Charged Residues Flanking the Transmembrane Domain of Two Related Toxin–Antitoxin System Toxins Affect Host Response

Abstract

A majority of toxins produced by type I toxin–antitoxin (TA-1) systems are small membrane-localized proteins that were initially proposed to kill cells by forming non-specific pores in the cytoplasmic membrane. The examination of the effects of numerous TA-1 systems indicates that this is not the mechanism of action of many of these proteins. Enterococcus faecalis produces two toxins of the Fst/Ldr family, one encoded on pheromone-responsive conjugative plasmids (FstpAD1) and the other on the chromosome, FstEF0409. Previous results demonstrated that overexpression of the toxins produced a differential transcriptomic response in E. faecalis cells. In this report, we identify the specific amino acid differences between the two toxins responsible for the differential response of a gene highly induced by FstpAD1 but not FstEF0409. In addition, we demonstrate that a transporter protein that is genetically linked to the chromosomal version of the TA-1 system functions to limit the toxicity of the protein.

Regulation of mannitol metabolism inEnterococcus faecalisand association withparEF0409toxin-antitoxin locus function

“…The origin and function of the parEF0409 TA-1 system remains unclear, but several pieces of circumstantial evidence suggest that its function may be coordinated with that of surrounding genes. First, as previously reported (33), mutation of an efflux pump located two Kbp downstream of mtlD results in hypersensitivity to FstEF0409, the parEF0409 toxin. Located between the mtlA2 operon and the genes for the efflux system are genes annotated as NAD binding proteins that could conceivably be involved in recycling the NAD+ cofactor required for MtlD function.…”

“…In-frame, markerless deletions of mltA, mtlR, mtlF and mltA2 were constructed in OG1RF by allelic exchange using vector pJH086 (39) as previously described (33, 40). The mutant alleles were synthesized by Blue Heron Biotech, LLC, (Bothell, WA, USA) and contained the first 5 and last 5 codons of the corresponding gene and approximately 900 base pairs of flanking DNA.…”

Charged Amino Acids Contribute to ZorO Toxicity

“…For some type I toxins, charged residues are critical for their toxicity and/or formation of larger complexes [ 23 , 31 , 32 ]. We thus examined the contribution of charged residues in ZorO activity by generating the specific point mutations ( Table S3 ) and monitoring the effects after overexpression.…”

“…Single amino acid substitutions or multiple deletion/truncation studies of type I toxins from both Gram-positive and Gram-negative bacteria have shown stretch of hydrophobic residues to be critical [ 24 , 35 ]. In the Fst type I toxin, the charged residues of the C-terminal tail are important for maximal function of the toxin [ 23 ]. In ZorO, two charged residues, glutamate at the 16th position and arginine at the 23rd position, are consistently predicted to be in the transmembrane domain regardless of the in silico approach used ( Figure S1 ).…”

“…For example, the overproduction of membrane-localizing type I toxins Fst or BsrG result in either nucleic acid condensation or inhibition of cell wall synthesis [ 20 , 21 , 22 ]. Specific regions within a type I toxin protein, such as a conserved hydrophobic domain of the Fst toxin within enterococci, are highly sensitive to mutation rendering them non-toxic [ 23 ]. Likewise, the highly hydrophobic center of the IbsC toxin was the determinant for the toxicity of this protein [ 24 ].…”