Characterization of two ornithine carbamoyltransferases from Pseudomonas syringae pv. phaseolicola, the producer of phaseolotoxin

Jahn, O.; Sauerstein, J.; Reuter, G.

doi:10.1007/bf00415280

Cited by 21 publications

(21 citation statements)

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“…4). These values are in close agreement with the size of other prokaryotic OCTs and the reported molecular mass of 110 kDa for the native (presumably trimeric) form of the enzyme (34). The predicted amino acid sequence of the 327-residue polypeptide aligns well with those of other OCTs (Fig.…”

Section: Resultssupporting

confidence: 85%

“…The high level of ROCT activity in leaf extracts from transgenic plants expressing argK RNA, together with the distinct shift in the pH activity profile of the OCT from transformant 21A, clearly establish that ROCT was expressed in a fully functional form in the plants. This conclusion is further supported by the demonstration of ROCT activity in the prog- (34), plant OCTs are multimeric (trimeric [76] or tetrameric [1]), and some bacterial OCTs can form functional heterotrimers (8), the lack of coincident maxima in the three pH activity profiles raises the possibility of hybrid plant-bacterium enzyme multimers.…”

Section: Discussionmentioning

confidence: 78%

“…phaseolicola lacks a functional arginine deiminase (75). Other investigators (34) reported that ROCTs from other P. syringae pv. phaseolicola strains do not catalyze the reverse reaction in vitro.…”

Section: Discussionmentioning

confidence: 99%

“…The occurrence of alanine at position 106 in ROCT has precedent in a site-directed mutant of the P. aeruginosa ArcB protein (E-105 -3 A in our numbering system) which reduces the enzyme's cooperative binding of CP and enables the enzyme to function as both catabolic and anabolic in vivo (3). It is unlikely that ROCT functions catabolically in vivo since the enzyme's Km for CP (0.0 mM [82] or 2.8 mM [34]) is uncharacteristic of the high Km of ArcB for this substrate (38 mM [3]) and P. syringae pv. phaseolicola lacks a functional arginine deiminase (75).…”

Section: Discussionmentioning

confidence: 99%

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Origin, structure, and regulation of argK, encoding the phaseolotoxin-resistant ornithine carbamoyltransferase in Pseudomonas syringae pv. phaseolicola, and functional expression of argK in transgenic tobacco

Hatziloukas

Panopoulos

1992

J Bacteriol

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Pseudomonas syringae pv. phaseolicola produces the tripeptide N8(N'-sulfo-diaminophosphinyl)-ornithylalanyl-homoarginine (phaseolotoxin), which functions as a chlorosis-inducing toxin in the bean halo blight disease by inhibiting ornithine carbamoyltransferase (OCT). The bacterium possesses duplicate OCT genes, one of which, argK, encodes a toxin-resistant enzyme (ROCT) and imparts resistance to phaseolotoxin. We sequenced the argK gene from strain NPS3121, defined its promoter region, analyzed its regulation, and characterized its transcripts. The gene probably originated from another organism, since it is very distantly related to the argF gene encoding the housekeeping toxin-sensitive OCT and has low G+C content compared with the bacterial genome as a whole and with other protein-coding genes from P. syringae pv. phaseolicola. Optimized alignments of 13 OCT sequences allowed us to define key residues that may be responsible for toxin resistance and to identify a distinct prokaryotic amino acid signature in ROCT, which argues for a prokaryotic origin of argK. An in-frame fusion of the argK coding region with the chloroplast transit peptide segment of the pea rbcS gene was introduced in Nicoiana tabacum byAgwbacterium-mediated transformation. The presence of an ROCT activity in transgenic plants was demonstrated by in vitro and in vivo assays. Some plants were toxin resistant, suggesting that pathogen-derived resistance to the toxin should be feasible in the pathogen's host.Bacterial pathogens of agronomic plants often produce low-molecular-weight toxins that function as virulence or pathogenicity determinants (15,53,61,62). Toxigenic strains (Tox+) of Pseudomonas syringae pv. phaseolicola growing at temperatures of 18 to 22°C produce phaseolotoxin [N6(N'-sulfo-diaminophosphinyl)-ornithyl-alanyl-homoarginine (55)], which functions as a chlorosis-and growth retardationinducing toxin in the bean halo blight disease (53). This toxin and its nonpeptide derivative [N6(N'-sulfo-diaminophosphinyl)-ornithine, trivial name octicidine (55)] are potent inhibitors of ornithine carbamoyltransferases (OCTs) (15,53). Octicidine is a transition-state analog, causing irreversible inhibition of OCT, while phaseolotoxin inhibits the enzyme reversibly (53, 82). A total of 17 different OCTs that have been tested (7 bacterial, 9 from plants, and the rat liver enzyme) are all sensitive to octicidine and/or to phaseolotoxin in vitro and/or in vivo (18).Toxigenic strains ofP. syringae pv. phaseolicola possess a unique form of OCIT that is tolerant to phaseolotoxin and octicidine, in addition to an OCT that is sensitive to these inhibitors (15,19,79). The two enzymes (here designated ROCT and SOCT, respectively) are encoded by different genes, which were previously cloned in this laboratory (66).Several lines of evidence suggest that the chief function of ROCT is to confer phaseolotoxin tolerance in the producing strains. These strains grow normally in media lacking arginine, even when they produce phaseolotoxin at maximal rates (at 18°...

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Section: Resultssupporting

confidence: 85%

Section: Discussionmentioning

confidence: 78%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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Origin, structure, and regulation of argK, encoding the phaseolotoxin-resistant ornithine carbamoyltransferase in Pseudomonas syringae pv. phaseolicola, and functional expression of argK in transgenic tobacco

Hatziloukas

Panopoulos

1992

J Bacteriol

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“…To protect itself from its own toxin, P. syringae pv. phaseolicola synthesizes a phaseolotoxin-resistant OCTase (ROCT) (6,10,11,25,34).…”

mentioning

confidence: 99%

In Pseudomonas syringae pv. phaseolicola, Expression of the argK Gene, Encoding the Phaseolotoxin-Resistant Ornithine Carbamoyltransferase, Is Regulated Indirectly by Temperature and Directly by a Precursor Resembling Carbamoylphosphate

et al. 2004

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Pseudomonas syringae pv. phaseolicola synthesizes a non-host-specific toxin, phaseolotoxin, and also synthesizes a phaseolotoxin-resistant ornithine carbamoyltransferase (ROCT) to protect itself from its own toxin. ROCT is encoded by argK, which is expressed coordinately with phaseolotoxin synthesis at 18°C. To investigate the regulatory mechanisms of this system, null mutants were constructed for argK, argF (encoding the phaseolotoxin-sensitive OCTase [SOCT]), and amtA (encoding an amidinotransferase involved in phaseolotoxin synthesis). The argF mutant did not exhibit arginine auxotrophy when grown in M9 medium at 28°C, because under this condition SOCT was replaced by ROCT. This loss of thermoregulation of argK was apparently caused by accumulation of carbamoylphosphate, one of the substrates of SOCT. Carbamoylphosphate, which has a structure similar to that of the inorganic moiety of phaseolotoxin, was used in induction assays with wild-type P. syringae pv. phaseolicola and was shown to be able to induce argK expression in M9 medium at 28°C. These results indicate that argK expression is independent of temperature and is regulated directly by a compound resembling the inorganic moiety of phaseolotoxin.

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Detection of an insensitive ornithine‐carbamoyltransferase in strains of Pseudomonas syringae pv. phaseolicola with different phytotoxin‐generating capacities

Jahn

Sauerstein

Laplace

et al. 1989

J. Basic Microbiol.

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Independently of their capacity to produce phytotoxins, strains of Pseudomonas syringae pv. phaseolicola contain two ornithine carbamoyltransferases (OCT, EC 2.1.3.3) which differ in resistance to phaseolotoxin and Orn-P(O) (NH2)-NH-SO3 H (PNSOrn). At 18 degrees C, the optimal temperature for product formation, the balance of the two types of OCT was shifted in favour of the insensitive type in phaseolotoxin producing strains, and in favour of the sensitive one in strains with little or no toxin production. The results suggest a causal relationship between the existence of an insensitive enzyme and the synthesis of toxins.

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Characterization of two ornithine carbamoyltransferases from Pseudomonas syringae pv. phaseolicola, the producer of phaseolotoxin

Cited by 21 publications

References 21 publications

Origin, structure, and regulation of argK, encoding the phaseolotoxin-resistant ornithine carbamoyltransferase in Pseudomonas syringae pv. phaseolicola, and functional expression of argK in transgenic tobacco

Origin, structure, and regulation of argK, encoding the phaseolotoxin-resistant ornithine carbamoyltransferase in Pseudomonas syringae pv. phaseolicola, and functional expression of argK in transgenic tobacco

In Pseudomonas syringae pv. phaseolicola, Expression of the argK Gene, Encoding the Phaseolotoxin-Resistant Ornithine Carbamoyltransferase, Is Regulated Indirectly by Temperature and Directly by a Precursor Resembling Carbamoylphosphate

Detection of an insensitive ornithine‐carbamoyltransferase in strains of Pseudomonas syringae pv. phaseolicola with different phytotoxin‐generating capacities

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