INTRODUCTION

Top Abstract Introduction Materials & Methods Results & Discussion References

Styrene is used in large quantities by the chemical industry, but it can also occur naturally, mostly by decarboxylation of cinnamic acid (42). Airborne emissions of styrene, even at low concentrations, often cause a problem because of their malodorous character and their toxic and carcinogenic effects (42). The removal of styrene from industrial waste gases could be accomplished by using styrene-degrading bacteria as biocatalysts; however, little is known concerning the microbial metabolism of styrene (6, 42). Two main routes of aerobic styrene breakdown have been described: (i) oxidation of the vinyl side chain with the formation of phenylacetate and (ii) initial oxidation of the aromatic nucleus (42).

The only information about the organization of the styrene catabolic genes has been recently obtained for Pseudomonas fluorescens ST (6, 25). This strain degrades styrene by oxidation of its lateral chain, and it has been shown that the upper pathway for the conversion of styrene to phenylacetate is encoded by four catabolic genes, styABCD (Fig. 1A). Although 2-phenylethanol accumulates in styrene-grown cells (25), genetic and biochemical analyses of the styABCD cluster have suggested that this compound is not an intermediate of the styrene catabolism in P. fluorescens ST (6). However, 2-phenylethanol appears to be an intermediate of styrene catabolism in Corynebacterium sp. strain ST-10 (19). In this sense, it has been also proposed that Pseudomonas sp. strain Y2 degrades styrene via 2-phenylethanol and phenylacetate (40). Therefore, it appeared interesting to investigate if the styrene upper catabolic pathway of this microorganism was different from that of P. fluorescens ST. Moreover, although there is now some biochemical and genetic information about the catabolism of styrene via lateral chain oxidation (6, 10, 16, 17, 25, 29, 40, 42), there are still many aspects that need to be studied. For instance, there are no genetic data about the regulation of the styrene upper pathway, and the genes responsible for the catabolism of phenylacetate (lower pathway) remain to be investigated.

View larger version (26K): [in this window] [in a new window]   FIG. 1.   Pathway for the catabolism of styrene in Pseudomonas sp. strain Y2 and genetic organization of the corresponding structural and regulatory genes. (A) Biochemistry of the pathway. Metabolites: styrene (compound I), epoxystyrene (compound II), phenylacetaldehyde (compound III), phenylacetate (compound IV), and phenylacetyl-coenzyme A (compound V). Enzymes: StyAB, styrene monooxygenase; StyC, epoxystyrene isomerase; StyD, phenylacetaldehyde dehydrogenase; PaaK, phenylacetyl-coenzyme A ligase. (B) Physical and genetic map of the chromosomal region encoding styrene catabolism. Locations of the genes are shown relative to those of some relevant restriction sites. Arrows indicate the direction of gene transcription. The cloned fragments (thick line) and their orientations with respect to the lacZ promoter (bent arrow) of plasmid pUC18 or pUC19 are indicated. Restriction sites: B, BamHI; Bg, BglII; C, ClaI; E, EcoRI; P, PstI; S, SmaI; Sa, SalI. Asterisks mean that other identical restriction sites are present.

This report describes the genetic characterization of the styrene upper catabolic cluster of Pseudomonas sp. strain Y2 and provides evidence of a mechanism of regulation that is unusual for the catabolism of aromatic compounds. In addition, the gene encoding the first step for phenylacetate degradation has been located and characterized.

	MATERIALS AND METHODS

Top Abstract Introduction Materials & Methods Results & Discussion References

Bacterial strains and plasmids. The bacterial strains used were Escherichia coli W ATCC 11105 (13), E. coli W14 (an E. coli W mutant deficient in the catabolism of phenylacetate) (13), E. coli DH5 (36), and Pseudomonas sp. strain Y2 (40). Plasmids pUC18 and pUC19 were used for cloning purposes (36). Bacteria were grown with shaking at 30 or 37°C in Luria-Bertani (LB) medium (36) or at 30°C in minimal medium M63 (27), using as carbon source 20 mM glycerol or a styrene-saturated atmosphere. Where appropriate, ampicillin (100 µg/ml), thiamine (1 µg/ml), vitamin B₁₂ (5 ng/ml), and 1 mM indole were added.

DNA and RNA manipulations. DNA and RNA manipulations and other molecular biology techniques were carried out essentially as described elsewhere (36). Total RNA was extracted as previously described (1). Southern and Northern blotting as well as colony hybridization analyses were performed as previously reported (36), using as probes DNA fragments labeled with digoxygenin or [-³²P]dCTP by using a Dig Luminescent Detection kit (Boehringer) or the random primer method (Pharmacia), respectively. Primer extension reactions were carried out with avian myeloblastosis virus reverse transcriptase (34). Pulsed-field gel electrophoresis was performed as previously described (37). Nucleotide sequences were determined by using a model 377 automated DNA sequencer (Applied Biosystem Inc.). Nucleotide and protein sequence similarity searches were done with the BLASP, BLASTN, and BLASTX programs (2) via the National Institute for Biotechnology Information server. Pairwise and multiple protein sequence alignments were done with the ALIGN (44) and CLUSTAL W (38) programs, respectively, at the Baylor College of Medicine-Human Genome Center server.

Phenylacetate production assay. E. coli W14(pSTY) cells were incubated overnight at 30°C in minimal medium M63 containing 20 mM glycerol, ampicillin, and vitamin B₁₂, in a styrene-saturated atmosphere. Products accumulated in the culture medium were analyzed spectrophotometrically at 220 nm with Gilson high-pressure liquid chromatography (HPLC) equipment, using a Lichrosphere SRP-8 column (150 by 4.6 mm) (mobile phase, 40% methanol; flow rate, 1 ml/min).

Production of PaaK protein in E. coli. The paaK gene was PCR amplified from plasmid pSTY by using oligonucleotides K5 (5'-GGGAATTCCACCAGCTATCGGCGCTCTTC-3') (the EcoRI site introduced is underlined) and S5 (5'-CCCAACACTTCGAACGGAG-3') as primers. The 1.6-kb amplified fragment was digested with EcoRI and ligated to EcoRI-HincII double-digested pUC18, such that in the resulting plasmid pPAAK (Fig. 1), expression of the paaK gene was under the control of the lac promoter. To determine the production of PaaK, E. coli W14(pPAAK) cells were grown overnight at 30°C in LB medium containing 0.5 mM isopropylthiogalactopyranoside. Culture was harvested by centrifugation, washed, and resuspended in 0.05 volume of 0.5 M potassium phosphate buffer (pH 8.0) prior to disruption by passage through a French press. The cell debris was removed by centrifugation, and the clear supernatant fluid was used as a crude extract.

Phenylacetyl-coenzyme A ligase assay. Phenylacetyl-coenzyme A ligase was assayed as previously described (28). One unit of enzyme activity is defined as the catalytic activity leading to the formation of 1 nmol of phenylacetylhydroxamate in 1 min. Protein concentration was determined by the method of Bradford (8), using bovine serum albumin as standard.

Indigo production assay. To quantify styrene monooxygenase activity, indigo production was assayed in resting cells essentially as previously described (6) but using chloroform instead of ethyl acetate to extract the culture samples.

Nucleotide sequence accession number. The nucleotide sequence reported in this study (nucleotides [nt] 1 to 10743) has been submitted to the GenBank/EMBL data bank (accession no. AJ000330).

	RESULTS AND DISCUSSION

Top Abstract Introduction Materials & Methods Results & Discussion References

Cloning and expression of a gene cluster for the catabolism of styrene. We had observed that styrene induces in Pseudomonas sp. strain Y2 an activity that oxidizes indole to the blue dye indigo. To isolate the genes involved in the first steps of the styrene catabolism in Pseudomonas sp. strain Y2, a screening strategy based on the appearance of a blue phenotype in the presence of indole was followed (20, 25, 30). Thus, an EcoRI DNA library of Pseudomonas sp. strain Y2 was constructed in E. coli DH5 by using EcoRI-digested pUC19 vector; and after 2 days of growth on indole-containing LB medium at 30°C, three indigo-positive clones were isolated. All of them showed a plasmid, pUE14, that contained a 12.2-kb DNA insert (Fig. 1). The subcloning of this fragment revealed that the gene(s) encoding the oxygenase activity were localized within the 2.6-kb PstI fragment of pUE14 (plasmid pIP27 [Fig. 1]).

Sequence analysis. To genetically characterize the styrene upper catabolic cluster of Pseudomonas sp. strain Y2, the 15.5-kb insert of plasmid pSTY was sequenced. The nucleotide sequence of the 10,743-bp right region of the insert is shown in Fig. 2. Computer analysis of this sequence revealed the presence of eight open reading frames (ORFs) which may encode the putative PaaK, StyS, StyR, StyA, StyB, StyC, StyD, and PorA proteins (Fig. 2). Databases were searched for similar proteins, and those showing the highest similarity values were then retrieved and compared with the sequences obtained in this study (Table 1). An overall analysis revealed that genes styABCD were nearly identical to the styABCD genes of the styrene catabolic cluster from P. fluorescens ST (Table 1), and the order of these genes corresponded to that of the catabolic steps (Fig. 1). Two regulatory genes (stySR), an ORF (porA) encoding a potential truncated outer membrane protein, and the paaK gene, which codes for a phenylacetyl-coenzyme A ligase, were also identified (Fig. 1 and 2).

View larger version (54K): [in this window] [in a new window]   FIG. 2.   Nucleotide and derived amino acid sequences of the styrene catabolic pathway. Only the sequences of the 5'- and 3'-end coding regions of the genes are shown. Small arrows show the direction of gene transcription. Asterisks indicate the stop codons. Sequence comparison between the region upstream of porA and the equivalent region in the sty operon from P. fluorescens ST (6) revealed that in Pseudomonas sp. strain Y2, a duplication of seven nucleotides (GCGAGCCgcgagcc) at position 9469 could be responsible for the truncation of a longer reading frame that probably started at nt 9357 (large open arrow). The putative transcription termination sequences are underlined. The proposed extended 10 promoter box is double underlined. +1 indicates the transcription start site of the sty operon. The postulated StyR-binding site is boxed.

(i) Catabolic genes. The styA gene encodes a protein of 46,639 Da (415 amino acids [aa] long) (Fig. 2) that shows similarity to several bacterial flavin-type aromatic hydroxylases (Table 1) and contains the three flavin adenine dinucleotide-binding regions also observed in other flavin monooxygenases (11, 43).

(ii) Regulatory genes. The styS and styR genes encode two proteins that show a high similarity with members of the superfamily of two-component signal transduction systems found both in eukaryotes and prokaryotes (3, 32, 35). The styS gene shows two putative ATG start codons at nt 1618 and 1639, respectively (Fig. 2). The stop codon of styS was located at nt 4564 and overlaps the putative ATG start codon of styR (Fig. 2), suggesting that the two genes are likely expressed in a translationally coupled fashion. The large version of StyS is a protein of 108,758 Da (982 aa long) that shows similarity to many sensor histidine kinase proteins (Table 1). Specially relevant was the high similarity between StyS and the TodS and TutC sensor hybrid histidine kinases that regulate the aerobic and anaerobic catabolism of toluene in P. putida F1 (23) and Thauera sp. strain T1 (9), respectively (Table 1 and Fig. 3). These three proteins, together with the BpdS sensor kinase from Rhodococcus sp. strain M5, which responds to biphenyl and polychlorobiphenyl (22), are the unique sensors of the two-component regulatory systems involved in the metabolism of aromatic compounds that have been described so far. The N terminus of StyS (aa 1 to 75) resembles the basic region leucine zipper motif that mediates protein dimerization and DNA binding of TodS (23) (Fig. 3). Another interesting feature of StyS, shared only by TodS and TutC, is the presence of a receiver domain flanked by two canonical kinase domains (Fig. 3). The sequence (aa 570 to 754; Fig. 3, INPUT2) that resembles the putative oxygen-sensing domain of TodS (23) contains the highly conserved S1 and S2 sensory boxes present in PAS domains (47) (Fig. 3), suggesting that this region may act as an input domain in StyS. On the other hand, the N-terminal location of the input domains in most sensor kinases (32, 35) points to the existence of another input domain in StyS (Fig. 3, INPUT1; aa 76 to 187).

View larger version (94K): [in this window] [in a new window]   FIG. 3.   Schematic domain structure of StyS (A) and alignment of its amino acid sequence with those of TodS and TutC sensor kinases (B). Amino acid residues, indicated by the standard one-letter code, are numbered on the right. Separate domains are indicated by differently shaded rectangles. A putative leucine zipper (ZIP) characterized by the repeating heptads (MLLLI) is shown. HK1 and HK2 are two histidine kinase domains characterized by the conserved amino acid blocks known as H, N, G1, F, and G2 (32). Histidine residues that may be phosphorylated in HK1 and HK2 are underlined and in boldface. The receiver domain (RECEIVER) contains the conserved DDSK residues characteristic of bacterial response regulators (4, 32, 41); the conserved serine (S) is often replaced by threonine (T) (Fig. 4). The aspartic acid residue that may be phosphorylated is underlined and indicated in boldface. The rigid group of the -turn loop is shown (). The putative input domains are represented as INPUT1 and INPUT2. Asterisks show the position of the most relevant conserved residues detailed in the StyS domain structure. S1 and S2 indicate the locations of the sensory boxes (47).

View larger version (50K): [in this window] [in a new window]   FIG. 4.   Schematic domain structure of StyR (A) and alignment of its amino acid sequence with that of other bacterial response regulators (B). The sources of TodT, TutB, NodW, and FixJ sequences are indicated in Table 1. Amino acid residues, indicated by the standard one-letter code, are numbered on the right. Separate domains are shown by differently shaded rectangles. Shading code corresponds to that of the bars at the top of the aligned sequences. The receiver domain (RECEIVER) contains the conserved DDTK residues characteristic of bacterial response regulators and are located in the alignment by asterisks (4, 32, 41). The aspartic acid residue that may be phosphorylated is underlined and indicated in boldface. The rigid group of the -turn loop is also shown (). A predicted Q-Linker region (45) between the receiver and DNA-binding domains is indicated. Amino acids underlined by solid bars correspond to the three internal -strands (1, 3, and 4) of the prototype CheY response regulator structure (4). The consensus sequence (Cons) of putative DNA-binding domains in the family 3 response regulators as tabulated by Pao and Saier (31) is in boldface italics. The -helix (a)/turn (t)/-helix (a) secondary structure typical of DNA-binding domains is also indicated.

(iii) The first gene of the styrene lower pathway. The paaK gene shows two putative ATG start codons at nt 39 and 45, but only the first one is preceded by a putative RBS (Fig. 2). The paaK stop codon was located at nt 1350, and 23 bp downstream of this codon a 32-bp palindromic sequence (G = 17.2 kcal/mol) that may act as a transcription terminator can be found (Fig. 2). The large version of PaaK is a protein of 49,074 Da (437 aa long) that shows high similarity to the phenylacetyl-coenzyme A ligase (Pcl) of P. putida U, an enzyme that catalyzes the first step, i.e., the activation of phenylacetate to phenylacetyl-coenzyme A, in the aerobic phenylacetate degradation pathway of this strain (28). The AMP-binding site typical of acyl-coenzymeA-activating enzymes (28) can be also found in the primary structure of PaaK (96-SSGTTGKPTV-105).

Transcription analyses of the styrene catabolic genes. The genetic arrangement of the styABCD coding sequences suggests the possible cotranscription of these genes. To determine whether the genes styABCD form part of the same transcription unit, a Northern blot analysis was performed by using a styAB-specific probe and total RNA isolated from Pseudomonas sp. strain Y2 cells grown minimal medium containing glycerol or styrene as the sole carbon source. While three RNA bands of about 3.9, 1.8, and 1.2 kb were observed in styrene-grown cells, no hybridizing band was detected in the RNA sample isolated from cells cultivated in the absence of styrene (Fig. 5). The hybridizing band of 3.9 kb perfectly matches a styrene-induced transcription unit encompassing the styABCD genes. This 3.9-kb transcript is consistent with the presence of a palindromic sequence (G = 21.2 kcal/mol) at the 3' end of the styD gene (Fig. 2) which could act as a transcriptional terminator. Interestingly, two putative stem-loop structures showing G values of 25.3 and 27.0 kcal/mol were found downstream of the styA and styB genes, respectively (Fig. 2). Thus, the existence of potential styA and styAB transcripts could explain the 1.2- and 1.8-kb hybridizing bands (Fig. 5), respectively. Nevertheless, the possibility that these two bands were the result of incomplete transcription or of RNA degradation or processing cannot be ruled out.

View larger version (41K): [in this window] [in a new window]   FIG. 5.   Northern analysis of the Pseudomonas sp. strain Y2 styABCD genes. Total RNA (10 µg) was isolated from cells grown at 30°C in minimal medium M63 containing 20 mM glycerol (lane 1) or styrene (lane 2) as the sole carbon and energy source and then probed with a radioactively labeled SalI-PstI 1.5-kb fragment of plasmid pISM7 containing the styAB genes (Fig. 1). The sizes of RNA molecular weight markers (Promega) are indicated on the left; arrows on the right show the sizes and positions of the major transcripts.

View larger version (47K): [in this window] [in a new window]   FIG. 6.   Identification of the 5' transcription start site of the sty operon. Total RNA from Pseudomonas sp. strain Y2 was purified from cells grown at 30°C in minimal medium M63 containing styrene (lane 1) or 20 mM glycerol (lane 2) as the sole carbon source. Total RNA from E. coli DH5(pSTY) was purified from cells grown at 30°C in LB medium in the absence (lane 3) or presence (lane 4) of styrene. The size of the extended products was determined by comparison with a DNA-sequencing ladder of the Psty promoter region (T, G, C, and A), using plasmid pSTY as the template. The primer extension and sequencing reactions were performed with the same primer (5'-CGATCAGTGTACACAGTGACGTCG-3'), which hybridized at 80 nt downstream of the styA start codon. To the right, an expanded view of the nucleotide sequence surrounding the transcription initiation site (+1) is shown. Note that the sequence corresponds to the coding strand.

Regulation of the styrene catabolic cluster. Experimental evidence that the genes styS and styR were required for expression of the styABCD catabolic operon emerged from the genetic studies involving E. coli cells harboring plasmids pUPAB18 (pUC18 derivative that harbors a 2.2-kb BglII-SmaI fragment containing the styAB genes) (Fig. 1) and pISM7 (pUC18 derivative that harbors a 6.6-kb SmaI fragment containing the stySR and styAB genes) (Fig. 1). Despite the presence of the styrene monooxygenase-encoding genes (styAB) necessary for the indole-indigo conversion, the production of indigo was negligible in E. coli DH5(pUPAB18) cells grown in the presence or absence of styrene (Fig. 7). However, when genes styS and styR were provided in cis, E. coli DH5(pISM7) cells showed a high production of indigo that was inducible by styrene (Fig. 7). These results indicate that the StySR two-component system is functional in E. coli and behaves as a positive regulator of the sty promoter. A similar finding was observed with the homologous TodST system that also activates the expression of the tod structural genes in P. putida F1 (23).

View larger version (22K): [in this window] [in a new window]   FIG. 7.   Assessment of styrene monooxygenase expression in E. coli. Expression of the styAB genes in the presence (plasmid pISM7) or absence (plasmid pUPAB18) of stySR genes was monitored by measuring indigo production. E. coli DH5(pUPAB) and E. coli DH5(pISM7) cells were grown in LB medium at 30°C until the cultures reached an A600 of 0.4. Styrene was then added to half of each culture, and growth was resumed until cultures reached an A600 of 0.8. Cells were collected by centrifugation, washed with 0.1 M sodium phosphate buffer (pH 7.0), and resuspended in the same buffer (0.05 ml of buffer/ml of culture). The cell suspension was incubated with stirring at 30°C in the presence of 1 mM indole. After 60 min of incubation, indigo produced was assayed.