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Ornithine Transcarbamylase ArgK Plays a Dual role for the Self-defense of Phaseolotoxin Producing Pseudomonas syringae pv. phaseolicola.

Chen L, Li P, Deng Z, Zhao C - Sci Rep (2015)

Bottom Line: The tripeptide, Cit-Ala-hArg, was identified to be a by-product of PHT biosynthetic pathway in this report.Formation of Cit-Ala-hArg was catalyzed by ArgK with tripeptide Orn-Ala-hArg and carbamyl phosphate as substrates.It showed that ArgK not only provided alternative Arginine source as reported previously, but also controlled the production of PHTs by converting PHT biosynthetic precursors to nontoxic Cit-Ala-hArg reservoir for producers' self-defense.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Combinatorial Biosynthesis and Drug Discovery, Ministry of Education, and School of Pharmaceutical Sciences, Wuhan University, Wuhan, 430071, China.

ABSTRACT
Pseudomonas syringae is a phytopathogenic bacterium widely spread on terrestrial plants. Sulfodiaminophosphinyl tripeptide Phaseolotoxins (PHTs), produced by P. syringae pv. phaseolicola and P. syringae pv. actinidiae, represent a kind of antimetabolic phytotoxins. PHTs inhibit host cell Ornithine transcarbamylase (OTCase) activity and induce Arginine auxotrophic phenotype. The biosynthesis of PHT is temperature dependent, being optically produced at around 18 °C, while blocked above 28 °C. PHT resistant OTCase ArgK acts as a functional replacement of housekeeping OTCase ArgF, which is the acting target of PHT, to confer PHT producers with self-resistance. It was postulated that argK might be regulated directly by a PHT biosynthetic precursor and indirectly by temperature with an unknown manner. Neither transcriptional regulator nor thermal regulation related protein encoding gene was detected from PHT biosynthetic gene cluster. The tripeptide, Cit-Ala-hArg, was identified to be a by-product of PHT biosynthetic pathway in this report. Formation of Cit-Ala-hArg was catalyzed by ArgK with tripeptide Orn-Ala-hArg and carbamyl phosphate as substrates. It showed that ArgK not only provided alternative Arginine source as reported previously, but also controlled the production of PHTs by converting PHT biosynthetic precursors to nontoxic Cit-Ala-hArg reservoir for producers' self-defense.

No MeSH data available.


Related in: MedlinePlus

HR MS spectra of PSOrn.(A) Extracted ion chromatogram of PSOrn, with a tolerance of 5 ppm. (B) high resolution mass spectrum of PSOrn (inset is fragmentation pattern of PSOrn); (C) tandem mass (MS2) spectrum of PSOrn.
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f3: HR MS spectra of PSOrn.(A) Extracted ion chromatogram of PSOrn, with a tolerance of 5 ppm. (B) high resolution mass spectrum of PSOrn (inset is fragmentation pattern of PSOrn); (C) tandem mass (MS2) spectrum of PSOrn.

Mentions: To determine the biosynthetic relationship between tripeptides Cit-Ala-hArg and PHTs, P. syringae pv. phaseolicola 1448A genes phtU, phtQ and phtL were in-frame deleted (Figure S2). Neither Cit-Ala-hArg nor PHT was detected from the culture supernatants of phtU- mutant CL001 strain. Gene phtU complementation strain CL004 regained the ability to produce PHTs (Figure S3). Authentic L-Orn- L-Ala- L-hArg standards feeding restored phtU- mutants with the ability to produce PHTs at 18 °C, which is the temperature permissive for PHTs synthesis in wild type producers (Fig. 2B). Simultaneously, feeding of L-Cit- L-Ala- L-hArg and L-Ala- L-hArg restored phtU- mutants with the ability to produce PHTs as well (Figure S3). To the phtQ in-frame deletion, neither Cit-Ala-hArg nor PHT was detected from the culture supernatants of phtQ- mutant strain CL002. Surprisingly, PSOrn was detected from the culture supernatants of phtQ- mutants. HR tandem MS spectra of PSOrn were shown in Fig. 3. None of the authentic oligopeptides described here restored phtQ- mutants with the ability to produce PHTs (Fig. 2C). Meanwhile, gene phtQ complementation strain CL005 regained the ability to produce PHTs (Figure S3). Gene phtL knockout did not abolish phtL- mutant strain CL003 the ability to produce tripeptides Cit-Ala-hArg at 18 °C (Fig. 2D). PhtL is necessary for PHTs biosynthesis since phtL- mutants did not produce any PHT. The accumulated amounts of Cit-Ala-hArg were increased when PHT biosynthesis pathway was blocked in P. syringae pv. phaseolicola phtL- mutants (Fig. 2D).


Ornithine Transcarbamylase ArgK Plays a Dual role for the Self-defense of Phaseolotoxin Producing Pseudomonas syringae pv. phaseolicola.

Chen L, Li P, Deng Z, Zhao C - Sci Rep (2015)

HR MS spectra of PSOrn.(A) Extracted ion chromatogram of PSOrn, with a tolerance of 5 ppm. (B) high resolution mass spectrum of PSOrn (inset is fragmentation pattern of PSOrn); (C) tandem mass (MS2) spectrum of PSOrn.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4530439&req=5

f3: HR MS spectra of PSOrn.(A) Extracted ion chromatogram of PSOrn, with a tolerance of 5 ppm. (B) high resolution mass spectrum of PSOrn (inset is fragmentation pattern of PSOrn); (C) tandem mass (MS2) spectrum of PSOrn.
Mentions: To determine the biosynthetic relationship between tripeptides Cit-Ala-hArg and PHTs, P. syringae pv. phaseolicola 1448A genes phtU, phtQ and phtL were in-frame deleted (Figure S2). Neither Cit-Ala-hArg nor PHT was detected from the culture supernatants of phtU- mutant CL001 strain. Gene phtU complementation strain CL004 regained the ability to produce PHTs (Figure S3). Authentic L-Orn- L-Ala- L-hArg standards feeding restored phtU- mutants with the ability to produce PHTs at 18 °C, which is the temperature permissive for PHTs synthesis in wild type producers (Fig. 2B). Simultaneously, feeding of L-Cit- L-Ala- L-hArg and L-Ala- L-hArg restored phtU- mutants with the ability to produce PHTs as well (Figure S3). To the phtQ in-frame deletion, neither Cit-Ala-hArg nor PHT was detected from the culture supernatants of phtQ- mutant strain CL002. Surprisingly, PSOrn was detected from the culture supernatants of phtQ- mutants. HR tandem MS spectra of PSOrn were shown in Fig. 3. None of the authentic oligopeptides described here restored phtQ- mutants with the ability to produce PHTs (Fig. 2C). Meanwhile, gene phtQ complementation strain CL005 regained the ability to produce PHTs (Figure S3). Gene phtL knockout did not abolish phtL- mutant strain CL003 the ability to produce tripeptides Cit-Ala-hArg at 18 °C (Fig. 2D). PhtL is necessary for PHTs biosynthesis since phtL- mutants did not produce any PHT. The accumulated amounts of Cit-Ala-hArg were increased when PHT biosynthesis pathway was blocked in P. syringae pv. phaseolicola phtL- mutants (Fig. 2D).

Bottom Line: The tripeptide, Cit-Ala-hArg, was identified to be a by-product of PHT biosynthetic pathway in this report.Formation of Cit-Ala-hArg was catalyzed by ArgK with tripeptide Orn-Ala-hArg and carbamyl phosphate as substrates.It showed that ArgK not only provided alternative Arginine source as reported previously, but also controlled the production of PHTs by converting PHT biosynthetic precursors to nontoxic Cit-Ala-hArg reservoir for producers' self-defense.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Combinatorial Biosynthesis and Drug Discovery, Ministry of Education, and School of Pharmaceutical Sciences, Wuhan University, Wuhan, 430071, China.

ABSTRACT
Pseudomonas syringae is a phytopathogenic bacterium widely spread on terrestrial plants. Sulfodiaminophosphinyl tripeptide Phaseolotoxins (PHTs), produced by P. syringae pv. phaseolicola and P. syringae pv. actinidiae, represent a kind of antimetabolic phytotoxins. PHTs inhibit host cell Ornithine transcarbamylase (OTCase) activity and induce Arginine auxotrophic phenotype. The biosynthesis of PHT is temperature dependent, being optically produced at around 18 °C, while blocked above 28 °C. PHT resistant OTCase ArgK acts as a functional replacement of housekeeping OTCase ArgF, which is the acting target of PHT, to confer PHT producers with self-resistance. It was postulated that argK might be regulated directly by a PHT biosynthetic precursor and indirectly by temperature with an unknown manner. Neither transcriptional regulator nor thermal regulation related protein encoding gene was detected from PHT biosynthetic gene cluster. The tripeptide, Cit-Ala-hArg, was identified to be a by-product of PHT biosynthetic pathway in this report. Formation of Cit-Ala-hArg was catalyzed by ArgK with tripeptide Orn-Ala-hArg and carbamyl phosphate as substrates. It showed that ArgK not only provided alternative Arginine source as reported previously, but also controlled the production of PHTs by converting PHT biosynthetic precursors to nontoxic Cit-Ala-hArg reservoir for producers' self-defense.

No MeSH data available.


Related in: MedlinePlus