An Isoflavonoid-Inducible Efflux Pump in <i>Agrobacterium tumefaciens</i> Is Involved in Competitive Colonization of Roots

Agrobacterium tumefaciens 1D1609, which was originally isolated from alfalfa (Medicago sativa L.), contains genes that increase competitive root colonization on that plant by reducing the accumulation of alfalfa isoflavonoids in the bacterial cells. Mutant strain I-1 was isolated by its isoflavonoid-inducible neomycin resistance following mutagenesis with the transposable promoter probe Tn5-B30. Nucleotide sequence analysis showed the transposon had inserted in the first open reading frame, ifeA, of a three-gene locus (ifeA, ifeB, and ifeR), which shows high homology to bacterial efflux pump operons. Assays on alfalfa showed that mutant strain I-1 colonized roots normally in single-strain tests but was impaired significantly (P < or = 0.01) in competition against wild-type strain 1D1609. Site-directed mutagenesis experiments, which produced strains I-4 (ifeA::gusA) and I-6 (ifeA::omega-Tc), confirmed the importance of ifeA for competitive root colonization. Exposure to the isoflavonoid coumestrol increased beta-glucuronidase activity in strain I-4 21-fold during the period when coumestrol accumulation in wild-type cells declined. In the same test, coumestrol accumulation in mutant strain I-6 did not decline. Expression of the ifeA-gusA reporter was also induced by the alfalfa root isoflavonoids formononetin and medicarpin but not by two triterpenoids present in alfalfa. These results show that an efflux pump can confer measurable ecological benefits on A. tumefaciens in an environment where the inducing molecules are known to be present.