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Applied and Environmental Microbiology, September 2004, p. 5102-5110, Vol. 70, No. 9
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.9.5102-5110.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

The gnyRDBHAL Cluster Is Involved in Acyclic Isoprenoid Degradation in Pseudomonas aeruginosa

A. L. Díaz-Pérez, A. N. Zavala-Hernández, C. Cervantes, and J. Campos-García*

Instituto de Investigaciones Químico-Biológicas, Universidad Michoacana de San Nicolás de Hidalgo, Morelia, Michoacán, México

Received 8 March 2004/ Accepted 12 May 2004


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ABSTRACT
 
Pseudomonas aeruginosa PAO1 mutants affected in the ability to degrade acyclic isoprenoids were isolated with transposon mutagenesis. The gny cluster (for geranoyl), which encodes the enzymes involved in the lower pathway of acyclic isoprenoid degradation, was identified. The gny cluster is constituted by five probable structural genes, gnyDBHAL, and a possible regulatory gene, gnyR. Mutations in the gnyD, gnyB, gnyA, or gnyL gene caused inability to assimilate acyclic isoprenoids of the citronellol family of compounds. Transcriptional analysis showed that expression of the gnyB gene was induced by citronellol and repressed by glucose, whereas expression of the gnyR gene had the opposite behavior. Western blot analysis of citronellol-grown cultures showed induction of biotinylated proteins of 70 and 73 kDa, which probably correspond to 3-methylcrotonoyl-coenzyme A (CoA) carboxylase and geranoyl-CoA carboxylase (GCCase) alpha subunits, respectively. The 73-kDa biotinylated protein, identified as the {alpha}-GCCase subunit, is encoded by gnyA. Intermediary metabolites of the isoprenoid pathway, citronellic and geranic acids, were shown to accumulate in gnyB and gnyA mutants. Our data suggest that the protein products encoded in the gny cluster are the ß and {alpha} subunits of geranoyl-CoA carboxylase (GnyB and GnyA), the citronelloyl-CoA dehydrogenase (GnyD), the {gamma}-carboxygeranoyl-CoA hydratase (GnyH), and the 3-hydroxy-{gamma}-carboxygeranoyl-CoA lyase (GnyL). We conclude that the gnyRDBHAL cluster is involved in isoprenoid catabolism.


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INTRODUCTION
 
Hydrocarbon degradation by microorganisms has been the subject of many studies, especially with relation to the environmental impact of oil spills (27) and as a natural phenomenon in soil and water (24). Biodegradation of n-alkanes proceeds by the classical fatty acid oxidation pathway (30), whereas branched-chain alkanes are generally less susceptible to biodegradation (6, 19, 23). Brevibacterium erythrogenes has been reported to assimilate branched-chain alkanes with {alpha}, {omega}, ß, and ß-modified oxidation mechanisms for pristane (2,6,10,14-tetramethylpentadecane) degradation (19, 20). However, when a 3-methyl-branched alkane such as the acyclic isoprenoid citronellol (3,7-dimethyl-6-octen-1-ol) is found, ß-oxidation is prevented (23). Several bacterial strains have been isolated with the ability of 3-methyl-branched alkane degradation (3, 6, 11, 12).

The acyclic isoprenoids of the citronellol kind such as citronellol, geraniol, nerol, citronellal, citral, citronellic acid, and geranic acid contain a 3-methyl substitution in the principal hydrocarbon chain; this substitution is related to the environmental recalcitrance of these compounds (6, 23). Several species of Pseudomonas (P. citronellolis, P. aeruginosa, and P. mendocina) can use 3-methyl-branched alkanes as the sole carbon source (5). The pathway for the degradation of acyclic isoprenoids in P. citronellolis has been reported (5, 6), but the genes and enzymes involved are uncharacterized.

The general route for citronellol degradation proposed in P. citronellolis involves the oxidation of the alcohol to aldehyde and then to citronellic acid (called the upper pathway). Biochemical studies suggest that degradation of acyclic isoprenoids in both microorganisms and plants involves at least three unique enzymes (called the lower pathway): geranoyl-coenzyme A (CoA) carboxylase, an enzyme homologous to the 3-methylcrotonoyl-CoA carboxylase, which activates the 3-methyl group of the substrate by CO2 fixation; an enoyl-CoA hydratase, which introduces a water molecule; and a 3-hydroxy-{gamma}-carboxygeranoyl-CoA lyase homologue, which catalyzes the removal of the activated ß-carboxymethyl group as acetyl-CoA, generating 3-oxo-7-methyl-octenoate, a suitable substrate for the ß-oxidation pathway (5, 6, 7, 9). Such a route in P. citronellolis has been proposed to also occur in P. aeruginosa (4, 5). In this report, we identify the P. aeruginosa genes (called the gny cluster for the isoprenoid metabolite geranoyl-CoA) that encode the enzymes involved in the lower pathway of acyclic isoprenoid degradation and suggest that the {alpha}-geranoyl-CoA carboxylase subunit is encoded by the gnyA gene.


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MATERIALS AND METHODS
 
Microbiological methods and plasmids.
The bacterial strains and plasmids used in this work are shown in Table 1. P. aeruginosa and Escherichia coli strains were grown at 30°C in M9 minimal medium with 0.1 to 0.2% of the carbon source indicated or in Luria broth (22). Solid media were prepared by adding 1.5% agar. Antibiotic concentrations used (µg/ml) for P. aeruginosa strains were streptomycin at 200 µg/ml, carbenicillin at 100 µg/ml, gentamicin at 100 µg/ml, and tetracycline at 100 µg/ml; for E. coli strains they were ampicillin at 100 µg/ml, gentamicin at 20 µg/ml, and tetracycline at 15 µg/ml.


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  		TABLE 1. Strains and plasmids used in this work

Transposon mutagenesis and screening.
Transposon insertion mutants were generated by mobilizing the suicidal plasmid pFAC from E. coli S17-1 to P. aeruginosa PAO1SM as described by Wong and Mekalanos (31). The PAO1SM strain was grown overnight in Luria broth at 42°C with shaking, and the donor E. coli strain was grown at 37°C to the exponential phase (optical density at 600 nm = 0.6). A 2:1 proportion of donor to recipient was mixed on nylon filters, placed on Luria agar plates, and incubated for 12 h at 37°C. The bacteria were suspended in Luria broth, and dilutions were spread on plates containing streptomycin and gentamicin. Five thousand streptomycin- and gentamicin-resistant mutants, from a library of about 106 clones, were tested for their ability to grow on M9 agar plates supplemented with one of the following carbon sources (added as vapors): citronellol, geraniol, nerol, citronellal, citral, n-octanol, 0.1% citronellic acid, geranic acid, 6-methyl-5-hepten-2-one, or 0.2% glucose (obtained from Aldrich, Sigma, or Merck Co.).

Nucleic acid procedures.
DNA isolation, molecular cloning, Southern blot hybridization, and PCR were carried out as previously described (22). Recombinant plasmids were analyzed by PCR with the oligonucleotide MarIN2 (5'-GATCTAACAGGTTGGCTGATAAGTCCCCGGTCT-3') (31). DNA probes were labeled with the AlkaPhos direct labeling and detection system with ECF (Amersham Pharmacia Biotech) following the provider's recommendations.

Sequencing of transposon insertion fragments.
Sequencing was done with oligonucleotide GM2, designed from the gentamicin resistance gene contained in the Himar1::Gmr transposon system (5'-GGGCGTCACCGAGAGATATGTTTC-3'), and the M13/pUC forward primer from the pBluescript SK plasmid (5'-CCAGTCACGACGTTGTAAAACG-3'). Sequencing reactions were done with the ABI Prism BigDye terminator v3.0 ready reaction cycle sequencing kit (Applied Biosystems), and the products were analyzed in an automatic ABI Prism 310 genetic analyzer sequencer (Perkin-Elmer). Sequence data were analyzed with data from the Pseudomonas Genome Project (PGP; http://www.pseudomonas.com), the NCBI home page (http://www.ncbi.nlm.nih.gov), and the DNAstar program.

Disruption of the gnyB, gnyA, gnyD, and gnyL genes.
The gnyB and gnyL genes were obtained by PCR amplification of open reading frames (ORFs) PA2014 and PA2011 from P. aeruginosa PAO1SM genomic DNA with oligonucleotides gnyB1 (5'-CACCTACTATCCGCTGACCGTGAAG-3') versus gnyB2 (5'-CTGGTCTACTCGTCACATGCG-3') and gnyL1 (5'-GGTAGTGAAGGCGCTGTATTGCAG-3') versus gnyL2 (5'-GTATGGATCTCCAGGCCGTTCAG-3'), respectively. The PCR fragments obtained (1.28 and 0.9 kb, respectively) were cloned into the pGEM-T vector (Promega). For gene disruption, a gentamicin resistance cassette (0.9 kb) from the pBSL148 plasmid (1) was introduced for gnyB into the unique BamHI restriction site (pAAB plasmid) and for gnyL into the PstI site (pAAL). From the constructed plasmids, PstI restriction fragments (1.7 and 1.52 kb, respectively) containing gnyB::Gmr or gnyL::Gmr were subcloned into the suicide pKOK4 vector (15), yielding pKAAB and pKAAL, respectively. These plasmids were mobilized by triparental conjugation into the PAO1SM strain with the pRK2013 helper plasmid (8). Transconjugants were selected in plates with streptomycin and gentamicin. The gnyB gene disruption was characterized by PCR analysis with oligonucleotides gnyR1 (5'-AGCAGCGTTTGTAGGTCATCAG-3') and gnyB2 (see above).

The gnyA and gnyD disruptions were obtained by cloning PstI fragments from pMO013850 (2.19 and 2.3 kb, respectively) into the pKOK4 vector. Plasmids were digested at KpnI sites, located inside the gnyA and gnyD genes, and a gentamicin resistance cassette from plasmid pBSL148 (1) was cloned, yielding pKAAA and pKAAD, respectively. These plasmids were mobilized as above.

RT-PCR.
The strains were grown in M9 agar plates with the appropriate carbon source and incubated for 24 h at 30°C. Cultures were suspended in M9 salts solution and washed twice. Total RNA was isolated with the RNA Protect Bacteria Reagent and RNeasy Total RNA mini kit (Qiagen) following the provider's recommendations. After the RNA quality was verified by agarose gel electrophoresis, remaining DNA was removed with DNase I treatment (22). The reverse transcription (RT)-PCR assay was done with the gnyB2 and gnyD1 (5'-GCGGAGCATGTCGATGGTTTCG-3') oligonucleotides in the RT reactions with the Omniscript RT kit (Qiagen) as indicated by the provider. The PCR was done with 1/10 of the RT volume reaction as the template and the oligonucleotide pairs gnyB1 with gnyB2, and gnyR2 (5'CACCTACACCATCTCCGATCTCG-3') with gnyD1, respectively, with the Platinum Pfx DNA polymerase (Invitrogen).

Transcriptional fusions.
Transcriptional fusion of the P1 promoter region (P1gnyR::lacZ) was constructed by cloning a 1,840-bp PCR fragment obtained with an oligonucleotide starting 130 bp upstream of the ATG codon of the gnyR gene (gnyR3, 5'-AGCAGCGTCCTGTAGGTCATCAG-3'), and an oligonucleotide located in the 3' region of gnyD gene (gnyD2, 5'-CAGCGGGTTTCGTTGAACAGC-3') into the pGEM-T vector (Promega). A PstI fragment of 774 bp of this construct was cloned into the pLP170 vector (21), yielding pANP1. For the P2 promoter transcriptional fusion (P2gnyB::lacZ) a 650-bp fragment obtained from pAL-22 plasmid, containing an EcoRI site 443 bp upstream of the ATG codon of the gnyB gene and a XhoI site 207 bp downstream, was cloned in pLP170, producing the pANP2 plasmid. Plasmids pANP1 and pANP2 were transferred to strain PAO1SM by heat shock transformation as described (22), and transformants were selected in Luria broth plates with carbenicillin.

ß-Galactosidase assays of P. aeruginosa strains harboring the fusions were performed with cultures grown to the exponential phase (optical density at 600 nm = 0.6) in M9 medium supplemented with glucose (0.2%); cells were harvested by centrifugation and washed twice with M9 salts solution. For induction assays, cell suspensions were incubated at 30°C with adequate carbon source, aliquots were taken, and ß-galactosidase activity was measured as described (22).

Genetic complementation.
The complete wild-type operon involved in citronellol degradation was obtained from a cosmid genomic library from P. aeruginosa PAO1 (Pseudomonas Genetic Stock Center of PGP). The pMO013850 cosmid has a 25-kb PAO1 chromosomal DNA fragment contained in the pLA2917 cosmid vector. A HindIII-EcoRV fragment (7.42 kb) from pMO013850 was subcloned into the pUCP20 vector, yielding plasmid pAL-22, which contains the structural genes of the gny operon. Complementation was assayed by mating the donor E. coli(pMO013850) strain with the mutant strains, with the pRK2013 plasmid as a helper, and selecting transconjugants in plates with tetracycline. The mutants were transformed with the pAL-22 plasmid, and clones were selected in plates with carbenicillin.

Identification of biotin-containing proteins.
Free cell extracts were obtained from cultures of P. aeruginosa grown at 30°C for 24 h in M9 with an adequate carbon source. Bacterial cells were disrupted by sonication, and the crude extracts were centrifuged at 10,000 x g for 10 min at 4°C to remove undisrupted cells. Samples (approximately 100 µg of total protein) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE, 10%) and electrophoretically transferred to nitrocellulose membranes. The Western blotting conditions were as indicated by the provider (Bio-Rad). The membranes were blotted with the antibody avidin horseradish peroxidase conjugate-avidin (Bio-Rad), and horseradish peroxidase color development was done with 4-chloro-1-naphthol and H2O2 (Sigma). Biotinylated SDS-PAGE standards (Bio-Rad) and a BenchMark prestained protein ladder (Invitrogen) were used as molecular size markers.

Metabolite analyses.
The strains were grown in 100 ml of M9 medium with 0.2% (wt/vol) succinic acid and 0.05% (wt/vol) casein peptone at 30°C under shaking for 12 h. Cells were harvested by centrifugation and washed twice with M9 salts solution. The pellet was suspended in the original volume with M9 salts, and citronellol was added to a final concentration of 0.1% (vol/vol). The suspension was incubated at 30°C under shaking, and aliquots of 25 ml were withdrawn at intervals and centrifuged at 10,000 x g for 10 min. Supernatants were saturated with NaCl (7.5 g) and extracted three times with 10 ml of ethyl acetate. The samples were collected, dried with anhydrous Na2SO4, concentrated by evaporation in a fume hood at 50°C, and suspended in 500 µl of methanol. Samples of 1 µl were analyzed with gas chromatography and mass-spectrometry (Hewlett Packard Series II gas chromatograph 5890 and HP 5989B mass spectrometer, with a DB-5MS column, 30 m long, 0.25 mm inner diameter, film 0.25 µm; J&W Scientific). The analyses were carried out at 60°C for 2 min, increased to 135°C (25°C/min), and then to 210°C (5°C/min). Commercial citronellol, citronellal, citronellic acid, geraniol, citral, and geranic acid were used in the gas chromatography-mass spectrometry as standard identification compounds.

Nucleotide sequence accession numbers.
The nucleotide and amino acid sequences analyzed are denoted as ORFs PA2016 to PA2011 in PGP (26) and were renamed in this work as the gnyRDBHAL genes, respectively.


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RESULTS AND DISCUSSION
 
Isolation and characterization of mutants affected in acyclic isoprenoid assimilation.
P. aeruginosa is able to use acyclic isoprenoids such as citronellol, geraniol, nerol, and their metabolites as the sole carbon source. To identify the genes involved in the acyclic isoprenoid degradation pathway, a library of about 106 P. aeruginosa gentamicin-resistant mutants was generated with the mariner-based transposon Himar1::Gmr from plasmid pFAC (31). About 5,000 mutants were screened for their ability to grow on a variety of isoprenoid derivatives as the sole carbon source. Twenty-one mutants were selected and classified according to their growth phenotype (data not shown). Chromosomal DNA (PstI digested) from these strains was analyzed by Southern blot hybridization with the gentamicin resistance cassette from the pFAC plasmid as a probe.

The hybridization band patterns obtained with the mutants (data not shown) suggested that the mutations were caused by a single transposition event. The genomic regions interrupted by the transposon in the mutants were cloned in the PstI site of the pBluescript SK vector, and the flanking DNA fragments were sequenced. After sequence analysis, two mutants, PAE80 and PAA447, were selected for further study (see below). These mutants were unable to grow on citronellol and citronellic acid as the sole carbon source but were able to grow on n-octanol (Table 2). The presence of the transposon was confirmed by PCR, showing a size increase of about 1 kb in the amplified fragment (data not shown). Sequence analysis of PAE80 and PA447 strains showed that transposon insertion had occurred in the PA2012 and PA2014 ORFs, respectively, from the PGP database (26).


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  		TABLE 2. Phenotype analysis of P. aeruginosa strainsa

Identification of the gny cluster from P. aeruginosa.
The regions flanking ORFs PA2012 and PA2014 in the P. aeruginosa PAO1 genome were analyzed. A putative cluster constituted of six ORFs (PA2011 to PA2016) was identified and named the gny operon for its relation with the isoprenoid geranic acid assimilation pathway. This cluster is located at 35 min on the genomic map of P. aeruginosa (26). ORFs located upstream and downstream of this cluster are probably not related to gny operon function. Therefore, the gny cluster may be constituted of the gnyRDBHAL genes, which are transcribed in the same direction (Fig. 1). A potential transcriptional terminator was identified just downstream of the last gene, gnyL (Fig. 1).



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  		FIG. 1. Schematic representation of the genetic arrangement of the gny cluster from P. aeruginosa PAO1. The locations of transposon insertions and disruption mutations are indicated with open and shaded arrowheads, respectively. The gene arrangement of the gny cluster genes and the transcription direction are shown by open arrows. ORF numbers corresponding to the PGP designations (PA2016 to PA2010) are shown below. Probable promoter regions P1 and P2 are shown as bold arrowheads. A putative transcriptional terminator is indicated with a hairpin. Numbers above the map indicate the distance in base pairs between adjacent genes; a negative number indicates that the genes overlap. A HindIII-EcoRV restriction fragment (7.42 kb) subcloned in the pAL-22 plasmid is shown below. RT-PCR1 and RT-PCR2 indicate the fragments amplified in the RT-PCR analysis.

Disruption of the gnyD, gnyB, gnyA, and gnyL genes.
To further establish that the gny cluster is involved in isoprenoid degradation of the citronellol kind, disruption mutations of the gnyD, gnyB, gnyA, and gnyL genes (ORFs PA2015, PA2014, PA2012, and PA2011, respectively) were constructed as described in Materials and Methods. PCR analysis of chromosomal DNA from mutants PAMgnyD, PAMgnyB, PAMgnyA and PAMgnyL (Fig. 1) showed DNA inserts about 1 kb larger than that amplified from the PAO1SM strain (data not shown), corresponding to the gentamicin resistance cassette inserted after homologous recombination. The mutants obtained by gene disruption were unable to grow on the isoprenoids tested as the sole carbon source (Table 2). These data strongly suggest that the genes contained in the gny cluster are involved in the isoprenoid degradation pathway in P. aeruginosa. PAO1 mutant 2009 (13), containing a transposon insertion in ORF PA2016 (here named gnyR), was unable to grow on isoprenoids (Table 2), suggesting that the gnyR gene is also involved in this catabolic pathway, probably playing a regulatory role in the gny cluster.

Sequence analysis of the gny cluster.
Nucleotide sequence analysis of the gny cluster showed that it is constituted of five probable structural genes (gnyDBHAL) and a putative regulatory gene (gnyR) (Fig. 1). The GC content of the gny cluster ranged from 66.4 to 71.0%, with the exception of gnyR (59.4%), resembling the GC content of the P. aeruginosa genome (61 to 67%). Also, the use of codons in the gny cluster is in agreement with the codon usage of P. aeruginosa (28).

Sequence alignment of the gnyR product showed high similarity with putative transcriptional regulators of the MerR family (Table 3). The 5' end of the gnyR gene has a putative promoter, P1, that could be the starting point of gny cluster transcription (Fig. 1). The protein encoded by the gnyD gene has a high similarity to putative isovaleryl-CoA dehydrogenase enzymes (Table 3). This suggests that the GnyD protein might be involved in the dehydrogenation of citronelloyl-CoA to cis-geranoyl-CoA (Fig. 2), a metabolic step originally proposed by Cantwell et al. (5).


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  		TABLE 3. Proteins showing significant amino acid identity with gny cluster gene products



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  		FIG. 2. Proposed degradation pathway and putative function of the gny cluster products from P. aeruginosa. GnyD, citronelloyl-CoA dehydrogenase; GCCase, geranoyl-CoA carboxylase; GnyA and GnyB, alpha and beta subunits of geranoyl-CoA carboxylase, respectively; GnyH, {gamma}-carboxygeranoyl-CoA hydratase; GnyL, 3-hydroxy-{gamma}-carboxygeranoyl-CoA lyase; MCCase, 3-methylcrotonoyl-CoA carboxylase; MG-CoA hydratase, methylglutaryl-CoA hydratase; HMG-CoA lyase, hydroxymethylglutaryl-CoA lyase. (Adapted from references 5 and 9.)

The gnyB gene is 122 bp downstream of the stop codon from gnyD. The start codon of the gnyA gene overlaps the stop codon of gnyH (Fig. 1). A probable promoter sequence, P2, was found by nucleotide sequence visual analysis in the 5' region of gnyB (Fig. 1), suggesting the presence of a second transcriptional unit comprising the gnyBHAL genes. Multiple sequence alignments of the gnyA and gnyB products showed a high similarity with the {alpha} and ß subunits of the acyl-CoA carboxylase family, respectively. The alignment of the ß-subunit of acyl-CoA carboxylases with GnyB showed two principal domains, the acyl-CoA-binding and the carboxybiotin-binding domains. The ß-subunit of acyl-CoA carboxylases has been proposed to transfer the CO2 molecule to the acyl-CoA substrate (14). Sequence alignment of GnyA showed the presence of four highly conserved domains in several {alpha}-subunits of acyl-CoA carboxylases (14, 16, 25): the ATP-binding site (GGGGKGM); a CO2 fixation domain (RDCS); the catalytic site of biotin-dependent carboxylase family (EMNTR); and a biotin-carboxyl carrier domain (AMKM). Our data suggest that the gnyA and gnyB genes from P. aeruginosa may encode the geranoyl-CoA carboxylase involved in acyclic isoprenoid degradation.

The protein encoded by gnyH (Fig. 1) is highly similar to enoyl-CoA hydratase enzymes (Table 3), suggesting that GnyH could be involved in the hydration of {gamma}-carboxygeranoyl-CoA to 3-hydroxy-{gamma}-carboxygeranoyl-CoA (Fig. 2). The product encoded by the sixth gene, gnyL, shows high similarity to 3-hydroxy-3-methylglutaryl-CoA lyase enzymes (Table 3). The GnyL protein probably catalyzes the reaction of 3-hydroxy-{gamma}-carboxygeranoyl-CoA to produce acetyl-CoA and 3-oxo-7-methyl-6-octenoyl-CoA, an intermediary suitable for the ß-oxidation pathway (Fig. 2).

From these data, it may be concluded that the gny cluster of the P. aeruginosa chromosome constitutes a catabolic operon involved in the degradation of isoprenoids of the citronellol kind.

Complementation of mutants affected in acyclic isoprenoid degradation.
Mutant strains affected in the gnyD, gnyB, gnyA, or gnyL gene were transformed with wild-type genes from the pMO013850 or pAL-22 plasmid, containing the complete gny cluster, or only the structural genes from P. aeruginosa PAO1; the ability of transformant clones to grow on acyclic isoprenoids was tested. Table 2 shows that the inability of the mutants affected in the gny genes to grow on isoprenoids was complemented by wild-type genes from the pAL-22 plasmid except for the gnyD mutant. This behavior suggests that gnyD is transcribed from the P1 promoter located upstream of the gnyR gene (Fig. 1). When tested in liquid medium with citronellol as the sole carbon source, the mutants were not able to grow, but these mutant strains transformed with the pMO013850 cosmid showed a doubling time similar to that of the wild-type strain (data not shown). The mutant strains transformed with the pAL-22 plasmid also recovered their growth ability, although with a longer doubling time (data not shown), probably due to lack of regulatory elements. In addition, gny mutants were not affected in their ability to grow on 6-methyl-5-hepten-2-one, a 3-methyl-branched alkane compound that theoretically concurs on 3-methycrotonoyl-CoA, suggesting that the gny cluster is involved in isoprenoid catabolism but does not encode the enzymes involved in 3-methylcrotonoyl-CoA catabolism (Fig. 2). However, the enzymatic nonspecificity of geranoyl-CoA carboxylase, as was described to occur in P. citronellolis (10), may be involved in both catabolic pathways.

Transcriptional analysis of the gny cluster.
When the PAO1 strain was incubated in M9 liquid medium supplemented with 0.1% succinic acid plus 0.05% citronellic acid and then inoculated in medium with 0.1% citronellic acid as the sole carbon source, growth was faster than in non-pretreated cultures (data not shown), suggesting induction of the isoprenoid catabolic genes. To explore this response at the transcriptional level, the expression of the gny cluster was first analyzed by a slot blot Northern DNA/RNA hybridization assay with a DNA fragment containing the gnyB gene as a probe. RNA obtained from citronellic acid-induced PAO1 cultures showed a 10-fold increase in hybridization signal compared to RNA from glucose-grown cultures (data not shown). In RT-PCR analysis, more of an amplified fragment containing the gnyR-D genes was found in RNA from glucose-grown cultures than in RNA from cultures grown in citronellol. In contrast, an amplified fragment of the gnyB gene was observed only in RNA obtained from PAO1 cultures grown in citronellol (data not shown). The RT-PCR data, sequence analysis, and complementation results suggest that the gny cluster is transcribed as two polycistronic mRNAs.

Validation of the functionality of the regulatory regions was further tested with LacZ transcriptional fusions. The results showed that, in cultures grown in glucose, P1 promoted transcription of the lacZ gene, whereas expression from the P2 promoter was repressed (Fig. 3). Upon addition of citronellol, this behavior reverted (Fig. 3). These results confirm that the gny cluster is expressed as two transcriptional units, one starting from the P1 promoter and containing the gnyRD genes, and the other starting from the P2 promoter and comprising the gnyBHAL cluster (Fig. 1). These data also suggest that the gnyR gene product may be the repressor of the gnyB promoter in the presence of glucose. Similar situations in which catabolic operons or genes are repressed by glucose or succinate have been described (17, 18).



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  		FIG. 3. Transcriptional analysis of the P1 and P2 promoters of the gny cluster from P. aeruginosa PAO1. Open and solid bars correspond to the ß-galactosidase activity of cultures of the strains with the indicated plasmids, respectively. Cells were grown in M9 medium with glucose (open bars) or glucose plus citronellol (filled bars), and ß-galactosidase was measured after 1 h of induction, as described in the text. Data given are the averages of two independent experiments; the standard deviation was less than 10% of the given value.

Detection of biotinylated proteins.
As the gny operon includes a biotinylated carboxylase (Fig. 2), a Western blot assay based on the detection of biotin-containing proteins was used to identify this enzyme. In PAO1 glucose-grown cultures, two major biotinylated proteins appeared with molecular masses of 62 kDa and 22 kDa (bands III and IV, respectively, in Fig. 4). Sequence analysis of the PAO1 genome suggests that these proteins might correspond to biotinylated carboxylases encoded by ORFs PA5435 (66,095 Da) (26) and PA4847 (16,851 Da) (2), respectively. Cultures grown in minimal medium with citronellol as the sole carbon source, however, showed two additional biotinylated protein bands of 73 and 70 kDa (bands I and II, respectively, in Fig. 4). Similar results have been described in P. citronellolis, where the biotinylated subunits of geranoyl-CoA carboxylase and 3-methylcrotonoyl-CoA carboxylase showed molecular masses at pH 7.2 of 75 and 73 kDa, respectively, and at pH 8.9 of 70 and 68 kDa, respectively (7, 10). These proteins induced by citronellol may correspond to geranoyl-CoA ({alpha}-geranoyl-CoA carboxylase, ORF PA2012, here named GnyA) and 3-methylcrotonoyl-CoA ({alpha}-3-methylcrotonoyl-CoA carboxylase, ORF PA2891) biotinylated subunits of their respective carboxylases.



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  		FIG. 4. Western blot analysis of biotinylated proteins in P. aeruginosa PAO1. Lanes 1 to 3, cell extracts from the PAO1 strain grown in glucose, citronellol, or glucose plus citronellol, respectively; lane 4, protein markers. I to IV, proteins identified with antibiotin antibodies with molecular masses of 73, 70, 62, and 22 kDa, respectively.

In agreement with data from the RT-PCR and transcriptional fusion assays (Fig. 3), the expression of both {alpha}-geranoyl-CoA carboxylase and {alpha}-3-methylcrotonoyl-CoA carboxylase showed repression by glucose (Fig. 4, lane 1), even in the presence of citronellol (Fig. 4, lane 3). The induction-repression behavior of carboxylase subunit expression was further confirmed by a Western blot assay of cultures induced at different times. Glucose-grown cultures did not show expression of the 73- and 70-kDa proteins even after 48 h of incubation, whereas in citronellol these proteins appeared at 24 h (data not shown). These findings confirm the involvement of both carboxylases in the isoprenoid degradation pathway shown in Fig. 2.

The expression of biotinylated proteins in PAO1 mutant strains was tested. Western blot assays showed that in mutants affected in the gnyA gene (PAE80 and PAMgnyA), the 73-kDa biotinylated protein was not present (Fig. 5A), suggesting that the {alpha}-geranoyl-CoA carboxylase subunit was not expressed. In the gnyB mutant (PAMgnyB), the GnyA protein was also absent (Fig. 5A), probably due to a truncated transcript caused by disruption of the gentamicin resistance cassette in the homologous recombination event.



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  		FIG. 5. Expression of the GnyA biotinylated protein. (A) Expression of GnyA in P. aeruginosa. Lanes: 1, PAO1 culture grown in glucose; 2 to 5, cultures grown in glucose plus citronellol, 2, PAO1; 3, PAMgnyB; 4, PAE80; 5, PAMgnyA; 6, biotinylated protein band marker of 66 kDa. I to III, biotinylated proteins of 73, 70, and 62 kDa, respectively. (B) Expression of GnyA in (lane 1) P. fluorescens strain L1 grown in glucose plus citronellol; (lane 2) Pf-L1 pMO013850 grown in glucose plus citronellol; (lane 3) strain PAO1 grown in glucose plus citronellol; (lane 4) biotinylated protein standard. Arrows indicate the positions of the biotinylated protein band of 66 kDa (right) and of the GnyA protein (left).

To establish that the gnyA gene encodes the {alpha}-geranoyl-CoA carboxylase and that it corresponds to the 73-kDa biotinylated protein identified, the pMO013850 cosmid was transferred to P. fluorescens L1, a strain unable to grow on acyclic isoprenoids and with a different profile of biotinylated proteins. In the transformed strain, the 73-kDa protein band was clearly observed (Fig. 5B). Although this strain was unable to grow on isoprenoids, suggesting that the pMO013850 cosmid was not sufficient to promote isoprenoid catabolism in P. fluorescens, these results confirm that gnyA encodes the {alpha}-geranoyl-CoA carboxylase subunit.

Metabolite accumulation in mutant strains.
The effect of mutations on isoprenoid catabolism in the PAO1 derivative strains was analyzed by measuring metabolite accumulation. Solvent-soluble compounds were extracted from cultures grown with citronellol and assayed by gas chromatography-mass spectrometry. Accumulation of four major compounds was observed in the supernatants of cultures from the gnyB and gnyA mutants compared to cultures of the wild-type strain (Fig. 6). Accumulated compounds corresponded by mass spectrum to citronellol, citral, citronellic acid, and geranic acid. These compounds were found in significantly lower amount in supernatants from the gnyB and gnyA mutants complemented with the pMO013850 cosmid (Fig. 6).



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  		FIG. 6. Metabolite accumulation in cultures of P. aeruginosa grown in citronellol. Cultures were grown in M9 medium with succinic acid as the carbon source for 12 h, and citronellol was then added. The metabolites were extracted from the supernatants and analyzed at 48 h after the addition of citronellol as described in the text (gas chroma-tography-mass spectrometry). Extracts were from (A) PAO1 (wild-type strain); (B and C) PAA447 and PAE80 strains (gnyB and gnyA mutants, respectively), unable to grow on citronellol; (D and E) PAA447 and PAE80 mutants transformed with the pMO013850 cosmid, able to grow on citronellol. The peaks with retention times of 7.4, 7.5, 8.4, and 9.6 min correspond to citronellol (m/z = 156), citral and citronellal mix (m/z = 152 and 154, respectively), citronellic acid (m/z = 170), and geranic acid (m/z = 168), respectively.

Rate studies also showed a higher accumulation rate of geranic acid in both the gnyB and gnyA mutants; in contrast, the PAO1 strain and the cosmid-complemented mutant showed a lower accumulation proportion and the disappearance of this compound (Table 4). The higher accumulation of geranic acid in the mutants tested is probably due to the inactivation of the geranoyl-CoA carboxylase encoded by the gnyB and gnyA genes. These results suggest that in the isoprenoid degradation upper pathway, citronellol is oxidized to citronellic and geranic acids during the first hours of incubation, whereas the second phase of degradation (the lower pathway) occurs more slowly. These data also confirm that inactivation of the gnyB or gnyA gene impaired the lower pathway, causing metabolite accumulation.


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  		TABLE 4. Metabolite accumulation in culturesa

In conclusion, the mutagenesis, transcriptional analysis, and expression results suggest that the lower pathway of acyclic isoprenoid degradation in P. aeruginosa is encoded by the gny cluster. These analyses also showed that the products of the gnyB and gnyA genes constitute the geranoyl-CoA carboxylase, of which biochemical characterization is currently in process.


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ACKNOWLEDGMENTS
 
We thank J. E. López Meza and J. L. Salvador Hernández for technical assistance in sequencing and gas chromatography-mass spectrometry analysis, respectively. We also thank the Pseudomonas Genome Project, the Pseudomonas Genetic Stock Center, and the Pseudomonas aeruginosa Community Annotation Project for the use of the updated database of annotation information of the website and for cosmid donation. We thank S. M. Wong and J. J. Mekalanos for donation of pFAC.

This research was funded by CONACYT (J35095-B) and C.I.C./UMSNH grants. A.L.D.-P. held a postgraduate CONACYT scholarship during the development of this work.


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FOOTNOTES
 
* Corresponding author. Mailing address: Instituto de Investigaciones Químico-Biológicas, Universidad Michoacana, Edif. B-3, Ciudad Universitaria, CP 58030, Morelia, Michoacán, México. Phone and fax: (52) 443 3265788. E-mail: jcgarcia{at}zeus.umich.mx. Back


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Applied and Environmental Microbiology, September 2004, p. 5102-5110, Vol. 70, No. 9
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.9.5102-5110.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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