Microbiology Department, National University of Ireland, Cork, Ireland1
Department of Industrial Microbiology, National University of Ireland, Dublin, Ireland2
Institut für Biotechnologie, ETH Hönggerberg, CH-8093 Zurich, Switzerland3
Author for correspondence: Alan D. W. Dobson. Tel: +353 21 4902743. Fax: +353 21 4903101. e-mail: a.dobson{at}ucc.ie
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ABSTRACT |
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Keywords: Pseudomonas putida, styrene, induction, catabolite repression
Abbreviations: PAA, phenylacetic acid; PACoA, phenylacetateCoA; SMO, styrene monooxygenase
The GenBank accession number for the sequence determined in this work is AF257095.
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INTRODUCTION |
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Styrene lacks true xenobiotic status however as it is naturally produced in the environment by the decarboxylation of cinnamic acid in decaying plant material (Shirai & Hisatsuka, 1979 ). Given the potential long-term exposure of various microbial communities present in the environment to styrene, it is not surprising perhaps that a variety of microbial species capable of degrading this aromatic compound have been isolated and identified (Baggi et al., 1983
; Hartmans et al., 1989
; OConnor et al., 1995; Warhurst et al., 1994
; Panke et al., 1998; Cox et al., 1993
). Two major routes of aerobic styrene degradation are known to exist: (1) initial oxidation of the vinyl side-chain and (2) direct cleavage of the aromatic nucleus (Baggi et al., 1983
; Hartmans et al., 1990; Warhurst et al., 1994
; OConnor et al., 1995). Several intermediates are sequentially produced during side-chain oxidation, which proceed through phenylacetic acid (PAA) (Baggi et al., 1983
; Hartmans et al., 1990
; OConnor et al., 1995
). Ring oxidation results in the formation of styrene cis-glycol and 3-vinyl catechol (Warhurst et al., 1994
). It has been demonstrated previously that styrene degradation by Pseudomonas putida CA-3 proceeds via initial side-chain oxidation and can be divided into an upper pathway involving styrene, styrene oxide and PAA, and a lower pathway which begins with PAA (OConnor et al., 1995
).
Genetic studies have identified the genes involved in the upper pathway conversion of styrene to PAA in a number of styrene degraders of the genus Pseudomonas (Panke et al., 1998 ; Beltrametti et al., 1997
; Marconi et al., 1996
; Velasco et al., 1998
). However, information regarding the lower pathway involved in PAA degradation is limited despite the identification of the first gene which encodes a phenylacetateCoA (PACoA) ligase enzyme in a number of different Pseudomonas strains (Martinez-Blanco et al., 1990
; Vitovski, 1993
; Minambres et al., 1996
; Velasco et al., 1998
; Ferrandez et al., 1998). Potential regulatory genes styS and styR have also been isolated and sequence homology analyses have suggested strong links between these genes and those involved in other two-component regulatory systems (Velasco et al., 1998
).
Despite the fact that much of the styrene side-chain oxidation degradative pathway has been elucidated both at the biochemical and genetic level, little attention has focused on studying the physiological factors affecting the regulation of the pathway. Information such as this may help to facilitate the potential use of styrene-degrading strains in biological filters with the potential to convert styrene, present in a variety of industrial emissions, to less recalcitrant or innocuous pathway intermediates. In addition, it may help in the manipulation of the metabolic pathways for biotransformation applications, such as in the production of optically pure chemicals with broad chemical versatility (Panke et al., 1998 ; Di Gennaro et al., 1999
).
In this study, we report on the inducive and repressive effects of various culture conditions on the styrene catabolic pathway(s) of P. putida CA-3 at both the physiological and genetic level by examining variations in catabolic enzyme activities, and in the transcription levels of genes encoding these enzymes, under different culture conditions.
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METHODS |
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Enzyme assays.
Styrene monooxygenase (SMO) activity was monitored using the indole to indigo assay as previously described (OConnor et al., 1997 ). PACoA ligase activity was measured using the assay method of Martinez-Blanco et al. (1990)
. Activities are expressed as nmol product formed min-1 (mg protein)-1 for both assays. Cells were harvested at mid-exponential phase unless otherwise stated.
Physiology and induction/repression studies.
P. putida CA-3 was cultured solely on styrene, PAA, citrate and glucose, and the effects of these various carbon sources on the degradative pathway(s) examined. In the catabolite repression studies, cells were cultured in the presence of styrene+citrate, styrene+glucose, PAA+citrate, PAA+glucose and styrene+PAA. All carbon sources were added at the concentrations previously outlined. In experiments to assess the effect of adding PAA to a styrene-growing culture, PAA was added in early exponential phase (OD540) to a final concentration of 10 mM. Cultures were then incubated for a further 30 min, after which time cells were harvested and subjected to enzyme assays and RNA isolation for RT-PCR. A second styrene-growing culture, to which no PAA was added, acted as a control.
HPLC analysis.
In the catabolite repression studies, samples were taken for HPLC analysis to monitor concentrations of the repressing carbon sources. Sampling was performed by taking 1 ml of culture and filtering through a 0·45 µm sterile syringe filter (Schleicher & Schuell) into a HPLC vial stored on ice. Samples were then analysed on an LKB Bromma 2150 HPLC system with a Shodex R1-71 refractive index detector and a Highchrom heating block. A Rezex 8m%H organic acid column (300x7·8 mm; Phenomenex) was used with 0·005 M H2SO4 as the elution fluid, at a flow rate of 0·6 ml min-1. The temperature of the column was maintained at 65 °C. Peaks and concentrations were determined by comparison of retention times with known standards.
Nucleic acid isolation and manipulation.
Genomic DNA, isolated from P. putida CA-3 by the method of Ausubel et al. (1987) , was used together with oligonucleotide primer pairs in the PCR cloning steps. The following primer pairs were successful in identifying target gene homologues in our strain: S51/K51 (S51, 5'-GGTTGAGCATGTAGGACGGT-3', and K51, 5'-GCCAATACCGCCTTGCTTGA-3', produced a 540 bp fragment of the paak gene); stySR1/F1 (R1, 5'-TGCGGGCAGCTCTACTTGGAAAAT-3', and F1, 5'-CTGGCGGAAGGGCGGAACATC-3', generated a 750 bp styS gene fragment); styRR1/F1 (R1, 5'-CGCCCCTTTCAAACGATTCAT-3', and F1, 5'-ATGACCACAAAGCCCACAGTA-3', generated a 590 bp styR gene fragment); smaR1/F1 (R1, 5'-GGCCGCGATAGTCGGTGCGTA-3', and F1, 5'-AGAAAAAGCGTATCGGTATT-3', generated the complete 1247 bp styA gene); styDR1/F1 (R1, 5'-GTAGGCGATAACCAACGAGCG-3', and F1, 5'-ATGACAAGGAGCCTAACCATGAAC-3', amplified the complete 1508 bp styD gene); crcR1/F1 (R1, 5'-GCGGCGCATGCTGGGAGAA-3', and F1, 5'-TGTGATCAGCGGCTTAGGTTT-3', generated a 900 bp fragment of the crc gene). These PCR products were cloned into Topo TA vector (Invitrogen), according to the manufacturers instructions. Sequencing reactions were performed via CEQ 2000 dye terminator cycle sequencing (Beckman Coulter) and analysed on a 373 DNA stretch sequencer (Perkin Elmer Biosystems).
The above primers (with the exception of crcR1/F1) were also utilized in the analysis of gene transcription levels by RT-PCR. RNA was isolated according to Ausubel et al. (1987) and 1 µg reverse-transcribed with 1 µl 10 mM Random Primer (Boerhinger Mannheim), 1 µl 10 mM dNTPs (Boerhinger Mannheim), 2 µl BSA (1 mg ml-1), 4 µl 5x buffer (Promega), 40 U RNasin (Promega) and 200 U MMLV-RT (Promega). Reactions were made up to 20 µl with diethylpyrocarbonate (DEPC)-treated demineralized water and incubated for 1 h at 37 °C to generate cDNA. Two microlitres of the RT reaction was then used as a template for subsequent PCR with the appropriate primer pair(s). The number of amplification cycles used was optimized to avoid reaching a point at which band intensities, representing differing gene expression levels within cells, would be misleading due to a plateau of amplification having been reached.
Selected oligonucleotide primers were recombined into pairs suitable for PCR analysis of genomic DNA and cDNAs generated by the reverse transcription process. The recombined pairs were: K51/stySR1, stySF1/styRR1, styRF1/smaR1 and smaF1/styDR1. PCR products amplified were partially sequenced to establish their identity.
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RESULTS |
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When CA-3 genomic DNA was subjected to PCR analysis using recombined oligonucleotide primers the following observations were made. K51/stySR1 generated a 2329 bp product containing N-terminal paak and C-terminal styS homologous regions while styRF1/smaR1 produced a 2052 bp fragment which, when subjected to nested PCR with the appropriate primers, was found to contain the styR and styA genes. Thus the genetic organization of the styrene catabolic operons in CA-3 appears identical to those of the highly homologous styrene-degrading Pseudomonas strains Pseudomonas fluorescens ST and Pseudomonas sp. strain Y2 (Fig. 1a).
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Induction studies
Table 1 shows the results obtained when an overnight culture of P. putida CA-3 was inoculated into MS media containing one of the following carbon sources: styrene, PAA, glucose or citrate. Both SMO and PACoA ligase activities were detected in the MS media containing styrene with mRNA transcripts being detected for the stySR regulatory genes as well as the styA and paak upper and lower pathway genes, respectively, by RT-PCR. While culturing on PAA did result in PACoA ligase activity, no detectable SMO activity was present. These effects were mirrored at the transcription level with only paak, and not styA or stySR, mRNA transcripts being detected under these culture conditions. Growth of the organism on glucose or citrate did not induce any detectable enzymic activity or expression at the transcriptional level from the styrene catabolic operon.
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DISCUSSION |
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PCR analysis of the CA-3 genome with various primer combinations has allowed us to map the structural organization of the styrene degradative pathway in this strain. Fig. 1(a) illustrates the high degree of structural conservation between the styrene catabolic operons of P. putida CA-3, P. fluorescens ST and Pseudomonas sp. strain Y2. One common structural feature of these pathways is that all genes thus far identified are transcribed in the same direction. We therefore attempted to determine if discrete operons existed within the pathway. Total RNA from a styrene-grown culture of CA-3 was chosen for analysis as this growth condition allows for detection of paak, stySR and styA gene mRNA transcripts (Table 1
). Despite the success of the primer combinations K51/stySR1 and styRF1/smaR1 in amplifying appropriate regions of the CA-3 genome, their application in PCR analysis of cDNA produced from styrene-grown cells failed to generate products. Therefore, it appears that a transcriptional termination signal(s) exists between the paak and the styR genes which prevents readthrough transcription into the regulatory elements styS and styR. This conclusion is supported by recent work involving the promoter region of stySR from the styrene-degrading P. fluorescens ST (Santos et al., 2000
). Similarly, it would appear that a transcriptional termination signal(s) exists between styR and styA, which prevents the progress of RNA polymerase into the upper pathway genes beginning with styA. The primer pairs stySF1/styRR1 and smaF1/styDR1 generated RT-PCR products of 3571 and 3932 bp, respectively (Fig. 1b
). This indicates that styS and styR are co-transcribed and expression of the upper pathway genes involves a single polycistronic mRNA. The pathway therefore appears to be composed of at least three discrete operons from which transcription occurs.
Complementation studies in Escherichia coli with elements of the Pseudomonas sp. strain Y2 styrene degradative pathway have identified a key role for stySR in the positive regulation of the upper pathway genes (Velasco et al., 1998 ). The styS gene encodes a sensor kinase which becomes phosphorylated due to the intracellular presence of styrene. This results in a phosphorylation cascade event causing the response regulator, StyR, to become active. Amino acid comparisons of the gene products from the 3·5 kb stySR region of strain CA-3 with those of Pseudomonas sp. strain Y2 and P. fluorescens ST identified highly conserved regions consistent with functional domains identified in other two-component systems (Lau et al., 1997
; Coschigano & Young, 1997
). The probability that styR functions as a response regulator in CA-3 is further supported by the observation that, as in strain Y2, a potential DNA-binding site with the pallindromic sequence ATAAACCATGGTTTAT, centred at position -41 of the upper pathway promoter region, is also present in strain CA-3. As both strains lack a putative -35
-factor-binding site in the promoter region, it is likely that StyR exerts control over the upper pathway by binding at the -41 region and attracting RNA polymerase to the -10 TGTTAGCTT sequence upstream from styA (Barne et al., 1997
; Velasco et al., 1998
). This mechanism is very similar to the effect mediated by TodT, the response regulator of the tod operon (Lau et al., 1997
). Given the degree of sequence homology between CA-3 and strain Y2 and the highly conserved structural features of the respective catabolic operons (Fig. 1a
), a similar regulatory system is likely to function in our strain.
Induction experiments with strain CA-3 reveal the significance of styrene in expression of the upper and lower pathway enzymes. Table 1 clearly illustrates that detection of SMO and PACoA ligase activities occurs when cells are cultured on styrene, and not when glucose or citrate acts as the sole carbon source. Pathway induction in our strain is controlled at the transcriptional level since RT-PCR analysis of the respective genes indicated that transcription of paak, stySR and styA does not occur in the absence of the inducer styrene (Table 1
). These results also indicate a key role for stySR as the two-component mechanism positively regulating the upper pathway given that expression of the upper pathway genes is not observed in the absence of stySR transcription. It should also be noted that cells grown on PAA, while showing increased levels of PACoA ligase enzyme expression, failed to induce transcription of either the upper pathway enzymes or the stySR regulatory molecules. Therefore, while stySR appear essential for induction of the upper pathway genes, they do not play a role in lower pathway induction by PAA.
In P. putida CA-3 an additional level of control exists, where the presence of PAA in the growth medium results in complete repression of the upper pathway, even in the presence of the upper pathway inducer, styrene. RT-PCR analysis of cells grown under these conditions reveals that this effect is mediated by repressing transcription of the two-component stySR genes (Fig. 2b, c
). Thus, PAA acts as a negative regulator of the upper pathway genes. This is in contrast to the styrene-degrading strain Xanthobacter 124X, as growth of the bacterium on PAA results in detectable levels of activity from upper-pathway-associated enzymes styrene oxide isomerase and phenylacetaldehyde dehydrogenase (Hartmans et al., 1989
). Furthermore, a recent study with P. fluorescens ST suggested that stySR transcription is constitutive regardless of the carbon source (Santos et al., 2000). Therefore, while a common route for styrene catabolism is observed in many of the bacterial species studied to date, it is clear that there are significant differences in how these degradative pathways are regulated. The mechanism by which this repressive effect is elicited in strain CA-3 is as yet unknown. To our knowledge this is the first report of transcriptional repression of a two-component regulatory system controlling an aromatic hydrocarbon degradative pathway by an intermediate of the pathway.
We have previously reported on the repressive effect of citrate and other nonaromatic carbon sources such as glutamate on styrene degradation in P. putida CA-3, by assessing oxygen uptake rates by cell-free extracts (OConnor et al., 1995 ). Here, we demonstrate that the effect of catabolite repression is reduced transcription of both upper and lower pathway genes together with a reduction in stySR transcript levels. HPLC analysis of cultures grown in the presence of both styrene and citrate reveals that this repressive effect is sustained only while citrate is present in the media (data not shown). Depletion of the alternative carbon source coincides with increased upper and lower pathway gene expression, together with detectable levels of SMO and PACoA ligase enzyme activity, indicating growth on styrene (Fig. 4a
, b
). However, catabolite repression by citrate does not result in complete inhibition of gene transcription as paak, styS, styR and styA mRNA transcripts were detected during the early growth phase (Fig. 4ad
). Despite the low levels of gene transcripts, no enzyme activities were detectable. These observations contrast with those made when CA-3 is cultured on styrene and PAA, where complete inhibition of the stySR regulatory genes, and subsequently the upper pathway genes, occurs (Fig. 2c
). Therefore the repression of the upper pathway by PAA appears to be exerted by a different mechanism to catabolite repression mediated by citrate. PAA specifically inhibits expression of the StySR regulatory molecules while citrate affects transcription of both upper and lower pathway genes. Citrate also exerts a similar repressive effect on PAA metabolism in strain CA-3. Reduced gene transcription is observed in cultures grown on PAA and citrate as long as citrate is present in the media (data not shown). This suggests that catabolite repression involves a more general cellular regulation mechanism rather than inhibition of specific targets as exhibited during growth of CA-3 on styrene and PAA. Using PCR primers based on the P. putida crc gene, a 900 bp crc homologue has been isolated from the P. putida CA-3 genome. The catabolite repression control (Crc) protein has previously been shown in P. putida and Pseudomonas aeruginosa (Hester et al., 2000
) to act as a structure-specific ribonuclease which down-regulates the expression of a branched-chain keto acid pathway, by degrading mRNA of branched-chain keto acid dehydrogenase, encoded by bkdR, the positive regulator of the pathway. Therefore, given that reduced mRNA transcript levels for both styS and styR regulatory genes are observed during catabolite repression, and that a crc homologue is present, the possibility exists that a similar mechanism of control is being exerted in our strain. Further work is currently under way to explore this possibility.
In conclusion, the results presented here on the transcriptional repression of the styrene catabolic operon by metabolic intermediates of the pathway, as well as by nonaromatic carbon sources, may have implications regarding the suitability of this, and other, styrene-degrading strains for use in a variety of biotechnological applications.
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Received 13 September 2000;
accepted 4 December 2000.