Edited by R.Y.C. Lo
A genetically engineered strain of Pseudomonas putida as a useful tool for identifying new therapeutic herbicides
Article first published online: 9 JAN 2006
FEMS Microbiology Letters
Volume 249, Issue 2, pages 297–302, August 2005
How to Cite
Arias-Barrau, E., Sandoval, Á., Arcos, M., Arias, S., Naharro, G., Olivera, E. R. and Luengo, J. M. (2005), A genetically engineered strain of Pseudomonas putida as a useful tool for identifying new therapeutic herbicides. FEMS Microbiology Letters, 249: 297–302. doi: 10.1016/j.femsle.2005.06.027
- Issue published online: 9 JAN 2006
- Article first published online: 9 JAN 2006
- Received 1 April 2005, Revised 16 May 2005, Accepted 13 June 2005
- Homogentisic acid;
- Pseudomonas putida;
A genetically engineered strain of Pseudomonas putida U designed for the identification of new therapeutic herbicides has been obtained. In this bacterium, deletion of the homogentisate gene cluster (hmgRABC) confers upon this mutant huge biotechnological possibilities since it can be used: (i) as a target for testing new specific herbicides (p-hydroxy-phenylpyruvate dioxygenase inhibitors); (ii) to identify new therapeutic drugs-effective in the treatment of alkaptonuria and other related tyrosinemia – and (iii) as a source of homogentisic acid in a plant–bacterium association.
Homogentisic acid (Hmg) is a central metabolite involved in many important cellular functions in different organisms [1–3]. In higher plants, blue and green algae and in photosynthetic bacteria, Hmg is the precursor of plastoquinones and other photosynthetic pigments, tocopherols, α-tocopherolquinones, tocotrienols as well as other important antioxidant molecules . In other organisms (several microbes and different eukaryotes) Hmg is the first intermediate in the melanogenetic pathway, underscoring the physiological importance of this compound in the prevention of cellular damage caused by oxidative stress, radiations or other harmful physico-chemical agents [1,2].
Many acute illnesses generated as inborn metabolic errors  affecting the catabolism of phenylalanine and tyrosine are related to the accumulation of homogentisic acid. Alkaptonuria, a rare inherited disorder of metabolism characterized by the fact that the patient's urine turns black (or dark) when exposed to air [2,5], is caused by a mutation in the gene encoding the homogentisate dioxygenase (E.C.188.8.131.52) [6,7]. Patients with this illness accumulate Hmg in collagenous tissue, leading to severe degeneration of the cartilage, of the spine and other major points of the body, usually causing osteoarthritis in adulthood [2,6].
Taking into account the metabolic importance of Hmg in different organisms, study of the enzymatic steps involved in the synthesis of this compound as well as identification of new drugs that inhibit its biosynthesis have attracted much interest in recent years. Many investigators have approached the characterization of specific inhibitors of 4-hydroxy-phenylpyruvate dioxygenase (Hpd) that could act as effective drugs in the treatment of alkaptonuria and related tyrosinemias, and as potent herbicides by preventing the synthesis of tocopherols and hence abolishing the lack of protection against oxidative stress caused by these compounds on biological membranes . Nevertheless, the identification of new herbicides (or antityrosinemic drugs) usually requires multiple enzymatic assays, which hinders the selection procedure and does not ensure that the molecules selected will be effective in vivo (i.e. they cannot be taken up by the target cells). The design of new strategies for the rapid and effective identification of these kinds of inhibitors could facilitate the isolation of new therapeutic herbicides.
2Materials and methods
Molecular biology products were supplied by Amersham Pharmacia Biotech Gmbh (España). Commercial vectors used for cloning or subcloning genomic fragments were from Stratagene (USA) and Promega (USA) Aromatic compounds were obtained from Merck, (Germany), Lancaster (France) or Sigma (USA). Mesotrione, sulcotrione were purchased from Fluka (Pestanal?) (USA). Biochemical and reagents were supplied by different commercial firms. All other products employed were of analytical quality or HPLC grade.
2.2Microorganisms and vectors
Pseudomonas putida U strain (Coleccion Española de Cultivos Tipo, CECT4848) was from our collection . This strain was originally obtained from R.A. Cooper (Department of Biochemistry, University of Leicester, UK). Escherichia coli DH5α′ (Life Technologies, Rockville, MD, USA) and E. coli DH10B were used for plasmid propagation . E. coli strain NM538 was employed for the amplifications of genomic libraries. E. coli (pRK600) was used as a helper strain in the triparental filter mating .
The commercial plasmid pGEM-T Easy (Promega, USA) were used for subcloning genomic fragments, and the pK18::mob and the pJQ200KS  were employed to induce specific gene disruption. DNA manipulations and sequence analyses were performed as indicated elsewhere [13,14]. For complementation studies in P. putida strains, different genes belonging to the hmg cluster were cloned into the broad-host-range pBBR1-MCS3 plasmid (TcR) . In these experiments, the required genes were amplified by PCR using the following primers: 5′-GCCAGCAACtagTCAGTCAGAGCCCGGAGG-3′ (from 1089 to 1118) and 5′-GTTCCCTTCACTGAGCCGGGCTG-3′ (from 4373 to 4351) (AY168855 GenBank/EBI Data Bank). Small letters indicate a modified sequence to generate a restriction site. The PCR products were cloned into pGEM-T Easy (Promega, USA), subcloned into pBBR1-MCS3 and this construction was used to transform P. putida mutants lacking the homogentisate pathway.
2.3Culture media and growth conditions
P. putida U was maintained on Trypticase Soy Agar (Difco) and growth slants (12 h at 30°C) were used to inoculate liquid media. Five hundred millilitres Erlenmeyer flasks containing 100 ml of a chemically defined medium (MM)  were inoculated with 2 ml each of a bacterial suspension (OD540= 0.5). Incubations were carried out in a rotary shaker (250 rpm) at 30°C for the time required in each set of experiments. The carbon sources used for culture were phenylacetic acid (PhAc, 10 mM), 3-OH-PhAc (10 mM), l-tyrosine (10 mM), succinic acid (42 mM) or combinations thereof. In some experiments different antibiotics (rifampicin, 25 μg ml−1; ampicillin, 100 μg ml−1; gentamicin, 30 μg ml−1 and tetracycline, 35 μg ml−1) were also supplied. When required, mesotrione or sulcotrione were added to the media at a final concentration of 10 mM.
In all the experiments in which solid media were employed, 25 g l−1 Difco agar was added.
2.4Isolation of mutants handicapped in the degradation of homogentisic acid
The isolation of mutants unable to catabolise l-tyr, l-Phe and 3-OH-PhAc was carried out by eliminating the hmg catabolic genes . Deletion of these genes was accomplished by using the methodology described by other authors, which involves a double-recombination event and the selection of the required mutant by expression of the lethal sac B gene [12,16]. All mutants were analysed by PCR. To confirm the position and extent of the deletion the DNA-fragments amplified were sequenced using the following primers: 5′-GGATGCGCGGGAAGCTGTCGAGG-3′ (from position 4283–4261 in the sequence AY168855 – GenBank/EBI Data Bank -) and 5′-GCCAGCAACGGCTCAGTCAGAG-CCCGGAGG-3′ (from 1089 to 1118, in the same sequence).
Along the text, deletions are indicated as Δ followed by the abbreviation of the name of the gene(s) that have been eliminated (Δgene).
2.5HPLC equipment and chromatographic procedure
The intermediates accumulated by the different mutants were analysed by HPLC. Samples taken at different times from the culture broths of the mutants were centrifuged (31,000g, 20 min) to eliminate the bacteria and were filtered through a Millipore filter (0.45-μm pore size). Aliquots of 50 μl were taken and analysed using a high-performance liquid chromatograph (Spectra Physics SP8800) equipped with a variable-wavelength UV/vis detector (SP8450), a computing integrator (SP4290) and a microparticulate (10 μm particle size, 1 μm pore size) reverse-phase column (nucleosil C18, 250 mm × 4.6 mm inner diameter; Phenomenex Laboratories, USA). The mobile phase was 0.05 M K2HPO4 (pH and acetonitrile (99:1 by vol.). The flow rate was 2.5 ml min−1 and the eluate was monitored at 254 nm. Under these conditions the retention times for l-tyrosine (l-Tyr), homogentisic acid (2,5-dihydroxyphenylacetic acid, 2,5-diOH-PhAc), 3,4-diOH-PhAc, 4-OH-PhAc and 3-hydroxyphenylacetic acid (3-OH-PhAc) were 3, 7, 11, 20 and 23 min, respectively.
The degradation of l-phenylalanine (l-Phe), l-tyrosine (l-Tyr) and 3-hydroxyphenylacetic acid (3-OH-PhAc) in P. putida U involves the participation of two convergent pathways that catalyses the transformation of these molecules into homogentisic acid (Hmg, 2,5-di-OH-PhAc) (Fig. 1). Through one of them l-Phe is hydroxylated to l-Tyr by an enzymatic complex integrated by two catalytic enzymes (phenylalanine hydroxylase and carbinolamine dehydratase) and by a putative transcriptional activator (PhhR) [8,17]. These proteins are encoded by the genes belonging to the bicistronic operon (phhAB) and by the gene phhR, which is divergently translated (see Fig. 1). Later, l-Tyr undergoes deamination, catalysed by an unknown tyrosine-aminotransferase that converts this amino acid into p-hydroxyphenylpyruvic acid (Hp), which is finally transformed into Hmg by the enzyme Hpd (E.C.184.108.40.206) [8,17]. The other pathway, that involved in the degradation of 3-OH-PhAc, catalyses – in a single reaction – the hydroxylation of this aromatic acid to Hmg, which is the common intermediate of both routes (Fig. 1). Once synthesized, Hmg induces in P. putida U the three enzymes required for the transformation of this compound into general metabolites (homogentisate dioxygenase -E.C. 220.127.116.11-, 4-maleylacetoacetate cis-trans-isomerase-E.C. 18.104.22.168- and 4-fumarylacetoacetate fumarylhydrolase-E.C. 22.214.171.124-) as has been reported for different prokaryotic and eukaryotic organisms [7,18,19].
The use of microbial systems as molecular models to study inherited metabolic disorder in humans being has been approached in some depth by several authors [4,6,7,19–21]. Taking into account the versatility of bacteria, and in particular P. putida strains, here we designed a genetically engineered strain of P. putida that can be used efficiently for the identification of new therapeutic herbicides. As indicated in Fig. 1, the catabolism of homogentisic acid requires the enzymes encoded in the hmg cluster. Thus, deletion of the genes hmg ABC by means of a single procedure involving a double recombination event [12,16], led to the introduction of a blockage in the catabolic pathway (see Fig. 2), in such a way that the homogentisic acid generated from l-Phe, l-Tyr or 3-OH-PhAc in P. putida U Δhmg ABC began to be accumulated intracellularly, later being released to the broths. As occurs in the urine of patient with alkaptonuria, in the presence of oxygen Hmg appeared as a reddish-brown water-soluble pigment that could be easily identified (see Fig. 4(b)). On the other hand, the transformation of this mutant (P. putida U Δhmg ABC) with the plasmid pBBR1-MCS3 carrying the hmg ABC genes, restored in the recombinant strain the ability for growing in chemical defined medium containing l-Phe, l-Tyr or 3-OH-PhAc. Furthermore, this strain, as occurred in the wild type (P. putida U), did not accumulated homogentisic acid in the broth (Fig. 3).
The potential usefulness of this strain can be seen in Fig. 4. When P. putida U Δhmg was cultured in a chemically defined media containing l-Tyr (5 mM) as a source of Hmg and an additional carbon source to support bacterial growth, it accumulated a dark-red pigment in the medium (see Fig. 4(b)). However, when this bacterium was cultured in the same medium and conditions but with a supply of the triketone herbicide sulcotrione, mesotrione, or other inhibitors of the enzyme 4-hydroxyphenylpyruvate dioxygenase [3,22–25] in the culture, bacterial growth was not affected but the red-dark pigment was not accumulated at all, indicating that Hmg had not been synthesized (see Fig. 4(a)). By contrast, when P. putida U Δhmg was cultured in MM + 3-OH-PhAc (5 mM) + succinic acid (42 mM) either in the presence or in the absence of sulcotrione (or mesotrione), this mutant accumulated in both cases the dark-red product pigment. This is because in the genesis of homogentisic acid from 3-OH-PhAc does not require the 4-hydroxyphenylpyruvate dioxygenase activity but an hydroxylase which is not sensitive to these inhibitors (see Fig. 1). HPLC analysis of the culture broths of P. putida U Δhmg revealed that true Hmg (see Section 2) was excreted and accumulated when this mutant was cultured in MM +l-Tyr (or l-Phe) (5 mM) + succinic acid (42 mM), whereas in the presence of sulcotrione or mesotrione (5 mM) homogentisic acid was not produced even after long time of incubations (more than a week). However, as above indicated, HPLC analysis of the culture broth of P. putida U Δhmg cultured in MM + 3-OH-PhAc (5 mM) + succinic acid (42 mM) showed that Hmg was always produced (in the presence or in the absence of the 4-hydroxyphenylpyruvate dioxygenase inhibitors tested).
We surmise that this genetically engineered bacterium could be a useful tool for the identification of new inhibitors of Hpd as well as others affecting the synthesis of Hmg in this bacterium. Thus, culture of this strain either in Petri dishes or in liquid media supplemented with l-Tyr and different potential inhibitors of these enzymes could facilitate the identification of new therapeutic herbicides by mere observation of the colour of the culture media (see Fig. 4).
It could be argued that use of this new method has an important limitation with respect to others based on the determination of the effect caused by the putative inhibitor on Hpd activity in vitro. Although it is clear that using the bacterial model, all the compounds that are extremely toxic, that affect bacterial growth or that cannot be transported by the microbe will not be selected, it is equally true that the apparent limitation is in fact an additional advantage, since our system allows the selection of quite specific inhibitors that do not affect other important functions in the plants, bacteria or other organisms entering in contact with the herbicide . In conclusion, the greater the specific inhibition, the greater the therapeutic value of the molecule selected and the easier its use in human therapy.
Additionally, deletion of the hmg cluster in symbiotic bacteria could represent an important model for studying the evolutionary and environmental advantages that these microbes afford to the plant. Thus, it is a priori expected that the presence of these microbes in a bacterium–plant association could represent an extra source of Hmg; this will promote the synthesis of photosynthetic pigments, antioxidants and may also contribute to increasing resistance to herbicides that, like those described here, block the synthesis of homogentisic acid.
This investigation was supported by the Comisión Interministerial de Ciencia y Tecnología (CICYT), Madrid, España, Grant BIO2003-05309-C04–01 and by a Grant of Excma Diputación de León (2005). AS, EA, MA, SA and ERO are recipients of fellowships from the Universidad de León (AS) and CICYT (EA, MA, SA and ERO), respectively.
- 1902) The incidence of alkaptonuria: a study on clinical individuality. Lancet 2, 1616–1620.(
- La Du, B.N. (1995) Alkaptonuria. In: The Metabolic and Molecular Bases of Inherited Diseases (Scriver, C.R., Beaudet, A.L., Sly, W., Valle, D., Eds.), pp.1371–1386 McGraw-Hill, New York.
- Sambrook, J., Fritsch, E.F., Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, second ed. Cold Spring Harbor Laboratory Press, Plainview, New York.
- 1995) Enzymes involved in conversion of tyrosine to acetoacetate. Meth. Enzymol. 2, 287–300., (