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J Bacteriol, March 1998, p. 1063-1071, Vol. 180, No. 5
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Genetic and Functional Analysis of the Styrene
Catabolic Cluster of Pseudomonas sp. Strain Y2
Ana
Velasco,1,2
Sergio
Alonso,2
José L.
García,1,*
J.
Perera,2 and
Eduardo
Díaz1
Department of Molecular Microbiology, Centro
de Investigaciones Biológicas, CSIC, 28006 Madrid,1 and
Department of Biochemistry
and Molecular Biology, Facultad de Biología, Universidad
Complutense, 28040 Madrid,2 Spain
Received 6 October 1997/Accepted 6 December 1997
 |
ABSTRACT |
The chromosomal region of Pseudomonas sp. strain Y2
involved in the conversion of styrene to phenylacetate (upper catabolic pathway) has been cloned and sequenced. Four catabolic genes, styABCD, and two regulatory genes, stySR, were
identified. This gene cluster when transferred to Escherichia
coli W confers to this phenylacetate-degrading host the ability
to grow on styrene as the sole carbon and energy source. Genes
styABCD are homologous to those encoding the styrene upper
catabolic pathway in Pseudomonas fluorescens ST. Northern
blot analyses have confirmed that genes styABCD constitute
a transcription unit. The transcription start site of the
sty operon was mapped 33 nucleotides upstream of the styA translational start codon. The styS and
styR genes, which form an independent transcriptional unit,
are located upstream of the styABCD operon, and their gene
products show high similarity to members of the superfamily of
two-component signal transduction systems. The styS gene
product is homologous to histidine kinase proteins, whereas the
styR gene product exhibits similarity at its N-terminal
domain with cluster 1 of receiver modules and at its C terminus with
the LuxR/FixJ family 3 of DNA-binding domains. Expression of the
catabolic operon decreased significantly in the absence of the
stySR genes and was restored when the stySR genes were provided in trans in the presence of styrene,
suggesting that the stySR system behaves as a
styrene-inducible positive regulator of the styABCD operon.
Finally, a gene encoding a phenylacetyl-coenzyme A ligase that
catalyzes the first step in the phenylacetate catabolism (styrene lower
catabolic pathway) has been identified upstream of the styS
gene. This activity was found to be induced in Pseudomonas sp. strain Y2 cells grown on styrene but not present in cells grown on
glycerol. These results strongly suggest that the genes responsible for
the complete mineralization of styrene are clustered in the chromosome
of Pseudomonas sp. strain Y2.
 |
INTRODUCTION |
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.

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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.
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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.
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MATERIALS AND METHODS |
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).
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RESULTS AND DISCUSSION |
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]).
When total DNA from Pseudomonas sp. strain Y2 was analyzed
by pulsed-field electrophoresis, no plasmid was observed. The
chromosomal location of the EcoRI fragment was confirmed by
Southern blot experiments. Therefore, the styrene catabolic pathway of
Pseudomonas sp. strain Y2 seems to be, as in the case of
P. fluorescens ST (25), chromosome encoded.
To determine whether the cloned 12.2-kb
EcoRI fragment
encoded the complete styrene upper catabolic pathway, we checked the
ability of plasmid pUE14 to confer to
E. coli the capacity
to
grow on styrene as the sole carbon and energy source. Since
E. coli DH5

cannot use phenylacetate as a carbon source, we
transformed
plasmid pUE14 into
E. coli W, a strain able to
mineralize this
intermediate in the catabolism of styrene. However,
E. coli W(pUE14)
cells were unable to grow on styrene as the
sole carbon source,
suggesting that the cloned
EcoRI
fragment probably did not contain
all of the genes needed for the
conversion of styrene to phenylacetate.
Since the gene(s) encoding the
putative styrene monooxygenase
was located at the right end of the
EcoRI fragment (Fig.
1; see
above), we decided to clone the
contiguous chromosomal region
of
Pseudomonas sp. strain Y2.
A
ClaI DNA library of
Pseudomonas sp. strain Y2
was constructed in pUC19 and its screening by colony
hybridization
using the terminal 0.6-kb
BamHI-
EcoRI fragment of
pUE14 (Fig.
1) as a probe allowed us to isolate plasmid pUCL50,
which
harbored a 4.6-kb
ClaI insert (Fig.
1). To reconstruct the
15.5-kb chromosomal region shown in Fig.
1, plasmid pSTY was engineered
by inserting the purified 12.2-kb
EcoRI fragment of pUE14
into
EcoRI-digested pUCL50. Remarkably,
E. coli W
harboring plasmid
pSTY was now able to grow on styrene as the sole
carbon and energy
source. Moreover, when plasmid pSTY was transformed
into
E. coli W14 (an
E. coli W mutant having a
complete deletion of the phenylacetate
catabolic pathway)
(
13) and the recombinant cells were grown
on glycerol in the
presence of styrene, we observed the accumulation
in the culture medium
of large amounts of a compound that cochromatographed
on HPLC with
standard phenylacetate. Gas chromatography-mass spectrometry
analyses
of the accumulated product confirmed its identity with
phenylacetate.
Taken together, all of these results suggested
that plasmid pSTY
contained the complete gene cluster of the styrene
upper catabolic
pathway involved in the transformation of styrene
into phenylacetate.
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).

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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.
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(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).
The
styB gene encodes a protein of 18,364 Da (170 aa long)
(Fig.
2) that shows similarity to the SnaC, ActRV, NtaB, and NmoB
flavin mononucleotide (FMN) oxidoreductases (Table
1) (
7,
21,
39,
46). On the other hand, a significant similarity
was also
observed between StyB and the coupling proteins of the
two-component
4-hydroxyphenylacetate-3 monooxygenases from
E. coli
(
33) and
Klebsiella pneumoniae (
14)
(Table
1). Although
no enzymatic activity has been ascribed to these
coupling proteins,
they appear to play an important role to
discriminate substrate
analogs that can be hydroxylated by the
flavin-containing component
(
33). Remarkably, it has been
shown that StyB is essential for
the complete activity of styrene
monooxygenase (
6). Therefore,
all of these data strongly
suggest that StyB may be the small
subunit of a two-component
monooxygenase.
The
styC gene encodes a protein of 18,028 Da (169 aa long)
(Fig.
2) which shows no significant similarity to other known proteins
with the exception of the epoxystyrene isomerase of
P. fluorescens ST (
6). The lack of similarity between StyC
and the isomerases
of other aromatic catabolic pathways could be
ascribed to the
unusual substrate of this enzyme.
The
styD gene shows two putative ATG start codons at nt 7759 and 7777. However, while the second start codon was preceded
by a
putative ribosome-binding site (RBS) (AAGGAG), the first
one
overlaps the stop codon of the preceding
styC gene (Fig.
2).
The
styD stop codon was located at nt 9265 (Fig.
2). The
putative
largest version of StyD is a protein of 53,502 Da (502 aa
long)
that shows similarity to many prokaryotic and eukaryotic aldehyde
dehydrogenases (Table
1). Surprisingly, the similarity between
StyD and
the PadA and FeaB phenylacetaldehyde dehydrogenases recently
characterized in
E. coli W (
13) and
E. coli K-12 (
15), respectively,
is not higher than that
observed between StyD and other aldehyde
dehydrogenases with different
substrate specificity. The StyD
sequences 249-FTGSTEVG-256
and 298-AIFFNHGQV
CTA-309, respectively,
match the consensus
NAD
+-or NADP
+-binding site motif and the
active-site motif spanning the catalytic
cysteine (underlined) of
aldehyde dehydrogenases (
18,
24).
Moreover, the conserved
glutamic acid residue that has been shown
to bind to the adenine ribose
of NAD
+ (
24) is also found (Glu-201) in StyD.
The ORF named
porA shows a potential ATG start codon, which
it is not preceded by a typical RBS, and a stop codon at nt 9434
and
10733, respectively (Fig.
2). The deduced PorA is a protein
of 45,906 Da (433 aa long) that shows a significant similarity
to some outer
membrane proteins (Table
1), although it lacks
the typical N-terminal
signal sequence involved in secretion and
integration of the protein
into the membrane. This fact, together
with the observation that a
similar ORF is completely truncated
in
P. fluorescens ST,
strongly suggests that PorA may not play
a relevant role in the
catabolism of styrene in these bacteria.
It had been proposed that styrene was catabolized via 2-phenylethanol
in
Pseudomonas sp. strain Y2 (
40). However, the
similarity
of the
sty catabolic pathway reported here and
that of
P. fluorescens ST (
6) suggests that
styrene in strain Y2 can be degraded by
a pathway that does not involve
2-phenylethanol as an intermediate.
(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).

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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).
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The
styR gene encodes a protein of 23,343 Da (207 aa long)
(Fig.
2) that shows a significant similarity to many response
regulators
of two-component systems (Table
1). Amino acid sequence
alignments
revealed that StyR was highly similar to the response
regulators
TodT (
23) and TutB (
9) (Table
1 and
Fig.
4), a result that
is in agreement
with the high similarity found among the cognate
sensory proteins StyS,
TodS, and TutC (see above). The residues
Asp-11, Asp-12, Asp-55,
Thr-83, and Lys-105 of StyR are highly
conserved in the receiver
domains of other response regulators
(
4,
32,
41) (Fig.
4).
By analogy with TodT (
23) and other
receiver modules,
Asp-11, Asp-12, and Asp-55 are predicted to
form an acid pocket where
Asp-55 may be the acceptor of the phosphoryl
group from the
phosphorylated StyS sensor. The C terminus of StyR
contains a sequence
(aa 143 to 185) that has 17 of the 19 consensus
residues of the
DNA-binding domain of the LuxR/FixJ family 3 DNA-binding
domains
(
31,
35) (Fig.
4). A putative Q-linker (aa 121 to
136)
(
45) between the receiver and DNA-binding modules was also
observed in StyR (Fig.
4). Interestingly, StyR shows a significant
similarity with NodW and FixJ (Table
1 and Fig.
4), two response
regulators that have been classified within the cluster 1 receiver
modules that contain family 3 DNA-binding domains (
35).
Thus,
StyR could be considered another example of domain shuffling that
occurred during the parallel evolution of family 3 DNA-binding
domains
with cluster 1 receiver modules (
35).

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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.
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(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).
To experimentally demonstrate that
paaK encodes a
phenylacetyl-coenzyme A ligase, we have cloned the gene under the
control
of the
lac promoter in plasmid pUC18. Expression of
paaK from
the resulting plasmid pPAAK (Fig.
1) was assayed
in
E. coli W14,
a mutant of
E. coli W devoid of
the endogenous phenylacetyl-coenzyme
A ligase activity (
12,
13). Interestingly, while
E. coli W14(pPAAK)
cell
extracts showed a high phenylacetyl-coenzyme A ligase activity
(130 U/mg of protein), control extracts of
E. coli W14(pUC18)
cells exhibited no detectable activity. This result confirmed
that PaaK
is involved in the activation of phenylacetate to phenylacetyl-coenzyme
A. On the other hand, while
Pseudomonas sp. strain Y2
cultured
in 20 mM glycerol-containing medium had no detectable
phenylacetyl-coenzyme
A ligase activity, cells grown in a
styrene-saturated atmosphere
or with 5 mM phenylacetate as the sole
carbon source showed a
significant activity (25 to 50 U/mg of protein).
Thus, these results
strongly suggest that a phenylacetyl-coenzyme A
ligase is involved
in the catabolism of styrene in
Pseudomonas sp. strain Y2.
It is worth noting that PaaK shows also a high similarity to the
putative products of the open reading frames o437 from
E. coli K-12 and
paaK from
E. coli W (Table
1).
The
paaK gene of
E. coli W has been located
within the gene cluster encoding a
phenylacetate catabolic pathway
(
12). This cluster has been
cloned in a plasmid (pFA2)
(
13) which confers to the phenylacetate-minus
mutant
E. coli W14 strain the capacity to use this compound as
the
sole carbon and energy source (
13). Analysis of the sequence
upstream of
paaK in
Pseudomonas sp. strain Y2
revealed that this
region encodes several proteins of unknown function
that are also
coded by plasmid pFA2 and that have been found to be
essential
for phenylacetate catabolism in
E. coli W
(
12). All of these
results strongly suggest that the styrene
lower and upper catabolic
pathways are contiguous in the chromosome of
Pseudomonas sp. strain
Y2, PaaK being the
phenylacetyl-coenzyme A ligase that catalyzes
the first step in the
styrene lower pathway.
Although the overall G+C content (57.7%) of the 10.7-kb DNA fragment
(Fig.
2) is close to that of the genus
Pseudomonas (60%)
(codon usage tabulated from GenBank), there is a difference between
the
G+C content of the
stySRABCDporA genes (Table
1) and that
of
paaK (Table
1) and the upstream genes present in plasmid
pSTY.
The G+C content (87.6%) at the third position of the
paaK codons
is also higher than that of the
sty
codons (66.4%). Therefore,
these values may suggest that the styrene
upper and lower clusters
have not evolved together.
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.

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|
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.
|
|
To determine the transcription initiation site of the
styABCD catabolic operon, primer extension analyses were
performed with
total RNA isolated from styrene-induced and uninduced
E. coli DH5

(pUE14) and
Pseudomonas sp. strain
Y2 cells. Under induced
conditions, a transcription initiation site
located 33 nt upstream
from the ATG translation initiation codon of the
styA gene was
identified both in the homologous and
heterologous hosts (Fig.
6). In contrast
no transcription initiation site of the
sty operon
was
observed in the absence of styrene (Fig.
6). Therefore, these
results
revealed not only that expression of the styrene catabolic
genes was
very low in the absence of inducer but also that it
was inducible in an
heterologous host. On the other hand, all
attempts to determine the
transcription start site of the
stySR operon were
unfruitful, probably due to the low expression level
of these genes,
and further research needs to be done.

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|
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.
|
|
The low G+C content (36.4%) of the intergenic region upstream of
styA is in agreement with the existence of a functional
promoter.
Thus, upstream of the identified transcriptional start site,
a
putative extended

10 box (TGTTAGCTT) (
5) was
observed (Fig.
2). The absence of a consensus

35 box typical of
70-dependent promoters would agree with StyR being a
transcriptional
regulator that binds to the
sty promoter. In
this sense, StyR
contains a DNA-binding domain similar to that of the
class 3 response
regulators which appear to interact with RNA
polymerases containing
various sigma factors (
35).
Furthermore, a palindromic sequence
(ATAAACCATGGTTTAT)
centered at position

41 from the
styA
transcriptional
start site (Fig.
2) was found to be nearly identical to
the inverted
repeat (ATAAACCATcGTTTAT) (lowercase letter
indicates a mismatch)
that binds to the analogous TodT response
regulator in the
tod operon (
tod box)
(
23). This observation strongly suggests that
the 8-bp
inverted repeat in the
sty promoter could be the
StyR-binding
site (
sty box). However, a striking difference
between the
sty and
tod boxes is that whereas the
latter is centered at

105 bp
from the transcriptional start site
(
23), the former overlaps
the putative

35 promoter region.
Hence, it can be predicted that
in the
tod and
sty operons there are different mechanisms of interaction
between the RNA polymerase and the TodT and StyR regulators,
respectively.
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).

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|
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.
|
|
It is worth noting that even in the absence of styrene,
E. coli DH5

(pISM7) cells produced higher amounts of indigo than
E. coli DH5

(pUPAB18) cells (Fig.
7). This observation can
be explained
by a low induction effect of indole on styrene
monooxygenase expression,
as it has been shown in
P. putida
CA-3 (
30). Nevertheless, we
cannot rule out the possibility
that StyR can be phosphorylated
by metabolic cross talk carried out
either by a noncognate host
sensor or by a chemical phosphorylating
agent such as phosphoramidate
or acetyl phosphate (
32). The
same argument has been used to
explain the
trans-acting
effect of TodT in
E. coli as a positive
regulator of the
tod operon in the absence of an intact
todS gene
(
23). Finally, it is also known that positive regulators can
activate their controlled genes in the absence of inducer when
they are
overproduced from high-copy-number vectors (
26).
In summary, the results presented here represent the first report on
the mechanism of regulation of a styrene catabolic pathway.
A
complex two-component regulatory system (StySR) has been
identified
and shown to be functional in a heterologous host.
Engineering
chimeric regulatory proteins by interchanging equivalent
domains
of the StySR, TodST, and TutCB systems will provide new
insights
into the structure-function relationships of these domains. On
the other hand, we have shown here that the styrene upper catabolic
cluster of
Pseudomonas sp. strain Y2 confers to
E. coli W the
ability to grow on styrene as the sole carbon source.
Thus, it
will be of biotechnological interest engineering the styrene
upper
and lower catabolic pathways as a transposable DNA cassette to
expand the catabolic abilities of environmentally relevant
microorganisms
endowed with a high solvent tolerance for styrene
removal.
 |
ACKNOWLEDGMENTS |
We are indebted to M. Yakimov for providing
Pseudomonas sp. strain Y2. We thank A. Ferrández for
his support with the HPLC analyses. The help of A. Díaz and G. Porras with sequencing is gratefully acknowledged.
This work was supported by grants from CICYT (AMB94-1038-C02-02 and
AMB97-063-C02-02) and from Comunidad Autónoma de Madrid (06M/029/96). E. Díaz was the recipient of a Contrato Temporal de Investigadores from the CSIC.
 |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Molecular Microbiology, Centro de Investigaciones Biológicas,
Velázquez 144, 28006 Madrid, Spain. Phone: 34-1-5611800. Fax:
34-1-5627518. E-mail: cibg160{at}fresno.csic.es.
 |
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