Chromosomally Encoded hok-sok Toxin-Antitoxin System in the Fire Blight Pathogen Erwinia amylovora: Identification and Functional Characterization

Abstract

Toxin-antitoxin (TA) systems are genetic elements composed of a protein toxin and a counteracting antitoxin that is either a noncoding RNA or protein.In type I TA systems, the antitoxin is a noncoding small RNA (sRNA) that base pairs with the cognate toxin mRNA interfering with its translation. Although type I TA systems have been extensively studied in Escherichia coli and a few human or animal bacterial pathogens, they have not been characterized in plant-pathogenic bacteria. In this study, we characterized a chromosomal locus in the plant pathogen Erwinia amylovora Ea1189 that is homologous to the hok-sok type I TA system previously identified in the Enterobacteriaceae-restricted plasmid R1. Phylogenetic analysis indicated that the chromosomal location of the hok-sok locus is, thus far, unique to E. amylovora. We demonstrated that ectopic overexpression of hok is highly toxic to E. amylovora and that the sRNA sok reversed the toxicity of hok through mok, a reading frame presumably translationally coupled with hok. We also identified the region that is essential for maintenance of the main toxicity of Hok. Through a hok-sok deletion mutant (Ea1189Δhok-sok), we determined the contribution of the hok-sok locus to cellular growth, micromorphology, and catalase activity. Combined, our findings indicate that the hok-sok TA system, besides being potentially self-toxic, provides fitness advantages to E. amylovora. IMPORTANCE Bacterial toxin-antitoxin systems have received great attention because of their potential as targets for antimicrobial development and as tools for biotechnology. Erwinia amylovora, the causal agent of fire blight disease on pome fruit trees, is a major plant-pathogenic bacterium. In this study, we identified and functionally characterized a unique chromosomally encoded hok-sok toxin-antitoxin system in E. amylovora that resembles the plasmid-encoded copies of this system in other Enterobacteriaceae. This study of a type I toxin-antitoxin system in a plantpathogenic bacterium provides the basis to further understand the involvement of toxin-antitoxin systems during infection by a plant-pathogenic bacterium. The new linkage between the hok-sok toxin-antitoxin system and the catalase-mediated oxidative stress response leads to additional considerations of targeting this system for antimicrobial development.

Activation of metabolic and stress responses during subtoxic expression of the type I toxin hok in Erwinia amylovora

“…Moderate overexpression of hok does not suppress bacterial growth. A hok overexpression construct, pOE-hok, was previously generated by cloning the E. amylovora Ea1189 hok gene into the lac promoter-containing plasmid pEVS143 (27). The lac promoter allows low levels of transcription in the absence of the inducer isopropyl b-D-1-thiogalactopyranoside (IPTG) (35).…”

Activation of metabolic and stress responses during subtoxic expression of the type I toxin hok in Erwinia amylovora

“…Moderate overexpression of hok does not suppress bacterial growth. A hok overexpression construct, pOE-hok, was previously generated by cloning the E. amylovora Ea1189 hok gene into the lac promoter-containing plasmid pEVS143 (27). The lac promoter allows low levels of transcription in the absence of the inducer isopropyl β-D-1-thiogalactopyranoside (IPTG) (35).…”

“…Recently, we identi ed a chromosomally encoded hok-sok type I TA system in Erwinia amylovora (27), a model enterobacterial plantpathogenic bacterium that causes the destructive re blight disease of pome fruit trees including apple (Malus sp.) and pear (Pyrus sp.)…”

“…(28,29). Episomal overexpression of the hok gene caused massive killing of E. amylovora cells and arrested cell division after septa were formed (27). We proposed that cell death due to hok induction at toxic levels in E. amylovora is likely to be associated with the disturbance of essential functions of the cell membrane.…”

Activation of metabolic and stress responses during subtoxic expression of the type I toxin hok in Erwinia amylovora

“…Recently, we identified a chromosomally encoded hok - sok type I TA system in Erwinia amylovora [ 27 ], a model enterobacterial plant-pathogenic bacterium that causes the destructive fire blight disease of pome fruit trees including apple ( Malus sp.) and pear ( Pyrus sp.)…”