PCR primer (Table 1) hybridization locations and directions are indicated. Genome nucleotide positions are numbered according to their homologous positions in the complete genome sequence of E. (A) Illustration of the argD gene and flanking genes yhfK and pabA showing the genome nucleotide position of the Tn 5 insertion in the argD( 1000)::Tn 5 mutant and the genomic DNA (gDNA) segment used for complementation. amylovora cannot obtain sufficient arginine from apple and pear fruit tissues or from apple vegetative tissues, either at the beginning of the infection process or after the infection has progressed to an advanced state.Ĭopyright © 2014, American Society for Microbiology. Nevertheless, these results are informative about the parasitic nature of the fire blight disease interaction, since they indicate that E. amylovora virulence factor because the argD(1000)::Tn5 mutant was auxotrophic and had a primary metabolism defect. The ArgD protein cannot be considered an E. amylovora growing in planta, without concern about losing the plasmid over time. Therefore, the pCR2.1-argD complementation plasmid could be useful for the expression of genes, markers, and reporters in E. Furthermore, the pCR2.1-argD complementation plasmid was stably maintained in the argD(1000)::Tn5 mutant growing in host tissues without any antibiotic selection. amylovora cells and inoculated onto immature apple fruit, the argD(1000)::Tn5 mutant still failed to grow, while the virulent strain grew and caused disease. However, even when mixed with virulent E. A plasmid-borne copy of the wild-type argD gene complemented both the nonpathogenic and the arginine auxotrophic phenotypes of the argD(1000)::Tn5 mutant. amylovora argD gene encodes a predicted N-acetylornithine aminotransferase enzyme, which is involved in the production of the amino acid arginine. amylovora argD(1000)::Tn5, an argD Tn5 transposon mutant that has the Tn5 transposon inserted after nucleotide 999 in the argD gene-coding region, was an arginine auxotroph that did not cause fire blight in apple and had reduced virulence in immature pear fruits. Fire blight is caused by Erwinia amylovora and is the most destructive bacterial disease of apples and pears worldwide.
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