Extragenic Suppression of Elongation Factor P Gene Mutant Phenotypes in Erwinia amylovora

Abstract

Elongation factor P (EF-P) facilitates the translation of certain peptide motifs, including those with multiple proline residues. EF-P must be posttranslationally modified for full functionality; in enterobacteria, this is accomplished by two enzymes, namely, EpmA and EpmB, which catalyze the β-lysylation of EF-P at a conserved lysine position. Mutations to efp or its modifying enzymes produce pleiotropic phenotypes, including decreases in virulence, swimming motility, and extracellular polysaccharide production, as well as proteomic perturbations. Here, we generated targeted deletion mutants of the efp, epmA, and epmB genes in the Gram-negative bacterium Erwinia amylovora, which causes fire blight, an economically important disease of apples and pears. As expected, the Δefp, ΔepmA, and ΔepmB mutants were all defective in virulence on apples, and all three mutants were complemented in trans with plasmids bearing wild-type copies of the corresponding genes. By analyzing spontaneous suppressor mutants, we found that mutations in the hrpA3 gene partially or completely suppressed the colony size, extracellular polysaccharide production, and virulence phenotypes in apple fruits and apple tree shoots but not the swimming motility phenotypes of the Δefp, ΔepmA, and ΔepmB mutants. The deletion of hrpA3 alone did not produce any alterations in any characteristics measured, indicating that the HrpA3 protein is not essential for any of the processes examined. The hrpA3 gene encodes a putative DEAH-box ATP-dependent RNA helicase. These results suggest that the loss of the HrpA3 protein at least partially compensates for the lack of the EF-P protein or β-lysylated EF-P.IMPORTANCE Fire blight disease has relatively few management options, with antibiotic application at bloom time being chief among them. As modification to elongation factor P (EF-P) is vital to virulence in several species, both EF-P and its modifying enzymes make attractive targets for novel antibiotics. However, it will be useful to understand how bacteria might overcome the hindrance of EF-P function so that we may be better prepared to anticipate bacterial adaptation to such antibiotics. The present study indicates that the mutation of hrpA3 could provide a partial offset for the loss of EF-P activity. In addition, little is known about EF-P functional interactions or the HrpA3 predicted RNA helicase, and our genetic approach allowed us to discern a novel gene associated with EF-P function.

Keywords: DEAH-box RNA helicase; Erwinia amylovora; apple; elongation factor P; fire blight; ribosome.

FIG 1

Deletion of hrpA3 restores colony size to that of the wild type in Δefp, ΔepmA, and ΔepmB genetic backgrounds. LB plates are shown after 72 hours of growth at 28°C. Genotypes are indicated on the figure. “Inset” represents a magnification of the boxed area in the Δefp panel and contains an example spontaneous suppressor colony. Plates are 100- by 15-mm petri dishes.

FIG 2

Quantification of colony sizes shown in Fig. 1. Colony sizes for the indicated strains were measured after 72 hours of growth on LB plates at 28°C. Colony size was quantified using NIH ImageJ to measure the area of each colony. Bars represent the average areas of 10 colonies for each genotype from a single representative experiment, and error bars indicate standard error (SE). Strains sharing a letter have no statistically significant difference based on a Tukey test. The entire experiment was performed at least four times with similar results each time.

FIG 3

Polymorphisms in the hrpA3 gene detected in seven independent suppressors of the Δefp and ΔepmB small-colony phenotype. Δefp^L1 to Δefp^L6 are from six independent Δefp suppressors; ΔepmB^L1 is from a ΔempB suppressor. For the frameshift mutations, alterations to the predicted protein product of the hrpA3 gene are indicated as the position of the first altered amino acid, along with the length of additional, aberrant amino acids produced by the frameshift. N and C indicate the amino and carboxy termini, respectively, of the HrpA3 protein.

FIG 4

Deletion of hrpA3 restores the amylovoran extracellular polysaccharide production level to that of the wild type in Δefp, ΔepmA, and ΔepmB genetic backgrounds. Amylovoran production in the indicated strains was assayed via a cetylpyridinium chloride precipitation assay, where the precipitate OD at 600 nm is measured using a spectrophotometer. The experiment was repeated three times, with similar results each time. Error bars represent SE. Strains sharing the same letter have no statistically significant difference based on a Tukey test.

FIG 5

Deletion of hrpA3 does not increase swimming motility in Δefp, ΔepmA, and ΔepmB genetic backgrounds. Bars represent average diameters of visible bacteria for the indicated strains on M9 minimal medium plates after 48 hours at 28°C. The experiment was repeated four times with similar results each time. Error bars represent SE. Strains sharing the same letter have no statistically significant difference based on a Tukey test.

FIG 6

Deletion of hrpA3 partially restores virulence in the Δefp, ΔepmA, and ΔepmB genetic backgrounds. (A) Symptom development in apple fruitlets at 7 days after inoculation with 2 × 10⁶ cells of the indicated E. amylovora strains. (B) Growth of the indicated bacterial strains in apple fruitlets inoculated as in panel A, over the first 2 days of infection, expressed as CFU per gram of apple tissue. The experiment was repeated three times, with similar results each time. Within a given time point, bars sharing a letter have no statistically significant difference according to a Tukey test, and error bars represent SE.

FIG 7

Δefp suppressor mutants have increased virulence in apple tree shoots compared with that of the Δefp parent strain. Apple trees were inoculated through shoot tip wounds with the indicated E. amylovora strains, and the extent of resulting necrosis in the inoculated shoots was measured at 1-week intervals. Bars represent the extent of necrosis in 25 shoots per strain; error bars indicate SE. Within a given time point, bars sharing a letter have no statistically significant difference according to a Tukey test. This experiment was performed twice with similar results each time; results of one representative experiment are shown.