Citrate Synthase Mutants of Agrobacterium Are Attenuated in Virulence and Display Reduced vir Gene Induction

Abstract

A citrate synthase (CS) deletion mutant of Agrobacterium tumefaciens C58 is highly attenuated in virulence. The identity of the mutant was initially determined from its amino acid sequence, which is 68% identical to Escherichia coli and 77% identical to Brucella melitensis. The mutant lost all CS enzymatic activity, and a cloned CS gene complemented a CS mutation in Sinorhizobium. The CS mutation resulted in a 10-fold reduction in vir gene expression, which likely accounts for the attenuated virulence. When a plasmid containing a constitutive virG [virG(Con)] locus was introduced into this mutant, the level of vir gene induction was restored to nearly wild-type level. Further, the virG(Con)-complemented CS mutant strain induced tumors that were similar in size and number to those induced by the parental strain. The CS mutation resulted in only a minor reduction in growth rate in a glucose-salts medium. Both the CS mutant and the virG(Con)-complemented CS strain displayed similar growth deficiencies in a glucose-salts medium, indicating that the reduced growth rate of the CS mutant could not be responsible for the attenuated virulence. A search of the genome of A. tumefaciens C58 revealed four proteins, encoded on different replicons, with conserved CS motifs. However, only the locus that when mutated resulted in an attenuated phenotype has CS activity. Mutations in the other three loci did not result in attenuated virulence and any loss of CS activity, and none were able to complement the CS mutation in Sinorhizobium. The function of these loci remains unknown.

Phosphoenolpyruvate Carboxykinase Is an Acid-Induced, Chromosomally Encoded Virulence Factor in Agrobacterium tumefaciens

“…A readily testable possibility is that the mutation reduces the level of vir gene induction in some way. This seemed like a real possibility, since so many other mutations (ivr211, miaA, and chvD), and another gene in carbohydrate metabolism (38), also reduced vir gene induction. The plasmid pSM243cd containing the virB::lacZ translational fusion was introduced into the pckA deletion mutant and the wild-type C58 strain.…”

“…In none of these cases is the basis for this reduction understood. We have observed recently that a mutation in citrate synthase also results in reduced vir gene induction (38). The question of whether the reductions in vir gene induction in the two mutations in carbohydrate metabolism share a common basis awaits further study.…”

Roles of Agrobacterium tumefaciens RirA in Iron Regulation, Oxidative Stress Response, and Virulence

“…There are reports that the attenuated virulence of A. tumefaciens mutants results from defects in vir gene induction (3,27). Thus, we sought to determine whether a mutation in rirA affected vir gene induction.…”

Multiple Superoxide Dismutases in Agrobacterium tumefaciens : Functional Analysis, Gene Regulation, and Influence on Tumorigenesis

“…This observation indicates that ROS play important roles in the A. tumefaciens/plant interaction and tumorigenesis. Thus, we tested various sod mutants containing the pTiC58 plasmid pCMA1 (27) for their abilities to induce tumors on tobacco leaf discs (44). The sodBI mutant was markedly reduced (80%) in its ability to induce tumors on the plant leaf discs (Fig.…”