Detection of two ornithine carbamoyltransferases in a phaseolotoxin‐producing strain of <i>Pseudomonas syringae pv. phaseolicola</i>

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

doi:10.1002/jobm.3620250821

Cited by 23 publications

(18 citation statements)

References 8 publications

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“…First, increase in temperature from 21 to 28°C caused a 28% decrease in the level of PhoA. This reduction was smaller in magnitude than previously reported for ROCT activity caused by elevated temperature (73% from 22 to 30°C [33]). Although reporter protein activity is not a direct measure of transcription, it is possible that the amplitude of temperature regulation differs between strains.…”

Section: Discussionmentioning

confidence: 57%

“…phaseolicola (19,33,79,82) showed that growth temperature modulates the level of ROCT specific activity, which decreases as the growth temperature is raised from 18 to 30'C. Templeton et al (83) further showed that arginine, which causes repression of anabolic OCTs in most microorganisms, did not have a significant effect on the specific activity of ROCT from P. syringae pv.…”

Section: Resultsmentioning

confidence: 99%

“…It is possible that multiple transcription is a common mechanism for regulatory fine tuning in this bacterium. In the case of argK, we examined conditions that are known to regulate the level of ROCT (growth temperature [33,79,83]) or other bacterial OCTs (medium composition [8]). Growth at 21°C in MM or at 28°C in KB medium did not change the number or size of the transcripts (data not shown) or the transcription start point detected by the K5 primer (Fig.…”

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: Discussionmentioning

confidence: 57%

Section: Resultsmentioning

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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“…However, P. syringae pv. phaseolicola is insensitive to the effect of its own toxin due to the presence of a phaseolotoxinresistant OCTase (7,37), which is produced under conditions leading to the synthesis of phaseolotoxin (13,14) and is encoded by the argK gene (10,26).…”

mentioning

confidence: 99%

Functional Characterization of the Gene Cluster from Pseudomonas syringae pv. phaseolicola NPS3121 Involved in Synthesis of Phaseolotoxin

Aguilera¹,

López‐López

Nieto³

et al. 2007

J Bacteriol

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Pseudomonas syringae pv. phaseolicola is the causal agent of halo blight disease of beans (Phaseolus vulgaris L.), which is characterized by water-soaked lesions surrounded by a chlorotic halo resulting from the action of a non-host-specific toxin known as phaseolotoxin. This phytotoxin inhibits the enzyme ornithine carbamoyltransferase involved in arginine biosynthesis. Different evidence suggested that genes involved in phaseolotoxin production were clustered. Two genes had been previously identified in our laboratory within this cluster: argK, which is involved in the immunity of the bacterium to its own toxin, and amtA, which is involved in the synthesis of homoarginine. We sequenced the region around argK and amtA in P. syringae pv. phaseolicola NPS3121 to determine the limits of the putative phaseolotoxin gene cluster and to determine the transcriptional pattern of the genes comprising it. We report that the phaseolotoxin cluster (Pht cluster) is composed of 23 genes and is flanked by insertion sequences and transposases. The mutation of 14 of the genes within the cluster lead to a Tox ؊ phenotype for 11 of them, while three mutants exhibited low levels of toxin production. The analysis of fusions of selected DNA fragments to uidA, Northern probing, and reverse transcription-PCR indicate the presence of five transcriptional units, two monocistronic and three polycistronic; one is internal to a larger operon. The site for transcription initiation has been determined for each promoter, and the putative promoter regions were identified. Preliminary results also indicate that the gene product of phtL is involved in the regulation of the synthesis of phaseolotoxin.Pseudomonas syringae pv. phaseolicola is the causal agent of halo blight disease of beans (Phaseolus vulgaris L.), which is characterized by water-soaked lesions surrounded by a chlorotic halo (23). This halo results from the action of a non-hostspecific toxin known as phaseolotoxin [N ␦

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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).…”

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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 two ornithine carbamoyltransferases in a phaseolotoxin‐producing strain of Pseudomonas syringae pv. phaseolicola

Cited by 23 publications

References 8 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

Functional Characterization of the Gene Cluster from Pseudomonas syringae pv. phaseolicola NPS3121 Involved in Synthesis of Phaseolotoxin

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

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