NieR is the repressor of a NaOCl-inducible efflux system in Agrobacterium tumefaciens C58

Abstract

The Agrobacterium tumefaciens atu4217 gene, which encodes a TetR family transcription regulator, is a repressor of the atu4218-atu4219-atu4220 operon. The Atu4218 and Atu4219 proteins belong to the HlyD family (membrane fusion protein) and the AcrB/AcrD/AcrF family (inner membrane transporter), respectively, and may form an efflux pump. The atu4220 gene encodes a short-chain dehydrogenase. Quantitative real-time PCR analysis showed induction of atu4217 and atu4218 by NaOCl but not by N-ethylmaleimide or reactive oxygen species (ROS) including H2O2, menadione and cumene hydroperoxide; therefore, the atu4218 and atu4219 were named NaOCl-inducible efflux genes nieA and nieB, respectively. The atu4217 gene, which was named nieR, serves as a repressor of nieA and nieB. DNase I footprinting assays identified 20-bp imperfect inverted repeat (IR, underlined) motifs 5′-TAGATTTAGGATGCAATCTA-3′ (box A) and 5′-TAGATTTCACTTGACATCTA-3′ (box R) in the intergenic region of the divergent nieA and nieR genes; these motifs were recognized by the NieR protein. Electrophoretic mobility shift assays demonstrated that NieR specifically binds to the 20-bp IR motifs and that NaOCl prevents this NieR-DNA interaction. Promoter-lacZ fusions and mutagenesis of the NieR boxes (A and R) showed a more dominant role for box A than for box R in the repression of the nieA and nieR promoters. However, full repression of either promoter required both operators. The nieR mutant strain exhibited a small colony phenotype and was more sensitive than the wild-type to NaOCl and antibiotics, including ciprofloxacin, nalidixic acid, novobiocin, and tetracycline. By contrast, the nieAB mutant strain showed no phenotype changes under the tested conditions.