Charged Amino Acids Contribute to ZorO Toxicity

Abstract

Chromosomally encoded toxin-antitoxin systems have been increasingly identified and characterized across bacterial species over the past two decades. Overproduction of the toxin gene results in cell growth stasis or death for the producing cell, but co-expression of its antitoxin can repress the toxic effects. For the subcategory of type I toxin-antitoxin systems, many of the described toxin genes encode a small, hydrophobic protein with several charged residues distributed across the sequence of the toxic protein. Though these charged residues are hypothesized to be critical for the toxic effects of the protein, they have not been studied broadly across different type I toxins. Herein, we mutated codons encoding charged residues in the type I toxin zorO, from the zor-orz toxin-antitoxin system, to determine their impacts on growth inhibition, membrane depolarization, ATP depletion, and the localization of this small protein. The non-toxic variants of ZorO accumulated both in the membrane and cytoplasm, indicating that membrane localization alone is not sufficient for its toxicity. While mutation of a charged residue could result in altered toxicity, this was dependent not only on the position of the amino acid within the protein but also on the residue to which it was converted, suggesting a complex role of charged residues in ZorO-mediated toxicity. A previous study indicated that additional copies of the zor-orz system improved growth in aminoglycosides: within, we note that this improved growth is independent of ZorO toxicity. By increasing the copy number of the zorO gene fused with a FLAG-tag, we were able to detect the protein expressed from its native promoter elements: an important step for future studies of toxin expression and function.

A simple BLASTn-based approach generates novel insights into the regulation and biological function of type I toxin-antitoxins

“…This EAP region is required for robust translation but is also where the OrzO antitoxin binds. Multiple copies of the zor-orz locus from EDL933 or just the zorO-orzO pair increased the minimum inhibitory concentration of aminoglycoside antibiotics and reduced lag in sublethal levels of kanamycin, though the mechanism is unknown ( 9 , 26 ). We initially examined zor/orz sequence conservation with zorO/orzO from EDL933 as a query (depicted in Fig.…”

Mechanism of action of sprG1-encoded type I toxins in Staphylococcus aureus: from membrane alterations to mesosome-like structures formation and bacterial lysis

“…The α-helical region appears to be critical for membrane interaction in most type I toxins, including Fst, TisB, IsbC, SprA1 and AapA1 ( Göbl et al, 2010 ; Mok et al, 2010 ; Steinbrecher et al, 2012 ; Solecki et al, 2015 ; Korkut et al, 2020 ). Additionally, specific charged amino acids in the predicted α-helical transmembrane domain are crucial for ZorO-mediated toxicity ( Bogati et al, 2022a ). We also found that the SprG1 31 sequence can be shortened to 25 amino acids with a minimum of 16 hydrophobic residues to retain toxicity, likely by maintaining the helix translocation across the membrane.…”

Effects of C-Terminal Lys-Arg Residue of AapA1 Protein on Toxicity and Structural Mechanism

“…These toxins also exhibit a membrane toxic effect, indicating the vital role of these basic C-terminal residues in the toxicity process. Moreover, charged amino acids in type I TA toxin protein ZorO are important for its toxicity and how it affects remain unknown [ 31 ]. Therefore, it is important to analyze the structural characteristics of AapA1-28 in the E. coli membrane and compare them with those of the AapA1 toxin protein.…”