Utilization of geraniol is dependent on molybdenum in Pseudomonas aeruginosa: evidence for different metabolic routes for oxidation of geraniol and citronellol
Birgit Höschle and
Dieter Jendrossek
Institut für Mikrobiologie, Universität Stuttgart, Allmandring 31, 70550 Stuttgart, Germany
Correspondence
Dieter Jendrossek
dieter.jendrossek{at}imb.uni-stuttgart.de
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ABSTRACT
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Mini-Tn5-induced mutants with defects in utilization of linear terpenes such as citronellol, geraniol, citronellate and/or geranylate were isolated from Pseudomonas aeruginosa. One mutant was unable to utilize geraniol but showed wild-type growth with the three other acyclic terpenes tested. The Tn5 insertion site of the mutant was determined by DNA sequencing. Comparison with the P. aeruginosa genome sequence revealed that PA3028, an ORF with high similarity on the amino acid level to molybdenum cofactor biosynthesis protein A2 (encoded by moeA2), was the target of mini-Tn5 in the mutant. Disruption of moeA2 in P. aeruginosa PAO1 wild-type by insertion mutagenesis resulted in the same geraniol-minus phenotype. The ability to utilize geraniol was restored to the mutant by conjugative transfer of PCR-cloned wild-type moeA2 on a broad-host-range plasmid. Growth of P. aeruginosa PAO1 on geraniol and geranial, but not on citronellol, citronellate or geranylate, was inhibited by the presence of 10 mM tungstate, a molybdenum-specific inhibitor. Inhibition by tungstate was prevented by addition of molybdate. The results indicate that at least one step in the oxidation of geraniol to geranic acid (geranial oxidation) is a molybdenum-dependent reaction in P. aeruginosa and is different from the molybdenum-independent oxidation of citronellol to citronellate.
Abbreviations: Moco, molybdenum cofactor
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INTRODUCTION
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Terpenes are widespread compounds in nature and frequently appear in the form of aroma compounds such as citronellol (linear terpene) and camphor (aromatic terpene). Terpenes consist of two isoprene units (monoterpenes) and frequently are functionalized at one end of the molecule. Terpenes represent one of several subgroups of natural compounds harbouring methyl-branched molecular structures derived from isoprene. Examples of other subgroups are carotenoids, steroids and polyisoprene (rubber). Citronellol (3,7-dimethyl-6-octen-1-ol) is a model compound for linear monoterpenes naturally occurring in citrus plants, where it is responsible for the characteristic flavour of its fruits. Citronellol is also of industrial interest due to its commercial use in food (flavour) and the perfume industry (odour). Moreover, citronellol is one of very few naturally occurring insect (mosquito) repellents and is a component of commercially available anti-insect outdoor candles. Geraniol is structurally related to citronellol and differs from the latter only by the presence of an additional double bond (Fig. 1
). Geraniol causes the typical flavour of plants belonging to the genus Geranium.

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Fig. 1. Degradation pathway of citronellol and geraniol in P. citronellolis according to Seubert & Fass (1964b) .
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Utilization of linear terpenes as a carbon source by micro-organisms was first studied in the early 1960s by Seubert and coworkers (Seubert & Fass, 1964b
). Seubert isolated Pseudomonas citronellolis by its ability to utilize citronellol and related compounds as the sole source of carbon and energy (Seubert, 1960
). Linear terpenes are difficult to metabolize due to the presence of
-methyl groups that inhibit
-oxidation. The first steps of the catabolic pathway of citronellol and geraniol are the oxidation of the alcohols to the corresponding aldehydes and acids [citronellal, citronellate and geranial (citral), and geranylate] and subsequent activation to the corresponding CoA esters citronellyl-CoA and geranyl-CoA. Because of the structural similarity of citronellol and geraniol, it is assumed that oxidation of citronellol and geraniol is catalysed by the same enzymes (Fig. 1
, Cantwell et al., 1978
). Citronellyl-CoA can be converted to geranyl-CoA by a dehydrogenase step, and all subsequent reactions are the same for citronellol and geraniol utilization. A key enzyme of the citronellol/geraniol degradation pathway is geranyl-CoA-carboxylase (Seubert et al., 1963
). Geranyl-CoA-carboxylase converts the branched-chain
-methyl group of geranyl-CoA to an acetate function. After hydratization of the carboxylated intermediate isohexenylglutaconyl-CoA by isohexenylglutaconyl-CoA hydratase the acetate side group is cleaved off by 3-hydroxy-3-isohexenylglutaryl-CoA lyase (Seubert & Fass, 1964a
). The major cleavage product of the lyase reaction (7-methyl-3-oxo-6-octenoyl-CoA) can be degraded by two rounds of subsequent
-oxidation reactions without hindrance by
-methyl groups. The resulting metabolite (3-methylcrotonyl-CoA) is most probably degraded via the leucine degradation pathway, which involves another carboxylase that is unable to utilize geranyl-CoA as a substrate but that carboxylates methylcrotonyl-CoA (Seubert & Fass, 1964b
; Hector & Fall, 1976
). For an overview of the postulated citronellol/geraniol degradation pathway, see Fig. 1
. The two branched-chain specific carboxylases of P. citronellolis, geranyl-CoA-carboxylase and methylcrotonyl-CoA carboxylase, have been purified and biochemically characterized (Seubert et al., 1963
; Fall & Hector, 1977
). P. aeruginosa and Pseudomonas mendocina are two additional bacteria with the capacity to utilize citronellol and other linear terpenes (Cantwell et al., 1978
). In this study, we initiated experiments to identify genes involved in the degradation pathway of geraniol in P. aeruginosa.
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METHODS
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Bacterial strains, plasmids and culture conditions.
The bacterial strains and plasmids used in this study are shown in Table 1
. Cultures of P. aeruginosa PAO1 were routinely grown in nutrient broth (NB) or in mineral salt medium (Schlegel et al., 1961
) containing different carbon sources [0·4 % (w/v) sodium succinate, 0·1 % (w/v) sodium citronellate, 0·1 % (w/v) sodium geranylate] at 30 °C. Water-insoluble carbon sources (citronellol, geraniol) were added in the space between the Petri dish and the agar (20 µl per plate) and the plates were sealed with Parafilm. Growth on solid media with liquid carbon sources was performed in separate incubators to avoid cross-contamination by vapours. Liquid cultures with citronellol or geraniol as carbon source additionally contained 4 % (v/v) heptamethylnonane to reduce the toxic effect of the terpenes. Heptamethylnonane solublizes citronellol and geraniol; it is water immiscible, non-toxic and not metabolized.
Growth experiments with tungstate and molybdate were done in Tris-buffered mineral salt medium (Mergeay et al., 1985
) with 10 mM sodium tungstate and 12 mM sodium molybdate. Escherichia coli strains were grown in LuriaBertani (LB) media at 37 °C. Solid media additionally contained 1·5 % (w/v) agar.
A mutant of P. aeruginosa PAO1 resistant to 500 µg streptomycin ml1 (P. aeruginosa PAO1 Smr) was isolated by two rounds of repeated transfer to NB media containing increasing amounts of streptomycin. For conjugation experiments, P. aeruginosa PAO1 Smr and E. coli S17-1 containing the respective plasmids were grown in NB medium and LB medium, respectively. Equal volumes of the donor and of the recipient culture were mixed and spotted onto a NB agar plate and incubated overnight at 30 °C. The cells were resuspended in 3 ml of 10 mM MgSO4, plated on selection agar containing the appropriate amounts of antibiotics and incubated for 2 days at 30 °C. Selected transconjugants were screened for utilization of different carbon sources.
Identification and sequencing of transposon insertion fragments.
PstI- or XhoI-digested genomic DNA of selected transconjugants was analysed by Southern blot hybridization with a DIG-labelled PCR probe (forward primer, 5'-AATGCGCTCATCGTCATCCTCGG-3'; backward primer, 5'-CGATCCTTGAAGCTGTCCCTGA-3') specific for the tetracycline resistance gene of the pUT mini-Tn5-Tc plasmid. Tn5-Tc-containing DNA fragments of the size of interest (2·5±0·5 kb) were isolated from agarose gel, cloned in PstI- or XhoI-digested pBluescriptSK+ and transformed into E. coli JM109. Clones with plasmids harbouring the mini-Tn5-Tc fragment were selected on LB plates containing 12 µg tetracycline ml1.
Sequencing of the isolated recombinant plasmids containing the mini-Tn5-Tc fragment was done with synthetic oligonucleotides specific for the respective I- and O-ends (5'-AGTGAGGGTTTGCAACTGC-3' and 5'-TTAAGCGTGCATAATAAGC-3'), and T3- and T7-primers specific for the multiple cloning site of the pBluescript SK+ vector. The mini-Tn5-Tc insertion site of the respective mutants was identified by comparison of the DNA sequences obtained with the updated database of the Pseudomonas Genome Project (www.pseudomonas.com).
Disruption of moeA2.
Disruption of moeA2 was carried out using pKnockout-G for rapid gene inactivation in P. aeruginosa (Windgassen et al., 2000
). A 3'- and 5'-truncated fragment of the moeA2 gene was obtained by PCR-mediated amplification of the ORF PA3028 from P. aeruginosa genomic DNA using the synthetic oligonucleotides PA3028-fwd (5'-GGAATTCCCCGAACAGCGCCATGGACGGCTAC-3') and PA3028-rev (5'-GGAATTCCTGCTCCAGGCGACCACGCAGGTACTC-3') as moeA2-specific primers. The EcoRI-digested purified 904 bp PCR fragment was cloned in the EcoRI site of pKnockout-G and transformed in E. coli S17-1. The plasmid pKnockout-G : : PA3028 was transferred to P. aeruginosa PAO1 Smr via conjugation and selection on LB agar containing 500 µg streptomycin ml1 and 50 µg gentamicin ml1. The disruption of moeA2 was verified by PCR using one moeA2-specific and one pKnockout-specific primer and by two Southern blot hybridization experiments of chromosomal mutant DNA with a DIG-labelled probe specific for the gentamicin resistance gene and a DIG-labelled probe specific for moeA2, respectively.
Genetic complementation.
For genetic complementation of the moeA2 mutant, moeA2 was amplified from P. aeruginosa PAO1 genomic DNA with the synthetic oligonucleotides 3028-fwd (5'-CGGAATTCCGGTCATCGCCCGTGCAACTTCG-3') and 3028-rev (5'-CGGGATCCCGCCGGCGGCGCAATGAGAAC-3'). The purified BamHI/EcoRI-digested 1332 bp fragment was cloned in the BamHI/EcoRI opened vector pBBR1MCS-5 and transformed in E. coli S17-1. E. coli clones harbouring the recombinant plasmid were used for conjugative transfer of pBBR1MCS-5 : : moeA2 to P. aeruginosa.
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RESULTS
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Isolation of mini-Tn5-Tc-induced mutants impaired in geraniol utilization
Mini-Tn5-Tc-induced mutants of P. aeruginosa PAO Smr were generated by conjugation with E. coli S17-1 (pUT mini-Tn5-Tc), as described in Methods, and selected on NB-Sm-Tc agar. About 8500 of the mutants obtained were tested for utilization of citronellol, citronellate, geraniol and geranylate. In total, about 253 mutants showed significantly reduced growth on at least one of the four terpenes tested. Thirty-five additional mutants apparently represented auxotrophs, because they grew normally on complex media but showed no growth on mineral salt media independent of a carbon source. Auxotrophs were not further analysed. Thirty-nine of the 253 mutants (1·5 %) specifically affected in the utilization of linear terpenes were strongly reduced in growth on geraniol but showed normal growth on the three other terpenes. Two hundred and fourteen mutants showed different combinations of more or less severe growth defects on one or more terpenes, including 129 mutants with partial growth reduction on geraniol. Only three mutants were unable to utilize any of the four tested terpenes. The high frequency of mutants that were defective in geraniol utilization but that showed normal growth on geranylate, citronellol and citronellate was unexpected, because it was assumed that citronellol and geraniol were oxidized to the corresponding acids by the same enzymes (Seubert & Fass, 1964b
; Cantwell et al., 1978
). Citronellol and geraniol differ only by the presence of one double bond (Fig. 1
). Southern blot analysis of the mutants specifically defective in growth on geraniol revealed that different gene loci were affected by mini-Tn5 insertion in many of the mutants, indicating that several genes are involved in the oxidation of geraniol and that oxidation of geraniol occurs independently from oxidation of citronellol (data not shown).
Identification of the mini-Tn5 insertion site in mutant #11-10-5
Mutant #11-10-5 (geraniol, citronellol+, citronellate+, geranylate+) was selected for further analysis. A 2·8 kbp PstI fragment was identified as the site of mini-Tn5 insertion by Southern hybridization of chromosomal DNA of the mutants with a mini-Tn5-specific DNA probe (data not shown). The corresponding DNA fragment was cloned in E. coli by ligation of chromosomal PstI fragments in pBluescriptSK+ and selection for Tc-resistant transformants. The site of mini-Tn5 insertion was identified by DNA sequencing of the respective recombinant plasmid and by comparing the obtained sequence information with the DNA sequences of mini-Tn5 and with the P. aeruginosa genome database (www.pseudomonas.com). It turned out that mini-Tn5 was inserted at position 3 392 394 within ORF PA3028 of the P. aeruginosa genome. PA3028 (moeA2) encodes a protein that is highly similar to molybdenum cofactor (Moco) biosynthesis proteins of Pseudomonas syringae (65 % identity on amino acid level), Pseudomonas putida, Pseudomonas fluorescens (each 65 %), P. aeruginosa (MoeA1, 51 %), E. coli (41 %) and to related proteins of many other bacteria.
Inactivation of moeA2 in P. aeruginosa by insertion mutagenesis results in the geraniol-minus phenotype that can be restored by expression of an intact moeA2 gene
To verify that the insertion of mini-Tn5 in mutant #11-10-5 was solely responsible for the observed geraniol-minus phenotype, chromosomal moeA2 was inactivated using a truncated moeA2 gene that had been PCR-amplified from chromosomal DNA and that had been cloned into pKnockout-G by conjugative insertion mutagenesis, as described in Methods. The success of chromosomal inactivation of moeA2 was verified by Southern blot and PCR analysis (data not shown). When the resulting insertion mutant P. aeruginosa Smr-
3028 was tested for utilization of linear terpenes, the same phenotype (geraniol, citronellol+, citronellate+, geranylate+) was obtained as for the mini-Tn5 mutant #11-10-5. The DNA sequence of PA3028 (moeA2) including 84 bp of the 5'-upstream region was PCR-amplified and cloned in pBBR1MCS-5 to yield pBBR1MCS-5 : : moeA2. When pBBR1MCS-5 : : moeA2 was conjugatively transferred to P. aeruginosa #11-10-5, the ability to utilize geraniol as a sole source of carbon and energy was restored (Fig. 2
).

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Fig. 2. Growth of the P. aeruginosa PAO1 wild-type (WT), the transposon mutant #11-10-5, the insertion mutant P. aeruginosa Smr- 3028 and the restored mutant P. aeruginosa #11-10-5K harbouring pBBR1MCS-5 : : moeA2 on agar plates containing citronellol or geraniol as sole carbon source after 3 days of incubation at 30 °C.
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Growth of P. aeruginosa on geraniol is dependent on molybdenum
The identification of moeA2 as a target of mini-Tn5 insertion in mutant #11-10-5 suggests that growth of P. aeruginosa on geraniol might depend on molybdenum and might require intact Moco. Fig. 3
(A) shows growth of P. aeruginosa PAO1 wild-type in liquid culture on succinate and on the four linear terpenes. The highest doubling time was obtained on succinate (td, 1·8 h), followed by citronellate (td, 2·3 h), citronellol (td, 2·9 h), geraniol (td, 9 h) and geranylate (td, 11 h). When the same growth experiment was repeated in the presence of 10 mM tungstate, a strong competitive inhibitor of molybdenum-dependent processes, almost identical doubling times were recorded for succinate, citronellol, citronellic acid and geranylate (Fig. 3B
). However, growth on geraniol was very poor and stopped after one to two doublings in the presence of 10 mM tungstate. To investigate whether tungstate was a specific inhibitor of a molybdenum-dependent process or whether it was toxic for the cells during growth on geraniol, the experiment was repeated in the presence of 10 mM tungstate plus 12 mM molybdate (Fig. 3C
). If tungstate acts as a general toxic compound, the addition of molybdate should not restore growth on geraniol. If, however, tungstate specifically inhibits molybdenum-dependent processes, the addition of high concentrations of molybdate should complement the inhibitory effect of tungstate. Exactly this was found, as shown in Fig. 3(C)
: growth of P. aeruginosa PAO1 in the presence of tungstate and molybdate could not be differentiated from growth in the absence of tungstate and molybdate. In conclusion, growth on geraniol is a molybdenum-requiring process in P. aeruginosa. The experiments were repeated on solid media and the same principal results were obtained (data not shown). P. aeruginosa is also able to utilize geranial as a carbon source. When growth of P. aeruginosa on geranial was tested, tungstate (10 mM) clearly inhibited the growth of the bacteria. Growth inhibition by tungstate was reversible by the addition of 12 mM molybdate. These results indicate that the oxidation of geranial to geranic acid is a molybdenum-dependent step.
Utilization of geraniol in P. citronellolis and P. mendocina is not inhibited by tungstate
P. citronellolis and P. mendocina are close relatives of P. aeruginosa and are the only other validly described species capable of the utilization of linear terpenes such as citronellol and geraniol (Cantwell et al., 1978
). The biochemical pathway of linear terpene utilization in P. citronellolis and P. mendocina is assumed to be identical or very similar to that of P. aeruginosa. When the effect of tungstate (10 mM) on the growth of P. citronellolis and P. mendocina with citronellol, geraniol, citronellate or geranylate as carbon sources was analysed, no growth inhibition by tungstate on citronellol, geraniol and citronellate was found, and only slight growth inhibition on geraniol was observed. Apparently, the utilization of geraniol is different from that of P. aeruginosa in P. citronellolis and P. mendocina.
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DISCUSSION
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In this study, we investigated the catabolism of linear terpenes in P. aeruginosa by mini-Tn5 mutagenesis. To our surprise, a high frequency of mutants was obtained (39 mutants, 1·5 %) that were specifically impaired in the utilization of geraniol but showed wild-type phenotypes with respect to the utilization of geranylate, citronellol and citronellate. The site of mini-Tn5 insertion in one of these mutants (#11-10-5) was within moeA2. MoeA2 is a protein involved in the Moco biosynthesis of many bacteria. One other mutant with the same phenotype as #11-10-5 also harboured mini-Tn5 within moeA2 (data not shown). In all other 37 mutants with growth reduction on geraniol, mini-Tn5 had integrated in different gene loci, as revealed by the different signal sizes in the Southern blots of chromosomal DNA of the mutants. In E. coli and in Rhodobacter capsulatus, MoeA is essential for the ligation of molybdate to molypdopterin (Leimkühler et al., 1999
; Nichols & Rajagopalan, 2005
). The involvement of molybdenum in the oxidation of geraniol was confirmed by specific inhibition of the growth of P. aeruginosa on geraniol by tungstate and by the competitive reduction of growth inhibition by molybdate. Since growth on geranial was also sensitive to tungstate, we assume that the oxidation of geranial to geranylate by a geranial dehydrogenase is the molybdenum-dependent step. However, our experiments do not exclude the possibility that the oxidation of geraniol to geranial is also molybdenum dependent. The sensitivity of geraniol utilization to tungstate was restricted to P. aeruginosa and was only partially observed in P. citronellolis or P. mendocina. These results indicate that the oxidation of geraniol to geranylate is a molybdenum-dependent step, and that the respective oxidation reactions could be catalysed by different enzymes in P. aeruginosa on the one hand and in P. citronellolis and P. mendocina on the other, or can be partially replaced by molybdenum-independent isoenzymes in the latter two species. The involvement of molybdenum-dependent steps is supported by the high frequency of the geraniol-minus phenotype in P. aeruginosa mini-Tn5 mutants: synthesis of active Moco requires several enzymic steps and the combined action of many gene products (Nichols & Rajagopalan, 2002
; Wuebbens & Rajagopalan, 2003
). Mutation in only one of these steps prevents the synthesis of active Moco, thus leading to a geraniol-minus phenotype. We assume that at least some of the other geraniol-minus mutants have defects in other genes of Moco synthesis. Since utilization of citronellol was neither reduced in nor sensitive to the presence of tungstate in both moeA2 mutants (mini-Tn5 mutant #11-10-5 and the moeA2 insertion mutant), we conclude that oxidation of citronellol is not molybdenum dependent in P. aeruginosa and therefore is apparently catalysed by different enzymes compared to oxidation of geraniol, and also that these different sets of enzymes cannot substitute for each other in P. aeruginosa. Other well-studied examples of molybdenum-dependent oxidation reactions are molybdenum hydroxylases (e.g. xanthin dehydrogenase), eukaryotic oxotransferases (e.g. sufite oxidase, nitrate reductase) and bacterial oxotransferases (e.g. formate dehydrogenase, DMSO reductase) (Hille, 1999
; Moura et al., 2004
and references cited therein).
Recently, a gene cluster comprising two putative operons (PA2011 to PA2014 and PA2015 to PA2016) has been identified to be involved in the degradation of linear terpenes in P. aeruginosa (Diaz-Perez et al., 2004
). Insertion mutagenesis in five of the six genes resulted in the inability of the respective mutants to utilize citronellol, geraniol and related linear terpenes. The cluster contains putative genes for carboxylase subunits and other genes apparently involved in the degradation of acyclic terpenes, but it contains no genes with apparent function in Moco synthesis. Conjugative transfer of the cluster to P. fluorescens did not result in the ability of the transconjugants to utilize linear terpenes. The results of Diaz-Perez et al. (2004)
and our studies indicate that the ability to utilize linear terpenes is complex, depends on more genes than have yet been identified and may be partially different in P. aeruginosa and P. citronellolis.
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ACKNOWLEDGEMENTS
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We thank R. Schäfer for assistance in mutant isolation and K. E. Jaeger for providing pKnockout.
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Received 9 February 2005;
revised 15 April 2005;
accepted 18 April 2005.
Copyright © 2005 Society for General Microbiology.