Biosynthesis of the proteasome inhibitor syringolin A: the ureido group joining two amino acids originates from bicarbonate
© Ramel et al; licensee BioMed Central Ltd. 2009
Received: 21 August 2009
Accepted: 28 October 2009
Published: 28 October 2009
Syringolin A, an important virulence factor in the interaction of the phytopathogenic bacterium Pseudomonas syringae pv. syringae B728a with its host plant Phaseolus vulgaris (bean), was recently shown to irreversibly inhibit eukaryotic proteasomes by a novel mechanism. Syringolin A is synthesized by a mixed non-ribosomal peptide synthetase/polyketide synthetase and consists of a tripeptide part including a twelve-membered ring with an N-terminal valine that is joined to a second valine via a very unusual ureido group. Analysis of sequence and architecture of the syringolin A synthetase gene cluster with the five open reading frames sylA-sylE allowed to formulate a biosynthesis model that explained all structural features of the tripeptide part of syringolin A but left the biosynthesis of the unusual ureido group unaccounted for.
We have cloned a 22 kb genomic fragment containing the sylA-sylE gene cluster but no other complete gene into the broad host range cosmid pLAFR3. Transfer of the recombinant cosmid into Pseudomonas putida and P. syringae pv. syringae SM was sufficient to direct the biosynthesis of bona fide syringolin A in these heterologous organisms whose genomes do not contain homologous genes. NMR analysis of syringolin A isolated from cultures grown in the presence of NaH13CO3 revealed preferential 13C-labeling at the ureido carbonyl position.
The results show that no additional syringolin A-specific genes were needed for the biosynthesis of the enigmatic ureido group joining two amino acids. They reveal the source of the ureido carbonyl group to be bicarbonate/carbon dioxide, which we hypothesize is incorporated by carbamylation of valine mediated by the sylC gene product(s). A similar mechanism may also play a role in the biosynthesis of other ureido-group-containing NRPS products known largely from cyanobacteria.
Syringolins are a family of closely related cyclic peptide derivatives that are secreted by many strains of the phytopathogenic bacterium Pseudomonas syringae pv. syringae (Pss) in planta and under certain culture conditions [1, 2]. Syringolin A, the major variant, was shown not only to induce acquired resistance in rice and wheat after spray application, but also to trigger hypersensitive cell death at infection sites of wheat and Arabidopsis plants infected by compatible powdery mildew fungi [3, 4]. Recently, syringolin A was shown to be an important virulence factor in the interaction of Pss B728a with its host plant Phaseolus vulgaris (bean), and its cellular target has been identified. Syringolin A irreversibly inhibits the eukaryotic proteasome by a novel mechanism, representing a new structural class of proteasome inhibitors [5, 6].
Here we show that the genes sylA-sylE are sufficient to direct the biosynthesis of bona fide syringolin A when heterologously expressed in Pseudomonas putida and Pss SM, two organisms which do not produce syringolin A and have no syl gene cluster homolog in their genomes. Thus, biosynthesis of the ureido group with its attached terminal valine is achieved without additional syringolin A-specific genes (i. e. genes with no other function than in syringolin A biosynthesis). We hypothesized that biosynthesis of the ureido group would most likely be accomplished by the product of the sylC gene, which would, in addition to the extracyclic peptidyl valine, also activate the terminal valine and join the two residues by incorporation of a carbonyl group derived from hydrogen carbonate/carbon dioxide, thus forming the ureido moiety. We demonstrate by NMR spectroscopic analysis of syringolin A isolated from Pss cultures grown in the presence of NaH13CO3 that the 13C isotope is preferentially found at the position of the ureido carbonyl atom. These results support our hypothesis, which may be of relevance for the hitherto unknown biosynthesis of other ureido-group-containing NRPS products largely known to be produced by cyanobacteria [13–20].
Biosynthesis of syringolin A in heterologous organisms
In order to test whether the sylA-sylE gene cluster was sufficient to direct syringolin A biosynthesis, we constructed a cosmid containing the sylA-sylE genes but no other complete gene by taking advantage of Asc I and Not I restriction sites flanking the syl gene cluster (Figure 1B). Southern blot analysis of Asc I/Not I-digested genomic DNA of Pss B301D-R probed with a sylA gene fragment labeled the expected 22 kb fragment and thus confirmed the uniqueness of the restriction sites in the relevant genome region (data not shown). Thus, Pss B301D-R genomic DNA digested with Asc I and Not I was separated by agarose gel electrophoresis. Fragments in the 20-23 kb size range were eluted and cloned into the wide host range cosmid pLAFR3 . After packaging into lambda phages and transfection into E. coli XL-1Blue, the library was screened with a radiolabeled sylA gene probe. Positive clones were isolated and confirmed to contain the complete syl gene cluster by PCR amplification and sequencing of the expected insert ends. One of the confirmed clones was designated pPL3syl and chosen for further work.
Next we wanted to mobilize pPL3syl into Pseudomonas strains not carrying syl gene homologs and lacking syringolin A production as evidenced by PCR, DNA gel blot analysis of genomic DNA, high performance liquid chromatography (HPLC) analysis of culture supernatants with regard to syringolin A content, infiltration into rice leaves followed by monitoring of Pir7b transcript accumulation, and whole genome sequence comparisons where possible (data not shown). After repeated unsuccessful attempts to transfer pPL3syl into the P. syringae pv. tomato DC3000 strain (all tetracycline-resistant putative transformants analyzed contained deletion variants of pPL3syl), the cosmid was successfully transferred into the non-pathogenic bacterium P. putida P3  and Pss SM, a strain originally isolated from wheat [23, 25]. Gel blot analysis of RNA extracted from rice leaves infiltrated with parental and transformed strains showed that, as expected, P. putida P3 and Pss SM did not induce Pir7b transcript accumulation. In contrast, both strains lead to Pir7b gene induction when carrying the pPL3syl cosmid (Figure 2B), suggesting that pPL3syl conferred the ability for syringolin A biosynthesis to these strains.
The ureido carbonyl group of syringolin A is incorporated from bicarbonate/carbon dioxide
The above results raised the question of how the ureido-valine is synthesized and incorporated into syringolin A. We hypothesized that this would most likely be accomplished by the product of the sylC gene, which would, in addition to the N-terminal valine of the tripeptide part of syringolin A, also activate the second valine and join the two residues via their amino groups formally by amidation of carbonic acid, thus forming the ureido moiety. If true, feeding syringolin A-producing cultures with 13C-labeled hydrogen carbonate should result in syringolin A that is preferentially labeled with 13C at the ureido carbonyl position. Thus, Pss B301D-R transformed with pOEAC, a plasmid carrying the sylA transcriptional activator gene under the control of the lacZ promoter, was grown in SRMAF medium. After 48 h, 13C-labeled sodium hydrogen carbonate was added to a final concentration of 70 mM and the culture was further grown for 20 h. Syringolin A was isolated from conditioned medium as described  and subjected to 13C NMR analysis.
We have demonstrated that the syl gene cluster is sufficient to direct syringolin A synthesis in heterologous organisms. Although the biosynthesis model presented earlier  plausibly explained every structural feature of the syringolin A tripeptide part through the enzymatic actions of the sylB, sylC, and sylD gene products, the generation and condensation of the ureido valine remained enigmatic. As the other genes present in the syl cluster encode a transcriptional activator (sylA gene) and an exporter (sylE gene), a plausible hypothesis was that the sylC-encoded NRPS module not only activated the N-terminal peptidyl valine, but also the ureido valine, and that the ureido carbonyl moiety is incorporated from hydrogen carbonate/carbon dioxide. As shown above, in vivo labeling of syringolin A with 13C-hydrogen carbonate supports this hypothesis. Currently, we can only speculate how this is achieved. One possibility is that the quaternary syringolin A synthetase complex may contain two (not necessarily identical) molecules derived from the sylC gene per SylD polypeptide. Both sylC gene products would activate valine, or, to a certain degree, isoleucine in minor syringolin variants . The first valine would then be carbamylated by HCO3-/CO2, perhaps without the action of another enzyme, as has been reported for the carbamylation of a catalytic lysine residue in β-lactamases of class D [27, 28]. The ureido moiety would then be formed by amide bond formation between the carbamylated valine and the second valine. In this scenario, it remains unclear how the first valine, which, like the second one, is envisioned to be bound to the peptide carrier protein domain by a thioester bond, is released upon ureido bond formation. It is also conceivable that ureido bond formation is achieved by a single SylC protein, which contains a condensation domain usually absent from starter modules that may be involved. To clarify these issues, more structural information about the large syringolin A synthetase and the SylC module must be obtained. The reconstitution of the enzymatic activities of the module(s) derived from the sylC gene in vitro will be challenging.
In addition to the syringolin family of compounds, a number of other natural cyclic peptides mostly isolated from cyanobacteria have been described in the literature that contain extracyclic ureido groups linking two different amino acids. These include anabaenopeptins from Anabaena, Oscillatoria, and Planktothrix species [13–16], ferintoic acids from Mycrocystis aeruginosa , pompanopeptins from Lyngbya confervoides , as well as mozamides and brunsvicamides, compounds of presumably cyanobacterial origin isolated from sponges [19, 20]. Bicarbonate/CO2 may also be the source of the ureido carbonyl group joining two extracyclic amino acids in the biosynthesis of these compounds, which, to our knowledge, has not been elucidated so far.
Our results show that the syl biosynthesis gene cluster was sufficient to direct the biosynthesis of bona fide syringolin A, including the enigmatic ureido group joining two amino acids. They reveal the source of the ureido carbonyl group to be bicarbonate/carbon dioxide, which we hypothesize is incorporated by carbamylation of valine mediated by the sylC gene product(s). A similar mechanism may also play a role in the biosynthesis of other ureido-group-containing NRPS products known largely from cyanobacteria.
Construction and expression of pPL3syl
Unless stated otherwise, standard protocols were used . Genomic DNA from Pss B301D-R was isolated and 11 μg were digested with the restriction enzymes Asc I and Not I. Asc I and Not I sites are both unique in the syl gene region (GenBank: AJ548826) located at position 2052 and 24124, respectively, within the ORFs flanking the sylA-sylE ORFs (3507-23596). A DNA gel blot was prepared with 1 μg of the digested DNA and probed with a 32P-labeled sylA gene fragment PCR-amplified from genomic DNA with primers P1 (5'-ccatcgatggagtagagtgatggc) and P2 (5'-ggaattcttacaaaattcccatcttg). The rest of the digested DNA was separated on a 0.4% agarose gel and the DNA in the 20-23 kb size range was cut out, electrophoretically eluted into a dialysis bag (10 kDa cutoff), extracted first with 1 volume of phenol and then with 1 volume of phenol-chloroform-isoamylalcohol (25:24:1), precipitated with ethanol and finally taken up in TE (10 mM Tris-HCl, pH 8; 1 mM EDTA). Fragments were ligated into the Hin dIII/Bam HI-cut broad host range cosmid vector pLAFR3  using adaptors prepared by annealing the oligonucleotide 5'-cgcgccaagcttcca with 5'-agcttggaaagcttgg (Asc I/Hin dIII adaptor) and 5'-ggccgctagtcaggag with 5'-gatcctcctgactagc (Not I/Bam HI adaptor), respectively. Ligation products were packaged into lambda phage particles using the Gigapack III Gold Packaging Kit (Stratagene, La Jolla, California) and the library was plated out on E. coli XL1-Blue (Stratagene) and screened according to the instructions of the manufacturer using the 32P-labeled sylA gene fragment described above as a probe. Positive clones were isolated and confirmed to contain the complete syl gene cluster by PCR amplification and sequencing of the insert end fragments using primers 5'-ccggcctacacgcattc (sylA end) and 5'-agcaacctggatgtacgg (sylE end) with the respective adaptor oligonucleotides (see above).
Construction of the syl gene cluster deletion mutant Δsyl
Two fragments of 783 bp and 655 bp length flanking the syl gene cluster on the 5' and 3' side, respectively, were amplified by PCR from Pss B301D-R genomic DNA using the primer pairs P3 (5'-cgggatcca acctgaaatgggagagtc; base given in bold at position 2297 in GenBank:AJ548826) and P4 (5'-agcgcgaggac tcaatgtgaaaacaacg; bold base at position 3072), and P5 (5'-tcacattgagt cctcgcgctggtaacc; bold base at position 23600) and P6 (5'-t tctgcagtcaagcctgacgaaaagc; bold base at position 24247), respectively. The two bands were isolated and joined by overlap extension PCR using primers P3 and P6 to yield a fragment flanked by Bam HI and Pst I restriction sites in which the syl gene cluster from position 3073-23599 (GenBank:AJ548826) was missing. The deletion is nearly identical with the one in the completely sequenced P. syringae pv. tomato DC3000 (GenBank:NC_004578.1), which does not contain a syl gene cluster. The fragment was cut with Bam HI and Pst I and cloned into the respective restriction sites in the cloning box of the suicide vector pME3087 (TcR, ColE1 replicon ). The recombinant plasmid was transformed into E. coli S17-1 (thi pro hsdR recA; chromosomal RP4 (Tra+ TcS KmS ApS; transfer gene-positive, tetracycline-sensitive, kanamycin-sensitive, ampicillin-sensitive) ) and mobilized into Pss B301D-R. Tetracycline-resistant colonies were grown in LB medium over night at 28°C on a rotary shaker (220 rpm). For selection of tetracycline-sensitive colonies, the overnight cultures were diluted 100-fold with LB. After 2 h of growth, tetracycline was added (20 μg/ml final concentration) and the cultures were grown for 1 h, after which the bactericide carbenicillin (2 mg/ml final concentration) was added for 3 h. The bacteria were then collected by centrifugation, and after washing them twice in LB, the selection procedure was repeated another 3 times. The cultures were then replica-plated on LB plates with and without tetracycline (10 μg/ml) and tetracycline-sensitive colonies were isolated (about 2-3%). The desired deletion mutants were distinguished from wild-type revertants and verified by sequencing of a 1.7 kb DNA fragment amplified from genomic DNA by PCR using primers 5'-attactcgaccagttccg and 5'-ttacgcaatggtatgatgc which are located outside the fragment cloned into the suicide vector pME3087 at position 2113 and 24385 (GenBank:NC_007005.1), respectively.
Construction of pOEAC
The sylA ORF was amplified from genomic DNA using the primers P7 (5'-ccatcgat ggagtagagtgatg gc; Cla I site in italics, translation initiation codon indicated in bold) and P8 (5'-ggaattc tta caaaattcccatcttg; reverse primer; Eco RI site in italics, reverse stop codon in bold), digested with Cla I and Eco RI, and cloned into the respective polylinker sites of the pME6001 (GmR) expression vector , thereby placing it under the control of the lacZ promoter. The resulting plasmid was named pOEA. As it turned out that pOEA did not confer gentamycin resistance in SRMAF medium, pOEAC was used, a derivative of pME6014 (TetR) , which, in addition to the lacZ::sylA chimeric gene, contained a sylC::lacZ reporter fusion gene in opposite orientation (the reporter gene is of no relevance in the present context). To construct pOEAC, the lacZ::sylA fusion gene was amplified from pOEA with primers P8 (see above) and P9 (5'-accgtccaaca ttaatgcagctgg; upstream of lac promoter; bold base complementary to position 987 of pBluescript vector (GenBank:X52329)) and joined with a sylC promoter fragment (position 5409-5649 of GenBank:AJ548826) that was amplified with primers P10 (5'-ctgcattaatg ttggacggtctgc; bold base at position 5409) and P11 (5'-aactgcag t catgacggcctcggat; Pst I site in italics, bold base at position 5649) by overlap extension PCR using primers P8 and P11. The resulting fragment was digested with Eco RI and Pst I and cloned between the respective sites in the polylinker of pME6014.
Bacterial infiltration of rice leaves and RNA gel blot analysis
Bacterial strains were grown on a rotary shaker (220 rpm) over night at 28°C in LB containing, where appropriate, 10 μg/ml tetracycline. Bacteria were pelleted by centrifugation, washed twice in distilled water, resuspended in distilled water at an optical density at 600 nm (OD600) of 0.4 (approximately 108 cfu), and infiltrated into first leaves of 14-day-old rice plants (Oryza sativa cv. Loto; supplied by Terreni alla Maggia, Ascona, Switzerland) as described previously . RNA was extracted from infiltrated leaves 16 h after infiltration and subjected to gel blot analysis using a 32P-labeled Pir7b cDNA probe (GenBank:Z34270 ) according to standard procedures .
HPLC analysis and mass spectrometry of syringolin A
To analyze conditioned media with regard to syringolin A content, Pseudomonas strains were grown in SRMAF medium [36, 37] at 28°C for 60 h on a rotary shaker (220 rpm). Bacteria were pelleted by centrifugation and the supernatant was sterile filtered (0.22 μm pore size). Two-hundred-microliter aliquots were acidified with trifluoroacetic acid (TFA; 0.3% final concentration) and subjected to reverse-phase HPLC with a Reprosil 100-5 C18 250/4.6 column (Dr. Maisch GmbH, Ammerbuch-Entringen, Germany) on a Dionex UltiMate 3000 system (Dionex Corporation, Sunnyvale, CA). Elution was performed isocratically with 20% acetonitrile and 0.06% TFA in water at a flow rate of 1 ml/min.
High-resolution electrospray mass spectra were recorded on a Bruker maXis QTOF-MS instrument (Bruker Daltonics GmbH, Bremen, Germany). The samples were dissolved in MeOH and analyzed via continuous flow injection at 3 μl/min. The mass spectrometer was operated in positive ion mode with a capillary voltage of 4 kV, an endplate offset of -500 V, nebulizer pressure of 5.8 psig, and a drying gas flow rate of 4 l/min at 180°C. The instrument was calibrated with a Fluka electrospray calibration solution (Sigma-Aldrich, Buchs, Switzerland) that was 100 times diluted with acetonitrile. The resolution was optimized at 30'000 FWHM in the active focus mode. The accuracy was better than 2 ppm in a mass range between m/z 118 and 2721. All solvents used were purchased in best LC-MS qualities.
13C-labeling and NMR Spectroscopy
Pss B301D-R was transformed with pOEAC and grown in LB containing 10 μg/ml tetracycline on a shaker at 28°C until an OD600 of approximately 0.5 was reached. Bacteria were collected by centrifugation, washed twice with SRMAF medium, and taken up in SRMAF medium at an OD600 of 0.3. Fifty-ml cultures were inoculated with 0.01 volume of the bacterial suspension and incubated at 28°C on a shaker (220 rpm). After 48 h, NaH13CO3 (98%; Sigma-Aldrich, Buchs, Switzerland) was added to a final concentration of 70 mM and incubation was continued for 20 h. Bacteria were pelleted and syringolin A was isolated from sterile-filtrated conditioned media as described .
1H broadband decoupled 13C NMR spectra were recorded at 25°C on a Bruker Avance III 600 MHz spectrometer equipped with a cryogenic 5 mm CPDCH probe head optimized for 13C detection. Two samples were prepared by dissolving 200 μg of labeled syringolin A in 130 μl DMSO-d6 and 5 mg of unlabeled syringolin A in 750 μl DMSO-d6, respectively. The labeled sample was transferred to a 3 mm Shigemi tube, the unlabeled sample was transferred to a regular 5 mm NMR tube. The spectral width in both spectra was 248.5 ppm, the transmitter was set to 100 ppm. The excitation pulse angle was set to 45°. The acquisition time was 2.1 s with a waiting time of 0.3 s between two scans. Both spectra were 1H broadband decoupled using the waltz16 composite-pulse decoupling scheme. The resulting fid consisted of 157890 total data points. For the unlabeled syringolin A sample 4000 scans were accumulated. For the labeled syringolin A sample 29605 scans were accumulated. Both spectra were zero filled to 131072 complex data points and processed using an exponential line broadening of 2 Hz. The samples contained no internal chemical shift reference and the spectra were referenced to the solvent peak (39.5 ppm). By comparison with chemical shifts listed in  the signals at 157.8 ppm and 132.8 ppm were assigned to the ureido CO group and the olefinic C at position 4 in the 3,4-dehydrolysine moiety, respectively.
We thank Zsuzsa Hasenkamp for expert technical assistance, and Enrico Martinoia, Stefan Hörtensteiner, Markus Kaiser, and André Bachmann for discussions and helpful comments on the manuscript. Financial support by the Swiss National Science Foundation (grant 3100A0-115970 to RD) is acknowledged.
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