BACKGROUND
syringolins are a family of closely related cyclic peptide derivatives that are secreted by many strains of the phytopathogenic bacterium pseudomonas syringae pv.
syringae  in planta and under certain culture conditions  <cit> .
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  <cit> .
recently, syringolin a was shown to be an important virulence factor in the interaction of pss b728a with its host plant phaseolus vulgaris , 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  <cit> .
structure elucidation revealed that syringolin a is a tripeptide derivative consisting of an n-terminal valine followed by the two non-proteinogenic amino acids  <dig> -dehydrolysine and 5-methyl-4-amino-2-hexenoic acid, the latter two forming a twelve-membered macrolactam ring.
the n-terminal valine is in turn linked to a second valine via an unusual ureido group .
the minor variants syringolin b to syringolin f differ from syringolin a by the substitution of one or both valines with isoleucine residues, by the substitution of  <dig> -dehydrolysine with lysine, and by combinations thereof  <cit> .
the structure of syringolin a suggested that it was synthesized by a non-ribosomal peptide synthetase , large modular enzymes that activate and condense amino acids according to the thiotemplate mechanism .
we previously cloned and delimited by mutational analysis a genomic region from pss b301d-r containing five open reading frames  necessary for syringolin biosynthesis .
whereas syla and syle encode a putative transcription activator and an exporter, respectively, sylc encodes a typical nrps module predicted to activate valine, whereas syld codes for two additional nrps modules  and a type i polyketide synthetase  module.
type i pks are also modular enzymes that, similar to fatty acid synthesis, extend a starter molecule by condensation/decarboxylation of malonate extender units .
the analysis of the structure and architecture of the syl gene cluster led to the postulation of a model that completely accounts for the biosynthesis of the tripeptide part of syringolin a, including its ring structure with the 5-methyl-4-amino-2-hexenoic acid and the  <dig> -dehydrolysine .
however, although the addition of the ureido group and its attached second valine could not be explained by the model, the syl gene cluster did not contain additional open reading frames, which, if present, could potentially have been involved in the biosynthesis of this unexplained part.
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 .
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 nah13co <dig> 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  <cit> .
RESULTS
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 asci and noti restriction sites flanking the syl gene cluster .
southern blot analysis of asci/noti-digested genomic dna of pss b301d-r probed with a syla gene fragment labeled the expected  <dig> kb fragment and thus confirmed the uniqueness of the restriction sites in the relevant genome region .
thus, pss b301d-r genomic dna digested with asci and noti was separated by agarose gel electrophoresis.
fragments in the 20- <dig> kb size range were eluted and cloned into the wide host range cosmid plafr <dig>  <cit> .
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.
to test the functionality of ppl3syl, the markerless pss b301d-r mutant Δsyl was constructed in which the complete syl gene cluster was deleted.
the ppl3syl cosmid was then mobilized into the Δsyl deletion mutant by triparental mating.
we previously showed that infiltration of syringolin a-producing pss strains or isolated syringolin a into rice leaves leads to the accumulation of transcripts corresponding to the defense-related pir7b gene , whereas strains or mutants unable to synthesize syringolin a do not activate this gene  <cit> .
syringolin a was originally identified and isolated based on its action on the pir7b gene in rice  <cit> .
we thus infiltrated the b301d-r wild-type strain, the syringolin-negative mutants Δsyl and syla_ko , as well as Δsyl , the deletion mutant complemented with ppl3syl, into rice leaves.
rna was extracted and subjected to gel blot analysis with regard to pir7b transcript accumulation.
as expected and in contrast to the wild type, the syringolin a-negative mutants did not induce pir7b transcript accumulation, whereas the deletion mutant complemented with the ppl3syl cosmid led to a much stronger induction of the pir7b gene .
this strongly suggested that ppl3syl contained a functional syl gene cluster able to direct syringolin a synthesis in the Δsyl deletion mutant.
this does not exclude the possibility that genes not present in the syl gene cluster are necessary for syringolin a production because such genes would also be present in the Δsyl mutant background.
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  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 .
after repeated unsuccessful attempts to transfer ppl3syl into the p. syringae pv.
tomato dc <dig> strain , the cosmid was successfully transferred into the non-pathogenic bacterium p. putida p <dig>  <cit>  and pss sm, a strain originally isolated from wheat  <cit> .
gel blot analysis of rna extracted from rice leaves infiltrated with parental and transformed strains showed that, as expected, p. putida p <dig> and pss sm did not induce pir7b transcript accumulation.
in contrast, both strains lead to pir7b gene induction when carrying the ppl3syl cosmid , suggesting that ppl3syl conferred the ability for syringolin a biosynthesis to these strains.
to confirm this, the transformed strains were grown in shaken cultures in srmaf medium and conditioned media were analyzed by hplc.
as shown in figure  <dig>  both strains produced a compound eluting at  <dig>  min, the elution time of the syringolin a standard.
peaks were collected from multiple hplc runs and subjected to mass spectrometry.
hplc-high resolution-electrospray ionization-mass spectrometry  of the peaks from pss sm and p. putida p <dig> carrying ppl3syl, and the pss b301d-r wild type showed quasi-molecular ions + at m/z  <dig>  ,  <dig>  , and  <dig>  , respectively, matching the empirical formula c24h40n5o6+ .
we conclude from these experiments that the syl genes contained in ppl3syl are sufficient to direct syringolin a biosynthesis in these heterologous strains and no further syringolin a-specific genes, i.e.
genes that exclusively function in syringolin a biosynthesis, are necessary.
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  <dig> h, 13c-labeled sodium hydrogen carbonate was added to a final concentration of  <dig> mm and the culture was further grown for  <dig> h. syringolin a was isolated from conditioned medium as described  <cit>  and subjected to 13c nmr analysis.
the spectrum of labeled syringolin a was normalized in order to get the same signal intensities for the valine methyl groups as in the unlabeled sample.
comparison of the normalized nmr spectra revealed that the signal from the ureido carbon atom in 13c-labeled syringolin a was 45-fold stronger than the corresponding signal from unlabeled syringolin a .
inspection of the resolved 13c satellite of the valine methyl group at lowest field in the 1h spectrum of labeled syringolin a  suggests a 13c content close to natural abundance.
therefore, the 45-fold signal enhancement in labeled syringolin a directly corresponds to the absolute 13c enrichment at this site.
the normalized signal strengths of all other c atoms were equal in labeled and unlabeled syringolin a, with the exception of the c <dig> position of  <dig> -dehydrolysine, whose signal was enhanced approximately 16-fold in 13c-labeled syringolin a .
inspection of biosynthetic pathways using the kegg database  <cit>  revealed that this can be attributed to a carboxylation reaction in the biosynthesis of lysine.
the c <dig> atom of lysine represents the c <dig> atom of l-aspartate-4-semialdehyde, a derivative of aspartate, which is condensed to pyruvate to yield the intermediary compound l- <dig> -dihydrodipicolinate in bacterial lysine biosynthesis.
the c <dig> atom of aspartate in turn originates from the carboxylation of pyruvate to oxaloacetate, an intermediary compound in the tricarboxylic acid cycle, which is transaminated to aspartate.
thus, enhanced 13c-labeling of lysine with h13co3- at the c <dig> position is to be expected.
we note that malonate will also be labeled by h13co3- as it is derived from acetate by carboxylation.
however, the label will be removed by the condensation/decarboxylation of malonate to the peptide chain during syringolin a biosynthesis.
we conclude from this analysis that our hypothesis is correct, i.e.
that the ureido carbonyl moiety in syringolin a originates from the incorporation of hydrogen carbonate/carbon dioxide.
discussion
we have demonstrated that the syl gene cluster is sufficient to direct syringolin a synthesis in heterologous organisms.
although the biosynthesis model presented earlier  <cit>  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  and an exporter , 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  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  <cit> .
the first valine would then be carbamylated by hco3-/co <dig>  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  <cit> .
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 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  <cit> , ferintoic acids from mycrocystis aeruginosa  <cit> , pompanopeptins from lyngbya confervoides  <cit> , as well as mozamides and brunsvicamides, compounds of presumably cyanobacterial origin isolated from sponges  <cit> .
bicarbonate/co <dig> 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.
CONCLUSIONS
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.
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 in planta and under certain culture conditions. 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. recently, syringolin a was shown to be an important virulence factor in the interaction of pss b728a with its host plant phaseolus vulgaris, 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. structure elucidation revealed that syringolin a is a tripeptide derivative consisting of an n-terminal valine followed by the two non-proteinogenic amino acids -dehydrolysine and 5-methyl-4-amino-2-hexenoic acid, the latter two forming a twelve-membered macrolactam ring. the n-terminal valine is in turn linked to a second valine via an unusual ureido group. the minor variants syringolin b to syringolin f differ from syringolin a by the substitution of one or both valines with isoleucine residues, by the substitution of -dehydrolysine with lysine, and by combinations thereof. the structure of syringolin a suggested that it was synthesized by a non-ribosomal peptide synthetase, large modular enzymes that activate and condense amino acids according to the thiotemplate mechanism. we previously cloned and delimited by mutational analysis a genomic region from pss b301d-r containing five open reading frames necessary for syringolin biosynthesis. whereas syla and syle encode a putative transcription activator and an exporter, respectively, sylc encodes a typical nrps module predicted to activate valine, whereas syld codes for two additional nrps modules and a type i polyketide synthetase module. type i pks are also modular enzymes that, similar to fatty acid synthesis, extend a starter molecule by condensation/decarboxylation of malonate extender units. the analysis of the structure and architecture of the syl gene cluster led to the postulation of a model that completely accounts for the biosynthesis of the tripeptide part of syringolin a, including its ring structure with the 5-methyl-4-amino-2-hexenoic acid and the -dehydrolysine. however, although the addition of the ureido group and its attached second valine could not be explained by the model, the syl gene cluster did not contain additional open reading frames, which, if present, could potentially have been involved in the biosynthesis of this unexplained part. 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. 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 nah13co 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.  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 asci and noti restriction sites flanking the syl gene cluster. southern blot analysis of asci/noti-digested genomic dna of pss b301d-r probed with a syla gene fragment labeled the expected kb fragment and thus confirmed the uniqueness of the restriction sites in the relevant genome region. thus, pss b301d-r genomic dna digested with asci and noti was separated by agarose gel electrophoresis. fragments in the 20- kb size range were eluted and cloned into the wide host range cosmid plafr. 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. to test the functionality of ppl3syl, the markerless pss b301d-r mutant Δsyl was constructed in which the complete syl gene cluster was deleted. the ppl3syl cosmid was then mobilized into the Δsyl deletion mutant by triparental mating. we previously showed that infiltration of syringolin a-producing pss strains or isolated syringolin a into rice leaves leads to the accumulation of transcripts corresponding to the defense-related pir7b gene, whereas strains or mutants unable to synthesize syringolin a do not activate this gene. syringolin a was originally identified and isolated based on its action on the pir7b gene in rice. we thus infiltrated the b301d-r wild-type strain, the syringolin-negative mutants Δsyl and syla_ko, as well as Δsyl, the deletion mutant complemented with ppl3syl, into rice leaves. rna was extracted and subjected to gel blot analysis with regard to pir7b transcript accumulation. as expected and in contrast to the wild type, the syringolin a-negative mutants did not induce pir7b transcript accumulation, whereas the deletion mutant complemented with the ppl3syl cosmid led to a much stronger induction of the pir7b gene. this strongly suggested that ppl3syl contained a functional syl gene cluster able to direct syringolin a synthesis in the Δsyl deletion mutant. this does not exclude the possibility that genes not present in the syl gene cluster are necessary for syringolin a production because such genes would also be present in the Δsyl mutant background. 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 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. after repeated unsuccessful attempts to transfer ppl3syl into the p. syringae pv. tomato dc strain, the cosmid was successfully transferred into the non-pathogenic bacterium p. putida p and pss sm, a strain originally isolated from wheat. gel blot analysis of rna extracted from rice leaves infiltrated with parental and transformed strains showed that, as expected, p. putida p and pss sm did not induce pir7b transcript accumulation. in contrast, both strains lead to pir7b gene induction when carrying the ppl3syl cosmid, suggesting that ppl3syl conferred the ability for syringolin a biosynthesis to these strains. to confirm this, the transformed strains were grown in shaken cultures in srmaf medium and conditioned media were analyzed by hplc. as shown in figure both strains produced a compound eluting at min, the elution time of the syringolin a standard. peaks were collected from multiple hplc runs and subjected to mass spectrometry. hplc-high resolution-electrospray ionization-mass spectrometry of the peaks from pss sm and p. putida p carrying ppl3syl, and the pss b301d-r wild type showed quasi-molecular ions + at m/z,, and, respectively, matching the empirical formula c24h40n5o6+. we conclude from these experiments that the syl genes contained in ppl3syl are sufficient to direct syringolin a biosynthesis in these heterologous strains and no further syringolin a-specific genes, i.e. genes that exclusively function in syringolin a biosynthesis, are necessary. 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 h, 13c-labeled sodium hydrogen carbonate was added to a final concentration of mm and the culture was further grown for h. syringolin a was isolated from conditioned medium as described and subjected to 13c nmr analysis. the spectrum of labeled syringolin a was normalized in order to get the same signal intensities for the valine methyl groups as in the unlabeled sample. comparison of the normalized nmr spectra revealed that the signal from the ureido carbon atom in 13c-labeled syringolin a was 45-fold stronger than the corresponding signal from unlabeled syringolin a. inspection of the resolved 13c satellite of the valine methyl group at lowest field in the 1h spectrum of labeled syringolin a suggests a 13c content close to natural abundance. therefore, the 45-fold signal enhancement in labeled syringolin a directly corresponds to the absolute 13c enrichment at this site. the normalized signal strengths of all other c atoms were equal in labeled and unlabeled syringolin a, with the exception of the c position of -dehydrolysine, whose signal was enhanced approximately 16-fold in 13c-labeled syringolin a. inspection of biosynthetic pathways using the kegg database revealed that this can be attributed to a carboxylation reaction in the biosynthesis of lysine. the c atom of lysine represents the c atom of l-aspartate-4-semialdehyde, a derivative of aspartate, which is condensed to pyruvate to yield the intermediary compound l- -dihydrodipicolinate in bacterial lysine biosynthesis. the c atom of aspartate in turn originates from the carboxylation of pyruvate to oxaloacetate, an intermediary compound in the tricarboxylic acid cycle, which is transaminated to aspartate. thus, enhanced 13c-labeling of lysine with h13co3- at the c position is to be expected. we note that malonate will also be labeled by h13co3- as it is derived from acetate by carboxylation. however, the label will be removed by the condensation/decarboxylation of malonate to the peptide chain during syringolin a biosynthesis. we conclude from this analysis that our hypothesis is correct, i.e. that the ureido carbonyl moiety in syringolin a originates from the incorporation of hydrogen carbonate/carbon dioxide. discussion 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 and an exporter, 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 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-/co 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. 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 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, ferintoic acids from mycrocystis aeruginosa, pompanopeptins from lyngbya confervoides, as well as mozamides and brunsvicamides, compounds of presumably cyanobacterial origin isolated from sponges. bicarbonate/co 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. a similar mechanism may also play a role in the biosynthesis of other ureido-group-containing nrps products known largely from cyanobacteria. 
we have cloned a kb genomic fragment containing the syla-syle gene cluster but no other complete gene into the broad host range cosmid plafr transfer of the recombinant cosmid into pseudomonas putida and p. syringae pv. syringae b728a with its host plant phaseolus vulgaris, was recently shown to irreversibly inhibit eukaryotic proteasomes by a novel mechanism. the results show that no additional syringolin a-specific genes were needed for the biosynthesis of the enigmatic ureido group joining two amino acids. a similar mechanism may also play a role in the biosynthesis of other ureido-group-containing nrps products known largely from cyanobacteria. 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. syringae sm was sufficient to direct the biosynthesis of bona fide syringolin a in these heterologous organisms whose genomes do not contain homologous genes. 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. nmr analysis of syringolin a isolated from cultures grown in the presence of nah13co revealed preferential 13c-labeling at the ureido carbonyl position. syringolin a, an important virulence factor in the interaction of the phytopathogenic bacterium pseudomonas syringae pv.
 syringolin a, an important virulence factor in the interaction of the phytopathogenic bacterium pseudomonas syringae pv. syringae b728a with its host plant phaseolus vulgaris, 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 kb genomic fragment containing the syla-syle gene cluster but no other complete gene into the broad host range cosmid plafr 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 nah13co 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. a similar mechanism may also play a role in the biosynthesis of other ureido-group-containing nrps products known largely from cyanobacteria. 