Article Figures & Data
Figures
- FIG 1
Genomic map showing names and positions of genes identified in this study as having an auxotrophic mutant phenotype in Erwinia amylovora strain HKN06P1. Numbers around the circle indicate base pair positions in the E. amylovora CFBP 1430 genome.
- FIG 2
Metabolic pathway map. Positions of disruptions identified in the auxotrophic mutant screen are indicated with red Xs. CoA, coenzyme A.
- FIG 3
Erwinia amylovora auxotrophic mutant disease severity in apple fruitlets, growth in fruitlet medium, and correlations. (A) Disease severity in apple fruitlets at 7 days after inoculation. The disease severity ratio (dsr) represents the ratio of disease severity in fruitlets inoculated with the mutant divided by the disease severity of fruitlets inoculated with wild type. A dsr of 1 indicates that the disease severity of the mutant equals that of the wild type, and values less than 1 indicate that the mutant caused less severe disease than did the wild type. The dsr values represent averages of all available alleles for each gene; error bars indicate the standard error. Affected metabolic and biosynthetic pathways for the various gene categories are indicted. Asterisks and triangles indicate statistically significant differences from the wild type using Student's t test, with P values of <0.01 and <0.05, respectively. (B) Growth in fruitlet medium, as measured by optical density at 600 nm after 20 h; error bars indicate standard error. The prototroph is a fully virulent E. amylovora strain carrying a Tn5 transposon in a nongenic region. Asterisks indicate statistically significant difference from the prototroph using Student's t test, P < 0.0001; n = 3 per strain. (C) Scatter plot correlation of growth in fruitlet medium with dsr including all mutants. Fuchsia points represent cysteine auxotrophs; green points represent glycolysis and PTS mutants. All other mutants are indicated by blue points. (D) Scatter plot correlation of growth in fruitlet medium with dsr, not including the cysteine, glycolysis, and PTS mutants.
- FIG 4
Disease severity caused by auxotrophic Erwinia amylovora mutants in 'Gala' apple tree shoots. Eight selected Erwinia amylovora mutants were inoculated on 'Gala' apple tree shoots by shoot tip wounding, and the percentage of total shoot length showing necrosis was measured over the course of 3 weeks. Error bars indicate standard error; n = 20 shoots per strain. Within each time point, bars sharing the same letter are not statistically different according to an ANOVA (α = 0.05). The entire experiment was performed twice with similar results; results from a representative experiment are shown.
- FIG 5
Hypersensitive responses in tobacco. Suspensions of the indicated Erwinia amylovora strains at an OD600 of 0.1 in 10 mM MgCl2 were infiltrated into Nicotiana tabacum cv. ‘Glurk’ leaf segments. Tissue collapse was photographed at 48 h postinoculation. MgCl2, 10 mM MgCl2 without bacteria; n.i., not infiltrated. The experiment was performed twice. Four leaves were infiltrated in each experiment, with similar results on all leaves. Photo shows a representative leaf.
- FIG 6
Exometabolomic heatmap of fruitlet medium before and after culturing with Erwinia amylovora. The indicated E. amylovora strains were cultured for 20 h in fruitlet medium. Each column represents a distinct biological replicate. Wild-type+Tet represents the supernatant of wild-type E. amylovora grown in the presence of 2 μm tetracycline as a bacteriostatic. Values are the log2 fold change compared to uncultured (fresh) fruitlet medium. Clustering was performed in R using a Ward clustering algorithm and Pearson distance measures. The four metabolites highlighted in red are directly related to predicted pathway disruptions in the four auxotrophic mutants grown for the metabolomics assay.
- FIG 7
Addition and depletion of all detected amino acids in spent fruitlet medium from the indicated Erwinia amylovora strains. Data are derived from the same data set as those shown in Fig. 6. Mutant strains were designated grower, partial grower, or nongrower based on their previously observed ability to grow in fruitlet medium. Depletion and addition are shown relative to amino acid levels in uncultured fruitlet medium. Error bars indicate the standard error.
- FIG 8
Wild-type Erwinia amylovora growth on detached apple flowers pretreated with nonvirulent bacterial suspensions. Flowers were pretreated by spraying with control 10 mM MgCl2 or the indicated bacterial suspensions 4 h prior to inoculation with wild-type E. amylovora at 103 CFU per flower. Wild-type E. amylovora populations per flower were determined at 72 h postinoculation. (A) Growth of wild-type E. amylovora on apple flowers pretreated with an E. amylovora argD mutant at 1010 CFU ml−1. (B) Growth of wild-type E. amylovora on apple flowers pretreated with an E. amylovora argD mutant at 109 CFU ml−1. (C) Representative apple flowers at 72 h following the indicated pretreatment. (D) Growth of wild-type E. amylovora on apple flowers pretreated with heat-killed E. amylovora argD mutant cells at 1010 CFU ml−1. (E) Growth of wild-type E. amylovora on apple flowers pretreated with E. coli SM10(λpir) at 1010 CFU ml−1. Error bars indicate standard error. Statistical significance was determined using Student’s t test, n = 4 flowers per treatment per experiment (Expt).
Tables
- TABLE 1
Genes identified as producing auxotrophic phenotypes in Erwinia amylovora
Gene(s) No. of mutant alleles Supplement(s) rescuing growtha argC, argD, argE, argG, argH, argI 12 Arginine asnB 3 Asparagine carA, carB 6 Arginine plus uracil cysB, cysE, cysI1, cysI3, cysJ 13 Cysteine glmS 1 Acetylglucosamine gltA, gltB 12 Glutamic acid guaB 1 Guanine hisA, hisB, hisD, hisG, hisH 8 Histidine ilvA, ilvC, ilvD, ilvE1 9 Isoleucine plus valine leuA, leuB, leuC 8 Leucine lysA 1 Lysine metA, metB, metE, metF, metR 12 Methionine pfkA 1 Succinate pgi 3 Succinate ppc 7 Succinate proA, proC 4 Proline ptsH, ptsI 2 Succinate plus glutamic acid purA, purC, purD1, purF, purH, purK, purL, purM 16 Adenine pyrB, pyrC, pyrD 9 Uracil serA 1 Glutamic acid srlD 1 Glucose thrA, thrB 6 Threonine trpB, trpC, trpE, trpG 11 Tryptophan ↵a Supplement or supplement combination that supports the growth of the indicated mutant when added to liquid M9.
Supplemental material
- Supplemental file 1 -
Fig. S1 to S3, legends to Data Sets S1 and S2
PDF, 690K
- Supplemental file 2 -
Data Set S1
XLSX, 20K
- Supplemental file 3 -
Data Set S2
XLSX, 43K
- Supplemental file 1 -
