) may be somewhat increased in stationary-phase cells.
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Exponential silencing of the pheBA and catBCA promoters is dependent on the nature of the culture medium
The role of growth medium in the regulation of gene expression has been shown in many cases. For example, growth medium composition (either rich or minimal) determines the level of transcription from the Po promoter of the operon (dmp) encoding dimethylphenol degradation (Sze & Shingler, 1999 ; Sze et al., 1996
), from the Pu promoter of the TOL plasmid pWW0 (Cases et al., 1996
; de Lorenzo et al., 1993
) and from the PalkB promoter from the Pseudomonas oleovorans alkane degradation pathway (Yuste et al., 1998
). We found that transcription from the pheBA and catBCA promoters was rapidly activated when bacteria were grown in MOPS minimal medium (Fig. 4
). Measurement of ß-Gal activity in cells sampled at different time points of an exponentially growing culture of P. putida PaW85 allowed us to detect the enzyme activity as early as 20 min after the addition of sodium benzoate.
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Transcription from the 54-dependent Po promoter of the dimethylphenol (dmp)-degradation pathway is silenced in rich medium until amino acids become limiting (Sze & Schingler, 1999
). The requirement of (p)ppGpp for activation of transcription from the Po promoter was demonstrated using overexpression of RelA and E. coli (p)ppGpp-deficient mutants (Sze & Schingler, 1999
). We were not able to demonstrate the positive role of (p)ppGpp on transcriptional activation from the pheBA and catBCA promoters. The absence of an effect of (p)ppGpp on transcription from these promoters was confirmed by experiments where the (p)ppGpp level was artificially increased either by adding 1 mM serine hydroxamate into the growth medium or by using RelA overexpression plasmid pVI536 (Sze & Schingler, 1999
) in P. putida cells carrying the pheBAlacZ transcriptional fusion in the chromosome (data not shown).
In the (p)ppGpp-dependent Po regulatory system, distinct groups of amino acids were not able to mediate the delay in transcription (Sze & Schingler, 1999 ). We found that specific groups of amino acids could cause partial silencing of transcription from the pheBA and catBCA promoters. Bacteria were grown in MOPS minimal medium containing glucose for 60 min in the presence of sodium benzoate. No amino acids or a different number of amino acids (all 20, 15 or 5) were added into the growth medium. The sets of amino acids were designed according to their biosynthetic pathways. The third group consisted of 5 amino acids (Asp, Asn, Glu, Gln and Ser) that are precursors for several other amino acids, and the second group contained the other 15 amino acids. In the absence of amino acids, the level of ß-Gal activity was 950±44 Miller units in the case of the pheBA promoter and 397±50 Miller units in the case of the catBCA promoter. In the case of the pheBA promoter, no expression of ß-Gal activity could be detected when all amino acids were added, but the presence of 5 and 15 amino acids allowed partial expression of this promoter: the ß-Gal activities were 91±3 and 196±20 Miller units, respectively. The repressive effect of amino acids on transcription from the catBCA promoter was lower than that observed with the pheBA promoter. The ß-Gal activity was 12±2·5 Miller units when all amino acids were added into the growth medium and it was approximately half of that observed without amino acids: 178±18 and 199±50 Miller units in the presence of 5 and 15 amino acids, respectively. This also indicates that the physiological control on the pheBA and catBCA promoters mediated by the presence of amino acids might be different from the mechanism related to stringent response. The occurrence of the partial silencing effect by different groups of amino acids on the transcription from the pheBA and catBCA promoters excludes the possibility that one particular amino acid could mediate this effect.
At this stage the mechanism by which the presence of amino acids causes repression of these two promoters is unclear. Data presented in Fig. 4 and results obtained in experiments with different sets of amino acids show that the exponential silencing of the pheBA promoter is stronger than that of the catBCA promoter. The nucleotide sequences of the pheBA and catBCA promoters are similar, but not identical. Differences become most apparent on sequences located downstream from the CatR-binding sites and -35 hexamers of the promoters (Fig. 1
). Analysis of the locations of regulatory sites of
70-dependent promoters has revealed that repressors usually bind downstream from -30 (Gralla et al., 1996
). It is possible that a hypothetical repressor protein could bind to the pheBA and catBCA promoters with different efficiency due to sequence differences of the target DNA. However, despite the minor differences in the expression level of the pheBA and catBCA promoters under certain growth conditions, the general mechanisms for physiological control of these promoters seem to be well conserved to coordinate the expression of the hybrid plasmid-chromosome-encoded pathway for phenol degradation in P. putida.
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ACKNOWLEDGEMENTS |
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Received 11 December 2000;
revised 19 March 2001;
accepted 12 April 2001.