An isoflavonoid-inducible efflux pump in Agrobacterium tumefaciens is involved in competitive colonization of roots

Abstract

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.

FIG. 1

Identification of an A. tumefaciens strain mutated in an isoflavonoid-inducible locus by the promoterless reporter gene nptII which confers neomycin resistance. (A) Growth of mutant strain I-1 on neomycin-containing medium with (right) or without (left) 10 μM formononetin. (B) Growth of mutant strain I-1 on neomycin-containing medium with (right) or without (left) 1 μM coumestrol. AB medium was supplemented with 0.2% methanol as a control (left) for the isoflavonoid solutions (right). All plates were inoculated with 40 replicate colonies of mutant strain I-1 (top five rows) and eight replicate colonies of a constitutively neomycin-resistant mutant (bottom rows).

FIG. 2

Alfalfa root colonization by wild-type A. tumefaciens 1D1609 and mutant strain I-1. Strains were inoculated separately (A) or as a 1:1 mixture (B) on sterile alfalfa seedlings at the time of germination. Root-colonizing bacteria were recovered and counted by dilution plating at the times indicated. Bacterial counts are reported as means ± standard errors from 7 to 10 replicate plants. Standard error bars are obscured by symbols in some cases.

FIG. 3

Restriction map of the 7.5-kb PstI fragment containing the Tn5-B30 insertion (▾) in A. tumefaciens mutant strain I-1. Restriction sites are represented as C, ClaI; E, EcoRI; K, KpnI; N, NotI; P, PstI; S, SalI; or X, XhoI. Arrows below the map indicate open reading frames predicted from nucleotide sequence analysis.

FIG. 4

Coumestrol accumulation and ifeA expression in A. tumefaciens. Coumestrol accumulation in wild-type strain 1D1609 (•) and mutant strain I-6 (□) was measured before and after the addition of 50 μM coumestrol (left axis). Corresponding expression of ifeA was measured as GUS activity (bar graph) in mutant strain I-4 (right axis). Data represent means ± standard errors from three replicates.