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Tabtoxinine-f-lactam, a hydrolytic product of tabtoxin produced by Pseudoonas syringae pv. tabaci, apparently inactvates pea seed glutamine synthetase. Inhibition of the enzyme's initial velocity is linear over a range of 0.5 to 5 minhmmolar tabtoxinne-/-lactam in the presence of 10 m_mlar glutamate. A method for the purification of glutamine synthetase from dried peas is presented which gives a 30% yield with a 2,000-fold Increase in specific activity. A method for obtaining highly puied tabtoxinine-f8-lactam and tabtoxin in good yields Is also presented. The authenticity and purity of tabtoxinine*lactam and tabtoxin were verified by chromatography, biological activity, and 1H and 13C nuclear magnetic resonance spectroscopy.The phytopathogenic bacterium Pseudomonas syringae pv. tabaci and certain other closely related pathovars produce a toxin which induces chlorosis in plants (5,11,12,16). This toxin, called tabtoxin, is a dipeptide composed of either threonine or serine and the novelf,-lactam-containing amino acid, tabtoxinine-,8-lactam (2-amino-4-[3-hydroxy-2-oxo-azacyclobutan-3-yl]-butanoic acid).Originally, it was hypothesized that chlorosis was caused by a rapid buildup of ammonium, resulting from the inhibition of GS' (EC 6.3.1.2) by tabtoxin. Semipurified tabtoxin did, in fact, inhibit the activity of crude GS from pea (14). This hypothesis was also supported by the following observations: (a) MSO, which was already known to inhibit GS, also caused chlorosis and ammonium accumulation (7, 10); (b) tabtoxin-and MSO-induced chlorosis and ammonium accumulation are light-dependent reactions (3) which can be inhibited by dichlorodimethyl phenylurea (Durbin, unpublished); and (c) glutamine negated the inhibitory effects of both MSO and tabtoxin in several biological systems (2). Preliminary studies using purified materials failed to show inhibition of GS by tabtoxin (19). This has been clarified by the present study and the recent observation that unidentified peptidases in tobacco will hydrolyze tabtoxin to yield tabtoxinine-,flactam (19). Inasmuch as this compound can also cause chlorosis and, more important, appeared to inhibit GS, we postulated that chlorosis development via an inhibition of this enzyme depended upon the generation of tabtoxinine-,B-lactam.This report establishes that purified tabtoxinine-fl-lactam, but not tabtoxin, inhibits purified GS from pea by apparently irreversibly inactivating the enzyme. A simple, rapid procedure for the purification of tabtoxin and tabtoxinine-f,-lactam is also reported.'Abbreviations: GS, glutamine synthetase; MSO, L-methionine-S-sulfoximine; NMR, nuclear magnetic resonance. MATERIALS AND METHODSEnzyme Purification. Commercially obtained, dried split peas (1 kg) were milled to a fine powder and soaked 24 h at 4°C in 2 L 20 mm imidazole (pH 7.5), 10 mM MgC12, and 1 mM ,B-mercaptoethanol. The crude enzyme preparation was clarified by centrifugation (14,000g for 30 min), and a fraction containing the enzyme activity was precipitated by adjusting the pH to ...

An approximate doubling in plant growth, total plant nitrogen, nodulation, and overall dinitrogen fixation of alfalfa are the consequences of the action of a toxin delivered by a Pseudomonas infesting the alfalfa rhizosphere. The toxin, tabtoxinine-beta-lactam, inactivates selectively one form of glutamine synthetase in the nodules. Thus, normal glutamine synthetase-catalyzed ammonia assimilation is significantly impaired; yet these plants assimilated about twice the normal amount of nitrogen. How plants regulate dinitrogen fixing symbiotic associations is an important and unresolved question; the current results imply that the glutamine synthetase-catalyzed step in ammonia assimilation, a plant function, strongly influences overall dinitrogen fixation in legumes.

Selected pathovars of Pseudomonas syringae produce an extracellular phytotoxin, tabtoxinine-p-lactam, that irreversibly inhibits its known physiological target, glutamine synthetase (GS). Pseudomonas syringae subsp. "tabaci" retains significant amounts of glutamine synthetase activity during toxin production in culture. As part of our investigation of the self-protection mechanism(s) used by these pathovars, we have determined that GS becomes adenylylated after toxin production is initiated and that the serine released from the zinc-activated hydrolysis of tabtoxin is a factor in the initiation of this adenylylation. The adenylylation state of this GS was estimated to range from E5. 7.*5. The irreversible inactivation by tabtoxinine-B-lactam of unadenylylated and adenylylated glutamine synthetase purified from P. syringae subsp. "tabaci" was investigated. Adenylylated GS was inactivated by tabtoxinine-p-lactam at a slower rate than was unadenylylated enzyme. Adenylylated GS (E7.510.5) was significantly protected from this inactivation in the presence of the enzyme effectors, AMP, Ala, Gly, His, and Ser. Thus, the combination of the adenylylation of GS after toxin production is initiated and the presence of the enzyme effectors in vivo could provide part of the self-protection mechanism used by subsp. "tabaci".

The effects of tabtoxinine-,8-lactam (T-,i-L) on nitrate uptake and glutamine synthetase (GS) and nitrate reductase (NR) activities in roots of Avena sativa seedlings were determined. Seven-day-old oat seedlings placed in a 10 mm KNO3 and 0.5 mm T-,B-L solution for 24 hours took up T-f,-L and lost approximately 90% of their root GS activity. 1H1T-,B-L taken up by roots of seven-day-old oat seedlings was associated with GS immunoprecipitated from the extract of these roots. Total nitrate uptake and in vivo NR activity were decreased approximately 50% in the T-,B-L treated roots. However, T-,6-L uptake did not affect the induction phases of nitrate uptake or reduction, nor did it inhibit in vitro NR activity. Thus, the decrease in nitrate uptake and reduction is a secondary effect of T-Ot-L action. Roots of seven-day-old oat seedlings were inoculated with Pseudomonas syringae pv tabaci (Tox+) and the pathogen population in the rhizosphere was estimated by dilution plate count; 6 x 10" bacteria were recovered after 3 days, as compared to the original inoculation with 7 x 10' bacteria, indicating a significant growth of the pathogen in the rhizosphere. The bacteria recovered from the rhizosphere caused chlorosis in tobacco leaves and produced T-,j-L in culture; I x 10i4 bacteria were recovered from roots of seedlings inoculated with P. syringae pv tabaci (Tox-) using the same inoculation and assay procedure as for the pv tabaci(Tox+). Extracts of surface-sterilized roots previously inoculated with P. syringae pv tabaci (Tox+) did not produce viable bacterial cultures when plated out on a complete medium. Oat seedlings growing in sand culture and inoculated with P. syringae pv tabaci (Tox+) had developed chlorosis, and root GS activity had declined to less than 10% of controls after 3 days. Conversely, seedlings inoculated with P. syringae pv tabaci (Tox-) never developed chlorosis and maintained normal levels of GS activity. All oat plants inoculated with P. syringae pv tabaci (Tox+) died within 7 days after inoculation as compared to the plants inoculated with P. syringae pv tabaci (Tox-) which grew to maturity.The phytopathogenic bacterium Pseudomonas syringae pv tabaci and several closely related pathovars produce tabtoxin which when hydrolyzed to tabtoxinine-#-lactam (18) (2-amino-4-[3-hydroxy-2-oxo-azacyclobutan-3-yl]-butanoic acid) (T-,B-L)2 is an irreversible inhibitor of GS (EC 6.3.2.1.) in vivo (7, 16). T-,B-L is not a host-selective toxin and inactivates GS purified from a variety of sources (4, 7).