Role of glutamine synthetase adenylylation in the self-protection of Pseudomonas syringae subsp. "tabaci" from its toxin, tabtoxinine-beta-lactam

Abstract

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".

Self-protection of Pseudomonas syringae pv. "tabaci" from its toxin, tabtoxinine-beta-lactam

“…GS is irreversibly inactivated by T,L in vitro (6,7,19). We report data showing that in vivo GS activity is not affected by the presence of TPL in liquid culture of a Tox+ strain.…”

“…We have shown that adenylylation of GS offers partial protection for GS against T,3L by slowing the rate of inactivation (6). Adenylylation of GS is correlated with the accumulation of serine that accompanies the enzymatic hydrolysis of tabtoxin to release serine and T,L (6).…”

“…Adenylylation of GS is correlated with the accumulation of serine that accompanies the enzymatic hydrolysis of tabtoxin to release serine and T,L (6). Adenylylated GS has a reduced affinity for TiL, but this GS is eventually, although very slowly, inactivated by T,L (6). Additional protection mechanism(s) must therefore be employed by P. syringae pv.…”

Cloning and Expression ofPseudomonas taetrolensY-30 Gene Encoding Glutamine Synthetase: An Enzyme Available for Theanine Production by Coupled Fermentation with Energy Transfer

“…It usually brings about changes in the reactivity of the enzyme, 35) and several Pseudomonas GS are known to be adenylylated in response to culture conditions. 23,29) …”

“…In the case of whole cells as an enzyme preparation, cetyltrimethylammonium bromide (CTAB, 0.1 mg/ml) was added to the reaction mixture. 28,29) (3) -Glutamyl-transferring reaction (GT-reaction): One unit of the activity (GTactivity) was defined as the amount of enzyme forming 1 mmol of -glutamylhydroxamate per min.…”