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.

Virulence Genetics of anErwinia amylovoraPutative Polysaccharide Transporter Family Member

“…Tn5 mutants used throughout were generated and identified using the EZ-Tn5 Kan-2 kit (Lucigen, Middleton, WI) according to the manufacturer's instructions. Targeted deletions of genes were generated by overlap extension PCR as described previously (66). Potential clones were identified by PCR and confirmed by sequencing.…”

De-novo designed β-lysine derivatives can both augment and diminish the proliferation rates of E. coli through the action of Elongation Factor P

“…37 In some organisms, including the increasingly problematic human pathogen Mycobacterium tuberculosis, EFP is reported to be essential. 21,38 Given the ribosome stall-alleviating role of EFP (inter alia [39][40][41][42][43] ) and since it has been shown that i) EFP binds during most (if not all) elongation cycles 44 (and not just during polyproline-associated stalling events), ii) the Lys34 hydroxyl unit (not necessary for function 17,18,28 ) and the amino group from the ligated -lysine residue form hydrogen bonds with the ribosyl-backbone of the CCA P-site tRNA polynucleotide, while the -lysine carbonyland amine moieties bind to the conserved nucleotide A2439 of the 23S rRNA 31 and iii) the -amino acid modification protrudes close in space to the peptide transfer centre; 29,31 we postulated that if PoxA could decorate EFP with -amino acids other than 2, that it would offer an opportunity to influence eubacterial translation very close to the peptide-forming event either in dramatic or subtle ways, depending on the substrates incorporated into EFP and the downstream phenotypic effects of same.…”