NorM, an Erwinia amylovora multidrug efflux pump involved in in vitro competition with other epiphytic bacteria

Abstract

Blossoms are important sites of infection for Erwinia amylovora, the causal agent of fire blight of rosaceous plants. Before entering the tissue, the pathogen colonizes the stigmatic surface and has to compete for space and nutrient resources within the epiphytic community. Several epiphytes are capable of synthesizing antibiotics with which they antagonize phytopathogenic bacteria. Here, we report that a multidrug efflux transporter, designated NorM, of E. amylovora confers tolerance to the toxin(s) produced by epiphytic bacteria cocolonizing plant blossoms. According to sequence comparisons, the single-component efflux pump NorM is a member of the multidrug and toxic compound extrusion protein family. The corresponding gene is widely distributed among E. amylovora strains and related plant-associated bacteria. NorM mediated resistance to the hydrophobic cationic compounds norfloxacin, ethidium bromide, and berberine. A norM mutant was constructed and exhibited full virulence on apple rootstock MM 106. However, it was susceptible to antibiotics produced by epiphytes isolated from apple and quince blossoms. The epiphytes were identified as Pantoea agglomerans by 16S rRNA analysis and were isolated from one-third of all trees examined. The promoter activity of norM was twofold greater at 18 degrees C than at 28 degrees C. The lower temperature seems to be beneficial for host infection because of the availability of moisture necessary for movement of the pathogen to the infection sites. Thus, E. amylovora might employ NorM for successful competition with other epiphytic microbes to reach high population densities, particularly at a lower temperature.

FIG. 1.

Temperature-dependent promoter activity of norM from E. amylovora as determined by using a transcriptional fusion with the reporter gene uidA. E. amylovora mutant Ea225 carrying uidA on a mini-Tn5 transposable element inserted into the genome was used to determine expression of GUS during growth in liquid AMM2. (A) Growth kinetics of Ea255 (norM′-uidA⁺); (B) GUS activity of Ea225 (norM′-uidA⁺). OD 600 nm, optical density at 600 nm.

FIG. 2.

Restriction map of plasmid pBBR.nor6 carrying the norM locus of E. amylovora. Restriction sites used for subcloning are underlined. Cm, chloramphenicol acetyltransferase; mob, gene region required for plasmid mobilization; rep, gene region required for plasmid replication; lacZ′, gene encoding the first 146 amino acids of β-galactosidase.

FIG. 3.

Rooted phylogenetic tree for NorM and its homologues. The dendrogram was generated with MegAlign, version 5.0 (DNASTAR Inc.), by using the ClustalW method. NorM of E. amylovora belongs to a cluster containing multidrug efflux transporters from members of the Eubacteria.

FIG. 4.

Southern blot analysis of E. amylovora insertion mutants. ClaI-digested genomic DNA was hybridized with a 1.0-kb digoxigenin-labeled norM probe and a 2.1-kb digoxigenin-labeled probe containing the chloramphenicol resistance cassette. Lane 1, Ea1189 (wild type); lane 2, Ea1189-14; lane 3, Ea1189-22; lane 4, Ea1189-23; lane 5, Ea1189-29; lane 6, Ea1189-34. The hybridization patterns indicated that in the mutants the chloramphenicol resistance cassette was inserted into norM by double crossover.

FIG. 5.

Growth inhibition of the E. amylovora wild type and mutant Ea1189-34 (norM::Cm) caused by the supernatant of P. agglomerans Bel2/7. The antimicrobial activity of strain Bel2/7 was determined by agar plate diffusion assays carried out at 18 and 28°C. Well 1, supernatant of MM1 culture of strain Bel2/7; well 2, spectinomycin (5 μg/ml).

FIG. 6.

Virulence assay with apple rootstock MM 106. The E. amylovora wild type and mutant Ea1189-34 (norM::Cm) were inoculated by the prick technique in shoot tips. The establishment of a population was determined by reisolating the bacteria 24 h after inoculation.

FIG. 7.

Distribution of norM-like genes among plant-associated enterobacteria. Southern blot analysis was performed by using total chromosomal DNA digested with ClaI and probed with a digoxigenin-labeled 1.0-kb PCR product of the norM gene from E. amylovora. Hybridization was carried out at 50°C. Lane 1, E. amylovora GSPB 1189; lane 2, Erwinia pyrifoliae Ep1/96; lane 3, Erwinia rhapontici GSPB 454; lane 4, Erwinia rhapontici DSM 4484; lane 5, Erwinia persicina CFBP 3622; lane 6, Erwinia persicina GSPB 2443; lane 7, Erwinia mallotivora CFBP 2503; lane 8, Erwinia psidii CFBP 3627; lane 9, Erwinia herbicola pv. gypsophilae 824-1; lane 10, P. agglomerans 2b/89; lane 11, P. agglomerans 52c/90; lane 12, Pantoea stewartii SW2; lane 13, Pantoea stewartii SS104; lane 14, Pectobacterium chrysanthemi AC4150; lane 15, Pectobacterium chrysanthemi 3937; lane 16, Pectobacterium carotovorum pv. carotovora 90; lane 17, Brenneria quercina CFBP 3617; lane 18, Brenneria salicis CFBP 802; lane 19, E. coli TG1.