1 Institute of Biotechnology INBIOTEC, Parque Científico de León, Avda. del Real, no. 1, 24006 León, Spain
2 Area of Microbiology, Faculty of Biology, University of León, 24071 León, Spain
Correspondence
Juan F. Martín
degjmm{at}unileon.es
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ABSTRACT |
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INTRODUCTION |
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Very little is known about the biosynthetic pathway of vinylglycines and the only information available, from Burkholderia andropogonis (Mitchell & Coddington 1991), suggests that rhizobitoxine is derived from amino acids of the aspartic acid family, probably through a homoserine intermediate. This result suggests that the vinylglycine pathway should branch somewhere from the threonine biosynthetic pathway. Additionally, hydroxythreonine has been proposed as a likely biosynthetic intermediate in the pathway from homoserine to rhizobitoxine (Mitchell & Frey, 1988
). On the other hand, studies with Bradyrhizobium elkanii have led to the cloning of a gene, rtxA, whose product is putatively involved (i) in serinol production in the other branch of the route; and (ii) in the condensation of serinol and homoserine as a dihydrorhizobitoxine synthase (Ruan et al., 1993
; Yasuta et al., 2001
).
Recently, another gene, rtxC, encoding a putative dihydrorhizobitoxine desaturase has been proposed for the final step of rhizobitoxine formation (Yasuta et al., 2001). The biosyntheses of AVG and methoxyvinylglycine, however, remain totally obscure, although they should have some steps in common with that of rhizobitoxine since the main part of the molecule is shared.
In a previous paper we cloned and sequenced the gene cluster responsible for the three last steps of threonine biosynthesis in the AVG-producing Streptomyces strain NRRL 5331 (Fernández et al., 2002). Here we report the functional analyses of the three genes encoding the enzymes involved in the conversion of aspartate semialdehyde to threonine in order to provide new insights into AVG biosynthesis in this bacterium.
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METHODS |
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Bacterial strains, cloning vectors and cultivation.
Streptomyces sp. NRRL 5331 was used throughout this study. Escherichia coli strain XL-1 Blue MR was used as a host for subcloning in plasmids pBluescript (Stratagene) and pUC19. Streptomyces sp. NRRL 5331 was routinely grown in YEME medium without sucrose (Kieser et al., 2000) or YEPEG medium (Fernández et al., 2002
). Sporulation was achieved in TBO medium (Aparicio et al., 2000
) at 30 °C. Auxotrophs were tested on Streptomyces minimal medium (MM) (Kieser et al., 2000
). L-Threonine, L-methionine or L-homoserine (each 0·4 mM) were added when required to the MM. Partial purification of AVG was performed from cells grown in asparagine MM (AMM) (L-asparagine, 2 g l1; K2HPO4, 0·5 g l1; MgSO4.7H2O, 0·2 g l1; FeSO4.7H2O, 0·01 g l1; glucose, 2·5 g l1).
Genetic procedures.
Standard genetic techniques with E. coli and in vitro DNA manipulations were as described by Sambrook & Russell (2001). For Streptomyces transformation, plasmids were previously introduced into E. coli ET12567 (dam-13 : : Tn9 dcm-6 hsdM; MacNeil et al., 1992
). Recombinant DNA techniques in Streptomyces species and isolation of Streptomyces total and plasmid DNA were performed as described by Kieser et al. (2000)
. Southern hybridizations were carried out with probes labelled with digoxigenin by using the DIG DNA labelling kit (Roche Biochemicals).
AVG determination.
AVG production was assessed by bioassay using Bacillus subtilis as test organism as described previously (Fernández et al., 2002). Solutions of pure AVG (Sigma) were used as reference values for halo formation. In the mutant strains, kanamycin, an aminoglycoside used for selection, was inactivated by treatment with amyloglucosidase as follows. A 2 ml sample of culture was incubated with 50 µg amyloglucosidase at 60 °C for 20 min and the mixture was extracted with 4 ml water-saturated butanol. The polar phase was then concentrated to dryness under vacuum and resuspended in 100 µl H2O. To confirm the identity of AVG, cells were grown in AMM at 30 °C for 48 h. The broth obtained was then extracted with water-saturated butanol and the polar phase was concentrated to dryness under vacuum and resuspended in 1/100 vol. H2O. The solution was then applied onto a Sephadex G10 column and active fractions were analysed by reverse-phase HPLC chromatography after derivatization with FMOC (fluorenylmethyl chloroformiate). We used a Waters 600 unit coupled to a PDA 996 detector equipped with a Polarity C18 column (3·9x150 mm; particle size, 5 µm). Elution was with a gradient (0·7 ml min1) of 100 % acetonitrile (acetonitrile concentration from 1 to 80 % for 08 min; 80 % for 810 min; down to 1 % for 1015 min).
Construction of a suicide vector and disruption of the genes.
The suicide vector for gene disruption was constructed as follows. A 1·3 kb BamHI fragment encompassing the neomycin phosphotransferase gene from transposon Tn5 was cloned into a BamHI-cut pUC19 vector to yield pUKM. This plasmid lacks the Streptomyces origin of replication and, therefore, cannot self-replicate in this bacterium. Its ability to remain in Streptomyces cultures depends largely on putative homologous recombination into the chromosome driven by inserted DNA fragments.
The DNA fragments used to promote such homologous recombination were as follows. For the hom gene, we used a 532 bp Ecl136IIMslI fragment internal to the gene (from nt 120 to 652) that was cloned into Ecl136II-cut pUKM vector to yield pUKMHDH. In a similar fashion, for the thrB gene we used a 529 bp SmaIHincII internal fragment (from nt 71 to 600) cloned into Ecl136II-cut pUKM vector to yield pUKMHK, and for the thrC gene we used a 555 bp SmaI fragment (from nt 226 to 781) cloned into Ecl136II-cut pUKM to yield pUKMTS. These plasmids were then used for transformation of the wild-type strain. Transformants were selected by kanamycin resistance on R5 medium and confirmed by genomic Southern hybridization.
The suicide vector for thrB gene replacement was constructed as follows. A 12 kb NotI-fragment containing the hom-thrC-thrB cluster was cloned into NotI-cut pBluescript vector to yield pBN37. This plasmid was then digested with StuI and EcoRV (the EcoRV site belongs to the pBluescript polylinker) and the resulting 3 kb DNA fragment containing the thrB gene was cloned into SmaI-cut pUC19 to yield pUHK. A SmaI site located in the coding region of the thrB gene, 0·8 kb from the 5' end of the DNA fragment and 2 kb from its 3' end, was used for cloning of a 1·3 kb SmaI fragment encompassing the neomycin phosphotransferase gene from transposon Tn5 (Beck et al., 1982). The construction with the direction of transcription of the neomycin gene against that of the thrB gene was selected from the two possible orientations to yield pUHKKM. After transformation of the wild-type strain, transformants that had undergone a recombination event at both sites of the kanamycin resistance cassette were selected by kanamycin resistance and threonine auxotrophy on MM, and confirmed by genomic Southern hybridization.
Partial purification of homoserine kinase (HK) activity.
HK was purified by a procedure derived from that described by Ramos et al. (1991) for yeast cells. Streptomyces NRRL5331 cells were grown at 30 °C in YEME medium and the mycelial pellet was sonicated at 14 µm frequency for 30 s periods, until complete rupture, in extraction buffer (50 mM Tris/HCl, pH 8·0, 100 mM NaCl, 1 mM EDTA). The ratio between the weight of the mycelium sample and the volume of the extraction buffer was kept constant throughout the experiment. Cell debris was removed by centrifugation at 40 000 g for 1 h at 4 °C. Protamine sulphate, pH 7·0 [5 mg (g wet wt)1] was added to the crude extract and, after 1 h stirring at 4 °C, the precipitate was removed by two consecutive centrifugations at 4 °C, one at 10 000 g for 20 min and the second at 25 000 g for 2 h. Ammonium sulphate was added to bring the supernatant to 30 % saturation and the mixture was stirred for 30 min at 4 °C. After centrifugation (see above) the precipitate was discarded and the supernatant was brought to 45 % saturation with ammonium sulphate. After 30 min stirring at 4 °C and centrifugation, the pellet was dissolved in buffer T (50 mM triethanolamine, pH 7·5, 10 mM MgCl2).
HK activity assay.
HK activity was assessed by monitoring ADP production using a pyruvate kinase/lactate dehydrogenase coupled assay (Burr et al., 1976) in which HK phosphorylates homoserine to homoserine phosphate, yielding ADP. The latter reacts with phosphoenolpyruvate in the presence of pyruvate kinase to yield pyruvate, which in turn is converted to lactate by the lactate dehydrogenase with the concomitant oxidation of NADH to NAD+. All these reactions are equimolecular. The standard reaction mixture contained 50 mM triethanolamine, pH 7·5, 5 mM L-homoserine, 0·25 mM NADH, 0·3 mM phosphoenolpyruvate, 25 mM ATP, 10 mM MgCl2, 3 U pyruvate kinase, 6 U lactate dehydrogenase and an appropriate amount of HK solution in a total volume of 1 ml. Reaction mixtures lacking homoserine were used as controls. Reactions were incubated at 30 °C. Enzyme activity was determined by quantifying the disappearance of NADH at 340 nm. One unit of activity is defined as the amount of enzyme required to produce a decrease in A340 of 0·01 min1. Protein concentrations were estimated using the Bradford (1976)
method.
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RESULTS |
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Disruption of the thrB gene
Following a similar strategy to the one used for hom disruption, the disruption of thrB was carried out using pUKMHK (see Methods). One of the disrupted mutants was randomly selected and named HK. The identity of the mutant was confirmed by Southern hybridization. Chromosomal DNAs isolated from the parental strain and mutant
HK and digested with SacI were probed with the 530 bp SmaIHincII fragment used to construct the pUC19 derivative utilized for gene disruption (see Methods). A hybridizing band of 3·0 kb was found for the wild-type as expected (not shown). However, in the disrupted mutant, an additional band of 4·5 kb was also detected (not shown), indicating that a single crossover event had occurred with the pUC19 derivative. Mutant
HK required added threonine to sustain growth in MM as expected.
HK activity is blocked in the HK mutant
To corroborate the functional knock out of HK in the HK mutant, we measured HK activity (see Methods) of the mutant and compared it to the parental strain. In the wild-type strain, the disappearance of NADH, measured by the decrease in A340, was 84 units min1, while in the mutant
A340 was 21 units min1 (Fig. 3
). No significant difference could be observed in the decrease of NADH produced by the enzyme preparation of mutant
HK when compared to that produced in a control which contains the wild-type enzymic preparation containing no homoserine (Fig. 3
), thus suggesting that the basal NADH decrease observed in both reactions is likely to be due to other NADH oxidases, and that the
HK mutant does not possess any HK activity. Similarly, mutant
HK(Km) (see below) did not show any HK activity.
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Effect of hom, thrB or thrC gene disruptions on AVG production
To determine the branching point of the aspartic-acid-derived biosynthetic route of AVG in Streptomyces sp. NRRL 5331, the different mutants generated were characterized by their ability to produce AVG. The parental strain and the mutants were grown in both MM and YEME until the beginning of the stationary phase of growth (48 and 72 h, respectively) and the AVG produced was measured (Fig. 4). MM was supplemented with 0·4 mM threonine or 0·4 mM threonine and methionine when required. The addition of such concentrations of amino acids did not have any effect on the yields of AVG attained by the parental strain (not shown).
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Interestingly, when we analysed the ability of mutant HK to produce AVG, we found a 4050 % yield decrease, in both MM and YEME, when compared with the wild-type (Fig. 4
). To discard any possible polar effects on other genes as a consequence of thrB disruption, we performed a thrB gene replacement using a pUC19 plasmid derivative, pUHKKM, constructed as described in Methods. After transformation of strain NRRL 5331, several transformants were obtained by selection for kanamycin resistance. One of the mutants out of 96 displayed a threonine auxotroph phenotype when grown in MM. Its identity was then confirmed by Southern analysis showing a hybridization pattern indicating that a double recombination event had occurred at both sites of the kanamycin cassette, thus inactivating the thrB gene. This mutant was selected and named
HK(Km). Fig. 5
shows chromosomal DNAs isolated from Streptomyces sp. NRRL 5331 and mutant
HK(Km) digested with BamHI and probed with a 600 bp SmaIBamHI fragment internal to the thrB gene. One hybridizing band of 3·5 kb was found for the wild-type as expected, while a single band of 0·6 kb was detected in the mutant, indicating that a double crossover event had occurred (Fig. 5
). Hybridizing bands corresponded exactly to those expected according to the integration shown in Fig. 5
.
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Effect of end products of the threonine pathway on AVG production
To check whether the significant decrease of AVG formation displayed by the HK (inactivated by single disruption) and
HK(Km) (inactivated by gene replacement) mutants was due to homoserine phosphate acting as an intermediate of AVG biosynthesis together with homoserine, or just a reflection of the alteration of the regulatory circuits that control the biosynthesis of amino acids of the aspartic acid family upon the chromosomal disruption of thrB, we tested the effect of the addition of the end products of the threonine pathway on the production of AVG by the wild-type strain in MM. As shown in Fig. 6
, while the addition of 5 mM threonine or isoleucine did not produce any effect on AVG production, in the presence of 5 mM homoserine or methionine the yield of AVG was drastically reduced (about 50 %). This result suggests that the decreased levels of AVG produced by mutants
HK and
HK(Km) are more likely to be due to the negative effect that homoserine and methionine exert on AVG biosynthesis rather than homoserine phosphate being a branching point of the AVG pathway.
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DISCUSSION |
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The low level of AVG production displayed by the two different thrB gene mutants might suggest that homoserine phosphate could also constitute a branching point of the route together with homoserine. If this were true, the disruption of thrC in mutant TS should have produced an accumulation of homoserine phosphate that in turn should have resulted in an increase in AVG production. However, mutant
TS did not show increased levels of AVG.
Bearing this in mind, we attributed the decreased levels of AVG produced by the HK-deficient mutants to the alteration of the regulatory circuits that control the biosynthesis of amino acids of the aspartic acid family upon the chromosomal disruption of thrB. To check this hypothesis, we tested the effect of the addition of several amino acids of the threonine pathway on the production of AVG by the wild-type strain in MM. Our results indicated that both homoserine and methionine drastically decreased AVG production in the culture (Fig. 6). This suggests that in the mutants lacking functional HK, the accumulation of homoserine (and indirectly of methionine) may explain the observed decrease in AVG production.
Whether the observed negative effect on AVG production of homoserine is exerted directly by itself or through the accumulation of methionine remains to be established. None of these amino acids affect the transcription of the genes studied (not shown); however, it is important to mention that methionine strongly represses the transcription of the first gene of the route, the aspartate-kinase-encoding gene, ask (M. Fernández, Y. Cuadrado, J. F. Aparicio & J. F. Martín, unpublished results). ask and asd (aspartate-semialdehyde-dehydrogenase-encoding gene) of Streptomyces sp. NRRL5331 have been characterized recently and found to form an operon (Cuadrado et al., 2004). Hence, the effect of methionine on AVG biosynthesis could be attributed to its effect on the first two steps of AVG biosynthesis, namely, the conversion of aspartate into aspartate semialdehyde. Homoserine and methionine have also been described as inhibitors of HDH activity in Streptomyces clavuligerus (Mendelovitz & Aharonowitz, 1982
) and if this is the case in Streptomyces sp. NRRL 5331 such inhibition could also contribute to the diminished AVG levels detected in the mutants lacking a functional HK.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 21 October 2003;
revised 8 January 2004;
accepted 12 January 2004.
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